Fundamental Immunology [ch 28-48, Seventh ed.] 1451117833, 9781451117837

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s: s: c: Coadministration of lysozyme in PBS or alum increases CTLA4-/- numbers in pancreatic node sixfold, and precipitates diabetes. lysozyme in CFA has similar effect for CTLA4+/+ cells.

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All, ala nina transaminasa;APC, anligan-prasanting call; ID, cluster of diffaranliation; CFA, complata Freund's adjuvant; CFSE, carboxylluorascain diacatsta succinimidyl astar; rn, cytotoxic T·lymphocyta; DC, dendritic call; DNA, deoxyribonucleic acid; HA, hemagglutinin; HEL, hen egg lysozyme; HSV, IFA, incomplete Freund's adjuvant; IFN, interferon; II... interleukin; L.CMV,Iymphocytic choriomeningitis virus; LN, lymph node; LPS, lipopolysaccharide; MHC, major histocompatibility complex; mRNA, messenger ribonucleic acid; CNA, ovalbumin; PBS, Phosphate Buffered Saline; SEB, stephy1ococcal enterotoxin B; TCR, T-cell receptor; Tg, transgenic;TNF, tumor necrosis factor; VSV, vesicular stomatitis virus. Color codas danota studias involving the following: yallow,axaganous suparantigans; orange, axoganous conventional protain antigans; purpla, andoganous convantional protain antigans; blua, MHC 11-rastrictad CD4 T calls; graan, MHC l-i'astrictad COB T calls; red, in vivo evidence of functionally tolerant ("anergic") T calls.

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these studies was by Kyburz et al.,254 who compared the fate of TCR-transgenic CDS cells recognizing a dominant peptide from LCMV presented by the MHC class I molecule, H2Db, after injecting thymectomized TCR-transgenic mice with LCMV peptide in incomplete Freund's adjuvant (which provides no microbial costimulus but creates a reservoir of peptide). The specific CDS T cells became large, increased in number, and developed cytotoxic T-lymphocyte (CTL) activity transiently, peaking on day 2 when many appeared apoptotic, with a rapid loss of blast cells and ex vivo CTL activity by day 3 and a drop in overall numbers below starting levels on day 5 and decreasing further on days 10 and 20. This abortive activation was independent of CD2S, although CD2S deficiency made the response even more transient.256 The cells present after day 2 appeared functionally tolerant, as they could not be induced to proliferate or form CTLs in vitro from this time onwards, even when their subsequent deletion was abolished by transgenic expression of either Bcl-2 or Bcl-XL.255 Deletion of the activated T cells in this model thus resulted from activation of the intrinsic pathway of apoptosis, implying that the peptide-activated T-cell blasts received insufficient external signals for cell growth and metabolism, whereas the extrinsic apoptosis pathways triggered by Fas or TNFR1 had no measurable role. 257•275 The Fas11Jr mutation nevertheless interfered with peripheral deletion of CD4 T cells in a parallel study of peripheral tolerance induced by injecting a peptide from cytochrome c without adjuvant into transgenic mice expressing an MHC II-restricted TCR.137 Similar studies in TCR-transgenic mice expressing an influenza hemagglutinin (HA)-specific MHC II-restricted TCR found that deletion could only be blocked by injecting an antibody to TNFa combined with the Fas11Jr mutation.139 Likewise, other studies also indicate a role for Fas in peripheral tolerance in certain settings.276•277 Thus, like the studies previously mentioned with superantigens, the relative role of the intrinsic and extrinsic pathways for deleting activated T cells in response to conventional peptide antigens may vary depending upon the TCR specificity and/or the duration and strength ofTCR stimulation.

Peripheral Tolerance to Exogenous Peptide Antigens in Adoptively Transferred T-Cell Receptor-Transgenic TCells The studies previously mentioned analyzed peripheral T-cell tolerance in situ either in TCR-transgenic mice with very high initial frequencies of antigen-specific T cells or in superantigen treated mice where the starting frequency of Vi}8+ CD4 T cells was also very high. A large number of experiments have nevertheless reinforced and extended these results by tracking acquisition of peripheral tolerance in smaller numbers of antigen-specific T cells that have been adoptively transferred from TCR-transgenic mice into wildtype mice with a normal T-cell repertoire (see Table 32.1). In one of the first studies of this type, Kearney et al. 250 harvested lymph node cells from DOll.lO TCR-transgenic mice, where most of the CD4 T cells expressed a TCR that could be detected by a clonotypic antibody (KJl-26) and recognized OVA peptide presented by MHC II I-Ed molecules. A total of 2.5 million K]l-26+ CD4+ cells were

injected into the circulation of normal BALB/c mice so that the antigen-specific T cells could just be detected by flow cytometry of lymph nodes, at a frequency of 1 cell in 300. The recipients were then challenged with a tolerizing regime comprising OVA peptide given intravenously in saline or intraperitoneally in incomplete Freund's adjuvant so that the antigen was encountered without a bacterial costimulus. For comparison, other recipients were given antigen as an immunizing regime comprising the same amount of peptide given subcutaneously emulsified in complete Freund's adjuvant containing an extract of Mycobacterium tuberculosis. With either regime, a fraction of the T cells were initially induced into S, G2, and M phase of cell cycle, and increased in frequency and absolute number in brachial and axillary lymph node on day 2 and 3. However, the tolerizing treatment caused the T-cell number to peak at day 3 and drop precipitously by day 5, so that the frequency of antigenspecific CD4 cells fell below untreated control recipients by day 17. By contrast, in response to the immunizing regime the T cells accumulated to higher numbers at day 3 and sustained these numbers at day 5, then falling gradually by day 17 although still very elevated over untreated controls. In the immunized animals, most of the KJI-26+ T cells were in B-cell follicles on day 5, reflecting what is now known to be differentiation into T-follicular helper cells that are critical for antibody responses, 278' 279 whereas they remained outside the follicles in the T-cell zone in the tolerized recipients. On day 17, compared to untreated control recipients, the T cells in the immunized recipients were hyperresponsive to antigen as measured by recall proliferation in vitro, whereas those remaining in the tolerized animals were hyporesponsive as measured by proliferation or IL-2 production even adjusted for their lower frequency. The latter anergic T cells had diminished surface TCR expression, yet had been induced to proliferate initially based on labeling with BrdU during the tolerizing regime. 252 Profound hyporesponsiveness of the latter was also observed in vivo, upon immunization of the tolerized mice with peptide in complete Freund's adjuvant. Thus, peripheral tolerance to a foreign peptide reflected abortive proliferation of CD4 T cells, failure to differentiate into fully fledged helper cells, and deletion of most of the activated T cells and anergy in the residuum. Further studies in the DOll.lO adoptive transfer model analyzed the effect of adjuvant and role of CD2S costimulation in the decision between peripheral tolerance and immunization. The effect of complete Freund's adjuvant in increasing the numbers of OVA-specific T cells that accumulated and their persistence was reversed by blocking CD80 and CD86 with monoclonal antibodies or a CTLA4-Ig fusion protein, whereas neutralizing the competing receptor for CD28 ligands, CTLA-4, further enhanced T-cell accumulation. 251 These results are consistent with at least one of the effects of bacterial adjuvant being the induction of CDSO and CD86 to higher levels on DCs, although they contrast with studies of the response to bacterial superantigen where CTLA4-Ig treatment did not block the ability of LPS to sustain the numbers of activated CD4 cells, and instead the bacterially induced cytokine tumor necrosis factor-a had a partial role?80 More recent studies using adoptive transfer

CHAPTER 32

of TCR-transgenic, OT-1, CDS or OT-11, CD4 cells bearing other OVA-specific TCRs have demonstrated that two other bacterially induced cytokines, IL-12 and IL-l, have a direct impact on the induction ofT-cell proliferation and effector function, in addition to induction of CD2S ligands.m-216 Along similar lines, peripheral tolerance induced by peptide without adjuvant could not be explained simply by absence of a CD28 costimulus, because when CD28-deficient DOll.lO T cells were adoptively transferred and the recipients treated with the tolerizing regime, the specific T cells exhibited an even more truncated proliferative response?81 By adoptively transferring CFSE-labeled DOll.lO-transgenic Ragl-/- T cells and treating the recipients with the tolerizing peptide regime, Turka and colleagues282 showed that blocking CDSO and CDS6 with CTLA4-Ig had no effect on the percentage of specific T cells induced by the peptide to express CD69, diminished the fraction of T cells that were induced into division on day 3 by only 30%, and had little effect on induction ofiL-2 in these cells provided it was measured as a function of cell division. Like CD28 deficiency, 281 CTLA4-Ig treatment nevertheless greatly decreased the number of accumulating daughter cells that had made multiple divisions, and this effect was blocked by transgenic expression of Bcl-XL.283 Thus, peripheral tolerance in this setting involves receipt of a limiting CD2S costimulus that sustains the survival of divided progeny through several divisions but is insufficient to sustain them after more than four divisions and for more than a few days. This abortion of proliferation after several cell divisions parallels the results from analyzing peripheral tolerance to SEB. 248 Complementary studies using a similar adoptive transfer design with other T-cell specificities reinforce this conclusion (see Table 32.1). For example, when CFSE-labeled CD4 cells bearing a lysozyme-specific TCR were adoptively transferred from TCR-transgenic mice into normal BlO.BR mice, they underwent abortive proliferation for several divisions but most of the divided progeny disappeared by day 5, even when the lysozyme peptide was presented by activated B cells bearing high levels of CDS6284 or by DCs.226 These studies collectively demonstrate that when T cells encounter exogenous antigen in the absence of microbial or adjuvant costimuli, various stages ofT-cell induction occur but the response is limited or truncated, leading to the outcome of tolerance. Although CD2S ligands play an important role as costimulators favoring T-cell immunity over tolerance, they are not necessarily sufficient on their own nor are they the only costimuli that favor inununity. Accordingly, the presence or absence of a CD2S signal is not the sole determining factor for whether or not T-cell tolerance is induced.

Peripheral Tolerance to Endogenous Peptide Antigens These studies have the caveat that they studied acquisition of tolerance in animals exposed to an exogenous, foreign antigen. Parallel studies nevertheless reinforce these conclusions by analyzing specific T cells during acquisition of tolerance to antigens that are made endogenously by the animal, either as natural self-antigens or in most cases "neo-self-antigens" encoded by transgenes that are present

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in the test animals but absent from controls and from the TCR-transgenic donor mouse (see Table 32.1). In this way self-antigens could be chosen where the peptide that was presented in a certain MHC type was known and where it was possible to follow the fate of initially nai:ve peripheral T cells specific for a particular endogenous antigen in vivo. Although TCR-transgenic models have their own caveats,285 in general this was a tremendous step forward as researchers could now ask the question what happens to self-reactive T cells in mice expressing an "endogenous" self-antigen, as opposed to studying what happens when a foreign antigen is administered to mice in a tolerogenic way. Rocha and von Boehme~ 4 o,253 were among the first to follow this approach to peripheral T-cell tolerance, taking advantage of a natural self-antigen, HY, that is present in males but absent in females because it is encoded by the Smc:y gene on theY chromosome. They harvested HY+Db-specific CDS T cells from the spleen of female Tl.70 TCR-transgenic mice, where they had matured without any exposure to HY antigen. These were injected into male mice, where they would encounter HY expressed ubiquitously, or into female controls that lacked HY. To facilitate tracking of the transferred T cells, nude mice that lack T cells were used as the recipients. The T-celllymphopenia in these recipients also introduces a complication, however, because it profoundly alters signals for T-cell proliferation and survival. Nevertheless, the numbers of T1.70 TCR+ CDS cells remained steady in female recipients but in the male recipients these dramatically increased by day 5 and then declined to half this number by day 9 and 25% of the peak number by day 60. On day 5, and especially days 9 and 20, both CDS and TCR were decreased on the surface of the cells in male recipients, and the day 9 and day 20 T cells were completely unresponsive to restimulation with male antigen presenting cells in vitro. Secondary transfers to male or female recipients showed that maintenance of the unresponsive state required continued exposure to HY antigen. The authors concluded that peripheral T cells become tolerant through a sequence of activation, proliferation, anergy, and death. Subsequent analysis showed that the deletion of activated HY-specific T cells in these experiments was blocked if they expressed a Bcl2 transgene but was unaffected by the FasPT mutation, indicating it was due primarily to the intrinsic pathway of apoptosis.286 Bertolino and Miller287 were among the first to extend these studies to trace peripheral T-cell tolerance to endogenous antigens after adoptive transfer of TCR-transgenic T cells into animals with a "full" complement of otherwise normal T cells. These studies tracked CDS T cells bearing the DES TCR, which could be detected with a clonotypic antibody and recognized H2Kb with an unknown selfpeptide. While H2Kb is normally expressed ubiquitously and would induce thymic deletion of DES TCR+ cells, to study peripheral tolerance they engineered BlO.BR mice expressing H2Kb as a transgene controlled by the metallothionein (MT) promoter, which is predominantly expressed in liver hepatocytes although also at low levels in other cells in thymus and lymph nodes. Two complementary approaches were followed to ensure that they studied only peripheral T-cell tolerance. First, they surgically removed

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the MT-K" transgenic thymus and replaced it with a nontransgenic thymus and traced the development of DES TCRtransgenic cells as they emigrated from the thymus and encountered self-peptide+Kb in the liver and lymph nodes. Second, they adoptively transferred naive T cells from DES 'OCR-transgenic mice into MT-K" transgenic B10.BR mice. These experiments revealed that the specific CDS cells were stimulated to proliferate and accumulate transiently in the liver, resulting in hepatitis and release of the liver enzyme ALT into the circulation that was self-resolving and followed by apoptosis and deletion of the T cells in the liver and other tissues, establishing a state of tolerance. Subsequent studies using this model288-29o revealed that the tempo and transient phase of tissue damage preceding peripheral tolerance were dramatically affected by whether the CDS T cells had encountered endogenous antigen on hepatocytes or on DCs. When they only encountered selfpeptide plus Kb on hepatocytes, proliferation of DES TCR+ CDS cells was aborted quickly after a number of cell divisions and between days 2.5 and 6 as a result of Bim-dependent apoptosis, without acquisition of CTL activity or induction of liver damage. The divided CDS cells were rescued from deletion by Bim-deficiency, but these did not acquire CTL activity or induce liver damage yet also did not appear to have become anergic as they could be stimulated to divide in response to Kb in vitro. By contrast, when transferred DES TCR+ cells also encountered self-peptide plus Kb on hemopoietic cells in lymph nodes (presumably DCs), this induced 100-fold greater accumulation of divided progeny and acquisition of CTL activity and hepatic damage, although that response was also ultimately self-limiting presumably due to exhaustive differentiation. The efficient induction of peripheral tolerance by hepatocytes compared to other cells potentially explains how cotransplantation of liver with other organs improves acceptance of allografts. Abortive proliferation as a mechanism for peripheral T-cell tolerance to tissue-specific endogenous antigens has been elegantly analyzed by Heath's group. 291' 292 The rat insulin promoter (RIP) was used to express membrane bound OVA in the ~-islet cells of the pancreas in C57BU6 mice. Kurts et al. harvested naive CDS T cells from lymph nodes ofTCR-transgenic mice bearing the OT-I TCR against an OVA peptide and H2Kb, labeled the cells with CFSE, and adoptively transferred them into RIP-mOVA transgenic recipients and nontransgenic controls. They made the surprising discovery that when mature OT-1 CD8 T cells reached the draining nodes of the pancreas, they encountered antigen that had been captured from pancreatic beta cells and cross-presented by cells that were later shown to be immature DCs. Cross-presentation by immature DCs did not cause anergy at any point but instead activated the OT-1 cells into more than eight rounds of cell division during the first 3 days, accompanied in this case by acquisition of cytotoxic function that destroyed the pancreatic islet beta cells within 9 days if 5 million OT-1 CDS cells were transferred. If islet cell destruction was avoided by analyzing chimeric mice where a different H2Kbm1 molecule was expressed on the pancreatic islets, so that OVA could only be presented by DCs and not by the islet beta cells, proliferation

of OT-1 cells was followed S weeks later by the loss of most of the T cells, demonstrating peripheral T-cell deletion as a mechanism ofT-cell tolerance. Deletion of the activated OT-1 cells was blocked by a Bc/2 transgene or Bim deficiency but unaffected by the Fa;pr mutation,293 implying that insufficient survival signals or cytokines or acute cytokine withdrawal was the cause of peripheral tolerance in this setting. Like the DES-TCR into MT-Kb experiments where lymph node cells presented the self-antigen,288-290 abortive proliferation was extensive in the transferred OT-1 cells and was accompanied by CTL activity, yet a key finding here was that this was only sufficient to cause diabetes if a very large bolus of CDS cells was introduced synchronously into the animals. If 250,000 OT-1 cells were transferred instead of 5 million, their abortive activation was insufficient to cause diabetes. Physiologically, T cells that escape thymic deletion or anergy will emigrate to the periphery asynchronously in much lower steady-state frequencies, so that their transient proliferation and acquisition of effector activity before deletion may not create any measurable tissue dysfunction. In this regard, a key extension of the RIP-mOVA model was to cotransfer several million naive CD4 T cells expressing the OT-11 TCR, which recognized another OVA peptide presented by l-Ab. While the CD4 cells did not cause diabetes, nor did they appear to increase OT-1 cell division, they nevertheless prevented deletion of the activated OT-1 cells and allowed 250,000 transferred OT-I cells to be sufficient to precipitate diabetes. 294 The decision between peripheral tolerance and immunity in this setting corresponds closely to Cohn's idea39 that immunity requires coincident recognition of two epitopes on an antigen, one by a helper T cell and one-in this case-by a cytotoxic T cell. Precisely how helper T cells protected cytotoxic T cells from peripheral deletion remains to be fully elucidated, as it could not be mimicked in the OT-1 RIP-OVA experiments simply by activating or "licensing" the DCs using an injection of agonistic antibody to CD40?95 Parallel findings were also made in another TCR-transgenic adoptive transfer model developed by Sherman's group, 217' 259.296-298 tracking CDS T cells bearing the Clone 4 TCR that recognizes an influenza HA peptide presented by H2r. The Clone 4 T cells were transferred into normal BALBIc mice expressing a transgene encoding HA under the insulin promoter (InsHA transgenic mice), either alone or together with CD4 cells from another TCR-transgenic strain expressing the HNT TCR against an lAd-presented HA peptide. In the absence of helper cells, a subset of the HA-specific CDS cells were induced to divide several times in the pancreatic lymph node but the daughter cells failed to accumulate in increased numbers due to Bim-dependent apoptosis and failed to acquire CTL activity or interferon gamma expression. When the HA-specific CD4 cells were cotransferred, a subset of these also divided one to four times in the pancreatic node, but this abortive proliferation of the CD4 cells failed to help the CDS cells and did not alter their abortive proliferation. However if the HA-specific CD4 cells were activated by influenza-infected spleen cells in vitro for 3 days before adoptive transfer, these fully activated helper T cells greatly increased the accumulation and persistence of divided HA-specific CD8 cells over

CHAPTER 32

the following 4 to S days, inducing them to make interferon gamma and precipitating diabetes.217 Treating the InsHA recipients ofHA-specific CDS cells with an agonistic antibody to CD40 was shown to increase the number of DCs in the draining node and their expression of CDS6, but was insufficient to mimic the effect of activated helper cells unless IL-12 was also given. Taken together with other studies mentioned previously,211- 216 these results indicate that abortive T-cell proliferation leading to peripheral tolerance is only switched into sustained proliferation and effector differentiation and tissue destruction when the T cells receive multiple costimulatory signals via CD2S together with other cytokines like IL-12, interferon alpha, or IL-l, and potentially other cytokines that are made directly by appropriately activated helper T cells. The conclusion that peripheral T-cell tolerance is acquired through abortive proliferation and deletion because of insufficient costimulatory signals is nevertheless unable to explain the fate of peripheral T cells encountering endogenous antigens in all adoptive transfer models. A striking example comes from the experiments of Mayerova and Hogquist/60 who transferred OT-1 TCR-transgenic CDS cells into C57BL/6 mice that were either transgenic for a Keratin 14 promoter-OVA peptide minigene that was expressed in skin keratinocytes or were transgenic for an actin promotermOVA construct expressed systemically. Although CFSElabeled OT-1 cells divided in skin-draining lymph nodes in both recipients, there was little accumulation of the progeny in the systemic expressors, and the OT-1 cells underwent abortive proliferation, failed to acquire effector function, and the remaining cells after 21 days were anergic. By contrast in the K14-0VA animals, a 100- to 1000-fold increase in OT-1 cells was observed peaking on days 4 to 6 and accompanied by the acquisition of interferon gamma and CTL function. These cells induced autoimmune dermatitis and vitiligo that became lethal if more than half a million OT-1 cells were transferred. Remarkably, the OT-1 T-cell response to endogenous K14-0VA was comparable to that induced in B6 recipients infected with vaccinia virus expressing OVA. A partial explanation for such strong induction of immunity and not peripheral tolerance in K14-0VA recipients was that the OT-1 T cells were activated by antigen presented by mature Langerhans cells, which had migrated from the skin to the draining node and expressed high levels of CDS6 and MHC II. However, given the evidence discussed previously and in Table 32.1 that TCR and CD2S costimulatory signals are insufficient to drive accumulation of large numbers of divided T cells and acquisition of effector function, it is likely that the Langerhans cells were also expressing IL-12 or other cytokines because of their exposure to products of skin bacteria. One might expect the presence of pathogenic stimuli to exert a "dominant" effect, resulting in the induction of immunity when Langerhans cells and immature DCs were both presenting antigen in K14-0VA x actin-OVA double transgenic mice. However, the opposite was observed: OT-1 CDS cells encountering endogenous antigen from the two sources underwent abortive proliferation, did not acquire CTL activity or cause dermatitis, and became anergic. Like other studies where dividing T cells become anergic when

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continuously stimulated by endogenous antigen distributed systemically, 240.253,261,263,264,268 the "dominance" of tolerance over immunity in K14-0VA x actin-OVA double transgenic mice may also reflect desensitization of TCR signaling to critical pathways forT-cell growth and differentiation like RAS-ERK-APl or NF-lCB. When endogenous antigen is only available from rare DCs in particular sites, and especially if these antigen-bearing DCs are eliminated by abortively activated T cells,299 the frequency of TCR engagement may in this setting not be sufficient to activate TCR desensitization and anergy, explaining the absence of anergy in other examples of abortive proliferation.289•293• 297 Chronic TCR engagement during abortive T-cell proliferation to endogenous or exogenous antigens also induces inhibitory signaling molecules on the activated T cells, which play an important role in suppressing the accumulation of divided progeny and their acquisition of effector functions. This has been particularly well demonstrated by Probst et al.300 in the case of PD-1, a negative signaling receptor displayed by chronically stimulated T cells. 301- 305 They engineered an elegant transgenic mouse strain that endogenously expressed two dominant peptide epitopes of LCMV in a subset of DCs. Expression was controlled by a drug-inducible genetic switch, which allowed a burst of peptide-presenting DCs to be formed in adult mice that had a normal T-cell repertoire and had not been immunized or infected. In chimeric mice, where half of the T cells lacked the PD-1 inhibitory receptor and the other half were wild-type, switching on peptide display induced accumulation of high frequencies (-2%) of LCMV peptide-specific CDS T cells expressing interferon gamma, revealed by flow cytometric straining with p/MHC I tetramers. However, these were all derived selectively from the subset ofT cells that were PD-1 deficient, with no measurable accumulation of corresponding T cells from the wild-type T cells in the same animals. If wild-type T cells were exposed to the burst of peptidedisplaying DCs for 12 days, they became actively tolerant as revealed indirectly by infecting the mice with LCMV and observing a 90% decrease in the accumulation of tetramer positive CDS cells recognizing the two LCMV peptides that had been expressed by DCs endogenously but not a third LCMV peptide that had not been encountered as "self" in the 12 days before infection. By infecting animals with equal mixtures of tolerant and naive T cells, they excluded T,g induction as a mechanism. While the actual fate of antigenspecific T cells could not be visualized in this setting due to their very low frequency, these studies establish that presentation of antigen by immature DCs leads to actively acquired tolerance by peripheral T-cell deletion or anergy, and this requires induction ofinhibitory receptors on the responding T cells to abort their proliferation. While abortive T-cell activation generally leads to a cell autonomous process of peripheral tolerance that is limited to the T cells that are deleted or anergic, in some settings partially activated self-reactive T cells accumulate and suppress other self-reactive T cells. This has been revealed by elegant analyses of a prevalent population of CDS T cells in diabetes-prone NOD mice, which proliferate in response to a pancreatic autoantigen, islet-specific

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INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

glucose-6-phosphatase catalytic subunit-related protein (IGRP) ,306.307 CDS T cells bearing TCRs with intermediate affinity for an IGRP peptide are not rendered tolerant in the thymus but, in combination with CD4 cells, are stimulated to proliferate in the pancreatic lymph node and destroy the pancreatic islet beta cells. However, other CDS T cells with very similar TCRs recognizing IGRP peptide with low affinity are stimulated to accumulate in the lymph nodes in a partially activated CD44hi CD62L+ state expressing interferon gamma and granzyme B but not IL-2. These low affinity self-reactive T cells suppress the islet antigen-induced proliferation of high-affinity T cells, in part by eliminating antigen-presenting DCs and B cells through perform-mediated killing. This mechanism may explain observations in the 1970s and 19SOs of CDS+ suppressor T cells. It also suggests that one of the functions of thymic positive selection is to populate the peripheral T-cell repertoire with low-affinity self-reactive T cells that actively suppress activation of any high-affinity self-reactive T cells that had escaped thymic deletion. Collectively, the studies summarized in the previous sections demonstrate that peripheral T-cell tolerance induced by exogenous or endogenous antigens is a result of suboptimal T-cell stimulation, that generally leads to the tolerized T cell undergoing rounds of division, acquiring effector function in some cases, but following an abortive sequence that is generally self-limiting. One major distinction between T-cell proliferation to tolerogenic antigens and immunogenic foreign antigens is that the former does not normally leave a population of memory T cells with heightened responsiveness, but instead leaves either a "hole" in the repertoire or T cells with diminished responsiveness that may in some cases be actively suppressive. Costimulatory signals such as those through CD2S, and other important cytokine signals that promote T-cell activation, contribute to the overall signals that drive T-cell immunity versus tolerance. However, the ability of specific costimuli to influence T-cell fate varies substantially for reasons that are imperfectly understood.

T Cell Ignorance Toward Undetected Self-Antigens A number of transgenic models traced the fate of T cells expressing a well-defined transgenic TCR specific for a tissue-restricted antigen and found that tolerance mechanisms such as thymic or peripheral deletion or anergy were not observed.3°8-m In some cases, animals developed spontaneous autoimmunity and in other situations no spontaneous autoimmunity occurred. This situation has become known as T cell ignorance. In one model, the LCMV-gp was expressed using the RIP. This led to the expression of the LCMV-gp in the P-islet cells of the pancreas. These mice were bred with P14 TCR-transgenic mice, expressing thereceptor specific for the LCMV-gp. Importantly, these double transgenic P14/RIP-gp mice and single transgenic RIP-gp mice clearly showed that the islet-specific T cells were not deleted in the thymus or tolerized in the periphery. LCMV infection of either RIP-gp mice or P14/RIP-gp mice resulted in the induction of diabetes, due to the cytotoxic activity of

CDS+ gp-specific T cells. Infection with a control virus such as vaccinia or an LCMV strain that did not encode the major gp epitope did not induce diabetes. Studies have suggested that immunologic ignorance is a consequence of low levels of self-antigen presentation.312.313

Regulatory T Cell Populations Maintain Tolerance and Limit Autoimmunity Many studies have shown that a population of T,..g cells can also be induced in the periphery. These cells have become known as induced T,.s (iT,.g) cells. These are also an important population of cells that inhibits autoimmunity. Evidence has shown that transforming growth factor P is an important factor that promotes the differentiation of this population, and that IL-10 production is important for their inhibitory function. 314•315 Further analysis has shown that iT"'"' arise upon presentation of low levels of antigen on DCs together with suboptimal maturation signals. Transforming growth factor P promotes the generation ofiT"'81 in this model. 316 Both natural Trego and iTrego appear to use several molecules to suppress antigen presentation and costimulation of peripheral T cells, thereby contributing to actively acquired tolerance. One of these is IL-10,317•318 which has long been known to downregulate the surface display of both MHC II and CD86 on DCs and macro phages.319- 321 Recently, this action ofiL-10 has been shown to be through the induction of the ubiquitin ligase, March1, which attaches ubiquitin molecules to cytoplasmic lysines in MHC II and CD86 and tags these proteins for degradation.322' 323 When DCs mature in response to LPS or other bacterial or viral products, they dramatically increase expression of CD83, and this protein functions by blocking the effects ofiL-10 and March1 to tip the balance in favor ofT-cell stimulation.323 Another critical molecule for tolerance and Treg function in vivo is CTLA-4, which has recently been established to function by stripping CDS6 and CDSO from the surface of DCs and other activated antigen-presenting cells.124" 24- 326 CTLA-4 and IL-10 from T,..gs thus work in concert to suppress delivery of signal1 and signal2 to helper and cytotoxic T cells in the absence of microbial costimuli. However, when large numbers of MHC Ilhigh CD86wgh CDS3high mature DCs accumulate in a lymph node because of a microbial stimulus, these will exceed the inhibitory capacity of CTLA-4 and IL-10 from Tross and favor T-cell activation. CTLA-4 and IL-10 are also produced by activated Foxp3- T cells under various circumstances, providing a potential feedback loop whereby clonal expansion and differentiation of activated T cells may similarly dampen signal1 and signal 2.

Fate of Auto reactive Mature T Cells These different mechanisms of peripheral tolerance act in different situations (Fig. 32.2). Autoimmunity may be prevented when self-reactive T-cell activity is limited by the influence of Tross. Alternatively, if self-antigen is presented at a level that is detectable by the T cell, then tolerance may occur either by deletion or anergy. If there is no detectable self-antigen, then T cells remain ignorant of the antigen.

CHAPTER 32 IMMUNOLOGIC TOLERANCE

I

715

Autoreactive Mature T cell

FIG. 32.2. Fata af Potantially Autoreactin Mature T Cells. If an autoreactive T cell does not encounter self-antigen in the thymus, then autoimmunity may be avoided if the T cell is silenced by a number of peripheral tolerance mechanisms. Regulatory T cells may inhibit the activity of the autoreactive T cell. Alternativelv, if the T cell engages cross-presemed self-antigen on a resting amigen-preseming cell, this would result in T-cell tolerance either by rendering the T cell anergic or by deleting the T cell. If the T cell does not encounter self-antigen, this would be known as T-cell ignorance and the T cell would persist as a na'ive T cell in the repertoire.

~No

/

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.....

I I \

/

'

......

~

/

-- ' __ ,

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1

\

I I

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Studies have shown that the level of self-antigen determines the fate of the self-reactive T cell.'13 Further studies have also provided evidence that low-avidity tissue-specific T cells can escape central and peripheral tolerance and potentially cause autoimmune disease.m-m

Why do Anergic T Cells Exist? Although T-cell anergy has been defined in vitro and in vivo, the idea that nonfunctional T cells remain in the T-cell repertoire is intuitively not attractive. How could the host benefit &om having nonfunctional T cells in the repertoire? Several groups have proposed a model where T-cell activation thresholds could be modulated, such that the resting threshold is set by tonic signals in the environment, and TCR engagement with "stronget" antigens could lead toT-cell activation.~ In this way, the "anergic'" T cells would actually be responsive to other antigens, thereby diversifying the T-cell repertoire. Experimental evidence has been published that supports this model.92-"l.m Others have suggested a model called adaptive tolerance.)» However, the concept that T cells have tunable activation thresholds still remains unclear. Importantly, Ta:p have often been considered to be anergic cells. Consistent with the classic definition of anergy, Tn:p can proliferate only in the presence of higher doses of IL-2 and cannot themselves produce IL-2. Whether the mechanism of anergy exists solely for the capacity for Tn:c function is currently unknown. However, studies have been reported that . ~"* evaluate connections between anergy and suppression. Molecular Pathways Involved in T-Cell Anergy Rfgardless of the functional reason why anergy exists, a tremendous number of studies have addressed the molecular pathway that programs the T-cell unresponsive state.

Anergy

Ignorance

Understanding the molecular basis for anergy may be important for manipulating this state in order to promote antitumor immunity or limit autoimmunity. The signaling events that lead to T-cell anergy have been studied from multiple perspectives. Studies have investigated pathways that fa~ to be activated in anergic cells. Others have evaluated vanous knockout animals to determine whether the induction ofanergy or activation is impaired. Importantly, inconsistencies may stem from the model that is used to induce T-cell anergy. Experiments that rely on chemicals to induce anergy, such as treating T cells with ionomycin alone, are evaluating a distinct way to disable signaling events in T ceUs. Furthermore, it is possible that CD4 T-cell anergy is different from pathways that induce CDS T-cell anergy. Madan and colleagues have proposed that anergy occurs when NFAT is preferentiaUy activated in the absence or reduced activation of AP-1 and NF-lCB. Accordingly, NFATde:ficient T cells cannot be anergized. By inhibiting the formation ofNFAT-AP-1 complexes, the cells can be shifted from activation to anergy."6-"7 In anergic cells, NFAT induces genes that inhibit TCR signaling cascades as well as inhibit cytokine production. Importantly, NFAT-dependent genes include Itch, Cbl-b, Grail, and caspase-3. Studies using a CD4-dependent system demonstrate that caspase-3 is essential for the development of anergy in T cells." 3 However, other studies demonstrate that caspase-3 is not required for ' duction ' ofCDS anergy m . VTVO. . lSI t hem Because CD2S is a key costimulatory molecule that promotes full T-cell activation, signaling pathways downstream of CD2S should be critical in limiting anergy. Evidence from several studies has shown that PKC9 is triggered downstream of TCR and CD2S stimulation.'~ 41 Importantly, anergy of CDS+ T ceUs can be readily induced in CD28and PKC9-deficient mice, and is regulated via the c-Rel

786

I

SECTION VI

INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

subunit ofNF-KB but not NF-KBl (p50).342' 343 Other models have also supported a role for PKC9 in preventing anergy and promoting T-cell activation.344 Early studies using a variety of models have also shown that the activation of the transcription factor AP-1, which is downstream of PKC9,34s,346 is important for regulating anergy.347 Evidence also supports a role for the Erk pathway in the activation of AP-1 and accordingly has also been shown to play a role in T-cell anergy. 348' 349 These and other studies support an important role for NF-KB and AP-1 in regulating CD4+ T-cell anergy.3so Early studies examining anergy suggested that this state is an actively maintained state and that continuous TCRderived signals are necessary.232' 253 Therefore, researchers have looked for molecules that are important in maintaining the anergic state. These molecules would be predicted to be upregulated in anergic cells. One candidate is known as Tob. Tob is a member of the APRO (antiproliferative) family and is expressed at low levels in quiescent cells.351 Another example is diacylglycerol kinase-a. Diacylglycerol kinasea is upregulated in anergic cells and has been shown to associate with RasGRP-Ras complexes and thereby inhibit signaling via the Ras-GRP/Ras complex and ERK activation.352 Accordingly, the induction of anergy is impaired in diacylglycerol kinase-a-deficient T cells or in the presence of pharmacologic inhibitors. 353 Several E3 ubiquitin ligases are involved in regulating anergy. As protein degradation is regulated by ubiquitination, it is possible that a functional T-cell response is regulated by the degradation of proteins involved in signaling and function ofT cells. Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that was identified using an in vitro screen for anergy. A T-cell clone specific for sperm whale myoglobin was stimulated with a fibroblast line transduced with the appropriate MHC molecule and/or CD80 and with or without antigen. In this way, the authors evaluated the expression of genes when the T-cell clone was stimulated with antigen and costimulation (CD80).354 Grail is expressed in resting CD4 mouse and human T cells and is upregulated in anergic cells. It is required for the induction of anergy in mouse CD4 T cells. 355- 357 Another E3 ubiquitin ligase that is important for regulating anergy is Cbl-b. Initial interest in this molecule was piqued when Cbl-b knockout mice were generated.358•359 Studies showed that the absence of Cbl-b rendered T-cell activation to be independent of CD28. Therefore, it appeared that Cbl-b negatively regulated costimulatory signals. Although Cblb-null mice generally do not develop spontaneous autoimmunity, they were more susceptible to experimentally induced autoimmunity. Interestingly, Cblb was also identified as a key gene that was mutated in the KDP rat strain that spontaneously develops type 1 diabetes.360 Experiments have also shown that Cbl-b-deficient T cells are unable to be anergized in vivo. 344'361 While Cblbdeficient B10.BR mice exhibit no evidence of autoimmunity, when it was combined with Aire-deficiency (which also caused little autoimmunity on its own), the combination resulted in autoimmunity directed specifically at the

exocrine pancreas and submandibular salivary gland that was lethal by 20 to 30 days of age.362 These results underscore the importance of peripheral T-cell tolerance through Cbl-b as a failsafe when self-reactive T cells escape thymic deletion. Importantly, studies have also shown that the absence of Cbl-b also results in T cells that are resistant to the regulatory properties of Treg•363-365 However, if the Cbl-b-deficient T cells were resistant to Treg activity, then one prediction would be that these mice develop autoimmunity in a similar way to Tre,-deficient mice. This, however, does not occur. One ofthe hallmarks of anergy is the reduced proliferative capacity of anergic T cells. This has prompted many groups to evaluate the importance of cell cycle regulation in anergy. Several important studies have been done with anergic cells that demonstrate alterations in the molecules involved in cell cycle progression. For example, rapamycin inhibits the mammalian target of rapamycin pathway, which is important for cell division and can induce anergy in T cells.366'367 Other studies have focused on a molecule known as p27kip that inhibits cell cycle progression. Forced upregulation of p27kip induces an anergy-like state, and accordingly anergy cannot be induced in p27kip-deficient cells. 368•369 Further studies of the p27kip pathway has identified a role for Smad3 in the induction of anergy.370 In general, T-cell anergy is a consequence of impaired signaling capacity in T cells, which has been shown to be regulated at many levels.371- 374 It is not yet clear how these pathways are interconnected in anergic cells or what internal programming is required to maintain anergy.

Breaking T-Cell Tolerance Autoimmunity can arise by many different mechanisms, some of which include the failure of tolerance mechanisms described previously. There is much interest in uncovering strategies to break tolerance, either to promote antitumor immunity or limit autoimmunity. In order to reverse an anergic state, several approaches have been taken, including stimulation of T cells through the common y chain of the IL-2R or triggering costimulatory molecules.375-378 Many approaches have also been taken to appropriately activate ignorant T cells. These strategies generally involve activation of the immune system with viruses, the administration of antigen together with other costimulatory signals, or other stimuli that lead to DC maturation.224•308.379-381 Notably, tumor growth can generate sufficient amounts of antigen to activate ignorant cells and promote immune surveillance in animal models.382' 383 One can speculate that similar tissue-specific "ignorant" cells may be responsible for providing immune surveillance in patients. Correlative studies have been reported by many groups that demonstrate that patients with a defined T-cell signature have a better prognosis.384- 387 It is compelling to speculate that there are ignorant T cells that exist in the T-cell repertoire that are activated to tumor antigens and can prolong survival of patients by promoting an antitumor response.

CHAPTER 32 IMMUNOLOGIC TOLERANCE

Studies have dearly shown that the costimulatory/ coinhibitory family of molecules play an important role in T-cell activation and also maintaining tolerance (Pig. 32.3). The absence of CfLA4, PD-1, or PD-Ll results in spontaneous autoimmunity and has been proposed to act largely by controlling autoreactive T-cell activity.!Oih'IIXZ,)l.t,m,,u PD-1 has two lisands, PD-Ll (B7-H1) and PD-L2 (B7-DC). PD-Ll is expressed on T cells, B cells, DCs, macrophages, as well as parenchymal cells, whereas PD-L2 expression is induced on DCs and macrophages. Importantly, studies have shown that the expression of PD-Ll on parenchymal cells also plays a role in maintaining peripheral tolerance. ~0

Dendritic Cell MHC

Parenchymal cell MHC 87-

family?

T ceiJ FIG. 32.3. Regulating Tolerance and h1111unity by Costimulation and Cainhibition. A:. lmeractions between naTve T cells and mature dendritic cells are critical for inducing the adaptive immune response but are also critical for maintaining tolerance. The costimulatory molecule cluster of differemiation (CD)28, binds to its ligands 87-1 (CD801 and 87-2 {CD86). The coinhibitory molecule CTlA4 is induced after T-een activation and competitively binds to the same ligands, with a higher affinity. Other members of the immunoglobulin superfamily also regulate these processes. PD-1 binds to PD-LI (87-Hl) and PD-L2 (87-DCiand sends an inhibitory signal toT cells. B: Members of the 87 family are expressed on a variety of cells other than the hematopoietic system, such as the islets cells in the pancreas. It is possible that the 87 family of molecules contribute to tissue homeostasis in a variety of settings.

I

717

There may be many unexplored molecules expressed by tissues that are critical for maintaining tolerance.

B-CEU. TOLERANCE: AcnvELY ACQUIRED TOLERANCE IN ntE PREIMMUNE B·CELL REPERTOIRE Actively Acquired B-Cell Sell-Tolerance Varies witla Antibody Affinity, Antigen Abundance, and Antigen Fonn Tracing the fate of self-reactive B cells accurately and controlling for the confoUll.ding effects of antibody heterogeneity, polyspecificity, and affinity, as discussed in the history section, has depended on engineering transgenic mice with rearranged Is heavy (IgH) and light (IgL) chain genes that encode antibodies of defined specificity and affinity. Initially, these transgenes were introduced in multiple copies cointegnted at a random chromosomal locus. Subsequently, the transgenes were targeted as single copies integrated into the endogenous IgH and IgL chromosomal loci. Collectively, these models have revealed that actively acquired tolerance in B cells is extensive. The first use of this approach by Goodnow and colleagues in 1988 employed an antibody against hen egg lysozyme, HyHEL10.~1 This antibody-antigen pair had been intensely characterized by Smith-Gill and Davies, including highresolution crystal structures, and exhibited the monospeci:ficity and affinity typical of highly selected, hypermutated antibodies that dominate long-tenn immunity against foreign protein antigens.m-'9' In transgenic mice inheriting both the rearranged lgH and lgL chain genes, most of the B cells that developed expressed cell surface IgM and IgD that only bound hen egg lysozyme. These foreign-specific B ceUs matured and recirculated amongst peripheral lymphoid tissue and, when mice were immunized with the foreign hen egg lysozyme antisen, these B cells were efficiently stimulated into clonal proliferation and antibody formation. Parallel sets of transgenic mice engineered to express hen egg lysozyme as a "neo-self" protein displayed actively acquired tolerance and were specifically unable to make antibody upon immunization with lysozyme.~1 In some lysozyme transgenic strains, only trace amounts of the antigen circulated in their blood (-10-10 M), which resulted in tolerance induction only in the helper T cell repertoire and not in B cells themselves.m,!l9s This greater sensitivity of T cells for acquiring tolerance, which was also observed in transgenic animals where lysozyme expression was limited to the pancreatic islets or thyroid gland, was later shown to be due to AIRB-independent and AIRB-dependent expression of the lysozyme gene in thymic medullary epithelial cells, which induced clonal deletion of lysozyme-specific CD4 T cells.1.67•596 However, T-cell tolerance could be bypassed and hish-affinity anubodies to lysozyme induced in these low-lysozyme animals by immunizing with lysozyme covalently coupled to horse red blood cells, establishing either absence or breakdown ofB cell tolerance to low-abundance, low-valency autoantigens.594 However, in mice with 10--9M or higher levels of circulating lysozyme, the same treatment

788

I

SECTION VI INDUCTION, REGULATION, AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

elicited only low-affinity antibody-secreting cells, implying that B-cell tolerance was induced as a result of sufficient exposure and affinity to the self-antigen and could not be overcome by strong T cell help to a linked foreign "'carrier." The existence ofactively acquired tolerance in individual B cells was clearly shown when lysozyme transgenic mice were crossed with the antilysozyme lg-transgenic mice, yielding four outcomes depending on the concentration and form of lysozyme expressed as a self-antigen (Fig. 32.4). Self-reactive B cells remained "ignorant" and exhibited little functional or numerical change when they developed in mice with -10-10 M circulating antigen in their blood, which was only sufficient to engage a few percent ofthe lysozyme binding receptors on the B cells.595 This circumstance included transgenic strains with tissue-specific lysozyme expression that was present at greater than Ilr M on thyroid epithelium or the pancreatic islet beta cells, because in these animals the preimmune B cells only encountered trace amounts oflysozyme that had been cleaved and released into the circulation.591 When lysozyme circulated at higher concentrations of 1~9 M or greater, the monomeric antigen continuously engaged> 40% of the binding sites in the lgM and IgD on the surface of the B cells, starting as soon as IgM was displayed on the surface of immature B cells in the bone marrow.!19l-'ll5."il& This did not delete or arrest the maturation ofthe B cells into ~ recirculating follicular B cells but caused them to selectively downregulate their surface IgM as they matured and induced a lasting functional change that made them much less responsive to lysozyme immunization as measured by proliferation and antibody secretion. Por simplicity, this state is referred to as B cell clonal anergy but, as with T cell donal anergy discussed previously, this single term should not be

Non-Tg

lg-Tg

ThyrHEL: lg-Tg

0 HEL antigen

blodlog

mistaken to connote a single mechanism. In fact, there are multiple separate inhibitory processes responsible for actively acquired tolerance in "anergic" B cells that are imperfectly understood, and these appear to vary depending on the nature of the autoantigen and antibody. .D:in:leric lysozyme, circulating at concentrations that ensaged an identical percentage of B-cell antigen receptors, triggered a more extreme surface IgM downregulation and arrested the maturation of the B cells at the immature 1'giY- CD21~aw CD24hi CD93+ CD62L- stage that characterizes their first arrival from the bone marrow to the spleen (see Pig. 32.4).!199 These cells were short-livedin the spleen, profoundly "anergic" not only to antigen but to LPS and, because of their lack of the CD62L lymph node bottling receptors, very few reached peripheral lymph nodes. Anergic B ceUs encountering monomeric lysozyme were similarly arrested as a shortlived IgM- IgD'- immature population in the spleen in mice that contained normal numbers of competing B cells!00 The extreme fate for self-reactive B cells was observed in transgenic mice displaying membrane-tethered lysozyme present on the surface of many bone marrow and blood cells, which resulted in almost complete absence of lysozymebinding B cells in the spleen and peripheral lymph nodes,401 including cells with very low affinity (-10-6) for the antigen.4oa This fate was first demonstrated forB cells in Ig-transgenic mice expressing an allo-antibody against MHC I proteins that were ubiquitously displayed on bone marrow cells•.w.404 The existence of this spectrum of actively tolerant states in B cells has been reinforced and extended through the analysis of Ig-transgenic mice expressing many clinically significant antibody specificities. These are summarized in Table 32.2 and specific mechanisms descnbed in more detail subsequently. sHEL: lg-Tg

6 6

sHEL2: lg-Tg

mHEL: lg-Tg

~

r ,;JI I_ I

l ·

Antigen receptors on cell surface

HG. 32.4. Tracing til aFata of Salf-Raactin B Calls in Transgenic Mice. Flow cytometric plots are gated on spleen B cells, with each dot rep· resenting one cell. These show on the x-axis the log1arelative abundance of surface immunoglobulin (lg) on each 8 cell, stained with antibody to kappa light chain. On they-axis is the log10 relative amount of hen egg lv5ozyme (HEll bound to each B cell, detected by staining the cells first with a saturating concentration of lysozyme and then a fluorescent antibody to lysozyme. In normal C57BIJ6 nontransgenic mice, the rare lysozyme-binding B cells detected are heterogeneous and probably represent poly-specific or nonspecific binding. In mice inheriting lgH and lgl transgenes, almost all the 8 cells bind lysozyme monospecifically, and cell-to-cell variation in binding is tightly correlated with variation in the amount of surface lg on each cell. Clonal ignorance is observed in double-transgenic mice expressing the lysozyme gene selectively in the thyroid epithelium: the frequency of lysozyme binding cells is unaltered and the amount of surface lg they display is decreased by less than twofold. In double transgenic mice where monomeric lysozyme accumulates to higher concentrations in the circulation and continuously engages -J.iO% of the antigen receptors on the developing B cells, actively acquired tolerance in the B cells is reflected by selective downregulation of their surface lgM receptors but the cells mature to the lgDhiOh recirculating stage. In a matched double transgenic strain where the same proportion of receptors is engaged by dimeric lysozyme, lgM and lgD surface antigen receptors are downregulated accompanied by developmental arrest atthe Tl-T2 stage, and edited B cells accumulate with normal densities of antigen receptors that do not bind lysozyme. Finally, in a double-transgenic strain where lysozyme is displayed in polyvalent form on the membrane of other blood and marrow stromal cells, lysozyme-binding B cells are almost completely eliminated and replaced by edited B cells.

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SECTION VI INDUCTION, REGULATION, AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

TABLE: •?'• Summary of Fate of Self-Reactive B Cells in Different Immunoglobulin-Transgenic Mouse Models (Cont} Fate af B Cells

Antigen

Antibody

Reference

Cardiolipin, histones, cytoplasmic and nuclear antigens Membrane phosphatidytserine Double-stranded DNA, phosphatidylserine Double-stranded DNA, nuclear antigens Double-stranded DNA higher affinity Double-stranded DNA Sialylated glycolipids, Sialyi·Lewis x

2F5 H and L antibody to HIV gp41 from chroni· cally infected patient 3H9 gennline VH 3H9 VH and Vk (Vk4t from MRLJipr lupus mouse

432

3H9 VH with Vlambda2* from MRLJipr lupus mouse 3H9 VH with S&R CDR2 mutation from MRl/lpr lupus mouse SP8 Hand L chain to TNP hapten GAS VHS-51 and Vk1 from Waldenstroms macroglobulinemia cryoglobulin

414,415,4:111 431,&18

411,52Q

m 4ZI

BCR, B-cell receptxr. CD, clllltllr of dilfwentiation; DNA. deoxyribonucleic acid; Ig. immunoglobulir~; LPS,Iipopolylaccharide; MHC. major hiltocompllllbilily compleJ,; RNA. ribonucleic acid.

Green mea111 •go-; yellow meena moderate warning {moderately seH·reactive); orange mea1111trong warning {ltronglv self·reaclival; and red mea111"stop- Ivery self·reaclivel.

Monospecificity. Polyspecificity. and the Need to Balance Immunity with SeH-Tolerance An overwhelming lesson from the studies summarized in Table 32.2 is that many self-reactive B cells are not eliminated in the bone marrow but reach the spleen and in many cases circulate to lymph nodes. Why are so many self-reactive B cells allowed to reach the spleen and circulation? The likely explanation is that the immune system cannot afford simply to discard antibodies that have unwanted self-reactivity and has the opportunity to remodel these antibodies

Nascent g repertoire in bone marrow ~

g.

Edited repertoire reaching the spleen

by hypermutation and purifying selection in germinal centers405.406 (Fis. 32.5). There are compelling mathematical arguments that germline antibodies need to be polyspecific to provide adequate coverage of microbial epitopes with a finite pool of preimmune B cells. Increasing the crossreactivity and polyspecifi.city of each antibody minimizes the chance of having a gap in the repertoire due to limited B cells but this is offset by progressively larger holes in the antibody repertoire due to these antibodies also binding self-antigens.407- 410 I nggenng Uevelopmental set-point - - arrest

r

Edit /

1

~Cell death

death

Adapted repertoire mediating lasting immunity

RG. 32.5. Gradual Ewlution of Antibadies with Minimal SeH~eactivity. Self-reactive B cells are eliminated in a series of steps that balances the need for self-tolerance against the need to maimain clones for immunity. (Top) Hypothetical distribution of clones are plotted by degree of autoreactivity (x axis) against clone frequency (y axis). The degree of autoreactivity is a function of the amount of autoantigen presented and the avidity with which it is bound by the clone. Most newly formed B·cell clones have low but appreciable self-reactivity, but only a subset appear to exceed an inherited set point and trigger either elimination in the bone marrow or editing to lower self-reactivity. Clones with less autoreactivity are exported to the periphery, illustrated by the hypothetical distribution of clones in the midd/6. By this time, repeated bind· ing of autoantigens has tuned down sulface immunoglobulin signaling in the more self-reactive end of the spectrum, illustrated by orsngs shading. Binding of autoantigen and competition for follicular niches also trigger exclusion and death of the more self-reactive clones in the T-cell zones, although these cells can potentially be rescued by T cells if they bind foreign antigens with much higher avidity than they bind to self. As a result of follicular competition, together with hypermutation and further selection in germinal centers, the B-een repertoire that recirculates for weeks or months among lymphoid tissues is skewed toward a small subset of B cells with the least autoreactivity (bottom). A much larger range of newly produced clones is nevertheless available in the T-cell zones of the spleen (middle) to be tested for its fit against microbial antigens and potemially recruited for transient antibody responses or remodeling in genninal centers. (Adapted from Goodno~).

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Indeed, a large fraction of the preimmune antibody repertoire does exhibit polyspecificity that includes binding to a variety of self-antigens.22- 28 This may reflect conformational flexibility in the binding sites of germline antibodies, which then become locked into a monospecific shape by point mutations arising during germinal center selection.411 Allowing many B cells bearing antibodies with self-reactivity to reach the peripheral lymphoid tissue may enable gradual refinement of these antibodies in germinal centers by hypermutation and the twin forces of purifying selection to "breed out" the negative trait of self-reactivity and positive selection for better binding to foreign microbes.405,4°6

B-Cell Receptor Editing in the Bone Marrow Nemazee's analysis of Ig-transgenic mice expressing an antibody to the MHC I protein, H2K\ initially suggested the B cells were deleted as soon as they expressed this antibody and bound the self-antigen on neighboring bone marrow cells, because no cells with surface IgM could be detected.403•404 However, more sensitive staining and cell mixing experiments in membrane lysozyme transgenic mice revealed that the immature B cells did not initiate apoptosis in the first day or two after binding the self-antigen but instead they actively downregulated their surface IgM to very low levels and arrested their maturation at a stage that was intermediate between immature B cells and small pre-B cells.412 This response was reversible if self-antigen ceased to bind to the arrested B cells before the onset of apoptosis: when removed from membrane lysozyme, the B cells regained high surface IgM and matured to the IgD positive stage. This seemed an oddly precarious way to eliminate forbidden clones of B cells. Nemazee413 and Weigert414 independently recognized that the transgenic mice had illuminated an entirely new mechanism for actively acquired tolerance: instead of clonal selection as envisaged by Burnet, this was a system for receptor selection. N emazee showed the B cells that had downregulated their surface IgM had also increased their expression of Rag2 mRNA, encoding the lg-gene recombinase, and contained abundant circular DNA excision products from rearrangements of the endogenous lambda light chain.413 He proposed that strong binding of self-antigens actively induced "receptor editing": by initiating another round oflight-chain rearrangement, the self-reactive antibody could be replaced by one that was less self-reactive. Indeed, a subset of the B cells with the self-reactive antibody to H2K or to double-stranded DNA in Weigert's experiments were salvaged from clonal deletion and matured by expressing less self-reactive antibodies comprised of the transgenic heavy chain paired with an "editor" light chain derived from an endogenous light chain gene rearrangement. The latter were highly selected and did not bind MHC I or double-stranded DNA. In the transgenic experiments, receptor editing was nevertheless inefficient because the transgenic light chain gene was located at a random chromosomal site where it was not flanked by upstream VK elements that could be joined to downstream JK elements. Second generation Ig-transgenic mice were then generated with the rearranged light chain VJK exons integrated into the endogenous light chain locus

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flanked by upstream VK and downstream JK elements.415--4 17 In these "knock-in'' Ig-transgenic animals, self-antigen binding triggered efficient replacement ofthe offending light chain by joining an upstream VK element to a downstream JK element. Indeed, when competitive bone marrow chimeras were established, bone marrow B cells that started out with prerearranged but self-reactive receptors nevertheless produced edited mature B cells as efficiently as competing B cells that needed to rearrange their H and L chain genes de novo.418 The efficiency with which a self-reactive antibody can be edited is likely to vary depending on what fraction of the light chain repertoire can serve as "editors" and exhibit sufficiently lower binding to self. Some H-chains have such a strong propensity to bind self-antigens like DNA, (for example, binding to DNA due to long CDR3loops and multiple charged arginines in their binding site) that a very small minority of oppositely charged light chains can salvage these H-chains.419.420 Analyses of mice with normal antibody genes indicate that 20% to 50% of productive light chain rearrangements are edited by a second light chain rearrangement.421--423 This is likely to be driven primarily by editing of self-reactivity because 55% ofthe antibodies produced by immature B cells with undetectably low surface IgM are poly-specific for self-antigens whereas only 7% of the antibodies in surface IgM+ immature B cells are poly-specific. The downregulation of surface IgM that is induced by self-antigen binding412 is comparable to the downregulation of non-self-reactive H-chain that occurs when they pair with surrogate light chains in pre-B cells that have yet to rearrange their light chain genes.424 When a light chain displaces surrogate light chain and IgM accumulates on the B cell surface, the receptor complex spontaneously signals through the PI3kinase pathway and inactivates the Foxol transcription factor, which in turn is required for Ragl and Rag2 gene expression. Downregulation of surface IgM by self-antigen allows Foxol to accumulate, reactivate Ragl and Rag2, and induce a second round of light chain recombination.425- 427 Dramatic downregulation of surface IgM, developmental arrest at the pre-B/immature B cell transition, followed by receptor editing or deletion, has been demonstrated in Istransgenic mice expressing many different antibodies (see Table 32.2). These include damaging antibodies to the blood group surface antigens alpha-Gal428 and sialyl-Lewis X,429 antibody with high affinity for lgG2a,430 a bispecific antibody that binds DNA and the foreign hapten TNP,431 antibody against membrane phosphatidylserine with low affinity for DNA,432 a demyelinating antibody to myelin oligodendrocyte glycoprotein that cross-reacts with an additional unknown selfantigen,433 nephrotoxic antibodies to glomerular basement collagen or laminin-the latter bispecific for DNN34.435 -and a broadly neutralizing antibody to human immunodeficiency virus (HIV) that is also polyspecific for cardiolipin, histones, cytoplasmic, and nuclear antigens. 420 The last example analyzing the 2F5 antibody to HIV provides a compelling clinical example of why the immune system cannot afford to discard antibodies with self-reactivity: in this case, it appears that cross-reactivity between a conserved epitope on the virus and multiple self-antigens stands in the way of developing broadly neutralizing immunity.

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Anergy and Peripheral Deletion of Self-Reactive B Cells Depending on their affinity and specificity, self-reactive B cells that escape or fall below the threshold required to induce developmental arrest and editing in the bone marrow follow two general fates in the spleen: 1) antigen/antibody pairs where the self-reactive B cells become mature follicular cells marked as IgDJUgh CD241.,. CD93- and L-selectin+, which recirculate to lymph nodes and may be relatively longlived; and 2) B cells that are very short-lived, mostly found in the spleen at the immature transitional stage marked as IgD~cw CD24JUgh CD93+ and L-selectin-, and few recirculate and reach the lymph nodes (see Table 32.2).

Anergy in Recirculating Immunoglobulin Dhitlll B Calls The longer-lived, recirculating self-reactive B cells are characterized by selective downregulation ofsurface IgM but high surface IgD. In Ig-transgenic mice with varying concentrations of soluble lysozyme, the extent ofigM downregulation is closely correlated with the fraction of antigen-engaged receptors and occurs after the cells have passed through an IgMhigh IgD~cw CD93+ CD24hl transitional stage. 395•393 This prompted the hypothesis that the broad range of surface IgM that exists from cell to cell among lgDhlgh L-selectin+ B cells in the normal repertoire reflects variable degrees of self-reactivity and that the 25% of IgD+ B cells with lowest surface IgM were anergic.395 Recent analyses in humans confirm this is indeed the case.436' 437 The IgM1.,. IgDJUgh fate has been observed forB cells in Ig-transgenic mice with antibodies that have relatively low affinity to single-stranded DNA and potentially other autoantigens,415' 438" 39 antibodies that are polyspecific for DNA and the foreign hapten arsonate,440--442 a lupus antibody to the Smith ribonucleoprotein antigen,443' 444 and an antibody to insulin.445'446 A similar fate occurs in normal humans for the -10% of naive B cells that use the VH4-34 heavy chain variable segment, which is almost invariably self-reactive to 1/i glycolipids in the membrane of erythrocytes.447 Selective downregulation of IgM but not IgD on anergic cells is due to rapid recycling of internalized antigen-IgD complexes back to the cell surface448 and to degradation of newly synthesized IgM in the endoplasmic reticulum. 449 The halt to IgM traffic probably results from being outcompeted for CD79 subunits with IgD, whose mRNA is increased in anergic cells,'50 and to changes in chaperone activity from depletion of endoplasmic reticulum calcium, due to constant release of calcium to the cytoplasm by inositol-3-phosphate. Selective downregulation of IgM occurs in the absence of antigen-binding when inositol-3-phosphate activity is not dampened by the enzyme inositol 1,4,5-trisphosphate 3-kinase B.451 The antigen receptors on the surface of anergic B cells are desensitized at a proximal point in their intracellular signaling cascade, activating less tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs in CD79alpha and beta and Syk kinase, with a concomitant decrease in the magnitude of intracellular calcium.440" 52--454 Desensitization of antigen receptors on anergic cells depends upon normal function of the SHP-1 (PTP1C) tyrosine

phosphatase455 and the antigen receptor-activated tyrosine kinase Lyn.456 Lyn initiates antigen receptor signaling but at the same time it activates SHP-1 as a negative feedback, by phosphorylating immunoreceptor tyrosine-based inhibitory motifs in inhibitory cell surface receptors such as CD22, Siglec-G, and CD72 that recruit SHP-1 to antigenbound receptors. 456- 461 Desensitization of the intracellular response to antigen in anergic B cells is nevertheless pathway specific. It selectively dampens intracellular signals that activate the NFKB transcription factor and the c-jun N-terminal kinase. 462 Low magnitude (-200 nM) spikes of intracellular calcium are repeatedly induced as long as anergic B cells continue to be exposed to self-antigen.462 While these are too low for NF-KB activation, they are sufficient to promote repeated cycles of calcineurin-mediated dephosphorylation of the NFATcl and NFATc2 transcription factors, triggering their nuclear translocation and inducing a subset of NFAT-target genes.450" 62" 63 Likewise, continuous activation of the MEKERK kinase pathway in anergic B cells induces target genes like Egrl and opposes induction of the plasma cell transcription factor Blimp-1.450.462.464.465 Rather than making no response to antigen, the anergic B cells induce a unique pattern of mRNAs when they encounter antigen compared to B cells that encounter the same antigen as a foreign protein.450 These early events of continuous calcium spiking and Egr1 induction cease if the B-cell antigen receptors stop being exposed to self-antigen!53•462 Recovery of the intracellular calcium flux in response to a new round of antigen-engagement occurred within minutes of removal from self-antigen in polyspecific DNA/TNP B cells,453 whereas it had not recovered after 36 hours in lysozyme-anergic B cells.452 In the latter, full recovery of surface IgM takes between 4 and 10 days, and the capacity to make a T-cell-dependent plasma cell response does not recover even 10 days after removal from antigen, as long as the B cells remain in a resting, follicular state and are not activated into cycle through other receptors such as TLRs or by primed helper T cells.466 Thus both short-lived and long-lived inhibitory feedbacks are at work in anergic B cells, and the latter are still poorly understood.

Short-lived. Immunoglobulin Mlow Immunoglobulin D111w Anergic B Calls The short-lived splenic fate has been observed for selfreactive B cells bearing antibodies with intermediate affinity to double-stranded and single-stranded DNA,441A67--470 ribonucleic acid, 471 high-affinity human rheumatoid factor antibody, 472--474 high-affinity arthritogenic antibodies to glucose-6-phosphate isomerase,475 hemolytic antibody to erythrocytes, 476--478 very-low-affinity autoantibody to a glyco-epitope on the T-cell membrane protein Thy1,479 membrane lysozyme or membrane H2Kb limited to 5% to 10% of blood cells,480'481 and membrane H2Kb MHC molecule or a kappa light chain crosslinking "superantigen'' limited to liver hepatocytes.482- 484 In each of these examples, the self-reactive B cells develop in the bone marrow with little downregulation of IgM and hence most cells are not edited at that point. The B cells are found in the spleen primarily as transitional immature cells

CHAPTER 32

with downregulated surface IgM and low IgD, low complement receptor CD21, little CD62L lymph node homing receptor, and high expression of CD24 and CD93. Anergic, self-reactive B cells with this phenotype represent -5% of the peripheral B cell pool in normal mice.485 The IgMiow B cells persist in the spleen for only 1 to 3 days before being eliminated by the intrinsic, Bcl2-regulated apoptosis pathway400 that reflects antigen-induced increase in the proapoptotic Bcl-2 inhibitor Bim.486•487 Anergic B cells in the spleen require higher concentrations of the anti-apoptotic cytokine BAFF for their survival compared to B cells that are not selfreactive,467 and their elimination can be delayed or blocked in animals that have increased BAFF.484•488 This heightened dependence upon BAFF provides a basis for the selective clinical benefits of BAFF antagonists in some people with autoantibody-mediated diseases.489 During the time that short-lived anergic B cells remain in the spleen, they are excluded from the B-cell follicles and concentrate at the junction with the T-cell zones.400·467·471•475 Concentration at the T-B junction is a general effect observed in B cells that have bound antigen, either self or foreign, and is due to BCR signals that increase CCR7 expression and enhance responsiveness to the CCL19 and CCL21 chemokines produced by stroma in the T zone.490 When B cells that have acutely bound foreign antigen are attracted to this site, they are also triggered to express high levels of the T-cell costimulatory molecule CD86 and to enter Gl of cell cycle. By contrast, self-reactive B cells have already been exposed to the same antigen in the bone marrow,398'445 and as a result have become desensitized and do not induce CD86 or enter cell cycle when antigen signals them to migrate towards the T zone in the spleen.452,491 Although they can present antigen to unprimed T cells, this leads to active killing of the anergic B cells through the sequential action ofT cell-derived CD40L and FasL. 284' 492.493

Salvaging Self-Reactive Antibodies from Anergic B Cells A key point is that desensitization to antigen is relative and not absolute. Anergic B cells can activate high intracellular calcium flux, induce CD86, and collaborate with naive T cells to proliferate and make antibody if their antigen receptors are stimulated by a much more potent agonist than the one to which they have grown accustomed. In other words, they are not unresponsive but have tuned down their responsiveness to antigen, similar to that suggested for selfreactive T cells (see previous discussion). This is illustrated by the calcium flux and plasma cell reaction generated when B cells anergic to monomeric soluble lysozyme are acutely stimulated by polymeric membrane lysozyme and provided with helper T cells that recognize foreign MHC II molecules on the B cells.452 Similarly, biotin-streptavidin polymerized lysozyme, but not monomeric lysozyme, was able to transduce signals that block Fas-mediated apoptosis in lysozyme anergic B cells, whereas both agonists delivered the protective signal in naive B cells.494 Primed T follicular helper cells276' 279 may also salvage anergic B cells that are multispecific and also bind foreign antigen. As antigen-presenting cells, anergic B cells are poor inducers of IL-4 and OX40 from unprimed helper T cells

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and this is a result of their lack of CD86, because anergic B cells that express CD86 constitutively recover the capacity to induce these helper T cell proteins, to evade Fas-killing, and to mount T-cell-dependent antibody responses. 495 IL-4 is well documented to override Fas-mediated apoptosis in B cells.494,496,497 Moreover, while anergic B cells make a muted proliferative response to CD40 stimulation alone, the combination of CD40 and IL-4 stimuli elicits heightened proliferation compared to naive B cells.452' 468'493 This switching capacity of IL-4 may enable primed follicular helper T cells to salvage B cells with antibodies that cross-react with foreign and self-antigens and induce their proliferation in germinal center reactions, where their antibodies can be remodeled to eliminate self-reactivity but preserve or enhance binding to foreign antigens.405 T cell-independent stimuli, in the form of foreign antigens linked to ligands for TLRs, can also divert short-lived anergic B cells from death to proliferation. This is best exemplified by the short-lived anergic B cells that result from an encounter with the H-2Kb protein in the liver while en route to the spleen.483 Potent cross-linking of their antigen receptors by bacteriophages bearing a mimotope of the Kb molecule rescued these anergic B cells from peripheral deletion in the spleen and stimulated them into autoantibody formation. Reversal from anergy in this instance may result from the absence ofliver-specific antigen in the spleen. Continuous exposure to self-antigen in lysozyme anergic B cells resulted in the sustained activation of the ERK signaling pathway, which blocked plasma cell differentiation and antibody secretion even when the B cells were induced to divide with TLR9 or TLR4 stirnulation.464.465 In addition, some self-antigen/antibody pairs result in a state of anergy in the short-lived splenic B cells that blocks the proliferative response to TLR4 ligands.46M 71 Collectively, these experiments indicate that abnormal processing of endogenous TLR ligands like DNA or ribonucleic acid, 499- 503 or an overactive response of TLRs, may cause aberrant rescue of selfreactive B cells.471' 504- 507 In several antigen/antibody pairs analyzed in transgenic mice, B cells bearing self-reactive antibodies are anergic and short-lived in the spleen, yet become expanded in the peritoneal cavity as Bl cells where they secrete autoantibodies. In Ig-transgenic mice expressing an antibody against erythrocytes, expansion of Bl cells depends upon their relative shielding from erythrocytes in this compartment476,477 and requires concurrent infection with pathogenic or commensal microorganisms.478 It is not known whether the infectious microorganisms present a cross-reactive foreign antigen that stirnulates the self-reactive B cells or ifthey simply provide potent TLR stimulation of B cells. In either case, the IgM antibody that is secreted binds with high avidity to erythrocytes and causes severe autoimmune hemolytic anemta. A similar situation occurs in Ig-transgenic mice expressing a low-affinity germline antibody against a carbohydrate epitope on the T-cell protein, Thyl, where the B cells are arrested as anergic immature cells in the spleen, few reach the lymph nodes, but they accumulate as Bl cells in the peritoneal cavity and secrete anti-Thyl IgM autoantibody.479

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In this case, the antibody binds with insufficient affinity to Thyl to cause any depletion of T cells. Remarkably, the induction of Bl cells and autoantibodies requires the Thyl autoantigen and is abolished in Thyl-deficient mice. It is not yet known if this also requires infection with commensal or pathogenic microbes, like the antierythrocyte Bl cells. When Thyl is restored at 10% of normal levels in the mice, it changes the fate of the self-reactive B cells again by inducing their maturation into marginal zone B cells that do not secrete autoantibody.508 These experiments support the view that some low-affinity polyspecific antibodies may be positively selected into the plasma cell, Bl, or marginal zone pool by their binding to self-antigens, because these antibodies are not harmful to self-tissues but represent protective antibodies against common microbial antigens.27

Clonal Ignorance in Self-Reactive B Cells The fate of "clonal ignorance," where self-reactive B cells are not tolerized because they do not bind sufficient autoantigen, has also been observed for a number of antibodies expressed in Ig-transgenic mice. These include a low affinity rheumatoid factor antibody against IgG/09' 510 a polyspecific VHl-69 germline antibody from a chronic lymphocytic leukemia,473' 511 an antibody against the lupus and SjOgren syndrome antigen SSB/La,512 and a low-affinity antibody against single-stranded DNA,469 although in this case the B cells displayed some level of anergy. 415' 438,4lll In the case of the low-affinity rheumatoid factor antibody, careful analysis revealed that the self-reactive B cells were not ignorant per se, but a small number were actively stimulated by selfantigen to form short-lived, nonproductive germinal center reactions.510 This has some parallels with the stimulation of autoantibody production by low-affinity binding of Thy-1 self-antigen, although in that case the B cells were excluded from the follicular/germinal center pathway and formed plasma cells via the Bl B-cell pathway.479

TOLERANCE FROM BENCH TO BEDSIDE-A PLAY IN THREE ACTS This chapter summarizes the enormous progress that has been made in understanding immunologic tolerance and the long and winding road involved. But it is fair to say that we are only in the early parts of a play of three acts. The first act has been to define the physiologic mechanisms of actively acquired tolerance. This has proved much more complex than any human mind could have dreamt, and while many mechanisms have been defined, the recent discoveries of Foxp3+ Tre8 s and AIRE indicate that even this first act is still in full swing. There are likely to be entirely new mechanisms revealed in the future, and answers are yet to come about fundamental questions like how lymphocytes choose alternative fates of death, survival, or growth at a given tolerance checkpoint.

The second act is to define the root cause of spontaneous autoimmune disease. Rapid progress is currently occurring in this area, particularly in simpler, monogenic forms of autoimmunity following on from autoimmune lymphoproliferative syndrome, APS-1, and IPEX. In the more common sporadic autoimmune diseases, MHC is the largest shared genetic factor, yet we still do not understand why particular MHC haplotypes are so strongly associated with particular autoimmune diseases and not with particular infectious diseases. With respect to the non-MHC heritable elements, unanswered questions remain as to what proportion are common versus rare genetic variants. Currently, this is an area of great activity, through genome-wide association studies of unparalleled size and the dawn of an exciting era of whole exome and genome sequencing in patient cohorts. The third act will be to use the knowledge gained in the first and second acts to develop specific agents that induce or restore physiologic mechanisms of actively acquired tolerance in autoimmunity, allergy, or transplantation, or agents that break tolerance to allow immune clearance of cancer cells or chronic infections like HIV. The success of bone marrow transplantation in children with primary immunodeficiency, where immunosuppressive agents can be withdrawn within months, represents progress directly from Medawar's bench to the modern bedside. The induction oftolerance to foreign RhD erythrocyte alloan tigens in primiparous RhD-negative mothers is the best present-day example of the ability to induce antigen-specific tolerance reliably, although in this case clinical development was empirical and we do not really understand why it works. Analysis of B- and T-cell anergy has revealed the limitations of cyclosporin and tacrolimus as blocking both lymphocyte activation and actively acquired tolerance, 236•450 and helped promote a shift to "tolerance sparing" immunosuppressive drugs like rapamycin and mycophenolate. Perhaps the most exciting evidence for what is to come is the clinical approval in 2011 of two human antibodies that really begin to use the amassed knowledge of tolerance for rational therapeutics. One is a blocking antibody to BAFF, belimumab (Benlysta), which is supported by the principle that self-reactive B cells require more BAFF than non-self-reactive B cells.400' 484' 487' 488 This heightened dependence upon BAFF provides a basis for the selective clinical benefits of BAFF antagonists in some people with autoantibody-mediated diseases. 489 The other milestone is the clinical approval for a blocking antibody to CTLA-4, ipilimumab (Yervoy),513•514 which works on the principle that T cells that recognize melanoma antigens are not all deleted but are unable to mount a beneficial autoimmune response. Neither Yervoy nor Benlysta represent a "penicillin moment" for clinical therapy. They are a small step for most patients, but the clinical response that occurs in a subset represents a giant leap for mankind. These agents mark the start of the age when we understand immunologic tolerance sufficiently to induce, restore, or suspend it in practical ways.

CHAPTER 32 REFERENCES

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CHAPTER 32 REFERENCES 495. Rathmell JC, Fournier S, Weintraub BC, Allison JP, Goodnow CC. Repression of B7.2 on self-reactive B cells is essential to prevent proliferation and allow Pas-mediated deletion by CD4( +) T cells. J Exp Med. 1998;188:651-659. http:/ /Www.ncbinlm.nih.gov/pubmed/9705947 496. Nakanishi K. Matsui K, Kashiwamura S, et a1 IL-4 and anti-CD40 protect against Fas-mediared B cell apoptosis and induce B cell growth and differentiation. Int ImmunoL 1996;8:791-798. http:/ /Www.ncbinlm.nih.gov/pubmed/8671668 497. Foote LC, Howard RG, Marshak-Rothstein A, Rothstein TL IL-4 induces Fas resistance in B cells. JImmuooL 1996; 157:2749-2753. http:/ /Www.ncbinlm.nih.gov/pubmed/8816375 498. Eris JM, Basten A, Brink R, Doherty K, Kehry MR, Hodgkin PD. Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigenreceptor-mediated functions. Proc Natl Acad Sci US A. 1994;91 :43924396. http://Www.ncbinlm.nih.gov/pubmed/7514304 499. Nagata S. Autoimmune diseases C811Sed by defects in clearing dead cells and nuclei expelled from erythroid precursors. Immunol R~. 2007; 220:237-250. http:/ twww.ncbinlm.nih.gov/pubmed! 17979851 500. Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG, Mcroy T. Features of systemic lupus erythematosus in Dnase !-deficient mice. Nat Gen. 2000;25:177-181. http:/ twww.ncbinlm.nih.gov/pubmed! 10835632 501. Yasutomo K, Horiuchi T, Kagami S, et al. Mutation of DNASE! in people with systemic lupus erythematosus. Nat Gm. 2001;28:313-314 http:/ twww.ncbinlm.nih.gov/pubmed! 11479590 502. Lee- Kirsch MA, Gong M, Chowdhury D, et al. Mutations in the gene encoding the 3'-5' DNA exonuclease TREXl are associated with systemic lupus erythematosus. Nat Gen. 2007;39: 1065-1067. http:/ twww.ncbinlm.nih.gov/pubmed! 17660818 503. Rice G, Newman WG, Dean J, et a1 Heterozygous mutations in TREXl cause familial chilblain lupus and dominant Aicardi-Goutieres syndrome. Am JHum Gen. 2007;80:811-815. http://www.ncbinlm.nih.gov/pubmed! 17357087 504. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature. 2002;416: 603-607. http://www.ncbinlm.nih.gov/pubmed! 11948342 505. Pisitkun P, Deane JA, Difilippantonio MJ, Tarasenko T, Satterthwaite AB, Bolland S. Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication. S~nce. 2006;312:1669-1672. http:/ twww.ncbinlm.nih.gov/pubmed! 16709748 506. Deane JA, Pisitkun P, Barrett RS, et a1 Control of toll-like receptor 7 expression is essential to restrict autoimmunity and dendritic cell proliferation. Immunity. 2007;27 :801-810. http:/ twww.ncbinlm.nih.gov/pubmed! 17997333 507. Subramanian S, Tus K.. Li QZ, et a1 A Tlr7 translocation accelerates systemic autoimmunity in murine lupus. Proc Natl Acad Sci U S A. 2006;103:9970-9975. http:/ twww.ncbinlm.nih.gov/pubmed! 16777955

508. Wen L, Brill-DashoffJ, Shinton SA, Asano M, Hardy RR, Hayakawa K. Evidence of marginal-zone B cell-positive selection in spleen. Immunity. 2005;23:297-308. http://www.ncbi.nlm.nih.gov/pubmed/16169502 509. Hannum LG, Ni D, Haberman AM, Weigert MG, Shlomchik MJ. A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease. JExp Med. 1996;184: 1269-1278. http://www.ncbi.nlm.nih.gov/pubmed/8879198 510. William J, Euler C. Primarolo N, Shlomchik MJ. B cell tolerance checkpoints that restrict pathways of antigen-dri~n differentiation. JImmunol. 2006;176:2142-2151. http://www.ncbi.nlm.nih.govtpubmed/16455970 511. Koenig-Marrony S, Soulas P, Julien S. et al. Natural autoreactive B cells in transgenic mice reproduce an apparent paradox to the clonal tolerance theory. JImmunoL 2001;166:1463-1470. http://www.ncbi.nlm.nih.gov/pubmed/11160185 512. Aplin BD, Keech CL, de Kauwe AL, Gordon TP, Cavill D, McCluskey J. Tolerance through indifference: autoreactive B cells to the nuclear antigen La show no evidence of tolerance in a transgenic model J Immunol. 2003;171:5890-5900. http://www.ncbi.nlm.nih.govtpubmed/14634099 513. Hodi FS, O'Day SJ, McDermott DF, et a1 Improved survival with ipilimumab in patients with metastatic melanoma. N Engl JMed. 2010;363: 711-723. http://www.ncbi.nlm.nih.gov/pubmed/20525992 514. Robert C. 1homas L, Bondarenko I, et al. Ipilimurnah plus dacarbazine for previously untreated metastatic melanoma.. N Engl JMed. 2011;364: 2517-2526. http://www.ncbi.nlm.nih.govtpubmed/21639810 515. Mamalaki C, Tanaka Y, Corbella P, Chandler P, Simpson E, Kioussis D. T cell deletion follows chronic antigen specific T cell activation in vivo. Int IimmunoL 1993;5:1285-1292. http://www.ncbi.nlm.nih.gov/pubmed/8268134 516. Kramer S, Mamalaki C, Horak I, Schimpl A, Kioussis D, Hung T. Thymic selection and peptide-induced activation of T cell receptortransgenic CDS T cells in interleukin-2-defi.cient mice. Eur J ImmunoL 1994;24:2317-2322. http://www.ncbi.nlm.nih.gov/pubmed/7925559 517. WaithmanJ, Allan RS, Kosaka H, etal. Skin-derived dendritic cells can mediate deletional tolerance of class !-restricted self-reactive T cells. J Immunol. 2007;179:4535-4541. http://www.ncbi.nlm.nih.gov/pubmed/17878350 518. Belz GT, Behrens GM, Smith CM, et a11he CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. JExp Med. 2002;196: 1099-1104. http://www.ncbi.nlm.nih.govtpubmed/12391021 519. Roark )H, Bui A, Nguyen KA, Mandik L, Erikson J. Pc:Iliistence of functionally compromised anti -double-stranded DNA B cells in the periphery of non-autoimmune mice. Int Immunol. 1997;9: 1615-1626. http://www.ncbi.nlm.nih.gov/pubmed/9418123 520. Chen C, Radic MZ, Erikson J, et al. Deletion and editing of B cells that express antibodies to DNA./ Immunol. 2004;152:1970-1982. http://www.ncbi.nlm.nih.gov/pubmed/812040 1

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CHAPTER 33

While T-bet is the master transcription factor controlling Th1 responses, it is also expressed by a subset of Treg cells and is required for Treg cell homeostasis and function during polarized Th1 responses. While IFNy can inhibits the peripheral generation of Foxp3+ Treg cells from naive CD4+ T cells,102 IFNy signaling through STAT1 activation drives T-bet expression in Treg cells resulting in control ofTh1-type immune responses. IFN"f may thus limit the magnitude and duration of Th1-type inflammatory responses. T-bet+ Treg accumulate at sites of Th1 inflammation and T-bet-/- Treg cells display impaired proliferation during Th1-type inflammation and do not control the expansion of IFN"(-producing Th1 cells when transferred into scurfy mice.236 The failure ofT-bet-/- Treg cells to control Th1-driven inflammatory responses may result from their inability to survive and proliferate in a highly polarized Th1-type environment. Lastly, conditional deletion of STAT3 in Treg cells results in the selective dysregulation of Th17 responses accompanied by development of a spontaneous fatal intestinal inflammation with elevated levels of IL-17 production. 237 The mechanisms by which T-bet, IRF4, and STAT3 control Treg cell activity during Th1, 2, and 17 responses are still unclear but likely involve control of Treg-cell migration, function, and homeostasis. Selective expression of transcription factors associated with a given Th responses may induce molecular changes needed to restain a particular response. This hypothesis has important implications for the therapeutic uses of Treg cells as it implies that specific subsets of Treg cells may be needed in treating Th1-, Th2-, and Th17mediated inflammatory diseases.

REGULATORY T CELL PLASTICITY The studies on Treg specialization have demonstrated the potential benefical effects ofT helper cell produced inflammatory cytokines on the ability of Tregs to control pathologic immune responses mediated by those cytokines. Other studies have challenged the notion of a committed Foxp3+ Treg cell lineage and raised the possibility that Treg cells can lose Foxp3 expression when present in an inflammatory milieu or following transfer to an immunodeficient environment and be "reprogrammed" to become pathogenic T effector cells. For example, during infection with Toxoplasma gondii, Treg cells lose Foxp3 expression and become IL-12 responsive. They can then express high levels ofT-bet, acquire Th1 effector characteristics, and produce IFN"f contributing to lethality. 238 Furthermore, when Treg cells were cultured under Th17 or Th1 polarizing conditions, a substantial fraction of IL-17+Foxp3+ and INF"(+ Foxp3+ T cells were observed.239 Thus, II...-6 may potentially convert Treg cells into proinflammatory Th17 cells in a proinflammatory environment.Z40' 241 No evidence of reciprocal conversion of Th17 cells to Treg was observed. The Treg and Th17 programs of differentiation are clearly linked, 101 and Foxp3 and ROR"(t transcription factors can interact with one another.101 Whereas transduction of naive CD4+ T cells with ROR"(t induced IL-17 expression, cotransduction with Foxp3 abrogated IL-17-producing capacity when Foxp3 contains its exon 2-encoded domain, which is

REGULATORY/SUPPRESSOR T CELLS

I

819

required for binding to to ROR"(t, After in vitro induction of ROR"(t and Foxp3 by TGF-1}, cells do not express IL-17 and have the potential to differentiate into either Th17 or Treg depending on the cytokine environment. In the presence of proinflammatory cytokines (IL-6, IL-21, or IL-23) and low concentrations of TGF-~, ROR"(t expression is further elevated and Foxp3 expression is reduced resulting in Th17 differentiation. Conversely, in the absence of proinflammatory cytokines and high concentrations of TGF-1}, Treg induction is promoted. IL-2 suppresses ROR"(t expression and promotes the induction of Tregs.242 Runx1 can also form a complex with ROR"(t to promote Th17 differentiation, but can also interact with Foxp3 to suppress IL-2 and IFN"f production, resulting in enhancement of Treg suppressor function. Functional plasticity in cells that coexpress Runx1 and ROR"(t may be governed by the ability ofRunx1 to cooperate with either transcription factor. 243 Foxp3+ Treg cells in Peyer patches differentiate into Tfh cells that can then participate in germinal center formation and IgA synthesis in the gut.244 The potential plasticity of Tregs is supported by epigenetic studies of cytokime gene expression. Wei et al. 245 have performed genome-wide examination of histone modifications and DNA methylation that accompany changes in gene expression in CD4+ T cell subsets. Trimethylation of histone H3lysine 4 (H3K4me3) is a permissive mark found in the promoters and enhancers of active genes, whereas trimethylation of histone H3lysine 27 (H3K27me3) is present in broad domains that encompass inactive genes. The epigenetic marks found at the IFN"f, IL-4, and IL-17 genes correlate precisely with Th1, Th2, and Th17 lineages. However, these genes in Tregs were bivalently modified, and this result is consistent with their potential plasticity. These bivalent modifications may allow specific lineage regulator gene loci to be activated under different polarizing conditions thus allowing reprogramming of Tregs into other lineages. In addition to having permissive epigenetic modification ofT effector cytokine genes, Tregs may express GATA3, the canonical Th2 transcription factor. GATA3 is highly expressed in Tregs at barrier sites such as the skin and the gut and is readily expressed in Tregs following TCR activation. Treg intrinsic expression of GATA3 is required for maintenance of high level of Foxp3 expression and promotes Treg accumulation at inflamed sites.246 Expression of GATA3 was not seen in Tregs expressing T-bet or ROR"(t. In one study, GATA3 expression by Tregs was required for Treg accumulation in inflammatory conditions such as GI infection or EAE and GATA3-/-. Tregs failed to control colitis due to impaired accumulation. Another study demonstrated a more profound defect in Treg function in Treg conditional GATA-1- mice, including the development of a generalized inflammatory disorder, decreased expression of Foxp3 and Foxp3 signature genes, and decreased suppressor function in vitro. 247 Two groups recently generated mice that were engineered to track the fate of Foxp3+ cells in vivo. In these systems, mice expressing the Cre recombinase in Treg cells under control of regulatory elements from the Foxp3 gene, were crossed with mice in which the ere-mediated recombination removes a stop codon from within a fluorescent protein

820

I

SECTION VI

INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

encoding a reporter gene that was knocked into the ubiquitously expressed Rosa 26 locus. Thus, in these animals, cells even transiently expressing Foxp3 are permanently marked, and their phenotypic properties can be spatially and temporarily examined. Using this system, Zhou et al.246 reported that a portion of CD4+Foxp3+ T cells downregulate Foxp3 and acquire the ability to produce effector cytokines such as IFN"f even in the absence of any experimental manipulation. The frequency of these ex-Treg cells was increased in the pancreatic islets of NOD mice, indicating that they may contribute to immune pathology. These studies suggest that highly polarized inflammatory environments can subvert Treg-cell function by converting them to Foxp3- effector T cells. The other study demonstrated that Foxp3 expression by Treg cells is remarkably stable, even in highly inflammatory settings.l49 The discrepancies in these studies may be due to differences in the inflammatory sytems used to examine Treg stability in vivo, or to differences in the way the reporter mice were constructed. Alternatively, Tregs that have lost Foxp3 may not be the product of reprogramming of committed Treg cells, but may represent the products of a minor population of uncommitted cells that transiently express Foxp3.250 These cells can accumulate by selective expansion and under the appropriate environmental conditons differentiate into Thl, Th2, or Thl7 T effector cells.251

FOXP3-- REGULATORY T CELLS Thymic-derived Foxp3+ Tregs or Foxp3+ Tregs induced in peripheral sites represent the major populations of Tregs that have been characterized in both normal physiologic studies and in disease models. A number of other Treg populations have been described in both mouse and man, and many of these also appear to exert potent Treg functions in certain defined situations. One important distinction between these induced populations of Tregs and the thymicderived Foxp3+ Tregs is that the TCR of the former are very frequently specific for known antigens, while the antigenspecificity of the Foxp3+ T cells are likely preferentially specific for self-peptide MHC class II.

Th3 Cells One of the first approaches used for the induction of Tregs was the administration of antigen via the oral route. Oral tolerance takes advantage of the normal physiologic process that is needed to prevent systemic immune responses to ingested proteins. Oral administration of antigen at low doses induces populations of Tregs that secrete suppressor cytokines, while higher antigen doses result in deletion or donal anergy of autoreactive precursors. Pretreatment with orally administered antigen-induced suppressor populations that inhibited pathology in a number of different animal models of autoimmunity including EAE, collagen-induced arthritis, and uveitis.252 Bulk T cells from orally tolerized animals can suppress active immune responses to other antigens in the microenvironment, a phenomenon called antigen-driven bystander suppression. The suppressor cells from orally tolerant mice have been termed Th3 cells and mediate their

suppressive effects primarily by secreting TGF-~. A major advance in our understanding of the function of regulatory cells following oral tolerance was the study by Chen et al.,253 which successfully isolated T-cell clones from the mesenteric lymph nodes of SJL strain mice that had been orally tolerized to myelin basic protein. These clones produced large amounts ofTGF-P and varying amounts ofiL-4 and IL-10. Most importantly, upon adoptive transfer to normal recipients, they suppressed EAE induced with either myelin basic protein or proteolipid protein. Their in vivo suppressive activity could be neutralized with anti-TGF-1}. The selective induction of Tregs via the oral route is thought to be secondary to certain poorly characterized properties of the gut mucosal microenvironment, most likely the type of resident APCs.254•255 Although the oral administration of antigen represents a potentially easy way to induce Tregs, progress in this area has been slow because it has been difficult to determine the antigen concentration that is capable of inducing Tregs, but which does not induce deletion. It has also been very difficult to isolate the types of clones described previously in the EAE model in other systems or to identify Treg cells that exclusively produce TGF-P in other models. The therapeutic utility of orally administered antigens in autoimmunity has primarily been demonstrated in pretreatment protocols, and oral administration of antigen was ineffective in treating animals once disease has been initiated. Nevertheless, in animal models, the oral administration of antigen can result in the induction of Foxp3+ antigen-specific Tregs that can modulate immune responses. These Foxp3+ Treg populations can produce TGF-1}, but the relationship of the Foxp3+ Tregs to what was formally described as Th3 cells remains unclear.

T Regulatory 1 (Tr1) Cells One important lesson that can be derived from the experiments on oral tolerance is that the milieu in which T cells are primed is critically important in determining whether regulatory rather than effector T cells will be generated. Decreased expression of costimulatory molecules on APCs or the presence of suppressor cytokines such as IL-10 and TGF-P may generate suppressor T cells rather than effector T cells. The production of these suppressor cytokines by Tregs may lead to the generation or expansion of additional regulatory cells via a positive feedback loop. One of the first studies demonstrating the potential importance of IL-10 in the generation of Tregs was derived from an analysis256 of patients with severe combined immunodeficiency (SCID) who received transplants of human leukocyte antigen (HLA) -mismatched hematopoietic stem cells. Complete immunologic reconstitution was achieved in the absence of graft versus host disease. CD4+ T-cell clones reactive with host MHC antigens from these patients produced IL-10, but not IL-2, after antigen-specific stimulation in vitro. It therefore seemed likely that endogenous IL-10 production in the transplanted patients was responsible for maintaining tolerance in vivo. The IL-10 may prevent the activation of host reactive T cells or suppress APC function and cytokine production by host APCs. The high IL-10

CHAPTER 33

production in vivo may reflect a chronic activation of donor T cells and host monocytes. IL-10 is a cytokine produced by numerous cell types induding activated T cells, B cells, mast cells, and macrophages, and primarily acts by inhibiting the maturation and function of APCs. The activation of CD4+ human T cells in the presence of IL-10 renders them nonresponsive or anergic:.257 Activation of human CD8+ T cells with allogeneic APC and IL-10 also results in reduced proliferation and cytotoxicity. T cells rendered anergic by the addition of exogenous IL-10 in an mixed leukocyte reaction (MLR) beoome unable to respond to a rechallenge with the same antigen. IL-10-iinduced anergy is strictly antigen-specific as treated T cells retain normal proliferative and cytotoxic: responses toward other protein antigens and third-party alloantigens. Collectively, these studies suggest that IL-10 itself is a major factor for the induction of suppressive IL-10producing T cells. Culture of mouse or human CD4+ T cells with antigen or alloantigen in the presence of IL-10 results in the generation of IL-10-producing T-cell dones. Most of these T-cell dones produce high levels ofiL-10 and TGF-~, moderate amounts of IFNy and IL-5, but no IL-2 or IL-4. CD4+ T cells generated in this manner have been termed T regulatory 1 (Trl) cells.258 Tr1 cells proliferate poorly following polyclonal TCR-mediated or antigen-specific activation and do not expand significantly under standard T-cell culture conditions. This low proliferative capacity is due in part to autocrine production of IL-10, as anti-IL-10 mAbs partially restore proliferative responses. The intrinsic low proliferative capacity ofTrl cells has been a major limitation and has hindered their detailed characterization. The ability to generate human Trl cells is enhanced by the addition of IFNo:.259 Trl cells can be generated from human cord blood with IFNo: alone, as cord blood T cells have the intrinsic: ability to produce IL-10. IFNo: and IL-10 do not act as general antiproliferative agents, but rather as factors that induce the differentiation ofTrl cells and inhibit the growth ofnonTrl cells in the culture. Tr1 cells do not express Foxp3 and can be generated in the absence of Foxp3+ Tregs. Both human and mouse Trl clones suppress immune responses in vitro. Antigeninduced proliferation of naive CD4+ T cells was dramatically reduced following ooculture with activated Trl clones that were separated from the responding cells by a trans-well insert. The capacity of either human or mouse Trl clones to suppress CD4+ T cell proliferation was reversed by the addition of anti-TGF-1} and IL-10 mAbs.258 Some IL-10producing Tr1 cells suppress the proliferation of naive CD4+ T cells by an IL-10-independent, cell contact-dependent mechanism.260' 261 Human Trl cell clones also suppress the production oflg by B cells, as well as the antigen-presenting capacity of monocytes and DCs. Most importantly, it could be shown258 that mouse Trl dones have regulatory effects in vivo and suppress Th1-mediated colitis induced by transfer of CD45RBhi cells into SCID mice. Suppression was only seen if the Trl clones were activated by antigen-specific stimulation via their TCR. Because the function ofTrl cells is mediated by IL-10 and TGF-~, these studies imply that Tr1 clones can suppress active immune responses to unknown antigens

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in the microenvironment by an antigen-driven bystander suppression mechanism similar to the mechanism proposed for Th3 cells in oral tolerance. Although IL-10 was originally described as a product of mouse Th2 cells, Trl clones are also capable of regulating Th2 responses including antigenspecific IgE production.261 Anti-lL-lOR antibodies reversed this inhibitory effect. An alternate approach to the generation of Trl cells in vitro has involved pharmacologic manipulation of the microenvironment during T-cell priming. 262 The immunosuppressive drug 1, 25(0H)2-vitamin D3 (VitD3) acts on APCs and activated T cells. VitD3 inhibits antigen-induced T-cell proliferation, cytokine production, and the maturation of human DCs, leading to inhibition of the expression of CD40/CD80/CD86 expression. Similarly, the glucocorticoid dexamethasone inhibits key transcription factors involved in the regulation of a number of inflammatory cytokine genes. When naive CD4+ T cells were stimulated through their TCR in the presence of the combination of VitD3 and dexamethasone, the primed T cells produced IL-10, but not IFNy, IL-4, or IL-5. The IL-10-producing cells developed independently of Thl (IL-12, IFNy) and Th2 (IL-4) polarizing cytokines, and addition of these cytokines inhibited the development of the IL-l 0-producing cells. Although the induction of these Tregs could not be induced by IL-10, endogenous IL-10 production was required because addition of anti-IL-lOR antibodies substantially reduced the number of IL-10-producing T cells. The development of the IL-10-producing T cells occurred under conditions where the expression and/or activation of key transcription factors involved in Thl (T-bet) and Th2 (GATA3) differentiation was minimal, suggesting that the IL-10 producers were completely unrelated to conventional Thl or Th2 cells. VitD3/dexamethasone-induced Trl cells are also capable of inhibiting the induction of Th1-mediated autoimmune disease in vivo. One common theme to emerge from the studies on the in vitro induction of Trl cells, either by the addition of IL-10 or of pharmacologic agents, is that both of these modalities are likely to inhibit the maturation and activation of DCs and to generate what has been termed "tolerogenic DCs."263 Clinical trials are ongoing to evaluate the potential therapeutic: effects of Trl cells in the prevention and treatment of GVHD after bone marrow transplantation. The clinical protocol involves the transfer of ex vivo generated Trl cells to patients with hematologic cancers treated with hematopoietic: stem cell transplantation. Treatment of patients with IL-10-anergized donor T cells has potential to reconstitute immunity, prevent GVHD, while guarding against infection and recurrence of cancer. The inabilityto expand Trl cells in large quantity has hampered progress in understanding the biology of these cells. The relationships between Trl cells that produce IL-10 and Foxp3+ Tregs that also produce IL-10 remain undear. IL-10 reporter mice have been generated264 in which expression of IL-10 was replaced with expression of a Thy-1.1 reporter, facilitating identification of cells producing IL-10 spontaneously in vivo. This mouse stably identifies all cells in which IL-10 alleles have been previously activated as well as those

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c:ells actively transcribing IL-10. Foxp3+IL-10- Treg c:ells were more frequenc:t in LN and spleen, while Foxp3+1L-10+ Treg cells were more frequent in the lymphoid tissues of the large intestine. In the small intestine and Peyer patches, Foxp3-IL-10+ Treg cells were the most predominant population and had a cytokine profile, proliferative response, and suppressive function typical ofTrl c:ells. The Foxp3-IL-10+ population developed from Foxp3- precursors in all tissues and its development was dependent on TGF-1} but not IL-10. The presence of these cells in the gut may explain why IL10-/- mice and mice treated with an anti-IL-10R succumb to intestinal inflammation despite having normal numbers of Foxp3+ c:ells in the intestines. Thus, IL-10 has a nonredundant function in maintaining immune homeostasis to the diverse intestinal microbiota. The relative contributions of the Foxp3+IL-10+ and Foxp3-IL-10+ producing subsets is still unclear. As the two populations are located in different parts of the intestinal tract, it remains possible that a distinct class of commensal organisms would preferentially induc:e both of the subsets. Rec:ent studies have demonstrated that IL-27, a member of the IL-12 cytokine family consisting of two subunits, p2S and EBI3, is a differentiation factor for the generation of IL-10-producing Trl cells.265' 266 IL-27 is secreted by tolerogenic DCs that were conditioned in vitro or in vivo by Foxp3+ Treg c:ells. These Treg-modified DCs express plasmacytoid-like markers. The addition of IL-27 to naive T cells induced expansion and differentiation of CD4+ T cells that secreted high levels of IL-10. 267 IL-27-driven Tr1 cells proliferate poorly following TCR-mediated activation and suppress T effector responses through IL-10 production. Granzyme B is also induced by IL-27 during Tr1 differentiation and may mediate suppression of T effector responses by cytolysis. IL-27 also c:an induc:e IL-10-produc:ing human Trl cells.268 Signaling via the arylhydrocarbon receptor may modulate Trl differentiation. Naive T cells differentiated in vitro with IL-27 in the presence of the arylhydrocarbon receptor ligand, TCDD, had enhanced secretion of both IL-10 and IL-21.

CDB+ Regulatory T Cells Most of the early studies on T suppressor cells in the mouse demonstrated that they were confined to the CDS+ subset. Almost all of the recent studies on suppressor/regulatory T cells in mouse or man have focused on CD4+ T cells. Several studies have suggested that potent CDS+ suppressor c:ells may also exist. Repeated stimulation of human T c:ells in the MLR resulted in a progressive decrease of the capacity of CD4+ T cells to proliferate when rechallenged with the APCs used for priming. The relative nonresponsiveness of the stimulated CD4+ T cells could be restored by depletion of CDS+CD2S-, but not CDS+CD2S+, T c:ells from these cultures. The stimulated CDS+CD2S- T-c:ell population was devoid of cytotoxic activity for either CD4+ T cells or the APCs used for priming. When the CDS+CD2S- T cells were added to mixtures of CD4 cells and APCs, they inhibited proliferation,269 but suppression was only observed when the stimulatory APCs shared at least one HLA class I allele with

the original stimulator population. The regulatory effect of the CDS+ suppressors was not restricted either by class I or class II MHC antigens expressed by the responder CD4+ T cells. Suppression mediated by CDS+CD2S- T c:ells required cell-c:ell contact and was not reversed by antisuppressor c:ytokine antibodies. Coincubation of the CDS+ suppressors with CD4+ responders had no effect, while coincubation of the CDS+ T c:ells with the APCs rendered the APCs unable to stimulate CD4+ proliferation. Phenotypic: analysis indicated that the CDS+ suppressors blocked the upregulation of costimulatory molecules such as CDSO/CDS6, CD54, and CD5S on the APCs. 270 The mechanism by which the CDS+ suppressors deactivate APC functions has been shown to involve upregulation of the genes encoding Ig-like transcript 3 (ILT3) and ILT4.271 These inhibitory receptors are structurally and functionally related to killer c:ell inhibitory receptors. Thus far, the biologic activity of these CDS+CD2S- suppressor populations has only been studied in vitro. Their potential roles in mediating immunosuppression in vivo as well as their relationship to CD4+ suppressor cells remain to be explored. It has been proposed that antigen-specific: MHC class I restricted CDS+CD2S- Tregs first induc:e ILT-expressing tolerogenic DC, which in turn generate CD4+ Tregs. 272 Regulatory CDS+ T cells have also been generated in vitro by stimulation with unique subpopulations ofDCs.273 When naive CDS+ T cells were stimulated in vitro with CD40Lac:tivated monocyte-derived DCs (DC1), the primed CDS+ T c:ells proliferated when restimulated with allogeneic: target cells, secreted large amounts of IFNy, and had potent cytotoxic activity. In contrast, when naive CDS+ T c:ells were stimulated with CD40L-activated plasmacytoid DCs (DC2), the primed CDS+ T cells proliferated poorly, displayed weak cytotoxic: activity, and secreted primarily IL-10. DC2-primed CDS+ T c:ells inhibited the ability of naive CDS+ T c:ells to proliferate to allogeneic monocytes, immature DCs, or mature DCs. Both the generation of CDS+ suppressor cells and their suppressor function could be markedly inhibited by anti-IL-10, but not by anti-TGF-1}. Dhodapkar et al. 274 have measured the ability of immature DC cells to modulate the immune response in vivo in humans. Injection of immature DC pulsed with influenza matrix peptide resulted in an expansion of antigen-specific tetramer binding CDS+ T cells. These CDS+ c:ells were capable of proliferating when stimulated with antigen in vitro, but were defective in IFNy secretion and lacked cytotoxic function. These findings indicate that immature DC c:an dampen preexisting antigen-specific: effector function in man. The potential suppressive function of the CDS+ T cells that responded to the iDCs was not studied. The relationship of these CDS+ T cells to those induced in vitro with DC2 cells is unknown. Wei et al. 275 have isolated plasmacytoid DCs from malignant ascites of patients with ovarian c:anc:er. These DCs, upon CD40L activation, c:ould induce antigen-specific: CDS+ IL-10-produc:ing Tregs that suppressed the responses of tumor antigen-specific CD4+ T cells. A unique subpopulation of mouse CDS+ T cells that express high levels of the IL-2R ~-chain (CD122) has been shown to have immunoregulatory activity?76 CDS+CD122- are Foxp3and inhibit the activation of both CDS+ and CD4+ T cells in vitro by an IL-10-dependent mechanism as CDS+CD122+ T

CHAPTER 33

cells from II..-10-/- could not suppress T-cell activation in vitro, but may exert some suppressive effects in vivo.277

Qa-1 Restricted CDB+ Regulatory T Cells Mice genetically deficient or depleted of CDS+ T cells have demonstrated a clear role for CDS+ T cells in regulating EAE. CDS+ T cells from mice that had recovered from EAE downregulated or killed some CD4+ neuroantigen-specific T-cell clones. Inhibition was blocked by antibodies to the MHC class Ib molecule Qa-1, but not by anti-MHC class Ia molecules. 278 It has been proposed that Qa-1 self-peptide complexes expressed by activated CD4+ T cells trigger the TCR on CDS+ cells. These CDS+ T cells then differentiate into suppressor cells that in turn suppress the CD4+ T cells that express the same Qa-1 self-peptide complex. The CDS+ Treg population can target a peptide from the conserved region of the TCR.m The mechanism of suppression has not been fully defined, although it may involve cytotoxicity or the secretion of suppressor cytokines. Human CDS+ T cells restricted by HLA-E, the human homologue of mouse Qa-1, may have similar functions.uo

Double Negative Regulatory T Cells The majority ofT cells in normal mice and humans that express the TCR-a.P chains also express either the CD4 or CDS coreceptor molecules. However, 1% to 5% of the peripheral TCR-a.J} population express CD3 but not CD4 or CDS. Double negative (DN) T cells can be divided into NKT cells and NK-marker negative DN Tregs. DN Tregs do not develop from CDS+ precursors, nor is CDS expression required for their development in vivo.281 Functional DN Tregs develop outside of the thymus and may mature from chronically activated CD4+ T cells. DN T cells have been shown to have immunoregulatory activity both in vivo and in vitro in both mouse and man.282' 213 DN Tregs have the ability to acquire alloantigens from APCs and present them to alloreactive syngenic effector T cells in human in vitro studies. DN suppressors generated in this way kill alloreactive CD4+ and CDS+ T responders that express functional Fas by a mechanism involving Fas/ Fas-L interactions. These activated DN cells were only able to kill syngeneic targets activated by the same alloantigenic stimulus. A significant difference was noted between the mean percentages of DN Tregs in patients who developed GVHD after stem cell transplantation when compared to those that did not. All patients in whom DN Tregs were expanded to > 1% did not develop GVHD, suggesting that the expansion of these cells may prevent the development of GVHD. 214

Regulatory T Cell-Induced Regulatory T CellsInfectious Tolerance One possible mechanism to increase the efficiency ofFoxp3+ Tregs is that Tregs induce anergy or suppressor activity in the Foxp3- responders as a form of infectious immunologic tolerance. It has been shown that human CD4+CD25+ T cells

REGULATORY/SUPPRESSOR T CELLS

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induce CD4+CD25- T cells to become Tregs that are capable suppressing by producing IL-l(f85 or TGF-J}, 286 thereby mediating suppression of naive CD4+ T cells. Similar studies in the mouse demonstrated that coculture ofTregs with Foxp3- responder cells resulted in the induction of both TCR and IL-2 unresponsiveness in the responder population.287 The responder cells remained Foxp3- and exerted suppressor activity in a cell contact-dependent manner that was partially abrogated by anti-TGF-Jl. One problem with these early studies is that it was difficult to determine if the regulatory populations induced by coculture with presumably Foxp3+ T cells actually were induced to express Foxp3. Recent studies have demonstrated that activation of Foxp3- T cells in the presence of activated Foxp3+ T cells resulted in the induction ofFoxp3 expression in the Foxp3- population.288 The induction ofFoxp3 expression was mediated by TGF-P produced by the Foxp3+ inducer population in a contact-dependent fashion. The newly induced Foxp3+ T cells mediated suppression both in vivo and in vitro. Foxp3+ T cells have also been shown to mediate infectious tolerance locally at the site of a transplanted skin allograft. The induced cells were suppressive and prevented potential efectors from rejecting grafts.Z89

REGULATORY T CELLS AND DISEASE Animal Models of Autoimmune Disease One of the most widely used experimental models for the study of Treg function is the animal model of IBD. Transfer of CD4+CD45RBhilh cells to SCID mouse recipients resulted in the development of a wasting disease and colitis 6 to S weeks after T-cell transfer. This disease was characterized pathologically by epithelial cell hyperplasia, goblet cell depletion, and transmural inflammation.190 There was a 20- to 30-fold accumulation ofTh1 cells in the intestine compared to normal mice. Treatment of recipients with anti-IFNy, anti-TNF-a, or anti-IL-12 inhibited the induction of disease. Transfer of CD45RB1'"" cells did not induce colitis, and cotransfer of RBh.i'b and RB1ow cells prevented the development of colitis. A ratio of 1:8 RBIDw to RBhiah was able to prevent disease. When CD45RB1ow cells were fractionated into CD25+ and CD25- fractions, control of intestinal inflammation was primarily mediated by the CD25+ (Foxp3+) fraction.Z7 CD45RB1""' CD25- cells did exert some suppressive function when transferred at high cell concentrations. Treatment of recipients of RBhiah cells with IL-10 inhibited the development of colitis (Table 33.7). This treatment inhibited the accumulation of Th1 cells in the intestine, but did not induce Tregs, as colitis developed when IL-10 administration ceased. Treg isolated from IL-10-/- mice are able to inhibit colitis induced by CD4+CD45RBh; cells, indicating that IL-10 secretion by Tregs is not required. However, control of colitis by CD25-CD45RBJow cells was found to be highly dependent on IL-10.291 In contrast, Treg-mediated suppression of autoimmune gastritis involves a cell contactdependent mechanism of suppression, and IL-10 and TGFp are not involved.Z92 One reason for this difference is that the pathogenesis of autoimmune gastritis and IBD are quite distinct. Bacteria play a required role in IBD, whereas

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SECTION VI

INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

tumors coinciding with tumor regression. In this model, aspirin and cyclooxygenase inhibitors also decreased the risk for colon cancer by inhibiting cyclooxygenase-2. As Tregs can suppress bacterially triggered inflammatory responses in the bowel of mice, the ability of Tregs to traffic and suppress inflammation likely explains their therapeutic efficacy in this model. As inflammatory mediators drive tumor development, Tregs and anti-inflammatory drugs exert their effect by modulating the levels of these molecules.

Immunity to Infectious Agents Treg cells dampen immune responses in a wide variety of infections caused by bacteria, fungi, parasites, and viruses, especially those that persist in the host. One of the most important roles of Tregs may, in fact, involve modulation of the immune response to infectious agents to prevent the lethal consequences of an overwhelming inflammatory response during the course of a productive immune response to an invading microorganism. Treg and effector cells must maintain equilibrium between no immunity at all and immunopathology. This critical role of Tregs is well illustrated by the immune response of mice to infection with Pneumocystis carinii (PC). When SCID mice chronically infected with PC are reconstituted with CD4+CD25- T cells, they develop a severe inflammatory response in their lungs that is ultimately fatal. Animals injected with CD25+ T cells alone did not become moribund and only manifested transient weight loss. Cotransfer of CD25+ T cells prevented the development of the PC-driven fatal pulmonary inflammation induced by CD25T cells but also suppressed the elimination of PC mediated by the CD25- T cells. Protective CD25+ T cells are needed to inhibit the lethal immunopathologic response mediated by the PC-specific CD4+CD25- T cells but they also inhibited complete clearing of the organism.352 Although Tregs may target CD4+ and CDS+ T cells responding to an infectious challenge, Tregs have also been shown to act on the innate immune system in the response to Helicobacter hepaticus.353 The role of Foxp3+ T cells in the immune response to infection includes more than suppression of inflammation.354' 355 Foxp3+ T cells also maintain persistence of infection and promote chronicity. The persistence of pathogens following clinical cure is a hallmark of certain viral, bacterial, and parasitic infections. In clinical and experimental forms of leishmaniasis, small numbers of viable organisms persist within lymphoid tissue and within the site of former skin lesions following self-cure or successful chemotherapy. As low numbers of parasites persisting in the dermis can be efficiently transmitted back to their vector sandflies, the expansion and/or recruitment of regulatory T cells to the site of Leishmania major infection might reflect a parasite adaptive strategy to maintain its transmission cycle in nature. Despite the absence of sterilizing immunity, these individuals maintain strong lifelong immunity to reinfection, a status similar to the concomitant immunity described in tumor models.356 In healed C57BL/6 mice, CD4+CD25+ Treg cells accumulate in sites of L. major infection in the skin. 357 These cells are exclusively derived from CD25+ T cells and not

from activated CD25- T cells. They suppress the expansion of and killing mediated by L. major-specific effector cells. Although IL-10 produced by CD25+ T cells is essential to the establishment of persistent infection, early in the infectious process CD25+ T cells can promote parasite survival and growth in an IL-10-independent manner. Later in the course of infection, IL-10 is absolutely required for development of the chronic lesion as recipients of CD25+ T cells from IL-10-/- mice ultimately healed and completely cleared the parasite from the site. IL-10 produced by Tregs contributes directly to parasite persistence by either modulating APC function, inhibiting cytokine production by Th1 cells, or by rendering macrophages refractory to IFN'y that is needed for intracellular killing. When rechallenge studies were performed in IL-10-/mice or in WT mice that were treated during the chronic stage of their primary infection with anti-IL-10R, conditions that result in complete clearance of parasites from the skin and draining LN, reinfection at a site distant from the initial infection resulted in parasite loads that were comparable to those following primary infection in naive mice. Because healed mice treated with control antibody maintained strong immunity to reinfection, the maintenance of a residual source of infection, secondary to IL-10 production by CD25+ T cells at the lesion site, is required for preservation of acquired immunity to L. major. Treg cell lines isolated from chronic L. major are able to respond to parasiteinfected DCs by proliferating and producing IL-10. 358 The cells that have undergone proliferation express and maintain Foxp3 expression. It appears that the parasite has specifically evolved to manipulate DCs in a manner that favors and sustains Treg proliferation. Most importantly, theL. major-specific cell lines maintain their suppressive functions in vivo, as transfer of the lines to chronically infected mice results in massive disease reactivation and dissemination. Thl effector cells are induced during the course of infection with Bordetella pertussis and ultimately play a critical role in the clearance of bacteria from the respiratory tract.359 Antigen-specific Th1 responses in the lung and local LNs are severely suppressed during the acute phase of infection. B. pertussis has evolved a number of strategies to circumvent protective immune responses. One bacterial component, filamentous HA, is capable of inhibiting LPS-driven IL-12 production by macrophages and DCs, and stimulating IL-10 production. Filamentous HA may contribute to the suppressed Thl responses during acute infection with B. pertussis by the induction of T cells with regulatory activity as a result of its interactions with cells of the innate immune system. Repeated stimulation of T cells from the lungs of mice acutely infected with B. pertussis resulted in the generation of Trl clones specific for filamentous HA. Trl cells could only be generated from the lungs of infected animals and not from spleen. These Tr1 cells secreted high levels of IL-10 and inhibited protective immune responses against B. pertussis in vivo and in vitro. Suppression was substantially reversed by anti-IL-10 in vivo. The capacity to induce Trl cells is thereby exploited by a respiratory pathogen to evade protective immunity and suppress protective Th1 responses at local sites of infection.

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Analysis of cytokine production by T cells from patients with chronic hepatitis C virus infection identified antigenspecific regulatory T cells that secreted IL-10 in addition to IFN'Y-producing circulating Th1 cells360 ; no IL-4-producing T cells were identified. IL-10-producing cells were detected in a higher proportion of patients with chronic infection than in those who had cleared the virus. Taken together with the studies on L. major and B. pertussis, these studies on hepatitis C virus strongly support the general concept that many infectious agents have evolved mechanisms for selective activation of either naturally occurring CD25+ T cells or the generation ofiL-10-producing Tr1 cells from CD25- T cells. The ultimate result is perpetuation of the chronic infectious state with incomplete dearing of the infection. Depending on the extent of suppression of effector T cells in the host, the consequences of the chronic state may be protective immunity (L. major) or continued pathogenmediated organ destruction (hepatitis C virus). Many viruses such as herpesviruses, hepatitis viruses, and retroviruses evade immunologic destruction during acute infection and establish chronic, persistent infections that may culminate in life-threatening diseases. Friend virus (FV) infection in mice has been used as an experimental model to study retrovirus-induced immunosuppression and may offer insights into our understanding of immunosuppression associated with human immunodeficiency virus {HIV). Mice chronically infected with FV are unable toreject both FV-induced and unrelated immunogenic tumors. CDS+ T cells from acutely infected mice produced perforin, granzyme A, and granzyme B, and display recent evidence of degranulation and in vivo cytotoxicity. Activated T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation and in vivo cytotoxicity. CD4+, but not CDS+, T cells from infected mice can transfer suppression to normal mice and can inhibit the generation of CTL in culture. 361 Suppression could be substantially reversed by the addition of antiCTLA-4, but not anti-IL-10R, to the cultures. FV-induced Tregs suppress CDS+ T cells in vitro regardless of the TCR specificity of the CDS+ T cells.362 It is unknown whether the Tregs in chronically infected mice are specific for any viral proteins. Depletion of Foxp3+ T cells early after infection resulted in an increase in virus-specific CDS+ T cells on day 10 postinfection, enhanced production of IFN'Y and TNFa, and a 10-fold reduction in viralload.363 As HIV infection in man in many respects mimics the animal models of chronic retroviral infections, several studies have begun to examine Treg function as different stages of HIV infection. Kinter et al.'64 found that in the majority of HIV-infected but still healthy individuals, CD25hi Treg cells significantly suppressed cellular proliferation and cytokine production by CD4+ and CDS+ T cells in response to HIV antigens in vitro. Suppression was cell contact-dependent and IL-10- and TGF-~-independent. Patients with strong HIVspecific suppression in vitro had lower levels of viremia and higher CD4/CD8 T-cell ratios than patients who did not have Treg activity. These data suggest that the suppression of CD4+ T-cell activation by Tregs may make HIV replication less favorable as the virus must replicate within the CD4+ T cell

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itself. Thus, in this chronic infection model, the suppressive functions of Tregs may actually be beneficial to the patient. The contribution of Tregs has been most clearly shown in responses to chronic infections. Much less is known about the role of Tregs in modulating acute viral infections. In the murine model of herpetic stromal keratitis, depletion ofTregs before infection resulted in lesions of greater severity and permitted the induction of disease with lower infecting doses of virus.365 Cotransfer of Tregs with CD25- T cells reduced lesion severity and diminished the Ag-specific cytokine response of splenic CD4+ T cells. The mechanism of action of Tregs in this model is not fully elucidated. Tregs inhibit the induction of virus specific CD4+ T cells, but may also modify the expression of homing molecules involved in T-cell migration to the ocular inflammatory site or of pathogenic T cells to extralymphoid inflammatory sites. Tregs can also control the intensity of secondary responses to herpes simplex virus and may also influence the magnitude of immunologic memory. Pathogen-specific Treg cells have been detected in a few studies using whole pathogen preparations as targets without the identication of specific target epitopes. The role of Treg cells in facilitating the persistence of infections with Mycobacterium tuberculosis is unclear. Rigorous depletion of Treg cells during early infection results in enhanced bacterial clearance.366 Early arrival in the lung of M. tuberculosisspecific T cells is associated with enhanced immune control and lower bacterial burdens; Treg cells slow this recruitment process. This result differs from studies of infection with herpesvirus concluded that Treg cells facilitate effector T-cell recruitment to tissue sites of infection.367 Studies using TCR-transgenic cells specific for an epitope derived from M. tuberculosis attempted to identify Tregs specific for the same epitope recognized. Treg cells specific for this epitope were not found. 366 Virus-specific Treg cells have been detected in mice infected with a strain of mouse hepatitis virus. Polyclonal Treg cells from normal mice will decrease weight loss, clinical scores, and demyelination when adoptively transferred into mice infected with this viral strain. Treg cells for two viral epitopes were detected using peptide-MHC class II tetramers 6 to 7 days after infection, at the same time as CD4+ effector T cells. The virus-specific Foxp3+ cells expressed both IFN'Y and IL-10 in the CNS after infection and suppressed T-cell proliferation of cognate epitope-specific CD4+ T cells.368 IFN'Y expression by these cells was maintained during both acute and chronic phases of infection. The identification of Tregs specific for a pathogen-derived epitope is consistent with the view that exposure and immunization may expand not only protective T-cell subsets, but also T-cell subsets that impede protection. Overall protection likely depends on the relative ratio of protective versus suppressive T-cell subsets.

Autoimmune Disease in Man A large body of data supports the existence of Foxp3 + T cells in humans,29- 36 and the in vitro characterization of human Foxp3+ cells suggests that they are identical to their murine counterparts. Tr1 cells have been readily induced in cultures of human T cells, and a number of studies have

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11 C'2 > C'3 > C'4), rather than according to their position in the reaction. C4 is a relatively abundant protein, present in plasma at around 0.5 g/1. It is a large (210 kDa), disulphide-bonded heterotrimer. Activated Cls captures C4 from the fluid phase, perhaps in part through interaction of its short consensus repeat (SCR) domains with C4, then cleaves the C4 o: chain at a single site near the amino terminus, releasing a 77 amino-acid fragment, C4a, and exposing in the cleaved a' chain of the large fragment C4b a labile thioester group {Fig. 36.2A). Although most of the nascent C4b formed will decay in the fluid phase through hydrolysis of the thioester, a small proportion will bind reactive hydroxyl or amino groups on the activating surface, creating a cluster of covalently bound C4b around the initiating IgG/Cl complex. Immobilized C4b binds the next component in the sequence, C2, in a magnesium-dependent complex. C2 is a single-chain plasma protein ofmass 100 kDa and plasma concentration around 25 mg/L; it is the most heat-labile of the complement proteins, destroyed by brief incubation of plasma at 56°C. C4b-bound C2 is cleaved 'Convention states that active complement enzymes such as Cls and C4b2a are shown with an ove:rline to denote active status. I have chosen here to ignore this convention because it is unnecessary, confusing, and difficult to achieve in most word-processing packages!

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by activated Cls in an adjacent IgG/Cl complex, releasing a 30 kDa fragment C2b, while the 70 kDa C2a fragment, an active serine protease, remains associated with C4b on the surface.t,20,21 The magnesium-dependent C4b2a complex is the CP C3 convertase, the next activation enzyme in the sequence. C2a in the C4b2a complex is an active serine protease that cleaves C3, a two-chain 190 kDa protein, homologous to C4 and the most abundant of the complement proteins at around 1 gil in plasma.22 Cleavage releases a 77 amino-acid fragment, C3a, from the amino terminus of the a chain of C3, exposing in the large fragment, C3b, a labile thioester group essentially as described previously for C4b (Fig. 36.2B). Again, most of the C3b formed decays bythioester hydrolysis, but a small fraction covalently binds the activating surface, clustering around the site of activation. Some of the C3b formed will directly bind C4b2a through its thioester to form a trimolecular complex, C4b2a3b; this binding is not a random event but occurs at a single specific site in C4b, placing C3b in the correct orientation for succeeding steps of activation. The C4b2a3b complex contains a binding site for CS involving interactions with both C4b and C3b in the complex. C5, another homologue of C4 and C3, is a 200 kDa, two-chain molecule present in plasma at about 100 mg/L; importantly, C5 lacks the critical thioester group and so cannot bind covalently to targets. Once bound to C4b2a3b, the CP C5 convertase, C5 is cleaved by C2a in the complex, releasing a 74 amino-acid fragment, C5a, from the 0: chain of C5 and leaving the large fragment, C5b, loosely attached to the convertase. Cleavage of C5 is the final enzymatic step in the CP (Fig. 36.3). Two features of CP activation are critical to its roles. First, amplification at each of the enzymatic steps is critical for efficient activation; thus, a single active IgG/Cl complex will deposit an abundance of C4b in the vicinity of the initiating 1Perhaps

the most confusing and controversial aspect of complement nomenclature surrounds the naming of the C2 cleavage products and the CP C3 convertase. Most early articles referred to the large protease fragment that remained associated with Colb as C2a and the complex as C4b2a, but this was arbitrarily changed in the 1990s in a popular immunology text book on the pretext that lr. p~o." ' -·~, 1•111t I I'd ... •""'• ,__claMrM Q ~crertirt 1111l.r liol:o.t 10 tlle CP 14 1hoo C!l> eo-llh>l """""" tlle 11!1« ..m fi!ollmo the -

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CHAPTER 36 COMPLEMENT

I

111

C5 convertase Membrane attack

complex

RG. 3&.6. Tanninal Pltlrway. C5b, still attached to the convertase, binds C6, then C7. The trimolec~la.r C5b67.complex is released to the fluid phase. A fraction of tfle complexes form~d attach through hydrophobJC.Jntera_ctJon to the membrane. Membrane bound C5b67 recruits C8, tflen multiple cop1es of C9. C9 monomers unfold, msert mto and through the membrane, and polymerize to form a transmembrane pore through which ions and water can freely flow. The inset shows an electron micrograph of a complement-tyzed cell; circular membrane attack complex lesions, a light protein rim surrounding a dark pore, are readily seen.

in an adjacent complex, and the resultant enzyme, C4b2a, continues activation through C3 and C5.

The Tenninal Pathway The terminal pathway (TP), sometimes referred to as the membrane attack pathway, is a final, common pathway for all activation routes. Cleavage of C5 is the last enzymatic step in the complement sequence and the final step of each activation pathway. The TP is a system almost unique in nature where five plasma proteins join to create an amphipathic membrane-inserted complex, the membrane attack complex (MAC}, that creates a lytic pore in the membrane (Fis. 36.6)." The TP begins with the binding of the next component in the sequence, C6, to C5b still in the grip of the C5 convertase. C6 is a 100 k:Da single-chain protein present in plasma at around 50 mg/L. Conformational changes during formation of the C5b6 complex weaken the grip of the convertase and create a binding site for the next component, C7, a 95 kDa single-chain molecule, plasma concentration about 90 mg/L. C6 and C7 are homologous molecules and are genetically linked. with genes adjacent on chromosome 5p. Incorporation of C7 causes further loosening of grip, releasing the trimolecular C5b67 complex into the fluid phase. The newly released C5b67 complexes shower down onto the lipid membrane surrounding the convertase and bind firmly to the surface via a hydrophobic site in the complex, thereby creating a nidus for continued assembly of the MAC. This is an inefficient process; the large majority of C5b67 complexes formed are inactivated in the fluid phase before they can bind membranes. Spontaneous inactivation occurs rapidly even when the C5b67 complex is assembled from pure proteins; in plasma, several proteins act as CSb-7 inhibitors to further accelerate inactivation. Those

C5b67 complexes that do bind membranes then recruit the next protein in the sequence, C8, a heterotrimeric molecule

(ex and pchains each approximately 61 k:Da, "(chain, 22 k:Da; ex and 'Y covalently linked. p noncovalently associated) present in plasma at about 80 q/L. Binding of C8 introduces additional hydrophobicity, causing the resultant CSb-8 complex to embed more firmly in the membrane. There is some evidence that the CSb-8 complex can cause membrane disruption and leakiness, but the major membrane disruption necessary to kill bacteria or other target cells requires the recruitment of multiple copies of the final component of the MAC, C9, a 70 k:Da single-chain protein present in plasma at around 60 mg/L. The first globular C9 molecule binds C8 in the CSb-8 complex and undergoes major conformational rearrangement, unfolding to reveal a hydrophobic face that allows insertion into and through the membrane lipid bilayer. As additional C9 molecules are recruited, they in turn unfold and insert, aligning with the first C9, like barrel staves; with the recruitment of about 10 C9 molecules, the barrel is completed, creating a protein-lined channel through the membrane, the MAC (see Fig. 36.6). The C9 hydrophobic faces tightly lock the MAC in the membrane while the opposite, hydrophilic faces create a channel through which water and ions can flow, the MAC pore."" In electron micrographs of complement-lyzed targets, the MAC is readily visible as a

~The precise IUitUI:e of the MAC "pore" wu another area of entertainiug debate thzough the 1980s. with some coute!ldiug that it wu a rigid chmnci ltrllcture made frommultipleC9moleculet, whileothencomidmd ita "leaky patch" ofmembrme daruption. AI. is often the cue. both camp& were J:isht in put. The complete MAC. vaible u circular lesion& on elec:tron micrtJSI~Lphs.likelr is .. J:isid protein pore. but ccllly3is dou not require 1:ha structure and complCD:.t coatainiDg u few u two or three C9 moleculu have lftic activity, medimd by the membrme-pertmbiDg prupertiet of the inserted C9molecules.

872

I

SECTION VI

INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

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CHAPTER 36 COMPLEMENT

Jtt''

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RG. 36.10. Reconry Mechanisms in Nucleltld Cells. A: Neutrophils prior to complement attack display a smooth, slightly ruffled appearance in scanning electron micrographs (SEMs). B: Wiltlin minutes of exposure to a nonlaltlal complement attack, the appearance is transformed: calls are covered in membrana projections. C: This frame shows SEM appearance of vesicles shad from attacked cells. D: After recovery, calls regain their tranquil appearance. E: This frame shows transmission electron micrographs of the same vesicles to show rich decoration with membrana attack complex (MACt pores. F: Nonlethal MAC attack is not without consequences for ltla cell; changes in call survival, proliferation, morphology, inflammatory mediator release, and many oltler events may be triggered in ltla cell.

was :first descnbed in neutrophils that, when attacked bycomplement, rapidly started to ..bleb,"' releasing multiple vesicles to the fluid phase.46 Electron microscopy and biochemical analyses showed that these vesicles were packed with MAC pores, while the rest of the cell membrane was free oflesions (Fis. 36.10). This packaging and removal was an active process and, when inlubited by metabolic poisons, neutrophil killing by MAC was increased. Similar mechanisms of MAC removal, either by shedding on vesicles or internalization, were soon discovered in many other nucleated cell lines and primary cells. Despite the fact that the recovery mechanism was discovered 25 years ago, the precise way in which the cell senses the MAC, packages MAC lesions into membrane patches, and sheds or internalizes the patches remain poorly defined. In neutrophils, calcium ion influx. through the MAC pore is a key early event, but the downstream signaling pathways leading to recovery are unknown.

COMPLEMENT RECEPTORS, OPSONIN$, AND ANAPHYLATOXIN$ Complement activation is a danger sisnal that alerts the host to infection or injury and initiates appropriate responses.

Complement activation products flag danger by physically binding to pathogens, immune complexes, and other to.xic bodies, and by their release into the surrounding milieu; subsequent events require the interaction of these fragments with specific receptors present on a variety of cell types {see Table 36.3).

Receptors for 1he Opsonic Fragmerrts of C3 and C4 C3 is the most abundant complement component and the most important source of complement fragments. C3b and its degradation products iC3b and C3d coat the target, a process called opsonization, thereby tagging the target for recognition by cells bearing complement receptors.47 CRl, described previously as a complement regulator, is an atypical receptor in that it modifies its own ligand. CRl binds C3b (and C4b) coating immune complexes {ICs) via its binding sites in LHRs I, 2, and 3, and catalyzes its cleavage by fl. Erythrocyte CRI plays a critical role in IC handling; the C3b-coated IC binds CRI on the erythrocyte and is held transiently until the lisated C3b is deaved by fi to iC3b/C3dg; binding affinity is lost and the complex is released, only to bind again via another C3b.4° This

F

876

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SECTION VI

INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

dynamic binding permits the efficient sequestration of ICs on erythrocytes but without immobilizing them in a way that inhibits their efficient transfer to complement receptor-bearing tissue macrophages in the spleen and liver for final disposal. CRI on other cell types also operates by binding and processing C3b-coated ICs. On dendritic cells, CRI localizes opsonized ICs for presentation of antigen to T cells, while on B cells, C3b coating the IC binds CR1 and is cleaved to C3d, a ligand for another complement receptor, complement receptor 2 (CR2; CD21), clustered with CRI and the B-cell antigen receptor (BCR). Simultaneous engagement of CR2 and the BCR markedly lowers the threshold for antibody response. CR2 is made up of either 15 or 16 SCR domains (alternative splicing removes SCRll in the smaller isoform), a transmembrane region, and a cytoplasmic tail with important roles in signalling.39 It is expressed on B cells, T-cell precursors and some mature T cells, dendritic cells, and some other cells involved in antigen presentation, basophils and epithelia. CR2 is a rather promiscuous receptor with numerous binding partners, including the IgE receptor (CD23), interferon-a, and the Epstein-Barr virus; its complement ligands are C3d or iC3b, binding at a single site in SCRI and SCR2. The key role of CR2 is to enhance the immune response to antigens contained within the IC. On B cells, CR2 is clustered with the B cell-specific signaling molecule CD19 in a complex held together by the tetraspanin CD81; this tri-molecular complex interacts with the BCR to modulate the B cell response to antigen. CR2 on dendritic cells contributes to IC trapping in lymph node germinal centres. Complement receptor 3 (CR3; CD11b/CD18) is a heterodimer comprising a 165 kDa ex-chain (CDllb) and a 95 kDa Jl-chain (CD18); it is a member of the ~-integrin family of leukocyte surface heterodimeric proteins sharing the common Jl-chain. CR3 is expressed on monocytes, neutrophils, mast cells, natural killer cells, dendritic cells, and some T cells.•8 It is notable for its promiscuity, binding adhesion molecules (intercellular adhesion molecule [ICAM]-1 and -2), coagulation proteins, microbial products, and carbohydrate antigens; its principal complement ligand is iC3b, although it binds weakly to C3d. Binding of an iC3b-coated particle to CR3 on phagocytic cells triggers the phagocytic process, leading to the elimination of the opsonized particle. Other phagocytic receptors, including the Ig Fe receptors and CD14, certainly contribute to the phagocytic process, and there is continuing debate around which of these triggers are required for and most important in the phagocytic process. Complement receptor 4 (CR4; CD11c/CD18), another j}.z-integrin expressed on myeloid cells, resembles CR3 with regard to distribution and complement ligand-binding properties. It is possible that CR4 plays important roles in some dendritic cell processes, although it remains something of an enigma. Complement receptor of the immunoglobulin superfamily is a recently described receptor for C3b/iC3b expressed exclusively on tissue-resident macrophages, including Kupffer cells in the liver.49 It plays important roles in the

capture and clearance of opsonized ICs and pathogens from the circulation.

Receptors for the Anaphylactic Fragments of C3and C5 Receptors for the small anaphylatoxin (AT) fragments released from C3 and C5 during activation are extremely important players in the recruitment of inflammatory cells and in a growing list of other events. C3, C4, and C5 are structurally similar molecules and are all activated in similar ways: a single cut by the activating enzyme that releases a peptide, C3a, C4a, and CSa, respectively, from the amino terminus of the ex-chain. Current opinion is that there are no receptors for C4a and that, as a consequence, it has no biological function. In contrast, specific receptors exist for the AT molecules C3a and C5a, which mediate important biological roles. Three AT receptors have been described to date, the C3a receptor (C3aR), the CSa receptor (C5aR; CD88), and the C5a receptor-like 2 (C5L2).50 They are homologous molecules, members of the G-protein-coupled receptor family of heptaspan membrane proteins. C3aR is expressed on all myeloid cells and some nonmyeloid, including activated T cells, astrocytes, endothelia, epithelia, and smooth muscle. C3aR binds C3a with nanomolar affinity but does not bind C3adesArg, C5a, or CSadesArg. Upon binding of C3a, intracellular signaling cascades are triggered through activation ofheterotrimeric G-proteins that in tum cause increased intracellular free calcium and other downstream events. CSaR binds C5a with nanomolar affinity and CSadesArg with tenfold lower affinity; it does not bind C3a/C3adesArg. C5aR is expressed on all myeloid cells and numerous nonmyeloid including endothelia, neurones, and astrocytes; expression on lymphoid cells has been suggested but current evidence does not support this. Binding of C5a or CSadesArg triggers activation via heterotrimeric G-proteins, essentially as described for C3aR. CSL2 was first described just 10 years ago and remains something of an enigma.5 1 It is broadly expressed on myeloid and nonmyeloid cells and is often co-expressed with C5aR in many tissues. C5L2 binds both CSa and CSadesArg with nanomolar affinity, although the binding sites for these two ligands are not identical, suggesting that they may have different effects. Some researchers contend that CSL2 also binds C3a and C3adseArg, although this remains controversial. Critically, C5L2, unlike the other AT receptors, is not G-protein coupled and, as a consequence, is considered by many as a nonfunctional decoy receptor that dampens down local responses to C5a. In one specific site, adipose tissue, CSL2 appears to be functionally important, strongly implicated as the receptor triggering fat mobilization in response to locally produced C3adesArg (termed "acylationstimulating protein" in this context).52 Clarification will likely soon follow in this active research area.

Receptors for C1q Identification of receptors for Clq has been a minefield of false leads because of the inherent stickiness of the protein. Several receptors have been reported over the years,

CHAPTER 36

although many have not stood the test of time and the physiological relevance of the others remains uncertain. The most convincing candidate receptor is the Clq receptor for phagocytic enhancement (ClqRp; CD93), a heavily glycosylated 120 kDa transmembrane sialoglycoprotein expressed on myeloid cells and endothelia. 53 Its extracellular region comprises a collagen recognition domain followed by five epidermal growth factor (EGF) domains; the collagen recognition domain binds the collagenous regions of C1q, MBL, and another lectin molecule, surfactant protein A (SpA).It is suggested that this interaction is important in the clearance of apoptotic cells; these spontaneously bind C1q, MBL, and likely SpA, tagging them for binding C1qRp and phagocytic clearance. Antibodies against C1qRp inhibit apoptotic cell clearance in vitro, and mice deficient in ClqRp or Clq display delayed apoptotic cell clearance. Several members of the integrin family of cell adhesion molecules have been implicated as C1q binders; of the several collagen-binding integrins, a2~1 stands out for its capacity to bind C1q and MBL. 54 A 60 kDa receptor for the collagenous tail of C1q, MBL, and other collectins, termed cClqR, was shown to be identical to calreticulin, a cytoplasmic chaperone protein, raising questions about how this molecule could associate with membranes and act as a C1q receptor. It is now suggested that calreticulin binds the extracellular domains of CD91 to create a receptor. Other molecules suggested to act as receptors for C1q (± MBL) include CR1 (binding the collagenous region through its membrane-proximal SCRs) and a 33 kDa protein that binds the C1q globular heads (gClqR).55 The physiological roles of these interactions are uncertain.

COMPLEMENT ONTOGENY, GENETICS, AND PROTEIN FAMILIES Complement is an evolutionarily ancient defense system; proteins with C3-like opsonic activities and fB-like enzymatic properties are present in the hemolymph of starfish, sea anemones, and even the horseshoe crab, but not in insects.56' 57 Early studies of primitive complement systems were limited because they relied upon the identification of opsonic or lytic activities in fluids derived from various species. A more recent approach to complement evolution has been to search for complement homologues in the genomes of primitive animals. 58 These studies confirm the very early arrival of C3- and fB-like molecules, show that C3 multiplication to yield C4 and C5 occurred prior to the emergence of cartilaginous fish, confirm that the fB/C2 duplication event preceded the split between Amphibia and Mammalia, and confirm the presence ofC1q-like, MBL-like, and MASP-like proteins in lamprey. All four terminal components are present in all mammals, birds, and amphibians, but no genetic evidence for terminal components is found in sharks despite early claims of a C9-like molecule and lytic activity in shark serum. Many of the complement components, regulators, and receptors share structural features with others, creating protein families within the complement system, usually a result of gene reduplication events during evolution.

COMPLEMENT

I

877

The Thioester Protein Family C3, C4, and CS, together with the abundant noncomplement plasma protease inhibitor, a2-macroglobulin (a-2M), comprise a family of homologous proteins each made up of multiple disulphide-linked chains derived from a single chain precursor and typified by the presence of an internal thioester, hence called the thioester protein family (TEP). These proteins have evolved from an ancestral protease inhibitor closely resembling a-2M56 and have utilized the thioester, part of the a-2M protease inhibition machinery, to confer capacity to covalently bind surfaces. Loss of the thioester in CS, a requirement for the evolution of a lytic TP, is a relatively late evolutionary event. With some of the complement protein families, their common evolutionary origins are evidenced by conservation of genetic linkagethe duplicated genes remain adjacent in the genome; however, for the TEP family, gene linkage has been lost, with each gene on a different chromosome; A2M on chromosome 12, C3 on chromosome 19, CS on chromosome 9, and C4 on chromosome 6. The C4 gene is particularly interesting. First, it is duplicated in man, the two genes, C4A and C4B, encoding highly homologous proteins, differing in just four amino acids clustered in the a-chain; second, it is situated in the major histocompatibility complex (MHC) on chromosome 6, linked to the genes encoding C2 and m, themselves homologous molecules, derived from a common ancestor.59 Co-location of these three genes into the MHC duster is seen in all higher vertebrates, indicating that it was an early event and suggesting that the common ancestor of the TEP family proteins was linked to fB/C2 from the earliest origins of the complement system. Whether there is any functional consequence of the location of these complement genes in the MHC is unclear; however, the C4 gene in zebrafish is not linked to MHC, 60 implying that its location within the MHC in higher vertebrates is an evolutionary accident of no functional importance.

The Terminal Pathway Protein Family The TP components, C6, C7, C8, and C9, are structural homologues derived from a common C9-like ancestor protein and show increasing complexity as one travels back from C9 to C6. C9 is the simplest, comprising, from the amino terminus, a thrombospondin (TSP) domain, a low-density lipoprotein-receptor A-type domain, a MAC-perforin domain, and an EGF domain. The C8~ and C8a chains each contain the same domains as C9, but with the addition of an extra TSP domain at the carboxy-terminus; C8y is a lipocalin family member with no homology to other complement proteins; C7 contains all the domains present in the C8 chains, but has an additional four domains at its carboxy-end, two SCRs and two factor I-MAC domains, also found as a single domain in fl.61 C6 closely resembles C7 but has acquired an additional TSP domain at its amino terminus. The MAC-perforin domain in the T cell pore-forming protein perforin is homologous to that in C9 and the other TP proteins, and is also found in many bacterial cytolysins, suggesting that it is a common feature of pore-forming proteins in nature.62 The genes encoding TP proteins are linked

878

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INDUCTION. REGULATION. AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

N

c

protease domain.65 The MASPl gene, on chromosome 3, is alternatively spliced to yield MASP-1 itself, MASP-3 in which the serine protease domain is larger, and Map44, a product truncated after the first SCR. Alternative splicing of the MASP2 gene, on chromosome 1, yields either MASP-2 itself or a truncated product, Map19, comprising the CUB and EGF domains with four unique amino acids at the carboxy-terminus. The common ancestor, present in ascidian worms, is a MASPl gene, although the precise pathway through which this gene reduplicated to yield the MASP2, ClR, and ClS genes is uncertain.66 The ClR and ClS genes are adjacent in a tail-totail arrangement on chromosome 12p, suggesting that these two genes arose relatively recently from a common precursor.

The Regulators of Complement Activation Family FIG. 36.11. Short Consensus Repeat Structure. The short consensus repeat comprises approximately 60 amino acids held in a rigid "bead" structure by intradomain disulphide bonds.

in two groups. C6, C7, and C9 are linked on chromosome 5. The first two are immediately adjacent to one another, albeit in opposite orientations, while C9 is more distantly linked. CBA and CBB are very closely linked and in opposite orientations on chromosome 1p.

The Collectin Family The colledin family includes C1q, MBL, and the ficolins. All have collagenous amino terminal stalks and globular recognition domains at their carboxy-termini.63 Evolutionary tracking of this family is complicated by the frequency of collagenous proteins in nature, most of which are unrelated to complement. Clade analysis strongly suggests that lampreys (agnathia; jawless fish) have a Clq gene that is a likely ancestor of the mammalian ClQA, ClQB, and ClqC genes. This finding indicates that Clq predates the evolution of antibody and adaptive immunity, likely as a pathogen recognition molecule. MBL genes are found even in the earliest vertebrate genomes, but ficolin genes appear to be more recent, present in tetrapod genomes but not in lower vertebrates.64 In man, the three ClQ genes are arranged in tandem in order A-C-B on chromosome 1p. The MBL gene resides on chromosome 10, clustered with the genes encoding the related collectins, surfactant proteins A and D (SFTPAl, SFTPA2, SFTPD). The genes encoding ficolins-1 and -2 are closely linked on chromosome 9q, while the ficolin-3 gene is on chromosome 1p.

The C1r/C1s/Mannan-Binding Lectin-Associated Serine Protease-2 Family The initiating enzymes of the CP and LP comprise another family (MASPs-1, -2, and -3; C1r; and C1s) sharing the same domain structure comprising, from the amino terminus, a C1r/C1s/Uegf/Bmp1 (CUB) domain, an EGF domain, a second CUB domain, two SCRs, and a carboxy-terminal serine

The last, and perhaps most interesting, of the complement protein families to be discussed in this section is the regulators of complement activation (RCA) family, characterized structurally by a preponderance of SCR domains and genetically by close linkage in the RCA cluster on chromosome 1q, suggesting that they have developed through serial duplication events.67 The SCR domain, the signature of the family, is a globular domain of around 60 amino acids, including a number of highly conserved tryptophans, prolines, and cysteines that give the SCR its shape (Fig. 36.11). The conserved cysteines, four in each SCR, form two invariant disulphide bonds to lock the structure, and joining of domains end-toend creates the elongated, "beads on a string'' appearance typical of this family. SCR domains are also found in other complement proteins (C6, C7, fl, C1r, C1s, MASPs, etc.) and some noncomplement proteins (eg, selectins), but only in RCA proteins is the dominance of this domain seen. RCA proteins in lower vertebrates, though fewer in number (six in chicken, two in frog), are also made up of SCR domains, and their genes are clustered. The frog genes are orthologs of the human DAF and C4BP genes, indicating that these are the common ancestors of other RCA proteins. The RCA cluster comprises two blocks of genes encoding complement regulators and receptors, interspersed with a number of unrelated noncomplement genes (Fig. 36.12). The first block comprises, in sequence from telomeric end, the genes for MCP (MCP), a CR1-like pseudogene (CRl-like), CR1 (CRl), CR2 (CR2), DAF (DAF), a C4bp ex-chain-like pseudogene (C4BPA-like), C4bp ex-chain (C4BPA), and C4bp P-chain (C4BPB); the pseudogenes have no protein product. The second block, some distance from the first in a centromeric direction, comprises, in sequence, the genes for fH (CFl{), fH-related 3 (CFHL3), fH-related 1 (CFHRl), fH-related 4 (CFHR4), fH-related 2 (CFHR2), and fH-related 5 (CFHRS). Alternative splicing of the CFH gene creates an additional protein product, fHlike 1 (fHL-1), comprising the first seven SCRs offHand an additional six unrelated carboxy-terminal amino acids.68

Expression Patterns of Complement Proteins The complement genes are not ubiquitously expressed; rather, they show cell type-specific expression patterns that are important in understanding how the system works.

CHAPTER 36

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FIG. 36..12. The Regulators of Complement Activation Gene Cluster. The regulators of complement activation cluster comprise a long region (up to 7Mb) on band q32 of human chromosome 1 that contains the genes for all the short consensus repeat-containing complement regulators and receptors. Two separate subclusters contain, respectively, the genes forfactor Hand the factor H-related proteins 1to 5, and the genes encoding, in order, the~ and a-chains of C4bp, decay accelerating factor, complement receptor 2. complement receptor 1, and membrane cofactor protein. The ntruew genes (blue) are interspersed with numerous pseudogenes (green) that have no protein product and several non related non complement genes (peach).

Restricted expression of complement receptors, described previously, ensures that the appropriate cell types respond to complement activation products. Regulators are broadly expressed, on most host cells and particularly those exposed to plasma, to protect from damage; however, there are notable exceptions where one or more regulators are absent or nearly so; for example, erythrocytes lack MCP, 38 while some cells in the brain lack CD59.69 Plasma contains an abundance of complement proteins, at concentrations varying from very high (1 g/L for C3) to very low (1 mg/L for fD). The major site of biosynthesis for most of these plasma components is the liver; abundant proteins like C3 and C4 are products of hepatocytes and, like the other hepatocytederived plasma complement proteins, behave as acute phase reactants, increasing in concentration in response to inflammatory cytokines. Hepatic synthesis of C3 and C4 can therefore increase two- or three-fold during inflammation, but plasma levels rarely increase as much because inflammation is also associated with increased complement activation and consumption of these proteins. A few complement proteins are mainly produced elsewhere; tissue macrophages are the major source of plasma C1q, adipocytes the primary source of fD, and leukocytes, likely neutrophils, the major source of plasma C7?0 Other cell types and tissues can contribute to plasma complement protein production, and this local production is likely to be very important for complement activity within organs and tissues, particularly when inflammation is present. The kidney is a secondary source of

claaalcal pathway

c1inh

1 Angloede

C1q C1 r C1s

MBLpathway

\ C4

/_

many complement proteins; indeed, transplantation studies have shown that the kidney produces up to 5% of plasma C3, increasing to as high as 10% with renal inflammation.71 There is very little complement synthesis in the noninflamed brain, fortunately so because brain cells are poorly protected from complement; however, inflammation switches on complement production in glia and neurons, contributing to brain cell loss in neurodegeneration.

COMPLEMENT MUTATIONS. POLYMORPHISM$. AND AUTOANTIBODIES Mutations in complement proteins are rare events that can be catastrophic and cause partial or total deficiency of the protein, or more subtle, altering the expression level or functional efficiency of the protein. Polymorphisms are common variations in protein composition that may or may not have functional consequences. The most dramatic outcome of a mutation in a complement protein is deficiency, which may involve a loss of protein product (Type I deficiency) or the production of a functionally inactive protein (Type II deficiency). For almost every complement protein, regulator, and receptor, individuals totally and/or partially deficient in the component have been described, experiments of nature that have informed understanding of the biological roles of different complement pathways; indeed, deficiencies are best understood when grouped according to pathway or family affected (Fig. 36.13).

alternative pathway

MBL MA~P1 MASP2 ·-.. C3(H 20)

~

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fB~·.......

C4

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I~ ···-..··..•_ fD proper~ ,...

~

!

C2 C2 Lupua-llke disease~ \. \.. / Severe, recunent ~C3! bacterial Infections .._

1

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Severe, recunent bactertallnfectlons

Nelaaertal Infection Recurrent Nelaaerlal

C6 c7 C8 C9~ Infection

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FIG. 36.13. Complement Deficiencies. Deficiencies of most of the components and plasma regulators of complement have been described in human subjects. The clinical presentations commonly associated with each of the deficiencies are summarized in this simplified figure.

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Classical Pathway Component Deficiencies Deficiencies of CP component proteins {C1, C4, C2) remove the capacity for efficient activation of complement on ICs and are strongly associated with lupus-like IC disease and infections.n Clq deficiency is rare (only a dozen or so families have been described, and all affected homozygous individuals develop severe lupus-like disease with rashes, renal failure, and other sequelae before adulthood). Deficiencies of either Clr or Cls are similarly rare and often combined because of linkage and coordinate expression of the genes; symptoms and penetrance are as for C1q deficiency. Total C4 deficiency is also rare because there are two C4 genes (C4A, C4B) each with two alleles, requiring co-inheritance of four mutations for complete deficiency. These rare individuals will all develop lupus-like disease early. Mutations leading to loss of expression of one of more C4 alleles (null alleles) are relatively common in the population: 35% have one null allele, 8% two null alleles, and 1% three null alleles; these latter individuals will have low plasma C4 levels and an increased tendency to develop IC disease, greater in those with more null alleles and in those null at the C4A gene.73 Genetic testing for C4 gene copy number is therefore a useful adjunct to measuring plasma levels in testing for C4 deficiency. C2 deficiency is the most common complement deficiency among Caucasians; C2 null alleles are present in some 1% of individuals, giving a predicted incidence of deficiency of 1:10,000 in the population. Although C2 deficiency does increase the risk of developing IC disease, the majority of C2-deficient individuals are healthy, detected at routine screening because their plasma lacks CP hemolytic activity. The strong association of classical pathway deficiencies with lupus-like disease has led to the assertion that lupus, not only in complement deficiency but also in classical systemic lupus erythematosus (SLE), is caused by the defective clearance of immune complexes and apoptotic cells which then become sources of autoantigens.74 This "waste disposal hypothesis" has altered perceptions of the disease and modified approaches to treatment.

Lectin Pathway Component Deficiencies Deficiencies of LP components, partial and total, have been described. Plasma levels of MBL vary widely in the population due to a complex set of mutations and polymorphisms in the constituent chains.75 Three mutations clustered in exon 1 of the MBL gene individually disrupt the capacity to form the higher oligomers necessary for MBL function and, in homozygosity or compound heterozygosity, result in complete deficiency of MBL. Complete MBL deficiency predisposes to bacterial infections, particularly in individuals who are otherwise compromised ( eg, neonates, the elderly, patients with cystic fibrosis, and patients with acquired immunodeficiency syndrome)?6 A number of common polymorphisms in MBL can also profoundly affect MBL plasma levels and oligomerization status, and those inheriting an extreme set of variants can have profoundly low MBL levels, with the same resultant problems as those totally deficient in MBL. Genetic mutations leading to deficiencies of fi.colin-2 and fi~olin-3 were recently reported and shown to

cause defective LP activation in plasma.30 Mutations in the MASP2 gene leading to complete or subtotal deficiency of this key LP enzyme MASP-2 were also described,77 but their clinical relevance remains unclear.

Cl Deficiency C3 is the most abundant and most critical of the complement proteins, essential for activation through all pathways. It is therefore not surprising that total C3 deficiency, a very rare finding restricted to a few dozen families, is devastating?8 Individuals present in childhood with severe, recurrent bacterial infections that, if not promptly treated, will progress to uncontrolled sepsis and death. Supportive care and antibiotics can permit survival into adulthood, but IC disease and renal injury then become apparent.

Alternative Pathway Component Deficiencies Deficiencies of the AP components are rare, with just a few individuals or families reported for each. Total deficiency of fB has not been reported, although a recent abstract described total or subtotal deficiency in a teenager with meningitis?9 Each of the ill-deficient cases have presented with Neisseria! infection, most often fulminant septicaemia, suggesting that AP amplification is particularly important in dealing with bacteria of this genus80; however, numbers of cases are small, and it is possible that AP deficiencies have broader consequences for susceptibility to bacterial infections. Deficiency of the AP stabilizer, properdin, is also associated with severe Neisseria! infections, meningitis, and septicaemia.81 Inherited in an X-linked manner, it is almost exclusively seen in young males, usually presenting in childhood. It is rare but probably underascertained. Properdin deficiency may be associated with a complete absence of the protein (Type 1), presence of very low levels of normal protein (Type II), or presence of near-normal levels of a nonfunctional protein (Type III) . Patients who survive the initial infectious episode tend to do reasonably well thereafter as anti-Neisserial antibodies develop, permitting recruitment of the CP in subsequent exposures.

Terminal Pathway Component Deficiencies Deficiencies of TP ~omponents (C5, C6, C7, C8, and C9) are relatively common and all predispose to Neisseria! infections. C5, though not a true TP component, is included here because consequences of deficiency are similar; C5 deficiency has been identified in a few dozen families from across the globe, perhaps more common in Africans and African Americans. Deficiency predisposes to Neisseria! infections, including meningitis and septicaemia, and perhaps other infections82; unlike AP deficiencies, infections tend to be recurrent and often involve unusual serotypes of Neisseria. C6 deficiency is the second most common complement deficiency in Caucasians, predicted from null allele frequency to have an incidence of around 1:10,000 in the population; in African Americans, it may be even more common, with a predicted incidence greater than 1:2,000.83 C6 deficiency is

CHAPTER 36

common among the Cape Coloured ethnic group in South Africa, believed to be the result of a founder mutation imported from Holland in the 17th century. Consequences are similar to those of CS deficiency: recurrent Neisseria! infections. Subtotal deficiency of C6 has also been described, with levels a few percent of normal and with or without linked deficiency of C7; the frequency and relevance of these is uncertain.84 C7 deficiency is also common in some racial groups, with a predicted incidence of 1:10,000 in Israelis of Moroccan Jewish descent.85 The frequency of total deficiency in Caucasians is not well defined, but personal experience suggests it is considerably less common that C6 deficiency in the United Kingdom. Clinical consequences are identical to C6 deficiency. Subtotal C7 deficiency is common in Caucasians, with a defined mutation at an allele frequency of about 1%.84 A few cases of combined deficiency of C6 and C7 have been described as a consequence of the close linkage of the genes.86 C8-deficient individuals can lack either C8ay or csp chain; the former is rare in Caucasians, the latter more common.87 In all C8ay-deficient individuals characterized to date, the mutation is in the C8A gene. Plasma contains no detectable C8ay and very low amounts of csp, indicating that this chain is labile in isolation in plasma. C8P-deficient individuals have no detectable csp in plasma but variable amounts of C8ay, often approaching normal. In either case, plasma haemolytic activity is completely lost. Presenting symptoms are identical to those of other TP deficiencies noted previously. C9 deficiency is rare in most ethnic groups with only a handful of cases reported in Caucasians, but it is by far the most common complement deficiency in rapanese and other Oriental races, the result of a single, ancient non-sense mutation (R95X) in the gene.88 Frequency of the null mutation is about 2% in Koreans and 1% in Chinese; however, in Japanese, null allele frequency is 6.7%, giving a predicted incidence ofC9 deficiency of about 1:260 of the population! This incredible frequency implies some selective advantage of the null allele in these populations; most compelling is the suggestion that C9 deficiency blunts the sometimes overvigorous complement response to infection, making C9-deficient individuals less susceptible to the development of sepsis.

Plasma Regulatory Protein Deficiencies Deficiencies of many of the complement regulatory proteins have been described. The most common by far is Clinh deficiency, which has a prevalence in Caucasian populations of 1:50,000. Deficiency of Clinh is common primarily because it presents as an autosomal dominant, heterozygous deficiency is sufficient to cause disease.89 Deficiency of Clinh causes the disease hereditary angioedema (HAE), characterized by recurrent, acute episodes of localized swelling of the skin and/or mucous membranes. As noted previously, Clinh is not only the sole plasma protease inhibitor of activated Clr/s and the MASP enzymes, but also inhibits enzyme cascades involved in coagulation, fibrinolysis, and the contact system. Inflammation or injury triggers activation of complement and these other proteolytic cascades in tissues, leading to the consumption of C1inh, a suicide inhibitor.

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In the presence of only a single normal CliNH gene, synthesis of C1inh cannot keep up with triggered consumption and plasma levels fall precipitously, leading to loss of control of the multiple cascades. Active products of the cascades, particularly the kinins, cause localized tissue edema, the hallmark symptom of HAE. Many different mutations in CliNH have been described in patients with HAE.90 In about 80% of cases, these are null mutations with no protein product (Type I); in about 20%, a protein is produced but is inactive (Type II). Deficiency of fl, the key enzyme responsible for the inactivation of the convertase enzymes of the AP and CP, is a rare but devastating problem.78•91 Tickover complement activation and AP amplification proceed unchecked, leading to complete consumption of C3; this secondary C3 deficiency predisposes to bacterial infections and IC disease, precisely as in primary C3 deficiency. The plasma regulator of the AP convertase, ffi, is essential for fluid-phase AP regulation. Complete deficiency offli is rare and, like fi deficiency, results in uncontrolled AP amplification and consumption of C3?8 Secondary C3 deficiency predisposes to infection and immune complex disease, but fli deficiency uniquely affects the kidney, causing a characteristic set of renal pathologies, including various subtypes of membranoproliferative glomerulonephritis and atypical hemolytic uremic syndrome (aHUS).92 aHUS is much more strongly associated with mutations in the carboxy-terminal SCRs of fli that do not cause deficiency but do reduce or ablate the capacity of fli to bind surfaces; these binding mutants of fli are the most common genetic finding in aHUS, responsible for up to half of all cases and causing disease even in heterozygosity.93 Deficiencies of other members of the fli gene family have recently been described and in some cases associated with disease. Combined deletion of the FHRl and FHR3 genes is a common finding, present in about 5% of Caucasians and even more common in some racial groups. Absence of fliRl and fliR3 is associated with aHUS, apparently because it predisposes to the development of anti-ffi autoantibodies (described in the following) 94; the same deficiency is protective for the common eye disease age-related macular degeneration (AMD). Deficiencies of other fliR proteins are much less common and of uncertain clinical relevance. DeficiencyofC4bp, the fluid phase regulator of the CP, is extremely rare with only two case reports to date; in neither case were the presenting symptoms compatible with a complement defect, suggesting that they were ascertainment artifacts.

Membrane Regulatory Protein Deficiencies Complete, global deficiencies of any of the cell surface regulators are very rare events. A single case of complete deficiency of CD59 was reported, a young Japanese male who presented with a severe hemolytic syndrome.95 A handful of families with complete deficiency of DAF have been described, apparently healthy apart from an apparent association with gut inflammation in some families. 96 Of note, both CD59 and DAF are GPI-anchored molecules and are missing from the GPI-negative hemopoietic clone in the hemolytic disorder paroxysmal nodurnal hemoglobinuria (PNH);

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absence of these complement regulators is the cause of the erythrocyte lysis and activation of platelets and leukocytes that typify this disorder.97 Complete defidency of MCP has not been reported, perhaps reflecting the fact that this molecule plays key roles outside of complement regulation. Deficiencies of complement receptors are similarly rare or nonexistent. No complete inherited deficiencies of CRI have been described, although acquired deficiencies are common in those with IC diseases such as SLE. No deficiencies ofCR2 have been reported, although again, acquired low levels are found in SLE. A specific deficiency of the CR3 a-chain was described in one individual presenting with severe SLE; deficiency of the common !}-chain causes loss of all four integrins that share this chain with profound consequences for leukocyte adhesion and immune function. No complete deficiencies of the AT receptors have been reported.

Complement Protein Point Mutations Point mutations that affect function of a complement protein in more subtle ways are also sometimes of clinical importance. Perhaps the best example is that of fH mentioned previously, where the carboxy-terminal SCRs appear to be "hotspots" for point mutations that have no direct effect on the complementregulating capacity of fH, but profoundly reduce its capacity to bind surfaces.98 Even in heterozygosity, these mutations predispose the individual to the development of aHUS, particularly when combined with other complement polymorphisms or mutations that alone have little effect. Another intriguing example is a recent report describing an unusual renal disease in Greek Cypriots; search for a genetic cause identified a mutation in FHR5.99 The mutant protein was present in plasma but was dysfunctional, unable to bind efficiently surface-bound C3b; quite how this functional deficit in an apparently minor member of the fH family causes such major pathology remains to be determined.

Complement Protein Polymorphisms Common polymorphisms in complement components and regulators have also been linked to disease, particularly chronic inflammatory diseases and infections. Among the inflammatory diseases linked to complement polymorphisms, two stand out; AMD, the most common cause of vision loss in the elderly in Western societies, and dense deposit disease (DDD), a rare renal disease. A common polymorphism in fH causing a single amino acid change in the seventh SCR (Y402H) was shown in many independent studies to be strongly linked to AMD, individuals homozygous for the less common H allele having a 6- to 10-fold higher risk of developing the disease than those homozygous for the more common Y allele.100 Vision loss in AMD is caused by a buildup of an amorphous material called "drusen" beneath the retina that disrupts retinal architecture, causing loss of vision. Analysis of drusen shows that it contains an abundance of complement fragments, indicating that it is a site of complement activation, driving inflammation in the eye. Recent biochemical studies have suggested that the Y402 variant of fH binds better to drusen than the H402 variant-more fH binding will result

in less complement activation, less inflammation, and less disease in Y402 homozygotes. A second fH polymorphism, V621, is also linked to AMD, the I allele being protective. This polymorphism increases the C3b binding and cofactor activities of fH, making a more efficient inhibitor and thus restricting AP activation.101 Other complement polymorphisms have also been linked to AMD; a common polymorphism in C3 (C3S/F; R102G) was associated with increased risk of AMD (two-fold higher for G allele homozygotes compared toR allele homozygotes), while a common polymorphism in factor B (R32Q) was associated with a near-two-fold decreased risk for Q allele homozygotes. The functional effects of the C3 and fB polymorphisms have been characterized102,103 ; in each case, the allele that is "risk" for AMD (C3102c; factor B3:nJ is more active in forming AP convertases and will hence drive more complement activation and inflammation in drusen. DDD is linked to both of the polymorphisrns in fH described previously, Y402H (H allele risk, as for AMD described previously), and V621 (I allele protective, as in AMD), and is also linked to the C3 Rl02G polymorphism.

The Complotype The "set" of complement polymorphisms that an individual inherits can have dramatic effects on complement activity in his or her plasma. An individual inheriting in homozygosity the more active variants of C3 and fB and the less inhibitory variants of fH can have an AP activation loop in plasma that is 10-fold more active than that of an individual inheriting the less active components and more inhibitory fH variants.103 This inherited "complotype" will markedly affect disease susceptibility-an individual with a more active AP will combat infection well but may be more susceptible to chronic inflammation, while those with a less active AP may be at risk of infection but protected from chronic inflammatory diseases (Fig. 36.14). To date, attention has focused on the AP; however, polymorphisms in any of the LP, CP, and TP components and regulators, that alter function and/or plasma levels, will impact upon the complotype of an individual.

Complement Autoantibodies Autoantibodies against complement components, regulators, and complexes have been found in and linked with disease. Nephritic factors (NeFs) are autoantibodies that bind and stabilize the AP (and rarely CP) C3 convertase. They are, as the name implies, associated with renal disease, usually present in patients with DDD, and sometimes found in other types of membranoproliferative glomerulonephritis.104 NeFs are also found in the rare disease partial lipodystrophy where peripheral adipose tissue is lost.105 NeF stabilizes the convertase both by slowing natural decay and by preventing accelerated decay mediated by fH and other regulators. Complement is therefore consumed systemically and/or locally, and this in tum causes renal pathology. It is not at all dear why an immune response to the convertase enzyme occurs in NeF-positive individuals. One due is provided by the demonstration that NeF are linked to the

CHAPTER 36 Activators

Protect from inflammation Predispose to infection

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Regulators

Predispose to inflammation Protect from infection

FIG. 36.14. Tbe Complotype. Despite the tendency of complement to spontaneously activate, the balance of activators and regulators ensures that homeostasis prevails. lnterindividual variability in terms of complement protein/regulator plasma levels, common polymorph isms, or rare mutations dictate that some individuals will have an intrinsically more active complement system (rightt, protective against infection but risk for chronic inflammation, while others have an intrinsically less active system (left), risky for infection but protective for chronic inflammatory disease.

convertase-stabilizing C3 polymorphism, C~02c (C3F) 103'106; perhaps a more stable convertase is more likely to be immunogenic because it lasts longer? Autoantibodies against fH are increasingly recognized in association with renal disease, particularly aHUS.94 Almost all anti-fH-positive aHUS patients lack fHRl and fHR3; this appears to be necessary but not sufficient for anti-fH production because most individuals with this common gene deletion do not make anti-fH antibodies. The anti-fH antibodies predominantly target the carboxy-terminal SCRs of fH and inhibit fH binding to surfaces, mimicking the effects of the aHUS-associated mutations in fH. Anti-Clq autoantibodies are associated with a number of autoimmune and renal disease, notably lupus nephritis and poststreptococcal glomerulonephritis. Anti-Clq antibodies are a useful marker of disease activity, but their contribution to the disease process is uncertain. Anti-Clinh autoantibodies are associated with a rare, acquired form of angioedema mimicking HAE107; the autoantibodies target the reactive center of Clinh thereby blocking its function.

COMPLEMENT IN DISEASE: ROLES AND TREATMENT OPPORTUNITIES As noted previously, complement deficiencies, mutations in complement proteins, polymorphic variants in complement, and complement autoantibodies can all cause disease. In addition to these specific circumstances, complement contributes to a large number of diseases, triggered by diverse events, by driving inflammation and tissue damage. Some diseases are clearly complement mediated, while others present a

mixed picture with multiple effectors contributing. The best example of the former is the hemolytic and thrombotic disorder PNH. A somatic mutation gives rise to a clonal population of hemopoietic cells that cannot make GPI anchors and thus lack all GPI-linked proteins from their surfaces.97 Key among these are the complement regulators DAF and CD59. Absence of DAF and CD59 on erythrocytes, which intrinsically lack the other membrane regulator MCP, renders them highly susceptible to autologous complement activation and hemolysis, resulting in the cardinal signs anemia and hemoglobinuria. Platelets and leukocytes derived from the clone are also deficient in regulators and prone to complement activation and injury, resulting in increased platelet aggregation and leukocyte activation, which in turn predispose to thrombotic stroke.

Complement in lschemia-Reperfusion lniuries Ischemia-reperfusion (IR) injuries (eg, ischemic stroke, myocardial ischemia, or post-bypass) are highly dependent on complement activation. The ischemic tissue, when reperfused spontaneously or through clinical intervention, activates complement through the AP and likely also the LP, leading to injury and inflammation, increasing the extent of the injury.108 Precisely why the ischemic tissue becomes an AP activator is still debated, although reactive oxygen species generated during anoxia are implicated and the ischemic tissue shows decreased expression of complement regulatory proteins. The earliest studies of the efficacy of anticomplement drugs were performed in animal models of IR injury;

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these, and many more since, have firmly demonstrated that complement inhibition is highly protective in this group of conditions.

Complement in Sepsis Sepsis is a devastating disease for which there are few therapeutic options. The best available drug treatments, steroids, and/or activated protein C, only modestly impact outcome, and for most patients, all that can be offered is life support. As a consequence, mortality exceeds 25% of cases and has remained stubbornly unchanged for decades. Therealization that sepsis was a consequence of overwhelming immune system activation provoked considerable interest and investment in anti-tumor necrosis factor and other agents targeting inflammatory cytokines; however, none have yet stood the test in clinical trials. There is an abundance of evidence implicating complement in sepsis. Serum complement levels fall, and complement activation products, including C3a, CSa, and TCC, are present at high levels and are predictive of outcome.109 Animal models of sepsis have added to the evidence; in many studies, inhibition of complement activation improves the clinical outcome, animals genetically deficient in complement components have improved survival. The activation fragment CSa appears to be particularly important in driving the disease in man and models, at least in part because of its effects on neutrophils. Initially, a powerful attractant and activator, C5a in excess causes suppression of neutrophil phagocytosis and mediator release.uo Antibodies that block C5a and several different agents that block the C5a receptor have been shown to be protective in sepsis models but have yet to be tested in man.

Complement in Transplant Rejection The immune system treats transplanted organs in much the same way that it treats other foreign invaders: it attacks. Complement is no exception in this respect, although the full force of complement-mediated rejection is seen only when the organ is very foreign. When organs are transplanted across species barriers, xenotransplantation, the host response is instant: hyperacute rejection occurs, destroying the organ graft within minutes of initiating perfusion. The primary cause of this explosive rejection is activation of complement in the graft.lll Pig-to-human transplantation is superficially attractive for anatomical, physiological, and ethical reasons; however, when pig organs are perfused with human blood, IgM natural antibodies in the plasma bind carbohydrate epitopes on the pig cells, activate complement, and destroy the graft. A decade ago, there was much interest in the production of pigs expressing transgenically the human complement regulators on their cells. This strategy, combined with a second genetic manipulation to modify the expressed carbohydrate epitopes, was successful in creating pig organs resistant to hyperacute rejection when perfused with human blood or transplanted into primates. This promising industry was mortally wounded by concerns about the transmission of pig viruses into the human population, but is slowly rising from the ashes on the back of even better pigs.112

Hyperacute rejection can also be seen in human-human (allo-) transplants where the donor and recipient are ABO blood group incompatible or where the recipient is presensitized to donor antigens.113 Here too, antibody binding activates complement in the graft, leading to its rapid destruction. As anticomplement drugs, described in the following, become more widely available, it is likely that they will be used to protect grafts transplanted into sensitized or incompatible individuals. Surprisingly, work in animal models suggests that even short-term complement inhibition can have long-term effects on graft survival, a phenomenon known as accommodation. Allotransplants are also subject to acute vascular rejection, a slower process starting days or weeks after the transplant and taking days or weeks. Long considered a cellmediated process, it is now clear that complement activation contributes; rejecting grafts stain for C4d, a marker of CP activation and inhibition of complement in animal models, is protective.

Complement in Autoimmunity In many autoimmune diseases, although the initiating event is the generation of an immune response against self antigens, much of the observed pathology requires complement activation. In some diseases, complement activation is essential for pathology. Myasthenia gravis offers a particularly good example. The initial event is an autoimmune response against motor end plate sequestered antigens, notably the acetylcholine receptor. Binding of antibody in itself causes little damage, but the resultant activation of complement causes the destruction of the end plate and failure of neuromuscular conduction.114 In the animal model, inhibition of complement completely prevents the development of weakness and paralysis.115 In multiple sclerosis, an immune response against central nervous system-sequestered antigens leads to breaching of the blood-brain barrier, influx of inflammatory and immune cells, and the generation of antibodies that bind myelin and other structures. Complement activation in the brain is particularly harmful because myelin and many brain cell types are poorly protected by complement regulators and are thus easily damaged or destroyed.69 Again, animal models have suggested that complement inhibition can suppress disease116; however, it is likely that multiple sclerosis is a heterogeneous disease, with complement as a major driver in only some patients.117 A related autoimmune neurological disease, neuromyelitis optica, is associated with autoantibodies against aquaporin 4, a water channel expressed on astrocytes and some other cell types. Complement activation is a prerequisite for the myelin loss and cell damage that typifies this disease. In the autoimmune peripheral neuropathy, GuillainBarre syndrome, antibodies target peripheral nerve myelin, fix complement, and drive axonal damage. Recurrent fetal loss is a distressing syndrome where women mount an immune response against fetal antigens leading to fetal rejection. Recent evidence has implicated antiphospholipid antibodies in a large proportion of affected women; evidence from human studies and mouse models implicates complement activation as the major effector of rejection in this syndrome.118

CHAPTER 36

COMPLEMENT

I

885

factor for AMD has focused attention; individuals homozygous for the risk allele (fH-H402) have up to 10-fold greater risk of developing AMD compared to those homozygous for the protective allele (fH-Y402). 99 Other AP protein polymorphisms have also been linked to AMD, leading many in the field to conclude that AMD is caused by AP dysregulation. Anticomplement therapies, described in the following section, are now being tested for treating AMD.

Complement in Renal Disease Many renal diseases are associated with abundant complement activation in the kidney. Some of these are autoimmune in origin, with antibody or immune complex deposition in the glomerulus being the trigger for complement activation. The glomerular injuries associated with aHUS, fHRS deficiency, and DDD were described previously. DDD, noted previously for its association with anti-C3 convertase autoantibodies (NeF), is an archetypal disease of complement dysregulation, linked not only to NeF but also to mutations and polymorphisms in complement proteins that predispose to a loss of fluid-phase control of activation.104 Precisely why these triggers target exclusively the kidney remains unknown. Other renal diseases in which complement activation plays a critical role include the various membranoproliferative glomerulonephritides, IgA nephropathy, antiglomerular basement membrane disease, lupus nephritis, and membranous nephritis.119 In the last of these, MAC-induced activation of glomerular podocytes is the principal cause of the renal injury.

ANTICOMPLEMENT THERAPIES General Principles Drugs targeting the complement system have come of age in the last 10 years. Twenty years ago there were, with the exception of Clinh, described in the following, no clinically feasible ways of modulating complement activation in man. Cobra venom factor, a C3-like molecule that forms a stable AP convertase in serum and rapidly consumes all available complement activity, was used as a proof-of-concept in animal models of disease, but caused shock syndromes and occasional deaths in animals through precipitous release of activation products, making it unviable clinically. The scene has changed markedly in recent years with the arrival of an armory of agents that inhibit complement in different ways and in different parts of the pathway (Fig. 36.15; Table 36.4).

Complement in Degenerative Diseases Complement also plays roles in many degenerative disorders, likely because of the accumulation of damage from low-grade complement activation over many years. The association of complement polymorphisms with AMD has been mentioned previously, a more active complement system increasing the risk of disease.99' 103 The brain and retina are particularly susceptible to this slow-burn injury, likely because these sites have poor waste disposal systems, allowing the accumulation of debris-drusen in AMD, plaques and tangles in Alzheimer's disease-that cause further complement activation thereby driving inflammation and injury. The recent demonstration that a common polymorphism in the AP regulator fH is the strongest known risk

Modified Membrane Regulators as Anticomplement Drugs The advent of clinically viable complement inhibitors came with the invention by Fearon and colleagues of the concept of modifying the naturally occurring membrane complement regulators to inhibit complement in plasma.120 They expressed a soluble form of CRl (sCRl) comprising the 30 SCRs of the extracellular portion and showed that it was a powerful fluid-phase inhibitor of C3 cleavage. When tested

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SECTION VI INDUCTION, REGULATION, AND EFFECTOR FUNCTIONS OF THE IMMUNE RESPONSE

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RG 37.6. The GranQme APathway of Cell Death. Reactive oxygen species generated by granzyme A (represemed by scisso~ cleavage of Ndufs3 in electron transport complex I in mitochondria drives the endoplasmic reticulum-associated SEJ complex into the nucleus. Granzyme A enters the nucleus by an unknown pathway. In the nucleus, Granzyme A cleaves three components of the SEJ complex (SEJ, HMGB2. and Apel) to activate two nucleases in the complex to make single-stranded deoxyribonucleic acid (DNA) lesions; NM13-H1 makes a nick. which is extended by the exonuclease Trexl. Granzyme A also degrades the lamins and the linker histone HI and removes the tails from the core histones. opening up chromatin and making it more accessible to these nucleases. DNA repair proteins Ku70 and PARP-1 are also targets. by damaged mitochondria drives an endoplasmic reticulum (ER)-associated oxidative stress response complex, called the SET complex, into the nucleus where it plays a critical role in granzyme A-induced nuclear damage.17Maz The SET complex: contains three nucleases (the base excision repair endonuclease Apel, an endonuclease NM23-Hl, and a 5'-3' exonuclease Trexl), the chromatin modifying proteins SET and pp32, which are also inlubitors of the tumor suppressor protein phosphatase 2A, and a DNA binding protein that recognizes distorted DNA, HMGB2.1.,_187 One of the normal functions of the complex. is to repair abasic sites in DNA generated by oxidatiw damage. Recent studies also implicate the cytosolic SET complex as binding to the human immunodeficiency virus preintegration complex. and facilitating human immunodeficiency virus infection.188 The SET complex exonuclease Trexl digests cytosolic DNA produced by endogenous

retroviruses and infectious viruses to inhibit the innate immune response to cytosolic DNA.11111- 192 Mutations in Trexl that inactivate its nuclease activity or cause its mislocalization are linked to human inflammatory and autoimmune diseases, including Aicardi-Goutiere syndrome and systemic lupus erythematosis.19'-197 Granzyme A, which traffics to the nucleus by an unknown mechanism, converts this DNA repair complex into an engine for DNA destruction by cleaving SET, an inhibitor of the endonuclease NM23-HI.I85 This allows NM23-Hl to nick DNA; the exonuclease Trexl then extends the break.184 At the same time, granzyme A cleaves and inactivates HMGB2 and .Apel to interfere with base excision repair.186.187 In addition to disabling base excision repair, granzyme A also interferes with DNA repair more generally by interfering with the recognition of damaged DNA by cleaving and inactivating Ku70'98 and PARP-1.199 Within the

CHAPTER 37 CELL-MEDIATED CYTOTOXICITY

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-~p~(MAo) tttldrboh!tot,atbn &7P'f to produce a necrotic granuloma. The necrotic granuloma may spontaneously heal or become sterilized by chemotherapy. Complete inactivation and scarring then ensues or in the event of incomplete clearance of persistent bacteria, reactivation of disease may occur later. In the case ofTB, the necrotic granuloma may also further develop to produce a large central caseous mass that eventual tv liquefies to give rise to a caseous granuloma. Absorption of bronchi by this structure allows egression of bacteria into the airway mediating transmission. IFNy, interferon gamma; IL interleukin; TNF-a., tumor necrosis factor-alpha.

CHAPTER 40

give insight into events governing how granulomas develop during the early stages of infection. These responses can be enhanced by early production of the cytokine IL-17 by activated yl) T cells.283 IL-17 enhances influx of Th1 cells produdng IFNy and TNF-a into the infected lung that are in turn able to control bacterial replication.380 Excessive IL-17 may also enhance host pathology due to enhanced Th1 responses and require counter measures of control.381 Such control could be provided by IL-10 as Th1 cell influx into the lung during murine TB is enhanced in its absence. 382 Furthermore, enhanced IL-17 production in the lung of mice after repeated application of M. bovis BCG during TB results in exacerbated disease, demonstrating the need to regulate inflammation to maintain a protective immune balance.383 In addition, IL-12 signalling of DCs represents a key event initiating Th1 responses, and this IL-12 signaling shows multifunctionality during the early granulomatous response to infection. Signaling of immature DCs via engagement ofiL-12 receptor f}1 chain by the IL-12p40 homodimer, IL-12p80, mediates DC migration to dLNs, a prerequisite for driving rapid T-cell responses in the lung.384 DCs can also produce an IL-12 receptor splice variant that not only drives naive T-cell proliferation in lymph nodes but also enhances migratory activity ofT cells.385 Chemokines, which play multiple roles in early immune responses, are also involved in granuloma formation. CCR7 is required for murine granuloma formation 386; CCL19 and CCL21 participate in development of the Thl responses while another homeostatic chemokine, CXCL13, is involved in the spatial construction of granulomatous lesions.387 CCL19 and CCL21 are ligands of CCR7 on DCs and also signal their trafficking to dLNs to prime T-cell responses.254 Therefore, homeostatic chemokine function demonstrates an intimate relationship between the need to drive T-cell responses in dLNs and the orchestration of granuloma formation in the lung during early stages of TB. During later stages of this disease, when granuloma formation has been fully established, T-cell activation can occur in the lung via ectopic or tertiary lymph node structures, such as inducible bronchus-associated lymphoid tissue.338 This ability to prime T cells directly in the lung may become more relevant in later stages of human disease when cavity formation and extracellular growth of M. tuberculosis results in the need to provide protective immunity in the face of increased bacterial loads in the lung. 388·3119 In addition, IL-17 produced by T cells enhances CXCL13 expression, which is required for inducible bronchus-associated lymphoid tissue formation, indicating an additional role for this cytokine in protective immunity.388•390

Contribution of Granuloma to Disease In addition to its central role in TB, granuloma formation and development represents a central feature of disease resulting from intracellular infection with brucellae.391 Brucellosis is a multiorgan disease in which granulomas contain largely epithelioid macrophages occurring in lymphatics, brain, lung, and bone. Hepatic granulomas can develop central necrosis. The structural similarity of these granulomas to those seen in other infections makes a definitive diagnosis of brucellosis complicated.392

IMMUNITY TO INTRACELLULAR BACTERIA

I

997

Melioidosis represents another systemic disease with an important granulomatous component in bone, brain, and lung.393 The disease in humans is caused by B. pseudomallei. Granulomas often show layers of epithelioid macrophages and presence of giant cells. These granulomas are often unable to contain bacteria because systemic spread of bacteria and septicemia can occur. Lymphogranuloma venereum, a sexually transmitted disease where granuloma formation is central, is caused by the Ll, L2, and L3 serotypes of C. trachomatis. Trachoma, which results from a granulomatous response of the upper eyelid due to C. trachomatis infection, is a leading cause of infectious blindness worldwide.20 In human infection with S. enterica serovars, 'JYphi and Paratyphi, bacteria are disseminated within MPs to the liver, spleen, and lymph nodes where granuloma are formed. 394 The definitive role of granuloma formation in development of active TB has been difficult to establish. A century ago, Anton Ghon defined latent subclinical TB in humans as the Ghon complex, which is composed of single subpleural caseous granulomas accompanied by caseation in the corresponding dLNs.395 Animal models currently in use are biased toward accelerated disease, where granuloma development consistently accompanies pathology, making understanding the role of the granuloma in protection difficult to discern. Key to initial control of M. tuberculosis very early in infection is how the infected cell dies. Should the cell die by apoptosis, bacterial growth is primarily prevented. Should the cell die to necrosis, where the host cell membrane is breached, M. tuberculosis can proliferate and disseminate.396'397 Central necrosis and caseation in granulomas could in some way reflect the balance between these mechanisms of cell death. Infected macrophages also employ antibacterial mechanisms to control intracellular bacterial growth, such as autophagr26' 142 and production of antimicrobial peptides. 398' 399 IFNy enhances these mechanisms, but intracellular bacteria are often incompletely eradicated requiring consecutive waves of specific T cells and monocytes to arrive in the lung or dLNs, which then arrange into the dassic stratified granuloma structure.374.4°0•401 The murine model of mycobacterial disease has addressed the contribution ofT-cell immunity to granuloma development using intravital imaging. These studies have shown that antigen presentation to T cells within granulomas leads to production ofTNF-a, which is critical to maintenance of granuloma structural integrity.402 Furthermore, the T-cell compartment within granulomas was shown to be highly dynamic with T cells entering and exiting lesions. T-cell effector function in this model could be potentiated by exogenously introduced antigen, and this strategy might be explored using therapeutic strategies to augment the protective properties of granulomas.403 The understanding of granuloma development has benefited remarkably from use of zebrafish infected with Mycobacterium marinum to simplify study of the early events of granuloma formation in vivo. In particular, intravital imaging of zebra fish embryos to model early events in granuloma formation in the absence of adaptive T-cell responses showed that infected macrophages traffic readily

998

I

SECTION VII

IMMUNITY TO INFECTIOUS AGENTS

between multiple nascent granulomas.404 This suggests that granuloma formation before the onset of adaptive immunity may in fact assist stable establishment of infection at an early stage. Later, T cells may be required to imprint on the granuloma the ability to contain mycobacterial growth. The zebrafish has been exploited for forward genetic screening revealing a critical function of Ita4h, whose gene product catalyses leukotriene B4 synthesis. Leukotrienes are eicosanoids derived from essential fatty acid metabolism that demonstrate chemotactic and anti-inflammatory activity. Increased leukotriene B4 synthesis was found to suppress TNF-a production, which in turn abrogated protection in nascent granulomas.300 This finding highlights the utility of using diverse models to address fundamental issues in granuloma biology. Other animal models ofTB, namely nonhuman primates (NHPs ), rabbits, and guinea pigs show some ability to model granuloma formation. In contrast to the murine model, granulomas formed in rabbits and guinea pigs demonstrate central caseation and hypoxia!05.4°6 The NHP represents the closest model to human granuloma formation. Infection of NHPs results in a portion of animals with latent M. tuberculosis infection. Unlike other animal models of TB, disease in NHPs transitions through formation of a Ghon complex, suggesting that this initial event may represent the very early response in all cases!07 The lymphatic lesion then undergoes necrosis and caseation as early as 3 to 4 weeks postinfection. These data and those seen in humans suggest that the Ghon complex indeed represents the site at which disease is either contained or progresses. Data from NHPs indicate that granuloma cellular composition and T-cell function can vary greatly in the same host and even in the same organ, for example, the lung. Additional studies in NHPs have established that early granuloma formation is accompanied by potent inflammatory responses, which are characterized by transcriptional networks controlled by the cytokines IFNy and TNF-a as well as the intracellular JAK and STAT signaling pathways, while later granulomas show transcriptional networks reflective of tight control of inflammation and chemokine production.408 This inhibitory property of the late-stage granuloma could reflect the necessity to limit host tissue damage resulting from a nonresolved inflammatory response.

Cellular Mechanisms Active within Granulomas In terms of understanding of cellular mechanisms operative in granulomas and how these impact granuloma biology, TB is the most widely studied granulomatous disease. Macrophages within granulomas demonstrate remarkable morphologic plasticity. Different macrophage morphologies in granulomas include epithelioid and foamy cells. In addition, macrophages can fuse to produce multinucleated giant cells.374 On the tissue scale, one of the most characteristic gross morphologic features of granulomas in TB is their propensity to form a region of central necrosis, which softens in end stages to become caseous or cheese-like. This white mass results from extensive MP cell death. 409 Central necrosis appears at the onset of vigorous T-cell immunity,

suggesting that it could represent a sacrifice of tissue to allow efficient killing of intracellular bacteria. At this stage, granulomas can become inert and eventually calcify. However, if intracellular bacteria continue to grow to up to hundreds of billions of microorganisms, granulomas in the lung become enlarged and can erupt into a bronchus with the central caseous mass liquefying. Coughing up the liquefied caseous mass containing bacteria allows transmission of TB. The molecular and cellular events that define these processes enable understanding of how TB is transmitted and offer important targets for therapy. In a model of granuloma caseation in the lung after dermal infection of knockout mice unable to produce RNis, central granuloma caseation is accompanied by a local increase in serine protease activity. The serine proteases cathepsin G and neutrophil elastase are both active at neutral pH. Although both serine proteases show antibacterial activity,39M 10 cathepsin G enhances necrosis of IFNy-activated macrophages infected with M. tuberculosis.410 Therefore, deployment of serine protease activity in the lung may represent a double-edged sword, reflecting at a molecular level the need to balance mycobacterial killing with macrophage cell death in the granuloma. Recent studies suggest that products of the sstl genetic locus in mice prevent development of necrotic lung lesions during murine TB. A protein product encoded at this locus termed intracellular pathogen resistance 1 shows ability to direct infected macrophages to undergo apoptosis rather than necrosis.411 For disease transmission to occur, the lung extracellular matrix must be remodeled. Fibrillar collagens, highly resistant to enzymatic degradation, lend the lung extracellular matrix its extraordinarily tensile strength. In matrix remodelling, matrix metalloproteinases (MMPs), which cleave collagen at neutral pH, appear to be critical players. Patients with TB demonstrate increased levels of MMP-1 activity in sputum, and a transgenic mouse expressing human MMP-1 suffered increased lung tissue destruction during TB, suggesting MMP-1 is a key proponent oflung remodeling.412 Tissue containing caseous granulomas shows an increase in host cell lipid metabolism.413 Foamy macrophages, containing multiple lipid droplets, are also found in regions surrounding central necrosis in granulomas.413 In addition, human macrophages infected in vitro with M. tuberculosis demonstrate upregulated lipid metabolism.414 M. tuberculosis in sputum from patients with TB are contained in lipid droplets and show reduced antibiotic susceptibility. 415 It is therefore possible that persistence of mycobacteria in foamy macrophages my represent a survival strategy in the face of drug therapy.

GENETIC CONTROL OF RESISTANCE AGAINST INTRACELLULAR BACTERIA Resistance against intracellular bacteria is genetically controlled, and inherited factors are of particular importance in chronic infections with broad clinical spectrum, such as TB and leprosy. Although the impact of host genetic mechanisms on the outcome of infectious disease has been recognized for a long time, our understanding of the underlying

CHAPTER 40

factors remains fragmented. 416' 417 First, resistance to infection is highly polygenic. Second, a marked heterogeneity exists within populations. Third, variability in the genome of the pathogen, as well as environmental factors such as the availability of nutrients, further affect the outcome of the host-pathogen relationship. However, technologic advances in recent years have markedly facilitated the progress of immunogenetic studies. Single nucleotide polymorphism arrays and DNA sequencing are frequently applied in such investigations and genome-wide association studies have allowed identification of distinct loci associated with susceptibility to infection.418-"'20 The significance of genetic factors is perhaps most dramatically illustrated by the Lubeck disaster in 1927, when 251 babies were accidentally vaccinated with viable M. tubercuwsis instead ofBCG. At the end of the 6-year observation period, six children {2%) still suffered from TB, 129 (51%) had become ill but recovered, 77 (31%) had died, and in 39 children (16%) clinical signs of TB had never developed. 421 The marked influence of ethnic differences on the prevalence ofTB further supports the role of genetic factors.422 In the 1940s, Lurie studied native resistance to TB in rabbits, and by selective inbreeding he succeeded in establishing strains of rabbits that differed remarkably in their susceptibility to infection with M. tuberculosis. 219 Similarly, congenic mouse strains that differ in their susceptibility to experimental infection with several intracellular bacteria have been developed.423 At least three levels of the host-pathogen relationship serve as potential targets for genetic control, which are briefly described in the following. 1. Genetic factors decide whether infection becomes abortive or establishes itself in a stable fonn. Convincing evidence for genetic control mechanisms at this level does not exist. 2. Genetic factors control transition from infection to disease. This control step distinguishes susceptible from resistant individuals. Such inherited influences are well proven in mice and begin to unfold in the human population. 3. Severity and/or fonn of disease are controlled by genetic factors.

Primary Immunodeficiencies Numerous single-gene (Mendelian) disorders that perturb immune functions (ie, currently> 300 primary immunodeficiencies) have been reported.424 These monogenic diseases are rare, arise from major functional aberrations at single genes, and confer predisposition to a certain type of infection. The most thoroughly characterized of these syndromes in context of infection with intracellular pathogens is the Mendelian susceptibility to mycobacterial diseases. 423 The IFNrRI deficiency was described first. 339' 425--427 Subsequently, deficiencies in other Thl-associated molecules, namely IFNrR2,428' 429 STAT1,430.431 IL-12B,432 and IL-12Bl433 were reported. More recently, IRF8 mutations, which affect development of monocytes and DCs, have been associated with susceptibility to mycobacterial diseases. 434 Patients with

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Mendelian susceptibility to mycobacterial diseases also develop infections with typhoidal and especially non typhoidal salmonellae with high frequencies.435

Multigenic Predispositions/Major Genes Linkage studies, and more recently genome-wide association studies, suggest polygenic characteristics of most infectious diseases. Studies in the mouse system have revealed a single dominant autosomal gene on chromosome 1, which is responsible for resistance against M. bovis BCG, M. lepraemurium, M. avium/M. intraceUulare, S. enterica, and the protozoan pathogen Leishmania donovani. In contrast, murine resistance against other intracellular bacteria, most remarkably M. tuberculosis, is apparently not controlled by this gene.436 The responsible gene has been named Nrampl for natural resistance-associated macrophage protein.437 The role ofNrampl in controlling infectious diseases in humans remains controversial.438,439 Other mouse gene loci affecting susceptibility to mycobacterial disease are Icbp/40 Trl-1,441 Trl-4,'42 Tbs1,'43' 444 and Iprl!LL·445 Certain loci impact on resistance against multiple intracellular bacteria. Iprl affects susceptibility to listeriae/LL whereas Icbp affects responses against salmonellae.'46 The contribution of human homologues of the previously mentioned loci to antibacterial resistance needs further in-depth investigations. Genomewide association studies have described susceptibility loci for TB on chromosome 18qll.2447 and chromosome 8ql2.'48 Susceptibility to leprosy is associated with multiple innate immune-relevant genes!49.450 Legionnaires' disease seems to be differently associated with polymorphisms in TLR-4 and TLR-5.451- 453 Single nucleotide polymorphism arrays, to analyze the association between clinical TB and certain PRRs {TLR-1, 2, 4, 6, 8, 9; DC-SIGN; NOD2; mannan-binding lectin) and adaptor molecules (TIR domain-containing adaptor protein), came to divergent results indicating a need for further studies.423.454 Polygenic predispositions and immune defects in innate elements are currently being investigated and seem to bear an unanticipated role in resistance against infectious diseases.455

Major Histocompatibility Complex Control of Severity and Form of Disease Segregation analyses in various human populations also indicate linkage ofHLA types with severity ofTB and leprosy. Strong evidence exists suggesting an influence of HLA on the development toward the tuberculoid or the lepromatous pole of leprosy. Although some linkage with MHC-I molecules has been observed in certain populations, MHC-11 control appears to be more important!56 Originally, it was found that HLA-DR2 subtypes are linked with increased incidences of lepromatous leprosy and that HLA-DR3 represents a linkage marker for tuberculoid leprosy. Recent population-based association studies, however, have provided evidence for an association between distinct HLA-DR2 alleles and susceptibility to tuberculoid leprosy. In TB, evidence for association of HLA-DR2 subtypes with pulmonary TB has been found. With more data from various population groups

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becoming available, it is increasingly dear that HLA-DR associations with distinct disease forms differ among population groups, thus making it impossible to extrapolate from one population to another. These discrepancies underline the polygenic nature of resistance to infectious diseases.

CONCLUSION AND OUTLOOK It is hoped that the reader of this chapter has not only be-

come familiar with the principal mechanisms underlying immunity against intracellular bacteria, but also realizes the great complexity at the interface between prokaryotic and mammalian eukaryotic systems. Understanding intracellular bacterial infections requires knowledge not only of immunology, but also of molecular biology of the infectious agent and biology of the target cell. In vitro analyses can only provide incomplete answers to the questions relevant to antibacterial immunity and must be complemented by in vivo experiments. Despite the high degree of complexity, such interdisciplinary research efforts certainly provide rewards. First, understanding the performance of the immune system in bacterial infections can provide clues to questions pertinent to basic immunology. Knowledge of the rules underlying the extraordinary plasticity and adaptability of the immune system required for coping with transmutable "viable antigens" that developed during millennia of coexistence will provide deeper insights into the immunoregulation and evolution of the immune system. Second, applied questions will benefit equally well from these approaches. With the increasing inadequacy of chemotherapy in the control of bacterial infections, the need for adjunctive immune

measures is gaining importance. Rational strategies toward vaccination and immunotherapy will benefit from the deeper understanding of the immune mechanisms operative in intracellular bacterial infections. With the elucidation of the genomes of major intracellular pathogens, as well as of the human and murine genomes, this type of interdisciplinary research has, in fact, entered a new phase and novel next-generation deep-sequencing technologies promise more rapid and less costly progress. Global analyses of the transcriptome and proteome down to the single-cell level will undoubtedly provide a comprehensive view of this dynamic interplay in the near future. The reader may find it ironic that the spirit of these investigations remains the same as it was at the early beginnings of immunology, which started as an approach to the intervention of bacterial infections.

ACKNOWLEDGMENTS The authors acknowledge financial support from the German Ministry of Science and Technology (Bundesministerium fftr Bildung und Forschung; BMBF grant nos. 01KA1010, 01KI1007C and 01KI0781, 01GS0814, 0313801K), The Bill & Melinda Gates foundation (BMGF GC6-74 37772), the European Union Framework Programme 7 (EU FP7 NEWTBVAC, PHAGOSYS, SysteMTB, TRANSVAC), the European and Developing Countries Clinical Trials Partnership (EDCTP projects AE-TBC and TB-TEA) and the National Institutes of Health (NIH grant no. 745090-HHSN272200800059C). We are grateful to M.L. Grossman for help preparing the chapter and to D. Schad for preparing graphics.

CHAPTER 40 REFERENCES

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CHAPTER 40 REFERENCES 393. Zumla A, James DG. Granulomatous infEctions: etiology and classification. Clin Infect Dis. 1996;23(1):146-158. http:f/www.ncbi.nlm.nih.gov/pubmed/8816144 394. Dougan G, John V, Palmer S, Mastroeni P. Immunity to salmonellosis. ImmunolRev. 2011;240(1):196-210. http:f/www.ncbi.nlm.nih.gov/pubmed/21349095 395. GhonA. Der primiire Lungenherd 1m der Tu~rkulose ckr Kinder (The primary lung luion ofchildhood tuben:ulosis). Berlin & Wien: Urban & Schwarzenberg; 1912. 396. Chen M, Divangahi M, Gan H. et al. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGF2 and LXA4 in the induction of macrophage death./ Exp Med. 2008;205(12):2791-2801. http:f/www.ncbi.nlm.nih.gov/pubmed/18955568 397. Divangahi M, Chen M, Gan H, et al. MycobactErium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair. Nat Immunol. 2009;10(8):899-906. http:f/www.ncbi.nlm.nih.gov/pubmed/19561612 398. Rivera-Marrero CA. Stewart J, Shafer WM, Roman J. The down-n:gulation of cathepsin G in TIIP-1 monocytes after infection with MycobactErium tuberculosis is assodat£d with increased intracellular survival of badlli. Infoct Immun. 2004;72(10):5712-5721. http:f/www.ncbi.nlm.nih.gov/pubmed/15385470 399. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediat£d human antimicrobial response. ~na. 2006;311(5768): 1770-1773. http:f/www.ncbi.nlm.nih.gov/pubmed/16497887 400. Kaplan G, Post FA, Moreira AL, et aL Mycobacterium tuberculosis growth at the cavity surface: a microenvironment with failed immunity. Infect Immun. 2003;71(12):7099-7108. http:f/www.ncbi.nlm.nih.gov/pubmed/14638800 401. Ulrichs T. Kosmiadi GA, Jorg S, et al. Differential organization of the local immune response in patients with active cavitary tuberculosis or with nonprogressive tuberculoma./ Infect Dis. 2005;192(1):89-97. http:f/www.ncbi.nlm.nih.gov/pubmed/15942898 402. Egen JG, Rothfuchs AG, Feng CG, Wmter N, Sher A, Germain RN. Macrophage and T cell dynamics during the development and disintEgration of mycobacterial granulomas. Immunity. 2008;28(2):271-284. http:f/www.ncbi.nlm.nih.gov/pubmed/18261937 403. Egen JG, Rothfuchs AG, Feng CG, Horwitz MA, Sher A, Germain RN. Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas. Immunity. 2011;34(5): 807-819. http:f/www.ncbi.nlm.nih.gov/pubmed!Z1596592 404. Davis JM, Ramakrishnan L The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell.. 2009;136(1):37-49. http:f/www.ncbi.nlm.nih.gov/pubmed/19135887 405. Ly LH, Barhoumi R, Cho SH, Franzblau SG, McMurray DN. Vaccinalion with Bacille-calmette Gtrerin promotes mycobacterial control in guinea pig macrophages infecred in vivo./ Infect Dis. 2008;198(5):768-771. http:f/www.ncbi.nlm.nih.gov/pubmed/18627245 406. Kjellsson MC, Via LE, Goh A, et aL Penetration of anti-tuberculosis agents in rabbit pulmonary lesions: a pharmacokinetic evaluation. AntimicrobAgents Chemother. 2012;56(1):446-457. http:f/www.ncbi.nlm.nih.gov/pubmed!Z1986820 407. Lin PL, Rodgers M, Smith L, et aL Quantitative comparison of active and latent tuberculosis in the cynomolgus macaque modeL Infect Immun. 2009;77(10):4631-4642. http:f/www.ncbi.nlm.nih.gov/pubmed/19620341 408. Mehra S, Pahar B, Dutta NK, et aL Transcriptional reprogramming in nonhuman primate (rhesus macaque) tuberculosis granulomas. PLoS One. 2010;5(8):e1ZZ66. http:f/www.ncbi.nlm.nih.gov/pubmed!Z08Z4205 409. Grosset J. Mycobacterium tuberculosis in the extracellular compartment an unden:stimated adversary. Antimicrob Agenl3 Chemother. 2003;47(3):833-836. http:f/www.ncbi.nlm.nih.gov/pubmed/12604509 410. Reece ST, Loddenkemper C. Askew DJ, et al. Serine protease activity contributes tn control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice./ Clin Invest. 2010;120(9):3365-3376. http:f/www.ncbi.nlm.nih.gov/pubmed/20679732 411. Pan H, Yan BS, Rojas M, et aL Ipr1 gene medial:£5 innat£ immunity tn tuberculosis. Nature. 2005;434(7034):767-772. http:f/www.ncbi.nlm.nih.gov/pubmed/15815631

412. Elkington P, Shiomi T, Breen R, et aL MMP-1 drives immunopathology in human tuberculosis and IIans~c mice./ Clin Invest. 20 11;1 21 (5): 1827-1833. http:l/www.ncbi.nlm.nih.gov/pubmed/21519144 413. Kim MJ, Wainwright HC. Loc.ketz M, et al. Caseation of human tuberculosis granulomas correlat£s with elevated host lipid metabolism. EMBO Mol Med. 2010;2(7):258-274. http:l/www.ncbi.nlm.nih.gov/pubmed!Z0597103 414. Peyron P, Vanbourgeix J, Paquet Y, et aL Foamy macrophages from tuberculous patients' granulomas coru;titute a nutrient-rich n:servoir forM. tuberculosis persistence 2987. PLoSPathog. 2008;4( 11 ):e1000Z04. http:f/www.ncbi.nlm.nih.gov/pubmed/1900ZZ41 415. Garton NJ, Waddell SJ, Sherratt AL. et aL Cytological and IIanscript analyses reveal fill: and lazy persister-like bacilli in tuberculous sputum. PLoS Med. 2008;5(4):e75. http:l/www.ncbi.nlm.nih.gov/pubmed/18384ZZ9 416. Alcais A, Abel L, Casanova JL Human genetics of infEctious diseases: between proof of principle and paradigm. TClin Invest. 2009;119(9): 2506-2514. http:f/www.ncbi.nlm.nih.gov/pubmed/19729848 417. Casanova JL, AbelL. Human genetics of infEctious diseases: a unified theory. EMBO /. 2007;26(4):915-922. http:l/www.ncbi.nlm.nih.gov/pubmed/17255931 418. Vannberg FO, Chapman SJ, Hill AV. Human genetic susceptibility to intracellular pathogens. Immunol Rtv. 2011;240(1):105-116. http:l/www.ncbi.nlm.nih.gov/pubmed!Z 1349089 419. Hill AV. Aspects of genetic susceptibility to human infectious diseases. Annu Rev Genet. 2006;40:469-486. http:l/www.ncbi.nlm.nih.gov/pubmed/17094741 420. Bellamy R. Genome-wide approaches to identifying genetic factors in host susceptibility to tuberculosis. Microbes Infect. 2006;8(4): 11191123. http:f/www.ncbi.nlm.nih.gov/pubmed/16513396 421. ReiterHE. Die Sauglinp~rkulose in Lubeck. Berlin, Germany: Julius Springer; 1935. 422. Stead WW, Senner JW, Reddick WT, Lofgren JP. Racial differences in susceptibility to infEction by Mycobacterium tuberculosis. N Engl J Med. 1990;322(7):422-427. http:f/www.ncbi.nlm.nih.gov/pubmed/2300 105 423. Fortin A, Abel L, Casanova JL, Gros P. Host genetics of mycobacterial diseases in mice and men: forward genetic studies of BCG-osis and tuberculosis. Annu Rev Genomic.s Hum Genet. 2007;8:163-192. http:l/www.ncbi.nlm.nih.gov/pubmed/17492906 424. Burgner D, Jamieson SE, Blackwell JM. Genetic susceptibility to infEctious diseases: big is beautiful, but will bigger be even better? Lancet Infect Dis. 2006;6(10):653~63. http:f/www.ncbi.nlm.nih.gov/pubmed/17008174 425. Jouanguy E, Lamhamedi-Cherradi S, Lammas D, et aL A human IFNGR1 small deletion hotspot assodat£d with dominant susceptibility to mycobacterial infection. Nat Genet. 1999;21(4):370-378. http:l/www.ncbi.nlm.nih.gov/pubmed/1 0192386 426. Jouanguy E, Lamhamedi-Cherradi S. Altare F, et al. Partial inter-ferongamma n:ceptor 1 defidency in a child with tuberculoid bacillus Calmette-Guerin infection and a sibling with clinical tuberculosis. TClin Invest. 1997; 100(11 ):2658-2664. http:l/www.ncbi.nlm.nih.gov/pubmed/9389728 427. Dorman SE, Picard C, Lammas D, et al. Clinical features of dominant and recessive interferon gamma n:ceptor 1 defidendes. Lancet. 2004;364(9451):2113-2 121. http:f/www.ncbi.nlm.nih.gov/pubmed/15589309 428. Dollinger R. Jouanguy E, Dupuis S, et al. Partial interferon-gamma receptor signaling chain deficiency in a patient with bacille CalmetteGuerin and Mycobacterium abscessus infEction. J Infoct Dis. 2000;181(1):379-384. http:f/www.ncbi.nlm.nih.gov/pubmed/1 0608793 429. Dorman SE, Holland SM. Mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection./ Clin Invest. 1998;101 (11):2364-2369. http:l/www.ncbi.nlm.nih.gov/pubmed/9616207 430. Dupuis S, Dargemont C. Fieschi C, et aL Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation. Scima. 2001 ;293 (5528):300-303. http:l/www.ncbi.nlm.nih.gov/pubmed/11452125

CHAPTER 40 REFERENCES 431. Chapgier A, Boisson-Dupuis S, Jouanguy E, et a1 Novel STAT1 alleles in otherwise healthy patients with mycobacterial disease. PLoS Genet. 2006;2(8):el31. http:f/www.ncbi.nlm.nih.gov/pubmed/16513400 1 432. Altare F, Durandy A. Lammas D, et a1 Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science. 151518;280(5368): 1432-1435. http:f/www.ncbi.nlm.nih.gov/pubmed/51603732 433. Altare F, Ensser A, Breiman A, et al. Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. l Infect Dis. ZOO 1; 184(2):231-236. http:f/www.ncbi.nlm.nih.gov/pubmed/11424023 434. Hambleton S, Salem S, Bustamante J, et a11RF8 mutations and human dendritic-cell immunodeficiency. N Eng!! M~d. 2011;365(2):127-138. http:f/www.ncbi.nlm.nih.gov/pubmed/21524210 435. MacLennan C, Fieschi C, Lammas DA. et al. Interleukin (IL)-12 and IL-23 are key cytnkines for immunity against Salmonella in humans. J InfoctDis. 2004;1510(10):1755-1757. http:f/www.ncbi.nlm.nih.gov/pubmed/15451515251 436. Gros P, Schurr E. Immunogenetics of the host immune response tn bacteria in mice. In: Kaufmann SHE, Sher A, Ahmed R, eds. Immunology of Infectious Diseases. Washington, DC: ASM Press; 2002: 407-420. 437. Vidal SM, Malo D, Vogan K, Skamene E, Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Beg. CelL 151513;73(3):4651--485. http:f/www.ncbi.nlm.nih.gov/pubmed/8490962 438. Abel L, Casanova JL Immunogenetics of the host response tn bacteria and piiiliSites in humans. In: Kaufmann SHE, Sher A. Ahmed R, eds. Immunology of Infoctious D~ases. Washington, DC: ASM Press; 2002:3515-406. 4351. Abel L, Sanchez FO, Oberti J, et al. Susceptibility to leprosy is linked to the human NRAMP1 gene./ Infect Dis.151518;177(1):133-145. http:f/www.ncbi.nlm.nih.gov/pubmed/514151180 440. Turcotte K, Gauthier S, Tuite A. Mulliclt A. Malo D, Gros P. A mutation in the Icsbp1 gene causes susceptibility tn infection and a chronic myeloid leukemia-like syndrome in BXH-2 mice. l Exp Med. 2005; 201(6):881-8510. http:f/www.ncbi.nlm.nih.gov/pubmed/15781580 441. Mitsos LM, Cardon LR, Fortin A, et al. Genetic control of susceptibility tn infection with Mycobacterium tuberculosis in mice. Ge~ Immun. 2000;1(8):467-477. http:f/www.ncbi.nlm.nih.gov/pubmed/111517687 442. Turcotte K, Gauthier S, Mitsos LM, et a1 Genetic control of myeloprolifurationin BXH-2 mice. Blood. 2004;103(6):2343-2350. http:f/www.ncbi.nlm.nih.gov/pubmed/146308151 443. Sanchez F, Radaeva Tv, Nikonenko BV, et a1 Multigenic control of disease severity after virulent Mycobacterium tuberculosis infection in mice. Infect Immun. 2003;71(1):126-131. http:f/www.ncbi.nlm.nih.gov/pubmed/124516157

444. Lavebratt C.. Apt AS, Nikonenko BV, Schalling M, Schurr E. Severity of tuberculosis in mice is linked to distal chromosome 3 and proximal chromosome 51. I Infect Dis. 1999; 180( 1): 150-155. http:f/www.nchi.nlm.nih.gov/pubmed/1 0353873 445. Kramnik L Dietrich WF, Demant P, Bloom BR. Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis. Proc Nafl Ac:ad Sci US A. 2000;97(15):8560-8565. http://www.ncbi.nlm.nih.gov/pubmed/1085105113 446. Turcotte K, Gauthier S, Malo D, Tam M, Stevenson MM, Gros P. Icsbp 1/ IRF-8 is required for innate and adaptive immune responses against intracellular pathogens. I Immunol. 2007; 1751(4):2467-2476. http://www.ncbi.nlm.nih.gov/pubmed/17675508 447. lhye T, Vannberg FQ Wong SH, et al. Genome-wide association analyses identifies a susceptibility locus for tuberculosis on chromosome 18q1l.Z. Nat Genet. 2010;42(51):7351-741. http://www.ncbi.nlm.nih.gov/pubmedf206514014 448. Baghdadi JE, Orlova M, Alter A, et al. An autosomal dominant major gene confers predisposition to pulmonary tuberculosis in adults. l Exp Med. 2006;203(7):16751-1684. http:f/www.ncbi.nlm.nih.gov/pubmed/1680 135151 445l. Zhang FR, Huang W, Chen SM, et a1 Genomewide association study of leprosy. N Engll Med. 20051;361(27):2609-2618. http://www.ncbi.nlm.nih.gov/pubmedf20018961 450. Wong SH, Gochhait S, Malhotra D, et al. Leprosy and the adaptation of human toll-like receptor 1. PLoS Pathog. 2010;6:e10009751. http://www.ncbi.nlm.nih.gov/pubmedf20617178 451. Hawn TR, Verbon A. Lettinga KD, et a1 A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease. I Exp Med. 2003;1518(10): 1563-1572. http://www.ncbi.nlm.nih.gov/pubmed/146235110 452. Dunstan SJ, Hawn TR, Hue NT, et al. Host susceptibility and clinical outcomes in toll-like receptor 5-defident patients with typhoid fever in Vietnam. l Infect Dis. 2005; 15l1(7): 1068-1 071. http://www.ncbi.nlm.nih.gov/pubmed/15747241 453. Hawn TR, Verbon A, Janer M, Zhao LP, Beutler B, Aderem A. Toll-like receptor 4 polymorphisms are associated with resil;tance to Legionnaires' disease. Proc Natl Ac:ad Sci US A. 2005;1 02(7):2487-24851. http:f/www.ncbi.nlm.nih.govtpubmed/1565151327 454. Kleinnijenhuis J, Oosting M, Joosten LA. Netea MG, Van CR. Innate immune recognition of Mycobacterium tuberculosis. Clin Dev ImmurwL 2011;2011:405310. http://www.ncbi.nlm.nih.gov/pubmedf21603213 455. Netea MG, van der Meer JW. lmmunodefidency and genetic defects of pattern-recognition receptors. N Eng! I Med. 2011;364(1):60-70. http://www.ncbi.nlm.nih.gov/pubmedf212081051 456. Hill AV.1he genomics and genetics ofhuman infectious disease susceptibility. Annu Rev Gmomics Hum Gend. 2001;2:373--400. http:f/www.ncbi.nlm.nih.govtpubmed/1170 1655

Immunity to Extracellular Bacteria Moaa H. Nalln • Jannat r.a

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CHAPTER 41 Most extracellular, as well as intracellular, pathogenic bacteria can be divided into two major groups (gram-negative and gram-positive) based on their response to staining with Gram stain. To illustrate the surface of the bacteria in the two groups, the surface structures of Streptococcus pneumoniae (Panel A) and Neisseria meningitidis (Panel B) are shown in Figure 41.1. Three layers are commonly recognized: cytoplasmic membrane, cell wall, and outer layer. Although these layers are described in detail in the following, it is important to note that these definitions are operational and that, in reality, the layers are not entirely distinct. Por instance, molecules anchored in the cytoplasmic membrane or cell wall may extend into or through other layers.

IMMUNITY TO EXTRACELLULAR BACTERIA

I

1003

It is also important to note that the capsule, 0 antigens, and cell wall are not contiguous shields; rather they are permeable enough to allow through secreted products and nutrients as well as some immunologic factors (eg, anu'bodies and complement). All bacteria have a cytoplasmic membrane, a non-sterolcontaining phospholipid bilayer. This membrane is an osmotic barrier and also forms a barrier for most molecules. The cytoplasmic membrane has various proteins, many of which function in transport. Some of these proteins, referred to as lipoproteins (eg, pneumococcal surface adhesion A, which is a manganese permease inS. pneumoniae), are noncovalently anchored to the membrane through

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RG. 41.1. Schematic representation of the surfaces of Strsptococcus pnsumoniss (AJ and Nsisssris msningitidis (B) as examples of gram-positive and gram-negative bacteria, respectively. The cell wall polysaccharide of S. pnsumonias is often called C-polysaccharide. The inset in B shows lipopolysaccharide anchored to the outer leaflet of the outer membrane.

1004

I

SECTION VII

IMMUNITY TO INFECTIOUS AGENTS

lipid modifications and are especially common among some bacteria (eg, Borrelia burgdorferi and Mycobacterium tuberculosis). Proteins not exposed on the surface generally display a greater degree of structural and functional conservation compared with those exposed to the selective pressure of host immunity. A cell wall is found in all of the pathogenic bacteria of both groups, with the exception of mollicutes (which include the genus Mycoplasma). The cell wall surrounds the cytoplasmic membrane and is made of peptidoglycan, which is a polymer of alternating sugars N -acetylglucosamine and N-acetylmuramic acid, the latter being connected to a stem peptide. The stem peptides include four to five Dand L-amina acids that are extensively cross-linked by bridges that provide rigidity to the cell wall and protect it from environmental extremes (especially differences in osmolarity). These cell wall peptides include atypical amino acids such as diamino pimelic acids, which are the anchoring site for Braun lipoprotein of gram- negative bacteria and are found in most gram-negative bacteria but in few gram-positive bacterial species. Peptidoglycan polymerization is carried out by enzymes, many of which are the target of ~-lactam antibiotics and are referred to as penicillinbinding proteins. Compared with gram-negative bacteria, gram-positive organisms may have different stem peptides and cross-linking as well as a thicker {20 to 30 nm compared with 2 to 4 nm) cell wall layer that can retain the Gram stain better. The thick cell wall of the gram-positive bacteria may be responsible for their greater resistance to complementmediated lysis. Other features of the cell wall such as the 0- acetylation of N-acetylmuramic acid or the deacetylation of N -acetylglucosamine5•6 found in some species mediate resistance to lysozyme, an enzyme that lyses bacteria by cleaving the peptidoglycan backbone.6 In addition to peptidoglycan, many gram-positive bacteria have polysaccharide (PS) associated with their cell walls, with this cell wall PS often extending into the outer layer. The structure of the cell wall PS of gram-positive bacteria varies between species but is relatively invariant within a species. Differences in the antigenicity of the cell wall PS have been used to distinguish species ( eg, separate streptococci into groups A, B, C, etc.).7' 8 Cell wall PS often has phosphate group(s) in repeating units of glycerol or ribitol in a structure known as teichoic acid. Teichoic acid may also be linked to lipid molecules and is then called LTA, which is anchored to the cytoplasmic membrane and extends out through the cell wall.9 In pneumococci, the overall PS structures of LTA and cell wall teichoic acid (also referred to as C-polysaccharide) are very similar, with the difference being their mode of attachment to the bacterial surface.10•11 Another major difference between the surface structures of gram-negative and gram-positive bacteria is the presence of an outer membrane on gram-negative bacteria. The outer membrane contains many proteins, including channel-forming porins. The outer membrane is an asymmetricallipid bilayer. The inner leaflet is comprised primarily of phospholipids while the outer leaflet contains lipid A, the hydrophobic component of LPS. LPS, also called endotoxin, is an amphipathic glycolipid with four distinct regions: lipid

A, the inner core, the outer core, and, in some species, the 0 antigen. Lipid A is composed of a dihexosamine backbone to which between five and seven saturated {12- to 16-carbon) fatty acids are attached through amide and ester linkages. Lipid A is the principal "toxin" associated with most gramnegative bacteria, although it is now clear that lipid A is not a true toxin. Rather, its ability to induce cytokines accounts for its potentially detrimental effects. The carbohydrate portion of the LPS, which makes a minimal contribution to its endotoxin activity, is attached to lipid A through a molecule unique to gram-negative bacteria called ketodeoxyoctanoate. Together with heptose moieties, this molecule forms the inner core of the LPS. The outer core is composed of 7 to 10 monosaccharide units whose arrangement is relatively conserved among gram-negative species.U In many gram-negative bacteria, the outer core of LPS is connected to a repeating series of carbohydrates called the 0 antigen. The 0 antigen forms a hydrophilic shield around the bacterium that provides a barrier to complement deposition on the bacterial cell surface. The 0 antigen is variable in length, is antigenically diverse, and confers serotypic specificity. The 0 antigens of Escherichia coli, Klebsiella, and Salmonella have as many as 30 repeating units composed of four to six sugars each.12 Members of the genera Neisseria and Haemophilus, on the other hand, lack LPS with 0 antigens but instead have lipooligosaccharides, which have short oligosaccharides that do not exceed 7000 daltons. For many pathogenic extracellular bacteria, PS components dominate the outer layer. In addition to the PS on LPS {gram-negative) and teichoic acid {gram-positive), there is often another thick layer of carbohydrate referred to as "capsule" that may account for more than half of the bacterial mass. An exception to this general rule is Bacillus anthracis, whose capsule is made of poly-D-glutamic acid rather than a polysaccharide.13 S. pneumoniae has capsular PS that is covalently attached to the cell wall in most (but not all) serotypes.14 In contrast, the capsule PSis anchored to the outer membrane by acyl chains in N. meningitidisl5 and Haemophilus influenzae type b. 16 Capsular PSs may be highly diverse both within and between species. In the case of S. pneumoniae, each strain expresses a single type of capsular PS, with members of this species being capable of synthesizing more than 90 structurally distinct types.17' 18 This diversity limits immune recognition until antibody is generated to the capsular PS of the infecting strain (antigenic variation). The outer layer is well developed in bacteria that cause extracellular infections and has many features that help the bacteria circumvent the host immune system. First, the outer layer has properties that reduce the attachment of extracellular bacteria to eukaryotic surfaces, including those of phagocytes. Generally, the PS capsules render the bacteria hydrophilic and negatively charged like eukaryotic cell surfaces, which are rich in sialic acid. By enhancing the degradation of C3b, the negatively charged surface makes the bacteria partly resistant to the deposition of complement by the alternative pathway.19 Second, in some cases, elicitation of antibody is minimized because the capsular PS or

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LPS mimics host antigens, as is more fully described in the section on Antigen-Specific Host Defense Response of this chapter. Third, by physically masking most of the other bacterial surface components, the outer layer minimizes the number of exposed epitopes that can be recognized by the antibody and complement. Although the capsule is porous to antibodies and complement, the binding of antibodies and fixing of complement beneath the capsule surface are relatively ineffective in promoting opsonophagocytosis and clearance.20

BACTERIAL VIRULENCE FACTORS Extracellular bacteria often elaborate molecules called "virulence factors" that are useful to their survival and proliferation in the host. For example, the shielding function of the outer layer is further augmented by the presence of surface proteins that can interfere with host clearance mechanisms. Proteins inhibiting effective complement deposition include M-protein in Streptococcus pyogenes,21 pneumococcal surface protein A (PspA), and pneumococcal surface protein C, which is alternatively called choline binding protein A or the C3-binding protein inS. pneumonia. 22' 23 An example of a protein that interferes with antibody is protein A, which is expressed on the surface of Staphylococcus aureus and binds immunoglobulin (Ig) in a manner that precludes recognition of its target antigen.24 In addition, many successful mucosal pathogens, including members of the Neisseria, Haemophilus, and Streptococcus genera, express proteases with specificity for the hinge region of human IgAl.25 These IgA1 proteases remove the Fe,. component required to promote the inflammatory process, leaving the organisms' antigens obscured by the binding of inert Faba fragments. By inhibiting the deposition of complement or antibody, many of these proteins act to diminish phagocytosis. The best-known virulence factors are toxins, which interrupt specific host functions. These proteinaceous molecules (also referred to as exotoxins to differentiate them from endotoxin) can be grouped on the basis of their molecular structure and their mechanism of action.26 The largest group are called A-B toxins, which are comprised of two subunits, each with a different function. The A subunit has enzymatic activity, and the B subunit targets the A subunit to the host cells. This group includes diphtheria toxin, cholera toxin, pertussis toxin, and two anthrax toxins (lethal factor and edema factor). For instance, the lethal factor of B. anthmcis behaves as the A subunit and requires a B subunit protein named "protective antigen" to enter into target cells. In some cases, the toxin alone is sufficient to account for the detrimental symptoms of its respective infection. Cholera toxin causes ADP ribosylation of G proteins, which stimulates adenylate cyclase and increases cyclic adenosine monophosphate in cells lining the gut. This results in the secretion of electrolytes and is responsible for a severe diarrhea, which promotes transmission of Vibrio cholerae but often also causes dehydration that, if not treated, may be fatal. Uptake of botulism toxin by nerve endings leads to retrograde transport that interrupts synaptic transmission and causes a flaccid paralysis.27 Staphylococcal enterotoxin

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A (a toxin that acts from the gut lumen), which is one of five membrane-damaging toxins produced by staphylococci, is the primary cause of staphylococcal food poisoning and plays a major role in invasive infections. 28 Some strains of E. coli produce a protein synthesis-inhibiting verotoxin, which may damage the microvasculature of the kidney and cause hemolytic uremic syndrome?9 Another group of proteins secreted by S. aureus and S. pyogenes have toxin-like effects but lack any enzymatic activity. These "superantigens" cause a nonclonal stimulation of T cells by bridging major histocompatibility complex (MHC) class II molecules (outside the antigen-binding site) on antigen-presenting cells with the V~ region of the T-cell receptor on T cells. The ensuing massive release of cytokines by localized release of a superantigen, such as the toxic shock syndrome toxin 1 expressed by some strains of S. aureus, results in systemic symptoms that are collectively known as "toxic shock syndrome."30 Another class of virulence factors neutralizes host defenses. As stated previously, many virulence factors interfere with complement deposition on bacteria. S. pyogenes and group B streptococci produce a CSa peptidase that inhibits the chemotaxic effects (recruitment of host phagocytes to the sites of infections) of CSa, a product of complement activation.31 Pneumolysin produced by S. pneumoniae is a member of a large class of cholesterol-dependent cytotoxins that oligomerize to fonn large pores, which interfere with a number of host cell functions or induce cell death when present in higher concentrations.32 Pneumolysin also depletes complement at a distance from the pneumococci and interferes with both the function of phagocytes and the development of protective immunity.33 Helicobacter pylori produces urease, which can generate ammonia that can neutralize acid in the stomach and thereby promotes the survival or the organism. S. aureus produces a pigment that makes the bacterium more resistant to oxidative stress and killing by neutrophils.34 While evasion of professional phagocytes is critical for extracellular pathogens, the ability to attach to other cell types, including both mucosal and nonmucosal surfaces, is important for their persistence. Many bacterial surface proteins have an adhesive function that confers a high affinity for binding to specific host cell receptors. Nasopharyngeal carriage of pneumococci is mediated largely by adherence to the host molecules N-acetylglucosamine ~143 galactose or N -acetylglucosamine 1}144 galactose. 35 Bacteria often mimic host ligands in order to coopt host receptors for their own purposes. Many pathogens of the airway express phosphorylcholine (phosphocholine [PC]) on their surfaces.36 This molecule, which is otherwise unusual in bacteria, is found on platelet-activating factor (PAF) and allows bacterial binding to its receptor (rPAF).37 To facilitate their attachment to host cells, many bacteria use pili (fimbriae), long filamentous structures extending from the organism. The Pap pilus of E. coli binds the galactose al44 galactose unit of cell surface globoside in urethral epithelial cells. 38 The V. cholerae pilus allows that bacterium to attach to the enterocyte for more efficient toxin delivery. 39' 40 Bordetella pertussis has three adherence factors-a filamentous hemagglutinin,

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pertactin, and a pilus-that allow it to attach to ciliated respiratory epithelial cells in the trachea and bronchi and thus resist the cleansing action of mucus flow.4M2 Another group of virulence factors helps bacteria acquire essential nutrients. Motile bacteria (eg, Pseudomonas aeruginosa) express flagellin, a complex motor apparatus that allows the bacterial cell to transit along a concentration gradient of nutrients.43 While in some cases the host and microbe provide nutrients for one another, for other nutrients there is fierce competition. Mucosal fluid and blood are low in free ferric iron due to the presence of iron-binding proteins such as lactoferrin and transferrin. To successfully compete with the host for this vital metabolite, N. meningitidis, Neisseria gonorrhoeae, and H. influenzae have complex surface transport systems that can obtain iron from human transferrin, lactoferrin, and hemoglobin.44 Other bacteria such as E. coli and salmonella use a different mechanism to acquire iron; they secrete small, high-affinityiron chelators, called siderophores, that remove iron from human proteins in the environment surrounding the bacteria. The iron-siderophore complex is then taken up by the bacterium, which then degrades the siderophore so that the iron can be freed for its use. 45 Production of virulence factors is often highly regulated by bacteria in order to adapt to different environments, such as the natural environment outside of a host, the mucosa of a host, or more invasive sites within a host. For staphylococci, it has been shown that the amount of capsule is regulated in response to environmental stimuli.46 One of the best studied of such regulatory systems is the BvgAS, a two-component regulatory system in B. pertussis.47 This system, which regulates the expression of adhesins, toxins, and other virulence factors, is controlled by external signals including Mi+, ternperature, and nicotinic acid. Two proteins are involved in this regulatory system: BvgS and BvgA. BvgS, the sensor, is a kinase and is able to autophosphorylate itself in response to the environmental signal. BvgA, the response regulator, is in turn phosphorylated by BvgS. Phosphorylated BvgA is able to activate transcription of virulence genes through a change in its interaction with a 70-bp consensus sequence repeated in bvg-regulated promoters.47 Analogous two-component regulatory systems in other pathogens are frequently used to regulate the expression of genes associated with virulence.48 Another strategy used by extracellular pathogens depends on selection among a heterogeneous population for those members with permissive characteristics. This heterogeneity in a population is commonly generated through genomic rearrangements, such as recombinational events or slipstranded mispairing in highly repetitive deoxyribonucleic acid (DNA) sequences.49 This latter mechanism allows for reversible on-off switching (phase variation) and is especially prevalent in genes encoding cell surface structures subject to immune pressure. For instance, the capsular PS on N. meningitidis is needed to protect the organism during invasive infection but inhibits adherence on the mucosal surface where complement is less abundant. Phase variation of a gene required for capsule synthesis allows for selection of organisms without capsule (phase-off) .50 This change facilitates the bacterial adhesion to the epithelial cells, perhaps by exposing the bacterial adhesins. Alternatively, by decreasing capsule

production, the bacteria become less hydrophilic and less negatively charged. This change facilitates their entry into the epithelial cells and their subsequent invasion into deeper tissues. Upon the emergence of the bacteria from the epithelial cells into the submucosa, capsule synthesis is restored (phase-on) because of the selective pressure of complementmediated clearance and the requirement for the capsule to survive where the concentration of complement is higher. The flexibility to express different surface properties helps bacteria successfully evade the host immune system and survive in many niches within the host. Bacteria-to-bacteria signaling is another important mechanism for the control of virulence factors. This phenomenon, called "quorum sensing,"51 has been shown to be operative in a large number of gram-negative and grampositive species. The signal transmitted between the bacteria can be an acylated lipid (eg, homoserine lactone) in gramnegative bacteria (eg, vibrios) or a peptide in gram-positive bacteria (eg, S. aureus). Quorum sensing has been shown to be important in biofilm formation in a number of bacterial species, in the expression of "competence" for the uptake and incorporation of exogenous DNA (transformation), and in the regulation of a number of virulence factors.52 Biofilms are communities of one or multiple bacterial species that adhere both to each other and to a target surface. Bacteria in biofims are particularly resistant to many host clearance mechanisms and to antibiotics that are effective against free-living (planktonic) bacteria. Biofilms, therefore, are often a contributing factor in more chronic bacterial infections such as those involving foreign bodies or chronic otitis media.53 An important characteristic of virulence factors is their structural polymorphism. For instance, there are at least 100 different serologic types of M proteins of S. pyogenes.54 Similarly, pneumococci have more than 90 serologically distinct capsular PSs.17' 13 The polymorphism in the structure of many virulence factors allows the bacteria making them to avoid antigen-specific host immunity. For example, antibodies to the immunodominant region on one serotype of M protein do not cross-react with M proteins of other serotypes and thus do not provide protection against strains expressing other serotypes. Similarly, newly acquired pneumococci can escape recognition by anticapsular antibodies produced in response to previous pneumococcal infections with other serotypes. The polymorphism in virulence factors is achieved by various genetic mechanisms. Variation in M proteins is the result of sequence differences in the N-terminal (but not C-terminal) half of M proteins.55 S. pneumoniae has the genes for synthesizing capsular PS as a "genetic cassette" that can be exchanged among different strains56 and may result in the shift in the serotype distribution following the use of vaccines designed to elicit serotype-specific protection.57' 53 Neisseria has genetic machinery for rapid gene rearrangement59 through gene conversion using the multiple "silent" pili genes with different sequences. This process, similar to gene rearrangements that generate specific lg, permits an individual bacterium to quickly produce progeny expressing pili with different characteristics. The number of potential

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pilus-antigen variants within the progeny of a single organism is estimated to be greater than 100,000.60 In addition, Neisseria contain large numbers of genes with tandem repeats that undergo phase variation through slip-stranded mispairing of these sequences. Based on predictions from whole genome sequencing, through this mechanism alone the organism may be able to generate more than 265 different phenotypic variants. 61 Sequencing of the entire genomes of bacteria has shown that the genes for virulence factors have generally originated from other organisms and exist as a part of large blocks of DNA containing multiple genes. These DNA blocks are called "pathogenicity islands" (PAis). For instance, strains of "enteropathogenic" E. coli contain a PAl encompassing about 41 genes encoding a surface ligand required for intimate association of the bacterial and host cells and for a bacterial secretion apparatus.62 This system (type IV secretion system) allows for delivery of the receptor for its own adhesin, encoded on the same PAl, into host cells. Elaborate secretion mechanisms (types III and IV) and pore-forming toxins are now known to be mechanisms whereby extracellular organisms gain access to the host cell cytoplasm to modify its activity to suit their needs. Another example is H. pylori, which injects cytotoxin-associated gene A (CagA) molecules directly into host cells using a syringe-like type IV secretory apparatus. CagA is then phosphorylated by the host cells, and the phosphorylated CagA alters host cell function, with the H. pylori strains producing the CagA molecules being more likely to cause ulcers.63 In the case of S. pyogenes, its pore-forming toxin, streptolysin 0, allows for translocation of an enzyme (NAD-glycohydrolase) that is capable of producing the potent cytoplasmic second messenger, cyclic ADP-ribose. 64

BACTERIAL INVASION OF THE HOST Both keratinized skin and mucosal surfaces have inherent nonimmune defense mechanisms that modulate bacterial growth and minimize the risk of invasion. Healthy human skin is an effective physical barrier to infection by most human extracellular and intracellular pathogens. The keratinization of fully differentiated skin epithelium results in a relatively impermeable surface. In addition, lysozymes, toxic lipids, and hydrogen ions secreted by cutaneous glands offer bacteriostatic protection for cutaneous pores and hair follicles. Occasionally, this defense can be breached by extracellular bacteria such as S. pyogenes or S. aureus, causing cellulitis and abscess. More commonly, bacterial invasion through intact skin requires physical damage, such as abrasions, burns, or other trauma. For instance, cutaneous anthrax develops when B. anthracis enters the body through a break in the skin. Staphylococcus epidermidis, a member of the commensal skin flora, can infect indwelling catheters by invading through the puncture site in the skin and may lead to bacteremia or colonization of prosthetic devices, including artificial heart valves and shunts. A major factor allowing these bacteria to cause disease is their ability to form a biofilm, which facilitates their adhesion, is antiphagocytic, and acts as a barrier to antibiotic penetration.65

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Unlike the skin, the mucosal epithelium is not keratinized. Instead, mucosal areas, such as the gastrointestinal tract, nasopharynx, upper airway, and vagina, are moist and nutritionally rich. Thus, it is not surprising that mucosal areas contain a large number of bacteria. In oral secretions and gastrointestinal products, 108 and lOu bacteria/mL may be found, respectively. To ensure their survival in the mucosal environment, extracellular bacteria elaborate many virulence factors required for the acquisition of essential nutrients or for their adherence to the host cells. In some cases, bacteria may subvert the host inflammatory response. Salmonella species can block the activation of NF-KB and the subsequent activation of the inflammatory response. They achieve this by preventing the degradation of IKB, which is essential for the translocation of NF-KB from the cytoplasm to the nudeus.66 In some cases, pathogens locate in a less well-protected microenvironment within the mucosal areas. H. pylori survives in the very acidic stomach by burying itself in the mucus, which protects it from direct exposure to the acid and from phagocytes. There are also few other species for H. pylori to compete with in this more hostile environment. Mucosal sites play host to an especially diverse array of bacterial species. Most of the bacteria species found at mucosal sites are harmless. In addition, polymerase chain reaction analysis of 16S ribosomal ribonucleic acid sequences suggests the presence of many additional unidentified (and so far unculturable) bacterial species on the mucosal surface. 67 Many potentially pathogenic bacterial strains are also often found in the mucosal areas of healthy individuals without causing symptoms. S. pneumoniae, N. meningitidis, H. influenzae, and S. aureus are examples of pathogenic extracellular bacteria that are frequently carried in the nasopharynx of healthy individuals. The carriage rate of pathogenic bacteria can be relatively high; for example, 50% to 60% of healthy young children may carry S. pneumoniae in their throats.63 Maintenance of species diversity in mucosa is dynamic. In some situations, collaboration among bacteria is essential for their successful colonization, as seen among the complex hierarchical communities adhering to tooth surfaces. In other situations, bacterial species compete and regulate diversity among themselves.36 Many bacterial species produce molecules that suppress the growth of other bacterial species. These molecules may include bacteriocins, small molecules that target members of the same or different species that do not express the same bacteriocins.69 Some species can take advantage of host responses to which they are resistant to outcompete another member of the same niche that is less resistant. 36 The host may also control the diversity of colonizing bacteria by modifying the pH or other environmental conditions in the mucosal area. Interference with these homeostatic mechanisms, as occurs with antibiotic therapy, may alter the flora and predispose the host to disease. As noted previously, stomach acid is an effective barrier to reaching the nutrient-rich environment of the gut. When stomach acid is pharmacologically reduced, the inoculum of organisms like V. cholerae required to infect the intestines is greatly diminished.

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Several explanations have been advanced to explain why many pathogens that colonize mucosal sites do not cause disease. One explanation is that the maintenance of diverse bacterial population is responsible for the prevention of the disease. For instance, the destruction of the normal gastrointestinal bacterial flora with some antibiotics can be associated with the selective expansion of Clostridium difficile and the development of pseudomembranous colitis/0 Another explanation may be that the pathogenic bacteria carried in healthy persons are different from those isolated in disease settings. For instance, during nonepidemic periods, approximately 5% to 10% of the population carries N. meningitidis, which are mostly nonencapsulated.71 During epidemics, 30% to 60% of the population may carry meningococci, which are mostly encapsulated and the majority of which are of the same capsular type as the case strain causing the epidemic.72 A third explanation is that the pathogenic bacteria are effectively confined to the mucosal surface where they do not cause damage or induce inflammation. Group B streptococci are carried asymptomatically in the lower intestine and the female genital tract. In the same host, in the setting of parturition, group B streptococci may access the bloodstream and cause septic infection. A fourth explanation is centered on the differences among hosts. Group B streptococci that colonize the mother may cause lifethreatening infection when the same strain is passed to the neonate at or before birth.73 Although pathogenic extracellular bacteria can exist asymptomatically in the mucosa, they can passively enter into less-well-defended sites and cause focal infections. For instance, E. coli, normally present in the gut, may enter the normally sterile urogenital tract and cause urinary tract infections. S. pneumoniae and H. influenzae are often carried in the nasopharyngeal space, but they can invade nearby normally sterile cavities (eg, lungs, sinuses, and the middle ear) and cause focal infections. Aspiration of bacteria from the nasopharynx into the lungs most likely occurs frequently with no ill effects; however, aspiration may lead to an infection when there is damage to the epithelial surface, particularly when the protective effects of mucociliary activity are lost, as often occurs in a smoker or during recent viral infection (respiratory syncytial virus or influenza).7 Some bacteria produce enzymes such as hyaluronidase/4 which may aid in their passage through tissue barriers. Bacteria can actively invade deeper tissues by multiple pathways. They can enter through specialized cells. Shigella, for example, can breach the gut mucosa by transcytosing through the M cells in the gut?5 Alternatively, extracellular bacteria can breach a cellular barrier (epithelium or endothelium) by going through (transcytosis) or between (paracellular pathway) the cells/5 Porphyromonas gingivalis, an organism associated with adult periodontitis, may breach the epithelial layer by the paracellular pathway through the production of enzymes useful in digesting the tight junction/6 Two different mechanisms of transcytosis have been described for pneumococci. In one, pneumococci may cross the bronchial epithelial cells by binding the polymeric Ig receptor of the epithelial cells and traveling

in a retrograde manner by the IgA secretory pathway.77 In the other, pneumococci may use PC to bind to rPAF, which is abundant on activated endothelial cells, epithelial cells, or pneumocytes.35' 78 In many cases, bacterial adhesion triggers changes in the host cell function, and these changes can assist transcytosis. For instance, nontypeable H. influenzae with LPS glycoform-containing PC can bind to rPAF on endothelial cells and initiate signaling through this receptor.79

ANTIGEN-NONSPECIFIC HOST DEFENSE RESPONSE To protect from infections caused by highly adaptable bacteria, the host employs a multilayered defense. This includes the mechanical barriers and iron sequestration described previously as well as phagocytes, complement fixation, lysozyme, and (cytokine-induced) local inflammation. In addition, the host is protected with antigen-specific antibody (see section on Antigen-Specific Host Defense Response) and T-eenmediated cellular immunity. Antigen-specific immunity, although exquisitely protective, takes several days to weeks to develop following exposure to a pathogen. As many extracellular pathogens are capable of causing overwhelming infection in periods of hours to days, other more rapidly acting forms of protection are needed. Consequently, the primary defense against bacteria during the early phase of infection remains antigen-nonspecific host immunity. The importance and significance of nonspecific immunity is readily demonstrated by the relative ease with which colonies of SCID mice, which lack antigen-specific immunity, can be maintained.80 This section describes several antigennonspecific host defense mechanisms, but see the chapter on innate immunity for additional information.

Mucosal Defense Although mucosal areas are rich with nutrients for bacteria, uncontrolled local proliferation ofbacteria is held in check by mechanical cleansing actions and the lack of available iron. In the gastrointestinal tract, normal peristaltic motility, the secretion of mucus, and the detergent action of bile limit the number of bacteria. The normally sterile lower respiratory tract is protected by the movement of mucus by cilia lining the airway, which continually remove aspirated bacteria. Normal epithelial and tissue architecture are essential for drainage and expulsion of bacteria, and disruption of this mechanism by smoking, viral infections (eg, influenza), or bacterial infection (eg, pertussis) makes the host markedly susceptible to infection by bacteria that otherwise exist only as commensals of the upper airway. The increased frequency of lower respiratory tract infections in the elderly is due, in large part, to the loss of function of the mucociliary elevator and the increased aspiration from the upper respiratory tract of secretions containing bacteria.81,82 In addition to the removal of bacteria by mucus flow, mucosal fluid contains many antibacterial products such as lactoferrin, lactoperoxidase, mucin, lysozyme, and defensins.83 Lactoferrin-found in various body fluids such as milk, saliva, and tears-binds iron and lowers the level

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of available iron (especially in areas with a low pH).84 Mucin traps microbes and facilitates their removal.85 IgA antibodies in mucosal fluid may inhibit colonization by interfering with microbial adherence or by inactivating toxins.86 In addition, the polymeric structure of secretory IgA promotes agglutination, which in turn facilitates removal by mucus. Lysozyme reduces the bacterial load by cleaving the l-44 linkage between N -acetylmuramic acid and N -acetylglucosamine of peptidoglycan. A number of antimicrobial peptides, including defensins, disrupt bacterial membranes. 87 In the intestine, Paneth cells at the base of intestinal crypts produce defensins that are important in defense against intestinal infections. 58 In the lungs, collectin-like surfactant proteins, such as SP-A and SP-D, may be important in host defense by opsonizing bacteria for alveolar macrophages.89 SP-A-deficient mice, for example, are more susceptible to group B streptococcal infection of the lungs.90 Epithelial cells that interface with the microbial world must exist in a quiescent state in response to colonizing organisms. If not, chronic inflammation may result in a disease (eg, chronic inflammatory bowel disease). When the epithelial barrier is breached, these cells are able to elaborate cytokines and chemokine as an early trigger to the inflammatory response.

Local Response to Bacterial Invasion (Acute lnflammationt Upon entry into the host, many bacteria initiate local inflammatory processes by providing various inflammatory products such as peptidoglycan, LPS, LTA, exotoxins, lipoproteins, and glycolipids.91 Antibiotics used for treatment may destroy bacteria and consequently release additional inflammatory products. These molecules are called pathogen-associated molecular patterns and trigger responses through their interaction with pathogen pattern recognition receptors (PRR), which often reside on the host cell membrane or in the cytoplasm. The best-known PRRs residing on the cell membrane are the toll-like receptors (TLRs), which have a transmembrane region that separates the cytoplasmic signaling domain from the extracellular ligand binding domain. TLRs are generally most abundant on inflammatory cells but are present in lower levels in the epithelial barrier, which is continuously exposed to microbial products.92 TLR2 detects lipoproteins and lipoteichoic acid and requires a binding partner (TLRl or 6) to transmit signals leading to cytokine production.93 Mice lacking the TLR2 gene are more susceptible to mucosal and systemic infection with staphylococci and streptococci.94.95 LPS binds to TLR4 with the help of MD2, cluster of differentiation (CD)l4, and lipid-binding proteins. Mice without functional TLR4 are unresponsive to LPS. Bacterial flagellin signals through TLR5.96 Bacterial DNA, rich in unmethylated CpG motif, is a potent inducer of inflammation through its binding to TLR9. TLR9 receptors are found inside the cells as they occur in endocytic vesicle membranes and react to phagocytosed bacteria. TLR activation increases expression of inflammatory cytokines (eg, tumor necrosis factor [TNF]-a) through increased transcription of their

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genes. The protective effect of TLR can be seen early in an infection. For instance, TLR9 can protect against pneumonia caused by S. pneumoniae even before circulating inflammatory cells enter into the lungs.97 Bacterial invasion is also detected by various intracellular PRRs. The best-known PRR family may be the nucleotide oligomerization domain (NOD)-like receptors (NLRs). At least 23 NLR family members have been identified in humans, with each member having a central nucleotide-binding domain (NACHT domain), anN-terminal effector domain, and a C-terminal receptor domain with leucine-rich repeats. Ligands may bind the leucine-rich repeat domain and initiate oligomerization of the NATCH domain to form a signaling platform that allows the binding of adaptor and effector molecules, ultimately leading to activation of caspase 1 and then interleukin (IL) -11}.98-100 This molecular assembly is named an "inflammasome,»~ 01 with the best-known infammasome being Nlrp3 (also known as NALP3 or cyropyrin), which can sense various bacterial products as well as alum, silica, and urate crystals.101 Another inflammasome is Nlrc4 (also known as CARD12 or IPAF), which can detect flagellin. Flagellin, however, is also detected by TLR5.102 Other well-known NLRs are NODI and NOD2. NODI is expressed in various cell types, but NOD2 is expressed primarily among the epithelial cells of the lungs and intestine, macrophages, and dendritic cells (DCs).103-106 NODI recognizes peptidoglycan fragments containing meso-diaminopimelic acid primarily from gram-negative bacteria,107 but NOD2 recognizes muramyl dipeptide, the minimal motif of peptidoglycan that is shared by both gram-positive and gram-negative bacteria.108 Interestingly, the lack of signaling by muramyl dipeptide results from the NOD2 gene mutation involved in Crohn disease.9s,108•109 During the initial phase of inflammation following a bacterial invasion, many cell types residing in the mucosa or skin (eg, keratinocytes) may produce molecules important in controlling infections. Several studies revealed that mast cells are one of the important resident host cells involved in the innate immune response. Mast cells, classically known for their stores of histamine and serotonin,110 are abundant along the bronchial tree and the epidermis of the skin. They are now known to both contain preformed TN F-a as well as be a major source of various cytokines. Mast cells account for 90% of IL-4- and IL-6-producing cells in the nasal cavity.m Upon exposure to various bacterial products (eg, LPS), mast cells release these cytokines, which are essential for the recruitment of neutrophils to the site of inflammation. The absence of mast cells can increase the susceptibility of animals to bacterial infections in the peritoneum or the lungs; their absence can be partially compensated for by administration ofTNF-a.112 As the inflammatory process persists, additional cell types come to the site of inflammation. In the case of experimental pneumococcal pneumonia, neutrophils come to the lungs in 12 to 24 hours, followed by the appearance of monocytes and macrophages in 48 hours.113 Similar phagocyte entry sequence was observed for nasopharyngeal

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colonization by S. pneumoniae. 114 Few lymphocytes are observed in the lungs during this time period. Upon their arrival at the site of infection, neutrophils and macrophages, which can rapidly phagocytize and kill the bacteria, become activated by the bacterial products (eg, LPS) and chemokines (eg, IL-8). Phagocytosis can occur when phagocytic cells recognize certain native molecular structures of the bacteria such as lectins, PS, andpeptides (RGD sequence), 115 or aided by their CR3 and Fe receptors, recognize the host opsonins on the bacterial surface. Inflammatory processes trigger the cascade of chemokine and cytokine release at the site of inflammation. Sequential appearance of chemokines has been noted in the pneumonia model.113 The peak levels of chemokines macrophage inflammatory protein (MIP) -2 and KC are achieved in the lungs less than 6 hours after infection. The peak levels of MIP-1a and MCP-1 are observed in 12 to 24 hours. Neutralizing MIP-1a and MCP-1 along with RANTES reduces macrophage recruitment. The cytokines produced during acute inflammation can be divided into two groups: proinflammatory cytokines (eg, IL-l and TNF -o:) and anti-inflammatory cytokines (eg, IL-4). The molecules produced during inflammation can induce the expression of ELAM, ICAM, and VCAM on endothelial cells and of selectins and integrins on leukocytes, thereby modifying the properties of the cells (cell adhesion, vascular permeability, etc.) at the site of inflammation. Inflammation also draws phagocytes to the site of pathogen invasion, where the phagocytes generally efficiently recognize, ingest, and kill the extracellular pathogens. Among many surface receptors, CR3 may be the most relevant to recognizing extracellular bacteria. Indeed, persons with CR3 deficiency (leukocyte adhesion deficiency type 1) have leukocytes ineffective in phagocytic killing.116 The phagocytic killing occurs rapidly (generally within 15 minutes) when phagosomes fuse with lysosomes and the ingested bacteria are exposed to lysosomal enzymes, although some extracellular bacteria can survive in phagocytes for a significant period.111 In addition to this classic killing mechanism, a new mechanism called "autophagy" has been described. This process is characterized by the engulfment of portions of the cytosol into a characteristic double-membrane vacuole called an "autophagosome."118' 119 After maturation, autophagosomes fuse with lysosomes followed by degradation of the sequestered structures and recycling of the degraded products. 120 While the autophage is important in controlling infections by intracellular pathogens (eg, virus or intracellular bacteria), it may also be involved in defense against extracellular pathogens. S. pyogenes may be killed by autophagy.l21' 122 However, S. aureus may also exploit autophages to its advantage as S. aureus activates an autophagic response to enter the double-membraned autophagosomes but prevents autophagosome maturation.123•124 Another phagocyte response involves neutrophil extracellular traps (NETs).125 In this situation, when phagocytes encounter pathogens, the phagocytes decondense the phagocytes' DNA and release their DNA as well as their cellular contents. This decondensed DNA forms a net with

cellular debris with the NET being able to capture and destroy many bacteria. However, some pathogens such as S. pneumoniae can escape the NET by destroying it with endonuclease.126

Systemic Response to Bacterial Invasion In response to inflammatory bacterial products, cytokines such as IL-l, TNF-a, and IL-6 are released into the systemic circulation and trigger many systemic changes, such as fever and accumulation of leukocytes at the sites of infection. The cytokines also trigger an acute-phase response by the liver.127' 128 The acute-phase response occurs when hepatocytes, in response to the cytokines, activate transcription factors such as NF-lCB and STAT3, 127' 126 and increase production and secretion of a variety of molecules that are termed acute-phase reactants, such as coagulation factors, serum amyloid A protein, C-reactive protein (CRP), TREM-1,129 and collectins. 130 CRP was named for its ability to bind to pneumococcal C-polysaccharide. Its serum level begins to rise 2 to 3 hours after infection and increases more than 1000-fold within 2 to 3 days after infection. 131 CRP recognizes pathogens by binding to PC, which is expressed on many respiratory tract pathogens, and activates complement on the bacteria or functions like anti-PC antibody by engaging FeR. Indeed, CRP can kill PC-expressing H. influenzae in vitro in the presence of complement. In addition, while many pathogens use PC to bind to the host cells via rPAF, CRP also blocks bacterial adhesion that involves this receptor.132 Transgenic mice expressing human CRP are more resistant to systemic pneumococcal infection.133 The liver produces mannan-binding lectin (MBL), which is a member of the collectin family. The structural hallmark of these family members is a cystine-rich N terminus, a collagen-like domain, and a C-type lectin domain. These molecular features allow MBL to assemble into a Clq-like structure. Although it is an acute-phase protein, MBL levels change only two- and threefold during the course of an infection. 134 However, the population distribution of MBL levels ranges between 100- and 1000-fold,13s. 136 with about 5% of Europeans being MBL deficient (< 100 ng/mL).137 MBL uses the lectin domain to recognize the target carbohydrate on a microbe, activates MBL-associated protease-2, and initiates the lectin pathway of the complement system. MBL is the innate opsonin for baker's yeasts, and MBL-deficient individuals may have an increased incidence of infection.133 MBL significantly reduces bacteremia from N. meningitides.139 Although this relationship is controversiaL140•141 MBL-deficient persons may be more susceptible to infections by S. pneumoniae with a low invasion index.142 Interestingly, transplant patients who received the liver from MBL-deficient donors are more susceptible to infections.143 The collectin family includes SP-A and SP-D, which may play important roles in lung immunity.144•145 The liver produces another group of innate opsonins, called ficolins. Humans produce three ficolins: L- and H-ficolins are from the liver/46 but M-ficolin is in the secretory granules of neutrophils, monocytes, and type II alveolar

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epithelial cells. 147 Ficolins have a fibrinogen-like domain at the C terminus instead of a C-type lectin domain, but they assume an overall structure similar to those of MBL and Clq. Ficolins also trigger the lectin pathway of the complement system by activating MBL-associated protease-2.148•149 L-ficolin can bind many structures with acetyl groups150 but many bacterial surface molecules that appear to be the natural ficolin ligands contain carbohydrate moieties. Thus, ficolins display lectin-like functional properties. Once extracellular bacteria enter the systemic circulation, these opsonins together with antibodies opsonize bacteria for rapid removal by the spleen or the liver.20'151 Persons lacking splenic function (due to sickle cell disease or splenectomy) are at an increased risk of overwhelming sepsis from encapsulated pathogens (eg, S. pneumoniae).133 Clearance of bacteria from the blood by these organs is facilitated because phagocytes are abundant and blood circulates slowly in these organs. S. pneumoniae serotype 27 produce capsular PS-containing PCs. CRP can bind and opsonize serotype 27 pneumococci for phagocytes,152 which may be why serotype 27 pneumococci are nonpathogenic.153 The liver is also the major source of transferrin, which increases iron storage by tissues and lowers the serum concentration of iron. Iron at the site of inflammation may be reduced by neutrophil-secreted lactoferrin. The reduction in the amount of iron available to bacteria can be a significant defensive measure.154 Moreover, even a moderate reduction in iron intake155 and the use of an iron chelator have both been shown to be beneficial against infections by extracellular bacteria. In contrast, an excess of iron may predispose an individual to infections. 156

ANTIGEN-SPECIFIC HOST DEFENSE RESPONSE Accompanying antigen-nonspecific responses, the host also mounts an adaptive, antigen-specific immune response. For protective responses to extracellular bacteria, B-cellmediated (but not T-cell-mediated) immune responses are critical, as shown by clinical observations of patients with Bruton agammaglobulinemia. These patients, who have relatively normal T-cell function but lack B cells, suffer primarily from infections caused by extracellular bacteria, infections that can be successfully treated with the passive administration of pooled garnmaglobulin.157 Consequently, protective B-cell responses are described in detail subsequently.

Responses of the Host (B CeiU Immune System to Bacteria Following an asymptomatic exposure to extracellular bacteria or to an infection, the host develops antibodies to many different bacterial antigens. For instance, the level of antibodies to various pneumococcal antigens increases in young children as they age, even if they never have clinical infections.158 However, the antibody levels remain low in those young children without evidence of asymptomatic nasopharyngeal carriage of pneumococci. This finding suggests that asymptomatic carriage of pneumococci is sufficient to raise antibody levels.158

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When an infection occurs, it presents the host with a large load of free antigens released from bacteria such as capsular PSs and proteins (eg, toxins). Bacterial proteins induce strong immune responses in a conventional T-celldependent manner; indeed, the antibody response induced by bacterial proteins has been used to diagnose infections.159 In addition, the released PSs that are readily detectable in the urine of many patients160 are used to diagnose pneumococcal pneumonia.161 The released PSs may neutralize the anti-PS antibodies in the host. For example, vaccination with hemophilus vaccines may neutralize preexisting antiPS antibody and briefly increase disease susceptibility immediately after the vaccination. 162 In contrast to proteins, bacterial PSs generally elicit antibody responses with minimal help from T cells.163 As bacterial PSs usually have many repeating units and multiple epitopes, they can efficiently cross-link B-cell receptors and stimulate B cells. The PSs primarily stimulate two subsets ofB cells: Bl B cells164 and marginal zone (MZ) B cells.165•166 These two types of B cells together with follicular B cells are the three recognized subsets of mature B cells with preferential anatomic locations. Bl B cells are associated with the peritoneum, MZ B cells are found in the splenic marginal zone, and follicular B cells are in splenic follicles. In mice, the subsets can be distinguished by their surface phenotypes. Follicular B cells are IgMto, IgDhl, CD23+, CD21int, and CDld1ow, whereas MZ B cells are IgMhi, IgDto, CD23Low, CD2lhi, and CD1dhi.167 Bl B cells express CDllb and B220, with CDS expression being used to divide them into Bla {CDS+) and Blb (CDS-) subsets.164 Furthermore, these subsets have distinct developmental requirements. MZ B cells require a proline-rich tyrosine kinase (Pyk-2)/65 Aiolos, and Notch2.167 Bl B-cell deficiency was noted in mice without the regulatory Bl subunit of calcineurin.168 BlaB cells are absent in CD19-deficient mice/69 and the development of Bl and follicular B cells requires BTK.170 Several observations support the contention that the antibody response to PS antigens is largely independent of T cells. Athyrnic mice can produce antibodies toPS antigens. PS antigens do not bind class II molecules as protein antigens do171 and may actually interfere with the presentation of protein antigens.172 In addition, they do not usually induce the formation of germinal centers,173 they elicit poor immune memory/74 and they easily tolerize B cells.175 Nonetheless, there have been past reports ofT-cell involvement in the antibody response toPS antigens,176 and recent studies suggest that CD40 is involved in this response.177 Because the PS antigens used for the studies may have had contaminants that affected their immune properties178 and as zwitter ionic PSs may behave differently from other PSs,179 additional studies with pure PSs would be needed to define the role of non-B cells in the antibody response toPS antigens. PS antigens commonly elicit oligoclonal antibodies, which utilize a restricted number of V region genes180•181 even among genetically unrelated humans. 182 In addition, the antibodies toPS exhibit few somatic mutations182•183 and generally have a low affinity to the antigen.184 However, because the capsular PS and LPS 0 antigens present repeating epitopes, even low-affinity antibodies can bind with enough

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avidity to fix complement and cause opsonization and/or bacteriolysis. Capsular PSs have been used as vaccines because antibodies specific for them are protective. Young children, however, do not produce antibodies to most PS antigens until they are several years old, 185 and they are particularly susceptible to infections by encapsulated bacteria during their first few years oflife.186 However, young children readily produce antibodies to PS when it is conjugated to a protein carrier. The clinical use of "conjugate" vaccines to induce antibodies to H. influenzae type b {Hib)-PS in young children has virtually eliminated Hib meningitis as well as oropharyngeal colonization by Hib.187 Similar "conjugate" vaccine approaches have been used to produce 4-valent meningococcal and 7-, 10-, and 13-valent pneumococcal conjugate vaccines.188-190 The pneumococcal conjugate vaccine has been used for young children since 2000 and has markedly reduced the incidence of invasive pneumococcal infections in both young children as well as old adults.191 The immunobiology of conjugate vaccines can differ based on the protein carriers used. Among hemophilus conjugate vaccines, Hib-PS conjugated to the meningococcal outer membrane protein complex can elicit antibody responses after only one immunization, 192 presumably because, unlike other protein molecules used as carriers, outer membrane protein complex stimulates TLR2.193 Not all bacterial antigens are presented to the host as free molecules, and some antigens remain associated with the bacteria. When bacteria enter the blood circulation, they preferentially localize at the marginal zone of the spleen. The marginal zone has features useful in capturing partides in the blood: the zone is where the terminal arteriole ends and empties into sinuses and has several characteristic macrophages and DCs.167 Following the localization of bacteria at the marginal zone of the spleen, even without T cells, within 2 to 3 days MZ B cells can be activated and become plasma cells secreting antibodies to bacterial PSs. 166 MZ B cells have additional unique characteristics. They are rapidly stimulated by LPS/94 and their maturation may be facilitated by other bacterial molecules as well. MZ B cells may facilitate the activation of follicular B cells as they can capture IgM immune complex and transport it to follicular DCs.l95,196 Although MZ B cells can mature and differentiate independently ofT cells, 166 other cells, including T cells, may help their maturation and antibody responses to various bacterial antigens. MZ B cells can present bacterial {protein) antigens to naive T cells.195 In addition to protein antigens, antibody responses to bacterial PSs require cofactors such as B7-2197 and CD40,197'198 and can be reduced with the simultaneous injection of anti-CD4 and anti-CD8 antibodies.199'200 Indeed, studies have shown antibody responses to PSs attached to bacteria to be T-cell-dependent.201 MZ B-cell response may depend on DCs as well. Upon taking up dead bacteria, CDllc1.,. CDllbb;gb DCs in the blood locate to the spleen and may provide transmembrane activator and CAML interactor ligand(s) helpful in MZ B-cell survivaP02 Adoptive transfer of live DCs after an in vitro exposure to dead pneumococci can transfer

antibody responses to pneumococcal proteins and PS antigens.Z03 Natural killer T cells may be involved in MZ B-cell maturation as MZ B cells express CD1 and may activate natural killer T cells. 195 Preimmune animals have antibodies that cross-react with many structurally unrelated antigens. These antibodies are often labeled as "natural antibodies." The majority of these antibodies is of the IgM isotype and frequently bind autologous antigens. Anti-PC antibodies may be an example of natural antibodies. Recent studies suggest that these natural antibodies are important in the early phase ofbacterial and viral infections.204 For instance, anti-PC antibodies react with a PC epitope found on S. pneumoniae, H. influenzae, and Wuchereria bancrofti (a tissue nematode) .20s--207 Anti-PC antibodies can reduce the susceptibility of mice to pneumococcal infections. 206 A recent study suggests that the natural antibodies are from BlaB cells. CD19-deficient mice lack B1a B cells and have a reduced number of MZ B cells. These mice also lack anti-PC antibodies and are susceptible to pneumococcal infections.169 In contrast, another mouse strain that exhibits both a reduced number of MZ B cells and a deficiency of B1b cells can produce anti-PC antibodies and is as resistant to pneumococcal infections as are normal mice.169 In addition to natural antibodies, animals often have preexisting antibodies to a PS that cross-react with structurally similar PSs,208- 210 probably because many PS molecules have very similar structures. Sometimes it is difficult to distinguish usual "anti-PS antibodies" from "natural antibodies." Cross-reactions may play an important role in protecting the host against its first exposure to a bacterial species. For instance, human adults carry detectable amounts of antibodies to the Hib-PS-even in the absence of vaccination-and are thus relatively resistant to H. influenzae infections.186 While some of the antibodies may be the result of immunization by subclinical infections, the majority of human preimmune (but not postimmune) anti-Hib-PS antibodies cross-react with E. coli KlOO, the PS capsule of which is an isopolymer of Hib-PS. 211 Experimental colonization of rats with E. coli KlOO can protect them against Hib.212 About 1% of human IgG binds a carbohydrate epitope (galactose [1--+3] galactose),213 and this antibody can kill Trypanosoma and Leishmania in vitro.Z14 Cross-reactive antibodies binding the LPS core components are thought to be responsible for the protection from bacteremic dissemination of gonococci in nonimmune patients, 215 although they cannot prevent infection of the genital tract.215 Normal gut flora may be the antigenic stimulus for many of the cross-reactive anti-PS antibodies. About 1% of the human population carries E. coli KlO 0 in their gut at any moment. 216 Antibodies to (galactose [1--+3] galactose) bind many species of bacteria isolated from normal stool specimens.213 The gut flora may have additional interesting impacts on the immune system. For example, in some transgenic mice, inflammatory bowel diseases develop in the presence of normal intestinal flora but not in the absence of gut flora. In addition, in some animals, such as chickens and rabbits, microbial colonization of the gut

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appears to be necessary for the normal development of antibody V region repertoires.217 Bacteria should therefore be considered as active participants in shaping the host immune system.

Protective Mechanisms of Antibodies Antibodies to virulence factors may act by neutralizing the function of those factors. Anti-toxin antibodies can protect a host by blocking the action of the toxins (eg, blocking the binding of the toxins to the host cell receptors) or by increasing the removal rate of the toxins. Antibodies to superantigens or tetanus toxin can inactivate them and thereby provide protection to the host. Antibodies to an E. coli adhesin can prevent experimental infections by E. coli.218 Antibodies to M protein neutralize its ability to interfere with complement and provide protection against S. pyogenes infections. Antibodies to LPS/19,220 and perhaps to LTA,221 can be protective. Antibodies to PspA, pneumolysin, autolysin, or pneumococcal surface protein C can protect animals from fatal pneumococcal sepsis. Although these antigens are being investigated as potential replacements for the expensive pneumococcal conjugate vaccines, the protective mechanisms that they employ are still unclear. The most recent hypothesis suggests that antibodies to PspA may inhibit its decomplementation properties and the antibodies may increase the complement fixation on pneumococci.33•222 Antibodies to IgAl protease or iron-transport systemsl23•224 can also protect against bacterial infections, most likely by neutralizing the nonnal functions of the target antigens. Finally, in the presence of antibodies and complement, the ability of the liver to remove bacteria increases significantly.20 Thus, another protection mechanism provided by antibodies may be to facilitate the in vivo removal of bacteria from circulation by enhancing the ability of the reticuloendothelial system to clear bacteria. Antibodies to capsule PS can provide protection by fixing complement on the surface of bacteria and by inducing bacteriolysis or opsonization. The bacteriolysis pathway can provide significant in vivo protection against gramnegative bacteria, as illustrated by the susceptibility toN. meningitidis infections of persons with deficiencies in CS-9 components.Z25 In contrast, antibodies and complement do not lyse gram-positive bacteria but opsonize them for phagocytic killing, as explained in the following. 226 Host phagocytes cannot readily recognize and kill the intact encapsulated gram-positive bacteria. However, once bacteria are coated with antibodies and complement, the host phagocytes can readily recognize the bacteria via various recognition receptors and engulf them for intracellular killing. The Fe receptor (CD16b) and the complement receptor (CR3) are some of the important recognition receptors. CR3, an integrin molecule, is a heterodimer of CDllb and CD18. Protection mediated by this antibody/complementmediated opsonization is probably important in vivo, as both complement deficiency and agammaglobulinemia predispose individuals to infections by many different extracellular bacteria.157'225 To be effective for opsonization, the epitope of the surface antigens must be exposed on the

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surface of the bacteria. Effective antibodies to the porins of N. meningitidis recognize the surface loop of the molecule.227 In most pneumococci, C-polysaccharide (C-PS) is mostly buried underneath the PS capsule. Although antibodies to the C-PS can fix complement,20 anti-C-PS antibodies were ineffective in protecting mice against most S. pneumoniae unlike antibodies to capsular PS, which are protective.Z28 However, a recent study found that purified human anti-C-PS antibodies can opsonize pneumococd29; thus, additional studies are needed. Because antibody-mediated opsonization and bacteriolysis are dependent upon the complement-fixing properties of the Fe region, the relative efficacies of antibodies of different Ig isotypes have been compared. IgM antibodies are produced early in the course of infections and should be important in the early phase of infections because they fix complement very efficiently and can opsonize bacteria. Selective deficiency of IgM antibodies was found to increase susceptibility to bacterial infections. 230 Studies found that specific IgM antibodies agglutinate erythrocytes, fix complement, and lyse erythrocytes more readily than IgG antibodies, 231 and IgM antibodies are more effective in complement-mediated bacteriolysis232 ; however, IgG antibodies are more effective than IgM antibodies in preventing pneumococci infections of mice233 or in opsonizing Hib in vitro.234 Moreover, antibodies of some IgG subclasses have been reported to be more protective against specific viraF35 and fungaF36 infections than antibodies of other subclasses. These results suggest that optimal opsonization requires not only complement receptors but also Fe receptors for IgG. In the absence of inflammation, IgM antibodies are confined to the intravascular space, whereas IgG antibodies can enter the extracellular space. However, inflammation can make the vessels at the infection site permeable, at which point, antibodies of all isotypes may enter the infection site. Compared with lgM antibodies, IgG antibodies may be especially efficient at neutralizing toxins because they have a longer half-life, generally have a higher affinity, and are already present in extravascular spaces prior to infection.237 IgG subclasses differ in their ability to fix complement and to bind Fe receptors.23a, 239 It has also been reported that IgGl mouse monoclonal antibody is protective against Cryptococcus neoformans but that IgG3 mouse antibody is not.236 Consequently, the fact that antibodies to bacterial PS are found to be largely restricted to a single IgG subclass (IgG2 in humans and IgG3 in mice) has led to many studies of the differences in the protective properties of anti-PS antibodies of different isotypes. Mouse IgG3 antibodies (but not antibodies of other IgG subclasses) can associate with each other through their Fe regions.240 This feature may make the IgG3 antibodies with a low affinity to PS more effective in binding the antigen than antibodies of other isotypes of the same affinity. Although these observations provide a theoretical advantage for mouse IgG3 antibodies, this same aggregation phenomenon has not been observed for human IgG2 antibodies even though some human IgG2 can form covalently joined dimers. 241 The full significance of IgG3 aggregation is not clear, however, as anti-PS antibodies of the IgG3 isotype have not been observed to be any more efficacious

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against pnewnococcal infections than antibodies of other isotypes.242 IgG2 antibody levels can be significant, however. People expressing the Gm23+ IgG2 allele have higher IgG2 antibody levels than people with the Gm23- allele. Among C2-deficient persons, Gm23- persons are more susceptible to bacterial infections than Gm23+ individuals.243 Moreover, in contrast to expectations, many studies found that hwnan IgG1 antibodies are slightly more effective at opsonization and bacteriolysis than are human IgG2 antibodies.239.244 Neither of these isotypes appears to be essential, however, as individuals lacking IgG1 and IgG2 subclass genes are healthy.245 Furthermore, human IgG2 antibodies bind less strongly to CD16, CD32, and CD64 than do IgG1 or IgG3 antibodies246 and may not be effective for neutrophil opsonization in individuals homozygous for a specific CD32 allele.244 These observations, taken together, strongly suggest that the human IgG2 (or mouse IgG3) subclass may not provide any unique advantage in defense against bacteria. IgA is highly heterogeneous in structure: it can exist as a monomer, a polymer, or in secretory forms. In addition, its function is still unclear. Although it has been reported that IgA can opsonize,247 fix complement,248 and facilitate the lysis of N. meningitides,249 other studies have found that IgA does not fix complement in vitra25° and may even inhibit IgG-mediated complement-dependent killing.251 The ability of IgA to fix complement may also depend upon its denaturation or its glycosylation status.252' 253 Nevertheless, other studies indicate that IgA antibodies may fix complement by the MBL pathwaf54 and that hwnan IgA antibodies against pneumococcal capsular PS can opsonize pneumococci for killing by neutrophils.255 Bacteria that commonly colonize or infect mucosal areas often produce IgA1 protease, and IgA antibody has been found to provide protection in at least some of these cases.256 These findings suggest that lgA may play an important role as a part of the complex mucosal immune defense. For example, IgA antibodies may be important in reducing nasopharyngeal colonization by bacteria inasmuch as the mice deficient in IgA or polymeric Ig receptor can carry pneumococci in the nasopharynx even after an immunization against pneumococci.257 IgA may function by aggregating the bacteria and facilitating their expulsion from mucosal areas. IgA may also block the invasion of bacteria through mucosal epithelial cells, as endocytosed IgA has been found to block the transport ofvirus through epithelial cells.258 However, IgA-deficient persons or mice are relatively healthy, and IgA-deficient mice can elicit normal protective immunity to experimental infections with influenza virus. IgM antibodies may function as secretory antibodies in IgAdeficient individuals.259

T-Celllmmune Responses to Extracellular Bacteria Although immune responses to toxins from extracellular bacteria are T-cell-dependent, antitoxin antibodies mediate protection; therefore, the protective immunity against extracellular bacteria is clearly centered on the B-cell responses. However, recent studies suggest additional roles for T cells in responses to extracellular bacteria and their products.

PS associated with lipid can stimulate T cells in association with CDl molecules.Z60 Also, studies of abscess formation in response to Bacteroides fragilis infections led to a discovery that zwitterionic PS can be taken up by antigen-presenting cells, which can process the PS via a nitric oxide-dependent mechanism and present it in association with MHC class II molecules179 to stimulate CD4+ T cells to produce IL-17.261 IL-17-producing CD4+ T cells are now named "Thl7 cells" and are found to be distinct from Thl and Th2 cells. Thl7 cells produce unique set of cytokines, such as IL-17 {IL-17A), IL-17F, IL-21, and IL-22, and require a distinct cytokine milieu {IL-l, IL-6, and TGF-1}) for their development.262- 265 Th17 cells may be involved in abscess formation by B. fragilis infections because Th17 cells are found in these abscess.266 Several studies found that IL-17 and Th17 cells are important in the nasopharyngeal carriage of S. pneumoniae in mice.114.267 A recent study showed that Thl7 cells may recruit monocytes and macrophages to the nasopharynx where the monocytes/macrophages may actually remove pneumococci. Clinical examples clearly demonstrate the importance of IL-17 on some extracellular bacterial infections in humans. Patients with autosomal dominant hyper-IgE syndrome are deficient in Th17 cells and are very susceptible to infections by fungus and S. aureus.268 Patients with mutations in IL-17F or IL-17 receptor A (IL-17RA) are susceptible to fungal and staphylococcal infections.269 In view of these new findings, some researchers are investigating the possibility of using antigens stimulating Th17 cells as vaccines against S. pneumoniae.

DELETERIOUS HOST RESPONSES Inflammatory responses by the host inevitably cause some tissue damage. In some bacterial infections such as pneumonia and meningitis, this damage plays a significant role in disease pathology and symptoms. For instance, animal models of meningitis have shown that inflammation associated with bacterial products (primarily bacterial cell walls) is the primary cause of neurologic damage. Treatment of animals with antibiotics alone can eradicate the bacteria, but it does not prevent neurologic damage. In contrast, when inflammation was controlled by steroids administered along with the antibiotics, neurologic damage was considerably reduced.Z70

Antigen-Nonspecific Deleterious Response Uncontrolled inflammation at the systemic level can produce septic shock, which can be triggered by several factors, including exotoxins (eg, staphylococcal enterotoxin B) of gram-positive bacteria, the combination of LTA and peptidoglycan from gram-positive bacteria/71 or LPS from gram-negative bacteria. The staphylococcal enterotoxin B superantigen binds the host's class II molecules of the MHC region and can stimulate large numbers of helper T cells to release cytokines. Septic shock can also be initiated when LPS from gram-negative bacteria binds CD14 and a TLR and stimulates macrophages or monocytes to secrete inflammatory cytokines. In addition to resulting in the release of cytokines, the stimulation of host cells by bacterial

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products leads to the release of other mediators of inflammation, such as arachidonic acid metabolites, activation of the complement cascade, and activation of the coagulation cascade. Excess release of the mediators leads to the failure of the vascular system and, finally, the failure of multiple organ systems. Studies using transgenic mice with defective genes have identified several molecules critical in developing septic shock, such as TNF-a, one of its receptors TNFRI, caspase 1, and ICAM-1.272 This approach also showed that CD14 and TLR4 are critical for LPS-induced septic shock and that CD28, aT-cell costimulation molecule, is necessary for superantigen-induced septic shock.272 Anthrax infections provide another example of uncontrolled host responses. "Lethal factor" binds the "protective antigen" immobilized on the macrophages and then stimulates the cells to secrete cytokines and reactive oxygen intermediates. These macrophage products are thought to kill the host, as the host dies even when the proliferation of the bacteria is controlled. When macrophage cells are removed from animals, the animals are resistant to anthrax toxins. 273 This suggests that the macrophage response to the toxins is actually responsible for the death of the host. Although inflammation is a significant cause of morbidity and mortality, it must also be regarded as the host's primary protection against bacterial infections. Evidence for this hypothesis comes from studies with TLR4-deficient mice, which, although nonreactive to LPS and completely resistant to LPS shock, are more susceptible to infection with gram-negative bacteria than are normal mice.274' 275 Perhaps LPS is "toxic" because the host has evolved to use this common bacterial component as a trigger for host responses.

Autoimmune Disorders Autoimmune diseases are characterized by an overactive host immune response toward self, the host's own cells and tissues. Various factors are involved in autoimmune diseases, including genetic predisposition and environmental triggers. The pathogenesis of autoimmune diseases has at its core the development of autoreactive effector lymphocytes, and these can involve, among others, T-cell bypass and molecular mimicry. T-cell bypass is based on the notion that activated T helper cells provide the necessary factors to activated B cells for the production of antibodies. Some microorganisms can provide the bypass with superantigens, which can bind to many T cells expressing certain types of vp regions and stimulate them to create a nonspecific polyclonal T-cell activation.276,277 The best-studied examples of superantigens are the exotoxins secreted by S. aureus and S. pyogenes. m-280 Molecular mimicry occurs when a bacterial antigen shares structural similarities with a host antigen, and hence, antibodies produced against the bacterial antigen could also bind to the host antigen, thereby casuing an autoimmune disease. For instance, the LPS of many strains of N. meningitidis, N. gonorrhoeae, H. influenzae, and Haemophilus ducreyi expresses the epitope of blood group antigen pK.281 The PS capsule of N. meningitidis group B mimics epitopes expressed in the central nervous system,282 such as the N -acetylneuramic acid epitope in the embryonic N -CAM.283

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While infection by these pathogens may cause autoimmune diseases, epidemiologic studies have clearly associated Campylobacter jejuni infections, a leading cause of acute gastroenteritis, with development of antiganglioside antibodies and autoimmune diseases: Guillain-Barre syndrom~84 or its variant Miller-Fisher syndrome. Patients with Miller-Fisher syndrome have ophthalmoplegia and generally have autoantibodies to GQlb, but Guillain-Barre syndrome is associated with autoantibodies to GMl or GDla.285 Production of the two different types of autoantibodies has been associated with an allelism of sialyl transferase II of C. jejuni. One allele produces lipooligosaccharide-mimicking GMl and GDla, and infection by C. jejuni with this allele has been associated with Guillain-Barre syndrome. On the other hand, the other allele produces lipooligosaccharide-mimicking GQlb, with its infection being associated with Miller-Fisher syndrome. Perhaps the most classical example of infection-associated autoimmunity may be rheumatic fever and acute glomerulonephritis associated with S. pyogenes infections. Studies found that S. pyogenes can be divided into two classes with a monoclonal antibody to M protein286 and that rheumatic fever develops only after infections with class I strains of S. pyogenes.286 Class I and class II strains of S. pyogenes can also be readily distinguished by the linkage relationship of the M protein genes with the genes encoding related surface proteins.286,287 M proteins from some class I S. pyogenes express epitopes highly cross-reactive with epitopes of cardiac myosin, tropomyosin, vimentin, laminin, and keratin. 288- 290 An antibody molecule may bind to all of these protein molecules because a major portion of these proteins is coiled-coil a-helix.290 The polyreactive antibodies to M protein may directly damage myocardial and endothelial cells.291 In addition to antibodies, CD4+ and CDS+ T cells are found at rheumatic heart valves, 292 and the T cells proliferate to M protein peptides and heart proteins. 293 These observations suggest that the T cells with crossreactivity between M protein and myosin may be involved in the pathogenesis of rheumatic fever as well.

CONCLUSION Because extracellular bacteria can grow rapidly and produce toxins, some are potent pathogens. To combat these bacteria, higher organisms primarily depend on two arms of the immune system: innate immunity and adaptive immunity centered on antibody molecules. The two arms of the immune system are comprised of multiple layers of protection. In the early stage of an infection, innate immunity involving pattern recognition receptors, complement, phagocytes, and natural antibodies cross-reacting with many antigens are important in host defense. During the late stage of an infection, pathogen-specific antibodies appear. These antibodies generally mediate the ultimate protection against extracellular bacteria by triggering the protective effects of complement and phagocytes. Nevertheless, innate and adaptive immune responses may cause damage instead of protection. A better understanding of how our immune system protects against each pathogen will aid in the development of more effective preventive and therapeutic measures against these pathogens.

CHAPTER 41 REFERENCES

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CHAPTER 41 REFERENCES 265. Weaver Cf, Hatton RD, Mangan PR, Harrington I.E. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev ImmunoL 2007;25:821--852. http:f/www.ncbi.nlm.nih.gov/pubmed/17201677 266. Chung DR, Kasper DL, Panzo RJ, et al. CD4+ T cells mediate abscess formation in intra-abdominal sepsis by an IL-17-dependent mechanism./ ImmuooL 2003; 170(4): 1958-1963. http:f/www.ncbi.nlm.nih.gov/pubmed/12574364 267. Lu YJ, Gross J, Bogaert D, et aL Inrerleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog. 2008;4(9): e1000159. http:f/www.ncbi.nlm.nih.gov/pubmed/18802458 268. Minegishi Y, Saito M, Nagasawa M, et al. Molecular explanation for the contradiction between systemic 1h17 defect and localized bactErial infection in hyper-IgE syndrome./ Exp Med. 2009;206: 1291-1301. http:f/www.ncbi.nlm.nih.gov/pubmed/19487419 269. Puel A, Cypowyj S, Bustamante J, et al. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science. 2011;332(6025):65-68. http:f/www.ncbi.nlm.nih.gov/pubmed/21350 122 270. Bhatt SM, Cabellos C. Nadol JB Jr, et aL 1he impact of dexamethasone on hearing loss in experimental pneumococcal meningitis. PediaiT Infoct Dis/. 1995;14(2):93-96. http:f/www.ncbi.nlm.nih.gov/pubmed/7746714 271. Kengatharan KM, De Kimpe S, Robson C, Foster SJ, 1hiemermann C. Mechanism ofgram-positive shoc.k: identification of peptidoglycan and lipoteichoic acid moieties essential in the induction of nitric oxide synthase, shock, and multiple organ failure./ E.xp Med. 1998;188(2): 305-315. http:f/www.ncbi.nlm.nih.gov/pubmed/9670043 272. Gutierrez-Ramos JC, Bluethmann H. Molecules and mechanisms operating in septic shock; lessons from knockout mice. Immunol TodaJ. 1997; 18:329-334. http:f/www.ncbi.nlm.nih.gov/pubmed/9238836 273. Hanna PC, Acosta D, Collier RJ. On the role of macrophages in antluax. ProcNatlAcad Sci USA. 1993;90(21):10198-10201. http:f/www.ncbi.nlm.nih.gov/pubmed/8234277 274. O'Brien AD, Rosenstreich DL, Scher I, Campbell GH, MacDermott RP, Formal SB. Genetic control of susceptibility to Salmonella typhimurium in mice: role of the Lps gene./ Immunol. 1980;124:20-24. http:f/www.ncbi.nlm.nih.gov/pubmed/6985638 275. Hagberg L, Briles DE, Eden CS. Evidence fur separate genetic defects in C3H/HeJ and C3HeB/FeJ mice, that affect susceptibility to gram-negative infections./ Immurwl. 1985;134:4118-4122. http:f/www.ncbi.nlm.nih.gov/pubmed/3886795 276. Llewelyn M, Cohen J. Superantigens: microbial agents that corrupt immunity. Lanat Inject Dis. 2002;2: 156-162. http:f/www.ncbi.nlm.nih.gov/pubmed/11944185 277. Perersson K, Forsberg G, Walse B. Interplay between superantigens and receptors. Scand l Immunol. 2004;59:345-355. http:f/www.ncbi.nlm.nih.gov/pubmed/15049778 278. AloufJE, Muller-AloufH. Staphylococcal and streptococcal superantigens: molecular, biological and clinical aspects. Int l Med Microbiol. 2003;292:429-440. http:f/www.ncbi.nlm.nih.gov/pubmed/12635926 279. Bachert C. Gevaert P, van Cauwenberge P. Staphylococcus aureus enterotoxins: a key in airway disease? Alkrgy. 2002;57 :480-487. http:f/www.ncbi.nlm.nih.gov/pubmed/12028112

280. Sriskandan S, Faulkner L, Hopkins P. Streptococcus pyogenes : Insight into the function ofthe streptococcal superantigen. Intll Biochem Cdl Bioi. 2007;39:12-19. http://www.ncbi.nlm.nih.gov/pubmed/17029999 281. Moran AP, Prendergast MM, Appelmelk BJ. Molecular mimicry of host structures by bacrerial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol. 1996; 16:105-115. http://www.ncbi.nlm.nih.gov/pubmed/8988391 282. Finne J, Leinonen M, Makela PH. Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet. 1983;2(8346):355-357. http://www.ncbi.nlm.nih.gov/pubmed/6135869 283. Rougon G, Dubois C, Buc.kley N, Magnani JL, Zollinger W A monodonal antibody against meningoccus group B polysaccharides distinguishes embryonic from adult N-CAM. l Cell Biol. 1986;103:24292437. http://www.ncbi.nlm.nih.gov/pubmed/3536966 284. Rees JH, Soudain SE, Gregson NA. Hughes RA Campylobactc:r jejuni infection and Guillain-Barre syndrome. N Eng J Med. 1995;333:13741379. http://www.ncbi.nlm.nih.gov/pubmed/7477117 285. Yuki N. Campylobacter sialyltransferase gene polymorphism directs clinical features of Guillain-Barre syndrome. J Neurochem. 2007; 103 (suppl1): 150-158. http://www.ncbi.nlm.nih.gov/pubmed/17986150 286. Bessen D, Jones KF, Fischetti VA. Evidence for two distinct classes of streptococcal M protein and their relationship to rheumatic fever./ E.xp Med. 1989;169:269-283. http://www.ncbi.nlm.nih.gov/pubmed/2642529 287. Hollingshead SK, Bessen DE. Evolution of the emm gene family: virulence gene clusters in group A streptococci. Dev Biol Standard. 1995;85:163-168. http://www.ncbi.nlm.nih.gov/pubmed/8586169 288. Cunningham MW; McCormack JM, Fenderson PG, Ho MK. Beachey EH, Dale JB. Human and murine antibodies cross-reactive with streptoooccal M protein and myosin recognize the sequence gln-lys-serlys-gln in M protein./ Immunol. 1989;143:2677-2683. http://www.ncbi.nlm.nih.gov/pubmed/2677144 289. Cunningham MW; Antone SM, Gulizia JM, McManus BA, Gauntt CJ. Alpha-helical coiled-coil molecules: a role in autoimmunity against the heart. Clin Immunol Immuoopathol. 1993 ;68:118-123. http://www.ncbi.nlm.nih.gov/pubmed/7689424 290. Cunningham MW Streptococci and rheumatic fever. In: Friedman H, Rose NR, Bendinelli M, eds. Microorganisms and Autoimmu~ Disease. New York. NY: Plenum Press; 1996:13-66. 291. Cunningham MW; Antone SM, Gulizia JM, McManus BM, Fischetti VA, Gauntt CJ. Cytotoxic and viral neutralizing antibodies crossreact with streptococcal M protein, enteroviruses and human cardiac myosin. Proc Nat.Actul Sci U SA.1992;89:1320-1324. http://www.ncbi.nlm.nih.gov/pubmed/1311095 292. Chow LH, Yuling Y, Linder J, McManus BM. Phenotypic analysis ofin1iltrating cells in human myocarditis. An immunohistochemical study in paraffin-embedded tissue. Arch Pathol Lab Med. 1989;113:1357-1362. http://www.ncbi.nlm.nih.gov/pubmed/2480099 293. Guilherme L, Chuna-Neto E, C

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CHAPTER 43

congenitally infected infants. Prevention of congenital disease is the main incentive for developing HCMV vaccines. A secondary objective would be to provide HCMV immunity to individuals prior to becoming immunosuppressed due to transplantation, HIV, or other reasons. In vivo, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, and macrophages can support replication of the virus while latent virus can be found in macrophages and other myeloid cells. Protection against HCMV is multifactorial and involves innate immunity (natural killer cells); neutralizing antibodies primarily to glycoproteins gB, gH, and gM; and pp65-, lEI-, and gB-specific CDS+ T cells. Approaches to HCMV vaccines have included live-attenuated and whole-killed viruses, vectored, DNA, protein, and peptide-based subunit vaccines.250 The live-attenuated Towne vaccine is the most widely evaluated HCMV vaccine. It was shown to be safe and well tolerated, induced seroconversion in healthy adults, and protected against challenge with the Toledo strain.251' 252 Furthermore, the Towne vaccine, albeit not preventing HCMV infection, reduced severe disease by 85% in renal transplant recipients,253'254 although it failed to provide protection to women naturally exposed to HCMV from their infant.255 Attempts were made at enhancing the immunogenicity of the Towne vaccine by adjuvanting it with IL-I2256 or by adding back gene regions from the more virulent Toledo strains that had been deleted in the Towne strain during the attenuation process. The recombinant Towne/Toledo appeared safe in seropositive adults.257 Subunit HCMV vaccines tested in the clinic predominantly target gB, pp65, and IEl. The most advanced subunit vaccine in human studies is gB/MF59 (developed by Sanofi Pasteur MSD, Lyon, France), a recombinant glycoprotein gB formulated with MF59 adjuvant. In two large phase I studies in healthy adults, gB/MF59 was found safe and immunogenic and induced higher anti-gB titers compared with alum gB recipients.258'259 A third study in toddlers revealed that immunization with gB/MF59 resulted in titers higher than found in vaccinated adults.260 A phase II efficacy study in seronegative women within 1 year of giving birth revealed a significant increase in the number of vaccine recipients that remained uninfected during the follow-up period.261 Another vaccine strategy used alphavirus-like replicon particles from Venezuelan equine encephalitis virus to express HCMV antigens. Alphavirus-like replicon particles have been shown to specifically target DCs in vivo and induce broad immune responses including neutralizing antibodies and CTLs. AVX60I, a bivalent alphavirus replicon vaccine expressing three HCMV proteins (gB, pp65, and lEI) (developed by AlphaVax Inc., Research Triangle Park, NC, U.S.A.), was found safe and induced neutralizing antibody and multifunctional CD4+ and CDS+ T-cell responses.262 The bivalent DNA vaccine, VCL-CBOl (developed by Vical Inc., San Diego, CA, U.S.A.), consisting of plasmids encoding for gB and pp65 formulated with poloxamer CRLI005 and benzalkonium chloride to enhance immune responses, was evaluated in HCMV-seropositive and -seronegative adults?63 VCL-CBOI was immunogenic as measured by IFN-y ELISpot in 45.5% of seronegative and

VACCINES

1061

25% of seropositive vaccine recipients. VCL-CBOl boosted pp65-specific T-cell responses but not anti-gB antibodies in seropositive individuals, suggesting that this vaccine is more effective at inducing T-cell than gB-specific antibodies. Finally, prime-boost strategies have been also evaluated. Trivalent HCMV DNA vaccine VCL-CT02 (developed by Vical Inc.), encoding gB, pp65, and lEI, was evaluated in three phase I studies using a DNA prime/Towne liveattenuated boost approach. 264 HCMV-specific memory T-cells were detected by IFN-y ELISpot in 20% and 60% of unprimed and primed subjects, respectively. The median time to first pp65 T-cell and gB antibody response after Towne was 14 days for DNA-primed subjects versus 28 days for controls administered Towne only. Similar observations were made with a canarypox gB prime/Towne live-attenuated boost.265

Tuberculosis TB caused by Mtb complex bacilli is one of the leading causes of death worldwide. The WHO has declared TB a global public health emergency and predicts that almost one billion people will be infected, with 35 million dying from the disease, by 2020. TB is prevalent in developing regions of the world such as sub-Saharan Africa and Southeast Asia, where it is often associated with the HIV epidemic. Upon exposure to Mtb, 30% to 40% of close contacts will develop TB infection, of whom 5% would be expected to develop active disease within a 24-month period, whereas the other 95% enter a state of controlled latent TB infection (LTBI), which can reactivate later in life following decreased immunocompetence of the host. Despite the availability of drugs against Mtb and the directly observed therapy short-course campaign initiated by the WHO, TB persists as a global health concern in part because infected individuals do not have access to point of care and/or are often noncompliant with the 6-month or longer drug treatment. This is particularly true in the developing world where more than 95% of infections occur. Treatment noncompliance has contributed to the current TB pandemic by increasing the probability of transmission and sustaining the development of multidrug-resistant strains of Mtb. Multidrug-resistant strains are resistant to the two most powerful first-line drugs: rifampicin and isoniazid. Since the discovery of multidrug-resistant TB in the 1990s, the resistance pattern of TB has continued to evolve, and isolates resistant to both first- and second-line drugs have been identified. Therefore, development of safe, effective, and affordable prophylactic vaccines that also provides long-lasting protection in BCG-immunized people is a critical step toward controlling TB. Promptly after inhalation, Mtb is engulfed by lung alveolar macrophages in an attempt by the host to destroy the invader. Mtb, however, has adapted to survive in macrophages by preventing phagosome acidification and fusion with lysosomes. T cells, both CD4+ Thi and CDS+, and IFN-'Y and tumor necrosis factor (TNF) cytokines play important roles in the prevention of active disease and the control of LTBI, as demonstrated by gene-knockout animal models and

1062

I

SECTION VII

IMMUNITY TO INFECTIOUS AGENTS

human subjects with mutations affecting the expression of these two cytokines.266 As a result of macrophage activation by these inflammatory cytokines, Mtb bacilli stop multiplying and enter a state of dormancy.Z67 Once the activation status of macrophages is lowered, however, mycobacteria can resuscitate, leading to disease reactivation. Current vaccine design strategies focuses on stimulation of a highly potent T-cell response in an attempt to contain or even eradicate Mtb after it has established itself in the phagosome of macrophages. BCG, the only licensed vaccine currently available against TB, is likely the most widely used vaccine in the world with more than four billion doses administered. BCG is a live-attenuated strain of Mycobacterium bovis that was developed by Albert Calmette and Camille Guerin by serial passage on media.268 During the attenuation process, BCG lost a large number of genes clustered in numerous regions of difference.269 The BCG vaccine strain was introduced in 1921 and was further distributed to numerous institutions all over the world. Today, due to differences in culture conditions over decades, the BCG vaccine exists in more than one genetic background, possibly resulting in differences in efficacy. BCG is administered to newborns and is effective in preventing severe childhood forms of TB such as miliary TB and TB meningitis, whereas its efficacy in adults against pulmonary TB has been highly variable and ranged from 0% to 80% protection.270 In adults, the lowest BCG efficacy was found in countries with the highest incidence of tuberculin skin test positivity due to prior exposure to Mtb or other environmental bacteria, suggesting that preexisting immune responses to mycobacterial antigens shared in BCG prevent necessary bacterial replication and vaccine take. Therefore, although safe in all immunocompetent individuals, BCG was efficacious only in the skin test negative population, primarily children.Z71 As a consequence, vaccine protection against adult pulmonary TB, the most common form ofTB, in high-endemic countries is very limited272 and demonstrates the urgency of developing novel TB vaccines. Other drawbacks of BCG include waning efficacy over time and interference with the tuberculin skin test diagnosis ofTB. Novel TB vaccines currently in development target different needs and individuals. Some efforts are made at improving the current BCG vaccine for newborns and nonimmunized tuberculin skin test-negative adults. In addition, a heterologous booster vaccine, consisting of an adjuvanted or vectored subunit vaccine, is a high priority for individuals who have already received BCG, as BCG efficacy wanes after 10 to 15 years and boosting with BCG has proven ineffective in both preclinical and clinical studies.273- 278 Ideally, this booster vaccine would be given during adolescence and possibly repeated during adulthood as necessary. In theory, such a booster vaccine could also boost the immunity of individuals already exposed to Mtb. Finally, a therapeutic vaccine as an adjunct to antibiotics in the therapy ofTB has been considered with the hope of reducing the overall duration of drug treatment. All novel TB vaccines that are being tested in humans or are about to enter clinical trials demonstrated immunogenicity and protective efficacy in mouse, guinea pig, and/or nonhuman primate models of TB.279

Vaccines Replacing Bacilla Calmatte-Guarin Due to the long history of BCG's safety and efficacy in children, it is likely to remain on the WHO Expanded Program on Immunization vaccination schedule. Therefore, a line of improved BCG vaccines was developed and showed superior efficacy compared to BCG in different animal models ofTB, including the stringent guinea pig infection model. The first two such vaccines to complete phase I clinical trials were rBCG30, a recombinant BCG overexpressing Mtb extracellular Ag85B antigen (developed by Dr. Horwitz's group at the University of California Los Angeles, CA, U.S.A.), and VPM1002, a urease-deficient BCG expressing listeriolysin (rBCGAureC::Hly+::Hyg+) that delivers mycobacterial antigens to the cytosol for enhanced presentation toT cells (developed by Dr. Kaufmann's group at the Max Plank Institute, Berlin, Germany) (Table 43.5). rBCG30 was as safe as nonrecombinant BCG and significantly enhanced the population of Ag85B-specific CD4+ and CDS+ T cells capable of proliferation and IFN-y secretion.Z80 Similarly, no safety concerns were reported for VPM1002 in studies including individuals with or without preexposure to BCG {Germany) and with preexposure to BCG in endemic areas {South Africa). Furthermore, VPM1002 induced antigenspecific multifunctional CD4+ and CDS+ T cells and showed a trend of superiority compared to BCG at equivalent dosage (for more information, see www.vakzine-manager.de/ en/resources/Produkte/VPM1002_en.pdf). Another approach still in preclinical evaluation is worth mentioning and consists of an attenuated Mtb strain resulting from the inactivation of the phoP virulence factor and has extensively been tested for safety and efficacy, including in the guinea pig model ofTB.231,282 Subunit Tuberculosis Vaccines Hundreds of Mtb proteins have been screened in experimental animal models of TB, including systematic functional genomic antigen discovery and prioritization.Z83- 285 Combinations of one or more among six protective Mtb antigens (Ag85A, Ag85B, ESAT-6, TB10.4, Rv0125, and Rvll96) comprise the subunit vaccines currently in human studies. Ag85A and 85B are major secreted Mtb proteins; ESAT-6 and TB10.4 are virulence factors belonging to the Esx family of proteins; Rv0125 is a serine protease; and Rvll96 belongs to the PE/PPE family of proteins. All but ESAT-6 antigens are present in BCG vaccine strains, allowing for heterologous prime-boost vaccination strategy using BCG as the prime and subunit as the boost. Three adjuvanted recombinant subunit TB vaccines have been tested in human studies. Mtb72F, a fusion protein made of Rv0125 and Rvll96 formulated with the AS02A adjuvant system has completed phase I. The Mtb72F/AS02A subunit vaccine was found safe and well tolerated in purified protein derivative-negative adults, with some mild reactogenicity. It induced good production of IL-2 and IFN-y in the ELISpot assay, and polyfunctional CD4+ T cells expressing CD40L, IL-2, TNF, and/or IFN-y were observed with intra-cellular staining while no CD8+ T-cell responses were detected.286•287 Mtb72F is currently being evaluated in phase II studies. H1, a fusion protein consisting of Ag85B and ESAT-6, has been

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The Burden of Autoimmune Diseases The existence of autoimmune diseases in humans has been known for 100 years. By now, autoimmune pathogenesis has been attributed to more than 80 human diseases, 40 yet it is still far from dear which features can conclusively prove an underlying autoimmune pathogenesis. It has been suggested, somewhat provocatively, that with knowledge about

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SECTION VIII

IMMUNOLOGIC MECHANISMS IN DISEASE

determining the relative ranking of autoimmune diseases in terms of mortality risk among women under the ages of 65. Remarkably, the collection of 24 autoimmune diseases specified by Jacobson et al. ranked within the top 10 causes of death. 48 Thus, autoimmune conditions not only decrease the quality of life among afflicted individuals but also due to their high prevalence constitute a major public health burden. In addition, a trend toward rising incidence rates among most autoimmune disease has been noticed over the past few decades. For example, the incidence of multiple sclerosis {MS) in Italy has doubled between 1981 and 2002,49 and in the United States, the incidence of celiac disease increased fivefold in 15 years. 50 These trends are not likely to abate, and predictions for the number of new type 1 diabetes (TID) cases in Europe are as high as 24,400 by 2020, whereas while the prevalence under the age of 15 years is estimated to rise from 94,000 in 2005 to 160,000 in 202051; in Finland, the country with the highest incidence of TID, the number of new cases diagnosed at or before the age of 14 years will likely double in the next 15 years.52 The economic challenges associated with these developments are indeed staggering as shown by the health care-related expenses in the United States and provided by the American Autoimmune Related Diseases Association for Crohn disease ($10.9 to $15.5 billion in 2008), rheumatoid arthritis (RA; $19.3 billion in 2005), or psoriasis {$11.2 in 2010). Treatment costs are expected to increase even further due to the nature of some of the more succesful therapeutic modalities developed for chronic autoimmune conditions, for example, the introduction of antitumor necrosis factor (TNF) biologics. While clearly an important advance and of great benefit to patients, these drugs do not promote a cure for the underlying disease, and the need for continuous treatment will exacerbate associated health care costs. These epidemiologic studies also permit several additionaL if not entirely unexpected, conclusions. Many autoimmune conditions are clearly understudied, and some of the most frequently studied diseases exhibit comparatively low prevalence rates. The cause for the seeming imbalance between the public health burden posed by some autoimmune disorders and their attraction as objects for scientific study remains to be elucidated but will likely include the presence or absence of effective therapy. Pernicious anemia, the sixth most common autoimmune disease in the United States, can be effectively managed, and therefore elicits only limited epidemiologic interest. In contrast, some rare conditions may pose a pronounced burden to afflicted individuals and thus warrant continued efforts to develop more effective prophylactic and therapeutic interventions. Further, the availability of certain models for autoimmune diseases, again not necessarily a reflection of the epidemiologic importance of the corresponding human autoimmune disease, will have an impact on choices made by researchers charting their field of study. Additionally, as in other areas of research or clinical medicine, the funds and resources available are the result of multiple factors that may or may not include the public health burden exerted by a particular autoimmune disease. Balancing these aspects to appropriately appreciate

and address the burden of autoimmune diseases, based on both the afflicted individual and society at large, is a challenge that will require our continued efforts to identify, investigate, inform, and, hopefully, improve the therapies for many autoimmune diseases.

Spectra and Continua: Organ-Specific and Systemic Autoimmune Disorders. Auto inflammatory Diseases. and the Challenges of Taxonomy A perennial approach in our quest to make sense of the complex phenomena we encounter is the establishment of dichotomies, however, fraught with shortcomings, inconsistencies, and exceptions to the rule. Steeped in clinical traditions and immediately intelligible, the distinction between systemic and organ-specific autoimmune diseases is as useful as it is inadequate. Given that our evolving understanding of autoimmune diseases requires a constant reevaluation of our concepts pertaining to etiopathogenesis and effective treatment modalities, it would be premature to abandon such a simple and still useful classification. Rather, that porous juncture between systemic and organ-specific disorders may reveal hitherto unappreciated aspects of pathogenesis. On the surface, the patterns of pathology result from the distribution of anatomic niches that provide a suitable environment to "interface" antigens and immune effectors. Leaving for the moment aside the difficulties pertaining to the identification of initiating autoantigens in many human autoimmune diseases and the challenging task to correlate markers of immunologic activity (eg, autoantibodies) with cause or consequence of tissue destruction, a particularly puzzling phenomenon is the seeming organ specificity of some disorders in the face of autoimmune responses that target ubiquitous antigens. For instance, the ribonucleoprotein antigens implicated in Sj()gren syndrome or the transfer ribonucleic acid (RNA) synthetases targeted in polymyositis are widely expressed intracellular antigens, yet the pathology of these diseases is relatively circumscribed. Another intriguing example is the K/BxN arthritis model in which pathogenic antibodies recognize the ubiquitous cytoplasmic enzyme glucose-6-phosphate isomerase. Here, the preferential involvement of the joints apparently results from unique properties of the regional vasculature that allow for an antibody-mediated increase of vasopermeability and amplification of pathology by extracellular glucose-6-phosphate isomerase deposition in the articular cavities.53•54 The observation that autoimmune damage is critically dependent on aspects of the local microanatomy emphasizes the importance to consider autoimmune processes in the larger context of interdependent organ systems. In addition, an examination of some animal models used for the study of particular organ-specific autoimmune disorders further challenges the simple notion of restricted pathology and may provide clues about etiologic commonalities of ostensibly disparate clinical autoimmune syndromes. The nonobese diabetic (NOD) mouse is the most widely used animal model for the study of TID, a severe condition caused by autoimmune destruction of

CHAPTER 44

the insulin-producing p cells in the pancreas.55•56 However, NOD mice also exhibit aspects of type 2 diabete~7 and are prone to autoimmune sialitis, thyroiditis, peripheral neuropathy, prostatitis, a lupus erythematosus-like syndrome that develops after exposure to killed mycobacteria, as well as, under certain circumstances, exocrine pancreatitis.56 Similar to the etiology of TlD, specific T cells are involved in the pathogenesis of all these disorders, although antigenic targets and requirements for costimulatory interactions are distinct. Thus, as in human TID, the NOD mouse combines a generalized genetic susceptibility to multiorgan autoimmunity that is focused on pancreatic p cells but not limited to endocrine organs. While the preceding considerations argue against a rigorous opposition of systemic versus organ-specific autoimmune diseases and rather support the notion of a spectrum of clinicopathologic features that variously emphasize prominent organ-specific and systemic features, even the conceptual distinction of autoimmune disorders at large is suffused with certain limitations {eg, absence of MHCs or autoantibody associations with some diseases tentatively labeled "autoimmune") that have prompted new taxonomic proposals for immunologic diseases in general. Perhaps most prominently, McGonagle and McDermott introduced the concept of autoinflammation defined as "self-directed tissue inflammation, where local factors at disease-prone sites determine the activation of the innate immune system."58 Accordingly, a continuum of immunologic diseases is demarcated by rare monogenic disorders that are purely autoimmune and demonstrate preferential involvement of adaptive immune responses (eg, autoimmune polyendocrine syndrome-1 resulting from mutations of the AIRE gene), and purely autoinflammatory conditions defined by mutations in cells or molecules of the innate immune system and localized pathologies that escape the traditional purview of "classical" autoimmune mechanisms {eg, TNF receptor-associated periodic syndrome). Between these boundaries, the vast majority of other immunologic disorders can be organized along a continuum of pathologies that range from classic polygenic autoimmune diseases to mixed pattern diseases and to polygenic autoinflammatory diseases.58 The major appeal of this expanded notion of autoimmunity lies in the recognition of innate immune mechanisms as an important component of autoimmune disorders (with inevitable echoes ofthe Ehrlich-Metchnikoff rivalry informing the current discussions) and in the provision of an inclusive classification of immunologic diseases.

Central and Peripheral Tolerance: Implementing an Operational Concept A detailed historical discussion of the concept of tolerance is beyond the scope of this chapter, but some aspects of the usage of the term tolerance require clarification at the outset. Tolerance in adaptive immunity, sensu stricto, is the absence of specific lymphocyte activity, the consequence of physical deletion, or functional silencing of specific T and B cells. Some researchers refer to these tolerance mechanisms

AUTOIMMUNITY AND AUTOIMMUNE DISEASES

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as "passive" or "recessive" tolerance to explicitly distinguish them from "active" or "dominant" tolerance. While the latter mechanisms constitute bona fide immune responses (therefore, other researchers do not categorize them as a mode of tolerance), their particular nature results in a phenotype that is comparable to that achieved by means of passive/recessive tolerance. Distinct effector mechanisms {eg, immunosuppressive cytokines) and possibly dedicated classes of immune cells (eg, "regulatory T" [T...,8] cells) assure that local or systemic autoimmunity is avoided. The concept of T-cell suppressors, first proposed in the early 1970s by Gershon and Kondo, 59 was resurrected in the form of "professional" duster of differentiation {CD)25+FoxP3+CD4+ and "adaptive" Tre8 s cells (TRl and other Tregs) and has since attracted considerable attention. However, while there is indeed a CD25+ lineage of T cells committed to regulatory activity in naive, nonimmunized mice, we wish to underscore that regulatory functions, including those that limit autoimmunity, are a feature of the immune system as a whole and can be exercised by other classes of immune cells as well (eg, CDS+ T cells, ~T-cell receptor (TCR) T cells, natural killer T cells, etc.). Thus, while CD25+CD4+ Tre8 cells occupy a distinct and important niche in the complex dynamic network of immune functions, not all T-cell regulators are CD25+CD4+ nor do all CD25+CD4+ T cells function as suppressors. Indeed, novel markers might characterize regulatory function better; among these are expression of the transcription factor FoxP360- 66 and secretion of cytokines with regulatory function such as interleukin (IL)-1067,68 or transforming growth factor (TGF) -p.69 The multiplicity of current efforts to understand the nature of CD25+CD4+ T,..8 cells has been expertly reviewed elsewhere/o,71 and we note in particular more recent observations on the transcriptional, phenotypic, and functional instability of Treg population.72' 73 This has important repercussions in the context of autoimmunity, as Tre8 s can lose their regulatory capacities when exposed to local conditions of autoimmune inflammation. On the one hand, these results suggest that aberrant conversion from regulatory to effector phenotype may partly underlie progression to overt autoimmune disease in susceptible individuals. On the other hand, such propensity may jeopardize the long-term efficacy of therapeutic strategies based on the induction or adoptive transfer of autoantigen-specific Trell' as these cells could potentially convert to effectors once they arrive in the inflamed tissue. Therefore, these recent insights dearly warrant caution regarding the popular endeavor of assigning T cells to defined subpopulations based on their "signature" transcription factors and functionalities and emphasize the importance of plasticity and mutability the mechanistic foundation and relevance of which remains to be explored in further detail. Lastly, we emphasize that changes in Treg functionality rather than a mere numerical decrease appear to constitute a correlate for some human autoimmune diseases.74 Thus, the simple quantification of peripheral Tu:g numbers as a measure of risk in autoimmunity-prone indviduals or as a biomarker in clinical studies will have to be complemented with functional assays to generate relevant data.

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T-Cell Tolerance Autoreactivity, by definition, designates a specific immune response to self-antigens. Antigen nonspecific responses such as inflammatory and innate immune processes should not be considered autoimmune in the strict sense, although they may accompany, enhance, or even trigger autoimmune processes proper. Thus, antigen-specific T-or B-cell immunity will have to underlie a genuine autoimmune disorder. Furthermore, for organ-specific autoimmune diseases, antigen specificity of primary effector lymphocytes must be largely restricted to autoantigens derived from defined organs or tissues. Once initiated, specific responses that precipitate or "drive" the localized autoimmune reaction may diversify to comprise additional specificities (determinant spreading) and pathogenic mechanisms. How does the adaptive immune system restrict generation and activation of autoreactive lymphocytes? The central process by which the generation of TCR diversity is limited is called thymic selection. Thymic selection is a developmental process that selects T cells with a biased repertoire for export into the periphery?5- 79 T cells that interact at least weakly with self-peptides presented in the context of MHC molecules are chosen in the course of positive selection,80'81 while those that do not effectively interact with MHC/peptide complexes die "by neglect." However, interactions above a certain avidity threshold result in elimination by negative selection and constitute the basis for "central tolerance."75' 79 Thus, central tolerance prevents widespread autoimmunity as a function of lymphocyte/antigen-complex avidity and preferentially selects T cells with specificity for antigens not expressed in thymic epithelium for export into the periphery. However, central tolerance is not a complete mechanism, and a sizable pool ofT cells with intermediate avidity can escape negative selection and constitutes most autoreactive T cells found in the peripheral immune system. The presence of these autoreactive T cells can be considered physiologic, and they are not noxious for two reasons: either they are usually not activated and exhibit a "naive" phenotype or they exhibit regulatory effector functions and act as adaptive TregS following activation. Thus, not all self-reactive lymphocytes need to necessarily exhibit an aggressive phenotype. Depending on their specific effector functions, autoreactive T cells may exhibit regulatory functions and may critically modulate or even abort local autoimmune processes. Such autoreactive regulators might occur physiologically and constitute most autoimmune responses present in healthy individuals. Only an encounter under appropriate stimulatory conditions (ie, presentation of autoantigen-derived peptides presented in the context ofMHC class I or II molecules accompanied by antigen-nonspecific costimulatory interactions and strong inflammatory signals) can lead to their full activation and detrimental effector functions in the periphery. As such "armed effectors," autoreactive T cells are now potentially very dangerous and may initiate specific autoimmunity, if they recognize the autoantigens or closely related proteins in a defined tissue. It is thought that a few autoaggressive "driver clones" with highly detrimental effector function

can sustain a localized autoimmune process. High receptor/MHC/self-peptide avidity likely, but not necessarily, predisposes to this phenotype.82- 84 The presence of autoreactive T cells in the periphery might suggest that detrimental autoimmunity should occur quite frequently if organ-specific autoantigens are not expressed in the thymus, or alternatively or in addition, such physiologically occurring autoimmunity is not of a regulatory nature. Yet, there are several additional mechanisms that maintain tolerance in the periphery. "Peripheral tolerance" involves a set of mechanisms that ensures that autoreactive T lymphocytes are not activated in the periphery. It should be noted that these mechanisms pertain to both autoreactive and "heteroreactive" T cells and involve the following pathways. First, it has been observed that naive T cells triggered by a strong signal through the TCR alone may lose the ability to proliferate, and some but not all effector functions become "anergic.'>as-87 Presence of certain cytokines or costimulatory interactions can avoid the induction of anergy or may reverse an anergic state. Second, highly activated T cells will eventually undergo activationinduced cell death (AICD).88 AICD is thought to be essential for the downmodulation of immune responses and the reestablishment of immune homeostasis. Impairment of AICD may lead to continued immune activation and generalized autoreactivity. For CD4 lymphocytes, AICD is FAS/FAS-L dependen~· 90 ; it is not clear which interactions precisely control AICD in CDS cells. Third, molecules that can deliver specific negative signals, such as the B7-binding cytotoxic T-lymphocyte antigen (CTLA)-4 are involved in "turning off" of antigen-specific T cells.91' 92 Finally, other factors such as regulatory lymphocytes and regulatory antigen-presenting cells (APCs) might play important roles in maintaining peripheral tolerance.59' 7o,93

B-Cell Tolerance Although they are not selected in the thymus, similar paradigms as those for T cells apply to autoreactive B lymphocytes. Clonal selection occurs after recognizing antigens, is avidity dependent, and allows the B cells to undergo further receptor editing. Both central (ie, bone marrow) as well as peripheral tolerance mechanisms are in place to avoid generating mature B cells with specificity for self-antigens. After B cells mature in the bone marrow, they clonally expand after recognizing antigens in the periphery. T-helper (Th) lymphocytes are needed for this process in response to most protein antigens, and these B-cell responses are therefore termed "thymus dependent" or, in other words, require T cells with specificity linked to an epitope on the antigen they are reacting with. Th cell-independent B-cell responses occur mostly to bacterial and lipid antigens, for example, to lipopolysaccharide, and are therefore rarely autoaggressive. Thus, T-cell tolerance directly controls B-cell reactivity to autoantigens. In general, systemic autoimmune disorders such as systemic lupus erythematosus (SLE) are B-cell dependent, and organ-specific diseases such as MS and TID are less dependent on autoantibodies, although

CtwnR 44 AUI'O!MMUNI1Y AND AUTOIMM~E DllllASES

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CHAPTER 44

a particularly strong association with early disease onset, relapse, and the eventual necessity of liver transplantation. In contrast, type 2 AIH is associated with carriage of HLA-DR7. Genes outside the HLA loci potentially conferring disease susceptibility include complement component C4, CTLA-4, vitamin D receptor, Fas receptor, and others.407,403,422 The association with hepatitis C, D, and E virus-induced hepatitis and autoimmunity remains very intriguing. However, at this point, the prevalence of hepatitis C virus, for example, exhibits drastic variations between studies performed in different countries, and autoantibody titers appear much higher in patients with hepatitis C virus-negative AIH. In a more recent study, it was found that sera of 38% of patients with chronic hepatitis C reacted specifically with CYP2D6, whereas none of the sera obtained from patients with chronic hepatitis B showed CYP2D6 reactivity.424 Furthermore, it was found that hepatitis C virus has the potential to induce autoreactive CDS+ T cells that crossreactively recognize the cytochrome P450 isoforms 2A6 and 2A7 that contain sequence homology to hepatitis C virus aa 178-187. In this context, it may be important to emphasize that molecular mimicry also seems to be an important factor in other immune-mediated diseases of the liver. Hence, trifluoroacetyl {TFA)-protein adducts, as generated during the metabolism of halothane by CYP2E1, confer molecular mimicry to the lipoic acid prosthetic group of the pyruvate dehydrogenase complex (PDC) and other members of the 2-ox.oacid dehydrogenase family, 32' 234' 295' 426 which in turn are major autoantigens in PBC. 115 Consequently, halothane hepatitis and PBC may be linked on the level of cross-reactive autoantibodies that recognize similar target antigens. It remains to be seen whether novel subtypes of hepatitis viruses can be found in patients with AIH and whether further studies will corroborate such an association. It is likely, however, that viruses play a mutifactorial role in AIH pathogenesis (similar to TID), and that no single virus will be identified as a specific cause for liver autoimmunity.

Animal Models. Over the past 30 years, many attempts have been made to develop animal models for AIH; however, to date, no reliable model is available that reproduces the spontaneously relapsing chronic disease course oftype 1 AIH. 427•423 In the hepatitis B virus surface antigen-transgenic mouse, the hepatitis B virus surface antigen is expressed in hepatocytes under control of the mouse albumin promoter. Induction of transient hepatitis is possible after adoptive transfer of activated T cells from hepatitis B virus surface antigen-primed donor mice.429.430 Although not designed to reproduce features of AIH, this model has been extraordinarily helpful in understanding the role of IFNs in inducing liver damage as well as dearing hepatitis B virus from the liver. Results have shown that IFNs, in the absence of cytotoxicity, can purge virus from infected hepatocytes. Furthermore, induction of hepatitis B virus-specific CD4+ and CDS+ T-cell responses can be evaluated in this model. The transgenic mice exhibit profound liver damage and infiltration after transfer of hepatitis B virus-specific T lymphocytes.

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In another model system, the MHC class I molecule H-2Kb is transgenically expressed in the liver of mice that also carry transgenic T cells specific for H-2Kb. Hepatitis induction was only successful when such mice were infected with a liver-specific pathogen, indicating that bystander activation within the liver microenvironment can be very potent in causing autoimmune damage.431 A similar approach was chosen in yet another model that combines liver-specific expression of a transgene (here, the immunodominant LCMV glycoprotein epitope [GP33]) and transgenic T cells specific for the transgene.432 These mice remain tolerant to the transgene even in the presence of transferred GP3rspecific T cells. However, under conditions of LCMV infection and TCR-transgenic T-cell transfer, these mice develop a transient form of hepatitis as evidenced by elevated alanine aminotransferase levels.432 Together, these findings emphasize the importance of both autoreactive T cells and generalized inflammatory alterations in the wake of microbial infection. The potential pathogenic contribution of autoantibodies was demonstrated in a new mouse model. Injection of a monoclonal antibody, derived from a patient with type 1 AIH and specific for an undefined 190 kDa cell surface protein, promotes predominantly perivenular hepatocellular necrosis in mice.433 However, the validity of this model remains to be critically evaluated as the pathology does not appear to closely resemble that of type 1 AIH. Arguably, more compelling models have been developed for the study of type 2 AIH. DNA immunization of mice with human CYPD6 and formiminotransferase cyclodeaminase antigens leads to the generation of LMK1 and LC1 autoantibodies (targeting CYPD6 and formiminotransferase cydodeaminase, respectively) and specific cytotoxic T cells, pronounced lymphocytic infiltration of the liver, and an elevation of alanine aminotransferase levels indicative of liver damage.434 Interestingly, work with this model has reproduced some aspects of the genetic predisposition exerted on disease development as C57BL/6 mice demonstrated more pronounced disease than 129/Sv mice, and no inflammation was found in BALB/c mice.435 Lastly, adenoviral infection with recombinant expressing the 2D6 antigen can lead to focal and confluent liver necrosis in mice resembling that of AIW36 (Fig. 44.12). In summary, the success of most mouse models has only been partial, as hepatitis was transient and induction of chronic disease appeared difficult to achieve. Figure 44.13 illustrates the possible pathogenetic mechanisms implicated in AIH.

lreatment and Prevention. Immunosuppression is the therapy of choice for AIH. Since their introduction in 1968,437 prednisolone and azathioprine have become standard treatment regimens.408' 438 The impressive success rate of this therapeutic approach (more than 85% of patients with AIH) may have, to a certain extent, removed the urgency to pursue basic AIH research.428 Immunosuppressive therapy also improves survival of patients with severe AIH, 439 but no guidelines are available for individuals with minimal symptoms. End-stage AIH is an important indication for liver transplantation.440 Recurrence of AIH has been reported after liver transplantation 441 but interestingly is not as frequent as observed for islet

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A

Focal necrosis with lnflltratfng CDS cells

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FIG. 44.12. Liver Necrosis in 2D6 Transgenic Mice Infected with an AdenDViral ReconiJinant Expressing 2D6. Courtesy of Urs Christen and Matthias von Herrath, University of Frankfurt, Germany, and La Jolla Institute for Allergy and Immunology, La Jolla, CA.

transplants in patients with TID. Therapeutic use of IFNs, effective for treatment of viral hepatitis, can worsen autoimmune liver disease424 and challenges the assumption that unknown chronic viral infections of the liver, while possible initiators, maintain the active disease process.

Primary Biliary Cinftosis Introduction and Disease Ducription. PBC is a slowly progressive hepatic autoimmune disorder that primarily affects women. The disease is typically associated with high titers of antimitochondrial antibodies (AMAs) that presage onset of clinical disease by 5 to 10 years; antinuclear antibodies (ANA) are found in about half of PBC patients. Histopathologic changes include portal inflammation and destruction of intrahepatic bile ducts that occur at various rates and degrees in different patients. The resulting d.eaease of bile secretion and retention of toxic substances within the liver leads to further hepatic damage, fibrosis, cirrhosis, and eventual liver failure.41o. 412

Autoimmune Features. Although speculation about the autoimmune pathogenesis of PBC centers around the usual

suspects of genetic and environmental/microbial factors, PBC may serve as an example how advanced knowledge about the targeted autoantigens can give rise to novel and intriguing hypotheses. The presence of AMA in PBC were first described in the 1950s, the autoantigens cloned in the 1980s and 1990s, and B cell, CD4+, and CDS+ T-cell epitopes of the major autoantisen PDC-E2 mapped in the 1990s and 2000s. More than 90% of patients with PBC have high titers of autoantibodies directed against the 2-oxoacid dehydrogenase complex E2 subunit, in particular the E2 component of the PDC. Mapping studies have identified determinants within the PDC-E2 lipoyl domains as a primary targets for antibody and T-cell reactivity, a motif that is also found in the E2 subunit of other 2-oxoacid dehydrogenase complex members (OGDC-E2, BCKD-E2; motif is also found in E3-BP).'111 Antibody reactivity to nuclear envelope proteins gp210 and p62 are found in 10% to 30% of patients and are apparently associated with more active disease. The obvious question emerging from the identification of these autoantigens pertains to the fact that they are 1) found in every cell type and 2) located on the inner surface of the inner mitochondrial membrane, thus separated

FIG. 44.11 Pathogenesis of Linr Autoimmunity aft.Virallnfaction in tile 2D6 Animal Modal. A:. Viral infection leads to expression of high cytokine levels in the liver. B: The infection will probably also activate resident macrophages (Kupffer cellst. Antiviral T cells react against virally infected cells and destroy them. This leads to release of autoamigens fom hepatocytes, which can be (cross~·presemed leading to pathogenic responses against autoamigens such as cytochrome P450 208. Chemokine and cytokine release (C) leads to enhanced inflammation and amigen presemation (D).

CHAPTER 44

from immune effectors by three membranes. In addition, the existence of a clinical entity known as AMA-negative PBC, a disease very similar to PBC but without detectable AMAs, appears to further argue against a pathogenic role of these autoantibodies.412 Yet, certain particularities of bile duct epithelial cells may predispose them as targets for humoral immune responses. These cells, just as salivary epithelial cells, express receptors involved in the transcytosis of IgA and high titers of PDC-E2-specific IgA are found in bile, saliva, and urine of patients with PBC. AMA IgA was shown to induce apoptosis, which in turn may lead to PDC accumulation in the cytoplasm and cell surface, as well as amplification of the immune response. Further speculation has focused on the possibility that failure of PDC glutathionylation specifically in epithelial cells may promote accumulation of immunogenic forms of PDC, a process that may be initiated by xenobiotic-modified PDC and molecular mimicry.411 In the "determinant density model,'>l42 potentially PDC-reactive T cells survive negative thymic selection due to low TCR avidity and remain "dormant" despite sporadic exposure to PDC-derived epitopes. However, strong inflammatory stimuli that allow for improved antigen presentation and activation of toll-like receptors {TLRs; monocytes from PBC patients exhibit enhanced sensitivity to TLR signals) {eg, through microbial molecular mimicry) may induce the breakdown of tolerance that precipitates the disease. It should be noted that the general validity of this hypothesis, including a key role for low-avidity T cells and TLRs, is fully supported by elegant studies in models for induction of autoimmune diabetes.114" 43 Finally, a model has been proposed in which a primary dysfunction of endothelial cells results in overproduction of endothelins, inflammatory alterations, and ischemic damage due to vasoconstriction and the generation of PDC-specific humoral and cellular responses as a secondary event.412 These hypotheses are not mutually exclusive and may be critically evaluated in two animal models.

Genetic Features and Environmental Factors. Familial clustering of PBC and a high concordance rate in monozygotic twins {63%) indicate a genetic component to PBC pathogenesis. A flurry of GWAS data has dramatically improved our insight into the genetics landscape of PBC. Before the implementation of GWAS technology, only class II HLA loci had been reproducibly shown to associate with disease. Non-HLA loci were reported on some occasions but replication often proved problematic. GWAS confirmed HLA as the strongest association and many additional non-HLA risk loci have been identified, with equivalent effect size to HLA, including IL-12A,444 IL-12RB2,444 STAT4,445 IRF5-TNP03,446 17ql2.21,446 MMEL1,'46 SPIB,'46 and CTLA-4.'44 As argued previously, the list of associated genes comprises many proteins with important roles in innate and adaptive immunity, and, thus, the existence of a monogenic pathway that leads to disease can be excluded. In correspondence with, for instance, TID and RA, CTLA-4 also confers risk for this condition and many of the candidate genes signal though NF-lCB, TLR, and TNF pathways, which are also important in many other autoimmune conditions. The future will tell whether

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this growing list of gene associations will be instumental in unraveling the immune pathology that underlies PBC.

Animal Models. The absence of a suitable animal model has long been a serious obstacle to PBC research. However, two publications document important progress on this front by describing the spontaneous onset of a PBC-like disease in two mouse strains. NOD.c3c4 mice congenically derived from the NOD strain generate PDC-E2-specific autoantibodies and exhibit biliary pathology characterized by destructive cholangitis, granuloma formation, and eosinophilic infiltration. Importantly, T cells are found in affected areas of the biliary epithelium, and transfer of splenocytes or CD4+ T cells induces disease in NOD.c3c4-scid recipients, suggesting a central role forT cells in pathogenesis.447 In the second model, abolition of TGF-~ signaling in T cells (achieved by T cellspecific expression of a dominant-negative TGF-J}RII) results in production of AMAs directed at PDC-E2, BCOADC-E2, and OGDC-E2 as well as lymphocytic liver infiltration with periportal inflammation analogous to the histologic profile in human PBC. These observations implicate the TGF-~ pathway in PBC pathogenesis and suggest that the activation of intrinsically self-reactive T cells is a consequence of impaired T,..8 cell function rather than molecular mimicry.448 Treatment and Prevention. It is noteworthy that despite its presumed autoimmune etiology, PBC is not ameliorated by immunosuppressive therapy. In the absence of optimized therapeutic protocols, treatment is tailored according to individual responsiveness of patients with PBC and includes management of disease symptoms {pruritus, osteoporosis, hyperlipidemia, portal hypertension) as well as therapy of the underlying disease by stepwise addition of ursodeoxycholic acid, colchicin, and methotrexate. Nevertheless, the use of colchicine and low-dose methotrexate, presumably acting as an immunomodulatory rather than antimetabolic agent, remains controversial. In cases of liver failure, orthotopic liver transplantation is the only effective treatment.410

Primary Sclerosing Cholangitis Introduction and Disease Description. PSC is a chronic cholestatic liver disease (cholestasis: suppression of biliary flow) frequently associated with inflammatory bowel disease {>75% of patients with PSC). It is characterized by fibrotic inflammation and destruction of large intrahepatic and extrahepatic bile ducts and may lead to the development of cholangiocarcinoma (in 10% to 30% of patients with PSC) and ultimately death by liver failure. Autoimmune Features. Although the etiology of PSC remains undefined, a complex multistep process has been delineated and involves cholangiocyte activation through bacterial pathogenassociated molecular patterns, production of proinflammatory cytokines in conjunction with aberrant chemokine expression and endothelial cell adhesion molecules, and the recruitment ofT cells presumably specific for enterocyte antigens and primed in gut-associated lymphoid tissues.414 The generation of autoantibodies is a common feature (see Table 44.2), but most antibodies are not specific for PSC and their contribution to disease pathogenesis remains at present unclear.422

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Genetic FeGtures und Environmental Fuctors. The relative risk of PSC among first-degree relatives of patients with PSC is almost 100-fold increased as compared to the general population, indicating that there is a strong genetic component to the condition.413 As in many other autoimmune conditions discussed here, GWAS have greatly contributed to our understanding of the array of genes that contribute to disease susceptibility. A first report revealed a similar genetic architecture as described in PBC, and it was confirmed that the association with HLA was even more pronounced.449 The authors also found one non-HLA association at chromosome 13q31 (locus also associated with MS), and the next assayed 15 established inflammatory bowel disease (which affects 50% to 80% of patients with PSC) susceptibility loci. They were able to identify two additional non-HLA susceptibility loci (chromosome 2q35 and chromosome 3p21) for which the G protein-coupled bile acid receptor 1 (TGR5) and macrophage-stimulating 1 (MSTl) were proposed as the likely disease genes. A subsequent study from the same group added two more non-HLA loci, 2q13 and 10p15, with presumable candidate genes BCL2-like 11 (BCL2Lll) and IL-2RA.450 Interestingly, IL2ra-/- mice spontaneously develop intestinal and biliary inflammation.451 Finally, three more inflammatory bowel disease susceptibility loci were found to be associated with PSC, encompassing the candidate genes REI., IL-2, and CARD9.452 Collectively, these genetic data offer strong, albeit still indirect, support for an important role for innate and adaptive immune components in PSC. Moreover, the shared genetic patterns between PSC and inflammatory bowel disease indicate a certain degree of common immunopathogenesis and once again suggest the potentially shared origin of what first seemed to be distinct autoimmune disorders. Animal Models. None of the animal models developed to date reproduce all pertinent aspects of human PSC.453 Rat models of small bowel bacterial overgrowth and administration of bacterial cell wall components have provided some support for an ethiopathogenetic role of pathogenassociated molecular patterns. Other models include injury of biliary epithelial or vascular endothelial cells (rodents, dog, monkey) and toxic, infectious, or intraluminal biliary injury (rodents) permitting the study of cytokines, chemokines, and inflammation in the development of diffuse bile duct sclerosis.453

'freutment und Prevention. There is currently no effective medical therapy available for PSC.454 Treatment with the hydrophilic bile acid ursodeoxycholic acid improves liver enzymes, but its effect on liver histology and prognosis remains inconclusive. Orthotopic liver transplant constitutes the only established long-term treatment for PSC with cumulative 5-year survival of about 70%.

TFA form of hepatic proteins. TFA proteins are generated during the oxidative metabolism of halothane (2-bromo2-chloro-1,1,1-trifluoroethane) and include cytochrome P450, protein disulfide isomerase, microsomal carboxylesterase, calreticulin, Erp72, GRP78 (BiP), and GRP94. Current evidence suggests that such TFA proteins arise in all individuals exposed to halothane. However, the vast majority of individuals appear to tolerate this covalent protein modification. The lack of immunologic responsiveness was suggested to occur due to tolerance induced through the presence of structures in the repertoire of self-determinants that immunochemically and structurally mimic TFA proteins very closely. In fact, lipoic acid, the prosthetic group of the constitutively expressed E2 subunits of members of the 2-oxoacid dehydrogenase complex family was demonstrated by immunochemical and molecular modeling analysis to perfectly mimic N6 -trifluoroacteyl-L-lysine, the major haptenic group of TFA proteins. Interestingly, a fraction of patients with halothane hepatitis exhibit irregularities in the hepatic expression levels of these cross-reactive proteins. Thus, molecular mimicry ofTFA lysine by lipoic acid, or the impairment thereof, can be considered a susceptibility factor of individuals for the development of halothane hepatitis.456 A small animal model for chemically induced liver diseases has been described.457

Perspective und Conclusion. The presence of autoantibodies in AIH, PBC, and PSC is indicative of autoimmune processes, but none of the antibodies described is liver specific and their contribution to pathogenesis remains unclear. While the complex role ofT cells is subject to current investigations, the same caveats of specificity and pathogenicity discussed previously apply for autoimmune liver diseases. 453 Although the success of immunosuppressive therapy, in fact a criterion for the diagnostic scoring system for AIH, 44•420 further supports autoimmune etiology for AIH, it is conceivable that a viral infection might initiate disease as a "hit-and-run" event. Following elimination of viral antigens, disease may be perpetuated by immune-mediated processes and preclude the identification of a particular virus as causative agent at the time of liver disease diagnosis. Again, the role of "foreign antigens" and "self-antigens" becomes blurred and their interactions with the immune system in terms of immunity or autoimmunity conceptually problematic. Future investigations should seek to improve animal models and focus on human autoantigens and the application of contemporary tools for identification and isolation of specific lymphocytes. On the basis of such developments, future in vivo tracking of auto aggressive lymphocytes with noninvasive methods should substantially improve our insight into disease pathogenesis.

Halothane Hepatitis

Renal Autoimmune Diseases

Halothane hepatitis is a severe, life-threatening form of hepatic damage that affects a small subset of individuals exposed to the anesthetic agent halothane455 and is thought to have an immunologic basis. Sera of afflicted individuals contain autoantibodies directed against the native and the

Glomerulonephritis ( GN) is the major cause of chronic renal disease and kidney failure and exhibits a wide spectrum of histopathologic alterations, disease severity, and clinical outcomes. In most cases, evidence for an immunomediated pathogenesis, including humoral and cellular responses, has

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CHAPTER 44

instrumental in delineating SLE pathophysiology (see full reviews in Kippel and Dieppe, 531 and Perry et al.538). Numerous genetically modified strains were found to exhibit some SLE-like features and are thus informative in the identification of gene function but are not commonly used to study SLE per se. One of the most widely adopted models is the New Zealand Black/White Fl mouse. This strain originates from a cross between the New Zealand Black and New Zealand White strains, of which only the former develops mild clinical signs of autoimmunity. The Fl hybrids, however, display a lupus-like phenotype including B-cell hyperactivity, development of ANA, and immune complex-mediated glomerulonephritis around 5 to 6 months of age. Mice typically suffer kidney failure and die around 10 to 12 months of age. As in humans, the disease develops more frequently in females and is hormone dependent. Extensive genetic analysis has been performed on related inbred strains that resulted from accidental backcrossing (New Zealand mixed strains) and led to the identification of multiple susceptibility loci. Several of these were also identified in human SLE subjects, including MHC class II alleles, Fey receptors, and components of the complement system. The MRL lpr/lpr strain is another spontaneous model that develops massive lymphadenopathy, high concentrations of autoantibodies such as ANAs, anti-dsDNA and anti-Sm, and renal disease. In this strain, both males and females are equally affected. The lpr mutation interferes with expression of the Fas receptor (CD95), an important mediator of apoptosis. As discussed previously, defective apoptosis may in part underlie SLE in humans, and it is known that aberrant Fas signaling can lead to the development of autoimmune lymphoproliferative syndrome, which shares many symptoms with SLE. SLE-like symptoms can be induced by the repeated injection of pristine, a compound found in mineral oil. These features include the development of ANAs, immune-complex deposition, and severe disease of the kidneys. This model can be reproduced in most mouse strains, although the extent of susceptibility varies. In strains such as the SJL/J mouse, females are more prone to disease development. Alternatively, some aspects of SLE can be mimicked by the experimental induction of graft-versus-host disease. Disease aspects consist of lymphoid hyperplasia, formation of ANAs, and development lupus nephritis leading to severe kidney dysfunction and mortality. Only certain strains are prone to induction, once again illustrating the requirement for a genetic contribution in addition to an environmental stimulus. Disease severity can be tailored by adjusting the number of allografted cells, and onset occurs from a known starting point relative to injection. ANA sare usually detectable only 2 weeks after injection, which makes it a suitable model for the more rapid assessment of pathogenic pathways and experimental therapies.539

Therapy The multiorgan nature of the condition and its course of relapses and remissions dictates that therapeutic management is in accordance with disease activity. In addition to diverse

AUTOIMMUNITY AND AUTOIMMUNE DISEASES

1111

clinical scoring systems, active disease often correlates with altered levels of immune markers such as increased antidsDNA and decreased serum complement. Nonsteroidal anti-inflammatory drugs are prescribed to treat musculoskeletal symptoms or SLE-associated fever. Acute symptoms are generally treated by some form of corticosteroid treatment, either topical in case of cutaneous flares or injected when major organ disease develops. Immunosuppressive agents such as azathioprine are used mostly when major organ disease threatens to considerably worsen the patient's condition. The antimalarial agent hydroxychloroquine is known to ameliorate disease by inhibiting the function of TLRs. It is thus dear that, in line with the disease's protean pattern, no disease-specific treatment options exist, and that the therapeutic goal often is to prevent where flares where possible (eg, avoid sun expo sure) and tackle them in a symptomatic fashion as they occur. Since the approval of an antimalarial and corticosteroids for treatment of SLE in 1955, no new agents have been introduced. After more than 50 years and many failed trials, the U.S. Food and Drug Administration finally approved a new drug for SLE. Belimumab (marketed as Benlysta® [GlaxoSmithKline, Brentford, UK]) is a fully human monodonal antibody that binds to soluble human B-lymphocyte stimulator and inhibits its biologic activity.540 B-lymphocyte stimulator was shown to be a key survival cytokine forB lymphocytes which, as argued previously, are a major pathogenic immune subset in SLE. In patients with SLE, B-lymphocyte stimulator concentrations reflect disease activity and correspond with anti-dsDNA antibody titers. 541' 542 It was found that the drug selectively decreases B-cell numbers and shortlived plasma cells as well as SLE-associated anti-dsDNA antibody titers. 543 In two phase III trials that had enrolled patients with serologically active disease, a modest clinical effect was demonstrated combined with a safety profile similar to that ofplacebo. 544 It was argued that both inclusion criteria (patients with SLE activity that actually stands to be improved) and optimized disease-assessment indices contributed significantly to the successful outcome of these studies.545 Initial studies with a humanized anti-CD22 monoclonal also had an encouraging initial outcome, further corroborating the idea of B cells as a suitable target cell in SLE. 546 The fact that not just any therapy that targets B cells has therapeutic potential was underscored by two recent phase III trials that failed to show efficacy of rituximab (anti-CD20) in SLE.547' 543 These recent advances, however, represent a glimmer of hope for patients affected by this particularly heterogenic and pathophysiologically complex condition.

CONCLUSION A common trait among diverse animal models for autoimmune diseases and the human conditions they aim to model appears to be the difficulty with which experimental autoimmunity is achieved. Breaking of tolerance to self-antigens requires in most instances strong inflammatory (eg, pertussis toxin and adjuvant in EAE or collagen-induced arthritis) or infectious (eg, RIP-LCMV model for TID or TMEV/ mouse hepatitis virus models for MS) stimuli. In addition,

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many animal models require a genetically susceptible background or have to rely on artificial autoantigen expression by means of transgenic technology. In contrast to the relatively easy detection of autoreactive lymphocytes in antigeninduced animal models, it has been a daunting endeavor in humans (as well as spontaneous disease models such as the NOD mouse) and data obtained frequently vary considerably between different patients as well as laboratories. What are the implications for our understanding of autoimmune diseases in humans given these challenges? First, it is unlikely that strong inflammatory stimuli can be provided under natural conditions in the absence of infectious disease. Thus, viral and bacterial infections remain prime candidates for causing secondary autoimmunity (see Miller TMEV model of APC-mediated determinant spreading to autoantigens). Infectious pathogens can provide the "danger signals"548a needed for propagation of extended inflammation leading to clinical disease. Nevertheless, proof of a causal relationship is exquisitely difficult (if not impossible), as traces of pathogens detected may have no relation to the underlying disease process or may be cleared from the system by the time of secondary clinical disease. In addition, disease is likely dependent on individual pathogen strains, making a very detailed immunologic profiling in prospective clinical trials necessary. It is possible that the introduction of new antivirals might unmask such an association in the future. Finally, the importance ofthe gut microbiome has attracted some attention in the context of autoimmune disease.549 This is not unexpected because the gut harbors the major part of our immune system, and immune tolerance can often be induced by passage of an antigen through the gastrointestinal system. We now have gained a more precise view on the vast diversity of commensals inside our bodies,550•551 and it appears that this composition affects the likelihood of a genetically susceptible subject to develop autoummunity.261 For instance, generation of the pathogenic Th17 subset described previously in many autoimmune conditions critically depends on the composition of microbiota.552 Second, a genetically susceptible background will likely be required to provide a "fertile field" for initiating a chronic inflammation involving autoantigens. This probably occurs via a multifaceted network of multiple susceptibility and protective genes, and it will be impossible to treat a respective disease just by analyzing the background genes involved. Last, autoreactive lymphocytes might predominantly be present in the affected organ or site and not in the peripheral blood, which makes their identification and characterization in humans rather difficult. A word of caution should be devoted to our interpretation of specific findings obtained in individual animal models. Animal models should serve to teach us paradigms of how a disease could develop kinetically in vivo. The precise parameters, targeted antigens, susceptibility genes, and effector molecules may be considerably different in humans. Thus, for example, if there is indication that insulin is a primary antigen in the NOD mouse, this may or may not have direct relevance to the human disease. Some evidence exists that leads us to believe that insulin, indeed, maybe

also a primary antigen in human autoimmune diabetes, but other antigens such as GAD might also play a role. The opportunities as well as limits of each animal model have to be delineated. Again, the NOD mouse appears to be prone toward multiorgan autoimmunity (induction of EAE,553 neuritis, 554 arthritis, and hepatitis have all been observed in NOD or NOD-congenic mouse strains) and exhibits, in addition to diabetes, thyroiditis, sialitis, and orchitis. Thus, diabetes in the NOD mouse is clearly different from typical human TID. Therefore, treatments capable of correcting the systemic immune dysregulation that predominates in the NOD model may not directly apply to human TID where no such pronounced systemic dysregulation is present. Based on these considerations, it is not surprising that out of the more than 140 therapeutic strategies that prevent diabetes in the NOD mouse, only a handful have made it to phase I human trials. An appealing solution that has shown promise in TID epitope discovery is the development of humanized NOD mice expressing HLA class I transgenic molecules for antigen presentation. Remarkably, several of the islet epitopes that become targeted under these conditions correspond to the disease-associated specificities found in peripheral blood mononuclear cells from patients with TID.555 When those epitopes are administrated in a tolerogenic fashion, the disease course can be significantly altered.556 While still animal data in principal, these results may have higher translational value when tolerogenic strategies are to be envisioned for humans. Experiments with human cells or materials should be undertaken in order to solidify the choice of molecules or target antigens. The successful story in respect to blockade of TNF-a to treat RA underlines the importance of this step. Employment of a multiplicity of models thus becomes imperative to evaluate potential candidate interventions, as does a careful proceeding, objective evaluation, and avoidance of premature conclusions. In addition to the researchers and clinicians, publishers as well as news media will have to share this responsibility. Continued research will undoubtedly provide us eventually with sufficient insight into the complexities of autoimmunity and autoimmune disorders, but patience and perseverance coupled with experimental objectivity will be required. Therapeutic progress remains variable using monoclonal antibodies to dampen autoimmune processes in humans. Succesful examples include anti-TNF, CTLA-4Ig, anti-CD20, and anti-IL-l treatments, while a highly anticipated non-Fc binding anti-CD3 antibody recently failed to clear phase III trials in recent-onset TID.557' 558 As discussed previously, many of the successful therapies have also shown efficacy in a diverse array of other autoimmune diseases such as in psoriasis, Crohn disease, RA, MS, and uveitis. Thus, the road map to therapeutic success might lie in a well-tuned combination of such therapeutics, likely in conjunction with autoantigenspecific approaches that mediate more long-term and lasting tolerance. Augmentation of adaptive as well as intrinsic Trees might be, in the end, the key for achieving site-specific tolerance (involving bystander suppression of effector responses and "infectious" induction of more Troes) and is being explored with encouraging developments in many preclinical models.

CHAPTER 44 REFERENCES

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1174. Blanche G, Gianello PR, Lorf T, Setal. Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine. Transplantation. 1997;63:26-33. http://www.ncbi.nlm.nih.gov/pubmed/9000656 1175. Yamada K, Gianello PR, Ierino Fl., et al. Influence of the thymus on tillll5plantation tolerance in miniature swine. Transplant Proc. 1997; 29:1076. http://www.ncbi.nlm.nih.gov/pubmed/9123206 1176. Yamada K, Gianello PR, Iereno FL, et al. Role of the thymus in transplantation tolerance in miniature swine: I. Requirement of the thymus fur rapid and stable induction of tolerance to cla!;s !-mismatched renal allografts. JExp Med. 1997;186:497-506. http://www.ncbi.nlm.nih.gov/pubmed/925464S 1177. Yamada K, Gianello PR, Ierono Fl., et al. Role of the thymw; in transplantation tolerance in miniature swine. II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts. Transplantation. 1997;67:45S-467. http://www.ncbi.nlm.nih.gov/pubmed/10030295 117S. Ierino FL, Yamada K, Hatch T, Rembert J, Sachs DH. Peripheral tolerance to class I mismatched renal allografts in miniature swine: donor antigen-activated peripheral blood lymphocytes from tolerant swine inhibit anti donor CI1. reactivity. JImmunal. 1999;162:550-559. http://www.ncbi.nlm.nih.gov/pubmed/9S86432 1179. Mezrich JD, Kesselheim JA, Johnston DR, Yamada K, Sachs DH, Madsen JC. The role of regulatory cells in miniature swine rendered tolerant to cardiac allografts by donor kidney cotransplantation. Am J Transplant. 2003;3:1107-1115. http://www.ncbi.nlm.nih.gov/pubmed/12919090 11SO. Wu A, Yamada K, Ierino FL, Vagefi PA, Sachs DH. Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine. TransplimmiDiol. 2003;11:367-374. http://www.ncbi.nlm.nih.gov/pubmed/129677S9 11S1. Oluwole SF, Jin MX, Chowdhury NC. Ohajewkwe OA. Effectiveness of intrathymic inoculation of soluble antigens in the induction of specific unresponsiveness to rat islet allografts without transient redpient immunosuppression. Transplantation. 1994;5S: 1077-10Sl. http://www.ncbi.nlm.nih.gov/pubmed/7974714 11S2. Sayegh MH, Perico N, Gallon L, et al. Mechanisms of acquired thymic unresponsiveness to renal allografts. Thymic recognition of immunedominant allo-MHC peptides induces peripheral T cell anergy. Transplantation. 1994;5S: 125-132. http://www.ncbi.nlm.nih.gov/pubmed/S042230 11S3. Odorico JS, O'Connor T, Campos L, Barker CF, Posselt AM, Naji A. Examination of the mechanisms responsible for tolerance indudion after intrathymic inoculation of allogeneic bone marrow. Ann Surg. 1993;21S:525-531. http://www.ncbi.nlm.nih.gov/pubmed/S215643 11S4. Agus DB, Surh CD, Sprent J. Reentry of T ceUs to the adult thymus is restricted to activated T cells. J Exp Med. 1991 ;173: 1039-1046. http://www.ncbi.nlm.nih.gov/pubmed/202291S 11S5. Ali A, Garrovillo M, Oluwole 00, et al. Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo allomhc peptide activated syngeneic T cells. Transplantation. 2001;71:1442-144S. http://www.ncbi.nlm.nih.gov/pubmed/11391233 11S6. Gopinathan R, DePaz HA, Oluwole 00, et al. Role of reentry of in vivo alloMHC peptide-activated T cells into the adult thymus in acquired systemic tolerance. Transplantation. 2001;72:1533-1541. http://www.ncbi.nlm.nih.gov/pubmed/11707742 11S7. Bonasio R, Scimone ML, Scha£rli P, et al. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat ImmiDiol. 2006;7:1092-1100. http://www.ncbi.nlm.nih.gov/pubmed/169516S7 11SS. Hadeiba H, Lahl K, Edalati A, et al. Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promore central tolerance. Immunity. 2012;36:43S-450. http://www.ncbi.nlm.nih.gov/pubmed/22444632 11S9. Pearce Nw, Spinelli A, Gurley KE, Hall BM Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. V: Dependence ofCD4+ suppressor cells on the presence of alloantigen and cytokines, including inrerleukin-2. 7'ransplantatian. 1993;55:374-379. http://www.ncbi.nlm.nih.gov/pubmed/S434390

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1206. Spitzer TR, MCafee S, Dey BR, et aL Non-myeloablative haploidentical stem cell transplantation using anti-CD2 monoclonal antibody (MEDI507)-based conditioning for refractory hematologic malignancies. Transplantation. 2003;75:1748-1751. http://www.ncbi.nlm.nih.gov/pubmed/12777868 1207. Locascio SA, Morokata T, Chittenden M, et al. Mixed chimerism, lymphocyte recovery, and evidence for early donor-specific unresponsiveness in patients receiving combined kidney and bone marrow transplantation to induce tolerance. Transplantation. 2010;90:1607-1615. http://www.ncbi.nlm.nih.gov/pubmed/2 1085064 1208. Kawai T, Cosimi AB, Spitzer TR. et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl I Med. 2008;358:353-361. http://www.ncbi.nlm.nih.gov/pubmed/18216355 1209. Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation. Sci Trans! Med. 2012;4:124ra28. http://www.ncbi.nlm.nih.gov/pubmed/22399264 1210. Li HW. Sykes M. Emerging concepts in haematopoietic cell transplantation. Nat Rev Immunol. 2012;12:403-416. http://www.ncbi.nlm.nih.gov/pubmed/22627859 1211. Strober S, Spitzer TR, Lowsky R, Sykes M. TI-anslational studies in hematopoietic cell transplantation: treatment of hematologic malignancies as a stepping stone to tolerance induction. Semin Immunol. 2011; 23:273-281. http://www.ncbi.nlm.nih.gov/pubmed/2 1705229 1212. U.S. Department of Health & Human Services. The Organ Pro curement and TI-ansplantation Network. 2007. http://optn.transplant.hrsa. gov. Accessed September 4, 2012. 1213. Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses. Am I Transplant. 2006;6:2121-2 133. http://www.ncbi.nlm.nih.gov/pubmed/16796719 1214. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance and chimerism after renal and hematopoietic-cell transplantation. N Eng! I Med. 2008;358:362-368. http://www.ncbi.nlm.nih.gov/pubmed/18216356 1215. Montgomery RA, Lonze BE, King KE, et aL Desensitization in HLAincompatible kidney recipients and survivaL N Engl I Med. 2011;365: 318-326. http://www.ncbi.nlm.nih.gov/pubmed/2 1793744 1216. Wong BS, Yamada K, Okumi M, et al. Allosensitization does not increase the risk of xenoreactivity to alpha1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists. Transplantation. 2006;82:314-319. http://www.ncbi.nlm.nih.gov/pubmed/16906027 1217. Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. The natural history of chronic allograft nephropathy. N Eng! I Med. 2003;349:2326-233 3. http://www.ncbi.nlm.nih.gov/pubmed/14668458 1218. Bishara A, Brautbar C. Eid A, Scherman L, Ilan Y, Safadi R.ls presensitiza.tion relevant to liver transplantation outcome? Hum ImmunoL 2002;63:742-750. http://www.ncbi.nlm.nih.gov/pubmed/12175728 1219. Goggins WC. Fisher RA, Kimball PM, et aL The impact of a positive crossmatch upon outcome after liver transplantation. Thlnsplantation. 1996;62:1794-1798. http://www.ncbi.nlm.nih.gov/pubmed/8990365 1220. Hathaway M, Gunson BK, Keogh AC, Briggs D, McMaster P, Neuberger JM. A positive crossmatch in liver transplantation-no effect or inappropriate analysis? A prospective study. Transplantation. 1997;64:54-59. http://www.ncbi.nlm.nih.gov/pubmed/923370 1 1221. Navarro v; Herrine S, Katopes C, Colombe B, Spain CV:. The effect of HLA class I (A and B) and class II (DR) compatibility on liver transplantation outcomes: an analysis of the OPTN database. Liver TranspL 2006; 12:652-658. http://www.ncbi.nlm.nih.gov/pubmed/16555339 1222. Taylor AL, Gibbs P, Bradley JA. Acute graft versus host disease following liver transplantation: the enemy within. Am I Transplant. 2004;4:466-474. http://www.ncbi.nlm.nih.gov/pubmed/15023138 1223. Snyder LD, Palmer SM. Immune mechanisms of lung allograft rejection. Semin RespiT Crit Care Med. 2006;27:534-543. http://www.ncbi.nlm.nih.gov/pubmed/17072801

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1277. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC. Perreault C. Adoptive transfer of minor histocompatibility antigen-specific T lymphocyres eradicates leukemia cells without causing graft-versus-host disease. Nat Med. 2001;7:789-794. http://www.ncbi.nlm.nih.gov/pubmed/11433342 1278. Anderson BE, McNiffJ, Yan J, et al. Memory GD4+ T cells do not induce graft-versus-host disease. I Clin Invest. 2003;112:101-108. http://www.ncbi.nlm.nih.gov/pubmed/12840064 1279. Zheng H, Matte-Martone C. Li H, et al. Effector memory GD4+ T cells mediate graft-versus-leukemia without inducing graft-versus-host disease. Blood. 2008;111 :2476-2484. http://www.ncbi.nlm.nih.gov/pubmed/18045967 1280. Caine RY. Organ transplantation between widely disparate species. Transplant Proc. 1970;2:550-556. http://www.ncbi.nlm.nih.gov/pubmed/5000236 1281. Sachs DH. The pig as a potential xenograft donor. V-

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Function Lack of tumor stroma drastically reduces tumorigenicity.47•w8•109 All cancers depend on stromal support and establish some type of paracrine loop.43- 45'52'11o-m Signals initiating the loop seem to be intrinsic to the cancer cells (ie, endogenous) and depend on oncogenic mutations in the cancer cells11._117 (see Cancer and Inflammation). The proinflammatory mediators attract mesenchymal, endothelial, myeloid, and lymphoid progenitors to the stroma from adjacent and systemic reservoirs; the mediators also induce these cells to make factors that stimulate the growth of the cancer cells.43•4s. 118-125 Cytokines released by transfected cancer cells can have powerful local effects on all components of tumor stroma including fibroblasts. 73•126- 143 Angiogenesis is a fundamental necessity for tumors to grow by allowing oxygenation and nutrients to diffuse from

the blood into the mass. Myeloid cells, including mast cells, neutrophils, eosinophils, Gr-1 +CD11b+ MDSC/monocytes, and tumor-associated macrophages, can promote tumor angiogenesis.144 However, cancer cells and tumor-associated fibroblasts can produce proangiogenic as well as growth stimulatory factors such as vascular endothelial growth factor (VEGF) and Bv8.145 Several studies have shown the importance of neutrophils and granulocyte-colony-stimulating factor (G-CSF) production by cancer cells in causing refractoriness to anti-VEGF therapy. 43- 45'146-149 Metalloprotease released from neutrophils and from Gr-1+CD1lb+ MDSC/ monocytes catalyzes the release of preexistent VEGF and transforming growth factor (TGF) -P from the ECM and activates latent TGF-P.150-153 Neutrophils are essential for mobilizing various types of stromal progenitor cells including the macrophages from bone marrow and other reservoirs in the body.154 Finally, neutrophils and Gr-1+ CD11b+ monocytes in the tumor stroma themselves can produce large amounts ofTGF-PL69' 80 ECM is an essential stromal component providing the cancer cells not only with a scaffold for adherence and structure but also with growth and antiapoptotic signals, thereby preventing anoikis.155- 160 TGF-P1 stimulates fibroblasts to produce ECM proteins, including collagen, fibronectin, and proteoglycans, and TGF-P1 prevents ECM degradation.161 Accordingly, transfecting cancer cells to produce TGF-P1 makes them more aggressive.128 ECM is also a major reservoir for binding and releasing growth factors, chemokines, and cytokines.162' 163 Cancer cells may release the ECM proteoglycan versican that helps attract and activate myeloid cells via toll-like receptors (TLRs) to release interleukin (IL) -6.164 At later stages, neoplasms often replace paracrine with autocrine loops, notably IL-6 activating signal transducer and activator of transcription (STAT)3. 123•165•166 However, all evidence suggests that even the most aggressive cancers still depend on some factors and ligands provided by tumor stroma. Knowing how essential stroma is for cancers to grow, it is somewhat surprising that cancer cells would not generate their own stroma. Indeed, epithelial cancer cells can form a "pseudo-stroma" by assuming a mesenchymal phenotype at the invading edges of the cancer.167'168 However, there is no evidence that this so-called epithelial-to-mesenchymal transition can replace the host-derived stroma (ie, the need of cancer cells to establish paracrine stimulatory loops with nonmalignant stroma169), Fusion of cancer cells with stromal cells, particularly macrophages, has been proposed as a major mechanism of cancer development and progression.170-175 However, we still lack conclusive experimental evidence supporting this attractive hypothesis formulated over a century ago.176'177

Reaction to Cancer Call Inoculation Careful studies showed decades ago that the many cancer cells that die on inoculation play a critical role in the establishment of the cancer. It was found that adding lethally irradiated cancer cells to an inoculum of viable cancer cells at a 100:1 or larger ratio can increase the take of a cancer cell inoculum by more than a 100-fold.178 The dead cancer

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cells have potent thromboplastic activity,179 and death of the cancer cells is required for this activity to form fibrin at the site ofinoculation.171 In addition, hypoxia induces VEGP-A andCXCL12 (stroma-derived factor [SDP]-1) in manytypes of cells, but many cancer cells produce VEGF even in normoxie conditions.'80 VEGF-A is a most potent inducer of vascular leakage of plasma proteins, including fibrinogen, that rapidly form a fibrin-fibronectin clot as a provisional tumor stroma.181 Fibrin deposited at the site of inoculation serves as primitive ECM for cancer cells to escape anoilds. The ECM then undergoes major remodeling during the first 2 weeks after inoculation. Remodeling the ECM microenvironment requires the activity of ECM-degrading enzymes such as matrix metalloproteinases.182 Thereby, these transplanted tumors acquire later the harder consistency typical for autochthonous tumors that evolve with stroma.

EXPERIMENTAL CANCER Key Principles Number of Cancer Calls Targeted A critical determinant for any cancer therapy is the number of the targeted cancer ce1ls that have proliferative potential ("cancer stem cells"). That number determines the likelihood of recurrence/relapse after most cancer cells have been destroyed by treatment. Even microscopic parts ofa cancer left behind by a surgeon often lead to recurrence. Nevertheless, the size of tumor-stem cell population is not the only factor determining the likelihood of relapse as other cancer cells, even when dead or lethally irradiated, can inaease the tumorigenicity of the remaining cancer cells by orders of magnitude.113 Most human tumors are not detected until they are 0.5 to 1 em in diameter, have a volume of -500 mm', and contain -1011 cancer ceUs.13 In leukemia, malignant cells generally do not form tumors, but the patient has also -109 cancer cells when the disease is clinically detected. It is not appropriate to adjust for the difference in host size when comparing cancer in man and mice. Not the size of the species but the size of the cancer cell population determines the chance of relapse because the latter correlates with the number of therapy-resistant variants causing relapse after therapy. Skipper, who pioneered combination chemotherapy ofchildhood leukemia in the mouse model of L1210 leukemia, targeted 109 cancer stem cells as the starting population. This was one major reason why the principles he established in an animal model were clinically relevant and led to the cure of most childhood leukemias. Cettular heterogeneity within a tumor becomes much more relevant when the tumor accumulates 1 billion (109) cancer cells, equivalent to a tumor with a diameter of 1 cm.18'-'&5 At this point, the acc.umulation of so many cancer cell variants makes it improbable that all cancer cells are susceptible to a single chemotherapeutic agent or specific T cell. Cancer cell variants can be considered analogous to drug-resistant bacteria or viruses (ie, the nature of the problem is fundamentally the same in both cases). Unfortunately, in most experimental studies on immunotherapy, almost fivefold smaller populations of cancer cells are being treated' (Fig. 47.1).

CANCER IMMUNOLOGY

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Duration of Growth Duration of growth of a cancer greatly influences experimental results. Summarizing three decades of studies on immunity to cancer, Woglom concluded in 1929 that immunotherapy is futile against an established tumor, and "nothing may accordingly be hoped for at present in respect to a successful therapy from this direction."186 Eighty-two years later, the outlook is not quite so grim, but unfortunately, the vast majority of research in animal models is still concentrating at treating malignant cell populations grown for an average of only S days after cancer cell inoculation' {see Fig. 47.1). The word "established" is not a scientific term, yet is frequently used to describe neoplastic lesions caused by recently implanted cancer cells,186 conveying the message that the malignancy being treated does not differ from what would be found in a human cancer patient. Just the opposite is true; transplanted tumors must grow for at least 2 weeks before they are histologically indistinguishable from autochthonous murine or human cancers.107 Most human cancers have resided in the patient for months if not years before being detected and treated, whether primary, metastatic, dormant, or relapsing. An additional problem of experimental models is that growth of many serially transplanted "standard" cancer lines is so rapid that death may occur so early that treatment has to be started before solid tumors have established a microenvironment even vaguely comparable to that of an autochthonous tumor. Measuring Growth and Destruction Thmors are masses and have weight and volumes best approximated by the formula of an eUipsoid (V = 1tlwh/6 or V =0.5236lwh or V- +2 lwh); length I, width w, and height h are the orthogonal diameters in the three perpendicular

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axes. As was carefully documented, the third dimension, height (or depth), has an inordinately large effec:t on errors in volume187 but c:an usually be determined ac:c:urately for subcutaneous tumors.187- 189 Describing tumors as areas is inc:orrec:t and illogical despite widely prac:tic:ed misuse. By definition, areas have no volume nor contain a single c:anc:er c:ell. Area measurements as commonly used by radiologists frequently disagree with volumetric: measurements.187•188•190 Depth of a tumor c:an greatly vary and determines malignancy and prognosis (eg, in melanoma). There are important variables when extrapolating the number of c:anc:er c:ells from a given tumor volume; for example, the human or murine adenocarcinoma of the pancreas usually consists predominantly (-90%) of nonmalignant stromal c:ells yet is highly aggressive, whereas in most tumors stromal and c:anc:er c:ells are more balanced in numbers. Furthermore, while it takes weeks for a tumor to disappear completely even after all c:anc:er c:ells have been eradicated, it is usually impossible to determine from volume measurements how many viable c:anc:er c:ells are left. Certain c:anc:ers release hormones that have systemic: effects such as insulin; even mic:rosc:opic: growth c:an have major systemic: effec:ts.191 While many experimental models utilize subcutaneous tumors, some c:anc:ers, particularly human tumor xenografts, may require transplantation to sites where it is more difficult to assess growth objec:tively.192 Canc:er c:ells have been transduced with ludferase to give a signal proportional to the tumor size when substrate is injected; this allows whole body sc:ans with a luminometer, though sensitivity decreases for fewer than 1,000 c:anc:er c:ells, and there is absorption by overlying tissues.193

Cure and Dormancy The effects of the immune system on c:anc:er c:an be read out in different ways. Certainly, cure ofthe c:anc:er is the obj ec:tive. There should be dear prolongation of survival and absence of relapse, but proving that all c:anc:er c:ells have been eradicated is often difficult. Unfortunately, for most c:anc:ers, we lac:k assays sensitive enough to detec:t and quantify remaining dormant c:anc:er c:ells. This information would indicate whether or not a patient requires further treatment. Only in a few types of c:anc:ers194- 196 does a negative diagnostic: polymerase chain reaction analysis of the tissue reservoir from where a c:anc:er may relapse suggest-though not provecomplete eradication. Experimental evidence for dormant c:anc:er c:ells may c:ome from provoking relapse by treating the animal with antibodies neutralizing factors or c:ells suspected of causing tumor dormancy. Experimentation should indude waiting for minimally 30 days after the tumor has disappeared completely (it is best to wait several months or longer). A c:ommon unac:c:eptable prac:tic:e is using the word eradication or c:ure when there is no follow-up after the c:anc:er becomes undetectable. Relapse of c:anc:ers may oc:c:ur within days, weeks, months, or even years after complete disappearance, and is one of the most important problems of c:anc:er therapy. Nevertheless, experimentalists commonly describe treatments as effective even when followed by rapid relapse.197 Eradication means tearing a tree out with its roots so it cannot regrow, and the term is synonymous with c:ure.

Thus, using the term eradication is only appropriate if the host does not harbor dormant cancer cells, comparable to "sterilizing immunity" in infections. 19 ~201

Inhibition, Arrest, Regression, and Equilibrium Dependent on the extent and type of destruction as well as when it occurred, tumors may be found to have smaller or larger volumes after treatment compared to controls. It is important to determine whether the rate of tumor growth has been altered or the rate of growth remained unaltered but the onset of growth has changed. Altered rates of growth require an ongoing process, whereas altered onset of outgrowth is usually c:aused by an event that happened at times of inoculation. Slower growth rates (growth inhibition) should be distinguished from shrinkage (regression) of a cancer. A steady size of a treated tumor compared to controls is referred to as growth arrest, equilibrium, or progression-free survival in c:ancer patients, an important goal when c:ure cannot be achieved.169, 202

Specificity Controls It is completely inappropriate yet quite customary to study immunologic: therapies specific: for a self-antigen on a human cancer in mouse models (human tumor xenograft) when the murine host does not express the same target. These irrelevant models often yield impressive results and mislead the reader that the targeted molecule is tumor-specific: when in the real situation it is not.203' 204 If someone subscribes to the widespread highly questionable perception that many, or even most, useful tumor antigens are self-antigens, then this investigator should also use appropriate experimentation. Either a mouse model must be used in whic:h normal tissues express the target molecules closely resembling expression patterns in man or there should be a dear warning to the reader. For example, anti-carcinoembryonic: antigen (CEA) immune responses caused toxicity in mic:e that expressed the target antigen also in normal tissues, as humans do, 205 predicting the severe toxicity later observed in a dinical study.206 Side effects often become apparent only when the treatment is intense enough to provide dinic:al efficacy.207 It is therefore questionable to dismiss results in proper animal models.208,209

Selection of Tumor Model General Considerations Experimentalists have to be able to translate their findings to clinicians and vice versa. There is no single human cancer, let alone single animal model, that c:an serve as appropriate model for all human cancers.28 Organ site as well as histologic: type of a canc:er may greatly influence the results. Thus the complexity of c:ancer makes it extremely important that the experimental model used to study canc:er immunity be relevant to the question asked; a single model, if carefully chosen, may be appropriate to answer a specific question. Nevertheless, it is essential that we uncover the broader principles underlying c:anc:er-host interactions. For example, the extensive clinical and experimental research on immunotherapy of melanoma has failed to answer the

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central question: why are we struggling to make similar advances in the immunotherapy of more common cancers such as breast, colon, prostate, and lung cancers? Weiss argued in 1980 that the failures of clinical immunotherapy were due to using irrelevant laboratory tumor models for extrapolating results for clinical application.210 One of the major reasons for translating results from animal models to clinical cancer immunotherapy being frustratingly evasive is the disregard of using truly established tumors for experimental therapy (see Fig. 47.1).4 Current experimentation in cancer immunology mostly uses young hosts to study the effects of the immune system on small, recently implanted inocula of cancer cells derived from tumors once induced in inbred mice (with homozygous genetic loci) and then passed in vivo for decades from animal to animal, whereas human cancers are already well established when first detected, have a very high probability of heterozygous loci, have never been transplanted, and develop in mostly older individuals.

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manipulations are driven by tissue- or cell lineage-specific promoters that are either constitutively active or inducible locally or systemically. While these mice become prone to develop cancer, the tumors they develop are autochthonous but by no means "spontaneous" despite widespread misuse of terminology.

Curcinogen-Induced (Physicul, Chemicul, or Virul). Many of the physical and chemical carcinogens involved in the induction of cancers are mutagens.16' 17 Since repeated application of coal tar was used to induce the first chemically induced cancers, 214 a very large number of chemicals such as polycyclic hydrocarbons and nitroso compounds has been identified with remarkably potent cancer-inducing activity.215 Cancers have been induced in many tissues and organs and several animal species. Ultraviolet light (UV) has potent skin cancer-inducing activity in man and mice,216•217 and many well-defined models of UV-induced tumors are now available.218' 219 The potential of ionizing radiation to cause cancer in humans was recognized soon after Roentgen's discovery of x-rays in 1895.220' 221 But study of radiation carcinogenesis in animals mostly occurred after World War II when large-scale tumor-induction studies were carried out in many species over the succeeding three decades in response to the threat of irradiation from nuclear reactors or bombs.221- 223 Finally, many models of viral cancer induction have been developed after Rous224 showed that viruses can also induce cancer in animals.

Autochthonous Tumors Autochthonous tumors originate in the place where they were found (autochthonous, in Greek, means indigenous). The antonym is transplanted tumors. Autochthonous tumors can be spontaneous, carcinogen-induced, or transgeneinduced. Even when induced in the same experiment by the same mode of induction, autochthonous tumors will differ genetically, biologically, and antigenically from one another, because additional but individually differing genetic changes are required for each tumor to develop. Thus autochthonous tumors lack the uniformity of well-defined transplantable models. An advantage of autochthonous over transplanted tumors, however, is that the host's immune system has been neither artificially primed nor altered by an inoculum. Unlike autochthonous tumors developing after exposure to physical, chemical, or viral carcinogens, transgeneinduced autochthonous tumors have the disadvantage that transgene expression in the thymus during development usually causes systemic tolerance to the transgenic proteins. Nevertheless, several excellent transgenic cancer models have been developed.225' 226 Some models are based on immunologic findings first made in patients.12 For example, the R24C mutation in the cyclin-dependent kinase 4, first identified as tumor-specific antigen by T cells of a melanoma patient, causes familial melanoma when in the human germline.227 Introducing this mutation in the germline makes mice highly susceptible to develop melanoma225 ; tumor development, however, requires additional carcinogenic insults followed by prolonged chemical promotion. Certain oncogenes such as SV40T are powerful because they inactivate several important suppressor pathways and may therefore require fewer additional mutations. In some transgenic cancer models, the transforming genetic event can be controlled, sometimes reversibly, by topical or systemic application of an inducer such as tamoxifen or tetracycline.193 Alternatively, systemic or topical application of Cre-recombinase may excise a "floxed" blocking element or attenuator of gene expression. 228 These temporal controls appear to be helpful for answering important questions on tolerance, because expression at birth causes neonatal tolerance to a highly antigenic oncogene such as SV40 T antigen. An approach that more closely mimics the sporadic nature of human cancer relies on a spontaneous mutational event activating an introduced floxed oncogene. The sporadic nature ofthis event unfortunately also makes time and site of tumor development less predictable.228

Trunsgene-Induced. Experimental cancers are produced artificially by inserting oncogenes into the germline of mice or by manipulating the mouse genome to allow excision of tumor suppressor genes. Genes used for these

Transplanted Tumors Most current experimental work in tumor immunology uses transplantable tumors. However, there are substantial differences among transplantable tumors, and it is

Modes of Induction Spontuneous. The term spontaneous cancer is defined as cancers arising "in the absence of any experimental manipulation."211 Spontaneous murine cancers develop without any known exposure to carcinogens or genetic (often transgenic) manipulations introducing oncogenes or eliminating tumor suppressor genes. For example, spontaneous tumors can be observed in many mouse strains with advancing age. All genes have a spontaneous rate of mutation, and genetic instability, though often a consequence, may not be a requirement for tumor development.212•213 As would be expected from the sporadic occurrence of spontaneous mutations, the occurrence of spontaneous tumors is random and unpredictable.

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important that the chosen cancer is appropriate for the particular question being asked. It is important to know that 24 hours after inoculation, the majority of the injected cancer cells usually have died,178 leaving only a shallow outer rim of viable cancer cells at the oxygenated margin.107 Histology or in vivo imaging reveals pronounced edema at the injection site. In fact, much of the "tumor growth" early after inoculation is due to tissue swelling caused by the inflammatory reaction. Cancer cells may well be invading surrounding normal tissues and be vascularized within 48 to 72 hours after transfer, thereby meeting standard criteria for malignancy. But experienced pathologists will immediately spot the abnormal inflammatory reaction to the transplantation injury and the necrotic center of the early inoculum. Thus, common terms such as "three-day established cancer" or "three-day established metastases" are highly misleading. Such necrosis would not be found in an autochthonous cancer of similar size. The inflammatory reaction progressively decreases with time, and at 14 days, the necrotic material has usually disappeared. The tumor then contains -109 cancer cells, measures -1 em diameter, and is histologically indistinguishable from primary autochthonous tumors. Ideally, therapy uses tumors 2 weeks or more after inoculation (see Fig. 47.1). Even though the terms tumor transplantation and tumor injection are old and commonly used, they are quite misleading, as they usually refer to inoculation of tumor fragments or a suspension of cancer cells. A common error is the belief that tumors can be injected or "transplanted" like a vascularized organ such as kidney or heart. Instead, much of the inoculum-whether a cancer cell suspension or fragments of a tumor-dies initially and needs about 2 weeks before it becomes histologically indistinguishable for autochthonous tumors. Tumors can be induced in animals by injecting cancer cell suspensions prepared from cultures or by mincing tumors into 1-mrn3 pieces and injecting the tumor fragments using a 12-gauge trocar. Cancer cells in fragments are 10to 100-fold more tumorigenic than stroma-free cancer cell suspensions.47'108 It had long been known that certain cancers would only grow in mice when transplanted as tumor fragments. This was erroneously thought to be due to more cancer cells being inoculated with fragments?29 Later analyses revealed that fragments contained fewer cancer cells than injected cell suspensions, yet produced a higher take or larger tumors earlier.47'108 Another erroneous explanation was that the stroma of tumor fragments provided a physical barrier preventing cancer cells from migrating to draining lymph nodes and priming a protective T-cell response.108•230 More likely, more cancer cells remain viable when embedded in tumor stroma (by preventing anoikis160 ) and therefore release less antigen than suspended cancer cells, most of which die. In any case, as long as cancer cells express sufficient levels of antigen, professional antigen-presenting cells in the tumor stroma pick up the antigen and travel to the draining lymph nodes where they present the antigen to naYve T cells.231 It is important to know that whether suspensions of cancer cells or tumor fragments are being used, a threshold

number of cells or fragments must be inoculated for tumors to develop. The threshold in T cell-deficient mice may be due to innate immunity or nonimmunologic mechanisms. 178,232 The increase in threshold in immunocompetent mice is probably due to adaptive immunity.

Cell Lines from Tumors Serially Passed in Mice. When choosing tumors that have been serially passed in animals for transplantation, it is critical to be aware of how such tumors were altered by the serial transplantation. As was discovered decades ago, even a single in vivo passage of a cancer can select for heritable cancer variants.45' 219•233- 237 Even half a century ago, investigators already noted that serial transplantations of these cancers "inevitably result in progression toward more rapid growth rate, loss of functional and histological differentiation, loss of responsiveness to extraneous stimuli"238 (p. 522l and a diminution of strain specificity, a problem shared by B16. 239 These serially transplanted cancers, such as B16, can easily be transferred in mice using cancer cell suspensions rather than tumor fragments. As a result of hundreds of passages in mice, 239 these cancer cells have become resistant to anoikis caused by lack of stroma by acquiring alternate signaling pathways that replace the prosurvival signals of ECM. 240 Many of these tumors grow and kill so fast that they must be treated early before they are truly established. Certainly, many of these tumors no longer resemble primary mouse or human tumors that virtually always grow at much slower rates and have been established for months or years before being treated. B16 melanoma was derived in C57BL/6J mice in 1954 and is the parent of many available B16 sublines.239 B16 had been transplanted serially through 328 mice for 13 years before it became available as a standard cancer cell line. Nevertheless, B16 still is arguably the "Escherichia coli" of tumor immunology with almost 1,000 entries in PubMed in 2011. The A/J-derived Sa 1 originated in 1947 in an A albino mouse and was serially transplanted in A/J mice for 1,017 generations for 19 years. Similarly, the A/J-derived neuroblastoma 1300 derived in 1940 was serially transplanted for decades from mouse to mouse. The Lewis lung carcinoma, isolated by Lewis, 241 arose spontaneously in 1951 at the Wistar Institute in a black C57 mouse (not a C57BL/6 mouse), was serially transplanted extensively, and is still being used in numerous studies as a model for exploring the immune responses to lung cancers and their metastases. When genetically inbred mouse strains became available as sources for murine cancers over half a century ago, serial transplantation was necessary for maintaining a tumor. Dependable long-term cryopreservation did not become available until the late 1960s and early 1970s.242- 244 Even after dependable cryopreservation became available, investigators still continued to use serial transplantations to propagate newly derived cancers such as the BALB/c-derived CT26 colon cancer,245 the C57BL/6-derived MC38 colon cancer,246 and the BALBI cCr-derived RENCA renal cancer.247 Many cell lines are renamed sublines of old parental cell lines. Thus, it is often overlooked that RMA, RMA-S, MBL-2, and EL-4 tumors are derived from the same single tumor line,248 most likely

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E.L.4 induced by 9:10-dimethylbenzanthracene in 1945 by Go~ 9 and serially transplanted for many decades from mouse to mouse. Importantly, referring to EL-4 as a C57BL/6 tumor is incorrect as this strain did not exist in 1945. Similarly, Neuro2a is a subline derived in 1969 from the A/J-derived neuroblastoma 1300 derived in 1940. Unfortunately, organizations providing these lines and/or publications often fail to cite the fac:t of long-term in vivo passage before cryopreservation.

CeU Lines from Autochthonous Mouse Thmors. Autochthonous cancers never need to be serially transplanted. Standard lines can be created by adapting the autochthonous cancer cells to culture and/or by freezing fragments or cells in liquid nitrogen.m To prove that antigens expressed by the malignant cancer are truly tumor-specific, nonmalignant cells, tissue and DNA from the host oftumor origin must be available.218'219 This approach is commonly used by researchers studying the antigenicity and genetics of human cancers. Carefully controlled experimental cancers exist.218•219 Hu'""n Thmor Xenogrufts. For certain purposes, human tumor xenograft:s are useful models. Human cancer cell lines grown in vitro can cause tumors when injected into T-, B-, and/or NK-deficient mice. It is important to remember that the stroma of such cancers is entirely of mouse origin and the cancer cell-stromal loops are defective because of multiple mismatches in ligand-receptor signaling.250 This problem can be partially alleviated by "humanizing" the recipient mouse. Humanizing usually refers to expressing certain human molecules such as receptors/cytokines in mic:e that lack murine T, B, and/or NK cells, and/or transferring human mesenchymal and/or hematopoietic: cells to such mic:e. Interestingly, human T- and B-lymphocytes and fibroblasts contained in human tumor fragments may coengraft thereby "humanizing" the mouse192 (eg, when injecting cell dusters or aggregates from human fresh ovarian cancers into nonobese diabetic-severe combined immunodeficiency IL2yR null mice). Progressive growth of these xenograft:s leads with great regularity to ascites formation, and pleural metastasis closely simulating classical tumor progression observed in patients with ovarian cancer.192 Despite continuous improvement, mic:e can never be completely humanized. Only very few cancer cells of the xenografts may be able to progress in the chimeric: milieu.30 Nevertheless, once a tumor grows, its sensitivity to potential therapeutic agents might reveal the sensitivity of the original cancer growing in the patient.

Selection of Recipient/Host The vast majority of human cancers develop in later midlife and old age,251-254 and there is dear evidence that, at comparable ages, mice have difficulties rejecting immunogenic: cancers. 25~253 Yet most experimental studies use young mic:e. Mic:e should have a dinic:ally relevant tumor burden or be selected for treatment when the bulk of cancer cells has been removed to undetectable levels by surgery or chemotherapy, but dormant cancer cells stay behind to cause later relapse. T cell-deficient mice have been used extensively

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as models for adoptive transfer ofT cells.259 However, cancer patients are usually T cell competent and capable of generating regulatory T (Tu:g) cells that may not allow an effective "take" of the transferred T cells unless the recipient is lymphodepleted.

CANCER ANTIGENS No term in cancer immunology is more important and confusing than the term cancer antigen (or tumor antigen). Any molecule detected with T cells or antibody on the surface or within cancer cells is commonly referred to as cancer antigen. The usefulness of a cancer antigen for detection and destruction depends on its specificity (ie, that the antigen is not common to normal cells). The first well-defined tumor antigens encoded by the MHC were discovered by Gorer. 8 Immunity to MHC antigens will kill the cancer cells but also the host because of the ubiquitous expression of MHC on normal cells.

General Aspects Early History

In the late 180 Os,260'261 it was discovered that in some instances tumors developing spontaneously in experimental animals could be transplanted into other animals of the same species and in this way could be propagated continuously. This finding provided an important experimental tool for cancer research.4o.262 Immediately, scientists began to investigate the possibility of immunizing against such transplantable cancers. Rodents exposed to a small nonlethal challenge of certain tumors became immune to subsequent challenge with large transplants of the same tumor that regularly killed nonimmunized recipients. Also, complete removal of the transplanted tumors, after initial growth, immunized animals against that tumor. These early results seemed to suggest that immunization against cancer was possible. Furthermore, there were certain other spontaneous tumors that were not readily transplantable, and this was taken as evidence for "natural resistance" or "natural immunity" to the cancers. Many years later, it became clear that no such conclusions could be drawn from these early studies, because outbred, or incompletely inbred, rats or mice had been used. The problem became apparent when it was realized that the immunization with tumor would also immunize the host against normal tissue of the donor and that normal tissues of the donor could also immunize the host against the tumor.186 These experiments brought the idea of tumorspecific antigens into disrepute but also started the search for antigens that caused rejection of normal transplanted tissue. This research eventually led to the discovery of the MHC and to the development of inbred mouse strains. s,263•264 Once inbred mouse strains became available, it was found that cancers transplanted within an inbred mouse strain usually grew so well that the existence of tumor-specific antigens seemed very unlikely. In fact, transplantability of tumors in syngeneic animals became (and still is) a diagnostic criterion for the malignant phenotype of an experimental tumor. This criterion was especially useful because many rodent

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tumors are cancers of nonepithelial origin (sarcomas) and also because a dear histologic demonstration of local invasive growth can be especially difficult in such cancers.

Proof of Specificity After discovery of the MHC and the development of inbred mouse strains, the gloom over tumor immunology ended with the discovery that inbred mice could be immunized against syngeneic sarcomas induced by the chemical carcinogen methylcholanthrene (MCA). The first demonstration of induced immunity to a transplantable MCAinduced sarcoma was by Gross in 1943265; however, it was not until the 1950s that more complete experiments provided unequivocal evidence for "tumor-specific" rejection of transplanted cancers.229' 266- 270 In particular, the experiments of Prehn and Main268 in 1957 made it likely that the rejection antigens on the MCA-induced sarcomas were functionally tumor-specific, because transplantation assays could not detect these antigens in normal tissues of the mice used. However, irrefutable evidence for the existence of tumorspecific antigens in autochthonous unmanipulated cancers only came after a 50-year-long search 3•5• 218 •219•236 •267~2 when in 1995, it was proven that tumor-specific antigens on cancer cells were encoded by somatic cancer-specific mutations absent in the normal cells of the host of tumor origin. 1~ 12 Even though it is clear that cancer-specific antigens are encoded by somatic cancer-specific mutations, the terms "tumor-specific" or "cancer-specific" are frequently used inappropriately, sometimes even used in conjunction as "relatively" tumor-specific. The discussion is far from being semantic. In reality, an antigen either is or is not tumor-specific. Germline controls are absolutely critical for proving mutations are somatic and tumor-specific. 283~86 Unfortunately, germline controls are missing from virtually all tumors used for experimental work today. Nevertheless, mouse tumors with proper autologous controls have also been used and are available for distribution.218.2 19 Notorious problems are genetic polymorphisms. For example, even after 20 backcross generations when a strain is arbitrarily pronounced "'inbred" (because it is then more than 99.9% genetically identical), about 373 polymorphic proteinencoding loci remain allogeneic.287 Any one of these loci could encode a pseudotumor-specific antigen when a tumor is transplanted into a mouse misperceived to be fully syngeneic. Mice respond preferentially to nonself- or mutantself-antigens whether caused by genetic polymorphism or tumor-specific somatic mutation.

Peptide Antigens and Major Histocompatibility Complex Affinity T cell-mediated destruction of cancer cells requires the interaction ofT-cell receptor (TCR), peptide, and MHC molecules. In this "'three body problem," two affinities simultaneously determine the interaction238' 289 : the peptide to the MHC and that of the TCR to the peptide-MHC complex. Even when the complex cell-cell interaction is reduced to the three molecules interacting in vitro, biochemical analysis is still too complex for analyzing physiologic interactions. TCR affinity to peptide-MHC is therefore usually

measured by plasmon resonance in the presence of saturating nonphysiologic amounts of the peptide, and TCR affinities measured this way range between (Kd) 1 to 100 ~M.238.289 This is a narrow range considering that affinities of peptides to the MHC range from 1 to more than 20,000 nM.290 This difference points at the peptide-MHC affinity probably being the greatest variable and emphasizes the paramount importance of choosing target peptides with highest possible affinity to the presenting MHC. Proper selection is particularly important as the amount of peptide produced by the cancer cell may be relatively small and always must compete for binding with all other peptides naturally present in the cancer or in the cells cross-presenting the antigen. Affinity of a peptide to a given MHC molecule is best measured empirically and many empirical affinities are already available (eg, http://tools.immuneepitope.org or www.syfpeithi.de).290- 292 However, even when a peptide binds with high affinity to MHC, it will only be expressed on the cell surface when it is naturally processed and present in sufficient amount.293•294

Aatigens Revealed by ·neverse Immunology'" "'Reverse immunology" is the attempt to predict T -cell epitopes within a given amino acid sequence. Traditionally, "reverse immunology" has focused on finding optimal T -cell antigens, properly referred to as peptide epitopes, on infectious agents for generating vaccines. More recently, self- or mutant proteins recognized by T cells or antibodies from cancer patients have been analyzed in the search for peptide epitopes that may be effective targets of T-cell immunity. Computerized algorithms (eg, http://tools.immuneepitope. org295•296 and www.syfpeithi.d~97 ) have been developed to predict the affinity of peptide-MHC binding, appropriate proteasomal cleavage, and transport by the transporter associated with antigen processing (TAP).~:m.297 Though these algorithms are useful, it appears that these tools cannot replace empirical biochemical measurements. Many of the antigens proposed by reverse immunology have affinities to MHC insufficient to serve as effective targets when tested in appropriate animal models. Together, high peptide-MHC affinity is essential but not sufficient to predict that a given peptide serves as an efficient target. Complete sequencings of cancer cell genomes from individual patients is becoming increasingly affordable and has revealed up to many thousands of mutations per cancer cell,298- 300 However< l% of these mutations cause amino acid substitutions. For example, of the 33,345 nucleic acid base substitutions found in a human melanoma, most of the mutations were intergenic, intronic, noncoding, silent, or truncating. Only 187 caused amino acid substitutions in coding genes,300 and only a few of these 187 coding substitutions are expected to lead to mutant peptides that serve as effective targets. To be effective, the mutant peptide 1) must bind with a high affinity (IC50 in the nM range) to the particular MHC molecules of that individual and 2) be naturally processed, 3) escape destruction by proteasome cleavage, and 4) be present in sufficient amounts. Only a few of the myriad of tumor-specific mutations identified in human or murine cancers give rise to antigens that fulfill these requirements. However, the frequency of unique tumor-specific targets in

CHAPTER 47 CANCER IMMUNOLOGY a cancer cell may be larger than predicted when considering only mutations in coding sequences, because mutations in intron sequences can be translated and also encode tumorspecific antigens.11 These mutations are over SO-fold more frequent than those in protein-encoding loci.

Antigens Recognized by Patients• T cells In the search for the best antigens to eradicate the cancer, T cells infiltrating tumors (TILs) have been recovered. expanded, and used therapeutically. However, TILs are removed from cancers that have not been destroyed. It is, therefore, a widespread misperception that the Tn.s were necessarily fighting cancer growth. Three questions become obvious: I) Are strong antigens retained because the host has been tolerized during very early stage of cancer growth? 2) CanT cells specific for the strongest antigens be recovered from the Tn.s? 3) CanT cells specific for weak antigens promote rather than inhibit tumor growth? It is possible that I) potent rejection antigens are retained because they tolerize the host very early during cancer development,22M 01 2) a few T cells have not been tolerized and can be propagated for therapy, and 3) tolerization is reversible, and competent effector cells can be obtained from TILs. Clinically, the patients who fare best have T cells in their peripheral blood that are specific for antigens encoded by somatic tumor-specific mutations.12.302.34» Whether the same T cells were infiltrating these cancers as TILs is unknown. In any case, it is likely that only a few (if any) of an

Cancer-Specific Antigens (Encoded by Mutant Genes) Prevalence All cancers in man and mouse that have been analyzed carefully express bona fide tumor-specific antigens that could be targeted by T cells. Because tumor-specific antigens arise from mutations,1o-12 they are usually unique. Each patient's cancer seems to have a unique set of mutations, and unique antigens can provoke powerful immune responses. In mice, immunization with one tumor protects only against the same tumor {Fig. 47.2).~7' Shared cancer-specific antigens do exist when the same mutations occur in several cancers, but by comparison, such antigens are relatively rare. Most cancer-specific mutations affect intracellular proteins that may be recognized by T cells as mutant peptide-MHC complex on the surface of viable cancer cells. Very few cancer-specific mutations affect surface proteins, such as the mutant epidermal growth factor receptor (EGFR), a shared tumor-specific antigen on glioma cells.306. 30s

Oncogenicity A major misconception is that unique cancer-specific antigens are caused by random mutations and are incidental to the oncogenic process. For example, a group of experts

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endless anay of antigens recognized on cancer cells by antibodies or tumor-infiltrating T cells may have significance as targets or diagnostic markers.

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Demonstration of Tumor Antigens by Transplantation Assays: Implantation of a viable tumor 7-14 days

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FIG. 47.2. Demonstration of the lndiYidual (Unique) Spec:iic:ity of Rejection Antigens an Independently DeriYed Methylc:holanthrene-lnduc:ed Tumors in Transplantation Experimems. For details, see Basombrio.3 Modified from Basombrio and Prehn.6

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stated in 2009306 (p.s326l that unique antigens come "from random mutations presumed to be present in all patients," implying mutations encoding unique tumor antigens have no functional significance in the malignant process. Nothing is further from the truth. Of course, all mutations caused by chemical and physical carcinogens are random, but virtually all of them are lost during cancer evolution except those that promote the neoplastic process. When it was first shown in 1995 that cancers harbor cancer-specific antigens caused by somatic tumor-specific mutations, 1(}-12 every one of the three antigens represented a mutation in a tumor suppressor locus.307- 3n For example, the mutation in the cycline-dependent kinase 4 reduces the binding to its inhibitor and tumor-suppressor protein p16INK4a,12 and the same mutation is found in the germline in cases of familial melanoma.227 The fact that selection for a tumorspecific antigen commonly represents a mutation in genes with functional significance in the malignancy of that cancer (oncogenes or tumor suppressor genes) has been shown in many other studies.219' 312-315 Remarkably, T cells against unique tumor antigens identify mostly novel, functionally important tumor-specific mutations and oncogenic proteins that would not have been easily detected by other technologies.10-12'219'307-309,316 Importantly, several of these unique tumor-specific antigens are excellent targets because they cannot be lost by immune selection. The reason is that some of these mutant proteins are not only oncogenic but are also needed to provide an essential household function no longer provided by the second allele due to Knudson-type loss317 or mutational inactivation.302'307- 309

Therapeutic Significance Another misperception surrounding unique tumor-specific antigens is that they have remained unexploited clinically, because truly personalized therapy would be required. Again, nothing is further from the truth. One of the most effective immunologic treatments of cancer today is the adoptive cell transfer of autologous tumor-infiltrating lymphocytes from patients with metastatic melanoma: response rates are in the range of 50% to 70% of the patients, and a few of these patients are cured.316 This truly personalized therapy involves reinfusion ofthe patients' own lymphocytes isolated from the patients' own melanomas and expanded in vitro. Because the TII..s response is dominated by T cells to unique tumor-specific antigens,303 it is likely that the success of the reinfused T cells depends on their reactivity to unique tumor-specific antigens. Similarly, clinical studies show that therapeutic vaccinations with autologous cancer cells are likely to be much more effective,319'320 confirming decades of experimental work.229'234' 268'269' 271'321'322 Self-antigens may serve as useful and effective targets in a very few instances (eg, CD20 and CD19), but industry supported by government panels and organizations focuses almost exclusively on self-antigens,306'323' 324 so far with very modest results. 325 Finally, it is a misperception that immunizing each patient to the unique antigen of the individual's tumor is impractical because of cost and should not be pursued. This is incorrect compared with other highly individualized treatments such as those used in renal transplantation. Thus, individualizing

cancer therapy could be affordable as other strategies used in the clinics.

Shared Tumor-Spacific Antigans Ideally, antigens targeted on cancers would be expressed exclusively on malignant cells but be shared by cancers of the same type or at least subtype. Several cancer-specific mutations have been identified that are shared between cancers,9'304' 326'327 but very few of them have so far been found to be an effective immunologic target. The main reason probably is that, aside from specificity, additional requirements for effective recognition by T cells are 1) high-affinity binding to the patients' MHC molecules and 2) expression of the protein at sufficient amounts to compete with other peptide for presentation. Unique tumor-specific antigens must fulfill the same requirements but, because these unique mutations are more abundant, chances are much higher to yield an effective antigen. A few examples of shared tumor-specific antigens are discussed in the following.

Mutant Epidermal Growth Factor Receptor. See further under Immunotherapy.

Fusion Proteins. Fusion proteins found in cancer cells are the result of internal deletions (see mutant receptor EGFRviii under Immunotherapy) or chromosomal translocations.9•326 New antigenic determinants can result from the juxtaposition of previously distant amino acid sequences, resulting in a mutant peptide sequence at the breakpoint and possibly a change in conformational structure. The same chromosomal breakpoints consistently recur in different individuals with the same cancer, therefore result in shared tumor-specific antigens. Fusion proteins encoded by these translocations are usually essential for cancer maintenance, making them ideal targets for pharmacologic and immunologic intervention because tumor cells may not easily escape therapy by losing expression of the fusion proteins.328 Pharmacologic approaches are exemplified by the drug imatinib targeted to the fusion protein of the 9;22 translocation in chronic myelogenous leukemia.9'294'329 The antikinase drug imatinib does not selectively inhibit the catalytic activity of the BCRABL fusion protein, and this results in toxicity to normal cells in the patient. The intracellular BCR/ABL fusion proteins can be recognized specifically by antibody but only in fixed cells.330 BCR/ABL fusion peptides can also be recognized by human CD4+ T cells in the context of MHC class 11331 and serve as targets for CDS+ human cytolytic T cells.332 Why do we then still lack effective immunologic therapies for targeting this and other fusion proteins? As explained previously, the predicted epitopes may 1) not be generated naturally by cells, 293 2) lack sufficient affinity to the particular MHC of that patient, or 3) not be produced in sufficient amounts. Mutant RAS. Point mutations in oncogenes can also be shared by several cancers and could encode useful antigens. For example, a valine for glycine substitution at position 12 of RAS is one of the most common mutations in human cancers and can be recognized by human CD4+ T cells. 333 The region of the mutant RAS protein from which

CHAPTER 47 the peptide was derived is identical for all the three members of the RAS proto-oncogene family, namely, H-RAS, K-RAS, and N-RAS, which have different prevalence in different cancers. In addition, about 90% of pancreatic adenocarcinomas, a very aggressive human cancer, have one of three to four different single amino add substitutions in codon 12 of the cellular Kirsten RAS gene. 334 Despite remarkable clinical and experimental efforts,335- 340 targeting mutant RAS-derived epitopes has so far met with little if any success, the likely reasons being the same as those mentioned under the section titled Fusion Proteins. Immunizing cancer-prone mice harboring "initiated" mutant ras-expressing cells with mutant ras peptide resulted in mutant ras-specific CD4+ T cells, antibodies to the mutant ras protein, and more tumors and faster tumor development.341 Possibly, the antibody, produced in response to antigen, activated myeloid cells via FcR-y to become tumor-promoting, and/ or antibody carrying TGF-~ downregulated CDS+ effector responses.342' 343

Self-Antigens (Encoded by Normal Genes) All antigens listed in this section are encoded by nonmutant cellular genes and expressed not only by cancer but also by at least some normal adult cells. Therefore, these antigens are not tumor-specific and are commonly referred to as tumor-associated antigens. The level of expression of these antigens can vary from widespread expression to restriction to a small population or a subset of normal cells. However, self-antigens should not be called "quasi-tumor-specific" because even very low levels and/or selective expression of a self-antigen may cause severe even lethal toxicity when targeted.204'206 Self-antigens expressed by tumor cells are used for destruction, inhibition, or detection of cancerous growth. When used as targets for destruction, T cells or antibodies must eliminate the cancer cells while not destroying normal cells expressing the same self-antigen, or destruction of selfantigen-expressing cells must be tolerated. Thus, no serious toxicity must occur even when the immunity is strong enough to destroy the cancer cells.195' 196 Antibodies against growth factors or their receptors can inhibit growth of cancer cells without destroying them. When self-antigens are used for diagnosis, background levels of antigen generated by normal cells complicate use of these antigens for early detection of cancer. However, changes in the amount of circulating self-antigens may indicate relapse of cancer after therapy. All normal individuals have so-called natural autoantibodies as well as T cells to a wide spectrum of self-antigens without causing pathology, but those B-cell receptors and TCRs have usually very low affinity. In fact, any immune receptor binds with some affinity to any particular antigen344 and immune receptors may bind to several molecularly unrelated structures,345 making the discussion of specificity seemingly useless. However, the antibody response of an individual to self- and nonself-antigens differs in affinity by many orders of magnitude, and therapeutically effective antiself-antibodies or TCRs are commonly raised in a nonself, usually xenogeneic, setting. Whether removing

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natural mechanisms that prevent autoimmunity is a general approach to achieve truly effective antitumor immunity needs to be substantiated. The problem of B-cell unresponsiveness can be overcome by immunizing mice with xenogeneic human antigen. Misleadingly, these high-affinity antixenogeneic antibodies are usually advertised as "fully human'' when they were made in mice in which the murine immunoglobulin (Ig) gene locus had been inactivated and replaced with the human Ig gene locus. 346 The toxicities of such "fully human" antibody will still be those of high-affinity antiself antibody: severe to lethal toxicity. These reactions may occur when the variable region of such antibodies (eg, anti-CEA or antihuman epidermal growth factor receptor [HER] -2204•206) is fused with the transmembrane and signaling domains and then transduced as chimeric antibody receptors (CARs) into T cells.347 T-cell tolerance to self can be overcome by making T cells in a host that is allogeneic to the MHC class I molecule presenting the targeted peptide.348 However, recent studies exposed that such allo-human leukocyte antigen (HLA)restricted high-affinity T cells can have severe "off-target" reactivity likely to cause toxicity when used in patients.349 T cells transduced with these TCRs kill each other (fratricide) when the self-antigen (eg, wild-type p53 or survivin) is expressed by the T cells.35o,351 High-affinity TCR cells can also be generated in knockout mice lacking the targeted selfpeptide (eg, wild-type p53 peptide sequences352), butT cells expressing these TCRs are lethal when given to normal mice because wild-type p53 is expressed in bone marrow and can also be expressed by most other normal cells.350

Overexpressed Molecules

Growth Factors and Their Receptors. See Immunotherapy. Survivin. Although survivin is overexpressed in many cancer cells, it is a problematic antigen particularly as a vaccine because it is widely expressed on lymphocytes causing fratricide.351 Not surprisingly, this antigen has been ineffective in vaccine trials. p53. Research using p53 as target for immunotherapy is

exemplary of the problems and persistent misconceptions of research on tumor-specific (unique or shared) and selfantigens. Mutations in the p53 suppressor gene are among the most common found in human and experimental cancers.353' 354 These mutations tend to cluster in evolutionarily conserved regions of the gene, but the exact locations are highly diverse in individual cancers. 355 Targeting these mutations would require an individualized therapy considered by many impractical. Therefore, researchers have attempted to exploit the fact that mutant p53 is usually overexpressed in cancers. Thus, major efforts have been made to target conserved, nonmutated regions of the p53 protein hoping for "relative tumor specificity" based on the usual belittlement for low-level expression of this protein by every normal cell including lymphocytes. As a result, T cells expressing highaffinity anti-p53 TCRs commit fratricide unless the anti-p53 TCRs are transduced into T cells from p53 knockout mice.

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However adoptive transfer of these T cells causes lethal hematopoietic ablation in normal mice.350 Mortality can be prevented by reconstitution of the recipients with bone marrow from p53 knockout mice,350 an option unavailable in humans. Not surprisingly, there is no evidence for clinical efficacy of targeting wild-type p53 in humans.

Prostate-Specific Membrane Anti~n. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein more correctly referred to as glutamate carboxypeptidase II. PSMA is an inappropriate misnomer because it is anything but specific for prostate; it is also expressed by duodenal mucosal cells, proximal renal tubule cells, and a subpopulation of neuroendocrine cells in colonic crypts,356 and has important functions in the brain. 357 Vaccination of mice with human PSMA induces antitumor effects without causing toxicity but because the mouse model lacks expression of human PSMA on normal tissues, the findings in this inappropriate model are meaningless.358 Similarly, a "fully humanized" anti-PSMA antibody had potent antitumor xenograft activity in mice that lacked expression of this antigen on normal cells.359 Therefore, this antibody may have severe, possibly lethal, toxicity in patients.

Diffarentiati on/Lineage-Specific Molecules Some antigens expressed on tumor cells are also expressed during at least some stage of differentiation on nonmalignant cells of the lineage from which the tumor developed. Differentiation antigens may therefore help to determine the organ or cell type of origin (lineage) of a cancer. 360 For example, B-cell tumors express surface Ig, and T-cellleukemias can be separated into helper and suppressor cell leukemias using T-cell subset-specific monoclonal antibodies to surface and intracellular antigens including transcription factors. For metastatic cancer of unknown origin, differentiation antigens can be important indicators of the histologic and organ site of the primary tumor.361 Careful diagnostic delineation of different subtypes of cancer is important because different tumor subtypes may have different prognoses and may be susceptible to different therapies. However, the use of differentiation markers for histologic or cytologic tumor classification has pitfalls. 361 Lineage-specific antigens represent a very diverse group of proteins: glycoproteins (including mucins) and glycolipids (carbohydrate, peptide, glycopeptide, or glycolipid epitopes). Several of these antigens are being explored as potential immunotherapeutic targets. However, the normal cell types that express these antigens will determine toxicity and usefulness of any antibody or agent for which these are targets.

Cluster of Differenti4tion 20 and Cluster of Differentiation 19. CD20 is a signature B-cell differentiation antigen targeted by a genetically engineered monoclonal antibody that is relatively effective in the treatment of B-cell lymphoma. Lineage restriction of this marker limits the cytocidal effects to long-term depletion of normal B cells. This depletion is usually well tolerated because patients can be protected by intravenous administration of IgG. While

antibodies to CD20 antibodies are therapeutically effective, relapse is common. CD19, but not CD20, appears to be expressed on the more immature malignant cells causing the relapse. Therefore, anti-CD19 Fv has been used in the killing of these cancer cells, either as a fusion protein with an antiCD3 Fv to engage T cells195 or as chimeric antibody receptor inserted into the patient's T cells. 196 These treatments may prevent relapse.

Melanocyte-Specific Differentiation Antigens. Several differentiation antigens (such as tyrosinase, the related brown locus protein or tyrosinase-related protein 1 (Trp-1), gp100, and Melan A/MART-e62' 363) appear to be restricted to melanocytes, and all of them are being explored as immunotherapeutic targets in melanoma.364--366 Immune recognition of the melanocyte differentiation antigens can lead to rejection of a tumor challenge but this self-antigen-specific immunotherapy not only targets the tumor cells but also normal cells expressing the shared antigens, 365•367 resulting in the depigmentation of normal skin (vitiligo) and possibly other, more serious toxicities (see following discussion). For example, mice immunized with syngeneic Trp-1 in various adjuvant settings fail to produce aT- orB-cell response.368 Xenogeneic or altered Trp-1, however, induces responses that cause vitiligo and protect against challenge with melanoma cells.368 Passive transfer of Trp-1-specific antibodies from these mice causes vitiligo and protects against metastatic spread of melanoma cells in mice when given at the time of seeding of the malignant cells. 367 Similar antibodies may have analogous beneficial effects in human melanoma patients,369 but evidence is lacking for such antibodies eliminating bulky human melanoma. Trp-1-deficient mice respond to Trp-1 as a foreign antigen and Trp-1-specific CD4+ T cells from these mice can eradicate large B16 melanoma 10 to 14 days after inoculation, 370' 371 cause autoimmune vitiligo, and damage the retina.370 Designating these T cells as "tumor-specific" when they are clearly not is incorrect and misleading.370 Trp-1 is expressed in all neurocrest-derived pigmented cells, not only those of the skin but also those of the eye (uvea), inner ear, and brain (substantia nigra, forebrain, and midbrain).372- 375 Other targets include gangliosides GD2 and GD3 that are also not only overexpressed in melanoma but are also found in other cells of neurocrest origin and in other tissues.376•377 Prostate-Specific Antigen. Prostate-specific antigen (PSA), also called kallikrein-3, is a chymotrypsin-like protease that digests semenogelin I and II to release motile sperm.378 Elevation of PSA above the normal range occurs in inflammation (prostatitis) and benign hypertrophy of the prostate as well as in prostate cancer. Using PSA levels for early detection of prostate cancer is controversial. The U.S. Preventive Services Task Force no longer recommends this test in healthy man. 379 However, detection of any PSA following complete surgical removal of the prostate indicates residual tumor cells and/or recurrence.380 Prostate-specific phosphatase is selectively expressed and secreted by the epithelial cells of the prostate gland.378•381 Sipuleucel-T, a vaccine targeting prostate-specific phosphatase

CHAPTER 47

and approved by the U.S. Food and Drug Administration for the treatment of castration-resistant prostate cancer, results in only a very modest improvement in overall survival.323 Furthermore, a recent analysis of intemal documents that became available after the U.S. Food and Drug Administration approval questions the adequacy of the trial and correctness of the conclusions drawn.382

Epithelial Cell Adhesion Molecule. Normal and malignant cells of epithelial origin express the transmembrane glycoprotein epithelial cell adhesion molecule (Epcam). 383 Anti-Epcam antibody 17-1N64 showed promising results in patients with colon cancer in several early studies,385 whereas subsequent randomized clinical trials consistently failed to show any benefit.386 A high-affinity engineered anti-Epcam antibody caused cases of acute pancreatitis and was discontinued.387 Other anti-Epcam antibodies in the form of bi- and trifunctional constructs may be more effective, but finding efficacy while keeping lethal toxicity under control presents a major hurdle.

Tumor Antigens Caused by Altered Glycosylation Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, 366•369 and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy.

Mudns. Mucins are normally heavily glycosylated glycoproteins (ie, containing complex 0-glycans) that protect the luminal mucous epithelial surfaces. Mucins of cancer cells often show decreased expression of the complex 0-glycans and increased expression of short oligosaccharides, the TF, sialyl-Tn, and Tn antigens390 (see following discussion). Thus, human adenocarcinomas of the pancreas, breast, and colon express mucins391 that can be recognized on cancer cells by MHC-unrestricted cytolytic T cells. These T cells apparently react specifically with repeated epitopes on the protein core of the mucin molecules, 392 exposed because of deficient glycosylation in the malignant cells.393 The epitopes are expressed at high density, do not require processing, have a stable conformation, and can directly bind to certain TCRs without being presented by MHC molecules. 394 Hypoglycosylated MUCl is expressed on about two-thirds of newly diagnosed cancers. However, multiple types of vaccines using the nonglycosylated tandem repeat peptides or tumor-associated saccharide antigens conjugated to carriers failed to immunize effectively.395'396 This finding is consistent with multiple lines of evidence that humans and MUCl-transgenic mice are tolerant to the unglycosylated long MUCl peptide. Numerous approaches have been developed capable of overcoming this tolerance such as using short and long synthetic glycopeptides or plant-expressed MUC1.397400 Many different mucin genes have been identified401 and antigens have been defined, but evidence for clinical efficacy with controllable toxicity is lacking.

T tJnd Tn Antigens. Tn antigen on human erythocytes, first

described by Moreau et al. in 1957,402 is the cause of a hemolytic autoimmune disorder, Tn syndrome.403 Tn ("T antigen nouvelle") is a Ser/Thr-0-linked N-acetylgalactosamine

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monosaccharide distinct from the disaccharide T (or TF) antigen galactose-1}1-3-N-acetylgalactose 0-linked to a Ser or Thr (Gali}1-3GalNAca1-Ser/Thr) described earlier by Huebner, Thomsen, and Friedenreich.404 Human cancers very frequently express TF and Tn antigens.405'406 Recently it was shown that the Tn syndrome is caused by somatic mutations in the chaperone COSMC.407 While TF antigen is an oncofetal antigen highly expressed in the embryo and fetus, 390 there is no evidence that Tn is also an oncofetal antigen.404 Mutational deletion of the chaperone COSMC leads to expression of the Tn antigen on virtually every embryonic cell and leads to early embryonic death. 408 Most adults naturally have anti-Tn as well as anti-TF antibodies due to antigenic stimulation by Tn and TF antigens expressed on the bacterial flora. 40M 10 Tn antigen is also expressed on human immunodeficiency virus-1 and pathogenic parasites. Thus, evidence is lacking for any expression of Tn antigen on adult or embryonic human or murine cells except by patients suffering from Tn syndrome and cancers that may or may not show a tumor-specific deletion of COSMC. There is no evidence that Tn is an effective therapeutic target. This may be due to anti-Tn antibodies being usually IgM or IgA of very low affinity, though recently developed IgG antibodies reduced slightly the growth rate of human cancer cells in vitro and in vivo.411

Glycopeptide Antigens Resulting from Tumor-Specific COSMC Mumtions. The remarkable characteristic of this new class of antigens is the exquisite tumor specificity of the antigen412-414 even though it is encoded by normal genes. Appearance of the antigen, however, depends on a tumor-specific somatic mutation that destroys the chaperone COSMC that is essential for any functioning of the core 11} (1-3) galactosyl-transferase, T-synthase. COSMC protects the newly synthesized T synthase from aggregation and subsequent endoplasmic reticulum-associated degradation.404 T-synthase is essential for extending 0-linked glycosylation beyond a single 0-linked N-acetylgalactosamine (ie, Tn antigen). Importantly, the antigen recognized by the high-affinity, tumor-specific antibody does not bind Tn alone but contains the Tn hapten (ie, the single 0-linked Nacetylgalactosamine on a threonine or serine). X-ray crystallography shows that the antibody completely envelops the carbohydrate moiety while interacting with the unique sequence of the peptide moiety in a shallow groove.413 Because Tn does not exist on nonnal embryonic or adult human or murine cells, there is no central or peripheral tolerance to these antigens allowing high-affinity destructive immune reactions to occur. This also explains the severity of the autoimmune disease associated with the Tn syndrome caused by somatic (not germline) mutations of the COSMC gene.407 Importantly, somatic tumor-specific mutations disabling the X chromosome encoded COSMC gene (for which one copy is naturally silenced) seem to be more common than originally assumed and occur in a wide variety of spontaneous or virally induced human and murine cancers.415 Nevertheless, it must be shown that high-affinity receptors when used as CARs on T cells or linked to anti-CD3 are effective against tumors but have little if any toxicity to

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normal tissues. This may be expected from the fact that the epitopes depend on somatic tumor-specific mutations.

Oncofetal. Carcinoembryoaic, aad Cancer-Gennline Antigens Over 50 years ago, human cancer cells were found to express antigens that serologically cross-react with normal embryonic tissue.' 16 Since then, it has been postulated repeatedly that certain normal genes that are completely silent in all nonmalignant cells may be activated exclusively in malignant cells.m Alternatively, it has been postulated that cancer cells may express proteins (or immature forms of a protein) that are only expressed in fetal but not in nonmalignant adult cells.418 There certainly is profound hope for finding a universal tumor rejection antigen that can be used for vaccination, prevention of cancer, and antitumor therapy. However, repeated claims of selective activation of normal genes or selective expression of immature forms of these proteins in cancer cells leading to tumor-specific antigens have not been substantiated. Expression of the same antigen by at least one normal cell type in the adult was later often disoovered.m__.22

Canc:er-Germline/Canc:er-Testis Antigens. Changes in gene methylation lead to the expression of a large number of genes encoding a group of antigens referred to "cancer-testis," "cancer-germline," or "cancer-spermatogonal" antigens. Many cancer-testis antigens have been described in humans such as MAGE, BAGE, GAGE, LAGE/NY-ES0-1, SAGE, HAGE, and BORIS.422- 430 All cancer-testis antigens are expressed at high levels in spermatocytes in the testis. And most, if not all, are also expressed in the thymus.'30-m Many, but not all, cancer-testis antigens are encoded on the X chromosome."' Most of cancer-testis antigens can be recognized by autologous cytotoxic T-lymphocyte (CfLs), but there is no evidence for these antigens being generally effective targets for cancer cell destruction in human.0 4' 05 PIA was the first cancer-testis antigen to be identified in the mouse mast cell tumor line P815.06' 437 PIA can be induced in a broad range of tumors of diverse histologic origins with the demethylating agent 5-aza-2'-deoxycytidine.433 Various normal tissues including thymus and premeiotic spermatocytes also express PIA.43M 39 While active immunization has only modest protective effects against cancer cell inoculation,440 adoptively transferred T cells (monoclonal or polydonal) targeting only the PIA antigen shrink large tumors(> I em in diameter) for several weeks followed by relapse caused by epitope-loss variants.44t For eradication, cancer cells must first be transfected to express costimulatory molecules, which probably leads to the induction ofT cells to recognize other tumor antigens.442 Remarkably, autoimmunity has not been reported to be associated with a response to this antigen. In humans, the HLA-A2-restricted NY-ESOI peptide has very poor affinity to its presenting MHC class I molecule, but high-affinity TCRs to this antigen have been generated and their efficacy in therapy is being explored.443 While many cancer-testis antigens have been described and studied extensively for two decades, their usefulness as targets eradicating human cancers still remains to be shown. Despite these uncertainties,

occasional successes have been observed. The hypothetical explanation: the relatively few T cells induced against the cancer-testis antigen served to elicit additional antitumor T-cell clones directed against other antigens that are actually responsible for the tumor regression by a poorly understood phenomenon referred to as epitope spreading.434' 444 Numerous cancer-testis antigens have been detected using sera of cancer patients for identifying antigens by recombinant expression cloning (SEREX). Importantly, sera from mice infected with cytopathic or noncytopathic viruses or injected with tumor celllysates also show an autoantibody response of broad specificity, and intriguingly the majority of the identified autoantigens have been previously described as autoantigens in humans.4 6 This suggests that human SEREX antigens may have to be regarded as afterglows of infection-associated immunopathology and/or tissue damage. This is consistent with the assumption that the human adult IgG autoantibody repertoire is the result of lifelong encounters with bacterial and viral agents and tissue damage. Together, usefulness of the SEREX-defined selfantigens remains to be demonstrated.

'M'

Carcinoembryonic: Antigen. CEA is a 200-kDa membraneassociated glycoprotein that is expressed not only in fetal but also in adult nonmalignant tissues such as normal colonic mucosa, lung, and lactating breast tissue.44M 48 It is released into surrounding fluids. At one time, it was hoped that CEA could be used as a marker for early diagnosis of gastrointestinal and other malignancies449 ; however, elevated serum levels of CEA are also found in the absence of malignancy (eg, in smokers and in inflammatory bowel diseases such as ulcerative colitis). Though serum levels of CEA are not useful for detecting early cancer, the level of CEA in the blood can be used to monitor the effects of therapy to indicate whether a cancer has been successfully eradicated or has recurred.450 Using CEA as immunotherapeutic target is highly problematic because of severe toxicity. Mice expressing the target antigen in normal tissues closely resembling expression in humans showed severe toxicity from anti-CEA T-cell responses,205 a result that predicted the severe adverse effects later observed in patients treated with anti-CEA CARs.206

Alpha-Fetoprotein. Alpha-fetoprotein (AFP) was the first defined oncofetal protein.451 Though produced by fetal liver and yolk sac cells, it is also present in small amounts in cells and the serum of normal adults. The amount of this protein is elevated in some patients with cancer of the liver or testis and also in some patients with various nonmalignant liver diseases. Therefore, similar to CEA, using AFP as a marker for the early diagnosis of cancer is of questionable use.452 Nevertheless, assays of AFP can detect primary liver cancer at a time when the cancer is treatable, and AFP assays are also used for monitoring patients after therapy.452 The use of AFP as target for active or passive immunotherapy is complicated by lack of specificity. 417•453

Clonal Antigens Clonal antigens are expressed only on the clone of cells from which the cancer originated.'54 Except for idiotypes of surface Ig-positive B-cell and T-cell malignancies, there

CHAPTER 47 CANCER IMMUNOLOGY

are presently no candidates for other clonal antigens. Immunization of animals against the idiotype induces an idiotype-specific transplantation immunity against the growth of cancers (myeloma. lymphoma, and leukemia) expressing the idiotype.m.•56 Furthermore, idiotype-specific anubodies prevented the growth ofsurface idiotype-positive murine malignant B cells in mice and guinea pigs in vivo and in vitro,4S7~o and occasionally induced the cancers to go into a long-lasting dormant state!61 Finally, idiotypespec::ific monoclonal antibodies have caused several partial remissions and one complete remission in patients with B-cell lymphoma.462 and targeting the idiotype on B-cell malignancies continues to be explored.w-165 lt is necessary to generate different monoclonal antibodies or idiotypic vaccines for each individual cancer to be treated. but the advantage of using such clonal antigens over less restricted tumor-associated antigens is that eliminating the few normal cells bearing the same antigen would probably not adversely affect the patient.

Viral Antigens (Encoded by Viral Genes) Cancer-causing tumor viruses such as SV40, polyoma viruses, human papillomavirus (HPV), hepatitis B virus, hepatitis C virus, human T-lymphotropic virus 1, Epstein-Barr virus (EBV), and other herpes viruses and their antigens are discussed in other parts of the chapter. Por discussion of a

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IMMUNOGENICI'IY OF AUTOCH1110NOUS CANCERS There is the widely held misconception that human cancers are usually less immunogenic than are murine tumors."18 However, convincing evidence indicates that many, probably all, human cancers are antigenic. Similarly, even the most antigenic murine UV-induced "regressor" tumors grow progressively and invariably kill the primary host218.479 (Fig. 47.3). Only transplantation of primary murine tumors into young, syngeneic, immunocompetent, tumor-free recipients reveals their antigenicity and imrnunogenicity. UVinduced regressors are so immunogenic that they are rejected byna'ive immunocompetent hosts. Many experimental cancersrequire preimmunization to be rejected upon transplantation, and the degree of immunogenicity usually refers to the relative strength of antigen-specific protection a tumor can induce against rechallenge with that tumor. Obviously, the resistance of the host to its autochthonous tumor cannot be great at the time it is clinically apparent and progressively growing.2211 Only weak reactions can be detec::ted~1 because resistance of the autologous host depends on vaccinations

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of tumor fragments into nude and normal mice

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70-k:Da glycoprotein (gp70)419' 420' 466' 461) and other important antigens"63-470 encoded by ribonucleic acid (RNA) tumor viruses (eg, murine leukemia virus, maize streak virus,471474 and mouse mammary tumor virus47$), see Coffin416 and Schretber.417

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HG. 47.3. Autachthanaus Cancers are Indistinguishable in the Primarr Hast Regardle• of Whither lbey are Highly Antigenic arNot Only subsequent transplantation into young syngeneic immunocompetent hosts defines these tumors as regressors or progressors, and shows their antigenicity and immunogenicity.

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with the autochthonous tumor following complete tumor removal. 269 Antigenicity refers to the antigens on a cancer cell. However, a cancer may have strong antigens yet fail to induce a response (ie, lack immunogenicity). A cancer may also be antigenic and immunogenic but resistant to destruction by the immune response it induces. Thus immunogenicity, antigenicity, and immunosensitivity must be distinguished. Progressively growing autochthonous cancers differ greatly in immunogenicity as determined by responses of syngeneic, tumor-free hosts. Autochthonous tumors induced in mice with UV are among the most immunogenic cancers; transplantation resistance to these so-called regressor tumors appears to be absolute rather than relative. Rejection by normal syngeneic mice is observed without prior immunization, even when the largest testable doses of tumor cells or fragments are used. 275 Most MCA-induced fibrosarcomas, unless induced in immunodeficient mice (see following discussion),'1110 display an intermediate degree of immunogenicity in normal mice.26a. 269•481 This is shown by the fact that induction of immunologic resistance to most chemically induced tumors requires prior immunization because the initial graft of the tumor generally produces progressive lethal growth.268 Importantly, failure of a cancer to induce a tumordestructive immune response does not mean it lacks either antigenicity or immunogenicity. (See below discussion of sporadic SV40-induced tumors under Immune Surveillance of Cancer28•301 .)

Age and Latency Experimentally, the length of the latency period of a tumor is usually inversely proportional to the dose of carcinogen. In humans, the doses of (ie, the levels of unintentional exposure to) carcinogens and promoters are believed to be usually relatively low. This means usually a long time is needed for the initiated cells to accumulate the multiple genetic events essential for a premalignant or cancerous lesion. Once the neoplastic cells have reached sufficient numbers to stimulate a response, the host may be too old to respond vigorously. The overwhelming majority of human cancers arise in individuals past 60 years of age,254 and it has been shown repeatedly in humans and mice that the immune response to new antigens declines with ag~8 ' 482--4 84 ; this should indude antigens on the developing tumors. Thus we do not know the extent of which cancers arising in old individuals may have been selected or retained antigenicity and immunogenicity. In stark contrast, most experimental cancers are induced at an age correlating to that of young middle-aged humans. If 2 years of mouse life roughly correlate to 60 years of human life, then most experimental cancers mirror cancers developing in humans 30 years old or less. Tumors induced by MCA or transgenic oncogenes are usually produced in mice less than 1 year old.485- 4811 The length of the latency period of these MCA-induced tumors correlates inversely with the degree of immunogenicity. 229.4110- 492 There is no such correlation in UV-induced tumor~75,.79 that begin to develop past 9 months of age, mostly in mice more than 1 year old. Old mice beginning at about 9 months of age

fail to reject various types of highly antigenic cancer cells that are regularly rejected by young mice.255- 258 Even though UV irradiation is immunosuppressive, young UV-irradiated mice remain immunocompetent long enough to select for antigen loss variants.235 This indicates that advanced age must contribute to allowing the developing cancers to remain immunogenic. An intriguing question is how many of the tumors that spontaneously arise in older animals would grow in younger syngeneic hosts. 480 Considerable but only indirect evidence makes it very unlikely that all tumors would grow.

Spontaneous versus Induced "Spontaneous" cancers that develop without any known exposure to carcinogens tend to be less immunogenic than cancers induced by DNA tumor viruses or by deliberate exposure to carcinogen.266' 267' 493--495 Unfortunately, serially transplanted tumors were used for most of these comparisons, and transplantation may have selected for less immunogenic variants, thereby confusing the results. If spontaneous murine cancers more closely resembled human cancers, this could suggest that human cancers are poorly immunogenic.495 However, most human cancers are not "spontaneous" but induced by environmental carcinogens, have never been transplanted, and develop mostly in old individuals that may not select for cancer variants. Most, if not all, carcinogens are mutagens17 and probably always cause the expression of tumor-specific antigens. However, it is not dear why tumors induced with the same dose of chemical or physical carcinogen may exhibit quite different degrees of immunogenicity.268' 479 One reason might be that the actual local dose of carcinogen that is delivered to a particular target cell or target tissue may vary greatly from animal to animal. Another reason might be that mutations are selected that favor malignant behavior irrespective of the degree of immunogenicity of that mutant protein. This is consistent with the observation that considerable differences in immunogenicity of primary UV-induced tumors only become apparent after transplantation into secondary hosts218.479 (see Fig. 47.3).

..Regressors'" from lmmunocompromised Hosts Immunocompetence of the host may not affect the cancer incidence but still influence the immunogenicity of the developing tumor. Conversely, immunosuppression or immune deficiency of the host during carcinogenesis should allow growth of highly immunogenic tumors in the absence of such selection.

Clinical Evidence Clinical support comes from the appearance of highly antigenic EBV-associated lymphomas in immunosuppressed transplant recipients that are virtually unknown to occur in immunocompetent humans.502 In addition, renal transplant patients experience a reduced risk of developing UVinduced skin cancer after immunosuppressive medications are stopped.503 It is tempting to suggest that many of these

CHAPTER 47

cancers developing in transplant patients are actually "regressor" tumors that have arisen only because host defenses were damaged.

Experimental Evidence In normal adult mice, Moloney sarcoma virus496 induces tumors larger than 2 em in diameter that then regress while tumors continue to grow and kill immunodeficient adult or newborn mice.497-499 The first experimental evidence for UV-induced regressors came from Kripke in 1974, 275 showing that UV irradiation made mice immunodeficient and induced cancers that were often "regressors," meaning that they were rejected with any testable size inoculum by naive syngeneic immunocompetent mice. Thmor transplants will grow for about a week and then disappear, though small numbers of the same tumor cells will grow and kill athymic nude mice. The same regressors grew after inoculation of few cancer cells in UV-irradiated mice. So, it may be highly relevant that some carcinogens are immunosuppressive. 500'501 Repeated exposures of mice to UV induce persistent immune suppression,502-504 leading to the development of highly immunogenic regressor tumors. In contrast, the single injection of MCA induces tumors in 100% of mice and only a short-lived state of immune suppression, 501 thus allowing a fully immunocompetent host to select for less immunogenic variants. The concept that immunocompetence of a host influences the immunogenicity of the developing tumor has been tested experimentally by Roberts and Daynes480 comparing the MCA-induced tumors occurring in immunodeficient UV-irradiated mice with cancers induced in normal mice. Indeed, cancers induced with MCA in mice immunosuppressed with UV were frequently regressor tumors, whereas none of tumors induced with MCA in immunocompetent mice were regressors.480 Decades later, this concept was confirmed with tumors induced with MCA in nude, severe combined immunodeficiency, or Rag-/- mice.487,so5,so6 Selection by the Immunocompetent Host Immunocompetent hosts can select for cancer variants. Thus, when regressors are transplanted into normal immunocompetent hosts, heritable progressor variants can escape. Some of these variants show antigen loss,218•219.507-509 but many others retain their antigenicity and grow faster than the parental tumor when their growth is compared in T, B, or NK cell-deficient hosts,43' 45'218,307'505'508'5m suggesting that mechanisms other than antigenicity must participate determining the differences in growth behavior between regressors and progressors. ..Regressors'" from Immunocompetent Hosts Human cancers are sporadic (ie, occur at irregular times and locations); this is in part due to the occasional genetic events that can cause malignant transformation in a single cell that expands to become a detectable tumor. A murine model of sporadic genetic events is the recombinational activation of a normally silenced transgene encoding the strongly oncogenic and strongly antigenic molecule SV40 T,

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thereby causing sporadic appearance of cancers. 228•301 These tumors are regressors. The tumors "snuck" through immune surveillance and immunoselection despite expressing such a powerful antigen and oncogene, but not unnoticed. The developing cancers did induce antibodies and proliferation of antigen-specific T cells during their early dormant phase of growth, but developing tumor-specific T cells were anergized in that they failed to produce interferon (IFN)y and kill tumor cells in vivo. In contrast, titers of the T antigen-specific IgG response increased with progressive tumor growth.

EFFECTOR MECHANISMS IN CANCER IMMUNITY Because cancer is not a single disease, it is not surprising that findings using one tumor model may not apply to other tumor models. Considering the antigenic diversity found in tumors, it is also not surprising that different types of innate and adaptive, humoral and cell-mediated immunity have been shown to play different roles in the destruction or enhancement of malignant cells in one or another of the numerous tumor models.

Assays to Study Effector Mechanisms in Vivo In principle, five different assays have been used to evaluate the importance of different effector mechanisms in vivo. The first type of assay involves transfer of effector cells, cytokines, or antibodies into sublethally irradiated, cyclophosphamide-pretreated, or normal animals challenged with tumor cells. There are certain limitations of this assay. Effector cells or molecules may not reach or localize in the tumor unless both the effector cells and cancer cells are injected intravenously, and both may be trapped in the lungs. Furthermore, if transferred cells or reagents are effective, the assay does not rule out that other effector mechanisms of the host may have been activated by the procedure. In a second procedure, called the Winn assay,229•269•511 tumor cells are mixed with effector cells or serum in vitro; the mixture is injected subcutaneously into an animal to determine whether tumor growth in vivo is prevented. Tumor cells may be killed within minutes before or shortly after the injection, though the readout takes much longer. Therefore, the Winn assay is, in part, an in vitro cytotoxicity assay, even though the host is used as a readout for viable cancer cells. A third method involves elimination of specific lymphocyte subsets or cytokines in vivo by treatment with antibodies specific for different lymphocyte subsets or cytokines. Failure ofthe host to resist a tumor challenge indicates that the particular subsets or cytokines are an essential component of the host resistance. A fourth method is to use mice genetically deficient in a certain effector mechanism, cell, or cytokine. An analysis of tumor variants that have escaped tumor destruction by the host provides a fifth way for determining the importance of immunologic effectors in vivo.43-45•218•235• 236•507·512 The phenotypic changes observed in these variants may indicate which effector mechanism was responsible for the selection. Therefore, the type of phenotypic change

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may give insight into the relative importance of a naturally occurring defense mechanism that may function in immunocompetent mice (analogous to deducing the action of an antibiotic from the type of change found in the bacterium that has become drug resistant). Host-selected variants from experimentally induced regressor tumors are used extensively for this approach.

Antibodies and B Cells The role of B cells in regulating tumor immunity continues to be studied extensively yet remains poorly understood. In a tumor model of leukemia in which CD4+ helper T cells were required for successful treatment, B cells were necessary for efficient T-cell priming.513 Conversely, in other tumor models, elimination of CD4+ T cells promoted tumor rejection by CDS+ cells, and an absence ofB cells improved CTL responses and tumor rejection.514- 516 Antibodies and B cells have been associated with enhanced papilloma formation and autochthonous cancer development.341.489•517- 519 The mechanism recently postulated is that regulatory B cells help the macrophage become tumor-promoting,520'521 or that they suppress surveillance by T cells that normally eliminate neoplastic cells with oncogenic mutations.519 There are other possible mechanisms.342•343 Also, none of these experiments identified the antigens involved, except one study showing that induction of mutant-ras specific-antibodies correlates with much more effective papilloma development in an autochthonous tumor model. 341 Human antisera and monoclonal antibodies reactive with autologous tumors have been isolated.420•522 However, a strong humoral response to tumor antigens does not correlate with demonstrable resistance of the host to the tumors. TL+ leukemias induce high titers of TL-specific antibodies that are cytotoxic to TL+ leukemia cells in vitro in the presence of heterologous complement,420 but TL+ leukemias grow equally well in immunized mice having high titers of TL antigen-specific antibody and in nonimmunized mice.523•524 Similarly, humoral immune response to MCA-induced sarcomas does not provide protective immunity against a tumor transplant.525 Obviously, the presence of antibodies for these kinds of tumors has no relevance in predicting whether the host will reject the tumor. Normal or malignant cells of hematopoietic origin are generally lysed quite effectively by antibody and heterologous complement in vitro; however, normal cells such as fibroblasts or malignant cells derived from solid tissues may be much less affected, even when expressing high levels of antigen. The reasons for this striking difference are still unclear. In vitro, some tumor cells are killed by a process involving coating with antibody, opsonization, and subsequent phagocytosis by macrophages; this process may be enhanced by the presence of heterologous complement. Alternatively, antibody-coated tumor cells may be killed in the absence of phagocytosis by antibody-dependent cellmediated cytotoxicity when cocultured with macrophages, NK cells, or neutrophils. The general relevance of these mechanisms for killing tumor cells in vivo is unclear. Exogenous antibodies that block growth factors and/or their

receptors or activate costimulatory molecules are useful for some kinds of immunotherapy (see Immunotherapy).

T-Lymphocytes It has been demonstrated convincingly that T cell-mediated immunity is critical for rejection ofvirallf26- 528 and chemically induced tumors, 229'26!1,529 or for the rejection of allogeneic530 and UV-induced tumors. 218' 275' 503'508 For example, in the model of murine MCA-induced tumors, it was shown that intravenous injection of immune cells, but not of immune serum, could transfer systemic tumor-specific immunity into sublethally x-irradiated mice.229 These findings were consistent with earlier work with allogeneic tumors showing that lymphocytes not serum were effective in transferring transplant resistance.531-533 In another study, transfer of immunity to a plasma-cell tumor was abolished by pretreatment of the immune cells by anti-T-cell antibodies and complement.529 The relative importance ofvarious T-cell subsets in tumor rejection (ie, Tel [type I CDS+ cytotoxic T cells]; Tc2 [type II CDS+ cytotoxic T cells]; T H1 [type I CD4+ helper T cells]; TH2 [type II CD4+ helper T cells], Tcl7 [IL-17-producing CDS+ T cells], etc.) have been the subject ofrepeated, and probably unnecessary, controversies. 534,s35 Different tumors are dissimilar enough so that differences would be expected in T-cell subset requirements. Also different therapeutic settings may require different subsets (eg, to prevent cancer development or destroy premalignant lesions), established solid cancers, malignant effusions, microdisseminated cancer cells, or leukemic cells. Dependent on their subtype, T cells produce and induce various cytokines and chemokines that may destroy tumors by direct effects on tumor vasculature or recruit neutrophils, macrophages, and NK and other innate effector cells that are needed for tumor eradication.536 Many recent studies employ overexpressed model antigens and TCR-transgenic T cells not available in humans; thus, results need to be confirmed using non-TCRtransgenic models and genuine tumor-specific antigens. Interestingly however, adoptive transfer of TCR-transgenic CDS+ T cells specific for a model antigen can eliminate large established tumors536-540 or artificial pulmonary metastases541 without the T cells needing perforin.536,s41 Release of IFNy and tumor necrosis factor (TNF) by the T cells seems to be critical; receptors for both cytokines must be expressed on bone marrow- and non-bone marrow-derived tumor stroma to kill T cell-resistant cancer variants as bystanders or relapse may occur.s37,538,54o,542 CD4+ T-cell subsets can influence antitumor immunity, and truly tumor-specific CD4+ T cell-recognized tumor antigens exist.1°CD4+ T cells seem to be critical at the effector phase of CDS+ T cells for cancer cell destruction in vivo,543' 544 for sustaining CDS+ T-cell memory/45 and for the survival of adoptively transferred CDS+ T cells.546•547 However, CD4+ T cells do not necessarily require recruitment of CDS+ T cells for eliminating cancer cells in vivo, 10•371•548- 551 even when the cancer cells are MHC class II negative and killing as well as presentation must be indirect.10'549'552'553 Destruction of the cancer cells

CHAPTER 47

in vivo requires IFNy, but occurs even when cancer cells lack the receptor for it.552 Thus the effects of CD4+ T cell-released IFNy must be on stroma, most likely tumor vasculature. 554' 555 Certain CD4+ subsets may also suppress tumor rejection, because elimination of the CD4+ T-cell subset may increase tumor resistance in certain tumor models. 514•556 Now tenned Treg cells, these Foxp3+ CD25+ T cells mostly suppress the induction of immune response to new antigens, reduce anticancer immunity particularly to self-antigens, and suggest poor prognosis when prevalent in excised cancers.557 However, it is uncertain whether these T,..gS can prevent adoptively transferred tumor-specific memory T cells from eradicating cancers.

Natural Killer. Lymphokine-Activated Killer. and Natural Killer T Cells NK cells are distinct subpopulations of lymphocytes that, without prior sensitization and without the requirement for MHC restriction, can kill some cancer cells, particularly while circulating in the bloodstream, as well as nonmalignant nonself cells553-565 (see Chapter on NK cells in this book). NK cells occur as 1) "resting" NK cells that nevertheless kill very sensitive targets such as YAC, 2) "activated NK" cells induced within hours by IFNaj} to become cytolytic but without proliferation (many conditions and microbial agents rapidly induce IFNaj}), and 3) lymphokine (IL-2)activated killer (LAK) cells developing after days of culture in high doses of IL-2 and requiring proliferation. NK cells can "recognize" the absence of self66 (ie, the missing MHC allele fails to provide an inhibitory signal to prevent the activation of NK cells to kill the target567 ). Therefore, cancer cells that fail to express at least one of the MHC class I alleles of the host are killed more effectively.568•569 Transformed cells often have decreased or lost MHC class I surface expression or have induced expression of ligands for activating receptors on NK cells and thus are targets of NK cells. In vivo, NK cells or NKG2D-mediated effects inhibited autochthonous and transplanted tumor formation and tumor recurrence, and reduced metastatic dissemination of intravenously injected cancer cells.570-574 However, NK cells were effective usually only at incipient stages.57s,576 In patients, intratumoral NK cells may reduce metastatic seeding, thereby leading to longer survival,577 and NK cells may be a key factor in the occasional cure of acute myeloid leukemia and childhood acute lymphoblastic leukemia by allotransplantation.578 Activation of peripheral blood cells in vitro with high doses of IL-2 induces LAK cell.579 Cancer cells, even when resistant to NK cells, are usually susceptible to killing by LAK cells in vitro, whereas most nonmalignant target cells have been reported to be resistant to killing by LAK cells.580 Intravenous injection of LAK cells early after intravenous seeding of cancer cells into mice reduces the metastatic tumor cell growth in the lungs; however, with this procedure, both LAK and cancer cells are trapped in the lungs.581' 582 Antitumor responses have also been reported in humans after adoptive transfer of LAK cells in

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patients with renal cell carcinoma and melanoma. 583 This selectivity is difficult to explain, considering the general susceptibility of cancer cells to LAK cells in vitro. In more recent studies, adoptive transfer of NK cells activated in vitro with IL-2 failed to cause tumor regression in patients with melanoma.584 The cells that mediate the killing in vitro of a broad range of malignant cells are more than 90% activated CD16+/CD3- NK cells/85•586 but which cells have antitumor activity in vivo is not fully established. Even though murine LAK cells can be generated from nude mouse spleen cells, 587 it has not been demonstrated that LAK cells from nude mice or normal mice have similar therapeutic effects against tumor cells in vivo. Other cell types, such as CD3+ lymphocytes, which are regularly present in every preparation of LAK cells, may contribute significantly to the killing of tumor cells in vivo, particularly because activated CDS+ T cells express the stimulatory lectin-like NKG2D receptor and can kill tumor cells expressing the ligands for the receptor. 588 Many surface receptors originally discovered in NK cells are expressed by subsets of T cells such as NKT cells, defined by the invariant Va14/24Ja18 TCR a chain.539 NKT cells, when treated with IL-12 at the time of cancer cell inoculation or after 1 day, inhibited tumor development and metastasis.590' 591 Also, the development of tumors induced by MCA was found to be reduced in some but not other studies.486,489' 592 In any case, clinical studies are exploring the usefulness of NKT cells in certain cancers.593-596 NKT cell infiltration of neuroblastoma is associated with a favorable outcome; NKT cells may target the tumor-promoting stromal macrophages rather than the neuroblastoma cells directly.5117 Together, previous studies have failed to provide evidence that NK, LAK, or NKT cells can eliminate well-established solid tumors casting continuing skepticism about the clinical usefulness of these cells in the therapy of advanced solid tumors. 575' 576' 598 A recent study, however, shows even very large solid tumors being eradicated by NK cells alone when the NK cells are properly activated by IL-15 in vivo at the site of the tumor.599

Macrophages and Granulocytes Neutrophils rapidly appear at the site of injury and respond very rapidly to diverse chemotactic and inflammatory stimuli.20° Furthermore, neutrophils often appear to pave the way for the influx of inflammatory monocytes that then mature to "angry" classically activated macrophages (or M1 macrophages) (eg, after "classical" activation by intraperitoneal/pleural injection of thioglycolate1os, 106•600-602). However, such inflammatory stimuli are usually absent in cancers. As a result, neutrophils can be relatively rare (0.1% to 0.4% of the total cells in solid tumors).603 Macrophages, however, are regularly found in the microenvironment of solid tumors and are usually at least 10 times more abundant than neutrophils,604 sometimes a third or more of the tumor mass. These tumor-associated macrophages differ from "angry:' tissue-destructive, "classically activated" macrophages (or Ml) but are "alternatively" activated macrophages (M2).

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"Alternatively" activated macrophages seem to be derived from resident tissue macrophages that proliferate.105•106 Cancers may not recruit tissue-destructive inflammatory cells (N1 neutrophils and M1 macrophages); apparently, cancer cells lack the signals necessary for activating innate immunity,605 but cancer cells transfected to express CD95L induce cancer cell-destructive neutrophils.606 Therapeutic interventions may aim at converting cells of innate immunity to a tumor-inhibitory "type 1" response pattern. IL-4, the prototypical "type 2" cytokine, can inhibit growth of cancer cells transfected to produce this cytokine by activating granulocytes73'127' 137' 138 and can activate macrophages to kill cancer cells in vitro,607 but may also interfere with eventual clearance leading to outgrowth of escape variants.142 IL-4-secreting tumors are heavily infiltrated by eosinophils,73'127 and many clinical and experimental studies reported eosinophilic tumor infiltrates.535 Nevertheless, the contribution of eosinophils to tumor destruction and tumor growth is still incompletely understood.138·535·608 Macrophages and neutrophils from normal donors are generally not cytotoxic to tumor cells or normal cells in vitro; however, macrophages and neutrophils can be activated by bacterial products and IFNy (or other cytokines) in vitro to cause selective cytolysis or cytostasis of malignant cells.609-617 Fully activated macrophages require long-term 16- to 72-hour assays to demonstrate in vitro tumoricidal activity in isotope-release assays or cytostatic activity in growth-inhibition assays. Some of the cytolytic or cytostatic effects of macrophages on tumor cells involve cell contact and/or the secretion of various cytotoxic substances, but phagocytosis may also play an important role.618 TNF-0:619,620 produced by thioglycolate-activated macrophages can account for all of the classical tumoricidal effects of macrophages against some cancer cells in vitro.621--&4 However, as might be expected because of the plethora of cytotoxic molecules that can be released by activated macrophages, 20o,625 other mechanisms such as reactive nitrogen intermediates can also be important mediators of killing of cancer cells in vitro.626'627 Systemic activation of macrophages reduces metastatic seeding,628 while a (nonactivated) colony-stimulating factor 1-dependent macrophage subset seems to help metastatic seeding.629 Because of the rather selective cytotoxicity of thioglycolate-activated macrophages against malignant cells, numerous studies have considered the potential role of this cell type in cancer (see Cancer and Inflammation, Immunotherapy, Factors Limiting Tumor Immunity for discussion of myeloid-derived suppressor cells). As would be expected, these in vitro-activated leukocytes mixed with cancer cells and injected into an animal prevent cancer development from the inoculum "in vivo" (a Winn assay, see previous discussion).630 Interestingly, dependent on the role neutrophils have in a particular cancer model, their transient elimination in vivo can either abrogate or enable T cell-mediated transplantation resistance.44•631 However, there is a major difficulty of effectively eliminating macrophages or neutrophils long-term in vivo. Therefore, there is, at present, no critical evidence to establish or refute the idea632 that macrophages and or granulocytes activated

in vivo destroy nascent tumors and therefore play a role in immune surveillance.

FACTORS LIMITING CANCER IMMUNITY Various mechanisms allow cancers to escape innate or adaptive host immunity, principally by 1) inducing a protective and supportive stromal microenvironment, 2) increasing resistance to direct attack, and 3) inducing T-cell anergy. Often, cancer cells seem to use a combination of the three, but numerous lines of evidences indicate that the first is key and virtually always part of tumor progression and escape. Unfortunately, most experimental tumor immunology depends on observations using cancers serially transplanted for decades. This is surprising, knowing that a single transplantation of an autochthonous tumor into an immunocompetent host dependably results in heritable variants that avoid destruction by the host43-45'218' 219' 508 (see above under Experimental Cancer, Key Principles, and Selection of Tumor Model).

Tumor Microenvironment Many escape variants keep their antigens but induce a stroma more effectively than the parental cells for better support of growth and protection against destruction by the host (also see Cancer Stroma). Experiments have shown that stroma is critical for preventing or permitting immunologic destruction of cancer,47 and it is likely that cancer stroma is also an important factor in causing very early cancers to resist therapeutic immunization.633 Local factors must explain why tumor-bearing mice, while failing to reject a primary tumor transplant, reject a later implant of small numbers of the same cancer cells at second sites, a phenomenon called concomitant immunity (see discussion below under Cancer and Inflammation, Facilitation of Inhibition of Metastasis). Local factors particular to the tumor environment must also explain why mice bearing malignant grafts fail to reject the established tumors but reject nonmalignant grafts expressing the same rejection antigen634; T cells in these tumorbearing mice are neither clonally exhausted nor systemically anergic.635 Antigenic stroma, as it exists in nonmalignant allografts635 or when strong tumor-specific antigens are crosspresented by tumor stroma,537-540'542 can help T cells to eradicate cancers. In addition, recent work in two tumor models suggest that lack of "help" at the site of cancer growth may be an important reason for the failure of cancer cells to be rejected by CDS+ T cells, thereby stressing the importance of CD4+ T cells cooperating with CDS+ T cells in the effector phase.543' 544 The situation might be somewhat analogous to transgenic mice that express allo-MHC class I molecules as self-antigen on islet cells and have autoreactive T cells that infiltrate the islets,636 but even after priming, the autoreactive cells fail to destroy the islet cells unless local help is provided in the form of IL-2.636 Antigen-specific T cells can infiltrate even tumors growing in immunologically privileged sites, but proper differentiation of the infiltrating T cells is prevented.301.637~ Several different models have shown that the

CHAPTER 47 milieu of large tumors can inhibit the function of adoptively transferred T cells.640- 646 Lack of costimulatory molecules or expression ofFas ligand by the cancer cells may lead to peripheral anergy.647~49 Suppression in the tumor microenvironment may also be the result of macrophages long known for abrogating immune responses when prevalent in culture.650 In the stroma, the macrophages are alternatively activated and produce indolamine-2,3-dioxygenase, inducible nitric oxide synthetase, lactate dehydrogenase-A, and myeloid cellderived arginase.651- 653 Autochthonous cancers transplanted once and reisolated are usually heritable variants that grow more progressively, more effectively attract macrophages and neutrophils that are pro-angiogenic {see Cancer Stroma), and more rapidly induce a protective and supportive stromal microenvironment.43-45 Treatment with RB6-8C5 allows mice to reject a lethal tumor cell inoculum,43--45 thereby indicating the profound role that Gr1+ CDllb+ leukocytes have in helping tumors to escape. The significance of tumor-infiltrating Treg cells is discussed separately in the following. Finally, tumor-bearing mice and cancer patients may have alterations in the signal-transduction machinery in T cells, particularly in those infiltrating the tumors but also in T cells from draining lymph nodes or, at later stages, even in circulating T cells.654- 656 NK cells can be similarly affected.654 A decrease in NF-lCB p65 at an early stage is followed by loss ofTCR ~chain and p56lck after continued tumor growth.657~62 Activated macrophages can secrete substances that induce these structural abnormalities.663 As another example of subversion of host defenses in the tumor microenvironment, tumors are a privileged site for bacterial growth.664'665 Together, numerous immunosuppressive mechanisms have been shown to be functioning in the tumor microenvironment and the tumor draining lymph node. Their relative significance is difficult to judge because there is rarely a real "positive control" {ie, destruction of a truly long established large solid tumor in the absence of the implicated mechanism).

Resistance to Direct Attack Cancer variants can become resistant to direct attack by T cells by 1) loss/downregulation of MHC class I and II molecules, 2) losing expression of the rejection antigens, or 3) increased resistance to the killing pathways. Selective loss or downregulation of MHC class I molecules or the associated antigen-processing machinery allows cancer cells to resist direct attack by T cells.237•666- 692 These mechanisms allow cancer cells to escape while retaining the antigen that might be essential for cancer cell survival and malignant behavior.10'302'307' 693 As noticed already decades ago, MHC-heterozygous F1 tumors can escape destruction when transplanted into either parent by loss of the mismatched MHC antigens.694•695 This agrees with the commonly observed loss of the mismatched HLA after haploidentical hematopoietic stem cell transplantation.696•697 MHC homozygous tumors rarely lose MHC antigens and require MHC compatible strains for serial transplantation, and they were therefore an essential tool to define the MHC.696 Two cancers are known to be naturally transmitted

CANCER IMMUNOLOGY

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within a species as cells.699 The devil facial tumor disease, first observed in 1996, is a lethal cancer cell line serially transmitted by facial bites during fights among Tasmanian devils.700•701 These marsupial carnivores in Tasmania have very little MHC diversity and are in rapid decline due to spread of the cancer. Canine transmissible venereal tumor, first described in 1876,260 is passed between dogs through coitus and bites/02-704 The tumor cells express very little 1}2-microglobulin and MHC class I during the progressive growth phase followed by regression after -6 months unless the dog is immunosuppressed. Some cancer cells express low levels of MHC, but levels can be upregulated by cytokines. At least some of these changes seem to be due to epigenetic mechanisms.705' 706 In other cancer cells, irreversible loss ofMHC class I expression is caused by several molecular mechanisms, including mutations in the gene coding for 1}2-microglobulin.672'674'675'707 Loss of a single HLA class I allele was found more commonly than loss of all class I alleles.706 The E1A gene of the adenovirus {Ad) strain Adl2 suppresses MHC class I expression in transformed cells that are tumorigenic and escape T cell-mediated destruction/08 but there was no correlation between the level of MHC class I expression and tumorigenicity of Ad2 and Ads transformed cells. Oncogenes such as myc can cause locusspecific suppression of MHC class I antigen expression.709 Importantly, MHC class I molecules have now been shown to act as tumor suppressor genes.710 Thus, some of the observed changes in MHC expression may not be the result of immunoselection. Cancers also escape by loss of expression of the rejection antigens.218·219·236·507·508 Even when the antigen is necessary for cancer cell survival,10'302" 07'693 point mutations might preserve the oncogenic potential while rendering the protein nonantigenic. Mutations in SV40 large T that preserve the transformed phenotype in vitro have been selected for with CTL clones, but, in vivo, evidence is lacking for tumorigenicity or selection of such clones.711 Interestingly, mutational changes in E6 and E7, the transforming genes of HPV, have not been observed in cervical cancer while total or allelic loss of HLA class I expression is commonly observed in this cancer.712 Immune selection has been reported in patients at later stages of malignancy and after therapy.713- 715 Partial immune suppression may lead to a higher yield of antigen loss variants.235'716 UV-irradiated mice show a partial immune deficiency so that the generation of cytolytic T cells is delayed. 23s,717 Incomplete therapy of bacterial infections with antibiotic drugs also favors the outgrowth of variant bacterial strains that show heritable resistance to these drugs; by analogy, partial or incomplete immunotherapy of cancer-bearing individuals may lead to selection of antigenloss variants. When targeting cross-presented tumor antigen on tumor stroma only, T cells do not select for cancer cell variants.169 Thus, direct (or very short-range) cell-cell interactions appear to be essential for selecting antigen or MHC-loss variants. Antigenic modulation, a reversible antibody-induced loss of surface antigen, ' ' ~~, and cellular resistance to perforin/granzyme,72° FAS/CD95,721 and other pathways to evade destruction are not discussed. 523 524 71

719

1m

SECTION VIII

IMMUNOLOGIC MECHANISMS IN DISEASE

Anergy/Unresponsiveness/Neonatal Tolerance Antigens newly expressed on cancers in the adult may be ignored at the earliest stages of autochthonous cancer devel. amount sof antig'en. 191,223,301,722 . ffi cient opment because of msu But once the tumor grows and the amount of antigen increases the host responds. 723 Thus, immunologic ignorance is unlikely in cancer patients. Tumor transplantation induces acute inflammation that may influence the immune response to the transplanted tumors.724'725 By c?ntra.st, the antigens on sporadic autochthonous tumors fail to mduce tumor destruction.228'301 Importantly, these cancers grow progressively though they are not resistant to direct immune attack (eg, by adoptively transferred T cells). Furtherm~re, tumor antigen-specific CDS+ T cells are remarkably mcreased in number in mice bearing autochthonous cancers but failed to lyse antigen-specific targets in vivo and the production ofiFNywas low whereas the production ofTGF-1}1 was increased and high titers oflgG antibody specific for the rejection antigen were observed.223'301 Thus, antigen-specific B and T cells in mice bearing the autochthonous tumors responded to the rejection antigen but in a way that resulted in · t h e cancers. Thisaltered responsiVeness . ..7 26'127 failure to reJect must be distinguished from "peripheral" or "extrathymic'' tolerance that occurred under certain conditions when an antigen was presented to an adult in highly im~unogenic form with the consequence of temporary expansion of mature T cells followed by clonal elimination.728 The important distinction is that donal deletion would require adoptive T cell transfer whereas retention of antigen-specific T cells in the host bearing autochthonous cancers may allow rescue or induction of a tumor-specific destructive T-cell response. Thymic tolerance to self may explain why oncofeta.l and carcinoembryonic or oncospermatogonal self-antige.ns, all expressed in the thymus, induce weaker protect~on than is found in animals immunized with tumor-specific antigens.364.453•729•730 Nevertheless, at least certain cancers seemingly do not follow the rules that prevent immune r~c­ ognition of normal self-tissues. Thus, some tumor-bearmg hosts may readily recognize normal differentiation antigens on certain cancers such as melanomas. In this type of cancer, it appears to be possible to uncouple the mechanisms of autoimmunity from tumor immunity.731'732 However, it is unclear how effectively these T cells can contro1 tumor growth .733 Epigenetic memory can prevent self-reactive CD8+ T cells from escaping their tolerant fate.734 Because the thymus "selects the useful, neglects the useless and destroys the harmful" T cells,m(p.s7) only self-antigen-reactive T cells with t~ low avidity to cause destruction escape deletion and prormnently infiltrate tumors.736' 737

Age As already discussed (see Immunogenicity ofAutochthonous Tumors), most cancers developing in older individuals may be fully sensitive to tumor-specific T cells but for age-related reasons fail to induce an effective immune ~e­ sponse. Immunotherapy in older individuals may require rescuing the age-dependent immune deficiencies of the host

environmenf38 as well as the T cells484 because adoptively transferred lymphocytes from young, but not old, immunized mice eradicate large solid tumors. 258

Regulatory T Cells/8 Cells/Antibodies/ Blocking Factors Passively administered antibody can prevent the rejection of tumor allografts (homografts).739-'741 These anti'bo dies are also referred to as "enhancing antibodies." "Blocking factors" are complexes of antigen and antibodies that can . . m . v1'tro.742,743 C omp1exes suppress cell-me diated tmmumty of tumor antigen and antibody can induce suppressor T celli44 (now generally referred to as Tregs) that can suppress specific immune responses in vivo. Treatments of recipients with donor-type cells as antigen and homologous antibody to donor-type cells enhances acceptance of rat renal allografts.745' 746 The mechanisms responsible for these findings are incompletely understood. The induction ofT s required active TGF-1}; latent TGF-1} linked to IgG cau:s the suppression of sensitization/proliferation of CDS+ effector T cells. Recent and earlier experiments have pointed at a joint role of CD4+ T cells and B cells/ antibodies in preventing the rejection of transplanted tumors514•515 and enhancing the development of autochthonous cancers in mice.341' 517' 520 Eliminating B cells or CD4+ T cells can have virtually identical effects in preventing . . m . t h e same tumor mo del, 514,515 poi'ntm'g tumor reJection at the fact that B cells are required for multiple functions of CD4+ T cells and vice versa747-750 and that T,o:ss may not necessarily be generated or act independent of B cells and antibodies.751•752 "Suppressor T cells" can suppress tumor rejection.514·556·753-755 Residual cancer cells remaining after incomplete tumor removal can be sufficient for continuing the suppression/56 However, unlike what has been reported/56 suppression is short-lived. aft~r com~lete ~umor removal and may give way to specific 1mmumty Without further immunization.757 Unlike CD4+ helper T cells, T,o:ss usually express CD25 and Foxp3?58-762 The Foxp3+ CD~5+ T cells are essential in preventing autoimmune destruction of the hosf59 and suggest, as discussed previously, poor . when preva1ent m . excise . d cancers.763 TregS are prognosis sensitive to low-dose gamma irradiation and cyclophosphamide pretreatment.764'765 Preirradiation, cydophospham~de pretreatment, T-cell deficiency, or CD4+ T-cell deplet~on of the tumor-bearing host facilitates tumor destructiOn by adoptively transferred immune T cells.766'767 However, these treatments also cause homeostasis-driven expansion and activation of the transferred T cells/68 which may be sufficient or at least synergize with the effects of T,o:ss depletion. However, whether Trcgs can suppress memory T cells needs further study. In any case, these proposed mechanisms and observed effects confirm the early pivotal discovery that low dose whole-body x-irradiation and/or chemotherapy are an essential adjunct to adoptive tumor immunotherapy in mice.766'767 "Nonmyeloablative ly~­ phodepleting chemotherapy" is now standard treatment m melanoma patients before adoptive T-cell transfer.769

CHAPTER 47

Myeloid-Derived Suppressor Cells 75 79 -

68

MDSCs are a subset ofmonocytes that express CD11b and Ly6C (resulting in intermediate levels of Gr-1 (RB6/8C5) staining), but lack Ly6G (defined by 1A8) (see Components, under Cancer Stroma). They differ from alternatively activated tumor-associated M2 macrophages in well-established tumors 3 weeks after transplantation and from neutrophils. Neutrophils express high levels of Ly6G and Gr-1 and intermediate levels of Ly6C. Cells of myeloid origin are increased in numbers in the peripheral blood of tumor-bearing mice and cancer patients, especially in later stages. In the mouse, MDSCs come from bone marrow as well as spleen, which is a natural extramedullary blood-forming organ in this species. MDSCs and blood monocytes seem to be the predominant precursors of intratumoral macrophages not only in transplanted but also autochthonous tumors.6s.no A vast number of studies indicate that MDSCs isolated from the peripheral blood of cancer-bearing individuals are profoundly immunosuppressive in vitro. How this correlates to immune suppression in vivo remains an open question.n.78

IMMUNE SURVEILLANCE OF CANCER Types of Surveillance Mechanisms The term surveillance should be restricted to its meaning (ie, protection from cancer developmenf71). Early in the last century, Ehrlich wrote, "I am convinced that the development of aberrant [mutant] cells occurs very frequently during the extremely complicated fetal and postnatal development but that these foci luckily remain completely latent in most humans because of protective mechanisms in the host. If these mechanisms were not existent, cancer would probably develop with an incredible frequency."261 ll~ III~IVII!Iol'l

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C.M 8,000/,.U.), and granulocytosis is common in tumor-bearing mice.871•1183•919--925 It is long known that systemic effects, such as splenic enlargement,926 frequently accompany local growth of autochthonous or transplanted murine tumors. Although many tumors commonly used in experiments have been serially passed hundreds or more times through euthymic mice,239 a similar enlargement is also observed in response to growth of primary autochthonous tumors in nontransgenic mice.927 Enlargement is mostly caused by increased myeloid hematopoiesis with macrophages, monocytes, and neutrophils accounting for more than half of the splenocytes.770•928 G-CSF,

CHAPTER 47 GM-CSF, and/or IL-6,871'876'878' 883' 929'930 released by cancers, may be particularly important in inducing the systemic effects. STAT3 is persistently activated in many types of cancers, particularly in cancer cells at the invasive edge of tumors adjacent to inflammatory cells.849 IL-6 preferentially activates STAT3,931 causing cancer cells to resist apoptosis, sustain angiogenesis, and suppress acute STATl-driven inflammation849•931•932 and destructive T-cell responses. G-CSF plays a central role in neutrophil production. Importantly, G-CSF also induces stem cell mobilization by upregulating CXCR4 and decreasing its ligand SDF-1 (CXCL12) in the bone marrow/54'933 spleen, and local86.88 reservoirs for progenitors. Degradation of SDF-1 in the progenitor/stem cell reservoirs is probably a result of G-CSF-induced granulocytosis that causes an increase of the neutrophil-derived proteases (TIMP-free matrix metalloproteinase-9 and neutrophil elastase, specific products of neutrophils that degrade SDF-1934-936). Mobilization of progenitors expressing CXCR4, the receptor for SDF-1, includes the mesenchymal, hematopoietic, and angiopoietic progenitors and Treg cells. 146'937 Attraction of these cells into the tumor stroma of a neoplastic lesion occurs along a chemotactic gradient and depends on high local concentrations of SDF-1 produced by cancer cells87g,sso.938 and twnor-stromal myofibro blasts.939' 940 Given these mechanisms, it is not surprising that elevated neutrophil blood counts, neutrophil/lymphocyte ratio, matrix metalloproteinase-9, and plasma levels of C-reactive protein have been described as prognostic indicators of recurrence and reduced survival.941-945 However, systemic responses to cancer vary greatly between individuals depending on stage and type of cancer. Even when they are not detectable, the mechanistic loops outlined previously are likely needed locally for every malignant growth,

Facilitation or Inhibition of Metastasis A systemic increase in circulating factors such as IL-6 and progenitor cells may facilitate metastasis by creating "metastatic niches" (ie, a stroma in which disseminated cancer cells can successfully engraft).629' 946-954 Experimentally, there is little evidence that facilitation of metastasis (or secondary tumors developing at distant sites) is antigenspecific.757•884'955 However, a primary transplanted tumor may suppress the growth of a second inoculation with the same tumor. 261 The phenomenon can be caused by immunologic as well as nonimmunologic mechanisms. The phenomenon is therefore properly referred to as "concomitant twnor resistance."956 Following surgical removal of the primary tumor, accelerated seeding and/or growth of metastases will occur, obviously a troubling observation for patients undergoing cancer surgery. This phenomenon, first described a hundred years ago,957 occurs in different species and in several tumor models.958 As would be expected, the primary tumor during its growth must inhibit metastasis possibly by usurping most available progenitor cells and/ or consuming growth factors and nutrients (similar to what Ehrlich described nutrient deprivation or athrepsia261 ) and/ or by producing antimitotic factors.959 There is little evidence whether or not cancer patients inhibit metastasis by

CANCER IMMUNOLOGY

1229

antigen-specific immunity. Concomitant immunity refers to the observation that an individual bearing a primary transplanted tumor may be resistant to secondary challenge with the same tumor at a different location because of an antigen-specific immune response.958 However, this old extensively studied observation958'960-962 may well be the result of artificially priming the host by tumor transplantation.

IMMUNOPREVENTION There is convincing evidence that immunosurveillance can prevent or reduce the incidence of cancers associated with certain viruses. Therefore, active immunization against viral capsid proteins may prevent infection and thereby cancer induction. While this is expected, it is still too early to confirm this with results. However, a federally funded, extensive vaccination program began in Australia in April of 2007 with a quadrivalent HPV vaccine to provide protection against the high-risk HPV types 16 and 18, which cause cervical cancer, and also low-risk types 6 and 11, which cause genital warts.963' 964 There has been a highly significant decline in the diagnoses of genital warts and a significant decrease in high-grade cervical abnormalities only 3 years after implementation of the program.963 The latency period for cervical cancer is 15 to 25 years; therefore, evidence for a decline in cancer incidence cannot be expected until about 2027. Furthermore, only 70% of cervical cancers are caused by the HPV types that are in the present vaccine; thus, this vaccine can be expected to prevent only about 70% of cervix cancers. The effect might be somewhat higher because of cross-reactivity with the other HPV types, which may provide cross-protection with viral types 31, 33, and 45. Therapeutic HPV vaccines for treating already existent persistent infections or advanced cervical lesions are needed.965 The current vaccine has no effect against HPV infection once it has been acquired and must therefore be given before onset of sexual activity. Remarkably, there is a therapeutic vaccine that seems to be effective against HPV-induced vulvar preneoplastic lesions.966 The same vaccine has not been shown to be effective against high-grade premalignant cervical lesions. In general, premalignant lesions often persist for a very long time; destroying these lesions should prevent the development of cancer. After introduction of hepatitis B virus vaccines, a decline in the incidence and prevalence of hepatitis B virus infection occurred967; this should eventually lead to a decline of chronic hepatitis and hepatocellular carcinoma. Several strategies for vaccination against hepatitis C virus, human T-lymphotropic virus 1, and human herpesvirus 8 are being developed. Also, developing vaccines against Helicobacter pylori and Schistosoma infections remains extremely important. The major influences of diet and microbial flora on the incidence of colorectal cancer and probably other cancers suggest new approaches for immunoprevention of these cancers. It will be important to determine whether cancer can also be prevented by active immunization of cancer-prone individuals with predisposing inherited or acquired antigens resulting from mutation (eg, in K-ras968). An ever-increasing

1230

I

SECTION VIII IMMUNOLOGIC MECHANISMS IN DISEASE sequence of the treatments. These important issues have been described and discussed elsewb.efe5!19•97l-916 and previously in this chapter. But why are so few immunotherapies effective or the treatment of choice, except for melanoma and superficial bladder cancer? Extensive recent reviews of immunotherapy of cancers expose some complementary but several differing viewpoints.3()6,}2),3:U.977...jl() There is also an astounding repetition of findings made decades ago, and a tendency of overstating the translational potential of new findings!• 14' As already discussed (see Fig. 47.1), the focus of current experimental research remains treating very small transplanted lesions in mice < 2 weeks after cancer cell inoculation.4.SS,93l Very few publications report immunotherapy in animals with cancers of clinically relevant size (~ 109 cancer cells and -1 em average diameter) and duration of growth (> 14 days). Adoptive T-cell therapy and anti-CD20 antibody treatments were singled out as experimental therapies effective in causing regression at this stage (Fig. 47.6). Indeed, adoptive transfer of T cells (with endogenous or transduced TCRs) may be effective with longer established tumor loads.977'912.~ Because both of these therapies can also be effective in humans, there is no "disconnect" between preclinical models and clinical experience.4.107 Relapse after therapy, the main problem of cancer treatments, is rarely

number of predisposing inherited or acquired mutations are being identified; however, inducing immune responses against them may be problematic. For example, active immunization against an oncogenic viral protein became ineffective in preventing cancer when the immunization was begun in the later part of the latency period221.79!\%9,970 or after the oncogenic protein was expressed in premalignant host tissues301.?22.Wl for reasons that are poorly understood. Finally, we need to avoid stimulating cancer development when vaccinating cancer-prone individuals.'41

IMMUNOntERAPY General Aspects Multiple immunotherapeutic strategies involving innate or acquired immunity have been developed to control cancer; they include 1) local application of a live bacterial vaccine, BCG (see discussion under Cancer and Inflammation); 2) use of cytokines; 3) active immunization; 4) passive therapy with antibodies; and 5) adoptive transfer of effector cells. Some of these strategies are being combined with other forms of cancer treatment. lt is important to realize that chemo- and radiation therapy can synergize with or antagonize innate or adaptive immunity dependent on timing and

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RG. 47.6. Analpis of All Experimerfal Cancer lmiTM.InotlleraPJ Publications Listed in PubMed far April, June, and Ncrnmber of 2010 and of the Entire Year 1980. Most experimental immunotherapies published treat small tumors(< 500 mm'~ yet succeed only at slowing or delaying tumor growth. In saveral2010 reports, larger tumors are baing treated than in 1980. and a few reports indicate tumor regression. An affect size (E) of 1indicates the treabnent arrested tumor growttl. An E< 1 indicates that the treated tumor still grew progressively but slower than control. An E > 1 indicates tumor regression. Regression of tumors> 200 mm' is observed only after passive antibody or adoptive T-cell therapy. Modified from Wen et al.4

CHAPTER 47

considered; in fact, most animal studies break off before relapse could occur.

Therapeutic Vaccinations Active immunization of cancer-bearing mice or humans is a heroic approach that was abandoned in the clinical management of infectious diseases, except for rabies, which has very long incubation period.984' 965 Cancer cells have a much slower generation time than most infectious organisms, and most of the bulk of the tumor load can usually be removed by other therapy (eg, surgery). At the time when the antigen load is lowest, the suppressive environment may be removed, and conceivably active immunization might lead to an effective immune response. However, persistent antigen appears to be a major reason why cancers and chronic infectious diseases are ineffectively treated by therapeutic vaccination. Possibly, combining active immunization with blockade of inununoinhibitory pathways may lead to more effective therapeutic vaccinations.965 Certain cancer cells may be fully sensitive to tumorspecific T cells but for various reasons fail to induce a response. The reasons for the poor immunogenicity may vary; therefore, different methods must be used for different cancers to immunize effectively.492 Currently, most methods have been developed in tumor-free mice (preventive/prophylactic vaccination).986 Immunization with small numbers of viable tumor cells may cause cancer, but, if not, it can stimulate solid long-lived memory immunity that prevents growth. Dead and disrupted tumor cells, membrane fractions, or cell extracts may enhance the growth of the cancer, although they stimulate "immune" (ie, lymphocyte) responses. Destroying the proliferative potential of the tumor cells, while leaving the cells viable and metabolically active, may result in a prolonged exposure to the antigen that allows T-cell immunity to develop.987 There are no general rules for accomplishing this, but exposing cancer cells to gamma radiation or certain cytostatic chemicals, such as mitomycin C, can work. However, chemotherapy makes cancer cells more immunogenic when they can undergo autophagy.9116 In any case, chemotherapy of transplantable tumors is more efficient in immunocompetent than in immunodeficient mice.988 Because these methods alone are often insufficient to elicit a cytolytic T-cell response to cancer cells, many strategies including genetic engineering have been designed to increase the immunogenicity of the tumor-cell inoculum and/or stimulate innate immunity at the site of vaccinations by the use of chemical and/or bacterial agents. However, irrespective of which particular genetic engineering of the tumor cells is used, rejection of the modified tumor cells is often followed by T cell-mediated immunity against the unmodified tumor cells. Methods include infecting cancer cells with certain viruses969--991; somatic cell fusion with various nontumorigenic cells992--994 ; transfection of self- or foreign MHC class I or class II molecules995--997; hapten conjugation998' 999 ; exposure to mutagens1000; transfection of tumor cells to express the B7 ligand that can provide a costimulating signal to T cells1001•1002; attracting secondary lymphoid structures

CANCER IMMUNOLOGY

1231

to the cancer003; targeting herpesvirus entry mediator pathways965; combining tumor cells with killed bacteria, such as Corynebacterium parvum100\ recombinant vaccines of antigen expressed by vaccinia, listeria, or virus-like particles2'1005; transfection of tumor cells to produce certain cytokines such as 11-2, IFN'Y, 11-4, 11-6, I1-7, G-CSF, GM-CSF, or TNF-a134•138•141•535•1006; injecting naked DNA constructs encoding the tumor antigen (whereby the gene for GM-CSF may also be used to recruit dendritic cells) 456•1007-1009; vaccination with anti-idiotypic antibodies, which bear the internal image of a tumor antigen1010; inhibiting extracellular adenosine triphosphate-degrading enzymes to increase autophagy986 ; transfection of tumor cells to express antisense RNA of a required growth factor thereby inducing terminal differentiation1011 ; peptide vaccines combined with blocking IFN'Y action1012•1013 with peptides long enough not to tolerize987'1013'1014 ; loading peptides to heat-shock protein1015; siRNA-mediated inhibition of nonsense-mediated messenger RNA decay/016 which may trigger innate immunitf017 or the expression of new epitopes to which the host is not tolerant; and finally dendritic cells323' 101s, 1019 that can be loaded with 1) synthetic antigenic peptides, 2) recombinant proteins, 3) native peptides stripped from tumor cell surfaces, 4) tumor-derived, peptide-loaded heat-shock proteins, 5) tumor-derived messenger RNA, or 6) by fusion of tumor cells. One advantage of the latter three strategies is that immunity to (unique) individually distinct tumor antigens, as well as tumor-associated antigen may be induced without having to identify the antigens. The limitation is that the antigen dose cannot be standardized. Vaccines are also being used that target the mutant epidermal growth factor receptor EGFRviii, a truly tumor-specific antigen on the surface of human malignant glioma cells capable of inducing strong B- and T-cell responses. The goal is to prolong patients' relapse-free survival because this cancer, the most common primary brain malignancy, is untreatable by conventional therapy.305 The mutation in the EGFRviii occurs in about 40% of the glioblastomas and represents an internal deletion in the gene encoding the receptor.1020 About half of the patients with this variant receptor have the same deletion, which generates a new amino acid at the fusion point of the resulting fusion protein and new antigenic determinant recognized specifically by a monoclonal antibody.304 A healthy skepticism is needed to stimulate experiments determining whether therapeutic effects of active immunization are found when the experimental tumor is not in early stages of malignant growth (see Figs. 47.1 and 47.7). Until then, it remains uncertain whether any of these procedures will be effective against longer established or advanced stages of cancer including microdisseminated cancer cells.362'965

Therapy with Engineered Antibodies Targeting Cancer Calls Directly General Considerations. The major alternatives to therapeutic vaccinations are antibody therapy and adoptive transfer of tumor-specific T cells. As already mentioned, there is little evidence that antibody produced by the host

1232

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RG. 47.7. Tumors can Escape from Therapeutic Vaccinatians Once Trutr Established. Then, therapeutic vaccinations become ineffective even against small tumors. Note the volumes of the tumors at start of therapeutic vaccinations, at day 7, or at day 29, respectively, are virtually the same size. However, on tv when vaccinations against were started early after TC1 cancer cell inoculation, tumors are eradicated and outgrowth prevemed. t, mice in the naYve group were sacrificed. For human papillomavirus-18 E71isteria, vaccine U.O-E71eads to production of a fusion protein comaining the complete E7 sequence.12 TC-1 cancer cells are HPV16 E6 and E7 and Ha-Ras transduced cell.' Unpublished; courtesy of Zhen-Kun Pan and Yvonne Paterson, University of Pennsylvania, Philadelphia, PA, USA.

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CHAPTER 48

deficiency was the first genetic etiology of the severe fonns of pediatric tuberculosis to be identified.483--486 Such identification of the defects underlying MSMD has been very beneficial to patients. Poor producers of IFNy, due to IL-12 deficiency, for example, benefit from preventive or curative treatment with recombinant IFNy. Conversely, patients whose cells do not respond to IFNy, such as those with complete IFNyR1 deficiency, should undergo HSCT. These studies have also had important immunologic implications. These experiments of nature have shown that IFNy is essential for immunity against mycobacteria and a few other intracellular bacteria, fungi, and parasites that infect macrophages. Surprisingly, however, these patients are not prone to infections with other intracellular agents, including most viruses, in particular. The rare viral illnesses occurring in these patients may have been favored by mycobacterially induced immunosuppression. IFNy plays a more important role as a macrophage-activating factor than as an antiviral molecule.487 The "Th1" ann of immunity, if such a lineage exists in humans, is not as broadly potent in host defense as reported for the mouse model. The dissection of MSMD has, therefore, revealed the narrow role of the "Th1" branch ofT helper cell immunity. These studies have also suggested that mycobacterial diseases in other medical settings may result from the impairment of IFNy immunity. Finally, these studies lend weight to the idea that otherwise healthy children with other infectious diseases may suffer from single-gene inborn errors of immunity.70 No germline mutation affecting the "Th2" arm of immunity has yet been identified, but mutations impairing the recently described "Th17" arm of immunity have been identified during investigations of CMC disease (CMCD), which was clinically described in the late 1960s and shown to display AR or AD inheritance in the early 1970s.488--490 CMC is characterized by persistent or recurrent infections of the nails, skin, and oral and genital mucosae with the fungus C. albicans. It is common in patients with various inherited or acquired T-cell deficits that are also associated with other infections.491 In patients with AD HIES caused by dominant-negative STAT3 mutations, the two principal infectious threats are CMC and staphylococcal diseases.65• 492 These patients have a small proportion of IL-17-producing T cells.479•493•494 However, this does not formally demonstrate a role for impaired IL-17 immunity in the CMC observed in these patients, as this immunologic phenotype may be silent or even be responsible for other phenotypes, infectious or otherwise. Interestingly, however, patients with autoimmune polyendocrinopathy type I suffer from a single infectious disease, CMC, which is accompanied by numerous autoimmune signs.495 They have high titers of neutralizing auto-Abs against IL-17 cytokines.496.497 Again, this observation alone does not prove that impaired IL-17 immunity underlies CMC in such patients, as the autoAbs may be clinically silent or may attenuate autoimmune signs. However, these clinical observations, together with the results of studies in mice, suggest that impaired IL-17 immunity may generally underlie CMC.491 These studies paved the way for the discovery of patients with isolated

INBORN ERRORS OF IMMUNITY

I

1257

CMC (CMCD) due to inborn errors of IL-17F or IL-17RA immunity.76 These patients were otherwise healthy and normally resistant to other infections, with the exception of a few cutaneous staphylococcal infections. One patient displayed AR complete IL-17RA deficiency, whereas patients from another kindred displayed AD partial IL-17F deficiency. These findings provided the first genetic etiologies of CMCD and suggested that IL-17A and IL-17F were essential for mucocutaneous immunity against C. albicans, but otherwise largely redundant in host defense, at odds with findings for the mouse model. Moreover, the use of a genome-wide approach led to the discovery of GOF mutations of STATl in other patients with CMCD, some of whom also displayed features of autoimmunity.77.498 Previously described STATl mutations were LOF (null or hypomorphic) and associated with AD or AR predisposition to viral and/or mycobacterial diseases.499- 501 By contrast, the CMCD-causing STATl mutations are GOF. They prevent the nuclear dephosphorylation of activated STAncontaining complexes, thereby increasing transcriptional activity in response to IFNy, IFNa/~, IFNI., and IL-27, the biologic functions of which are highly dependent on STATl. Cytokines that activate STAT3 predominantly and STATl to a lesser extent, including IL-6, IL-21, and IL-23, also trigger enhanced STATl-dependent responses in these patients. Patients with GOF mutations in STATl have small proportions of IL-17 T cells, because IFNy, IFNa/P, IFNI., and IL-27 are inhibitors of IL-17 T-cell differentiation via STATl, or because IL-6, IL-21, and IL-23 are inducers of IL-17 T cells via STAT3 but not via STATl, or both.77 Impaired IL-17 immunity therefore underlies CMCD in these patients. Surprisingly, some CMC patients with STATl mutations were recently reported to suffer from reactivations of viral disease.50h This situation is reminiscent of the observation that patients with HIES that are heterozygous for STAT3 develop shingles due to impaired T-cell memory.502 Overall, STATl LOF alleles underlie viral diseases that occur during primary infection due to the impairment of antiviral IFN activity. By contrast, GOF STATl mutations may confer a predisposition to the reactivation viral diseases (including some caused by the same herpes viruses) due to insufficient T-cell memory. In any case, studies ofCMCD revealed that the "Th17" ann of immunity is apparently redundant for host defense against most microbes in humans, at odds with findings for the mouse model. Studies of MSMD and CMC have indicated that helper T-cell immunity is unlikely to be restricted to the "Th1" and "Th2" arms, even with the addition of the third partner "Th17." Indeed, mutations ofiFNyand IL-17 immunity underlie predispositions to only a few of the millions of microbes in the environment, including hundreds of known pathogens. Another step forward came with the investigation of children with invasive pneumococcal disease (IPD). Patients with inborn errors of IL-17 or IFNy immunity typically display recurrent or persistent infectious diseases, consistent with the production of these cytokines principally by T cells. Only patients with IL-12 and IL-12R deficiencies

1258

I

SECTION VIII

IMMUNOLOGIC MECHANISMS IN DISEASE

seem to display a single episode of mycobacterial disease, implying that these cytokines are necessary for immunity against primary infection with mycobacteria but not for immunity to latent or secondary infection. 78 IPD was long known to be favored by inborn errors of pneumococcal opsonization, such as defects of the classical complement pathway or of antibody responses to capsular glycans, and asplenia.503 Patients with any of these PIDs are actually susceptible to a wide range of encapsulated bacteria. Interest in the investigation of IPD was stimulated in 2001 by the genetic dissection of a rare PID, known as EDA-ID.504' 505 IPD is, by far, the most common infection in these patients, but susceptibility to other pyogenic bacteria, mycobacteria, and some fungi and viruses has been recorded. Inflammation is weak or delayed in the course of infection. The only consistent immunologic abnormality is a lack of antibodies directed against glycans. This finding is consistent with the observation that IPD is often recurrent in these patients, with recurrences in some cases even being caused by the same serotype. Inborn errors of NF-KB were identified as responsible for the disease: patients with XR-EDA-ID carry hypomorphic mutations in NEMO, 506 whereas patients with AD-EDA-ID carry hypermorphic mutations in IKBA. 507 The key role ofNF-lCB in developmental processes accounts for the developmental features of the disease in affected children. Impaired immunity has a broad basis, consistent with the broad role of the multiple TNF, IL-l, and TLR receptors downstream from NF-KB. The NEMO mutations underlying EDA-ID are hypomorphic; null alleles cause the death in utero of male embryos and incontinentia pigmenti in women.508 These hypomorphic mutations are associated with immunologic and infectious clinical phenotypes, some underlying IPD509 and others underlying mycobacterial diseases 481 in otherwise healthy patients. NEMO mutations probably define the broadest phenotypic diversity in the field of PIDs, ranging from death in utero or lifethreatening neonatal disease to a mild and transient B-cell deficiency in adults. The mechanisms underlying IPD and other infections in patients with NEMO or IKBA mutations are progressively being deciphered through the identification of patients with germline mutations in genes encoding products acting upstream or downstream from NEMO. Children with isolated IPD provide the best example, as some have been shown to suffer from IRAK-4 or MyD88 deficiency.510' 511 These children have no developmental phenotype and are normally resistant to viruses, fungi, and mycobacteria. They suffer from IPD and, more rarely, from invasive staphylococcal diseases. Gram-negative infections are rare, with the exception of those caused by Pseudomonas and, more rarely, Shigella. 512-su The patients display weak, delayed biologic and clinical signs of inflammation during infection. The patients' cells do not respond to agonists of most TLRs (other than TLR3 agonists) and IL-1Rs (including IL-l, IL-18, and IL-33). Surprisingly, these patients are susceptible to a few pyogenic bacteria, but normally resistant to other bacteria and parasites, fungi, and viruses. The clinical status of patients improves spontaneously from adolescence onwards, with no deaths or invasive infections observed in the absence of prophylaxis. This implies that

TLRs and IL-lRs are not required for protective T- and B-cell immunity to these infectious agents. This experiment of nature has not only confirmed that IPD may have a genetic basis, as previously shown in patients with inborn errors of opsonization, but has also revealed that TLR and IL-lR immunity is largely redundant in host defense. This is at odds with theoretical models attributing a broad role in host defense to TLRs, as microbial sensors or pathogenassociated molecular pattern receptors, and with experiments conducted in MyD88-deficient mice, which were found to be susceptible to more than 35 of the pathogens tested.75 However, this finding is entirely consistent with evolutionary genetic studies, which have shown surfaceexpressed human TLRs to be subject to weaker selection pressure than intracellular TLRs. 515 Impaired TLR immunity may even play no more than a modest role in the development ofinfections in MyD88- and IRAK-4-deficient patients. It is not inconceivable that impaired IL-l immunity alone accounts for the development of the few pyogenic bacterial infections observed in these patients. TLR3, the only TLR that does not signal via MyD88 and IRAK-4 but instead uses TRIF as its sole adaptor, was serendipitously found to be essential for protective immunity against HSV-1 in the central nervous system (CNS), in the course of primary infection, in at least some children (Fig. 48.3).75 The other intracellular TLRs-TLR7, TLR8, and TLR9-which are stimulated by nucleic acids and generally thought to play an important role in antiviral defense, were found to be redundant in humans against most viruses. 512 However, the strong purifying selection operating on these genes suggests that past pathogens or other physiologic processes have exerted selection pressure on these four receptors.515 The role of TLR3 in host defense was deciphered by investigations of children with HSV-1 encephalitis (HSE). 516 This disease is the most common sporadic viral encephalitis in western countries. In this terrible disease, the virus is restricted to the CNS. It is absent from the bloodstream and does not spread to other organs. HSE is neurotropic in terms of both the route it follows and its destination: it reaches the CNS via cranial nerves. Patients with the most severe myeloid and lymphoid PIDs, including children with no T cells, display no particular susceptibility to HSE. The disease is sporadic in the vast majority of cases, with only four multiplex kindreds reported in 60 years, but there is a high frequency of parental consanguinity (14% in the French survey) in these cases, suggesting that HSE may be due to single-gene inborn errors of immunity displaying incomplete clinical penetrance.516 The first genetic etiology of HSE was identified as AR UNC-93B deficiency, resulting in an impairment of cellular responses to the four intracellular TLRs, including TLR3.517 Involvement of the TLR3 pathway was then suspected, because IRAK-4- and MyD88deficient patients, whose cells do not respond to TLR7-9, are not prone to HSE. TLR3 was formally implicated in the disease when AD and AR TLR3 deficiencies were discovered in other patients with HSE.518' 519 The subsequent identification of children with AR or AD TRIF deficiency confirmed the role of TLR3-TRIF and further suggested that childhood HSE might result from a collection of highly diverse

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