[1st Edition] 9780128124109, 9780128124093

Table of contents :
Content:
Series PagePage ii
CopyrightPage iv
ContributorsPage vii
Chapter One - Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional ActivityPages 1-36J. Tur, T. Vico, J. Lloberas, A. Zorzano, A. Celada
Chapter Two - Molecular Mechanisms of Somatic Hypermutation and Class Switch RecombinationPages 37-87S.P. Methot, J.M. Di Noia
Chapter Three - Emerging Major Histocompatibility Complex Class I-Related Functions of NLRC5Pages 89-119S.T. Chelbi, A.T. Dang, G. Guarda
Chapter Four - Nucleic Acid ImmunityPages 121-169G. Hartmann
Chapter Five - About Training and Memory: NK-Cell Adaptation to Viral InfectionsPages 171-207Q. Hammer, C. Romagnani
IndexPages 209-215
Contents of Recent VolumesPages 217-233

Citation preview

ASSOCIATE EDITORS K. Frank Austen Harvard Medical School, Boston, Massachusetts, United States

Tasuku Honjo Kyoto University, Kyoto, Japan

Fritz Melchers University of Basel, Basel, Switzerland

Hidde Ploegh Massachusetts Institute of Technology, Massachusetts, United States

Kenneth M. Murphy Washington University, St. Louis, Missouri, United States

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Publisher: Zoe Kruze Acquisition Editor: Alex White Senior Editorial Project Manager: Helene Kabes Production Project Manager: Surya Narayanan Jayachandran Senior Cover Designer: Miles Hitchen Typeset by SPi Global, India

CONTRIBUTORS A. Celada Macrophage Biology Group, School of Biology, Universitat de Barcelona, Barcelona, Spain S.T. Chelbi University of Lausanne, Lausanne, Switzerland A.T. Dang University of Lausanne, Lausanne, Switzerland J.M. Di Noia Institut de Recherches Cliniques de Montreal (IRCM); Mcgill University; Universite de Montreal, Montreal, QC, Canada G. Guarda University of Lausanne, Lausanne, Switzerland Q. Hammer Deutsches Rheuma Forschungszentrum, a Leibniz Institute, Berlin, Germany G. Hartmann Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Bonn, Germany J. Lloberas Macrophage Biology Group, School of Biology, Universitat de Barcelona, Barcelona, Spain S.P. Methot Institut de Recherches Cliniques de Montreal (IRCM); Mcgill University, Montreal, QC, Canada C. Romagnani Deutsches Rheuma Forschungszentrum, a Leibniz Institute, Berlin, Germany J. Tur Macrophage Biology Group, School of Biology, Universitat de Barcelona, Barcelona, Spain T. Vico Macrophage Biology Group, School of Biology, Universitat de Barcelona, Barcelona, Spain A. Zorzano Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology; Facultat de Biologia, Universitat de Barcelona, Barcelona; Centro de Investigacio´n Biomedica en Red de Diabetes y Enfermedades Metabo´licas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain

Advances in Immunology, Volume 133 ISSN 0065-2776 http://dx.doi.org/10.1016/B978-0-12-812409-3.09990-5

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CHAPTER ONE

Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity J. Tur*, T. Vico*, J. Lloberas*, A. Zorzano†,{,§, A. Celada*,1 *Macrophage Biology Group, School of Biology, Universitat de Barcelona, Barcelona, Spain † Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain { Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain § Centro de Investigacio´n Biomedica en Red de Diabetes y Enfermedades Metabo´licas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Mitochondrial Metabolism Governs Macrophage Activation State 2.1 Metabolic Reprogramming in Proinflammatory Macrophages 2.2 Metabolic Profile in IL-4-Activated Macrophages 3. Mitochondrial ROS in Innate Immune Responses 3.1 mROS Are Crucial Components of Inflammatory Signaling Pathways 3.2 TLR Signaling Upregulates mROS Production 4. Mitochondrial-Mediated Antiviral Immunity 4.1 RIG-I-Like Receptors 4.2 Mitochondrial Antiviral Signaling 4.3 Proteins That Regulate MAVS Signaling 4.4 Mitochondrial Dynamics Regulate Antiviral Immunity 4.5 ROS Regulation of Antiviral Signaling 5. Inflammasome Activation and Mitochondria 5.1 The Mitochondrion Is a Scaffold for Inflammasome Activation 5.2 Mitochondrial Signals in Inflammasome Activation 5.3 Mitophagy Restrains Inflammasome Activation 6. Concluding Remarks Acknowledgments References

Advances in Immunology, Volume 133 ISSN 0065-2776 http://dx.doi.org/10.1016/bs.ai.2016.12.001

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Abstract Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

ABBREVIATIONS AIM2 absent in melanoma 4 ASC apoptosis-associated speck protein containing a CARD ATP adenosine triphosphate CARDs caspase activation and recruitment domains CARKL carbohydrate kinase-like protein CCCP cyanide m-chlorophenyl hydrazone cGAS GMP–AMP synthase COX5B cytochrome c oxidase complex subunit 5B DAMPs danger-associated molecular patterns DRP1 dynamin-related protein 1 dsRNA double-stranded RNA ECSIT evolutionarily conserved signaling intermediate in Toll pathways ER endoplasmic reticulum ERK extracellular signal-regulated kinase ETC electron transport chain FADD Fas-associated death domain FCCP carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone FIS1 fission 1 Glut1 glucose transporter 1 H2O2 hydrogen peroxide HIF-1α hypoxia-inducible factor-1α HIV human immunodeficiency virus HR1 heptad repeat region 1 HSP90 heat-shock protein 90 IFNs type I interferons IKKα IκB kinase-α IRE1α inositol-requiring enzyme 1α

Macrophages and Mitochondria

IRFs interferon regulatory factors Irg1 immune-responsive 1 homolog JNK c-Jun terminal kinase LGP2 laboratory of genetics and physiology 2 MAMs mitochondria-associated endoplasmic reticulum membranes MAPKs mitogen-activated protein kinases MAVS mitochondrial antiviral signaling MDA5 melanoma differentiation-associated gene 5 MFF mitochondrial fission factor Mfn mitofusin MKP MAPK phosphatases mROS mitochondrial ROS mtDNA mitochondrial DNA mΔΨ mitochondrial membrane potential NDUFS4 NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 NEMO NF-κB essential modulator NF-κB nuclear factor-kappa B NLRC4 NLR CARD-containing protein 4 NLRP1 pyrin domain-containing 1 NLRs NOD-like receptors NLRX1 NLR family member NOD nucleotide oligomerization domain NOS2 nitric oxide synthase II O2 •
superoxide OPA1 optic atrophy 1 OXPHOS oxidative phosphorylation PAMPs pathogen-associated molecular patterns PBMCs peripheral blood mononuclear cells PGC-1β PPARγ-coactivator-1β PGK phosphoglycerate kinase PPARγ peroxisome proliferator-activated receptor gamma PRRs pattern-recognition receptors RIG-I retinoic acid-inducible gene I RIP1 receptor-interacting protein 1 RLRs RIG-I-like receptors ROS reactive oxygen species SeV Sendai virus SOD superoxide dismutase ssRNA single-stranded RNA STAT6 signal transducer activator of transcription 6 STING stimulator of interferon genes TANK TRAF family membrane-associated NF-κB activator TBK1 TANK-binding kinase 1 TLRs Toll-like receptors TOM outer membrane TRADD TNFR1-associated death domain protein TRAPS TNF receptor-associated periodic syndrome

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UCP2 uncoupling protein 2 UDP-GlcNAc uridine diphosphate-N-acetyl-alpha-D-glucosamine uPFK2 isoform of phosphofructokinase-2 VDACs voltage-dependent anion-selective channels VSV vesicular stomatitis virus

1. INTRODUCTION Macrophages play a central role in inflammation and tissue homeostasis and they are also major effectors of immune responses against pathogens. Until recently, it was believed that circulating monocytes, generated in the bone marrow, continuously repopulate tissues with resident macrophages. However, several lineage-tracking studies have revealed that the contribution of circulating monocytes to tissue-resident macrophages is restricted to a few specific tissues, including gut, heart, pancreas, and dermis. In contrast, most tissue-resident macrophages are derived from embryonic precursors that seed the tissues before birth and are maintained mainly by local proliferation throughout the life of the organism (Ginhoux & Guilliams, 2016). Although there are discrepancies about the origin of macrophages in several tissues, there is consensus that microglia are produced locally (Sheng, Ruedl, & Karjalainen, 2015). In addition, upon inflammation, the Ly6Chi subpopulation of circulating monocytes migrates to the affected tissues and differentiates to macrophages. However, whether resident or recruited macrophages play a similar role during inflammation is still unclear (Epelman, Lavine, & Randolph, 2014; Scott, Henri, & Guilliams, 2014). Inflammation usually starts when macrophages and other immune cells detect microbial structures through pattern-recognition receptors (PRRs). These receptors sense highly conserved molecules known as pathogenassociated molecular patterns (PAMPs), including compounds that comprise the bacterial cell wall, nucleic acids, and proteins. Several families of PRRs are involved in PAMP recognition, namely, Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), scavenger receptors, and C-type lectin receptors, among others. Upon interaction with their ligands, these PRRs trigger multiple signaling pathways, including nuclear factor-kappa B (NF-κB), interferon regulatory factors (IRFs), and mitogenactivated protein kinases (MAPKs). These pathways activate the transcription of proinflammatory cytokines, chemokines, type I interferons (IFNs), and costimulatory molecules, which are necessary to generate robust immune

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responses (Takeuchi & Akira, 2010). Apart from sensing microbial PAMPs, immune cells are also able through PRRs to detect endogenous molecules released during cellular damage and stress and subsequently trigger inflammatory responses. These danger-associated molecular patterns (DAMPs) are endogenous factors that are usually sequestered within intracellular compartments. Consequently, under normal conditions, DAMPs cannot be accessed by PRRs. However, in a context of cellular stress or tissue injury, DAMPs can be released. These molecules include reactive oxygen species (ROS), mitochondrial DNA (mtDNA), N-formyl peptides, uric acid crystals, and adenosine triphosphate (ATP), among others. Notably, DAMPs can be released either in association with or in the absence of microbial infection (Latz, Xiao, & Stutz, 2013; Petrilli, Dostert, Muruve, & Tschopp, 2007). Mitochondria are maternally inherited organelles that are pivotal in a wide range of cellular functions, including energy generation, the biosynthesis of molecules, Ca2+ homeostasis, the production of ROS, and the regulation of cell death. Although these organelles are known to play a key role in metabolism, mounting evidence suggests that they are also master regulators of immune responses. First, immune signaling pathways are tightly linked to metabolism, a process that provides the energetic requirements and intermediate metabolites necessary in each situation (Ganeshan & Chawla, 2014; Mills & O’neill, 2016). Second, mitochondria are centrally positioned hubs that regulate innate immune signaling pathways, including TLR, RLR, and NLR. Finally, mitochondria are also able to sense and process cellular damage and stress signals by initiating an inflammatory response (Cloonan & Choi, 2013; Weinberg, Sena, & Chandel, 2015; West, Shadel, & Ghosh, 2011). Here we discuss the relationship between mitochondria and innate immune responses in macrophages.

2. MITOCHONDRIAL METABOLISM GOVERNS MACROPHAGE ACTIVATION STATE Both recruited and resident macrophages are highly plastic cells that can modify their activation state in response to a broad range of environmental modifications. Activation of this cell type is most widely characterized by proinflammatory macrophages (also known as classically activated or “M1like macrophages”), antiinflammatory macrophages (commonly known as alternatively activated or “M2-like macrophages”), and intermediate stages between these two. Inflammatory macrophages show a marked production

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of proinflammatory mediators and reactive oxygen and nitrogen species (ROS and RNS, respectively) after exposure to molecules such as LPS or IFN-γ. In contrast, M2-like macrophages display a profibrotic, antiinflammatory, and antiparasite phenotype. Although this classification is overly simplistic for the description of the full spectrum of macrophage activation states, it is nevertheless useful for studying how mitochondria affect macrophage function (Mosser & Edwards, 2008). Changes in the activation state of macrophages involve coordinated regulation at both the metabolic and transcriptional level (Ganeshan & Chawla, 2014; O’neill & Pearce, 2016; Weinberg et al., 2015). During inflammation, macrophages are required to destroy microorganisms and thus show a proinflammatory profile. In contrast, when tissue repair is required, they adopt an antiinflammatory phenotype (Arnold et al., 2007). Proinflammatory macrophages are metabolically characterized by increased glycolysis, lactate production, and decreased oxidative phosphorylation (OXPHOS), even in conditions of oxygen availability (the so-called Warburg effect) (Lunt & Vander Heiden, 2011). They are also characterized by having the capacity to induce the pentose phosphate pathway, which is crucial for the generation of NADPH, a molecule required to produce ROS and nitric oxide (respiratory burst associated with phagocytosis). In contrast, antiinflammatory and profibrotic macrophages show increased oxygen consumption, mitochondrial respiration, and fatty acid oxidation, as well as decreased glycolysis (Haschemi et al., 2012; Huang et al., 2014; Weinberg et al., 2015). The relevance of the differences between the metabolism of pro- and antiinflammatory macrophages has been demonstrated in vivo using mice deficient for NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4), a subunit of complex I of the electron transport chain (ETC) that is required for OXPHOS. These mice show increased systemic inflammation, abnormal bone density, and alopecia. The macrophages of these animals switch to a proinflammatory phenotype, thereby inducing increased glycolysis and decreased fatty acid oxidation. That study emphasized that the balance between glycolysis and OXPHOS affects macrophage function, altering the inflammatory balance in the organism. In contrast, inhibition of respiratory complex I exacerbates ROS production, which may lead to the proinflammatory macrophage phenotype. It would be pertinent to determine the extent to which changes in mitochondrial metabolism rather than ROS production drive the balance between the pro- and antiinflammatory activity of macrophages (Jin, Wei, Yang, Du, & Wan, 2014).

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The metabolic differences between pro- and antiinflammatory macrophages might be explained by swiftness of response. The former cells rely on glycolysis to fuel short, rapid, and intense bursts of activation at the sites of inflammation or infection. In contrast, the latter use fatty acid oxidation and OXPHOS to sustain the more long-term process of inflammation resolution, tissue repair, and parasite fighting (Ganeshan & Chawla, 2014).

2.1 Metabolic Reprogramming in Proinflammatory Macrophages Upon proinflammatory activation by LPS, macrophages express a highly active isoform of phosphofructokinase-2 (uPFK2) that strongly promotes glycolysis. In contrast, M2-like or inactivated macrophages express the much less active isoform PFKFB1 in response to this stimulus (Kelly & O’neill, 2015; Rodriguez-Prados et al., 2010). Hypoxia-inducible factor-1α (HIF-1α), a key mediator of the Warburg effect, is also upregulated in proinflammatory macrophages. This factor induces the expression of glucose transporter 1 (Glut1) and phosphoglycerate kinase (PGK), both responsible for enhanced glycolysis. Moreover, deletion of HIF-1α impairs the activation of proinflammatory macrophages, thereby demonstrating that glycolysis and inflammatory activation are coupled processes (Cramer et al., 2003; Peyssonnaux et al., 2005). Another feature necessary for the activation of proinflammatory macrophages is the downregulation of the carbohydrate kinase-like protein (CARKL), an enzyme that catalyzes the formation of sedoheptulose 7-phosphate, an inhibitor of the pentose phosphate pathway. Upon LPS stimulation, CARKL is strongly downregulated, thus increasing the pentose phosphate pathway and glycolysis. This increase is required for LPS-induced superoxide generation and the production of proinflammatory cytokines (Haschemi et al., 2012). Finally, a recent study by Jha et al. combining transcriptomics and metabolomics approaches has provided a much deeper understanding of the metabolic changes that occur during macrophage polarization. In proinflammatory macrophages, Krebs cycle activity is reduced by inhibition in two sites. Inhibition of the first step is caused by the severe downregulation of isocitrate dehydrogenase expression, which converts isocitrate to α-ketoglutarate. This reaction leads to an accumulation of citrate, which is redirected to the production of itaconic acid (Jha et al., 2015), a metabolite with antibacterial properties, particularly against Salmonella enterica and Mycobacterium tuberculosis (Michelucci et al., 2013).

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Interestingly, immune-responsive 1 homolog (Irg1), which catalyzes the production of itaconic acid, is also overexpressed, thereby further enhancing the production of this metabolite. Furthermore, accumulated citrate is also used for the synthesis of fatty acids, which can be used for prostaglandin production, another hallmark of the proinflammatory macrophage phenotype. The second step reduced in the Krebs cycle is at the level of succinate dehydrogenase, which converts succinate into fumarate. Accumulated succinate stabilizes HIF-1α (Jha et al., 2015), thereby inducing the expression of proinflammatory genes such as IL-1β (Tannahill et al., 2013), as well as glycolytic enzymes/transporters. Under these conditions, reduced succinate dehydrogenase expression occurs in the absence of changes in the concentrations of fumarate. This observation would indicate activation of the aspartate-argininosuccinate shunt, which involves the synthesis of argininosuccinate from citrulline (argininosuccinate synthase), and the degradation of the latter into arginine and fumarate (argininosuccinate lyase). This pathway has the advantage of regenerating L-arginine from citrulline, then L-arginine through nitric oxide synthase II (NOS2) generates nitric oxide. In turn, increased nitric oxide production also inhibits the ETC complexes, either by nitrosylation or by competing with oxygen, thereby effectively decreasing mitochondrial respiration (Everts et al., 2012; Jha et al., 2015). Taken together, the aforementioned observations indicate that, during the activation of proinflammatory macrophages, the gene expression of these cells is reprogrammed to promote Warburg metabolism, the pentose phosphate pathway, a decreased Krebs cycle, and reduced mitochondrial respiration. Moreover, these metabolic changes lead to the accumulation of intermediate metabolites that play a crucial role in the proinflammatory activity of macrophages (Fig. 1A).

2.2 Metabolic Profile in IL-4-Activated Macrophages In contrast to proinflammatory macrophages, in antiinflammatory ones (M2-like), the Krebs cycle is induced. When macrophages are polarized in vitro with IL-4, the signal transducer activator of transcription 6 (STAT6) and the peroxisome proliferator-activated receptor gamma (PPARγ)-coactivator-1β (PGC-1β) are activated. This process may participate in the upregulation of mitochondrial biogenesis, respiration, and fatty acid oxidation. Interestingly, the overexpression of PGC-1β is enough to trigger the antiinflammatory phenotype (Lelliott et al., 2006).

A

Glucose uPFK2

CARKL

Pentose phosphate pathway

NADPH

Warburg metabolism

NO

Arginino succinate shunt

Citrate

Krebs cycle

Itaconic acid

IDH

Succinate

Fatty acids Proinflammatory cytokine expression B

NO

Irg-1 SDH

HIF-1a

ROS

LPS-induced genes

G2 Prostaglandins

Proinflammatory products

LPS-repressed genes

Glucose CARKL

PFKFB1

Fatty acids

Warburg metabolism

Pentose phosphate pathway

UDPGInNAc

Fatty acid oxidation Krebs cycle

Protein N-glycosylation

PGC-1b STAT6 IL-4-induced genes

Fig. 1 The metabolic profile of macrophages depends on their activation state. The expression pattern of multiple genes involved in metabolic pathways differs in function of the macrophage activation state, thus affecting the metabolic profile. (A) Proinflammatory macrophages show predominance for aerobic glycolysis (Warburg metabolism), with a highly active pentose phosphate pathway, which produces NADPH, a molecule necessary for ROS and RNS generation. Furthermore, the TCA activity of these cells is reduced at two points (isocitrate dehydrogenase and succinate dehydrogenase), leading to the accumulation of citrate and succinate and the activation of the argininosuccinate shunt. The consequences of this activation are, on the one hand, the production of itaconic acid and prostablandins from citrate and the activation of HIF-1α as a result of its stabilization by succinate, and, on the other hand, an increase in NO production caused by argininosuccinate shunt activity. These factors together contribute to the production of inflammatory and antimicrobial mediators, which are necessary for the correct function of macrophages. (B) Antiinflammatory macrophages present reduced glycolysis and pentose phosphate pathway activity and show a preference for the Krebs cycle and OXPHOS. Fatty acid oxidation is enhanced by PGC-1β and STAT6, which are activated by IL-4, to support increased carbon demand from the Krebs cycle. Glucose is also converted to UDP-GlnNAc, which increases the glycosylation of several proteins, including CD306 and CD206.

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This increase in fatty acid oxidation is required to fuel the increased mitochondrial metabolism. These fatty acids derive from triglycerides, which are captured by the scavenger receptor CD36 and processed by lysosomal acid lipase. These two proteins are also induced after IL-4 stimulation and are required for the M2-like differentiation of macrophages (Huang et al., 2014). The inhibition of fatty acid oxidation is sufficient to suppress the M2-like gene program and induce a proinflammatory phenotype (Carroll, Viollet, & Suttles, 2013; Mounier et al., 2013; Vats et al., 2006). Similarly, uncoupling mitochondrial respiration by means of oligomycin and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) dramatically decreases the expression of M2-like genes, thereby abolishing the antiinflammatory effects of IL-4 (Vats et al., 2006). These observations suggest that these changes in the metabolic profile of M2-like phenotype are not only a consequence of STAT6 and PGC1-β signaling but also that they are the drivers of the M2-like phenotype. Additionally, antiinflammatory macrophages show transcriptional upregulation of the N-glycan synthesis pathway, resulting in increased production of uridine diphosphate-N-acetyl-alpha-D-glucosamine (UDPGlcNAc). The accumulation of this metabolite is required for the N-glycosylation of proteins, including mannose and lectin receptors (CD206 and CD301, respectively), which are required for the correct function of M2-like macrophages (Jha et al., 2015). To sum up, the differentiation of macrophages to an M2-like phenotype involves a change in their transcriptional program to favor an active Krebs cycle and enhanced fatty acid oxidation to fuel it. Furthermore, the N-glycosylation of key proteins in this phenotype is enhanced by the accumulation of UDP-GlcNAc (Fig. 1B).

3. MITOCHONDRIAL ROS IN INNATE IMMUNE RESPONSES One consequence of electron flow through the respiratory chain is the generation of mitochondrial ROS (mROS). Superoxide (O2 •
) is generated during OXPHOS when electrons leak from the ETC and are prematurely accepted by oxygen. This leakage takes place mainly at respiratory complexes I and III, but can also occur at complex II. Complexes I and II release superoxide into the matrix, whereas complex III does so into both the matrix and the intermembrane space. Superoxide can then evade mitochondria through voltage-dependent anion-selective channels (VDACs) or

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instead be converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD) 1 or 2, which, unlike superoxide, can freely cross mitochondrial membranes (Pelletier, Lepow, Billingham, Murphy, & Siegel, 2012; West, Shadel, et al., 2011) (Fig. 2). For many years, mROS were considered to be unwanted byproducts of metabolism that can damage cellular components via nonspecific oxidation. Although it is well known that ROS are crucial for the degradation of phagocytosed bacteria, mounting evidence suggests that they also act as second messengers in multiple signaling pathways (Pelletier et al., 2012; West, Shadel, et al., 2011). Professional phagocytes, such as neutrophils and macrophages, generate ROS primarily by the phagosomal NADPH oxidase; however, several studies propose that mROS are also a major source of ROS in these cells and that they are in fact crucial for innate immune responses. Here we will discuss the effects of mROS and how the TLR 1, 2, or 4

TRAF6

Degradation of phagocyted bacteria

H2O2

TRAF6 ECSIT

ETC O2•–

NF-kB JNK ERK p38

mROS UCP2

Inflammatory gene expression

Fig. 2 Mitochondrial ROS are required for innate immune responses. Mitochondria produce superoxide at complexes I, II, and III. This superoxide escapes the mitochondria by VDACs or is catabolized to hydrogen peroxide by SOD1/2 and is then released by diffusion. mROS contribute to the degradation of phagocytosed bacteria in the endosomal compartment. Furthermore, mROS can activate MAPKs and NF-κB signaling, inducing enhanced expression of inflammatory mediators. JNK and p38 also participate in a feed forward mechanism involving the repression of UCP2 expression. UCP2 is a mitochondrial protein that reduces electron flow through OXPHOS, effectively decreasing mROS production. PAMP signaling through TLR1, 2, and 4 also promotes an increase in mROS production. This effect is mediated by the activation of TRAF6 and its translocation to mitochondria and subsequent interaction with ECSIT, which increases the production of superoxide through respiratory complex I.

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production of these molecules is regulated. Also, mROS are involved in antiviral signaling and in inflammasome activation. Both effects will be discussed in depth in the appropriate sections.

3.1 mROS Are Crucial Components of Inflammatory Signaling Pathways ROS act as signaling molecules by targeting transcription factors and enzymes to modify their activity. Specifically, ROS tend to oxidize the thiol groups of cysteine and methionine residues. Once the redox signal ends, these oxidations can be reversed by the action of glutathione and thioredoxins (Nathan & Cunningham-Bussel, 2013; Pelletier et al., 2012). The most well-known mechanism of ROS-mediated activation of the inflammatory signaling pathway is through the inhibition of MAPK phosphatases (MKPs) by oxidation of their catalytic center, a process that allows the sustained activation of MAPKs (Hamanaka et al., 2013). Specifically, ROS have been shown to prevent the dephosphorylation of c-Jun terminal kinase (JNK) (Kamata et al., 2005), extracellular-regulated kinase (ERK) (Kim, Ullevig, Zamora, Lee, & Asmis, 2012; Levinthal & Defranco, 2005), and p38 (Kim et al., 2012) by inactivating their respective phosphatases—a process that may enhance inflammatory signaling. As macrophages produce ROS in two locations, mitochondria and plasma membranes through NADPH oxidase, it is difficult to evaluate the relative contribution of each source to the activation of inflammatory signaling pathways. In spite of limited knowledge in this regard, several studies have revealed that mROS are crucial molecules in the generation of inflammatory responses in macrophages. In the first place, Bulua et al. (2011) demonstrated that peripheral blood mononuclear cells (PBMCs) from patients with the TNF receptorassociated periodic syndrome (TRAPS) show increased mROS levels, as well as enhanced MAPK activation and inflammatory cytokine production. Treatment with antioxidants reversed the increased inflammatory phenotype, thereby demonstrating the central role of ROS. Furthermore, they confirmed that the effects are specifically attributable to mROS, as deletion of NADPH oxidase did not reverse the phenotype. Another study using NDUFS4-deficient macrophages found that, apart from having an altered metabolism (discussed earlier), these cells showed increased mROS in response to LPS, as well as expression of proinflammatory cytokines (Jin et al., 2014).

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Finally, a third study demonstrated that mROS are also involved in LPSmediated production of pro-IL-1β. LPS-activated macrophages treated either with metformin (a drug for type-2 diabetes) or with rotenone (a respiratory complex I inhibitor) showed decreased mROS generation and, consequently, a reduction in pro-IL-1β expression. Furthermore, treatment with MitoQ, a specific mROS scavenger, was found to inhibit pro-IL-1β expression. This observation thus further confirms the role of mROS in the inflammatory response (Kelly, Tannahill, Murphy, & O’neill, 2015). The specific role of mROS in inflammation has been further confirmed by modulation of uncoupling protein 2 (UCP2), a mitochondrial protein that is distributed ubiquitously but shows higher expression in macrophages. The modulation of UCP2 generates moderate mitochondrial uncoupling, with a reduction in electron leakage from OXPHOS, thereby resulting in a significant decrease in mROS generation (Emre & Nubel, 2010). UCP2 overexpression in macrophages produces a significant reduction in mROS production, which leads to a decrease in NOS2 and in the production of nitric oxide (Kizaki et al., 2002). In contrast, upon LPS stimulation, UCP2
/
macrophages generate more mROS, thus resulting in enhanced ERK, p38, and NF-κB signaling and an increased induction of proinflammatory mediators, such as nitric oxide, IL-6, and IL-1β. Moreover, the effects of UCP2 deficiency are reversed with ROS scavenging. This observation therefore supports the notion that mROS drive inflammatory signaling in macrophage (Bai et al., 2005; Rousset et al., 2006). UCP2 is also involved in mROS-mediated responses against pathogenic microorganisms. UCP2
/
mice exhibit enhanced resistance to Toxoplasma gondii (Arsenijevic et al., 2000) and Listeria monocytogenes infection (Rousset et al., 2006). Additionally, UCP2-deficient macrophages show increased toxoplamacidal activity, as well as increased bactericidal activity against Salmonella typhimurium, thanks to the increase in ROS production (Arsenijevic et al., 2000). In fact, other pathogens, such as Leishmania, have even developed survival strategies involving the induction of UCP2 to inhibit mROS generation. In a model of leishmaniosis, downregulation of UCP2 increases mROS production, thus leading to enhanced p38 and ERK activation, increased proinflammatory cytokine production, and decreased survival of the parasite (Basu Ball et al., 2011). Additionally, given its importance during inflammation and infection, UCP2 must be tightly regulated. Upon LPS stimulation, UCP2 is rapidly downregulated in a p38- and JNK-dependent manner (Emre et al., 2007; Kizaki et al., 2002). This downregulation leads to an increase in mROS

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production, which further activates p38, initiating a signaling amplification loop that enhances the inflammatory response (Emre et al., 2007) (Fig. 2).

3.2 TLR Signaling Upregulates mROS Production To facilitate the induction of innate immune responses, mROS production is simultaneously regulated by TLR signaling. Activation of TLR1, TLR2, and TLR4 by their ligands (peptidoglycans, lipopeptides, and LPS, respectively) leads to translocation of TNF receptor-associated factor 6 (TRAF6) to the mitochondrial membrane. Therefore, TRAF6 interacts with evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) (West, Brodsky, et al., 2011), which is involved in the assembly of respiratory chain complex I (Vogel et al., 2007). This engagement results in TRAF6mediated ubiquitination of ECSIT, causing an increase in the production of mROS. Additionally, upon TLR engagement, mitochondria are recruited to phagosomes, where they contribute to ROS generation to kill phagocytosed bacteria. Consistent with these observations, depletion of TRAF6 or ECSIT in macrophages results in decreased mROS generation and impaired microbicide activity. Similarly, inhibition of mROS by overexpression of the antioxidant enzyme catalase in mitochondria inhibits bacterial killing (West, Shadel, et al., 2011) (Fig. 2).

4. MITOCHONDRIAL-MEDIATED ANTIVIRAL IMMUNITY Several structural components of viruses, including double- and single-stranded RNA (dsRNA and ssRNA, respectively), DNA, and surface glycoproteins, can be sensed by the host PRRs. Three families of PRRs detect viral nucleotides, namely, RLRs and NLRs in the cytosol, and TLRs in the endosomal compartment (TLR3, TLR7, and TLR8, respectively) (Akira, Uematsu, & Takeuchi, 2006). Among these receptors, TLRs and RLRs induce the production of type I IFNs and inflammatory cytokines, whereas NLRs are involved, among other functions, in inflammasome assembly, which in turn induces the proteolytic process of IL-1β and IL-18 (Petrilli et al., 2007; Takeuchi & Akira, 2010).

4.1 RIG-I-Like Receptors The RLR family comprises three members: RIG-I, melanoma differentiationassociated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), this last detects cytosolic viral RNA. These receptors are

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characterized by a central DExD/H box RNA helicase core that allows the detection of viral cytosolic RNA. RIG-I and MDA5 each contains two N-terminal caspase activation and recruitment domains (CARDs), which are required for downstream signaling, whereas LGP2 does not have a CARD (Dixit & Kagan, 2013; Yoneyama et al., 2005). Although LGP2 was initially proposed to be a negative regulator of RIG-I and MDA5 responses (Rothenfusser et al., 2005), more recent studies suggest that it is a positive regulator of RIG-I (Satoh et al., 2010; Venkataraman et al., 2007), presumably by facilitating the binding of viral RNA to both receptors (Takahasi et al., 2009). RIG-I detects viruses of the Paramyxoviridae (i.e., Sendai virus and Newcastle disease virus), Flaviviridae (i.e., Japanese encephalitis virus and hepatitis C virus), and Rhabdoviridae (i.e., vesicular stomatitis virus) families. MDA5 recognizes viruses of the Picornaviridae family (i.e., Polio virus and encephalomyocarditis virus). Reovirus, Dengue virus, and West Nile virus can be detected by both MDA5 and RIG-I (Wu & Chen, 2014). The differences in the types of viruses that each receptor can detect can be explained by the differential preference for distinct RNA species. Both receptors can detect poly(I:C), a synthetic analog of dsRNA, but MDA5 preferentially recognizes long fragments (more than 4 kb), while RIG-I shows a preference for shorter fragments (around 300 bp) (Kato et al., 2008). RIG-I preferentially recognizes RNA with a 50 triphosphate group, a feature that allows discrimination between host and viral RNA, as this structure is absent in host mRNAs (Hornung et al., 2006; Pichlmair et al., 2006). Another structural requirement for RIG-I recognition is shorts blunt dsRNA (Schlee et al., 2009; Schmidt et al., 2009). MDA5 agonists are not as well characterized as those of RIG-1; however, these agonists have been observed to preferentially bind to long dsRNA with no 50 end specificity (Reikine, Nguyen, & Modis, 2014). Also, the two receptors differ in their mechanism of activation. In a steady state, RIG-I is autorepressed, thereby preventing signaling by CARDs. Upon RNA interaction, the conformation of RIG-I changes to expose its CARDs (Kowalinski et al., 2011). This conformational change calls for ATP hydrolysis (Kowalinski et al., 2011), followed by the binding of polyubiquitin chains to CARDs. All of these events induce the oligomerization of RIG-1 (Jiang et al., 2012). In contrast, the CARDs of MDA5 are not sequestered in the absence of the agonist, and the interaction of this receptor with its ligand directly causes the oligomerization of its CARDs—a process that is also dependent on polyubiquitination (Berke & Modis, 2012; Jiang et al., 2012). Another difference between the receptors is that ATP hydrolysis

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regulates the stability of the filaments of MDA5 by triggering the dissociation of this receptor from its RNA ligand (Peisley et al., 2011). In both cases, these changes allow the association of MDA5 and RIG-I with mitochondrial antiviral signaling protein (MAVS, also known as IPS1, CARDIF, or VISA) in a CARD-dependent way. This adapter molecule, which is located on the mitochondria surface, is required for correct RIG-I and MDA5 signaling (Kawai et al., 2005; Meylan et al., 2005; Xu et al., 2005). The presence of MAVS in mitochondria, as well as being crucial for antiviral signaling, places these organelles in a pivotal position in innate immune responses against viruses.

4.2 Mitochondrial Antiviral Signaling This 540-amino acid protein is located mostly in the mitochondrial outer membrane and in mitochondria-associated endoplasmic reticulum membranes (MAMs) (Horner, Liu, Park, Briley, & Gale, 2011). Also, a small pool of MAVS is located in peroxisome membranes, where it participates in rapid early antiviral responses before the engagement of mitochondrial MAVS (Dixit et al., 2010). MAVS has an N-terminal CARD, a proline-rich region, and a C-terminal transmembrane region (Kawai et al., 2005; Meylan et al., 2005; Xu et al., 2005). The CARD of MAVS interacts with RIG-I and MDA5 CARDs. This interaction is essential for antiviral responses because it activates the downstream signaling cascades of NF-κB and IRFs (Seth, Sun, Ea, & Chen, 2005). When this interaction takes place, MAVS is induced to form large polymers in a prion-like mechanism (Hou et al., 2011). This mechanism induces the recruitment of more MAVS and its conversion into prion-like filaments to form very large aggregates that allow the rapid and robust amplification of RLS signaling (Wu & Chen, 2014). This amplification leads to the recruitment of several E3 ubiquitin ligases that are members of the TRAF family to mitochondria. These ligases include TRAF2, TRAF3, TRAF5, and TRAF6, which bind to MAVS through several TRAF-binding domains (Belgnaoui, Paz, & Hiscott, 2011; Liu et al., 2013; Saha et al., 2006; Tang & Wang, 2010). TNFR1-associated death domain protein (TRADD) is also recruited to MAVS, where it forms a complex with receptor-interacting protein 1 (RIP1) and Fas-associated death domain (FADD). TRAF2, 5, and 6 promote ubiquitin ligation of RIP1, which is recognized by NF-κB essential modulator (NEMO), a scaffold for IκB kinase-α (IKKα) and IKKβ, thereby initiating NF-κB signaling. In addition,

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TRAF3 and TRAF family membrane-associated NF-κB activator (TANK) also forms another complex with TRAF. This second complex can activate IKKε and TANK-binding kinase 1 (TBK1), leading to the phosphorylation of IRF3 and IRF7, thus initiating type 1 IFN response (Liu et al., 2013; Michallet et al., 2008). A recent study described that TBK1 and IKKβ, in turn, also phosphorylate MAVS, which is a necessary condition for IRF3 recruitment (Liu et al., 2015). On the basis of all this information, we can conclude that after interacting with MDA5 and RIG-I, MAVS leads, on the one hand, to the production of type 1 IFNs by the IRF3 and IRF7 pathways, and, on the other hand, to the expression of proinflammatory cytokines mediated by NF-κB (Fig. 3).

4.3 Proteins That Regulate MAVS Signaling Owing to the location of MAVS at the mitochondrial membrane and MAMs, several mitochondrial and endoplasmic reticulum (ER) proteins can directly interact with it, thereby modulating its downstream signaling, either by promoting or by inhibiting it. One of the major modulators of MAVS signaling is stimulator of interferon genes (STING, also known as MITA, MPYS, and ERIS). This transmembrane protein is located mainly in the ER (Ishikawa & Barber, 2008; Sun et al., 2009); however, it has also been associated with MAMs (Zhong et al., 2008). The distribution of STING is maintained by ER-retention sequences, which are necessary for the function of this molecule (Sun et al., 2009). STING overexpression activates IRF3 and NF-κB and induces type 1 IFN expression, whereas STING deletion inhibits IRF3 and type 1 IFN production and increases susceptibility to viruses such as the vesicular stomatitis virus (VSV) (Ishikawa & Barber, 2008; Zhong et al., 2008). Interestingly, several types of RNA viruses have evolved to evade STING-mediated responses. This observation thus confirms the relevance of this mechanism in antiviral responses (Maringer & Fernandez-Sesma, 2014). In basal conditions, STING interacts with MAVS and IRF3. Upon viral infection, STING dimerizes, thereby allowing its downstream signaling (Sun et al., 2009). After that, TBK1 is recruited to STING to form a complex containing MAVS-STING-IRF3-TKB1. At this point, STING also interacts with RIG-I. Then TBK1 phosphorylates IRF3, triggering the expression of type 1 IFNs and cellular antiviral responses (Zhong et al., 2008). Overall, these data suggest that STING provides a scaffold for the assembly of the MAVS signalosome. Finally, it should be noted that apart

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Fig. 3 Mitochondria are signaling platforms for antiviral responses. Upon viral RNA recognition, cytosolic receptors RIG-I and MDA5 are recruited to the external mitochondrial membrane where they interact with MAVS. Then MAVS oligomerizes and acts as scaffold for several downstream signaling proteins that will finally activate IRF3, IRF7, and NF-κB. The expression of type 1 IFNs and inflammatory cytokines is induced by IRFs and NF-κB, respectively, producing an antiviral state in the cell. MAVS-mediated signaling is tightly modulated by a broad range of mitochondrial proteins, which either activate (green arrows) or inhibit it (red capped lines). In particular, STING, a protein located in the ER, interacts with MAVS, enhancing its capacity to activate antiviral signaling. The integrity of mitochondria also affects MAVS signaling. mROS production, mitochondrial membrane potential, and mitochondrial fusion are parameters that favor MAVS signaling. Mitophagy, the selective removal of dysfunctional mitochondria, can downregulate MAVS-mediated signaling by restricting mROS production.

from modulating MAVS signaling, STING is also involved in the activation of IRF3 in response to cytosolic DNA, activation that is mediated by GMP– AMP synthase (cGAS) (Radoshevich & Dussurget, 2016). This observation indicates that STING is a converging point in the production of type 1 IFNs mediated by RNA and DNA detection pathways. Another cofactor that facilitates MAVS signaling is the translocase of the outer membrane (TOM) complex. As indicated by its name, this

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multiprotein complex is located in the outer mitochondrial membrane, where it recognizes nuclear-encoded mitochondrial preproteins and imports them into the transmembrane space of mitochondria. TOM20 and TOM70 are the receptors responsible for initial recognition of the preproteins. In addition, TOM70 is associated with the chaperone heat-shock protein 90 (HSP90), which is necessary for the correct import of other preproteins to mitochondria (Schmidt, Pfanner, & Meisinger, 2010). In fact, HSP90 directly interacts with TBK1 and IRF3, and this interaction is crucial for a correct IRF3-mediated response (Yang et al., 2006). During RNA virus infection, TOM70 strongly interacts with MAVS through its clamp domain, recruiting TBK1 and IRF3 to mitochondria. This recruitment forms a complex on the mitochondrial surface composed by MAVS-TOM70-HSP90IRF3-TBK1. This complex, in turn, leads to IRF3 phosphorylation and consequently to the activation of the antiviral response (Liu, Wei, Shi, Shan, & Wang, 2010). Interestingly, it has been reported that HSP90 also interacts with the IKK complex. Given this finding, MAVS-mediated NF-κB activation could be explained by TOM70 (Chen, Cao, & Goeddel, 2002). There are also other cofactors that negatively regulate MAVS signaling, among these the NLR family member NLRX1. Located in the outer mitochondrial membrane, this factor interacts with MAVS through its CARD and disrupts NF-κB and IRF3 signaling during viral infections (Moore et al., 2008). Moreover, NLRX1 can also directly interfere with the TRAF6–IKK signaling pathway, thus affecting NF-κB activation. Interestingly, this last effect can also be observed in a canonical NF-κB signaling pathway, such as in LPS-TLR4 pathway (Allen et al., 2011; Xia et al., 2011). This finding indicates that NLRX1 plays a crucial role in preventing excessive inflammatory activation. Finally, a recent study showed that NLRX1 also interacts with STING to disrupt TBK1–IRF3 signaling. Human immunodeficiency virus (HIV) and other viruses take advantage of this inhibition to reduce type 1 IFN responses after cGAS detection (Guo et al., 2016). This last mechanism is a clear example of the interconnection between antiviral pathways. To end with, another negative regulator of MAVS is the receptor for globular domain of complement component 1q (gC1qR), which is found predominantly in mitochondria. Upon viral infection, gC1qR translocates to the outer mitochondrial membrane where it interacts with MAVS to disrupt type 1 IFN-mediated response (Xu, Xiao, Liu, Ren, & Gu, 2009) (Fig. 3).

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4.4 Mitochondrial Dynamics Regulate Antiviral Immunity Mitochondria are highly dynamic organelles that undergo fusion and division, thus constantly reshaping their morphology. This process, known as mitochondrial dynamics, is crucial for the maintenance of mitochondrial integrity, as reflected by its involvement in several key processes including apoptosis, autophagy, calcium homeostasis, metabolism, ROS production, and respiration (Chan, 2006; De Brito & Scorrano, 2008b; Detmer & Chan, 2007; Liesa & Shirihai, 2013; Zorzano, Hernandez-Alvarez, Sebastian, & Mun˜oz, 2015; Zorzano, Liesa, Sebastian, Segales, & Palacin, 2010). In mammals, mitochondrial fusion is mediated by mitofusin (Mfn) 1 and 2 and optic atrophy 1 (OPA1), whereas dynamin-related protein 1 (DRP1), fission 1 (FIS1), MiD49/51, and mitochondrial fission factor (MFF) regulate mitochondrial fission. Both Mfn1 and Mfn2 are located in the mitochondrial outer membrane (Chan, 2006; Westermann, 2010; Zorzano et al., 2010), although the latter can also be found in MAMs (De Brito & Scorrano, 2008a). Mfns govern the fusion of mitochondrial outer membranes from adjacent organelles through homodimeric or heterodimeric interactions (Chan, 2006; Westermann, 2010; Zorzano et al., 2010). In addition, Mfn2 also mediates the tethering of mitochondria and the ER (De Brito & Scorrano, 2008a; Sebastian et al., 2012; Mun˜oz et al., 2013). In recent years, several studies have demonstrated that mitochondrial dynamics play an important role in the regulation of RLR-mediated antiviral responses (Castanier, Garcin, Vazquez, & Arnoult, 2010; Koshiba, Yasukawa, Yanagi, & Kawabata, 2011; Onoguchi et al., 2010; Yasukawa et al., 2009; Yu et al., 2015). Koshiba’s (Yasukawa et al., 2009) group showed that the heptad repeat region 1 (HR1) of Mfn2 interacts with MAVS, sequestering it in a nonproductive state and inhibiting downstream signaling by NF-κB and IRF3 upon RIG-I activation. These effects are specific for Mfn2, as manipulation of its homolog Mfn1 was found to have no effect on RIG-I-mediated signaling. A year later, Castanier et al. (2010) reported that RIG-I-mediated signaling can be regulated through the manipulation of mitochondrial dynamics. The promotion of mitochondrial elongation by silencing DRP1 or FIS1 increases the phosphorylation of IRF3 and IκB upon viral infection. Similarly, the induction of mitochondrial fragmentation by silencing Mfn1 or OPA1 has the opposite effect. These data demonstrate that the elongation and fusion of mitochondria specifically enhance MAVS signaling, whereas mitochondrial fragmentation impairs it. Interestingly, apart from being

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regulated by mitochondrial dynamics, MAVS can regulate this process. It has been proposed that this regulation occurs when, under basal conditions, Mfn1 is sequestered by interaction with MAVS. However, when RIG-I engages MAVS, Mfn1 is released, allowing fusion between adjacent mitochondria—a process that favors antiviral signaling. Furthermore, this mitochondrial elongation triggers ER–mitochondria associations, thus promoting the interaction of MAVS with STING to further enhance the aforementioned signaling (Castanier et al., 2010). In line with the latter study, Onoguchi et al. (2010) also demonstrated that Mfn1 plays a critical role in RIG-I-induced antiviral signaling, as overexpression of Mfn1 increased IFN-β promoter activity, whereas its silencing abolished such activity. However, there are some discrepancies with the other two studies mentioned earlier. First, Onoguchi’s group found that Mfn2 overexpression had no effect on antiviral signaling and thus concluded that Mfn1 was the relevant form, in clear contradiction with the observations made by Yasukawa et al. (2009) and Onoguchi et al. (2010). While Castanier et al. described that silencing DRP1 increases antiviral signaling; this effect was not reproduced by Onoguchi’s or Yasukawa’s group. However, both groups concurred that knocking down OPA1 blocks antiviral signaling, thereby demonstrating a role for mitochondrial elongation in MAVS signaling. Furthermore, neither did they observe mitochondrial elongation mediated by viruses nor 50 ppp-RNA. They hypothesized that the elongation of mitochondria observed by Castanier’s group was the consequence of using a variant of the Sendai virus (SeV) (H4) that specifically elongates mitochondria, but that other viruses do not have the same effect (Castanier et al., 2010; Onoguchi et al., 2010). Also, Koshiba et al. reported that depletion of both Mfn1 and Mfn2 resulted in a decreased induction of type 1 IFNs and proinflammatory cytokines upon viral infection (Koshiba et al., 2011). Deficiency in only one Mfn did not have any effect, presumably due to complementation by its homolog cells lacking both Mfn forms showed a disrupted mitochondrial network, as well as a decreased mitochondrial membrane potential (mΔΨ). Those authors proposed that this decrease in mΔΨ is responsible for the defect in MAVS signaling, as treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, an uncoupler of oxidative phosphorylation that leads to widespread loss of mΔΨ) resulted in the same inhibition as that observed in the double Mfn KO animal. They reasoned that the loss of mitochondrial potential might prevent the structural rearrangement of the MAVS

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complex, thus inhibiting polymerization and consequently suppressing downstream signaling (Koshiba et al., 2011). Finally, a recent study further confirmed the relevance of mitochondrial dynamics in antiviral signaling (Yu et al., 2015). Mfn1 and Mfn2 participate in the immune response against Dengue virus but are degraded as a result of cleavage by a Dengue virus protease. Using knockdown and overexpression approaches, Yu et al. showed that the two Mfns exert distinct functions during Dengue infection. In this regard, Mfn1 is required for efficient RIG-I signaling, whereas Mfn2 maintains mΔΨ, thus preventing cell death (Yu et al., 2015). Although some discrepancies remain, it is widely accepted that mitochondrial dynamics play a major role in MAVS-mediated antiviral responses. The data gathered to date support the notion that mitochondrial fusion is required to ensure proper signaling through MAVS, either by maintaining mΔΨ, by facilitating the formation of the RIG-I-MAVS signalosome, or by allowing the MAVS–STING interaction. The exact role played by the two Mfns proteins in this process requires further clarification (Fig. 3).

4.5 ROS Regulation of Antiviral Signaling Several studies have confirmed that mROS positively regulate antiviral signaling. A paradigmatic example is the cytochrome c oxidase complex subunit 5B (COX5B), a mitochondrial protein that directly interacts with MAVS to suppress antiviral responses. Interestingly, COX5B functions as a terminal enzyme of the ETC and inhibits mROS production. This negative regulation of mROS is responsible for the impairment of antiviral signaling (Zhao et al., 2012). Another study involving the use of autophagy-deficient cells revealed enhanced RLR signaling (Tal et al., 2009). These cells accumulate dysfunctional mitochondria, which produce a large amount of mROS. Antioxidant treatment reverses increased antiviral signaling, thereby revealing that the amplification of RLR signaling is dependent on ROS. Additionally, increasing mROS production in wild-type cells by means of rotenone treatment also results in enhanced RLR signaling. This observation thus confirms that mROS are sufficient to enhance RLR signaling pathway (Tal et al., 2009). Finally, another study demonstrated that ROS are essential for RIG-Imediated IRF-3 phosphorylation and dimerization, and the subsequent production of IFN-β (Soucy-Faulkner et al., 2010). All in all, these observations confirm that mROS are also a necessary intermediate moderator in RLRmediated antiviral signaling (Fig. 3).

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5. INFLAMMASOME ACTIVATION AND MITOCHONDRIA Upon interaction of PAMPs with TLRs and RLRs, the molecules NF-κB, IRFs, and MAPKs trigger the transcription of proinflammatory genes (Takeuchi & Akira, 2010). Another group of PRRs uses a completely different mechanism to trigger transcription consisting of the assembly of multimericsignaling complexes known as inflammasomes. The basic structure of these complexes comprises a sensor molecule, the adaptor protein apoptosisassociated speck protein containing a CARD (ASC) and caspase-1. ASC has one pyrin domain, which interacts with the sensor molecule, and one CARD, which interacts with caspase-1. Upon assembly, caspase-1 proteolytically activates proinflammatory cytokines IL-1β and IL-18. The release of these cytokines is a two-step process. The first step involves the expression of the inflammasome components and the inactived form of both pro-IL-1β and pro-IL-18 in a TLR-dependent fashion. This is also known as the priming step. The second step comprises the assembly of the inflammasome upon recognition of its ligand, and proteolytic cleavage of IL-1β and IL-18 zymogens by caspase-1 (Latz et al., 2013; Schroder & Tschopp, 2010). In addition to triggering both cytokines, caspase-1 activation can also lead to pyroptosis, a highly inflammatory form of programmed cell death that occurs most frequently upon infection with intracellular pathogens (Miao, Rajan, & Aderem, 2011). Unlike apoptosis, pyroptosis results in plasma-membrane rupture and the release of DAMP molecules such as ATP, DNA, and ASC oligomers into the extracellular milieu. These DAMPs induce cytokines that recruit more immune cells and further perpetuate the inflammatory cascade in the tissue. There are several subtypes of canonical inflammasomes, each named after its sensor molecule and protein scaffold, the four most widely studied being: NOD-, LRR-, and pyrin domain-containing 1 (NLRP1), NLRP3, NLR CARD domain-containing protein 4 (NLRC4), and interferon-inducible protein (AIM2), the latter also known as absent in melanoma 4 (Lamkanfi & Dixit, 2014). NLRC4 and NLRP1 activate caspase-1 without ASC because they already have CARDs through which to recruit this caspase directly (Latz et al., 2013).

5.1 The Mitochondrion Is a Scaffold for Inflammasome Activation Recent studies suggest that there is a very tight relationship between mitochondria and inflammasome activation. Similarly to what happens with

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MAVS-mediated antiviral signaling, mitochondria act as a signaling platform for inflammasome activation (Cloonan & Choi, 2013). Under basal conditions, NLRP3 is located in the ER; however, upon activation, both NLRP3 and the adaptor protein ASC translocate to MAMs in the perinuclear region (Zhou, Yazdi, Menu, & Tschopp, 2011). One crucial step here is the spatial rearrangement of mitochondria around ER membranes. This rearrangement occurs through a microtubule- and dynein-mediated mechanism that is required for inflammasome assembly (Misawa et al., 2013). Furthermore, the antiviral signaling protein MAVS is also required for optimal NLRP3 inflammasome activity. In response to inflammasome activators, MAVS favors NLRP3 recruitment to mitochondria and the subsequent maturation of IL-1β (Subramanian, Natarajan, Clatworthy, Wang, & Germain, 2013). Additionally, in analogy to antiviral signaling, the integrity of the mitochondrial network is crucial for correct inflammasome signaling. Loss of mΔΨ dramatically reduces inflammasome activation and IL-1β production. Moreover, the mitochondrial fusion protein Mfn2 also interacts with both MAVS and NLRP3 in a mΔΨ-dependent manner. This interaction is required for the proper assembly and activation of the inflammasome at the mitochondrial surface (Yoshizumi et al., 2014). The observation that Mfn2 is necessary for inflammasome activation suggests that, as well as with antiviral signaling, mitochondrial elongation is a requisite for this activation. Using macrophages deficient for the mitochondrial fission protein DRP1, Park et al. (2015) recently confirmed this hypothesis. These macrophages show increased mitochondrial elongation and enhanced NLPR3 assembly and IL-1β secretion. Furthermore, mitochondrial elongation promotes ERK activation, which is a prerequisite for the recruitment of NLRP3 to mitochondria and thus for the activation of the inflammasome. All together, these data indicate that mitochondria are signaling platforms for inflammasome assembly and activation, and thus mΔΨ and mitochondrial elongation are critical factors that modulate the inflammasome (Fig. 4).

5.2 Mitochondrial Signals in Inflammasome Activation Several mitochondria-driven signals, such as mROS, cytosolic mDNA, cardiolipin, and mitochondrial Ca2+ influx, are associated with NLRP3 inflammasome activation. NLRP3 stimulation by most of its agonists, such as ATP, silica, and nigericin, induces and requires an increase in mROS

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Fig. 4 Mitochondria are heavily involved in inflammasome activation. TLR signaling induces the expression of multiple genes, including NLRP3, and the inactive forms of IL-1β and IL-18. In basal conditions, NLRP3 is located in the ER, but when inflammasome-activating signals are present, NLRP3 is assembled with ASC and caspase1, and it translocates to mitochondria. Some inflammasome agonists directly induce the NLRP3 inflammasome, but usually they trigger mitochondrial damage, releasing mitochondrial DAMPs such as mROS, calcium, mDNA, and cardiolipin. These DAMPs are recognized by NLRP3 inflammasome-activating caspase-1, which cleaves pro-IL-1β and pro-IL-18 into their mature forms. The ER is also involved in inflammasome activation, as IRE1α branch of the UPR response, enhances the release of mROS, and activating the inflammasome. Mitophagy, the specific form of autophagic removal of dysfunctional mitochondria, prevents the accumulation of damaged mitochondria, thus restricting mitochondrial DAMP release and consequently regulating inflammasome activation. There are positive and negative feedback mechanisms to tightly regulate inflammasome activation through autophagy. P62 is a protein expressed in response to TLR priming that promotes mitophagy and acts as brake for excessive inflammasome activation. On the other hand, once the inflammasome has been activated, caspase-1 can cleave and degrade Parkin, a protein involved in mitophagy, thus preventing clearance of damaged mitochondria and thus enhancing inflammasome activation.

generation. Experimental manipulation that decreases mROS production also results in attenuation of inflammasome responses, thereby suggesting that these molecules are necessary for inflammasome activation (Cruz et al., 2007; Zhou et al., 2011). Several mechanisms have been put forward to explain mROS-mediated activation of the inflammasome, including modification of endogenous molecules to generate DAMPs, direct oxidation of NLRP3, and induction of mitochondrial dysfunction—the latter allowing the release of mitochondrial components to the cytosol (Elliott & Sutterwala, 2015). However, there are some exceptions in

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which mROS are dispensable for NLRP3 activation, such as in activation either by viruses (Yoshizumi et al., 2014) or by the antibiotic linezolid (Iyer et al., 2013). In addition to mROS, mDNA is another mitochondria-derived molecule that activates the NLRP3 inflammasome. Upon mitochondrial dysfunction, oxidized mDNA is released to the cytosol, where it binds and activates NLRP3 (Nakahira et al., 2011; Shimada et al., 2012). Similarly, when mitochondria are damaged, cardiolipin, a phospholipid normally located in the inner mitochondrial membrane, is exposed to the cytosolfacing outer membrane, thus activating the NLRP3 inflammasome (Iyer et al., 2013). Ca2+ is another activator of this inflammasome that can also lead to mitochondrial disruption, causing the release of mROS and mDNA, which further increase inflammasome activation (Horng, 2014). In most circumstances, activation of the NLRP3 inflammasome is associated with mitochondrial dysfunction. Nonetheless, there is some controversy as to whether mitochondrial damage occurs upstream or downstream of caspase-1 activation. Yu et al. (2014) showed that AIM2 and NLRP3 activation leads to caspase-1-dependent mitochondrial dysfunction, including membrane depolarization and permeabilization. This finding would place mitochondrial damage downstream of caspase-1 activation. However, a number of studies have reached the opposite conclusion, concurring that mitochondrial dysfunction takes place upstream of caspase-1 activation (Elliott & Sutterwala, 2015). Recently, using two distinct models, Zhong’s (Zhong et al., 2016) group demonstrated that mitochondrial damage occurs upstream of caspase-1. First, they used macrophages deficient for NLRP3, ASC, and caspase-1. These cells exhibit the same level of mitochondrial dysfunction as control macrophages incubated with inflammasome agonists. They also used macrophages containing a constitutively activated variant of NLRP3 that releases IL-1β without stimulation by inflammasome agonists. These macrophages did not show mitochondrial damage when compared to control macrophages in the absence of NLRP3 agonists. These findings support the notion that mitochondrial dysfunction is independent and upstream of caspase-1 activation. Finally, ER stress can also trigger inflammasome activation through the inositol-requiring enzyme 1α (IRE1α) pathway. IRE1α increases mROS production, thereby mediating the recruitment of NLRP3 to mitochondria and its activation. NLRP3 then forms a noncanonical complex, which is associated with caspase-2 rather than ASC and caspase-1. NLRP3 and caspase-2 induce mitochondrial damage via Bid and the opening of the mitochondrial permeability transition

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pore, thus releasing mDNA to the cytosol. This cytosolic mDNA triggers canonical NLRP3 inflammasome responses. In this case, NLRP3 acts both upstream (associated with caspase-2) and downstream (associated with caspase-1 and ASC) of mitochondrial dysfunction (Bronner et al., 2015). Overall, current data support the notion that mitochondrial dysfunction and the release of DAMPs is a crucial step in inflammasome activation and that this damage probably occurs upstream of inflammasome activation itself. However, in some cases, NLRP3 may be activated upstream of mitochondrial damage, thus forming either a canonical or a noncanonical complex (Fig. 4).

5.3 Mitophagy Restrains Inflammasome Activation Mitophagy is a specific form of autophagy that selectively removes damaged mitochondria (Lazarou, 2015). It prevents excessive inflammasome activation by preserving mitochondrial integrity. Inhibition of mitophagy by depleting autophagy proteins results in the accumulation of dysfunctional mitochondria. These damaged organelles produce excessive amounts of mROS and release mDNA to the cytosol, thus triggering the inflammasome (Nakahira et al., 2011). Activation of caspase-1 can also degrade Parkin, a protein involved in mitophagy. This degradation increases mitochondrial damage and releases mitochondrial DAMPs, thus amplifying inflammasome activation in a positive forward loop (Yu et al., 2014). However, other proteins involved in mitophagy may play distinct roles in the activation of the inflammasome. Additionally, a negative loop that restricts excessive activation has recently been described. Upon the priming signal (i.e., TLR stimulation), NF-κB is activated, inducing the expression of NLRP3 and pro-IL-1β. However, as a safety mechanism, NF-κB also induces the expression of p62. This molecule mediates mitophagy-mediated removal of damaged mitochondria, thus preventing the release of NLRP3 inflammasome-activating signals and thereby regulating the activation of this molecule (Zhong et al., 2016). As an alternative mechanism to restrain inflammasome activation, autophagy can also eliminate active inflammasome complexes, as well as pro-IL-1β (Harris et al., 2011; Shi et al., 2012). In brief, most studies point to autophagy as the critical process in preventing excessive inflammasome activation. Although this regulation is usually achieved by eliminating damaged mitochondria, the removal of inflammasome components may also be a relevant mechanism (Fig. 4).

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6. CONCLUDING REMARKS The innate immune response is an extremely complex process that requires tight coordination between the recognition of pathogens and stress signals, and the activation of specific signaling pathways. Apart from the energy-producing role of mitochondria, it is clear now that these organelles are also hubs for innate immune signaling. They integrate multiple signals from the surrounding environment, thereby enabling the generation of appropriate responses. The crosstalk between metabolism and innate immune responses is crucial. In macrophages, the reprogramming of mitochondrial metabolism depends on the activation state of these organelles. Upon stimulation with LPS or IFN-γ, macrophages shift to a phenotype that favors the rapid production of energy and intermediate metabolites required to effectively fight infections. In contrast, stimulation with IL-4 triggers fatty acid oxidation and OXPHOS to sustain long-term tissue repair and inflammation resolution. An interesting point here is that these metabolic changes themselves determine the correct function of macrophages in each specific context. This observation opens the door to the possibility of enhancing or decreasing inflammatory responses by experimentally modifying metabolism. Such a strategy could be envisaged for the treatment of autoimmunity and chronic inflammatory diseases. The production of mROS is another very important feature in innate immune responses, as this molecule activates multiple signaling pathways through oxidation, including MAPK, NF-κB, and the NLRP3 inflammasome. It is now clear that mROS production can be regulated depending on circumstances, UCP2- and LPS-mediated TRAF6 regulation being clear examples. However, further research efforts are needed to identify the specific targets of mROS and how these species modulate signaling pathways in different contexts. In addition, in order to control immune responses by regulating the metabolic profile and mROS production, mitochondria directly participate in signaling by providing a physical scaffold for multiple proteins involved in immune signaling cascades. This is the case for MAVS in antiviral responses and NLRP3 in inflammasome activation. In these cases, in order to exert their function, the proteins must be located in the mitochondrial membrane, where they can interact with many more proteins. Indeed, in these cases, mitochondria are not merely physical scaffolds, but they also modulate

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the functionality of these proteins by modifying parameters such as mΔΨ or their fusion state. Although mitochondrial dynamics are crucial for the regulation of these pathways, the exact role of each Mfn is still unclear, and the apparent contradictions between studies require clarification. Finally, damaged mitochondria produce many signals that can activate the inflammasome. Also, autophagy plays a major role in this context by eliminating dysfunctional mitochondria, thus contributing to regulating the release of DAMPs and inflammasome activation. Interestingly, mitochondria act as sensors for several stressors that damage these organelles, leading to the release of DAMPs, which in turn activate the inflammasome. However, more research has to be done to clarify whether this mitochondrial damage occurs upstream of NLRP3 activation and to identify the factors that determine the contexts in which mitochondrial damage is required for this activation and those in which it is not.

ACKNOWLEDGMENTS This work was supported by grant SAF2014-52887-R awarded to J.L. and A.C. by the Ministerio de Ciencia y Tecnologı´a. J.T. was supported by Formacion del Profesorado Universitario grant AP2012-02327.

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Yasukawa, K., Oshiumi, H., Takeda, M., Ishihara, N., Yanagi, Y., Seya, T., et al. (2009). Mitofusin 2 inhibits mitochondrial antiviral signaling. Science Signaling, 2, ra47. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., et al. (2005). Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. Journal of Immunology, 175, 2851–2858. Yoshizumi, T., Ichinohe, T., Sasaki, O., Otera, H., Kawabata, S., Mihara, K., et al. (2014). Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity. Nature Communications, 5, 4713. Yu, C. Y., Liang, J. J., Li, J. K., Lee, Y. L., Chang, B. L., Su, C. I., et al. (2015). Dengue virus impairs mitochondrial fusion by cleaving mitofusins. PLoS Pathogens, 11, e1005350. Yu, J., Nagasu, H., Murakami, T., Hoang, H., Broderick, L., Hoffman, H. M., et al. (2014). Inflammasome activation leads to caspase-1-dependent mitochondrial damage and block of mitophagy. Proceedings of the National Academy of Sciences of the United States of America, 111, 15514–15519. Zhao, Y., Sun, X., Nie, X., Sun, L., Tang, T. S., Chen, D., et al. (2012). COX5B regulates MAVS-mediated antiviral signaling through interaction with ATG5 and repressing ROS production. PLoS Pathogens, 8, e1003086. Zhong, Z., Umemura, A., Sanchez-Lopez, E., Liang, S., Shalapour, S., Wong, J., et al. (2016). NF-kappaB restricts inflammasome activation via elimination of damaged mitochondria. Cell, 164, 896–910. Zhong, B., Yang, Y., Li, S., Wang, Y. Y., Li, Y., Diao, F., et al. (2008). The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity, 29, 538–550. Zhou, R., Yazdi, A. S., Menu, P., & Tschopp, J. (2011). A role for mitochondria in NLRP3 inflammasome activation. Nature, 469, 221–225. Zorzano, A., Hernandez-Alvarez, M. I., Sebastian, D., & Mun˜oz, J. P. (2015). Mitofusin 2 as a driver that controls energy metabolism and insulin signaling. Antioxidants & Redox Signaling, 22, 1020–1031. Zorzano, A., Liesa, M., Sebastian, D., Segales, J., & Palacin, M. (2010). Mitochondrial fusion proteins: Dual regulators of morphology and metabolism. Seminars in Cell & Developmental Biology, 21, 566–574.

CHAPTER TWO

Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination S.P. Methot*,†,1, J.M. Di Noia*,†,{,1 *Institut de Recherches Cliniques de Montr
eal (IRCM), Montreal, QC, Canada † Mcgill University, Montreal, QC, Canada { Universit
e de Montr
eal, Montreal, QC, Canada 1 Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. Antibody Diversification During the Humoral Response 2. Molecular Mechanisms of SHM and CSR 2.1 Overview 2.2 Relevance for Humoral Immunity 3. DNA Deamination at the Igs 3.1 Activation-Induced Deaminase 3.2 Molecular Regulation of AID Activity 3.3 Targeting AID to the Ig Loci 3.4 Phosphorylation Focuses AID Activity to the Ig Locus 4. AID Initiates a DNA Repair Cascade 4.1 Detecting Uracil 4.2 The UNG and Base Excision Repair Arm 4.3 The MutSα-Initiated Arm 4.4 Interactions Between BER and MMR During Antibody Diversification 4.5 DNA Nicks and Breaks 4.6 Repairing the Breaks 5. Postdeamination Roles of AID? 6. Perspectives Acknowledgments References

38 39 39 41 41 41 42 47 56 57 57 58 61 63 65 67 69 70 71 71

Abstract In order to promote an efficient humoral immune response, germinal center B cells modify both the antigen recognition and effector domains by programmed genetic alterations of their antibody genes. To do so, B cells use the enzyme activation-induced deaminase (AID), which transforms deoxycytidine into deoxyuridine at the immunoglobulin genes, triggering mutagenic DNA repair. Data accumulated during the past decade have significantly advanced our understanding of how AID activity is regulated

Advances in Immunology, Volume 133 ISSN 0065-2776 http://dx.doi.org/10.1016/bs.ai.2016.11.002

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2017 Elsevier Inc. All rights reserved.

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and preferentially targeted to the immunoglobulin genes. There is also a better understanding of the ways by which AID-catalyzed uracil is recognized and the ensuing downstream processing underpinning the mechanisms of somatic hypermutation and class switch recombination. Here, we critically review these advances in the context of their relevance for the humoral immune response. A detailed understanding of these molecular mechanisms is paramount to uncover the basis of B cell intrinsic immunodeficiency, as well as to suggest tools and strategies that might allow boosting antibody gene diversification in the context of immunizations or infections that require the elicitation of rare or highly mutated antibody variants.

1. ANTIBODY DIVERSIFICATION DURING THE HUMORAL RESPONSE Naı¨ve and memory B cells that engage cognate antigen through their surface B cell receptor can initiate genetic mechanisms to modify the immunoglobulin genes (Ig) encoding for the antibody. The purpose of these alterations, which happen most often inside the germinal center, is to alter the antibody affinity and function. This is accomplished by the processes of somatic hypermutation (SHM) and class switch recombination (CSR), respectively. Both SHM and CSR are initiated by the enzyme activationinduced deaminase (AID), which is induced in germinal center and some extrafollicular B cells (Cattoretti et al., 2006; Crouch et al., 2007; Muramatsu et al., 1999). SHM introduces single point mutations in the variable regions of the Ig loci (IgV), which can alter the antibody binding to its cognate antigen. Mutations that promote affinity for the antigen will be selected for, resulting in a progressive increase in the affinity of the antibody response. AID will also initiate DNA double strand breaks (DSBs) in the regions of the Ig heavy chain gene (Igh) that encode for the domains determining the antibody class. These DSBs trigger CSR, a recombination that swaps the antibody’s isotype and thereby its biological properties. Together, SHM and CSR are vital for a specific and high-affinity humoral immune response that is tailored to a particular pathogen and able to clear the infection. The frequency of SHM is estimated to be around 10
3 per base pair per generation at the IgV, which would amount to one mutation per cell division (Rajewsky, F€ orster, & Cumano, 1987). SHM levels in circulating antibodies are highly variable, depending on the antigen, route of exposure, selection, etc. Antibodies expressed in memory B cells, which have undergone affinity

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maturation, bear 10–20 mutations per IgV, typically clustered at the complementary determining regions (CDRs) (Tiller et al., 2007), which directly contact the antigen. However, highly selected neutralizing antibodies against influenza virus can accumulate 30–40 mutations; and broadly neutralizing antibodies against HIV up to >100 mutations, at the CDR as well as framework regions (Klein et al., 2013; Wrammert et al., 2008). Moreover, insertion and deletion events, which develop as byproducts of SHM, are also relatively common in neutralizing anti-HIV antibodies (Kepler et al., 2014). The unusually high levels of SHM and insertion/deletions suggests that these broadly neutralizing antibodies arise from rare variants and are generated over a long time and through multiple rounds of germinal center reactions. Given their relevance in neutralizing virus that have high antigenic variability and pose a heavy burden on human health, a large effort is underway to mimic this type of reaction by immunization, which requires multiple immunizations using an evolving antigen (Burton & Hangartner, 2016; Doria-Rose & Joyce, 2015). Understanding the molecular mechanisms of SHM and its regulation in detail is not only a fundamental quest but could also provide alternative or complementary strategies for the elicitation of rare neutralizing antibodies.

2. MOLECULAR MECHANISMS OF SHM AND CSR 2.1 Overview SHM and CSR can be described as biochemical pathways in which one enzyme generates the substrate of the next (Fig. 1). Some species, like chickens, use a recombination-based mechanism named Ig gene conversion in addition to or instead of SHM to diversify the IgV (for a review, see Tang & Martin, 2007). These three processes are mechanistically related and are initiated by AID through the same enzymatic reaction: the deamination of DNA cytosine bases to convert them to uracils (Fig. 1). This activity was foreseen in the DNA deamination model for SHM and CSR (Petersen-Mahrt, Harris, & Neuberger, 2002; Poltoratsky, Goodman, & Scharff, 2000). The model, for which there is overwhelming evidence, posits that the AID-catalyzed uracils in the DNA are recognized by either the uracil-DNA glycosylase UNG or, as a U:G mispairing by the mismatch recognition heterodimer MutSα, made of the MSH2 and MSH6 enzymes. UNG and MutSα precede AID in evolution and normally initiate error-free uracil repair pathways that would reestablish the original cytosine. However,

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AID expression and regulation

AID

Ag

Antibody B cell receptor

C AICDA

NH2

Ig locus C O

CN N O UNG

U N

AID targeting

NH

Deamination

O MutSa

Uracil recognition Uracil processing

SHM

CSR Repair

Resolution

Fig. 1 Scheme of the major steps during antibody diversification in B cells. This scheme provides the framework for the review. B cells express AID after recognizing the antigen (Ag) through surface antibody acting as the B cell receptor (BCR), to initiate SHM and CSR of the antibody genes. AID is regulated at multiple levels in the cytoplasm and in the nucleus. In the nucleus, AID must home to the Ig loci via its association with the transcription machinery in order to access the DNA. AID deaminates cytosine bases (C) in the DNA to generate uracils (U). The U is recognized by either the UNG or the MutSα heterodimer, and subsequently processed in several possible ways. Final resolution of the lesion results in either faithful repair or antibody diversification via SHM or CSR.

downstream from AID, UNG, and MutSα initiate uracil processing with various possible outcomes, many of which are mutagenic and underpin antibody gene diversification. Thus, the uracil in DNA can be replaced by other bases; or prompt further mutation in its vicinity that expands the spectrum of SHM to include mutations at A:T pairs, which AID cannot directly modify. Uracils can also be converted into DNA DSBs, which are necessary for CSR. Thus, the pathways of SHM and CSR become progressively more complex as they go downstream from AID, with multiple layers of regulation and competition between alternative pathways defining the levels of SHM and the efficiency of CSR. Accordingly, early factors in the cascade play more defining roles in Ig diversification and need to be more regulated, as dramatically exemplified by the multitude of pathways regulating AID activity. Competition between the canonical DNA repair roles and the subverted action of UNG and MutSα also shapes the antibody response. Here, we will review recent advances in this field using this general scheme as a framework (Fig. 1).

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2.2 Relevance for Humoral Immunity AID deficiency leads to dramatic immunodeficiency, with the whole antibody repertoire restricted to unmutated IgM (Muramatsu et al., 2000; Revy et al., 2000). While AID cannot trigger CSR on its own, it is sufficient for a limited version of SHM. Uracil in DNA is indistinguishable from thymine for most DNA polymerases; hence, AID can directly produce transition mutations at C:G pairs (from C:G to T:A, Fig. 1) and thereby diversify the antibody genes, as shown by simultaneously removing Ung and MutSα (Shen, Tanaka, Bozek, Nicolae, & Storb, 2006; Xue, Rada, & Neuberger, 2006). The importance of SHM for gut homeostasis was demonstrated using a mouse model expressing an AID variant that allows normal levels of IgA in the gut but display severely reduced SHM (Wei et al., 2011). However, it is difficult to test whether the restricted pattern of SHM produced only by the AID footprint would be enough for adaptive immunity because these mice completely lack CSR. However, since even germline sequences are able to protect from certain infections, it is likely that even SHM restricted to transitions at C:G would be advantageous for the response against some infections. For instance, SHM is largely restricted to C:G pairs in some species, such as Xenopus (Wilson et al., 1992). On the other hand, normal levels of SHM with very low levels of CSR can be obtained in Ung
/
mice upon acute immunization or infection (Zahn et al., 2013). These mice are capable of affinity maturation of the IgM response but their ability to fight infections has not been tested. Nonetheless, the severely reduced kinetics of CSR in Ung
/
mice (Zahn et al., 2013) and the profound immunodeficiency of UNG-deficient patients (Imai et al., 2003) highlight the critical role of UNG and CSR in immune responses. Patients lacking MutSα or its downstream factor PMS2 display cancer predisposition but also reduced CSR, though their immunodeficiency is milder than in patients lacking AID or UNG (P
eron et al., 2008; Whiteside et al., 2002).

3. DNA DEAMINATION AT THE IGS 3.1 Activation-Induced Deaminase AID was discovered as a factor upregulated in the mouse CH12F3 B cell lymphoma line upon stimulation for CSR (Muramatsu et al., 1999). Genetic deficiency then demonstrated that AID is essential for SHM and CSR in mice and humans (Muramatsu et al., 2000; Revy et al., 2000), as well as for Ig gene conversion in chicken DT40 B cells (Arakawa, Hauschild, &

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Buerstedde, 2002). The identification of AID was vital for unraveling the molecular mechanisms of secondary antibody gene diversification in the past 15 years. AID forms a family of polynucleotide deaminases with the APOBEC enzymes, most of which can catalyze the conversion of C to U in DNA (Conticello, Thomas, Petersen-Mahrt, & Neuberger, 2005; Harris, Petersen-Mahrt, & Neuberger, 2002; Petersen-Mahrt et al., 2002). In B cells, the deamination activity of AID is concentrated at the IgV and switch (S) regions of the Ig loci to initiate SHM and gene conversion or CSR, respectively. With a lower but consequential frequency, AID concomitantly targets a subset of genomic loci usually referred to as off-targets, which underpin its oncogenic activity. AID off-targeting has recently been reviewed in detail (Casellas et al., 2016). The molecular mechanism of SHM, gene conversion, and CSR shows many similarities, sharing several enzymes. Yet, AID is so far the only factor that is absolutely necessary for all these mechanisms. It is also the only vertebrate- and B cell-specific factor, with all other enzymes participating in SHM and CSR being ubiquitous in most cell types and species. Not surprisingly, AID has been studied the most and is tightly regulated. The regulation of the AID gene (AICDA) expression combines cis- and trans-acting factors, as well as micro-RNAs that regulate its mRNA stability. How these allow maximum expression of AID specifically in activated B cells has been reviewed (Zan & Casali, 2013). We will concentrate here on the properties and regulation of the AID protein (Fig. 2) that may influence its mutagenic activity and could contribute to AID deaminating the Ig loci at a much higher frequency than any of its off-target sites (Liu et al., 2008).

3.2 Molecular Regulation of AID Activity 3.2.1 Biochemical Activity of AID AID deaminates cytosine in single-stranded DNA (ssDNA) (Bransteitter, Pham, Scharff, & Goodman, 2003; Dickerson, Market, Besmer, & Papavasiliou, 2003), and most efficiently so within a short DNA bubble (Larijani et al., 2007). This type of structure resembles the transcription bubble and indeed transcription greatly enhances the biochemical activity of AID on duplex DNA (Chaudhuri, Khuong, & Alt, 2004; Chaudhuri et al., 2003; Ramiro, Stavropoulos, Jankovic, & Nussenzweig, 2003). The preference of AID for deaminating cytosine within the WRC motif context (W ¼ A/T, R ¼ A/G) in vitro (Pham, Bransteitter, Petruska, & Goodman, 2003) explains the existence of SHM “hot spots” with the same

43

Early Molecular Events During Antibody Diversification

Positive regulation Function

Enzymatic activity or deamination efficiency

Negative regulation

Mediator RPA

Specificity loop

Mediator

Function

PKC

Enzymatic activity or deamination efficiency

Catalyic site

H-89

PKA Fsk

PKC

T140 T27

Cytoplasmic stabilization

Nuclear import

GA FTI

Hsp90 DnaJa1 Importins GANP? CTNNLB1?

D187 D188

Reg-γ

NES

Nuclear destabilization

Ub-ligase T140

S38

S3

T27

eEF1A1

DidB CytA

Cytoplasmic retention

CRM1

LMB

Nuclear export

Fig. 2 Regulation of AID function. Mechanisms that positively (green) or negatively (red) affect AID function are indicated, along with the protein factors that mediate them (black). Drugs that can indirectly affect AID function by inhibiting or activating these factors are indicated (gray) connected to their target. AID structures are shown in the middle: Top, experimental structure of the AID catalytic core (PDB 5JJ4) (Pham et al., 2016). The catalytic site (teal), specificity loop (blue), catalytic pocket (gray surface), and Zn2+ ion (orange ball) are highlighted. Note that the N-terminal domain (pink) was altered to permit solubility and the C-terminus was truncated. Bottom, a model of AID, based on the APOBEC3C structure, including the C-terminal domain (pink) and native N-terminus (Methot et al., 2015). Residues D187 and D188 (blue) necessary for cytoplasmic retention and the hydrophobic nuclear export sequence (NES—dark green) are highlighted. The NES residues are buried within a hydrophobic groove in the catalytic core. Phosphorylation sites are indicated with balls on both structures: S3 and S38 are only indicated on the model because they are not present in the experimental structure.

consensus sequence within the IgV and S-regions (Di Noia & Neuberger, 2007). This sequence preference of AID is directed by a specificity loop in AID (Conticello, Langlois, & Neuberger, 2007; Kohli et al., 2009; Wang, Rada, & Neuberger, 2010) (Fig. 2), which is a conserved feature of the APOBEC family (Langlois, Beale, Conticello, & Neuberger, 2005). Altering this loop alters AID specificity and SHM preference in B cells (Wang et al., 2010), which together with the direct demonstration of uracil at the Ig (Maul et al., 2011), definitively established that AID directly deaminates the Ig genes. The structural basis of the AID sequence preference was recently elucidated by resolving AID crystal structure. This structure showed that the specificity loop of AID is particularly open, explaining its preference for a purine residue adjacent to the cytosine (Pham et al.,

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2016; Fig. 2). It is interesting that recombinant AID copurifies tightly associated to RNA, which inhibits its DNA deaminase activity (Bransteitter et al., 2003). This puzzling feature may reflect the recent proposal, based on structural modeling, that AID has multiple ways of accommodating single-stranded nucleic acid, with most of them precluding the access of deoxycytidine to the catalytic pocket as a way of dampening its activity (King et al., 2015). On the other hand, once properly attached to ssDNA AID can apparently catalyze multiple deamination events (Pham et al., 2003). Clusters of deaminated C in the same strand at S-regions can be found, suggesting that clustered deamination can happen in vivo (Xue et al., 2006). This “processivity” could help in forcing mutagenic repair or increasing the chances that the uracil escapes repair and is replicated over during the S-phase (see later). Thus, it is possible that the intrinsic enzymatic activity of AID is modulated according to its location and association with the substrate. The ability of AID to deaminate the genome is not without consequences. B cell malignancies represent 85–90% of all non-Hodgkin lymphoma, an overrepresentation compared to T cell malignancies that is likely due in part to the activity of AID. Indeed, normal AID expression has been directly linked to the development or progression of B cell lymphoma or leukemia in mouse models (Montamat-Sicotte et al., 2015; Pasqualucci et al., 2008; Robbiani et al., 2015; Robbiani & Nussenzweig, 2013; Swaminathan et al., 2015). AID expression has been also clearly associated with bad prognosis in multiple human hematological malignancies (Montamat-Sicotte, Palacios, Di Noia, & Oppezzo, 2013; Robbiani et al., 2015; Robbiani & Nussenzweig, 2013; Swaminathan et al., 2015). The oncogenic activity of AID is predominantly due to its ability to target and deaminate genes outside the Ig locus, causing mutations and chromosomal translocations (Casellas et al., 2016; Robbiani & Nussenzweig, 2013). The AID crystal structure (Pham et al., 2016) now affords unprecedented insight into the catalytic pocket of AID, which should facilitate the development of specific inhibitors that may be useful as adjunctive therapies. 3.2.2 Limiting the Levels of AID in the Nucleus Given that SHM and CSR are genetic modifications that happen in the nucleus, the initial observation that AID localized predominantly to the cytoplasm of B cells was puzzling (Rada, Jarvis, & Milstein, 2002). This distribution is actually the consequence of AID shuttling constantly between nucleus and cytoplasm (Brar, Watson, & Diaz, 2004; Ito et al., 2004;

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McBride, Barreto, Ramiro, Stavropoulos, & Nussenzweig, 2004). It has now been established that an intricate balance between mechanisms of nuclear import, nuclear export, retention in the cytoplasm and differential stability of the protein in each compartment, control a dynamic equilibrium in which only 10% of AID is in the nucleus (Fig. 2). The significance of such a complex regulation for AID subcellular localization is not fully understood, although at least in part it contributes to limit the access of AID to the genome for an optimal balance between Ig diversification and deleterious off-target activity. AID is actively imported into the nucleus despite being small enough to diffuse through the nuclear pores, because a mechanism of cytoplasmic retention prevents its diffusion (Patenaude et al., 2009). The nuclear import of AID is likely mediated by the interaction of AID with karyopherins, a family of nuclear importins (Hu et al., 2013; Patenaude et al., 2009). Experiments in the chicken B cell line DT40 showed that the karyopherin-like splicing factor CTNNBL1 was necessary for Ig gene conversion and contributed to AID nuclear import (Ganesh et al., 2011). However, CTNNBL1 ablation has little effect on AID function in mouse CH12F3 and primary B cells (Chandra, van Maldegem, Andrews, Neuberger, & Rada, 2013; Han, Masani, & Yu, 2010). So, either the karyopherins and CTNNBL1 are redundant for AID nuclear import, or the CTNNBL1 plays an unrelated, but also redundant, role in AID biology. Overexpression of GANP, an RNA-binding protein involved in mRNA shuttling, promotes AID accumulation in the nucleus (Maeda et al., 2010). However, this may actually reflect the role of GANP in assisting AID recruitment to the Ig loci (Singh et al., 2013). In any case, there is good evidence that GANP favors SHM and affinity maturation (Kuwahara et al., 2004). Two activities determine AID nuclear exclusion and both are mediated by the C-terminal domain of AID. This short tail of 17 amino acids, which is not conserved in the APOBECs (Methot et al., 2015), plays multiple functions. It is required for CSR and in some way favors the recruitment of downstream DNA repair enzymes (Barreto, Reina-San-Martin, Ramiro, McBride, & Nussenzweig, 2003; Ranjit et al., 2011; Sabouri et al., 2014; Shinkura et al., 2004; Zahn et al., 2014). It further contains a canonical leucine-rich nuclear export signal (NES) (Brar et al., 2004; Ito et al., 2004; McBride et al., 2004) and a motif necessary for cytoplasmic retention (Methot et al., 2015; Patenaude et al., 2009). The factors mediating AID nuclear exclusion are known (Fig. 2). Nuclear export of AID is mediated by the Ran-dependent nuclear exportin CRM1 (McBride et al., 2004).

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AID is retained in the cytoplasm by its association with a complex containing the translation elongation factor eEF1A1 (Hasler, Rada, & Neuberger, 2011; Methot et al., 2015). This role of eEF1A is unrelated to its function in protein biosynthesis. In fact, the binding of eEF1A to its tRNA cargo interferes with AID retention. Drugs stabilizing the eEF1A-tRNA association release AID from the cytoplasm, increase its nuclear concentration, and result in higher CSR, but also more chromosomal translocations (Methot et al., 2015). Interestingly, nuclear export and cytoplasmic retention cooperate to exclude AID from the nucleus (Methot et al., 2015), suggesting that these mechanisms have evolved together to finely tune AID access to the genome. The atomic structure of the C-terminal domain of the AID is not available because it had to be removed to generate soluble protein for crystallization (Pham et al., 2016). Yet, molecular modeling of this region suggest that the NES and cytoplasmic retention motif are exposed by mutually exclusive conformations of the C-terminus. The cytoplasmic retention motif depends on AID, adopting a conformation that is stabilized when the NES fits into a hydrophobic grove in the catalytic core of the enzyme (Methot et al., 2015). This model segregates the two functions in space, despite the close proximity of the motifs. Of note, truncating the C-terminus of AID or mutating the hydrophobic residues of the NES affects both nuclear export and cytoplasmic retention, which explains the nuclear localization of those mutants. AID localization is also affected by the protein stability because the halflife of AID is significantly shorter in the nucleus than in the cytoplasm (Aoufouchi et al., 2008; Fig. 2). On one hand, AID in the cytoplasm is protected from ubiquitin-dependent degradation by its association with the molecular chaperone HSP90 and cochaperone DnaJa1, which ensure that the optimal amount of AID protein is available (Orthwein et al., 2010, 2012). On the other hand, AID is destabilized in the nucleus, where it undergoes ubiquitin-dependent degradation by an unknown mechanism (Aoufouchi et al., 2008); as well as ubiquitin-independent degradation promoted by direct binding to the protein Reg-γ (Uchimura, Barton, Rada, & Neuberger, 2011). Whether the different nuclear degradation mechanisms act on distinct pools of AID is unknown. In any case, nuclear AID persists long enough to access and mutate the DNA for antibody diversification. Altogether, the shuttling and destabilization of AID create a complex mechanism to regulate its accumulation in the nucleus. This has a role in protecting the genome from a continuous barrage of mutations, permitting just the right amount of AID to access the genome. Indeed, forced accumulation of AID in the nucleus by either removing its C-terminus, knocking

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out Reg-γ, or inhibiting eEF1A1 results in higher antibody gene diversification but also higher levels of off-target mutations, oncogenic translocations, and/or cytotoxicity (Barreto et al., 2003; Methot et al., 2015; Shinkura et al., 2004; Uchimura et al., 2011; Zahn et al., 2014). Further work is required to determine whether each of these mechanisms is alternative means of limiting the amount of AID inside the nucleus or whether they might also have nonoverlapping roles. For instance, CRM1 and eEF1A may associate with distinct pools of AID that are not functionally equivalent (Methot et al., 2015). There are additional areas of AID stability and compartmentalization that require further work. AID seems to associate transiently with the nucleolus (Hu et al., 2013). It also seems that DNA damage can promote nuclear accumulation of AID, possibly via PARP signaling (Brar et al., 2004; Tepper et al., 2016). The significance of these observations is still unclear. In addition, it is likely that AID accumulation in the nucleus is cell cycle regulated, as suggested by observations in engineered systems that nuclear AID is relatively more stable in G1 than in S/G2 (Le & Maizels, 2015). The timing of deamination has been assumed to be G1, based on when DNA damage at the Ig loci is detected (Petersen et al., 2001) or when uracil excision by UNG favors SHM and CSR (Sharbeen, Yee, Smith, & Jolly, 2012). Determining at which stages(s) of the cell cycle AID deaminates would probably shed light onto the error-free vs mutagenic repair choice downstream from AID. One could also envision a scenario in which uracils made at different stages of the cell cycle are preferentially recognized by either UNG or MutSα (Li, Zhao, & Wang, 2013).

3.3 Targeting AID to the Ig Loci 3.3.1 AID Piggybacks on Transcription to Access the DNA AID needs to access ssDNA in the nucleus. This is no trivial task, as the genome is protected by chromatin, and ssDNA very rarely exposed to prevent genomic instability. The idea that AID accessed its target DNA via transcription preceded its discovery. This was based on the observation that IgV mutations are closely associated with the transcription start site, and mutations can be induced in the normally untouched Ig constant exons simply by cloning a promoter upstream of the domain (Peters & Storb, 1996). AIDmediated mutagenesis in bacteria was found to require an active promoter at the target genes (Chaudhuri et al., 2004; Ramiro et al., 2003). The link between transcription and accessibility of any given locus to AID has since been demonstrated by multiple evidences (Casellas et al., 2016; Storb, 2014).

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AID in B cells is found at transcriptionally active Ig regions and associates with the RNA polymerase II (RNAPII) complex, indicating a physical association with the transcription machinery (Nambu et al., 2003) that has been biochemically confirmed (Willmann et al., 2012; Fig. 3A). The ability of AID to deaminate both strands, as observed in vivo (Rada, Di Noia, & Neuberger, 2004; Xue et al., 2006), is also linked to transcription. It is promoted by the phosphorylation of AID by protein kinase A (PKA), which permits the binding of AID with replication protein A (RPA) (Basu et al., 2005; Chaudhuri et al., 2004). In addition, the association of AID with stalled or paused polymerase seems to favor deamination, presumably by allowing more time for this relatively inefficient enzyme. This has been shown by using an engineered IgV containing sequences promoting transcription stalling (Kodgire, Mukkawar, Ratnam, Martin, & Storb, 2013). In line with these data, the concomitant accumulation of RNAPII and AID at the IgV compared to constant regions of the Ig, and a correlation between this accumulation and mutation frequency, have been documented (Maul et al., 2014; Wang, Fan, Kalis, Wei, & Scharff, 2014). Furthermore, several factors A

V-region

AID

Sµ Ac

PKA

H3.3

FACT RPA

1-3

SF

SR

Me

S-transcript

PAF

GANP SPT6

Sγ1 Ph PAF

FACT Kap1

DSIF RNAPII

R-loop Exosome

SPT6 DSIF

14-3-3

FACT PTBP2

RNAPolII RNAPII

PAF SPT6 DSIF RNAPII

Exosome

B

53BP1

VDJ

Sμ

Cγ1

Sγ1 Mediator Cohesin

3⬘RR

Cμ

VDJ

Sμ–Sγ1

Sγ1–Sμ

Cγ1 3⬘RR

Cμ

Fig. 3 Topological and functional factors promoting AID targeting to the Ig locus. (A) Schematic representation of different factors that contribute to targeting AID activity during SHM or CSR at the IgV or S-regions, respectively (see text for details). Factors are color coded according to their function: histone chaperones (yellow), transcription elongation factors (red), RNA-processing factors (purple), RNA molecules (orange), factors promoting AID catalytic activity (brown), and factors promoting AID targeting (green). (B) Structural organization and contacts involved in Igh switch (S) region synapsing. The mediator and cohesin complexes are at least partially necessary to establish these contacts. The Igh variable (green) and constant exons (red, blue), and the 30 regulatory region (gray) are indicated. DSBs in the S, shown as gaps, must be recombined in the indicated orientation for CSR to occur, for which 53BP1 is important. Productive recombination is shown on the left.

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that interact with AID are known modulators of RNAPII elongation. AID associates with the transcription elongation factor SPT5, which is necessary for the association of AID with the transcription machinery (Pavri et al., 2010). As SPT5 stabilizes stalled RNAPII, this interactions link AID to transcription stalling, which would promote more efficient deamination (Wang et al., 2014). Another line of evidence linking the initiation of SHM and CSR to transcription stalling comes from the observation that knockdown of topoisomerase I (TOPI) can promote CSR and SHM, while its complete inhibition with camptothecin blocks both (Kobayashi et al., 2011; Maul, Saribasak, Cao, & Gearhart, 2015). It is likely that reducing TOPI levels causes RNAPII stalling as a result of unresolved transcriptional supercoiling, while the stable TOPI–DNA adducts caused by camptothecin would completely block transcription (Maul et al., 2015). AID also interacts with the RNA exosome, an RNA-processing complex that modulates transcription elongation by degrading antisense RNA, which is proposed to help recruit AID to its targets as well as exposing ssDNA. The RNA exosome would degrade the R-loops formed when the transcript remains hybridized to the template S-region strand, thereby allowing AID to target both strands of the transcribed duplex (Basu et al., 2011; Pefanis et al., 2014; Fig. 3A). It is interesting that chromatin immunoprecipitation shows AID associated with thousands of genetic loci in B cells (Yamane et al., 2011), but only a subset of 300 genes displays consistent AID-mediated damage (Meng et al., 2014; Qian et al., 2014). Most of these AID off-targets are linked to super enhancers and display convergent transcription (Meng et al., 2014; Pefanis et al., 2014, 2015; Qian et al., 2014), a transcriptional environment presumed to be more prone to stalling and could provide AID with ssDNA substrate and sufficient time for deamination. Thus, the evidence so far indicates that transcriptional stalling plays along with the biochemical characteristics of AID to favor deamination at the Ig loci, and possibly at AID off-targets (Casellas et al., 2016). 3.3.2 The Ig Loci Contribute to AID Activity The basis of the preferential targeting of AID to the Ig genes is the focus of current research to determine the contribution of the loci, sequenceintrinsic determinants, and trans-acting factors that might promote mutations. Interestingly, these are not necessarily the same for the S-regions and the IgV. The topology of the S-regions is important for CSR. Each S-region is associated with its own promoter/enhancer element, which only drives

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transcription and thereby recruits AID under the appropriate cytokine stimulation (Matthews, Zheng, DiMenna, & Chaudhuri, 2014). The Igh 30 regulatory region (30 RR) is a composite enhancer that physically interacts with the Eμ along with the appropriate enhancer for the transcriptionally active downstream S-region (Fig. 3B). This locus reorganization usually envisioned as looping of the DNA, brings the two recombining S-regions into proximity and promotes efficient recombination of the DSBs induced by AID (Wuerffel et al., 2007), and possibly also skews the reaction toward the deletional orientation (Dong et al., 2015). The Igh locus seems to be intrinsically able to promote long distance synapsing, since replacing S-regions with I-Sce-I recognition sites results in substantial CSR upon delivery of the I-Sce-I endonuclease (Zarrin et al., 2007). Factors affecting chromatin structure, such as the cohesin complex and mediator, contribute to this synaptic configuration (Thomas-Claudepierre et al., 2013). The DNA damage response factor 53BP1 plays a major role in CSR, since its ablation results in one of the most dramatic CSR deficiencies (Manis et al., 2004). 53BP1 seems to have several functions in CSR, which include promoting S–S synapsing (Wuerffel et al., 2007). In addition, 53BP1 plays a key role in the overwhelming preference of CSR to join two DSBs from two distant S-regions in a way that the intervening sequence is deleted, thereby leading to isotype switching, rather than being inverted, which would inactivate the gene (Dong et al., 2015; Fig. 3C). The recently proposed role of 53BP1 in ensuring that the Sμ region breaks before the downstream S-region may contribute to this directionality (Rocha et al., 2016). The sequence itself of the Igh S-regions promotes AID mutagenesis and CSR (Luby, Schrader, Stavnezer, & Selsing, 2001; Zarrin et al., 2004). The presence of multiple AGCT motifs is a universal feature of S-regions (Zarrin et al., 2004). The AGCT motif contains two overlapping WRC motifs with adjacent cytosines in opposite strands within the preferred sequence context for AID, the simultaneous deamination of which would increase the likelihood of a DSB (Han, Masani, & Yu, 2011; Zarrin et al., 2004). The S-regions of higher vertebrates contain additional G-clusters that make them prone to forming R-loops (Yu, Chedin, Hsieh, Wilson, & Lieber, 2003). R-loops are not strictly required for CSR, as shown by Xenopus laevis S-regions, which do not form R-loops (Zarrin et al., 2004), but they may increase the efficiency of CSR by promoting transcriptional stalling (Rajagopal et al., 2009). It is unclear whether R-loops contribute to SHM, but they may not be necessary (Romanello, Schiavone, Frey, & Sale, 2016).

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It has been more difficult to demonstrate whether the IgV sequence favors mutation. A local bias in the codon usage for serine in the IgV favors the occurrence of AGY sequences in the CDR of multiple species (Conticello et al., 2005; Wagner, Milstein, & Neuberger, 1995), which probably evolved to favor AID activity. Moreover, the presence of AGCT sequences with overlapping AID hotspots in the IgV is important for overall mutation load and was proposed to act as entry points for AID (Wei et al., 2015). Thus, CDRs usually concentrate IgV mutations but because these regions bind the antigen, separating intrinsic mutability from selection is not obvious when studying SHM in vivo. The approach of using cell lines and B cells stimulated ex vivo is limited because they accumulate very low levels of SHM. This problem can be addressed using nonproductive Ig transgenes (Betz, Rada, Pannell, Milstein, & Neuberger, 1993), which allows mutation without undergoing selection. This approach uncovered many intrinsic features of SHM, such as the spectrum of mutation and the existence of hotspots (reviewed in Di Noia & Neuberger, 2007), but was limited by the variable genomic context of each transgene. An important improvement of that strategy used a variable tester sequence in place of the endogenous IgV of one Igh allele while the other was productively rearranged to permit B cell development and germinal center formation. This strategy showed that AID targeted equally well the productive IgV and a similarly sized but unrelated sequence placed in the equivalent genomic position within the other allele (Yeap et al., 2015). This confirmed that the high levels of mutation observed at the IgV are driven by its genomic context and not by the actual sequence (Yeap et al., 2015). Nevertheless, in this system, certain hotspots in non-Ig tester sequences were targeted as frequently as the CDR hotspots (Yeap et al., 2015). Thus, several lines of evidence indicate that the base composition of CDRs evolved to recruit AID to some hotspots that are more frequently mutated and would act as “entry” sites from where mutations can spread (Wagner et al., 1995; Wei et al., 2015; Yeap et al., 2015). Evidence of spreading from an initial deamination at a C:G pair to A:T pairs nearby has been obtained in vitro and in vivo (Frieder, Larijani, Collins, Shulman, & Martin, 2009; Unniraman & Schatz, 2007). Cis-elements within the Ig genes can attract AID activity. Enhancers located at the 30 end of the loci have been studied extensively and found to promote SHM beyond their effect on transcription. The best characterized of these is the DIVAC, an enhancer containing several transcription factor-binding elements, originally described for the chicken Vλ locus but

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conserved in the mammalian Igh and Igκ loci (Blagodatski et al., 2009; Buerstedde, Alinikula, Arakawa, McDonald, & Schatz, 2014; Kothapalli, Norton, & Fugmann, 2008). These sequences can ectopically attract AID targeting to an unrelated, transcribed gene. The exact molecular mechanism involved in mediating this enhanced targeting of AID remains unclear but it is probably multipronged. The DIVAC contains many transcription factorbinding sites that are necessary for enhancing AID activity (Buerstedde et al., 2014; Kohler et al., 2012). While it has no obvious epigenetic effects on the target region, it may somehow promote RNAPII stalling (Kohler et al., 2012). The Igh 30 RR element, critical for CSR synapsis, can also drive efficient SHM to the IgV of transgenic Ig loci and to the endogenous Igh (Dunnick et al., 2009; Rouaud et al., 2013). Whether the DIVAC enhancer and 30 RR help recruiting AID via similar molecular mechanisms, or the exact role and possible redundancy of specific transcription factors in this process, is still unclear. Furthermore, whether they associate or cooperate with superenhancers, and/or promote convergent transcription remains to be tested. It is very likely that all these processes are mechanistically related and contribute to the high mutation frequency of the Ig loci compared to AID off-targets. 3.3.3 Recruiting AID Activity to the Ig Locus Is a Team Effort The genome-wide association of AID with chromatin and stalled RNAPII is much broader than the activity of AID, or at least the one that can be detected as DSBs (Meng et al., 2014; Pavri et al., 2010; Qian et al., 2014; Yamane et al., 2011). Thus, additional factors must specifically promote AID activity at and off the Ig loci. The emerging picture shows AID as part of a multiprotein complex at the chromatin (Fig. 3A) with the RNAPII as well as several associated complexes that modulate transcription, such as the DSIF (SPT5/SPT4); PAF and the exosome; histone chaperones FACT and SPT6; and chromatin remodelers like KAP1, cohesin, and INO80, all of which copurify with AID and are important for antibody diversification (Aida, Hamad, Stanlie, Begum, & Honjo, 2013; Basu et al., 2011; Begum, Stanlie, Nakata, Akiyama, & Honjo, 2012; Jeevan-Raj et al., 2011; Kracker et al., 2015; Maul et al., 2014; Okazaki et al., 2011; Stanlie, Begum, Akiyama, & Honjo, 2012; Thomas-Claudepierre et al., 2013; Willmann et al., 2012). Some of the factors associated to AID may play different roles at the IgV or S-regions, highlighting the importance of the interaction between the cisand trans-acting factors for AID function. For instance, cohesin and the

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INO80 chromatin remodeler likely support CSR by promoting S–S synapsing (Kracker et al., 2015; Thomas-Claudepierre et al., 2013). In a more complex example, transient knockdown of SPT5 reduces CSR dramatically (Pavri et al., 2010; Stanlie et al., 2012), but actually increases SHM (Wang et al., 2014). The disparity may reflect different characteristics of the V- and S-regions. SPT5, along with SPT4, is involved in both stabilizing stalled RNAPII and promoting transcription processivity (Wada et al., 1998). Thus, R-loops could favor polymerase stalling at the S-regions (Rajagopal et al., 2009) while the IgV do not form R-loops. Therefore, reducing SPT5 levels may limit stalled RNAPII stability in the S-regions but actually promote transcription stalling at the IgV (Wang et al., 2014). Depleting SPT6, which is important for both transcription processivity and nucleosome remodeling, reduces CSR but increases SHM (Okazaki et al., 2011). It is unclear which SPT6 activity mediates the observed effects on CSR and SHM, but since its knockdown phenocopies SPT5 depletion it may be its role in promoting RNAPII elongation. On the other hand, knockdown of the FACT complex, which is also implicated in transcription, elongation, and nucleosome shuttling, reduces both CSR and SHM (Aida et al., 2013; Stanlie, Aida, Muramatsu, Honjo, & Begum, 2010). Interestingly, this does not seem to be mediated by transcriptional effects, but correlates with changes in epigenetic marks and/or histone usage. FACT levels at the S- and V-regions correlate with content of histone variant H3.3, which correlate with AID mutagenic activity (Aida et al., 2013). Accordingly, H3.3-deficient DT40 cells are severely compromised for SHM and gene conversion (Romanello et al., 2016). This seems to involve an increased propensity of H3.3 bound DNA to become single stranded. The FACT complex helps maintain H3K4me3 levels at the S-region, which may be linked to DSB repair (Stanlie et al., 2010). Stable recruitment of both FACT and SPT6 to the transcription machinery requires the PAF complex, which acts as a scaffold to coordinate transcription elongation (Saunders, Core, & Lis, 2006). Coincidentally, the PAF complex is found at the Ig loci, apparently helping with AID recruitment. Knockdown of PAF components reduces AID targeting to the S-region and thereby reduces CSR. The PAF complex may also promote SHM since it is enriched at the IgV, but this has not been tested (Willmann et al., 2012). Interestingly, the PAF complex interacts with the another complex, SKI, in mammals (Zhu et al., 2005). SKI works with the cytoplasmic exosome during mRNA degradation but also cotranscriptionally and both SKI and PAF are required to maintain H3K4me3 levels (Zhu et al., 2005). PAF could thus be potentially linked

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to the exosome. The exosome is equally important for CSR and SHM (Basu et al., 2011; Wang et al., 2014), highlighting the need for AID to target both DNA strands during both these processes. Additional factors have been implicated in AID recruitment to and activity at the Ig loci that reveal links to the epigenetic landscape of the nucleosomes surrounding the target area. The epigenetics of SHM and CSR are complex and have been reviewed in detail elsewhere (Li, Zan, Xu, & Casali, 2013). Several histone modifications are associated to the diversifying Ig loci, likely serving as docking points for chromatin readers that could in turn serve as scaffolds for AID or DNA repair (see Section 4). For instance, the combination of H3K9ac and H3S10ph is specifically enriched at the transcriptionally activated S-region that will recombine for CSR and are necessary to recruit the 14-3-3 adaptors as well as AID (Li, White, et al., 2013). The 14-3-3 adaptors can bind to DNA-containing AGCT repeats and may help recruiting AID to S-regions (Xu et al., 2010). Interestingly, H3K9me3 at the donor S-region recruits the KAP1 and HP1 chromatin factors (Jeevan-Raj et al., 2011). KAP1 is yet another factor that is necessary for CSR but not SHM. The exact mechanism by which it works in CSR is unclear but since it tethers AID to the Sμ region only (Jeevan-Raj et al., 2011), it may help ensuring that the Sμ is broken before the acceptor S-region, which has recently been proposed as an important determinant of efficient CSR (Rocha et al., 2016). The requirement for H3K9me3 contrasts with the association of AID to H3K9Ac-rich super enhancers, because both marks are mutually exclusive, this is but one example of the complex epigenetic regulation that underpins CSR and SHM. 3.3.4 Contribution of RNA Processing to AID Targeting The link between CSR and RNA processing was first made by the observation that removing the splicing ability of the Sμ sterile transcript prevented CSR (Hein et al., 1998; Lorenz, Jung, & Radbruch, 1995). AID is physically and functionally linked to the splicing machinery and this is likely to occur cotranscriptionally, when most splicing takes place (Kornblihtt et al., 2013; Fig. 3). The association to AID to CTNNBL1 (Conticello et al., 2008) evidences the association to the RNA-processing machinery. The localization of nuclear AID to nucleoli, as well as nuclear bodies believed to be splicing centers, which contain CTNNBL1 and other splicing factors that coimmunoprecipitate with AID are further evidence (Hu et al., 2014, 2013), although it is not known whether the association of AID to these nuclear sites is linked to its activity. AID also associates with several factors

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implicated in regulating splicing. The interaction of AID with the polypyrimidine tract-binding protein 2 (PTBP2) was proposed to recruit AID to the S-regions and promotes CSR in CH12F3 cells (Nowak, Matthews, Zheng, & Chaudhuri, 2011). Since PTBP2 binds the S-regions transcript, it may function in the context of the association of AID with the spliced S-region transcript, which in some way promotes its targeting to the corresponding genomic site (Zheng et al., 2015). The observation that PTBP2 knockdown reduced AID occupancy at the S-region but increased it at the adjacent IgV indicates that both regions may compete for AID (Matthews, Husain, & Chaudhuri, 2014). This raises the intriguing possibility that different factors may regulate AID targeting to either the V- (SRSF1–3, GANP) or S-regions (PTBP2, spliced S-transcripts) depending on the timing during the germinal center reaction. AID also forms complexes with various hnRNPs (Hu, Begum, Mondal, Stanlie, & Honjo, 2015; Mondal, Begum, Hu, & Honjo, 2016). However, only some of these RNA-binding proteins affect SHM and/or CSR in cellular models. For instance, hnRNPK seems to be necessary for AID mutagenic activity both at the S- and V-regions, whereas hnRNPL is dispensable for mutagenic activity but is nonetheless required for CSR in CH12F3 cells (Hu et al., 2015). The association of hnRNPs with AID is dependent on RNA, suggesting they are part of a large ribonucleoprotein complex that may have different mechanistic functions during CSR or SHM, however, the details remain unclear. A specific isoform of the splicing factor SRSF1 (SRSF1–3) seems to be necessary for SHM in DT40 cells. Intriguingly, SRSF1–3 does not promote the recruitment of AID, but may somehow facilitate its access to ssDNA by regulating the Ig transcript splicing (Kanehiro et al., 2012). It was recently shown that AID could bind selectively to the spliced S-region transcripts (Zheng et al., 2015). This interaction requires a G-quadruplex structure formed by the spliced S-region transcript, which can then target AID to the S-regions in a sequence-specific fashion. Inhibiting the splicing machinery blocked formation of the G-quadruplex RNA structure and reduced CSR, which could actually be rescued by expression of the homologous spliced S-region transcript. The association of AID with G-quadruplexes may not be unique to the S-region transcripts. Telomeric transcripts form G-quadruplexes (Rhodes & Giraldo, 1995) and AID is recruited to and deaminates the B cell telomeres (Cortizas et al., 2016). It remains to be determined whether G-quadruplexes play a role in targeting AID to off-targets, or whether RNA-mediated targeting contributes to SHM at the Ig or elsewhere.

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Another RNA-binding protein involved in SHM is the multifunctional GANP, which may function as part of the RNA export machinery (Wickramasinghe et al., 2010). SHM is reduced in Ganp
/
mice and increased in transgenic mice overexpressing GANP (Kuwahara et al., 2004; Sakaguchi et al., 2005). The function of GANP in SHM seems to depend on its acetyltransferase activity, as GANP levels at the IgV are associated with activating histone acetylation, like H3K9Ac and H3K27Ac, which promote accessibility. This in turn promotes IgV transcription and possibly RNAPII stalling, which can promote AID recruitment and SHM (Singh et al., 2013). However, GANP levels in primary mouse B cells also directly correlate with AID nuclear levels in steady state (Maeda et al., 2010), so the exact mechanism by which GANP functions needs further investigation. Thus, the physical and functional link between AID and splicing are well established, but the mechanisms and specific roles of the partaking factors are still vague.

3.4 Phosphorylation Focuses AID Activity to the Ig Locus AID is modified by phosphorylation at various sites (Fig. 2). The best studied is serine 38, which is phosphorylated by PKA in vivo and modulates the activity of chromatin-associated AID. This modification greatly enhances AID function in SHM, gene conversion, and CSR, without affecting its catalytic activity on ssDNA (Basu et al., 2005; Chatterji, Unniraman, McBride, & Schatz, 2007; McBride et al., 2006; Pasqualucci, Kitaura, Gu, & Dalla-Favera, 2006). Indeed, increasing PKA activity in B cells enhances CSR (Pasqualucci et al., 2006), while mutating serine 38 in vivo greatly reduces SHM and CSR (Cheng et al., 2009; McBride et al., 2008). Interestingly, AID from bony fish lack this serine; however, they maintain binding to RPA via an aspartic acid at position 44 (Basu, Wang, & Alt, 2008). Because CSR is not present in fish, serine 38 and its phosphorylation are probably later adaptations to regulate DSB formation for CSR. Consistent with this, there is evidence indicating that serine 38 is phosphorylated at the chromatin level, after PKA is activated by the DNA damage response kinase ATM, which is activated by the initial DNA damage made by AID (Vuong et al., 2013). The recruitment of the endonuclease apurinic/ apyrimidinic endonuclease 1 (APE1) by phosphorylated AID seems to be behind this positive feedback loop that allows the amplification of the initial DNA damage at the S-regions without necessarily affecting the enzymatic activity of AID (Delker & Papavasiliou, 2013; Vuong et al., 2013).

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Other, less studied, phosphorylation sites exist for AID. Tyrosine 184 is phosphorylated in vivo in B cells (Basu et al., 2005; McBride et al., 2006). Despite being in the functionally important C-terminal tail of AID, mutation of this residue to alanine has not shown any effect on AID activity or function so far (Basu et al., 2005; Patenaude & Di Noia, 2010; Zahn et al., 2014). AID threonine 27 is another PKA substrate and may affect AID enzymatic activity (Basu et al., 2005). Serine 3 and threonine 140 are probably phosphorylated by PKC (Gazumyan et al., 2011; McBride et al., 2008). The former negatively regulates AID activity in B cells and seems especially important to limit off-target activity (Gazumyan et al., 2011). Unlike serine 38, which is critical to antibody diversification, phosphorylation of threonine 140 seems to be more important for fine tuning AID activity as its mutation to alanine in vivo has a modest effect on CSR but reduces SHM more severely (McBride et al., 2008). Intriguingly, the recruitment of RPA to the Igh locus seems to depend on phosphorylation of serine 38 and T140 (Yamane et al., 2011). Thus, AID may recruit RPA in a regulated manner, presumably to stabilize ssDNA and provide time for deamination. Alternatively, RPA deposited downstream of AID may reflect more abundant DSBs, a fraction of which could persist until S/G2 and be repaired by homologous recombination (Yamane et al., 2013). The context in which PKC phosphorylates AID, the relevant PKC isoform, and the exact function of these modifications remain unsolved. Overall, phosphorylation of AID seems to have evolved in order to promote AID activity specifically when and where it is needed, limiting its potential to mutate the genome.

4. AID INITIATES A DNA REPAIR CASCADE Extensive work has led to a reasonably detailed understanding of the cascade of biochemical reactions that start with the recognition of the AID-catalyzed uracils in DNA and underpin SHM and CSR. We will emphasize the early steps of these pathways and the emerging evidence of crosstalk between the pathways initiated by UNG and MutSα, as well as the balance between error-free and mutagenic repair.

4.1 Detecting Uracil As mentioned earlier, detection of the uracil lesion introduced by AID is fundamental for antibody diversification. This is exclusively performed by either the nuclear isoform of the uracil-DNA glycosylase UNG or the

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mismatch sensor MutSα. In the combined absence of both these enzymes, all AID-induced lesions will produce transition mutations, as the uracil will remain in the DNA until S-phase and act as template for the incorporation of dAMP during DNA replication, thereby creating a C:G to T:A mutation (Rada et al., 2004; Shen et al., 2006; Fig. 4). UNG can recognize uracil in single- or double-stranded DNA (either as U:G of U:A) (Krokan & Bjøra˚s, 2013). MutSα recognizes it only as a U:G mismatch (Jiricny, 2013; Larson, Bednarski, & Maizels, 2008). Importantly, both UNG and MutSα are evolutionary conserved enzymes that initiate faithful DNA repair pathways in most cells and species. Thus, despite their normally antimutagenic functions, when UNG or MutSα recognize AID-catalyzed uracils they frequently lead to further mutations or to DNA breaks to diversify the Ig genes (Fig. 4). How this dangerous but useful diversion from error-free DNA repair is achieved and regulated in B cells is not well understood.

4.2 The UNG and Base Excision Repair Arm UNG removes uracil from DNA, leaving an abasic site in its wake that is normally repaired by faithful base excision repair (BER) (Krokan & Bjøra˚s, 2013; Fig. 4). The abasic site is cleaved by an APE, determining a nick in the DNA strand that activates PARP1, which acts as a signal to recruit the scaffold XRCC1 protein, the high-fidelity DNA polymerase β (Polβ) and a ligase, to complete repair. Rather than this canonical pathway, mutagenic repair frequently follows after UNG excises AID-generated uracil. UNG deficiency in B cells affects the pattern of SHM, sharply reducing the proportion of transversion mutations at C:G pairs (Di Noia & Neuberger, 2002; Imai et al., 2003; Rada, Williams, et al., 2002). This indicates that a proportion of the abasic sites left by UNG serve as a noninformative template for DNA synthesis, which must be performed by a specialized DNA polymerase able to bypass this type of DNA damage because the replicative polymerases cannot. One such specialized polymerase is REV1, which only introduces dCMP into DNA, and is responsible for most transversion mutations at C:G pairs in chicken and mouse B cells (Jansen et al., 2006; Simpson & Sale, 2003). B cells from Rev1
/
mice have an approximately threefold reduction in CSR, which might be explained by the substantially reduced UNG activity in extracts from Rev1
/
B cells (Zan et al., 2012). It is unclear why REV1 deficiency would affect overall UNG activity, but the proposal that REV1 is necessary to recruit UNG for CSR is inconsistent

A

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Error-free DNA repair Mismatch repair

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Fig. 4 See legend on next page.

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with the much stronger defect on CSR seen in Ung
/
compared to Rev1
/
B cells (Krijger et al., 2013; Rada, Williams, et al., 2002; Zan et al., 2012). REV1 does have scaffold functions, which may stabilize UNG, but also recruit other translesion polymerases (Guo et al., 2003) (see later). Half of the mutations in SHM are at A:T pairs, and all are introduced by DNA polymerase η (Delbos, Aoufouchi, Faili, Weill, & Reynaud, 2007). In the absence of MSH2 or MSH6, the proportion of mutations at A: T are reduced to 5–25% (Rada et al., 2004; Shen et al., 2006). Ablating Polη does not eliminate all A:T mutations but the combined deficiency with Msh2 does (Delbos et al., 2007). It follows that while most mutations at A:T are made downstream from MutSα, a proportion is also generated by the UNG pathway recruiting Polη. Thus, mutagenic BER for SHM involves not only bypassing single abasic sites but also a version of long-path BER generating a DNA gap that is then filled in by Polη. UNG deficiency leads to somewhat higher mutation loads at the Ig and at AID off-targets (Di Noia & Neuberger, 2002; Liu et al., 2008; Rada et al., 2004). Thus, the promutagenic activity of UNG coexists or competes with its canonical activity in BER during SHM. The mechanism forcing mutagenic BER is unknown. Yet, mutations are more likely to occur when a deaminated WRC is in certain sequence contexts (P
erez-Dura´n et al., 2012). So, UNG may partly direct the choice between faithful and Fig. 4 Biochemical pathways of error-free repair, SHM, and CSR. (A) Canonical pathways for short patch base excision repair (BER, left) and mismatch repair (MMR, right). Canonical uracil excision repair is initiated by UNG (or another uracil-DNA glycosylase), creating an abasic site that is recognized by APE1 or APE2. APE1/2 nick the DNA 50 of the abasic site. PARP1 and XRCC1 are recruited to the nick and recruit PCNA and Polβ. Polβ can remove the remaining 50 deoxyribose and insert a single nucleotide, followed by ligation. Not shown is the long-patch BER that requires PCNA and Polδ or Polε that synthesize past the lesion and displace the original DNA strand. The ssDNA flap is processed by FEN1 before ligation. In MMR, MutSα recognizes the U:G mispair and recruits MutLα, which nicks the DNA 50 of the mismatch via PMS2. The PCNA-associated EXO1 50 –30 exonuclease activity creates an extended patch of ssDNA from the nick going past the mismatch site. PCNA recruits Polδ to replicate over the patch and Ligase 1 finalizes repair. (B) During SHM, uracil can act as a template for replication leading to a C–T transition mutation. Alternatively, noncanonical BER leads to transition and transversion mutations. MutSα initiates mutagenic repair affecting A:T pairs. Both pathways recruit lowfidelity polymerases (Polη, ζ, ι) through PCNA ubiquitination (PCNA-Ub). For CSR, noncanonical BER can lead to DSBs when two uracils in opposite strands are closely spaced. MMR can process distantly spaced uracils, leading to staggered DSBs. DSBs can also be produced by the collaboration of BER and MMR. Blunt DSBs are joined by C-NHEJ during CSR, whereas staggered breaks are preferentially repaired via A-EJ.

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mutagenic repair during SHM through its interaction with the sequence context. The timing of excision by UNG is another determinant of mutation vs repair. Elegant experiments using a UNG inhibitor restricted to specific stages of the cell cycle showed that productive SHM and CSR require UNG excision activity in G1 (Sharbeen et al., 2012). Finally, it is possible that the locus can contribute to define whether uracil processing is mutagenic or not. Thus, sequencing of multiple loci targeted by AID in normal mouse B cells showed that a fraction of them are protected by UNG, MSH2, or both, as shown by the increased mutation frequency when they were sequenced from the corresponding knockout mice (Liu et al., 2008). A proportion of the lesions processed by BER at the Ig loci are actually faithfully repaired, as shown by the increase in SHM or mutations at the S-region in mice haploinsufficient for XRCC1 or Polβ (Saribasak et al., 2011; Wu & Stavnezer, 2007). This suggests that competition between error-prone and error-free repair also occurs after the uracil is excised.

4.3 The MutSα-Initiated Arm Most mutations at A:T during SHM depend on MutSα and EXO1, which are components of the mismatch repair (MMR) pathway. There is evidence in vivo that mutations at A:T bases extend to either side of a C:G pair, most frequently when the C is in the top (coding) strand. This spreading is dependent on MSH2, likely by recognizing the AID-catalyzed U:G (Unniraman & Schatz, 2007). Interestingly, the proportion of mutations at A:T are not affected by UNG deficiency, indicating that additional U: G pairs do not lead to additional MutSα recognition or mutations (Longerich, Tanaka, Bozek, Nicolae, & Storb, 2005; Rada, Williams, et al., 2002). In canonical MMR (Fig. 4), MutSα does not remove the mismatched base directly but recruits the MLH1/PMS2 heterodimer MutLα. The endonuclease activity of PMS2 then cleaves the DNA 50 of the mismatch, creating a DNA nick that serves as point of entry for the exonuclease EXO1. EXO1 degrades a patch of the nicked strand in the 50 –30 direction, going past and removing the mismatch. A complex of PCNA and a high-fidelity DNA polymerase, such as Polδ, fills in the gap (Jiricny, 2013). At variance with canonical MMR, the mutagenic version operating during SHM uses MSH2 and MSH6 to recognize the AID-catalyzed U:G mismatch but PMS2 and MLH1 are dispensable, albeit they do participate in CSR (for a detailed recent review on the phenotype of each MMR-

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deficient mouse, see Zanotti & Gearhart, 2016). EXO1 participates in both SHM and CSR (Bardwell et al., 2004). PMS2 could generate nicks for EXO1 in CSR. However, during SHM another factor, probably a uracilDNA glycosylase (see later), must provide this nick for EXO1. To further complicate the matter, mice carrying a mutant EXO1 with no apparent exonuclease activity show normal CSR and SHM (Schaetzlein et al., 2013). Barring the chance that this EXO1 retains some activity, it is possible that EXO1 acts as a scaffold that may stabilize the nick, or recruit other repair factors that actually create the DNA gap that is then resynthesized by Polη. It is still unclear why during SHM the usually error-free BER and MMR pathways recruit translesion polymerases, which are low fidelity, instead of Polβ or Polδ. For mutations at A:T pairs, this role in normally monopolized by Polη. It is interesting to note that mutations at A:T are severely decreased at the IgV and S-regions in humans suffering of xeroderma pigmentosa because of Polη mutations as well as in Polη-deficient mice (Delbos et al., 2007, 2005; Li, Zhao, et al., 2013 Zeng et al., 2001). Mutations at A:T in the absence of Polη could be introduced by other low-fidelity polymerases, such as Polκ, Polζ, and Polι (Faili et al., 2009; Maul et al., 2016; Saribasak et al., 2012; Zanotti & Gearhart, 2016). These polymerases make minor contributions and their deficiency does not affect much the mutation spectra unless combined with Polη deficiency. Nevertheless, these data show that the MutSα pathway for SHM is conditioned to recruit low-fidelity polymerases, thereby becoming mutagenic. A key molecular event determining the recruitment of translesion polymerases is the monoubiquitination of PCNA at lysine 164 (PCNA-Ub) (Moldovan, Pfander, & Jentsch, 2007), which allows the interaction with Polη and REV1 (Guo et al., 2006; Kannouche, Wing, & Lehmann, 2004). This event is also important for SHM (Fig. 4). Accordingly, mutating lysine 164 in PCNA reduces SHM. Consistently with an important role in recruiting Polη, mice expressing PCNAK164A show low levels of SHM at A: T pairs, which is compensated by an increase in mutations at C:G (Langerak, Nygren, Krijger, van den Berk, & Jacobs, 2007; Roa et al., 2008). The defect is similar to that observed in MutSα-deficient mice. In the DT40 system, in which SHM is largely restricted to C:G pairs (Sale, Calandrini, Takata, Takeda, & Neuberger, 2001), the mutant PCNAK164R reduces the overall mutation frequency (Arakawa et al., 2006). It is unclear whether or not PCNAK164A affects SHM frequencies in mice (Langerak et al., 2007; Roa et al., 2008). Recruitment of Polη to PCNA-Ub after DNA damage depends on the Rad18 ubiquitin ligase (Kannouche et al., 2004). However,

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Rad18 knockout DT40 cells still have PCNA-Ub and showed a milder phenotype than the PCNAK164R mutant (Arakawa et al., 2006), suggesting there is another ubiquitin ligase that can contribute to SHM. Interestingly, mutations at A:T downstream from UNG also depend on PCNA-Ub (Krijger, Langerak, van den Berk, & Jacobs, 2009), so the version of longpatch BER functioning during SHM must be quite different from the canonical long path, which involves PCNA but not PCNA-Ub (Krokan & Bjøra˚s, 2013). The question remains of why uracil processing at the Ig loci signals for the ubiquitination of PCNA, thereby rendering the process mutagenic.

4.4 Interactions Between BER and MMR During Antibody Diversification The use of PCNA-Ub and Polη exemplifies the mechanistic contacts between the UNG- and MutSα-mediated pathways of SHM. However, these pathways seem to have more intimate mechanistic interactions, by collaborating during SHM and CSR. Some of these interactions could contribute to making the pathways mutagenic. One explanation as to why BER and MMR can become mutagenic during antibody diversification is that AID creates a localized high density of uracils. In contrast, spontaneous deamination or dUTP misincorporation is unlikely to create a cluster of uracils in DNA. Thus, AID would create a unique environment where BER and MMR can work in proximity and interact with one another. One such proposed interaction posits that AID deaminates cytosines exposed by the gap produced by the MutSα pathway (Frieder et al., 2009; Krijger et al., 2009). Uracil excision there would leave an abasic that could template transversion mutations when the gap is filled in. This model could explain why mice deficient in MutSα components show an increase in transition mutations at C:G (Roa et al., 2010). It was also noticed that UNG-dependent transversion mutations at C, are often linked to A:T mutations in the hypermutating human Ramos B cell lymphoma, and that UNG inhibition not only reduces the transversions at C but also the associated A:T mutations (Frieder et al., 2009). The latter finding would support the notion that the abasic site acts as a signal for recruiting low-fidelity polymerases, perhaps by triggering PCNA ubiquitination. If that was the case UNG deficiency would reduce the mutations at A:T, which does not happen. However, another glycosylase or APE1 could be playing that role (see later).

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A mutagenic noncanonical pathway of MMR has been described (Pen˜aDiaz et al., 2012). This pathway uses PCNA-Ub to recruit Polη and is dependent on the endonuclease activity of PMS2/MLH1. A different version must be at work for SHM, for which PMS2 is dispensable. It was shown that the MMR machinery can highjack the nick from ongoing BER to recruit EXO1 to initiate MMR repair from a nearby U:G (Schanz, Castor, Fischer, & Jiricny, 2009). If the environment of SHM promotes PCNA-Ub, Polη would be recruited and allow mutations. A similar mechanism could promote DSBs by the collaboration of UNG and MMR (Bregenhorn, Kallenberger, Artola-Bora´n, Pen˜a-Diaz, & Jiricny, 2016), although in the case of CSR the nick could be made by PMS2. Thus, it is possible that UNG can help mutagenic MMR in generating the ssDNA patch by creating EXO1 entry points. SMUG1, another uracil-DNA glycosylase, also triggers uracil BER (Krokan & Bjøra˚s, 2013). SMUG1 can produce some CSR in Ung
/
Msh2
/
mice, especially for the chronic responses detected as switched isotypes in the serum of nonimmunized mice; but is much less efficient than UNG (Di Noia, Rada, & Neuberger, 2006; Dingler, Kemmerich, Neuberger, & Rada, 2014). Accordingly, the loss of SMUG1 does not affect antibody diversification (Dingler et al., 2014), while UNG-deficient mice and humans display a major defect (Imai et al., 2003; Rada et al., 2004; Rada, Williams, et al., 2002; Zahn et al., 2013). However, the additive reduction in isotype switching and in mutations at A:T pairs observed in Ung
/
Smug1
/
compared to Ung
/
mice actually suggest that SMUG1 participates to some extent in the MutSα-mediated pathways of SHM and CSR, possibly by providing a nick for EXO1 (Dingler et al., 2014). Thus, while the UNG pathway plays the major role in CSR in vitro and in vivo (Imai et al., 2003; Rada, Williams, et al., 2002; Zahn et al., 2013), the residual CSR activity in Ung
/
cells indicates that MutSα can create DNA breaks, probably through PMS2 and/or EXO1, as mentioned. MMR contributes most notably to generating breaks outside the S-regions, where the uracils might be separated by a longer distance because of the lower density of AGCT motifs (Min, Rothlein, Schrader, Stavnezer, & Selsing, 2005). In the absence of MSH2, the DNA breaks associated with CSR are focused at AGCT motifs sequences (Rada, Ehrenstein, Neuberger, & Milstein, 1998), suggesting that, even when UNG is present, MMR displaces the break away from the initial deamination site. Indeed, B cells from mice deficient in either Msh2, Msh6, Pms2, or Mlh1 have a 50% drop in CSR efficiency

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in vitro (Zanotti & Gearhart, 2016). Given that CSR in Ung
/
B cells is only 5 wt%, the data demonstrate that MMR collaborates with the UNG pathway during CSR, instead of simply being nonoverlapping alternative pathways.

4.5 DNA Nicks and Breaks It must be noted that UNG and SMUG1 are monofunctional glycosylases that remove the uracil base but do not cleave the deoxyribose phosphate backbone (Krokan & Bjøra˚s, 2013). Creating a nick requires an additional enzymatic activity such as the APE enzymes that cleave abasic sites. APE1 largely fulfils this role in most tissues and mediates most CSR in CH12F3 cells (Masani, Han, & Yu, 2013). The initial activity of AID at the S-regions activates a signaling cascade by ATM and PKA that phosphorylates AID at serine 38, enhancing AID activity and allowing its interaction with APE1 (Vuong et al., 2013). This local recruitment of APE1 to the S-regions could favor further DNA breaking thus promoting CSR. Interestingly, APE1 is capable of cleaving dsDNA containing a U:G mismatch in vitro (Prorok et al., 2013). Thus, APE1 could bypass the need for UNG or SMUG1 to provide nicks at sites of AID deamination. However, this does not seem likely given the large CSR defect caused by UNG deficiency alone (Rada, Williams, et al., 2002). CH12F3 Ape1
/
cells retain 20% of CSR activity (Masani et al., 2013). In mice, APE1 is essential, and haploinsufficiency has little effect on CSR (Guikema et al., 2007). Thus, it cannot be ruled out that other factors, such Mre11 through its AP lyase activity (Larson, Cummings, Bednarski, & Maizels, 2005), contribute to make DNA breaks for CSR and/or SHM. The ability of APE1 to nick dsDNA at a U:G (Prorok et al., 2013) could in principle allow entry to MMR in the absence of UNG, possibly explaining why SHM at A:T is normal in Ung
/
B cells (Rada, Jarvis, et al., 2002). However, while APE1 is well expressed in B cells activated ex vivo and in CH12F3 cells, APE1 levels are actually reduced in germinal center B cells, where its homolog APE2 is induced instead (Stavnezer et al., 2014). APE2-deficient mice show normal CSR but reduced SHM, with the caveat that they have fewer germinal center B cells, so additional defects may be contributing to the phenotype (Sabouri et al., 2009; Stavnezer et al., 2014). More importantly, a change in the pattern was apparent in these mice, with mutations at A:T being disproportionately reduced. This led to propose that its endonuclease activity provides nicks for the polymerization step that

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creates A:T mutations (Stavnezer et al., 2014). Alternatively, the 30 –50 exonuclease activity of APE2 could contribute to make ssDNA gaps from a nicks, as also proposed (Sabouri et al., 2009). Surprisingly, loss of APE2 is additive to UNG deficiency with regards to reducing mutations at A:T, suggesting that APE2 also contributes to the CSR pathway initiated by MutSα (Stavnezer et al., 2014), probably after uracil excision. The switch from APE1 to APE2 expression in germinal center B cells has been proposed as another way to prevent faithful BER, since APE1 interacts with XRCC1 and Polβ, while APE2 does not. Thus, low levels of APE1 could prevent or delay BER, forcing more uracils and/or abasic sites to be mutagenically bypassed during DNA synthesis (Stavnezer et al., 2014). Of note, cleavage of abasic sites by either APE1, APE2, or Mre11 leaves the deoxyribose bound to one end of the break, which must be eliminated by a deoxyribose phosphate lyase activity to allow reintroducing a deoxynucleotide. It was recently shown that REV1 has such activity, which may be another role for it in SHM (Prasad, Poltoratsky, Hou, & Wilson, 2016). It has been proposed that the glycosylase activity of UNG is not necessary for CSR and that UNG would function as a scaffold to facilitate DNA repair (Begum et al., 2004, 2009; Yousif, Stanlie, Mondal, Honjo, & Begum, 2014). Indeed, mutants with highly reduced catalytic activity are capable of sustaining CSR levels to the same levels as wt UNG (Begum et al., 2004; Di Noia et al., 2007). The kinetic properties of the different UNG mutants used (Di Noia et al., 2007; Krusong, Carpenter, Bellamy, Savva, & Baldwin, 2006), combined with their overexpression, which would override the complex cell cycle regulation of UNG (Hagen et al., 2008; Haug et al., 1998), could explain to a significant extent the apparent efficiency of these mutants in retroviral complementation assays. On the other hand, ChIP experiments in Ung
/
B cells indicate that UNG suppresses the recruitment of MMR and translesion synthesis enzymes to the S-region (Yousif et al., 2014). Suppression of MMR recruitment to AIDcatalyzed uracils by UNG was recently observed at the telomeres (Cortizas et al., 2016). While AID targets the telomeres, UNG efficiently corrects this avoiding DNA damage. However, removing or inhibiting UNG leads to the MMR recruitment to the telomeres, followed by DNA damage that causes proliferation defects (Cortizas et al., 2016). It is possible that in the context of CSR, catalytically compromised UNG mutants cannot excise all uracils and favor a mix of U:G mismatches and nicks within the S-region that recruit MMR. Supporting this view, the

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UNGD145N mutant can sustain normal levels of CSR in Ung
/
B cells but it is much less efficient in Ung
/
Msh2
/
B cells (Di Noia et al., 2007). That mutants with absolutely no catalytic activity are incapable of CSR despite still being able to bind to the DNA also suggests that some excision activity is necessary and is consistent with MMR collaboration (Di Noia et al., 2007). The data reviewed earlier indicate clear interactions between the UNG and MutSα pathways during antibody diversification. Both pathways collaborate and rely on invoking translesion synthesis to produce the full spectrum of SHM. While the mutagenic versions of BER and MMR operating at the S-regions can generate breaks for CSR separately, they are more efficient when they work together. One notable exception to the interaction between UNG and MutSα is Ig gene conversion. In DT40 B cells, Ig gene conversion depends almost exclusively on UNG (Di Noia & Neuberger, 2004; Saribasak et al., 2006). MutSα does not play a role in Ig gene conversion. Moreover, chicken MutSα does not even seem to recognize U:G mismatches in DT40 cell extracts (Campo et al., 2013), which likely explains why SHM in DT40 cells is restricted to C:G pairs (Sale et al., 2001). It is possible that this is an adaptation to the use of homologous recombination for IgV diversification in this system, which would be counteracted by MMR (Jiricny, 2013).

4.6 Repairing the Breaks When ssDNA breaks originating from uracil excision and/or a MutSαinitiated gap coalesce at the Ig genes, they produce DNA DSBs that need to be repaired. DSBs at the IgV are not necessary for SHM, but byproducts that nevertheless happen frequently. These DSBs must be quickly repaired, thereby generating insertions and deletions that can still contribute to rare but valuable antibody specificities (Kepler et al., 2014; Sale & Neuberger, 1998; Yeap et al., 2015). On the contrary, breaks at the S-region have to be joined in one particular orientation to produce CSR instead of a nonproductive intra-S rejoining or, worse, an inactivating inversion (Alt, Zhang, Meng, Guo, & Schwer, 2013; Di Noia, 2015; Dong et al., 2015; Fig. 3B). DSBs at the Ig genes must also be prevented from joining to spontaneous or AID-generated breaks elsewhere in the genome that can lead to chromosomal rearrangements or translocations (Aiden & Casellas, 2015). Thus, DSB repair during CSR must be carefully choreographed and regulated to ensure the survival and utility of activated B cells.

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DNA DSBs resulting from AID activity at the Ig are detected by the DNA damage response signaling cascade, similarly to spontaneous DSBs. This involves a cascade of the DNA damage kinases ATM and DNA-PK recruited to the breaks by the Mre11–Rad50–Nbs1 complex, further recruitment of chromatin modifiers like the ubiquitin ligases RNF8 and RN168 and chromatin readers like 53BP1, which has been reviewed in detail (Alt et al., 2013; Daniel & Nussenzweig, 2013). 53BP1 plays a particularly important role in CSR, preventing the rejoining of intra S-region DSB and promoting deletional over inversional recombination (Dong et al., 2015; Manis et al., 2004; Reina-San-Martin, Chen, Nussenzweig, & Nussenzweig, 2007). CSR between S-regions proceeds via the nonhomologous end joining (NHEJ) pathway, involving the factors KU70, KU80, DNAPKcs, Artemis, XRCC4, and DNA ligase IV. The finding that in the absence of some of these core NHEJ factors there was still considerable CSR led to the discovery of an alternative end joining (A-EJ) pathway that is distinct from the classical (C-NHEJ) (Boboila, Alt, & Schwer, 2012; Boboila et al., 2010; Yan et al., 2007). A-EJ may be several incompletely defined pathways, in which PARP1, XRCC1, CtIP, Mre11, and ATM, have been implicated (Boboila et al., 2012). The fact that many of these factors are also important for other DNA repair pathways has prevented a more positive definition of the A-EJ; however, it includes at least a microhomology-mediated end joining mechanism that joins DNA ends sharing a few (1–10) base pairs of homology. The analysis of joins between S-regions indicates that this pathway is used for a proportion of the CSR events, even in the presence of C-NHEJ, and involves limited end resection (Boboila et al., 2012). A-EJ may preferentially join staggered breaks, such as those determined by uracils far apart in opposite DNA strands (Cortizas et al., 2013). Breaks generated by the MMR pathway of CSR seem to favor A-EJ, possibly by exposing microhomologies after exonucleolytic processing (Eccleston, Yan, Yuan, Alt, & Selsing, 2011; Stavnezer & Schrader, 2006). On the other hand, PMS2 and MLH1 seem to limit the amount of end resection since microhomologies at S–S joins increase in their absence (Chahwan et al., 2012; Ehrenstein, Rada, Jones, Milstein, & Neuberger, 2001). An interesting distinction for C-NHEJ and A-EJ choice during CSR is demonstrated by the opposite effects of knocking out PARP1 or PARP2 (Robert, Dantzer, & Reina-SanMartin, 2009). It would seem that PARP1 signaling is necessary to promote A-EJ, while PARP2 would somehow suppress it. The list of proteins and enzymes involved in end joining continues to grow and many of them are involved in CSR. Recent examples include

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the deubiquitinase activity of the SAGA complex, which is important for the early DNA damage response for C-NHEJ and A-EJ (Ramachandran et al., 2016); and Rif1, which is recruited by 53BP1 phosphorylation and prevents resection of the DSB end, thereby promoting end joining (Lukas & Lukas, 2013).

5. POSTDEAMINATION ROLES OF AID? The molecular mechanisms initiated by AID for SHM and CSR are similar but not equivalent. AID may be considered dispensable downstream from the deamination. Indeed, CSR can be triggered independently of AID by a site-specific endonuclease (Zarrin et al., 2007). However, the frequency of AID-independent CSR is lower than natural CSR and lacks the preferential orientation that makes CSR so efficient (Dong et al., 2015; Zarrin et al., 2007). This observation suggests that AID may contribute beyond deamination, at least during CSR. The latter possibility was also raised by the identification of functional domains in AID. Certain N-terminal residues seem to be dispensable for CSR but absolutely required for SHM (Shinkura et al., 2004), which remains unexplained. On the other hand, the C-terminal domain of AID is necessary for CSR (Barreto et al., 2003; Ta et al., 2003). Chromatin immunoprecipitation shows reduced recruitment of UNG to the S-regions when the breaks are made by AID variants bearing truncations or mutations within the 17 C-terminal amino acids (Ranjit et al., 2011; Sabouri et al., 2014; Zahn et al., 2014). Whether MSH2 is also affected is unclear (Ranjit et al., 2011; Sabouri et al., 2014). Because S-regions targeted by these variants show reduced occupancy of multiple DNA damage signaling and end joining factors (ATM, Nbs1, γH2AX, Ku80, XRCC4 53BP1) (Sabouri et al., 2014; Zahn et al., 2014), it is possible that the C-terminus of AID helps recruiting some of these factors. Biochemical data showing that AID binds with very high affinity to ssDNA (Larijani et al., 2007) suggests that AID may linger at the Ig loci after deamination, providing opportunity for interactions with DNA repair factors. Interactions of AID with DNA-PK and APE1 have been identified (Vuong et al., 2013; Wu, Geraldes, Platt, & Cascalho, 2005), albeit they may be indirect. However, the inability of C-terminally truncated AID for CSR could also reflect an indirect effect of the type or magnitude of damage produced by these hyperactive AID variants. Indeed, they produce breaks that are extensively resected and attract homologous recombination factors (Zahn et al., 2014). Interestingly, these

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AID C-terminal truncations are found in human patients with autosomal dominant Hyper IgM syndrome 2 (Imai et al., 2005). In vitro, these AID variants can act in a dominant-negative fashion to block the CSR activity of endogenous AID in normal B cells (Ucher et al., 2014; Zahn et al., 2014). More work is necessary to determine whether AID contributes more than its enzymatic activity.

6. PERSPECTIVES The functional description of AID targeting to the Ig loci has advanced significantly in the past 10 years. Many components have been identified but the picture integrating all of them is still blurry. Outstanding questions are: What are the molecular basis of the AID preference for superenhancers? Why are the Ig loci targeted much more frequently than other genes that are also linked to superenhancers and similarly transcribed? What is the role of transcription factors in AID targeting? What are the molecular basis of the locus contribution to AID targeting? The structure of the AID core has been solved but important regulatory regions are missing from the picture. Atomic level understanding of the interactions between AID and regulatory factors would elucidate fundamental aspects of its regulation and permit their rational manipulation. The interactions between the UNG and MSH2/6-initiated diversification pathways are also becoming evident. Both UNG and MMR are cell cycle regulated and there are emerging evidences that AID might also be. These are exciting avenues of research to understand the choice between canonical and mutagenic repair. In the larger context of the antibody response, it remains to be analyzed whether and how SHM and CSR might be regulated during the germinal center response for optimizing the immune response. Regulating the timing and magnitude of SHM and CSR can play a role in regulating the output of the germinal center in terms of effector B cells (Gitlin et al., 2016; Weisel, Zuccarino-Catania, Chikina, & Shlomchik, 2016). Since AID is necessary for both mechanisms, regulating AID activity by posttranslational modifications and/or the interaction with cofactors, as well modulating the accessibility to the V- vs S-regions, are more likely candidates to allow either SHM or CSR if necessary. The identification of factors regulating AID, SHM, and/or CSR and the elucidation of their mechanisms of action could offer opportunities to improve immunization, identify the molecular defect in B cell intrinsic immunodeficiencies, and offer insight into the oncogenic capacity of AID.

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ACKNOWLEDGMENTS Work by J.M.D.N. is supported by a Canada Research Chair tier 2, and grants from the Canadian Institutes of Health Research and the Cancer Research Society. S.P.M. was supported in part by a doctoral fellowship from the Fonds de Recherche en Sant
e de Qu
ebec.

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CHAPTER THREE

Emerging Major Histocompatibility Complex Class I-Related Functions of NLRC5 S.T. Chelbi1, A.T. Dang1, G. Guarda2 University of Lausanne, Lausanne, Switzerland 2 Corresponding author: e-mail address: [email protected]

Contents 1. Background 2. Lessons From CIITA 2.1 CIITA: The Master Transcriptional Regulator of MHC Class II Genes 2.2 Transcriptional Regulation of CIITA 2.3 Transcriptional Regulation by CIITA 2.4 Transcriptional Targets of CIITA 3. NLRC5 and Its Role in Regulating MHC Class I Levels 3.1 Complex Transcriptional Regulation of MHC Class I Genes 3.2 NLRC5: A Transcriptional Regulator of MHC Class I Genes 3.3 NLRC5 vs CIITA: Similarities and Differences 3.4 CIITA: A Paradigm for NLRC5 Transcriptional Activity 3.5 Transcriptional Targets of NLRC5 3.6 NLRC5 Tunes MHC Class I Gene Transcription in Specific Tissues and Conditions 4. NLRC5 and Its Emerging Roles in Health and Disease 4.1 Role of NLRC5 in CD8+ T Cell Selection and Maintenance 4.2 Role of NLRC5 in APCs 4.3 Further Considerations on the Role of NLRC5 in DCs 4.4 Role of NLRC5 in Infections 4.5 NLRC5 and Cancer 4.6 Further Considerations on the Role of NLRC5 in Cancer 5. Concluding Remarks Acknowledgments References

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Abstract Recent evidence demonstrates a key role for the nucleotide-binding oligomerization domain-like receptor (NLR) family member NLRC5 (NLR family, CARD domain 1

Contributed equally to this work.

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containing protein 5) in the transcriptional regulation of major histocompatibility complex (MHC) class I and related genes. Detailed information on NLRC5 target genes in various cell types and conditions is emerging. Thanks to its analogy to CIITA (class II major MHC transactivator), a NLR family member known for over 20 years to be the master regulator of MHC class II gene transcription, also the molecular mechanisms underlying NLRC5 function are being rapidly unraveled. MHC class I molecules are crucial in regulating innate and adaptive cytotoxic responses. Whereas CD8+ T cells detect antigens presented on MHC class I molecules by infected or transformed cells, natural killer (NK) lymphocytes eliminate target cells with downregulated MHC class I expression. Data uncovering the relevance of NLRC5 in homeostasis and activity of these two lymphocyte subsets have been recently reported. Given the importance of CD8+ T and NK cells in controlling infection and cancer, it is not surprising that NLRC5 is also starting to emerge as a central player in these diseases. This chapter summarizes and discusses novel insights into the molecular mechanisms underlying NLRC5 activity and its relevance to pathological conditions. A thorough understanding of both aspects is essential to evaluate the clinical significance and therapeutic potential of NLRC5.

1. BACKGROUND Nucleotide-binding oligomerization domain-like receptors (NLRs) are a family of intracellular proteins, which play a pivotal role in host defense, recognizing conserved pathogen-associated molecular patterns (PAMPs) from invading pathogens, but also danger-associated molecular patterns (DAMPs). NLRs have been ascribed diverse roles in innate immune signaling, such as the activation of NF-κB and mitogen-activated protein kinases (MAPKs), the nucleation of inflammasomes, and the induction cell death in response to selected PAMPs or DAMPs (Martinon, Mayor, & Tschopp, 2009; Schroder & Tschopp, 2010). So far, 22 NLR family members have been reported in humans and 34 in mice (Schroder & Tschopp, 2010). Yet, besides selected family members that are intensively studied, the majority of the NLRs remain functionally poorly characterized. NLRs typically contain a tripartite structure composed on an N-terminal effector domain, a central NACHT domain, which contains the nucleotidebinding domain (NBD), important for self-oligomerization, and C-terminal leucine-rich repeats (LRRs), which are believed to sense PAMPs or DAMPs and/or autoinhibit the protein (Martinon et al., 2009; Schroder & Tschopp, 2010). NLR family members are divided into three classes based on their N-terminal effector domain, which can be a caspase recruitment domain (CARD), a pyrin domain (PYD), or a baculovirus inhibitor repeat (BIR)

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Fig. 1 Structural comparison between CIITA and NLRC5. This figure schematically depicts the CIITA (MHC class II transactivator) isoform mainly expressed in myeloid cells (type I), lymphoid cells (type III), and upon interferon (IFN)-γ stimulation (type IV) as well as NLRC5 (NLR family CARD domain containing 5). The CARD domain of NLRC5 differs structurally from classical CARD domains and is therefore designated as “atypical.” CARD, caspase recruitment domain; HAT, histone acetyltransferase domain; LRR, leucine-rich repeats; NLS, nuclear localization signal; P/S/T, proline/serine/threonine-rich domain.

(Martinon et al., 2009; Schroder & Tschopp, 2010). Recently, it became clear that certain NLRs belonging to the first clade play functions beyond innate immunity (Fig. 1). In fact, the NLRs CIITA (class II major histocompatibility complex (MHC) transactivator) and NLRC5 (NLR family CARD domain containing 5) have been identified as master cotransactivators of MHC class II and class I genes, respectively (Kobayashi & van den Elsen, 2012; Neerincx, Castro, Guarda, & Kufer, 2013; Reith & Mach, 2001), an important step toward the global understanding of NLR functions.

2. LESSONS FROM CIITA 2.1 CIITA: The Master Transcriptional Regulator of MHC Class II Genes MHC class II molecules are heterodimeric glycoproteins composed of a α and a β chain, which are constitutively displayed at the surface of professional antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells. MHC class II molecules present antigens of exogenous origin to CD4+ T cells. These antigens have undergone phagocytosis and endosomal degradation to generate peptides that are loaded onto MHC class II molecules. The peptide:MHC class II complex is then recognized by the cognate

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T cell receptor (TCR) of helper T cells. This TCR–peptide:MHC class II engagement is central to the induction and regulation of adaptive immunity by selecting the mature CD4+ T cell repertoire in the thymus and activating these lymphocytes in the periphery. In humans, there are three pairs of MHC class II α and β chain genes called human leukocyte antigen (HLA)-DR, -DP, -DQ. In mice, the MHC class II chains are designated histocompatibility 2 (H2)-A and H2-E. In both humans and mice, MHC class II genes are located in the so-called “MHC locus,” an extremely polymorphic region found on chromosomes 6 and 17, respectively. The NLR protein CIITA has been demonstrated in 1993 to play a decisive role as master transcriptional regulator of MHC class II genes (Reith & Mach, 2001; Steimle, Otten, Zufferey, & Mach, 1993). Mach and coworkers discovered the essential function of CIITA through complementation cloning of MHC class II-negative cell lines from patients with bare lymphocyte syndrome (BLS), a severe genetic disorder characterized by the loss of MHC class II expression (Steimle et al., 1993). Loss-of-function mutations of CIITA, but also of the regulatory factor X (RFX) proteins, which are essential for CIITA activity (discussed in Section 2.3), cause the absence of MHC class II expression. This, in turn, leads to defective humoral and cellular responses due to impaired helper T cell selection and activation, highlighting the relevance of CIITA to human health (Steimle et al., 1993; Ting & Trowsdale, 2002).

2.2 Transcriptional Regulation of CIITA The tight regulation of CIITA expression is achieved transcriptionally by the usage of independent promoters (LeibundGut-Landmann et al., 2004). There are four promoters in humans, pI–pIV, and three in mouse, pI, pIII, and pIV; these are highly conserved between the two species. Each of these three promoters harbors different regulatory elements, enabling cell typespecific activity and gives rise to three isoforms of CIITA (types I, III, and IV) differing in their N-terminus (Fig. 1) (LeibundGut-Landmann et al., 2004; Muhlethaler-Mottet, Otten, Steimle, & Mach, 1997). Myeloid cells, such as conventional DCs, preferentially use the promoter pI. This gives rise to a CIITA form with an N-terminal CARD domain; however, if this domain confers additional properties to this CIITA isoform is still unclear (LeibundGut-Landmann et al., 2004; Zinzow-Kramer et al., 2012). B cells, plasmacytoid DCs (pDCs), and human T cells upon activation use the pIII promoter (Holling, van der Stoep, Quinten, & van den Elsen,

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2002; LeibundGut-Landmann et al., 2004; Lennon et al., 1997; Wong et al., 2002). The pIV promoter drives the constitutive expression of CIITA in thymic epithelial cells (mTECs and cTECs) and the interferon (IFN)-γinduced expression of CIITA in nonimmune cells such as endothelial and epithelial cells, astrocytes, and fibroblasts (Muhlethaler-Mottet et al., 1997; Steimle, Siegrist, Mottet, Lisowska-Grospierre, & Mach, 1994; Waldburger, Suter, Fontana, Acha-Orbea, & Reith, 2001). pII has only been found in humans, and its relevance remains to be established (LeibundGut-Landmann et al., 2004; Muhlethaler-Mottet et al., 1997).

2.3 Transcriptional Regulation by CIITA MHC class II promoters contain highly conserved cis-regulatory elements referred to as S, X1, X2, and Y boxes, or collectively named “SXY module” (van den Elsen, Holling, Kuipers, & van der Stoep, 2004). These regulatory elements are present in both mouse and human. The X1 box is occupied by the trimeric RFX complex containing RFX5, RFXAP, and RFXANK (also known as RFXB). The X2 motif is bound by cAMP response elementbinding protein/activating transcription factor (CREB/ATF) and the Y box is bound by the heterotrimeric nuclear factor Y (NFY) complex composed of NFYA, B, and C (Reith & Mach, 2001). The DNA-binding protein occupying the S box remains, to date, elusive. Altogether, these factors form a multiprotein complex, referred to as “enhanceosome,” serving as a docking site for CIITA, which itself lacks a DNA-binding domain (Fig. 2) (Jabrane-Ferrat, Nekrep, Tosi, Esserman, & Peterlin, 2003; Masternak et al., 2000; Zhu et al., 2000). Spacing and orientation of the S, X1, X2, and Y motifs in MHC class II promoters have been shown to be determinant for proper enhanceosome assembly and transactivation activity by CIITA (Reith & Mach, 2001). Once docked onto the enhanceosome, several lines of evidence demonstrate that CIITA coordinates the recruitment of factors that promote chromatin opening and transcription at MHC class II gene promoters (Fig. 2). Several lysine acetyltransferases enhancing histone acetylation (HATs) have been shown to interact with CIITA. These factors include p300/CREB-binding protein (CBP), p300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5) (Choi, Majumder, & Boss, 2011; Koues et al., 2008; Kretsovali et al., 1998). Interestingly, CIITA possesses intrinsic HAT activity, which maps to its N-terminal “activation domain” (AD) and can contribute to transcription

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Fig. 2 MHC class I and II enhanceosome complexes and gene transactivation. CIITA is recruited by a multiprotein complex, called MHC enhanceosome, on the conserved S, X1, X2, and Y motifs (SXY module) found in MHC class II (MHCII) promoters. The enhanceosome complex is composed of the following factors: the regulatory factor X complex (RFXANK, RFXAP, and RFX5), cAMP-responsive element-binding protein (CREB)/activating transcription factor (ATF), and the trimeric nuclear transcription factor Y complex (NFYA, B, C). The S box-binding protein is still unknown. To regulate transcription of MHC class II genes, CIITA interacts with factors involved in chromatin remodeling (such as Brahma-related gene 1 (BRG1), protease 26S subunit ATPase 5 (SUG1)), histone modification (including methyltransferases (HMTs), acetyltransferases (HATs), and demethylases (HDMs)), transcription initiation, and elongation. NLRC5 engages a similar enhanceosome complex at the SXY module of MHC class I promoters. Whereas, some enhanceosome factors have been experimentally identified others are presumed based on the similarity between NLRC5 and CIITA molecular function. Recently, the S box motif has emerged as a key determinant for selective recruitment of NLRC5 and gene transactivation specificity. In contrast to MHC class II promoters, MHC class I promoters contain additional regulatory elements: the enhancer A, an interferon (IFN)-stimulated response element (ISRE), and an additional poorly characterized CCAAT box. Factors colored in light violet have been validated in endogenous systems, those colored in dark gray have evidences in overexpression, light violet dotted ones are supported by undirect evidence, whereas gray dotted factors still need experimental proof. Pol II, polymerase II; TAFs, TBP-associated factors; TBP, TATA-binding protein.

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activation (Fontes, Jiang, & Peterlin, 1997; Raval et al., 2001). In addition, CIITA has been shown to recruit histone methyltransferases (HMTs) that appose active marks, and demethylases (HDMs) reducing repressive diand trimethylation modifications at the MHC class II promoter (Beresford & Boss, 2001; Koues et al., 2010). Along these lines, CIITA has also been reported to interact with chromatin remodeling factors, components of the basal transcription machinery, and transcription elongation factors (Fig. 2) (Fontes et al., 1997; Koues et al., 2008). Altogether, these interactions favor transcriptional competency at the MHC class II promoters, as reviewed in detail in Choi et al. (2011).

2.4 Transcriptional Targets of CIITA In addition to genes coding for classical MHC class II molecules, CIITA has been shown to regulate genes implicated in the MHC class II antigenpresentation pathway (Taxman, Cressman, & Ting, 2000; Westerheide, Louis-Plence, Ping, He, & Boss, 1997). These include the invariant chain (or CD74), HLA-DM, and HLA-DO, which encode proteins involved in loading MHC class II molecules. Interestingly, in their promoters these genes have sequences reminiscent of the SXY motif, thus mechanistically explaining their transactivation by CIITA. Recent in vitro and in vivo evidence demonstrated that also the butyrophilin (Btn) gene Btn2a2 is a transcriptional target of CIITA, displaying an SXY module in the promoter region (Krawczyk et al., 2008; Sarter et al., 2016). Btn2a2, which belongs to an emerging family of proteins with functions in immunity and beyond, codes for a surface molecule exerting immunomodulatory effects on T cells (Abeler-Dorner, Swamy, Williams, Hayday, & Bas, 2012). In order to define the transcriptional targets of CIITA comprehensively, several studies addressed this question by genome-wide approaches. Work by the group of Reith identified, besides the canonical MHC class II targets, only nine novel targets, of which seven were or could be linked to MHC class II biology, as RFX5 or RAB4B, a small GTPase involved in endocytic trafficking (Krawczyk et al., 2008). Opposite to that, recent genome-wide studies by groups of Knight and Boss identified over 800 and 400 binding intervals, respectively (Scharer et al., 2015; Wong et al., 2014). However, the latter work highlighted also that most of the genes found in proximity of newly identified CIITA-binding sites were not transcriptionally regulated by CIITA (Scharer et al., 2015). These data indicate that, whereas CIITA has been suggested to more broadly poise chromatin structure, its transcriptional

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activity remains highly focused on MHC class II and related genes (Krawczyk et al., 2008; Scharer et al., 2015). As a final note to this section, it is interesting to recall that CIITA has initially been reported to control not only MHC class II but also MHC class I genes (Gobin, Peijnenburg, Keijsers, & van den Elsen, 1997; Martin et al., 1997). Whereas this result was very consistent in overexpression systems, Ciita-knockout mice showed deficiency for MHC class II expression, but normal MHC class I levels (Chang, Guerder, Hong, van Ewijk, & Flavell, 1996; Ludigs et al., 2015; Williams et al., 1998). In agreement, the subgroup of BLS patients harboring CIITA mutations lacked MHC class II expression but exhibited normal MHC class I expression (Ludigs et al., 2015; Steimle et al., 1993). These data indicated that the regulation of MHC class I is not dependent on CIITA under physiological conditions. Nonetheless, it is induced by its enforced expression, suggesting the existence of a homologous protein transactivating MHC class I genes.

3. NLRC5 AND ITS ROLE IN REGULATING MHC CLASS I LEVELS 3.1 Complex Transcriptional Regulation of MHC Class I Genes MHC class I molecules are expressed on virtually all nucleated cells and consist of a α chain and the invariant subunit beta-2-microglobulin (B2m) (Neefjes, Jongsma, Paul, & Bakke, 2011; van den Elsen et al., 2004). MHC class I glycoproteins present antigens of endogenous origin to cognate TCRs of CD8+ T cells. Endogenous peptides derive from degradation of intracellular proteins, including therefore viral or tumor antigens in infected or transformed cells, through the proteasome. Degradation products translocate from the cytoplasm to the endoplasmatic reticulum (ER) where they are loaded on MHC class I molecules via the peptide-loading complex that includes the ER transporter associated with antigen processing (TAP1/2), tapasin, the oxidoreductase ERp57, and the chaperone protein calreticulin (Neefjes et al., 2011). Cellular components involved in the presentation of endogenous antigens, from proteasome subunits to the peptide-loading complex, are collectively referred to as antigen-processing machinery (APM). Highly polymorphic MHC class I molecules that present antigens to conventional CD8+ T cells are known as “classical MHC class I” or “MHC class Ia” (Neefjes et al., 2011; Rodgers & Cook, 2005; van den

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Elsen et al., 2004). Three genes, which are located in the MHC locus, code for classical MHC class I proteins; HLA-A, HLA-B, and HLA-C in humans; and H2-K, H2-D, and H2-L in mice (H2-L is however not found in the commonly used C57BL/6 background). Classical MHC class I genes are key determinant in immunity, but also—due to their high degree of polymorphism—in transplant rejection. Besides MHC class Ia, there are MHC class Ib genes, also referred to as “non-classical MHC class I,” which are phylogenetically heterogeneous (Rodgers & Cook, 2005). The features better distinguishing MHC class Ib genes are their limited polymorphism and restricted expression pattern. For the scope of this review, we will discuss only the evolutionary more recent MHC class Ib genes, which—alike classical MHC class I genes— are encoded in the MHC locus (Howcroft & Singer, 2003; Rodgers & Cook, 2005). In humans, these encompass three genes only: HLA-E, HLA-F, and HLA-G. In mice there are over 40 genes belonging to the H2-M, H2-T, and H2-Q families, although only part of these are productively expressed (Howcroft & Singer, 2003). Their products associate with B2m and play important roles in immunity and immune regulation, controlling responses by unconventional T cell subsets (Rodgers & Cook, 2005; van den Elsen et al., 2004). Tight regulation of MHC class I expression is fundamental for effective immunity. For this reason, MHC class I gene promoters exhibit multiple regulatory motifs, including NF-κB-binding sites, also known as “enhancer A,” and/or an interferon responsive element (van den Elsen et al., 2004; van den Elsen, Peijnenburg, van Eggermond, & Gobin, 1998). Importantly, an SXY module closely related to the one found in MHC class II gene promoters is found upstream of the transcription start site of MHC class I genes, suggesting analogous regulatory mechanisms across MHC genes.

3.2 NLRC5: A Transcriptional Regulator of MHC Class I Genes Recently, the NLR family member NLRC5 has been described by the group of Kobayashi to be a crucial factor in the regulation of MHC class I expression in vitro (Meissner et al., 2010). Shortly thereafter, the study of Nlrc5-deficient mice independently generated by five research groups, ours included, showed that Nlrc5 deficiency resulted in a strong loss of MHC class I and APM gene expression at the protein but also at the transcript level, particularly in immune cells (Biswas, Meissner, Kawai, &

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Kobayashi, 2012; Robbins et al., 2012; Staehli et al., 2012; Tong et al., 2012; Yao et al., 2012). NLRC5 is the largest NLR and has the typical tripartite structure with an N-terminal atypical CARD domain, the central NACHT domain sharing high homology with the one of CIITA, and a long C-terminal LRR stretch (Gutte, Jurt, Grutter, & Zerbe, 2014; Meissner et al., 2010). NLRC5 shuttles to the nucleus thanks to a bipartite nuclear localization sequence (NLS) located between the CARD-like and the NACHT domains (Fig. 1) (Meissner et al., 2010; Neerincx, Rodriguez, Steimle, & Kufer, 2012; Staehli et al., 2012). However, similar to CIITA, it also retains clear cytoplasmic localization, recalling its phylogenetic belonging to the cytoplasmic NLR family of receptors (Cressman, Chin, Taxman, & Ting, 1999; Spilianakis, Papamatheakis, & Kretsovali, 2000; Staehli et al., 2012). Owing to its homology to NOD1 and NOD2, which form NF-κB activating complexes upon recognition of specific bacterial moieties, it was conceivable that NLRC5 would fulfill similar functions (Meissner et al., 2010). Indeed, roles for NLRC5 in regulating NF-κB and other innate immune signaling pathways have been proposed, both using cell lines as well as Nlrc5-deficient mice (Benko, Magalhaes, Philpott, & Girardin, 2010; Cui et al., 2010; Kuenzel et al., 2010; Neerincx et al., 2010; Tong et al., 2012). These results are however debated, as certain papers reported a negative role for NLRC5 in regulating these cascades, and others a positive one (Benko et al., 2010; Cui et al., 2010; Kuenzel et al., 2010; Neerincx et al., 2010; Tong et al., 2012). Others and we could not prove these functions using knockout mouse models (Kumar et al., 2011; Robbins et al., 2012; Staehli et al., 2012). These aspects, which therefore require to be clarified by future investigations, have been covered in other reviews (Kobayashi & van den Elsen, 2012; Neerincx et al., 2013); here, we want to specifically focus on the MHC class I-related effects of NLRC5.

3.3 NLRC5 vs CIITA: Similarities and Differences As mentioned earlier, MHC class I promoters contain highly conserved S, X1, X2, and Y boxes, with similar spacing and orientation to those found in the MHC class II promoters (van den Elsen et al., 2004, 1998). Promoter reporter assays and chromatin immunoprecipitation (ChIP)-sequencing studies showed that NLRC5 regulates MHC class I target genes by occupying this SXY cis-regulatory element, reminiscent of CIITA for MHC class II

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gene regulation (Ludigs et al., 2015; Meissner et al., 2012; Neerincx et al., 2012). Overexpression of the X2 box-binding factor ATF1 enhanced NLRC5-driven MHC class I transcription, suggesting that it might be part of an “NLRC5 enhanceosome” (Meissner, Liu, et al., 2012). Furthermore, analysis of MHC class I expression in cells lacking the enhanceosome factors, RFX5, RFXANK, and RFXAP, and the use of Rfx5-deficient mice demonstrated the importance of these factors for MHC class I expression through NLRC5 recruitment (Ludigs et al., 2015; Meissner, Liu, et al., 2012). Finally, immunoprecipitation experiments showed that NLRC5 interacts with the RFX complex through RFXANK. Altogether, these data indicate that NLRC5 occupies the SXY module through an enhanceosome complex. Analyses of Nlrc5 and Ciita single and double-deficient mice confirmed that these two NLRs do not exert redundant functions, regulating MHC class I and class II genes, respectively (Ludigs et al., 2015; Robbins et al., 2012). Moreover, ChIP experiments in murine T and B cells revealed that NLRC5 and CIITA binding was specific to the promoters of their respective target genes. This is surprising, given the fact that these two NLRs are recruited to similar SXY consensus sequences and—at least in part— through common enhanceosome factors (Ludigs et al., 2015; Meissner, Liu, et al., 2012; Neerincx et al., 2012). However, ChIP-sequencing experiments enabled to pinpoint subtle divergences in the SXY motifs occupied by NLRC5 and CIITA, with the S box emerging as the key determinant to confer selective recruitment of NLRC5 and gene transactivation specificity (Ludigs et al., 2015; Meissner, Liu, et al., 2012). The identification of the S box-binding factor, which remains elusive to date, will help detail key differences in the multiprotein complexes specifically recruiting CIITA and NLRC5 (Fig. 2).

3.4 CIITA: A Paradigm for NLRC5 Transcriptional Activity The striking parallels between CIITA and NLRC5 suggest that the mechanisms regulating MHC gene transactivation are similar. In vitro, NLRC5mediated transactivation was synergistically enhanced by cooverexpression of transcriptional coactivators such as CBP, p300, GCN5, or PCAF, all previously shown to contribute to CIITA-mediated transactivation (Fig. 2) (Meissner, Liu, et al., 2012). However, ChIP analyses showed that total H3 acetylation at the MHC class I promoter of the H2-K gene was comparable between Nlrc5-deficient and control splenocytes, although specific

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acetylation marks have not been investigated so far (Robbins et al., 2012). Interestingly, removal of the repressive H3K27me3 mark at the H2-K gene promoter induced by IFNγ-stimulation was impaired in Nlrc5-deficient splenocytes (Robbins et al., 2012). This infers a role for histone demethylases in NLRC5-driven MHC class I gene expression. Altogether, these initial data indicate that NLRC5 acts as a platform for histone modifying enzymes and—by analogy to CIITA—for chromatin remodeling factors and basal transcriptional machinery (Fig. 2). However, which factors are important under physiological conditions is unclear to date.

3.5 Transcriptional Targets of NLRC5 Several studies in murine models demonstrated that NLRC5 plays a crucial role as a key transcriptional regulator of classical, selected nonclassical MHC class I genes, and APM genes, as summarized in Table 1 (Biswas et al., 2012; Ludigs et al., 2015; Robbins et al., 2012; Staehli et al., 2012; Yao et al., 2012). In addition, NLRC5 has been demonstrated to directly occupy the promoters of most of these targets (Ludigs et al., 2015; Staehli et al., 2012). Notably, the importance of NLRC5 to the transactivation of these genes varies. For instance in T cells, NLRC5 fully controls the expression of the H2-Q6/7 genes, while it contributes to roughly 80–90% of the classical H2-K and to 50–60% of B2m gene transcription (Ludigs et al., 2015). Even if the targets and the contribution of NLRC5 to their expression might somewhat change in different cells, tissues, or conditions, over the last years we have gained a satisfying picture of NLRC5 transcriptional targets in murine cells (Table 1). What we have learned is reminiscent of the functional specificity reported for CIITA, emphasizing in both cases the highly focused transcriptional regulatory activity. Because of the extreme degree of intra- and interspecies polymorphism of the MHC class I family genes, it is impossible to comprehensively define, based on these mouse data, the targets of NLRC5 in the human system. In human cell lines, the NLRC5 targets HLA-A, HLA-B, and HLA-C have been confirmed by both overexpression and knockdown approaches, but we miss exhaustive information (Table 2) (Meissner et al., 2010; Neerincx et al., 2012). Kobayashi and coworkers also showed direct binding by overexpressed NLRC5 to HLA-A/B/C/G/E/F gene promoters (Meissner et al., 2010). Even though it would be preferable to repeat these experiments performing ChIP-sequencing analyses on endogenous

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Table 1 Murine NLRC5 Targets Organism Methods Used for Tissue/Cell Used for Validation Type or Line Mouse Targets Analysis

B2m

C57BL/6, Nlrc5
/


ChIP, mRNA

Cell line mRNA (+NLRC5)

References

Spleen, thymus, T, NK, B

Biswas et al. (2012), Ludigs et al. (2015), Robbins et al. (2012), Staehli et al. (2012), and Yao et al. (2012)

B16–F10

Rodriguez et al. (2016)

C57BL/6, Nlrc5
/


ChIP mRNA, protein

Spleen, thymus, LN, liver, T, CD4, CD8, NK, B, NKT, γδT, cDC, Mϕ, BMDM, BMDC, mTEC/cTEC

Biswas et al. (2012), Ludigs et al. (2016, 2015), Robbins et al. (2012), Rota et al. (2016), Staehli et al. (2012), and Yao et al. (2012)

BALB/c, Nlrc5
/


Protein

T

Ludigs et al. (2016)

Cell line mRNA, (+NLRC5) protein

B16–F10

Rodriguez et al. (2016)

C57BL/6, Nlrc5
/


ChIP, mRNA, protein

Spleen, thymus, LN, liver, Ileum, kidneya, T, CD4, CD8, NK, B, NKT, γδT, cDC, Mϕ, BMDC, BMDM, mTEC/cTEC

Biswas et al. (2012), Ludigs et al. (2016, 2015), Robbins et al. (2012), Rota et al. (2016), Staehli et al. (2012), Tong et al. (2012), and Yao et al. (2012)

BALB/c, Nlrc5
/


Protein

T

Ludigs et al. (2016)

mRNA, Cell line (+NLRC5) protein

B16–F10

Neerincx et al. (2014) and Rodriguez et al. (2016)

H2-L

BALB/c, Nlrc5
/


Protein

T

Ludigs et al. (2016)

H2-M3

C57BL/6, Nlrc5
/


ChIP, mRNA

Spleen, thymus, T, CD8

Biswas et al. (2012), Ludigs et al. (2016, 2015), and Yao et al. (2012)

H2-D

H2-K

Continued

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Table 1 Murine NLRC5 Targets—cont’d Organism Methods Used for Tissue/Cell Used for Validation Type or Line Mouse Targets Analysis

References

H2-Q4

C57BL/6, Nlrc5
/


ChIP, mRNA

T, CD8

Ludigs et al. (2015)

H2-Q6

C57BL/6, Nlrc5
/


ChIP, protein (Qa2 epitope)

T, CD4, CD8, NK, B, NKT, cDC

Ludigs et al. (2016, 2015)

H2-Q7

C57BL/6, Nlrc5
/


ChIP, protein (Qa2 epitope)

T, CD4, CD8, NK, B, NKT, cDC

Ludigs et al. (2016, 2015)

H2-T10

C57BL/6, Nlrc5
/


ChIP

T

Ludigs et al. (2015)

H2-T18

C57BL/6, Nlrc5
/


mRNA

B

Robbins et al. (2012)

H2-T22

C57BL/6, Nlrc5
/


ChIP, mRNA

T, CD8

Ludigs et al. (2015)

H2-T23

C57BL/6, Nlrc5
/


mRNA

Spleen, thymus

Yao et al. (2012)

Psmb8

mRNA Cell line (+NLRC5)

B16–F10

Rodriguez et al. (2016)

Psmb9

C57BL/6, Nlrc5
/


Spleen, thymus, T, CD8

Biswas et al. (2012), Ludigs et al. (2015), and Yao et al. (2012)

B16–F10

Rodriguez et al. (2016)

Spleen, thymus, T, CD8

Biswas et al. (2012), Ludigs et al. (2015), and Yao et al. (2012)

B16–F10

Rodriguez et al. (2016)

ChIP, mRNA

Cell line mRNA (+NLRC5) Tap1

C57BL/6, Nlrc5
/


ChIP, mRNA

Cell line mRNA (+NLRC5) a

Not confirmed by the study of Ludigs et al. (2016). +NLRC5 ¼ Analysis performed after NLRC5 overexpression. ChIP: Chromatin immunoprecipitation of endogenous NLRC5 followed by qPCR and/or deep sequencing; mRNA: qRT–PCR analysis; protein: FACS analysis. LN: Lymph node; T: T cells; CD4: CD4+ T cells; CD8: CD8+ T cells; NK: natural killer cells; B: B cells; NKT: natural killer T cells; γδT: gamma delta T cells; cDC: conventional dendritic cells; Mϕ: macrophages; BMDC: bone marrow-derived dendritic cells; BMDM: bone marrow-derived macrophages; mTEC: medullary thymic epithelial cells; cTEC: cortical thymic epithelial cells.

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Table 2 Human NLRC5 Targets Human Targets +NLRC5 2NLRC5

B2M

Cell Lines

References

ChIP, mRNA, protein

Jurkat; 293T

Meissner et al. (2010)

HLA-A ChIP, mRNA, protein, reporter

Jurkat; 293T; HeLa

Meissner et al. (2010), Meissner, Li, Liu, Gagnon, and Kobayashi (2012), Meissner, Liu, et al. (2012), Neerincx et al. (2014), and Staehli et al. (2012)

HeLa

Meissner et al. (2010)

Jurkat; 293T; HeLa

Meissner et al. (2010), Meissner, Li, et al. (2012), Meissner, Liu, et al. (2012), Neerincx et al. (2014), and Staehli et al. (2012)

Protein HLA-B ChIP, mRNA, protein, reporter

mRNA, HeLa; protein THP1 HLA-C ChIP, mRNA, protein

Meissner et al. (2010) and Neerincx et al. (2014)

Jurkat; 293T

Meissner et al. (2010), Meissner, Li, et al. (2012) and Staehli et al. (2012)

HeLa

Meissner et al. (2010)

HLA-E ChIP, protein

293T

Meissner et al. (2010)

HLA-F ChIP

293T

Meissner et al. (2010)

HLA-G ChIP

293T

Meissner et al. (2010)

PSMB9 mRNA, protein

Jurkat

Meissner et al. (2010)

TAP1

Jurkat; 293T

Meissner et al. (2010)

Protein

ChIP, mRNA, protein

+NLRC5 ¼ analysis performed after NLRC5 overexpression;
NLRC5 ¼ analysis performed after NLRC5 knockdown by SiRNA. ChIP: Chromatin immunoprecipitation on tagged NLRC5 followed by qPCR; mRNA: qRT–PCR analysis; protein: FACS and/or Western blot analysis; reporter: luciferase assays. 293T: HEK 293T cells.

NLRC5, this remains unfortunately not possible, as a suitable antibody for human NLRC5 has not been reported thus far. Despite the great progress done in our understanding of NLRC5 activity, we therefore miss complete knowledge of NLRC5’s target genes in humans.

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3.6 NLRC5 Tunes MHC Class I Gene Transcription in Specific Tissues and Conditions NLRC5 is constitutively highly expressed in lymphoid cells such as T cells, intermediately in B cells, and less in myeloid cells such as DCs and macrophages (Benko et al., 2010; Biswas et al., 2012; Ludigs et al., 2016; Neerincx et al., 2010; Staehli et al., 2012). In agreement, its contribution to MHC class I expression is mainly observed in lymphoid cells and to a lower extent in DCs (Biswas et al., 2012; Ludigs et al., 2016; Robbins et al., 2012; Staehli et al., 2012). Given its prominent expression among hematopoietic cells, an accurate analysis of NLRC5 levels in nonimmune tissues would require to carefully discriminate infiltrating immune cells. Although we still miss such detailed analysis, we reported that expression and contribution of NLRC5 to MHC class I transcription in kidney or skin tissue are very low under steady-state conditions (Ludigs et al., 2016). Therefore, in contrast to MHC class I deficiency, mice deleted for Nlrc5 do not show an absent, but rather a mosaic of lowered and normal MHC class I expression on different cells and tissues. NLRC5 expression can be upregulated by various inflammatory stimuli, including viral infections and PAMPs, such as the toll-like receptor (TLR) 3 ligand polyinosinic:polycytidylic acid, the TLR4 agonist lipopolysaccharide (LPS), and oligodeoxynucleotides triggering TLR9 (Benko et al., 2010; Guo et al., 2015; Kuenzel et al., 2010; Neerincx et al., 2010; Staehli et al., 2012; Yao et al., 2012). In fact, NLRC5 induction mainly depends on the transcription factor STAT1 (signal transducer and activator of transcription 1) downstream of autocrine/paracrine signaling by type I interferon (IFN), which is induced by the above-mentioned stimuli (Staehli et al., 2012). Moreover, also type II IFN has the ability to increase NLRC5 levels through the same signaling cascade. Of note, the transcriptional regulation of NLRC5 in DCs differs significantly from that of CIITA. In fact, whereas PAMPs induce NLRC5 and MHC class I transcription, these stimuli are known to silence CIITA (Benko et al., 2010; Landmann et al., 2001; Rota et al., 2016; Staehli et al., 2012; Yao et al., 2012). This observation suggests that the relevance of de novo MHC transcription following DC activation might substantially differ between MHC class I- and II-mediated antigen-presentation pathways. Whereas exposure to these inflammatory stimuli increases the levels and contribution of NLRC5 to MHC class I gene transcription in immune cells

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and organs, their effect on nonhematopoietic tissues remains poorly explored (Rota et al., 2016; Yao et al., 2012).

4. NLRC5 AND ITS EMERGING ROLES IN HEALTH AND DISEASE 4.1 Role of NLRC5 in CD8+ T Cell Selection and Maintenance An important consequence of the “chimeric” MHC class I levels found on different Nlrc5
/
cells and tissues is that Nlrc5-deficient mice do have CD8+ T cells, whereas mice fully lacking MHC class I, such as B2m-knockout, do not. Indeed, Nlrc5
/
mice exhibit an almost normal CD8+ T cell population, albeit a reduction ranging from 10% to 30% is observed (Ludigs et al., 2016; Staehli et al., 2012; Yao et al., 2012). Peripheral CD8+ T cells are the result of MHC class I-dependent selection and maintenance processes, which take place in thymus and peripheral tissues, respectively. In the thymus, thymic epithelial cells (TECs) are responsible for the two-step selection of CD8+ T cells. Positive selection ensures that the lymphocytes are MHC class I restricted, whereas negative selection eliminates thymocytes strongly recognizing self-peptide:MHC class I complexes, to avoid autoimmunity (Palmer & Naeher, 2009). Whereas the complete absence of MHC class I abolishes these processes, Nlrc5-deficiency only partially reduces MHC class I expression on TECs (35–40% in average), allowing for the selection of virtually normal CD8+ T cell numbers (Ludigs et al., 2015; Staehli et al., 2012; Yao et al., 2012). In the periphery, MHC class I has been shown to maintain naı¨ve CD8+ T cells through “tonic” signals, that is low affinity signals driven by MHC class I bound to self-peptides (Surh & Sprent, 2005). Despite the exact nature of the cells contributing to this phenomenon remains elusive, we can assume that MHC class I expression on these cells is only modestly affected by Nlrc5deficiency, as the CD8+ T cell population is largely present (Ludigs et al., 2016; Staehli et al., 2012; Yao et al., 2012). Despite this, it still needs to be evaluated whether the TCR affinity of CD8+ T cells in Nlrc5-deficient mice is normal. It is conceivable that the TCR repertoire selected and maintained in the absence of Nlrc5 exhibits—in average—increased affinity to self-peptide:MHC class I complexes, compensating for the lower signal transmitted by reduced MHC class I on TECs and peripheral cells. This possibility might provide

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interesting insights for the understanding of selected autoimmune conditions.

4.2 Role of NLRC5 in APCs An outstanding question concerns the relevance of NLRC5 to cytotoxic T cell priming. Interestingly, Nlrc5
/
B cells pulsed with the ovalbuminderived antigen SIINFEKL were defective in activating transgenic OT-I CD8+ T cells as compared to control B cells (Biswas et al., 2012). Conversely, LPS-treated, ovalbumin-fed Nlrc5
/
bone marrow-derived macrophages (BMDMs) did not show an overt defect in their ability to prime CD8+ T cells (Staehli et al., 2012). Lending support to these findings, the relevance of NLRC5 in maintaining high levels of H2-K, which present SIINFEKL, is substantial in B cells and milder in BMDMs, particularly after activation by inflammatory stimuli (Biswas et al., 2012; Robbins et al., 2012; Staehli et al., 2012; Yao et al., 2012). Although macrophages and B cells contribute to CD8+ T cell priming, DCs are considered the most important and efficient professional APCs in most instances. DCs combine the ability to crosspresent antigens (i.e., the ability to present peptides derived from exogenous antigens on MHC class I molecules) with the ideal costimulatory capacity for activating T cells. Despite Nlrc5
/
conventional DCs and BMDCs exhibit a moderate reduction in the expression of H2-K molecules, the role of NLRC5 in antigen presentation by DCs has been studied in two complementary settings (Biswas et al., 2012; Ludigs et al., 2015; Rota et al., 2016; Staehli et al., 2012; Yao et al., 2012). In the first study, OT-I T cells were cocultured in vitro with peptide-pulsed BMDCs in the absence of maturation stimuli (Yao et al., 2012). Under such conditions, a clear defect in T cell activation by Nlrc5
/
BMDCs was observed. However, DCs acquire full potential for optimal antigen presentation and activation of T cells after exposure to inflammatory signals, such as LPS, as analyzed in a second study by our group. Following stimulation, NLRC5 contributed in the range of 50% to H2-K transcription in BMDCs and to the intracellular pool of mature MHC class I (Rota et al., 2016). In spite of that, surface levels of MHC class I were less affected, suggesting that MHC class I display is partially uncoupled from the abundance of the neosynthesized pool and indicating the existence of compensatory mechanisms rescuing surface levels. Of note, no substantial differences in T cell crosspriming were detected when using

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Fig. 3 Activated Nlrc5
/
BMDCs retain the ability to prime CD8+ T cells. Activated bone marrow-derived dendritic cells (BMDCs) prime CD8+ T cells by presenting peptides onto MHC class I molecules. The engagement of peptide:MHC class I to their cognate T cell receptor (TCR) and CD8 coreceptor as well as the interaction of costimulatory molecules, such as CD80/86, with CD28 stimulate CD8+ T cells to undergo differentiation and clonal expansion. Although activated Nlrc5
/
BMDCs have a moderate decrease in surface MHC class I expression, CD8+ T cell priming was found to be virtually unaltered, suggesting that costimulation and cytokines produced by Nlrc5
/
BMDCs overcame the partial defect in antigen presentation.

activated Nlrc5-deficient BMDCs in vitro or ovalbumin and adjuvant in Nlrc5-deficient mice (Fig. 3) (Rota et al., 2016). Interestingly, Nlrc5
/
BMDCs exhibited a 50% reduction in displaying SIINFEKL-H2-K complexes (as detected with the clone 25-D1.16) loaded through the endogenous route of antigen presentation, i.e., through the direct antigen-presentation pathway (Porgador, Yewdell, Deng, Bennink, & Germain, 1997; Rota et al., 2016; Wenger et al., 2012). Yet, T cell priming was not significantly altered, suggesting that

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costimulation and cytokines produced by Nlrc5
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BMDCs were able to compensate for the partial defect in antigen presentation (Fig. 3). Taken together, whereas NLRC5 contributes to CD8+ T cell priming by B cells, its role in DCs requires further characterization. The two reports addressing this question use different settings with regard to DC activation state and antigen loading, potentially accounting for the different conclusions reached (Rota et al., 2016; Yao et al., 2012). Therefore, the relevance of NLRC5 contribution in the afferent arm of the immune response still needs to be carefully addressed.

4.3 Further Considerations on the Role of NLRC5 in DCs The emerging complexity in DC ontogeny and function strongly suggests that more work shall be performed to deeply understand the role of NLRC5 in cytotoxic T cell priming (Guilliams et al., 2014). Selected DC subsets could present larger defects, worth exploring in more detail. Along this line, the reduction in intracellular MHC class I observed in conventional splenic DCs is greater than the one observed in BMDCs, and this could well translate into defective presentation of endogenous antigens, as discussed also in Section 4.4 (Rota et al., 2016).

4.4 Role of NLRC5 in Infections Given the recent discovery of NLRC5, our understanding of its physiological relevance is still limited. Two independent studies clearly demonstrated that NLRC5 is required for controlling Listeria monocytogenes infection (Biswas et al., 2012; Yao et al., 2012). Nlrc5-deficient mice showed significantly higher bacterial burdens in spleen and liver. Strikingly, the numbers of specific as well as total CD8+ T lymphocytes drastically dropped in the course of L. monocytogenes infection in Nlrc5-deficient animals, well explaining the impaired control of the infection. The reduction in CD8+ T cell percentages observed 1 week after infection was larger than the defect observed at steady state (discussed in Section 4.1) (Staehli et al., 2012; Yao et al., 2012), reaching a 60% reduction in spleen and liver. In addition, the defect in Listeria-specific CD8+ T cells was even more dramatic ranging between 60% and 80% (Biswas et al., 2012; Yao et al., 2012). This decrease is in good agreement with a suboptimal T cell activation by APCs as well as by infected monocytes, macrophages, or polymorphonuclear cells, the main targets of L. monocytogenes. It remains therefore open at which precise stage and in which cells NLRC5 plays this important role.

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Conditional knockout animals are instrumental to address these questions and already proved useful uncovering an unexpected T cell-intrinsic role for NLRC5 in maintaining CD8+ T cells (Ludigs et al., 2016). Following infection by lymphocytic choriomeningitis virus (LCMV) clone 13, which establishes a chronic infection, a progressive and global loss of Nlrc5-deficient CD8+ T cells was observed, leading to uncontrolled viremia. Recent evidence highlighted how, in inflammatory conditions, natural killer (NK) cells dampen responses by T cells through their elimination (Crome, Lang, Lang, & Ohashi, 2013; Pallmer & Oxenius, 2016; Schuster, Coudert, Andoniou, & Degli-Esposti, 2016). NK cell cytotoxic activity is regulated by integrating opposing signals by two sets of receptors: the activating receptors, which are engaged by several stress-induced molecules; and the inhibitory ones, recognizing—interestingly with regard to NLRC5—MHC class I molecules on target cells. Indeed, NK cell depletion in the context of LCMV clone 13 infection fully recovered naı¨ve Nlrc5
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CD8+ T cells and partially rescued activated Nlrc5
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CD8+ T cells (Ludigs et al., 2016), revealing an essential role of NLRC5 in protecting CD8+ T cells from NK cell-mediated rejection in inflammatory conditions (Fig. 4). However, the only partial rescue of activated Nlrc5
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CD8+ T cells indicated that NLRC5 also sustains antigen-specific effector T cells

Fig. 4 NLRC5 protects T cells from NK cell-mediated rejection under inflammatory conditions. NK cells integrate signals by inhibitory receptors, which engage MHC class I molecules, and by activating receptors, which recognize stress-inducible ligands on target cells. When activating signals outweigh inhibitory ones, NK lymphocytes unleash their cytotoxic activity killing the target cell. Interestingly, Nlrc5
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T lymphocytes, which exhibit a strong reduction in MHC class I expression, are not rejected by NK cells at steady state, suggesting that the inhibitory signals still can compensate the activating ones. However, upon inflammation, Nlrc5
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T cells are eliminated by NK cells. Such inflammatory conditions prime NK cells and increase activating signals, suggesting that the low inhibitory signals provided by MHC class I molecules on Nlrc5
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T cells do not compensate anymore the activating ones.

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independently of NK cells. NLRC5 is therefore a crucial factor for chronic cytotoxic responses through two complementary mechanisms; an NK cell-independent one requiring further investigation; and an NK celldependent one (Ludigs et al., 2016). Whereas the former is essential to control the infection, the latter prevents a general loss of CD8+ T cells, which might have fundamental consequences—through depletion of naı¨ve cells and pregressed cytotoxic memory cells—also for superinfections. Of note, recent analyses of human immunodeficiency virus (HIV)infected blood samples, identified two CpGs sites in NLRC5 promoter, which are significantly hypomethylated in infected subjects as compared to healthy controls (Zhang et al., 2016). These data suggest that in HIVinfected individuals NLRC5 expression might be upregulated, enhancing MHC class I gene transcription. As HIV-infected subjects were on combination antiretroviral therapy and had controlled viremia, it is difficult to link this observation to potential functional consequences. However, it is tempting to speculate that expressing higher NLRC5 levels might represent, also in this context of chronic infection, a selective advantage for immune cells. Altogether, these data from infectious conditions start uncovering why NLRC5 evolved to control MHC class I transcription in lymphocytes and, most prominently, in T cells—which are not per se APCs. Whether Nlrc5driven MHC class I expression delineates a novel regulatory mechanism fine-tuning the immune response also in autoimmune, antitumoral, and allogeneic responses, it remains to be established.

4.5 NLRC5 and Cancer Silencing or downregulation of the MHC class I pathway is documented in a wide range of cancers and constitutes the main strategy by which transformed cells escape CD8+ T cells antitumor response (Algarra, GarciaLora, Cabrera, Ruiz-Cabello, & Garrido, 2004; Seliger, 2012). Importantly, NLRC5-driven MHC class I expression on target cells is necessary to induce efficient killing by CD8+ T cells. Indeed, Nlrc5-deficient lymphocytes pulsed with the ovalbumin peptide SIINFEKL were less killed by OT-I CD8+ cytotoxic T cells than their wild-type counterparts in vitro (Fig. 5) (Staehli et al., 2012). Moreover, similar results were obtained using Nlrc5-deficient BMDCs as target cells (Yao et al., 2012). At the molecular level, MHC class I or APM irreversible genetic defects are relatively rare events suggesting that MHC class I loss in primary tumors is often driven by regulatory alterations (Seliger, 2012). Hence, it is

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Fig. 5 Nlrc5
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lymphocytes evade cytotoxic T cell-mediated killing. Cytotoxic T lymphocytes (CTL) recognize target cells through the interaction of the TCR with cognate peptide:MHC class I complex. This drives the elimination of the target cell through the release of cytotoxic molecules such as granzyme B and perforin. However, the low MHC class I expression on Nlrc5
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lymphocyte targets leads to reduced CTL-mediated killing, therefore favoring immune evasion.

conceivable that transformed cells evade immune responses through alterations of NLRC5, which present the advantage of globally affecting the MHC class I pathway. A first observation supporting this hypothesis is that several human and murine tumor cell lines express low NLRC5 (Neerincx et al., 2013; Staehli et al., 2012). By taking advantage of the TCGA datasets available on the Oncoming platform, Kobayashi and coworkers recently demonstrated that NLRC5 loss-of-function mutations, copy number loss, and promoter hypermethylation are commonly encountered in primary solid tumors (Yoshihama et al., 2016). Even though type and frequency of alterations vary among different types of cancer, they are more frequent in NLRC5 as compared to other classical MHC class I genes, B2M, or APM genes. In addition, NLRC5 mRNA levels tend to be lower in the majority of solid tumors examined, as compared to healthy tissue controls. Studies that addressed whether a significant correlation exists between NLRC5 levels or mutations and MHC class I expression in cancers have reported a positive outcome. One report, which evaluated by immunochemistry the positivity for NLRC5 and HLA-ABC in tumors, showed that

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nuclear expression of NLRC5 significantly correlated with MHC class I expression in six cancer types (prostate cancer, nonsmall cell lung cancer, gastric adenocarcinoma, malignant melanoma, liver cancer, and rectal cancer) out of eight analyzed (Li et al., 2015). Another study on prostate cancer, which assessed expression by quantitative polymerase chain reaction on microdissected biopsies, demonstrated that MHC class I-negative tumors express in average lower levels of NLRC5 compared to healthy tissues (Carretero et al., 2016). Finally, bioinformatics analyses of the TCGA datasets revealed a high correlation between NLRC5 and its target gene expression for 21 different solid tumor types (Yoshihama et al., 2016). Going a step forward, Rodriguez and colleagues recently demonstrated that stable overexpression of NLRC5 in the B16 melanoma cell line restored the MHC class I pathway, tumor immunogenicity, and the tumor-specific cytotoxic T cell response (Rodriguez et al., 2016). As a consequence, tumor growth was reduced following subcutaneous or intravenous injection into C57BL/6 hosts. Interestingly, analysis of the TCGA datasets also indicated that NLRC5 expression in melanoma, bladder, rectal, uterine, cervical, and head/neck cancers was associated with higher patient survival rates (Yoshihama et al., 2016). Instead, brain and nonsmall cell lung cancers were inversely correlated to patient’s survival (Li et al., 2015; Yoshihama et al., 2016). The identification of NLRC5 alterations and significant correlations with prognosis in primary tumors is a first, important step toward the evaluation of NLRC5 as a clinical biomarker.

4.6 Further Considerations on the Role of NLRC5 in Cancer Despite the encouraging results discussed earlier, additional efforts are necessary to refine and extend these findings. One major concern is that NLRC5 and MHC class I genes are highly expressed in lymphocytes as compared to other, especially nonimmune, cell types (Ludigs et al., 2016; Neerincx et al., 2010; Staehli et al., 2012). Unfortunately, studies on lymphoma or leukemia are missing to date. Moreover, the contribution of NLRC5 to the regulation of MHC class I expression in nonimmune cells is still poorly characterized, but has been found to be minor in selected tissues at steady state (Ludigs et al., 2016). With these premises, it is critical to differentiate immune- and tumor-intrinsic NLRC5 expression to achieve accurate biological interpretations. Contamination of tumor samples with

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noncancerous components, which are mainly infiltrating immune cells, is a common issue, especially when performing genomic and transcriptomic analyses (Aran, Sirota, & Butte, 2015). NLRC5 expression in many cancers was found to positively correlate not only with MHC class I transcripts, but also with CD8, GRZB, and PRF1 mRNAs, which are expressed by cytotoxic T cells (Yoshihama et al., 2016). Based on this data, a plausible conclusion is that NLRC5 expression in tumors triggers CD8+ T cells recruitment and activation. However, an alternative interpretation is that infiltrating CD8+ T cells directly contribute to NLRC5 and MHC class I expression in the total tumor sample (Yoshihama et al., 2016). Along these lines, despite NLRC5 levels were decreased in the majority of tumors, some exceptions and inconsistencies among studies were reported (Liu et al., 2015; Yoshihama et al., 2016). Whether this might rely on tumor purity, it shall be evaluated. Therefore, to conclude that loss of NLRC5 expression in tumors causes a reduction of MHC class I and CD8+ T cells influencing patient’s prognosis, it is essential to define to which extent these correlations are confounded by the elevated expression of NLRC5 and MHC class I in immune infiltrating cells. Lately a number of bioinformatics processes have been developed to normalize genomic, epigenomic, or transcriptomic data for tumor purity (Aran et al., 2015; Yadav & De, 2015). Furthermore, single-cell RNA-sequencing studies performed on different tumors are becoming increasingly available and will enable to examine the relationship between NLRC5 and MHC class I expression specifically in cancer cells (Tirosh et al., 2016). With the development of such computational methods and the advancement of “omic” technologies on single cells, we might be able to definitely infer the role of NLRC5 in cancer. The concomitant use of mouse models and tumor cell lines will help determine the specific contribution of NLRC5 in the tumor cells. The demonstration that the antitumor immune response can be improved by overexpression of NLRC5 in a melanoma cell line encourages evaluating its potential as a therapeutic target (Rodriguez et al., 2016). Indeed, this study, which provides the first experimental proof for the role of NLRC5 in countering tumor immune escape, also implies that rising NLRC5 expression in tumors, in any possible way, such as IFN treatment, could be envisaged to further improve cancer therapeutic success, especially in the context of immunotherapies (Chelbi & Guarda, 2016; Rodriguez et al., 2016).

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There are also additional factors to be considered when investigating how NLRC5 modulates the crosstalk between immune and malignant cells. First, tumor mutational signature is likely to influence NLRC5-mediated immunogenicity (Alexandrov et al., 2013). Of note, the highest correlation between NLRC5 and positive prognosis was found in melanoma, known to be highly immunogenic (Yoshihama et al., 2016). Second, the inflammation state of the tumor microenvironment should be evaluated, as it can impact on the levels of NLRC5 and MHC class I genes in both immune and tumor cells and influence the NK cell-mediated response, as discussed in Section 4.4 (Benko et al., 2010; Guo et al., 2015; Kuenzel et al., 2010; Ludigs et al., 2016; Neerincx et al., 2010; Staehli et al., 2012; Yao et al., 2012). Finally, MHC class I-deficient/low tumors constitute nowadays a major obstacle for the efficacy of T cell-based immunotherapies; it would be therefore timely to evaluate the predictive value of NLRC5 in the context of T cell transfer or checkpoint blockade.

5. CONCLUDING REMARKS NLRC5 emerges as a promising new player in the regulation of MHC class I transcription, cytotoxic responses, and immunological disorders. However, important questions still need to be addressed in order to genuinely appreciate the potential of NLRC5. First, we miss a thorough understanding of NLRC5 transcriptional contribution and targets in various conditions and tissues, including nonhematopoietic ones, in particular in humans. Second, the molecular mechanisms underlying NLRC5-driven transactivation and enabling its extreme specificity are only partially uncovered, though their knowledge is prerequisite to its manipulation. Finally, our understanding of the physiological relevance of NLRC5 is at its infancy. The existence of a transcriptional regulator of MHC class I so important in lymphocytes suggest specific functions in these cells that we are only starting to explore. Furthermore, emerging work on the role of NLRC5 in cancer encourages further investigations in that direction, which might indicate novel ways to improve immunotherapies.

ACKNOWLEDGMENTS Studies in the group of G.G. are funded by the Swiss National Science Foundation (PP00P3_139094 and PP00P3_165833) and the European Research Council (ERC2012-StG310890). Conflict of Interest: No conflicting financial interest to declare.

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CHAPTER FOUR

Nucleic Acid Immunity G. Hartmann1 Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Bonn, Germany 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction Principles of Nucleic Acid Immunity in Different Species Historic Overview of Different Fields Merging Into Nucleic Acid Immunity Functional Components of Nucleic Acid Immunity Innate and Adaptive Components in Nucleic Acid Immunity Innate and Adaptive Nucleic Acid Immunity in Prokaryotes Receptors and Nucleases Not Involving Classical Immune Functions 7.1 ADAR1 7.2 SAMHD1 7.3 PKR 7.4 IFIT1 and IFIT5 7.5 OAS 7.6 RNaseH 7.7 DNases 8. RNA Interference 9. Immune-Sensing Receptors 9.1 TLR3 9.2 TLR7 and TLR8 9.3 TLR9 9.4 RIG-I 9.5 MDA5 and LGP2 9.6 AIM2 9.7 cGAS/Sting 10. Conclusions Acknowledgments References

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Abstract Organisms throughout biology need to maintain the integrity of their genome. From bacteria to vertebrates, life has established sophisticated mechanisms to detect and eliminate foreign genetic material or to restrict its function and replication. Tremendous progress has been made in the understanding of these mechanisms which keep foreign

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or unwanted nucleic acids from viruses or phages in check. Mechanisms reach from restriction-modification systems and CRISPR/Cas in bacteria and archaea to RNA interference and immune sensing of nucleic acids, altogether integral parts of a system which is now appreciated as nucleic acid immunity. With inherited receptors and acquired sequence information, nucleic acid immunity comprises innate and adaptive components. Effector functions include diverse nuclease systems, intrinsic activities to directly restrict the function of foreign nucleic acids (e.g., PKR, ADAR1, IFIT1), and extrinsic pathways to alert the immune system and to elicit cytotoxic immune responses. These effects act in concert to restrict viral replication and to eliminate virusinfected cells. The principles of nucleic acid immunity are highly relevant for human disease. Besides its essential contribution to antiviral defense and restriction of endogenous retroelements, dysregulation of nucleic acid immunity can also lead to erroneous detection and response to self nucleic acids then causing sterile inflammation and autoimmunity. Even mechanisms of nucleic acid immunity which are not established in vertebrates are relevant for human disease when they are present in pathogens such as bacteria, parasites, or helminths or in pathogen-transmitting organisms such as insects. This review aims to provide an overview of the diverse mechanisms of nucleic acid immunity which mostly have been looked at separately in the past and to integrate them under the framework nucleic acid immunity as a basic principle of life, the understanding of which has great potential to advance medicine.

1. INTRODUCTION Immunology is typically categorized in innate and adaptive immunity. While the term innate is associated with conserved molecular patterns detected by germline-encoded receptors, adaptive immunity refers to T cells and B cells which use recombination and clonal selection to specifically adapt their immune receptors (T cell receptor, B cell receptors, and antibodies) in order to target foreign protein antigens. In this wellestablished concept, the innate immune system in the form of myeloid immune cells (macrophages, dendritic cells) provides information whether new protein antigens are associated with potential pathogens or damage. A limited number of germline-encoded innate immune receptors have been identified in the last two decades which are specialized to detect different classes of pathogen- or damage-associated molecules. Among them are several groups of immune receptors which are specialized on the detection of foreign or damage-associated nucleic acids. One of these groups of nucleic acid-sensing immune receptors are the Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9 (TLR13 not existent in humans) which are preferentially located in the endolysosomal compartment of distinct immune cell subsets and certain somatic cells. Nucleic acid-detecting immune receptors

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located in the cytosol include the RIG-I family of helicases (RIG-I, MDA5, LGP2), cGAS, and AIM2. Although these nucleic acid-sensing immune receptors as part of the innate immune system participate in the regulation of protein antigen-directed adaptive immunity, they are now appreciated as part of a larger system of nucleic acid-directed immunity which functions to detect and eliminate foreign nucleic acids, whereas protein-directed adaptive immunity evolved to eliminate foreign proteins. Although there are crossregulatory functions of both systems, nucleic acid-directed immunity has a purpose on its own. This is underlined by the fact that protein-directed adaptive immunity developed more recently in evolution, whereas nucleic acid-directed immunity dates back to the earliest forms of life represented by bacteria and archaea. With the additions of RNAi and the CRISPR/Cas system, specific nucleases including the restriction-modification (R-M) systems, and antiviral effector proteins partially discovered only recently in the context of rare hereditary inflammatory diseases, the new concept of nucleic acid immunity evolves. With CRISPR/Cas and RNAi, biology has established two mechanisms which acquire new sequence information of pathogens and memorize this information for later defense against the same type of pathogen, characteristics which functionally correspond to adaptive immunity of T cells and B cells. The various mechanisms comprising nucleic acid immunity are highly relevant for the understanding of many inflammatory and infectious diseases. This review summarizes the currently known nucleic acid recognition-based antiviral response strategies. Antiviral response strategies span from ancient sequence or nucleic acid modificationdependent degradation systems (R-M, CRISPR, RNAi) to modern innate immunity in vertebrates, in which innate nucleic acid-sensing receptors induce a broad spectrum of antiviral alarm and effector mechanisms as well as subsequent adaptive immune responses.

2. PRINCIPLES OF NUCLEIC ACID IMMUNITY IN DIFFERENT SPECIES Foreign nucleic acids can be introduced by viruses or bacteriophages. However, species differ in their arsenal of defense mechanisms against such foreign nucleic acid invaders (Fig. 1). All species from bacteria to humans have established different types of nucleases which cleave nucleic acids that have identified themselves as foreign by their specific structure, by abundance and localization. One of the earliest forms of nucleases are the R-M systems in bacteria and archaea. In this system, modification enzymes

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Insects

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Nucleases R-M systems CRISPR/Cas RNAi Nucleic acid receptors restricting nucleic acid function and replication Nucleic acid immune sensing receptors (type I IFN)

Fig. 1 Mechanisms of nucleic acid immunity in species relevant for human disease. Biology has evolved a number of mechanisms to detect and eliminate foreign nucleic acids as introduced by viruses or bacteriophages. All species from bacteria to humans have established nucleases to directly degrade nucleic acids with structural characteristics or localizations which allow to distinguish them from regular cellular self nucleic acids. Other mechanisms are predominant in certain groups of species. Restrictionmodification systems in bacteria and archaea apply sequence-specific modification of self nucleic acids which allows the specific detection and degradation of foreign nucleic acids (restriction endonucleases). Acquired sequence information is used by the CRISPR/Cas system in which new sequence information about pathogenic nucleic acids is integrated into the genome and thereby memorized in order to sequencespecifically degrade foreign nucleic acids. Sequence information is also used by RNA interference which serves antiviral nuclease functions (siRNA/DICER) as well as regulatory (microRNA) functions in higher multicellular organisms. In vertebrates, innate immune-sensing receptors including DICER-related helicases RIG-I and MDA5 dominate over RNAi as antiviral defense mechanism. While innate nucleic acid immune-sensing receptors elicit signaling pathways resulting in antiviral functions, a number of nucleic acid receptors (e.g., PKR, ADAR1, IFIT1) directly detect and restrict nucleic acid function and replication. Since the principles of nucleic acid immunity are either established in mammals themselves or in pathogens (bacteria, parasites, helminths) or pathogentransmitting insects (e.g., mosquitoes), nucleic acid immunity as such is highly relevant for human health and disease.

and restriction endonucleases (REases) are directed to certain DNA sequence motifs in self DNA. Since modified DNA is not cleaved by the corresponding nucleases, DNA sequence motifs without modification are identified as foreign and are degraded (for further details, see later). Besides the R-M systems, a variety of RNases and DNases are established in evolution. DNA outside the nucleus is degraded by DNases I, II, and III.

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The RNA in DNA–RNA hybrids is degraded by RNase H. Long doublestranded RNA in the cytosol is subject to Dicer which cleaves RNA down to short double-stranded oligoribonucleotides which enter the RNAi pathway. Another way to acquire sequence information for antiviral defense is used by the CRISPR/Cas system. In this system, nucleic acid sequences derived from pathogens are integrated into the genome which allows the sequence-specific identification of the same type of pathogen during a subsequent challenge. In vertebrates, a number of highly specialized innate immune-sensing receptors such as TLR9 or RIG-I evolved to detect pathogen-associated nucleic acids and to induce appropriate immune responses. While innate nucleic acid immune-sensing receptors elicit antiviral signaling pathways, a number of nucleic acid-detecting effector proteins (viral restriction factors, e.g., PKR, ADAR1, IFIT1) directly detect and restrict nucleic acid function and replication. Various principles of nucleic acid immunity apply to different species, with only a subset applying to mammals. However, if it comes to infectious diseases, pathogens (bacteria, parasites, helminths) and pathogen-transmitting insects (e.g., mosquitoes) use additional mechanisms not present in mammals, which contribute to the interaction of the pathogen with transmitting organisms, and thus represent potential prophylactic or therapeutic targets.

3. HISTORIC OVERVIEW OF DIFFERENT FIELDS MERGING INTO NUCLEIC ACID IMMUNITY Several fields initially developed as independent lines of research and only recently were appreciated to closely cooperate in a defense system specialized in the detection and elimination of foreign genetic material. Fig. 2 provides a rough time line of key discoveries of principles, receptors, and ligands emerging from different areas of research all in the context of nucleic acid immunity. This brief overview cannot be comprehensive or provide the exact timing of each single discovery. The idea rather is to provide a picture how different fields evolved over the years. For more detailed information on the different receptors and pathways, the reader is referred to the respective specific paragraphs of this chapter below (Figs. 5 and 6). Immune sensing of nucleic acids dates back to the early 1960s with the observation that nucleic acids such as long double-stranded RNA and specifically poly(I:C) can induce the antiviral factor type I interferon (Isaacs, Cox, & Rotem, 1963) which was first described in 1957 (Isaacs, 1957; Lindenmann, Burke, & Isaacs, 1957). Three decades later, it was reported

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CpG-DNA AIM2 cGAS TLR9 Bacterial DNA TLR3 TLR7/8 RIG-I/3pRNA MDA5

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PKR RNaseL OAS1

SAMHD1 ADAR1 IFIT1 IFIT5

Nucleases Trex1

DNase I DNase II

RNase H Antiviral RNAi DICER CRISPR Cas

Restriction-modification-systems Phage restriction

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Fig. 2 Overview of the time line of discoveries in nucleic acid immunity. This graph provides a noncomprehensive overview of the time lines when important principles, receptors, and ligands contributing to nucleic acid immunity have been described. Immune sensing of nucleic acids dates back to the early 1960s with the observation that nucleic acids such as long double-stranded RNA and specifically poly(I:C) can induce type I interferon. Later, it was appreciated that bacterial DNA is more active than vertebrate DNA. In 1995, the activity of bacterial DNA was attributed to a higher frequency of unmethylated CpG motifs in bacterial DNA. In 2000, TLR9 was identified as the immune receptor for the detection of unmethylated CpG motifs in DNA in the endosomal compartment. Sensing of cytoplasmic DNA remained unclear until in 2009 AIM2 and in 2012 cGAS were identified as the cytosolic receptors responsible for DNA-induced inflammasome activation and type I IFN induction, respectively. For immune sensing of RNA, the story of discoveries continued in 2001 with reports on TLR3-sensing long double-stranded RNA and was continued in 2004 with the appreciation of TLR7 and TLR8 as receptors sensing shorter forms of unmodified single and double-stranded RNA with great implications for the application of siRNA. Another milestone was reached with the immune sensing of cytoplasmic forms of RNA, specifically the detection of 50 -triphosphate short double-stranded forms of RNA by the cytosolic receptor RIG-I. The RIG-I-like receptor MDA5 added another cytosolic receptor which explained the induction of type I IFN by long double-stranded forms of RNA as observed early on in the 1960s. PKR identified in the late 1970s was the first of the receptors restricting nucleic acid function and replication without activating immunity and cytokines. SAMHD1

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that bacterial DNA induces type I IFN much more vigorously than genomic DNA of vertebrates (Yamamoto, Kuramoto, Shimada, & Tokunaga, 1988; Yamamoto, Yamamoto, Shimada, et al., 1992). It was speculated that bacterial DNA in Sir William Coley’s bacterial lysates was responsible for the antitumor activity seen using bacterial lysates for the treatment of tumor patients around 100 years earlier (Wiemann & Starnes, 1994). Efforts were undertaken to generate synthetic oligodeoxynucleotides which mimic the type I IFN-inducing activity of bacterial DNA. Palindromic DNA sequences were identified, and oligonucleotides containing such palindromes induced type I IFN in vitro (Yamamoto, Yamamoto, Kataoka, et al., 1992) but only showed weak activity in tumor models in vivo due to rapid degradation by DNases. In 1995, the immunological activity of bacterial DNA was attributed to a higher frequency of unmethylated CpG motifs in bacterial DNA (Krieg et al., 1995). Unmethylated CpG motifs were contained in the former palindromic sequences, but a palindromic sequence was not required for the type I IFN-inducing activity. The introduction of the phosphorothioate modification in DNA first described in 1977 (Vosberg & Eckstein, 1977) was used to stabilized these so-called CpG oligonucleotides which now could be successfully applied for treatment in experimental tumor models in vivo (Heckelsmiller et al., 2002). In 2000, TLR9 was identified as the innate immune receptor required for the detection of unmethylated CpG motifs in DNA (Hemmi et al., 2000). Notably, TLR9 was the first innate immune receptor reported to detect a specific type of nucleic acid and to induce an immune response. Despite intensive research, it took almost a decade to identify AIM2 as the next innate immune receptor-detecting DNA (Hornung & Latz, 2010). (depletion of dNTPs) and ADAR1 (A-to-I conversion in dsRNA) entered the field more recently in the context of genetic alterations in these genes identified in the context of inherited inflammatory syndromes (e.g., AGS). IFIT1 and IFIT5 are two other examples of more recently described receptors which inhibit the translation of mRNA. OAS1 was identified early on soon after PKR as a factor restricting viral replication by activating RNase L. Other nucleases contributing to nucleic acid immunity include RNase H structurally resolved in 2004, which degrades the RNA in DNA–RNA hybrids; furthermore, extracellular DNase I and endolysosomal DNase II are known since the mid-1950s. Knowledge around the function of the cytoplasmic DNase III which is also called Trex1 accumulated since 1999 and gained great impact on nucleic acid immunity like SAMHD1 and ADAR1 more recently in the context of inherited type I IFN-dependent inflammatory syndromes. Antiviral RNAi and the role of Dicer were first described in 2005, while the bacterial version of sequence-specific antiviral immunity, CRISPR/Cas, was identified in 2011. Restriction-modification systems are studied since the 1950s.

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However, since AIM2 activates the inflammasome but not type I IFN, the field of DNA sensing struggled until end of 2012, when the cytosolic DNAbinding enzyme cyclic GMP-AMP synthase (cGAS) was discovered which generates cGAMP as second messenger for the downstream signaling molecule Sting, resolving a big question mark in the field. At that time, Sting was already known to be required for immune sensing of cytosolic DNA resulting in IFN induction (Barber, 2014), but Sting was unable to bind DNA directly. Although RNA molecules were the first nucleic acids found to induce type IFN as described earlier, this line of research continued only in 2001 with the identification of double-stranded RNA as ligand for TLR3 (Alexopoulou, Holt, Medzhitov, & Flavell, 2001). Although the activation of TLR3 induces some type I IFN, it could not explain the massive amounts of type I IFN induced upon cytosolic delivery of the double-stranded RNA mimic poly(I:C). In parallel with the discovery of RNAi and siRNA, techniques of chemical synthesis of RNA rapidly progressed and highly pure synthetic RNA oligonucleotides at high quantities and reasonable costs became available. With easier access to highly pure synthetic RNA oligonucleotides, it was found that not only long double-stranded RNA but also single-stranded RNA stimulated type I IFN, specifically in immune cells, and the immune receptors involved were found to be TLR7 and TLR8 (TLR8 nonfunctional in mouse) (Diebold, Kaisho, Hemmi, Akira, & Reis e Sousa, 2004; Heil et al., 2004; Judge et al., 2005). Then it was noted that even siRNA induce type I IFN in TLR7 expressing immune cells, a surprising finding at that time since siRNA was thought to be short enough to not stimulate interferon responses (Hornung et al., 2005). This work also reported that the immune stimulatory activity of siRNA can be avoided by introducing chemical modifications such as 20 -O-methylation or by introducing pseudouridine which is used since then to avoid immunostimulation by siRNA applied in cells expressing TLR7 in vitro or in vivo (Hornung et al., 2005). At that time, a cheap way to generate siRNA was the use of in vitro transcription. However, it was soon realized that siRNA made by in vitro transcription induces high amounts of type I IFN when transfected even in cells not expressing TLR7, including human myeloid immune cells. This stimulated research on the molecular mechanism responsible for type I IFN induction by in vitro-transcribed siRNA in myeloid immune cells. Finally, the cytosolic helicase RIG-I was identified to detect 50 -triphosphate ends in short blunt end double-stranded RNA (Hornung et al., 2006;

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Pichlmair et al., 2006; Schlee, Roth, et al., 2009). RIG-I was reported earlier as immune receptor involved in antiviral responses (Kato et al., 2005). Notably, in vitro transcription but not chemical synthesis of siRNA generates such 50 -triphosphate ends. The presence of unmodified 50 -triphsophate ends in the cytosol indicates the presence of RNA polymerase activity in the cytosol which only occurs in the course of viral replication. Another member of the RIG-I-like helicase family of receptors is MDA5 which was found to be responsible for the long sought after type I IFN-inducing activity of cytosolic long double-stranded RNA including poly(I:C) (Yoneyama et al., 2005). As of today, most of the type I IFN-inducing activities of nucleic acids can be assigned to specific immune receptors. Future may still keep some surprises for the field, for example, in the context of immune sensing in the nucleus or in the context of DNA damage repair. While activation of the immune receptors described above results in the induction of immunologically active cytokines and immune responses, there is a group of nucleic acid receptors directly restricting nucleic acid function and replication largely without inducing an immune response. PKR was one of the first of these. Binding of long double-stranded RNA activates PKR to phosphorylate elF2a leading to the inhibition of ribosomal translation of mRNA to proteins (Clemens & Elia, 1997; Sen, Taira, & Lengyel, 1978; Thomis, Doohan, & Samuel, 1992; Thomis & Samuel, 1992). Soon after PKR, the 20 –50 -oligoadenylate synthetase (OAS) system was reported (Rebouillat & Hovanessian, 1999; Yang et al., 1981). In parallel to the OAS system, RNase L was identified (Baglioni, Minks, & Maroney, 1978; Brennan-Laun, Ezelle, Li, & Hassel, 2014). Upon binding of OAS1 to long double-stranded RNA, OAS1 generates 20 -50 -linked oligoadenylates (20 50 -OA). 20 50 -OA activate RNAse L which then cleaves cellular RNAs thereby restricting viral propagation. SAMHD1 (sterile alpha motif and histidine–aspartate-domaincontaining protein 1), originally described as IFN-inducible gene in 2000 (Li, Zhang, & Cao, 2000), has triphosphohydrolase activity that rapidly converts dNTPs to the corresponding deoxynucleoside and inorganic triphosphate, thereby depleting the supply of dNTP for reverse transcriptase activity of retroviruses. In 2009, it was learned that mutations in SAMHD1 cause inherited inflammatory syndromes with a type I IFN signature (e.g., AGS) (Rice et al., 2009), but the exact mechanism leading to type I IFN induction in this context is not well understood. Originally cloned in 1994 (Kim, Wang, Sanford, Zeng, & Nishikura, 1994), the RNA-editing enzyme ADAR1 binds to double-stranded RNA and converts A to I thereby contributing to self vs nonself recognition of RNA (Nishikura, 2016). In 2012, it was

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realized that mutations in ADAR1 cause inflammatory syndromes associated with a type I IFN signature (Rice et al., 2012). IFIT1 and IFIT5 are known for many years as type I IFN-inducible RNA-binding proteins which bind to single-stranded RNA lacking 20 -O-methylation at their 50 -end and inhibiting RNA translation (Hyde & Diamond, 2015). More recent work from 2011 (Pichlmair et al., 2011) added the information that IFIT1 and IFIT5 preferentially bind to viral RNA containing a 50 -triphosphate group, completing the picture how these proteins distinguish self from nonself single-stranded RNA. Of the proteins which function primarily as nucleases, extracellular DNase I and endolysosomal DNase II are known since the mid-1950s. The cytoplasmic exonuclease Trex1 has been identified decades later in 1999 (Hoss et al., 1999). Only since 2006, we know that loss of function in Trex1 causes the type I IFN-associated inflammatory syndrome AGS (Crow et al., 2006), suggesting that Trex1 is critically involved in the clearance self DNA within the cytoplasm of cells. RNases H are widely expressed enzymes that hydrolyze RNA in RNA/DNA hybrids (Cerritelli & Crouch, 2009). While reports on RNase H activity date back to 1969 (Stein & Hausen, 1969), the heterotrimeric functional complex in eukaryotes was only described in 2004 (Jeong, Backlund, Chen, Karavanov, & Crouch, 2004) and human in 2009 (Chon et al., 2009). In 2007, it was reported that mutations in any of the three subunits of human RNase H2 cause Aicardi–Gouti
eres syndrome (AGS) (Rice et al., 2007). Antiviral RNAi and the role of Dicer were first described in 2006 (Wang et al., 2006; Zambon, Vakharia, & Wu, 2006), while the role of RNA interference (RNAi) in mammalian innate immunity is still poorly understood. A bacterial counterpart of acquired sequence-specific antiviral immunity is the CRISPR/Cas system in prokaryotes first described in 2007 (Barrangou et al., 2007; Marraffini & Sontheimer, 2010). This prokaryotic immune system confers resistance to foreign genetic elements such as those present within plasmids and phages. Information around R-M systems accumulated since the early 1950s (Luria & Human, 1952) with REases first described in 1975 (Nathans & Smith, 1975).

4. FUNCTIONAL COMPONENTS OF NUCLEIC ACID IMMUNITY The concept of nucleic acid immunity integrates different functional components which have been studied and reviewed separately in the past. Only in the last few years and with the elegant genetic work on rare human

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inflammatory disorders associated with a type I IFN signature (of Crow and others (Crow & Rehwinkel, 2009)), it became evident that many of the antiviral restriction factors and various nuclease systems are tightly connected with innate immune sensing of nucleic acids inducing type I IFN. Altogether, biology has established a broad spectrum of effector functions which cover most of the molecular and mechanistic possibilities to restrict the propagation of foreign genetic material. Effector functions reach from direct actions on the detected nucleic acid to the elimination of cells containing foreign genetic material. Fig. 3 illustrates the functional components of nucleic acid immunity. Central to all components is the detection of foreign nucleic acids by the molecular interaction of a protein (immune-sensing receptor, restriction factor, or effector protein) or a specific nucleic acid (RNAi, CRISPR/Cas) with the targeted nucleic acid. The molecular challenge on this level is the specificity of the detection and the distinction of self vs foreign. A specific molecular signature of self nucleic acid (e.g., 20 -O-methylation at the N1 position in capped mRNA), compartmentalization of self nucleic acids, and the rapid clearance of surplus self nucleic acids are three examples which enable specific detection of foreign nucleic acid. Structural differences such as long double-stranded RNA not present under physiological circumstances allow the highest confidence level of detection. Upon detection, the system generates different types of responses. Detection of a foreign nucleic acid can trigger an intrinsic effect which acts directly on the detected nucleic acid. Examples are degradation (e.g., TREX1, CRISPR/Cas), structural modification (e.g., A-to-I conversion by ADAR1), or disabling the function (e.g., inhibition of translation of mRNA by IFIT1 or RNAi) (see Fig. 3, middle gray layer). Extrinsic effects upon detection of foreign nucleic acids require a signaling cascade finally resulting in an effect on the detected nucleic acid. Extrinsic effects can occur solely inside the same cell, or they can involve functions outside the cell. Extrinsic effects inside the same cell include degradation of the nucleic acid (e.g., RNase L activated by 20 –50 -OA generated by OAS1 upon binding of long double-stranded RNA). Extrinsic effect inside the same cell can also impact on the function of the detected nucleic acid. Examples for such functional effects are inhibition of translation (e.g., phosphorylation of elF2a by activated PKR) or inhibition of replication (e.g., restricting replication of retroviruses by depleting intracellular dNTP pools by IFN-inducible SAMHD1) (see Fig. 3, light gray layer). Extrinsic effects that involve functions outside the cell in which the nucleic acid is primarily detected appear as immune responses. They range from alarming neighboring cells

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Activity Extrinsic effect outside cell

(e Tra .g. n , P sla KR tio -e n lF2 a)

Function (e.g., IFIT1)

Nucleic acid

Alarming neighbor cells (e.g., cGAS-cGAMP)

- Function -

Detection Degradation (e.g., OAS1-RNaseL)

(e Re .g p ., lic SA at M ion H D 1)

Intrinsic direct effect

Degradation (e.g., TREX1)

Guiding immune cells (e.g.,TLR7-chemikines)

Extrinsic effect inside cell

Modification (e.g., ADAR1)

Activating immune cells (e.g., RIG-I-induced type I IFN) Response

Fig. 3 Overview of functional components in nucleic acid immunity. The primary detection of specific forms of nucleic acids by highly specialized proteins is the central part of nucleic acid immunity. Upon binding of nucleic acids, the participating specialized proteins can either exert intrinsic direct effects on the nucleic acid which they have bound, or they can have indirect extrinsic effects which require the participation of additional signaling. Extrinsic effects that restrict viral replication and function can be located inside or outside cells, or both. Intrinsic direct effects include degradation or structural modification of the bound nucleic acids, or direct inhibition of translation. Extrinsic indirect effects via signaling pathways include mechanisms that restrict translation or replication, or that lead to degradation of nucleic acids. Extrinsic effects with activities beyond the infected cell include alarming neighboring cells, activating immune effector cells, and guiding immune cells to the site of infection. Together, these intrinsic and extrinsic activities represent the repertoire of nucleic acid immunity to restrict viral infection.

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(e.g., secretion of type I IFN or release of cGAMP via gap junctions) to the guidance of immune cells to the respective cell (e.g., TLR7-induced release of IP-10) and the activation of immune cells at the local site and systemically (e.g., activation of NK cells by RIG-I). Intrinsic and extrinsic effects act in concert to minimize the danger associated with foreign nucleic acids.

5. INNATE AND ADAPTIVE COMPONENTS IN NUCLEIC ACID IMMUNITY In classical immunology, we distinguish innate and adaptive immunity. While innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later use. While adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information on pathogen-derived nucleic acid sequences is acquired and memorized (CRISPR/Cas and RNAi) (see Fig. 4). Although the adaptive part of nucleic acid immunity requires the participation of germline-encoded proteins such as DICER, RISC,

Innate

Adaptive

Innate information on structure

Acquired information on sequence

Nucleic acid receptors with effector functions

CRISPR/Cas

Nucleic acid receptors inducing immune functions

RNA interference

Fig. 4 Innate and adaptive components in nucleic acid immunity. In classical immunology, we distinguish innate and adaptive immunity. While innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later usage. While adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information about pathogen-derived nucleic acid sequences is acquired and memorized (CRISPR/Cas and RNA interference). Unlike the adaptive components, the innate components of nucleic acid immunity rely on germline-encoded receptors which detect certain structures indicating viral pathogens. Therefore, these receptors are identical throughout the life span, but regulation of receptor expression (e.g., by means of epigenetics) still allows adaptation to different environments (e.g., low or high burden of viral pathogens).

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or CRISPR and Cas, the detection of foreign nucleic acids is mediated via acquired sequence information. In contrast, the innate components of nucleic acid immunity solely rely on germline-encoded receptors which via protein–nucleic acid interaction detect certain structures which are characteristic of foreign genetic materials. Innate immune-sensing receptors are identical throughout the life span. However, it is important to note that epigenetic regulation of gene expression still allows to adapt quantitatively to different environments (e.g., low or high burden of viral pathogens).

6. INNATE AND ADAPTIVE NUCLEIC ACID IMMUNITY IN PROKARYOTES The perpetual arms race between bacteria and phages has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems include R-M systems and CRISPR–Cas. The prokaryotic DNA R-M systems are based on the contrasting enzymatic activities of a sequence-specific REase and a matching sequence-specific host methyltransferase (MTase) (Vasu & Nagaraja, 2013). By transferring a methyl group to the C-5 carbon or the N4 amino group of cytosine or to the N6 amino group of adenine host-specific MTases protect potential cleavage sites of host DNA from REases, which on the other hand recognize and cleave foreign unmethylated or “inappropriately” methylated DNA from invading phages. This R-M systems can be considered as an innate defense system. On the other hand, the CRISPR–Cas system in prokaryotes represents a highly sophisticated adaptive immune system in which short fragments of invading DNA are integrated into the CRISPR loci. After transcription and processing of these loci, short CRISPR RNAs (CrRNAs) are generated which guide the nuclease activity of Cas proteins to complementary DNA or RNA resulting in target cleavage (Goldfarb et al., 2015; van der Oost, Westra, Jackson, & Wiedenheft, 2014).

7. RECEPTORS AND NUCLEASES NOT INVOLVING CLASSICAL IMMUNE FUNCTIONS This chapter provides an overview of the well-established proteins targeting foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells.

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DNase I 3′– 5′–

– 3′ – 5′

Cell membrane Endolysosome DNase II

3′ – 5′–

–3′ – 5′ dsDNA –3′

5′– 3′ – 5′ –

– 3′ – 5′ dsDNA

ssDNA – 3′

5′– 5′–

–3′ –3′

5′–

OAS1

PKR

ADAR1 long dsRNA

TREX1 (DNase III)

SAMHD1 SAMHD1 dTTP dATP dT dA dCTP dC dG dGTP

RNase H

IFIT1

5′–

IFIT5

5′–

elF2a 2′–5′oligo A elF2a(p)

A-to-I conversion

RNase L

mRNA

Ribosome cap-

capmRNA

eIF4E Ribosome cap – No translation

mRNA

No translation Nucleus

Fig. 5 Receptors and nucleases restricting function and replication of foreign nucleic acids. This graph provides an overview of the proteins which target foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. Such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid. The endonuclease DNase I is the most abundant DNase in the extracellular space which degrades DNA down to tetramers. DNase II is the predominant endonuclease in the endolysosomal compartment of cells. The cytoplasmic DNase III (Trex1) is a 30 -to 50 exonuclease which degrades both double- and single-stranded DNA. The cytoplasmic RNase H recognizes DNA–RNA hybrids and cleaves the RNA in such hybrids. In contrast, RNase L is indirectly activated by oligoadenylates which are formed by OAS1 upon binding to long doublestranded RNA. Furthermore, ADAR1 modifies long double-stranded RNA by A-to-I conversions destabilizing the double strand resulting in changes in the coding sequence of proteins. SAMHD1 depletes the pool of dNTPs which is the prerequisite for DNA formation. SAMHD1 hydrolyzes the triphosphate in dNTPs resulting in deoxynucleosides. At the same time, SAMHD1 has been proposed to be a 30 -exonuclease for single-stranded DNA and RNA. PKR and IFIT1/5 inhibit mRNA translation by phosphorylation of the elF2a and by replacing elF4 in the ribosomal complex, respectively. While PKR is activated by long double-stranded RNA, IFIT1 and IFIT5 bind 50 -triphosphate ends of singlestranded RNA.

Such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid (see Fig. 5). For the family of APOBEC proteins which detect and modify viral nucleic acids, we refer to detailed reviews by others (Harris & Dudley, 2015).

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7.1 ADAR1 Adenosine to inosine (A-to-I) RNA editing was originally discovered as enzymatic activity unwinding double-stranded RNA in Xenopus laevis oocytes and embryos (Bass & Weintraub, 1987). Soon after, it became clear that this activity is carried out by an adenosine deaminase acting on RNA (ADAR) (Bass & Weintraub, 1988; Wagner, Smith, Cooperman, & Nishikura, 1989). Adenosine deaminases perform C6 deamination of adenosine in base-paired RNA structures resulting in A-to-I conversions, a process termed A-to-I RNA editing (Hogg, Paro, Keegan, & O’Connell, 2011). The type I IFN-inducible isoform of ADARs, ADAR1, first cloned in 1994 (Kim et al., 1994) contains three repeats of a double-stranded RNA-binding motif, and sequences conserved in the catalytic center of other deaminases. Transcription from separate promoters generates two isoforms of ADAR1, a full-length, interferon-inducible ADAR1p150 and a shorter and constitutively expressed ADAR1p110. Interestingly, both ADAR1p150 and ADAR1p110 isoforms shuttle between nucleus and cytoplasm (Nishikura, 2016). A-to-I editing frequently occurs in noncoding regions that contain inverted Alu repeats but can also occur in proteincoding regions of mRNAs resulting in the expression of altered proteins with sequences that are not encoded in the genome. Recent studies indicate that ADAR1 is also found in complex with Dicer to promote miRNA processing and RNAi efficacy (Ota et al., 2013), suggesting that both RNAi and ADAR are functionally related. Viral dsRNA formed at different stages of replication of many viruses are substrates for RNA editing by ADAR. It is well established that ADAR enzymes interfer with the virus–host interaction with ADARs acting as pro- or antiviral factors. The biological consequences of A-to-I changes during viral infection is not completely understood (Tomaselli, Galeano, Locatelli, & Gallo, 2015). The current concept is that two effects oppose each other: on the one hand, ADAR1-mediated A-to-I editing of viral double-stranded RNA directly restricts the correct function of the edited RNA and thus directly inhibits viral replication. On the other hand, A-to-I editing of double-stranded RNA destabilizes long double-stranded RNA thereby reducing the recognition of long double-stranded RNA by double-stranded RNA receptors such as MDA5. As a consequence, depending on the type of virus, ADAR1 has the potential to negatively interfer or to support viral replication and thus can act as proviral or antiviral factor (George, John, & Samuel, 2014).

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Independently of the presence of viral infection, the lack of ADAR1 in a cell results in the activation of MDA5 by endogenous RNA species. The most likely scenario is that A-to-I editing masks endogenous RNAs from detection by MDA5. The consequence is that if a virus actively inhibits the function of ADAR1, endogenous RNA ligands will form which activate MDA5 and thus induce a type I IFN response. Indeed, sequencing studies demonstrated that ADAR1-deficient cells display stretches of endogenous double-stranded RNA (Liddicoat et al., 2015). Thus, A-to-I editing of endogenous dsRNA is an essential function of ADAR1 preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

7.2 SAMHD1 SAMHD1 is composed of a SAM and a HD domain. While the SAM domain of SAMHD1 mediates protein–protein interactions, the HD domain possesses the triphosphohydrolase activity through which SAMHD1 hydrolyzes dNTPs to deoxynucleosides (Goldstone et al., 2011; Powell, Holland, Hollis, & Perrino, 2011). SAMHD1 expression has been demonstrated in monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells (PDCs), and CD4 T cells (Baldauf et al., 2012; Gelais et al., 2012; Kim, Nguyen, Daddacha, & Hollenbaugh, 2012; Laguette et al., 2011; Pauls et al., 2014). The involvement of SAMHD1 in innate immunity was initially proposed based on its mouse ortholog Mg11 which is IFN-inducible in macrophages and dendritic cells (Li et al., 2000), hence the alternative name dendritic cell-derived IFN-γ-induced protein. Subsequent studies showed an increased SAMHD1 expression upon stimulation of macrophages with double-stranded DNA (Rice et al., 2009) and its upregulation in the context of viral infections (Hartman et al., 2007). Mutations in SAMHD1 have been shown to be responsible for 5% of patients with AGS which is characterized by inappropriate and aberrant type I IFN secretion causing symptoms reminiscent of a congenital infection (Rice et al., 2009). A loss of function of SAMHD1 results in spontaneous type I IFN production in AGS patients and SAMHD1/ mice (Behrendt et al., 2013; Rehwinkel et al., 2013). SAMHD1 was identified as a potent restriction factor for HIV (Hrecka et al., 2011; Laguette et al., 2011; Simon, Bloch, & Landau, 2015), other nonhuman retroviruses (Gramberg et al., 2013), and herpesviruses, including HSV-1 (Hollenbaugh et al., 2013; Kim, White, Brandariz-Nunez, DiazGriffero, & Weitzman, 2013). The current model is that SAMHD1 through its function as dNTP triphosphohydrolase decreases intracellular dNTP pools

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in nondividing cells below the threshold level required for efficient viral reverse transcriptase or viral DNA polymerase activity (Lahouassa et al., 2012; Wu, 2013). The observation that functional loss of SAMHD1 leads to a spontaneous type I IFN response suggests that uncontrolled activity of endogenous retroelements may be a source of the IFN-inducing nucleic acids, but the identity of such endogenous ligands is unknown to date. Besides dNTP triphosphohydrolase activity, a metal-dependent 30 - to 50 -exonuclease activity of SAMHD1 for ssDNA and ssRNA was demonstrated (Beloglazova et al., 2013). The RNAse activity was reported to directly degrade HIV-1 RNA (Ryoo et al., 2014), but further work will be necessary to confirm and exactly characterize the proposed nuclease activity of SAMHD1 (Rice et al., 2009).

7.3 PKR The interferon-inducible, double-stranded RNA-activated protein kinase (protein kinase RNA-activated, PKR; also known as eukaryotic translation initiation factor 2-alpha kinase 2, EIF2AK2) was first cloned in 1992 and represents a key mediator of antiviral activities (Feng, Chong, Kumar, & Williams, 1992; Garcia et al., 2006; Thomis et al., 1992; Thomis & Samuel, 1992). PKR contains an N-terminal dsRNA-binding domain (dsRBD) which consists of two tandem copies of a conserved doublestranded RNA-binding motif, dsRBM1 and dsRBM2, and a C-terminal kinase domain. Binding of PKR to long double-stranded RNA (longer 30 bp) activates PKR by inducing dimerization and subsequent autophosphorylation. Activated PKR inhibits 50 -cap-dependent mRNA translation by phosphorylation of the eukaryotic translation initiation factor eIF2a thereby preventing viral protein synthesis (Farrell et al., 1978; Levin & London, 1978). Besides long double-stranded RNA, PKR has been shown to recognize RNA with limited secondary structures (RNA with a length of about 47 nt and weak structure; short stem-loops) containing uncapped 50 -triphosphates (Nallagatla et al., 2007). Antiviral functions of PKR beyond the mechanism of translation inhibition are still controversial. PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-κB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IkappaB and NF-κB nuclear translocation has been intensively studied. PKR was also reported to enhance but not being required for NF-κB-dependent type I IFN induction (Chu et al., 1999; Kumar, Haque, Lacoste, Hiscott, &

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Williams, 1994; Maggi et al., 2000). Involvement of PKR in inflammasome activation is controversial (He, Franchi, & Nunez, 2013; Lu et al., 2012). PKR-mediated 50 -cap-specific inhibition of translation is expected to perturb the proteome. Since PKR-activated elF2a is involved in the initiation of the translation from an AUG codon, the alternative non-AUG initiation takes place instead. An example of mRNAs using non-AUG initiation are mRNAs for the heat shock proteins. Another effect is the selected reduction of proteins with short half-life. Reduced translation of the NF-κB inhibitor protein IkappaB-alpha is one plausible explanation for the activation of the NF-κB pathway in response to PKR activation (McAllister, Taghavi, & Samuel, 2012). Other signaling pathways may be affected in the same way by the removal of an inhibitor with short half-life resulting in the activation of the pathway.

7.4 IFIT1 and IFIT5 Interferon-induced proteins with tetratricopeptide repeats (IFITs) are among the most abundantly expressed proteins of the group of interferonstimulated genes (ISGs). They represent innate immune effector molecules that confer antiviral defense downstream of type I IFN through disruption of the host translation initiation machinery (Daffis et al., 2010; Pichlmair et al., 2011). They are evolutionarily conserved from fish to mammals. In humans, there are four well-characterized paralogues, IFIT1 (p56/ISG56), IFIT2 (p54/ISG54), IFIT3 (p60/ISG60), and IFIT5 (p58/ISG58). Like RIG-I, productive binding of both IFIT1 and IFIT5 was shown to depend on the presence of cytosolic 50 -triphosphate RNA and is nonsequence specific. Unlike RIG-I, IFIT1 and IFIT5 preferentially bind to single-stranded RNA or to double-stranded RNA with a minimum three (IFIT5) or five (IFIT1)nucleotide overhangs containing an uncapped triphosphate group at the 50 -end of RNA (Abbas, Pichlmair, Gorna, Superti-Furga, & Nagar, 2013; Habjan et al., 2013; Kumar et al., 1994). Replacing the triphosphate with a 50 -cap, a 50 -monophosphate or 50 -OH diminishes the binding significantly (Abbas et al., 2013). IFIT1 competes with eIF4E, the endogenous 50 -cap binding and translation factor, in the 48S initiation complex formation. However, while in vitro competition experiments convincingly show that IFIT1 can compete with eIF4E for binding at completely unmethylated cap0 RNA, the out-competing of eIF4E from N7 methylated cap (cap0) structures is unclear. Binding of eIF4E to cap0 structures in lysates of IFN-primed cells is rather enhanced than reduced, suggesting additional

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mechanisms beyond eIF4E competition for 48S disruption (Habjan et al., 2013). A key role for IFIT1 in negative-strand RNA viruses (VSV, Influenza) and positive-strand RNA viruses (WNV, MHV) except picornaviruses was reported (Daffis et al., 2010; Habjan et al., 2013; Pichlmair et al., 2011).

7.5 OAS The human 20 –50 -OAS family comprise four type I IFN-inducible genes: OAS1, OAS2, OAS3, and OASL (OAS-like protein) (Melchjorsen et al., 2009). Upon binding to long double-stranded RNA, OAS1, OAS2, and OAS3 catalyze the formation of 20 –50 -OA, whereas OASL has no enzymatic activity but still has potent antiviral activity due to its coactivating role in the RIG-I pathway (Schoggins et al., 2011; Zhu et al., 2014). The formation of 20 –50 -oligomers of adenosine (20 –50 -OA) upon exposure to dsRNA and subsequent inhibition of translation has been described early on (Clemens & Williams, 1978; Farrell et al., 1978; Hovanessian, Brown, & Kerr, 1977; Zilberstein, Kimchi, Schmidt, & Revel, 1978). 20 –50 -OA function as second messenger of OAS binding to RNase L leading to dimer formation and subsequent degradation of cellular and viral RNA (Dong & Silverman, 1997). The structural mechanisms of RNase L activation by 20 –50 -OA and its dimer formation have recently been described (Han et al., 2014; Huang et al., 2014). All three human OAS isoforms are activated by dsRNA in vitro which is the presumed ligand in vivo as well. The full activation of the OAS system in virally infected cells leads to the inhibition of protein synthesis and the induction of apoptosis, thereby interfering with viral replication (Castelli et al., 1998). Activation of the OAS–RNase L system restricts replication of a variety of viruses, in particular positive-strand viruses (e.g., picornaviruses, flaviviruses, and alphaviruses) which produce high numbers of dsRNA during replication (Silverman, 2007). Virus-encoded inhibitors of the OAS– RNase L system such as the nonstructural protein 2 (NS2) of murine coronavirus or inhibitors expressed by picornaviruses support a key role of this system in the restriction of viruses. It is interesting to note that OAS and cGAS (see later) share similar structural features and enzymatic function. Both OAS and cGAS catalyze the uncommon 20 –50 phosphodiester linkage upon binding to a nucleic acid ligand (Hornung, Hartmann, Ablasser, & Hopfner, 2014).

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7.6 RNaseH RNases H are a family of widely expressed nonsequence-specific endonucleases that hydrolyze solely the RNA of RNA/DNA hybrids resulting in 30 -hydroxyl and 50 -phosphate terminated products and an intact DNA strand (Cerritelli & Crouch, 2009). RNases H play crucial roles in the biochemical processes associated with DNA replication, gene expression, and DNA repair where RNA/DNA hybrids can occur. Furthermore, RNases H degrade RNA/DNA hybrids generated during viral replication. Members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes. Unlike in prokaryotes and in single-cell eukaryotes, in higher eukaryotes RNases H are essential for development. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits share very little homology between human and Saccharomyces cerevisiae or bacteria. RNASEH2B and RNASEH2C serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2. Furthermore, they contain interaction sites with other proteins to support functions other than RNase H nuclease activity, but these functions are not well-defined yet. RNase H2 deficiency can cause a number of pathogenetic principles including the occurrence of ribonucleoside monophosphates accumulating in genomic DNA and activating the DNA damage-response pathway. RNase H2 deficiency leads to abundance of cytosolic RNA–DNA hybrids and to an increase in retroelements which both represent potential ligands for the cGAS–STING signaling pathway (Mankan et al., 2014; Rigby et al., 2014). In fact, mutations in any of the three subunits RNASEH2A-, RNASEH2B-, or RNASEH2C of human RNase H2 cause AGS, a human neurological disorder with devastating consequences (Rice et al., 2007). Mutations that impair RNase H2 are also associated with systemic lupus erythematosus (SLE). Pathogenicity is supported by mouse models of AGS-associated mutations of RNase H which show a spontaneous cGAS/STING-dependent type I IFN-driven phenotype (Mackenzie et al., 2016; Pokatayev et al., 2016).

7.7 DNases 7.7.1 DNase I Deoxyribonuclease I (DNase I) is an endonuclease which is secreted to cleave DNA in the extracellular space down to an average of tetranucleotides

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with 50 monophosphate and 30 hydroxyl DNA ends (Baranovskii, Buneva, & Nevinsky, 2004). Both single-stranded DNA and doublestranded DNA are degraded by DNase I. This nuclease appears to account for the major nucleolytic activity on DNA in serum and is responsible for the degradation of the majority of circulating DNA derived from apoptotic and necrotic cell death and from neutrophil extracellular traps. In addition to its role in the serum, it has been proposed as one of the deoxyribonucleases responsible for DNA fragmentation in the process of apoptosis (Samejima & Earnshaw, 2005). Notably, DNase1L3 complements the activity of DNase I. Although DNase1L3 harbors nuclear localization signals, its main function appears to be in the serum, where it can degrade proteincomplexed DNA (Napirei, Ludwig, Mezrhab, Klockl, & Mannherz, 2009). In the absence of DNase I, degradation of extracellular DNA is heavily reduced resulting in the activation of DNA-sensing immune receptors. Mice deficient in DNase I display a lupus-like phenotype with increased antinuclear antibody titers and glomerulonephritis (Napirei et al., 2000). Mutations in the human DNase I gene and factors inactivating DNase I have been associated with SLE (Hakkim et al., 2010; Yasutomo et al., 2001). In a subset of SLE patients, the presence of DNase I inhibitors or autoantibodies was associated with impaired DNA clearance and poor prognosis, suggesting that decreased DNase I activity can also be acquired (Hakkim et al., 2010). Notably, loss-of-function variants in DNase1L3 have also been associated with familiar SLE, supporting an important functional contribution of DNase1L3 in clearing DNA (Al-Mayouf et al., 2011). 7.7.2 DNase II DNase II is a mammalian endonuclease with highest activity at low pH and in the absence of divalent cations. DNase II cleaves DNA between 50 -phosphate and 30 -hydroxyl resulting in the formation of nucleoside-30 phosphates. DNase II is the predominant DNase located in lysosomes of cells in various tissues including macrophages (Evans & Aguilera, 2003; Yasuda et al., 1998). With its lysosomal localization and ubiquitous tissue distribution, this enzyme plays a pivotal role in the degradation of exogenous DNA encountered by endocytosis. It has been demonstrated that digestion of large DNA molecules and of CpG-A (Hartmann et al., 2003) by DNase II creates short DNA fragments which are sensed by TLR9 (Chan et al., 2015; Pawaria et al., 2015). DNase II deficiency is another example of a cell-intrinsic nuclease defect driving autoimmunity. Loss of DNase II leads to a defect in the disposal of

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DNA within lysosomal compartments (Kawane et al., 2006; Yoshida, Okabe, Kawane, Fukuyama, & Nagata, 2005). Accumulation of undigested DNA can result in the translocation of DNA into the cytoplasm which is then sensed by the cGAS–STING pathway (Ahn, Gutman, Saijo, & Barber, 2012; Gao et al., 2015) as well as the AIM2 inflammasome (Baum et al., 2015; Jakobs, Perner, & Hornung, 2015). Mice lacking DNase II display an inflammatory response that depends on both cGAS and AIM2. Besides cell-extrinsic sources of DNA (e.g., nuclei expelled from erythroid precursor cells), recent work in DNase II-deficient mice suggests that damaged nuclear DNA is also subject to DNase II degradation and might stimulate cytosolic DNA immune-sensing receptors if not properly degraded (Lan, Londono, Bouley, Rooney, & Hacohen, 2014). In a model of cardiacspecific deletion of DNase II, severe myocarditis and dilated cardiomyopathy developed which was attenuated if immune sensing of accumulating mitochondrial DNA by TLR9 was inhibited (Oka et al., 2012). 7.7.3 DNase III/Trex1 The cytoplasmic DNase III (30 -repair exonuclease 1, Trex1) has been identified decades later than DNase I and II (Hoss et al., 1999). Trex1 is a 30 - to 50 - exonuclease which degrades both double- and single-stranded DNA. Most DNA reaching the cytosol is promptly removed by Trex1. Modifications have been reported which render DNA resistant to Trex1. For example, oxidation of guanine bases to 8-hydroxydeoxyguanine (8-OHdG) protects DNA from TREX1-dependent degradation leading to accumulation and cGAS-mediated recognition of oxidized DNA in the cytosol (Gehrke et al., 2013). Only since 2006, it is known that loss of function in Trex1 causes the type I IFN-associated inflammatory syndrome AGS (Crow et al., 2006), suggesting that Trex1 is critically involved in the clearance self DNA within the cytoplasm of cells which otherwise is recognized by the immune sensor cGAS. Defects in Trex1 have been associated with SLE (Lee-Kirsch, Gong, et al., 2007) and with familial chilblain lupus (Lee-Kirsch, Chowdhury, et al., 2007); furthermore, genetic defects in Trex1 can cause retinal vasculopathy with cerebral leukodystrophy (Richards et al., 2007). Trex1-deficient mice develop severe autoimmunity (Gall et al., 2012; Morita et al., 2004). The pathology is fully rescued by additional genetic defects in cGAS (Ablasser et al., 2014; Gao et al., 2015; Gray, Treuting, Woodward, & Stetson, 2015) or the type I IFN system demonstrating the critical pathogenic role of the IFN response triggered by endogenous

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DNA species. The source and identity of this DNA remain controversial but may derive from endogenous retroelements (Beck-Engeser, Eilat, & Wabl, 2011; Stetson, Ko, Heidmann, & Medzhitov, 2008). Alternatively, DNA ligands originating during chromosomal replication have been proposed (Yang, Lindahl, & Barnes, 2007).

8. RNA INTERFERENCE RNAi is considered one of the major mechanism for sequencespecific detection and elimination of RNA genome-based viruses in plants and invertebrates (Szittya & Burgyan, 2013; Zhou & Rana, 2013). Besides its antiviral function, RNAi regulates gene expression in many organisms. By suppressing transcription or translation or by targeted degradation of mRNA, it controls many cellular developmental and physiological processes (Burger & Gullerova, 2015). RNAi is initiated by RNAse III family nucleases (nuclear Drosha and cytosolic Dicer) that cleave endogenous or exogenous double-stranded RNA to finally yield short 21–23 bp exogenous siRNA or endogenous miRNA (Bernstein, Caudy, Hammond, & Hannon, 2001; Elbashir, Lendeckel, & Tuschl, 2001; Lee et al., 2003). The miRNAs/siRNAs are then integrated in the RNA-induced silencing complex (RISC) which target complementary RNA for degradation or inhibition of translation (Iwakawa & Tomari, 2015). AGO family proteins in the RISC complex determine its effector function: perfectly matched mi/siRNAs mediate direct target cleavage by AGO2, while imperfectly matched mi/siRNAs inhibit translation of target mRNAs by AGO1, 3, or 4 and recruit additional effector proteins which in turn can degrade target RNA (Doench, Petersen, & Sharp, 2003; Meister et al., 2004). While an important role for RNAi for the antiviral responses in helminths, insects, and plants is well established, the contribution to antiviral immunity in vertebrates is under debate. Evidence accumulates for an antiviral role of RNAi in mammalian cells (Gantier, 2014), specifically if the otherwise dominating Dicer-related RIG-I-like helicases are inhibited. The major obstacle is that the contribution of a siRNA-mediated antiviral response cannot be studied by the knockout of Dicer which is lethal at early stages of mouse embryo development (Bernstein et al., 2003). It was reported that type I IFN-dominated innate immune responses suppress RNAi and vice versa (Seo et al., 2013). The absence of Dicer products from small RNA libraries of (+)ssRNA virus (YFV, HCV) infections strengthens this assumption (Pfeffer et al., 2005). However, more recent studies using

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the more sensitive next-generation sequencing indeed provide evidence for the generation of short virus-derived small RNAs (vsRNAs) in complex with AGO proteins and conforming to Dicer cleavage fragments of 24–31 bp (siRNA and piRNA) in vertebrate infection systems (Parameswaran et al., 2010). In a seminal work, suckling mice were infected by Nodamura virus (NoV) or a mutant virus lacking the NoV virus-encoded suppressor of RNAi, B2 (Li, Lu, Han, Fan, & Ding, 2013). NoV is a mosquitotransmissible (+)ssRNA virus, which is highly virulent to suckling mice and suckling hamsters. Loss of B2 leads to abundant occurrence of viral siRNAs and rendered mice completely resistant to NoV titers which are lethal in the presence of B2 (Li et al., 2013). Since B2 appears not to prevent recognition by RIG-I (Fan, Dong, Li, Ding, & Wang, 2015), an important innate immune sensor of (+)ssRNA-based viruses, the data suggest a strong role of virus RNA-specific RNAi during NoV infection. Still, an impact of B2 on endogenous miRNA networks or inhibition of other dsRNAsensing receptors (MDA5, PKR, OAS) as a major cause of lethality is not completely excluded in this work and requires further analysis. Nevertheless, this study adds to the concept that virus-encoded suppressors of RNAi mask the actual role of RNAi in antiviral defense of vertebrates (VA1 noncoding RNA, Influenza NS1, vaccinia virus E3L, Ebola virus VP35, primate foamy virus Tas, HIV-1 Tat West Nile virus sfRNA) (Bennasser, Le, Benkirane, & Jeang, 2005; Haasnoot et al., 2007; Lecellier et al., 2005; Li et al., 2004; Lu & Cullen, 2004; Schnettler et al., 2012; Svoboda, 2014). Importantly, murine or rat oocytes or embryonic stem cells in rat and mouse express a shortened form of Dicer (DicerO) with enhanced cleavage activity for long dsRNA which complicate the interpretation of results (Flemr et al., 2013). On the other hand, the finding that poly(I:C) stimulation or infection with DNA or RNA viruses leads to PARP13 induced poly-ADP-ribosylation of AGO2 which induces AGO2 degradation in all tested cells strongly indicates a competition rather than a cooperation between antiviral RNAi and nucleic acid immune-sensing pathways (Seo et al., 2013).

9. IMMUNE-SENSING RECEPTORS Although some recent work propose a role of RNAi in the antiviral defense of vertebrates as described earlier, the current concept is that immune-sensing receptors represent the major antiviral defense strategy in vertebrates. Immune-sensing receptors recognize characteristic features of foreign and invading nucleic acids such as unusual localization, specific

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structural elements, and modifications. Stimulation of nucleic acid-sensing receptors results in the induction of cytokines (e.g., type I interferons) and chemokines to alarm neighboring cells, in the recruitment of immune cells, in the activation of cell autonomous mechanisms interfering with virus assembly and protein translation, or in the induction of several types of cell death including apoptosis, necroptosis, and pyroptosis. This chapter provides an overview of the most important immune-sensing receptors of nucleic acids for which robust evidence exists regarding the molecular mechanism of detection, the structural aspects of receptor ligand interaction, and the downstream signaling pathways (Fig. 6).

9.1 TLR3 TLR3 detects long double-stranded RNA (Alexopoulou et al., 2001), and unlike other nucleic acid-sensing TLRs, besides its endolysosomal localization, it is also expressed on the surface of certain cell types (Matsumoto, Kikkawa, Kohase, Miyake, & Seya, 2002; Pohar, Pirher, Bencina, Mancek-Keber, & Jerala, 2013). TLR3 detects dsRNA longer than 40 bp. The ectodomains of two TLR3 molecules bind one dsRNA molecule in a way that the cytoplasmic C-terminal signaling domains are juxtaposed to each other resulting in downstream signaling (Liu et al., 2008). TLR3 interacts with the ribose-phosphate backbone of dsRNA and has no specific sequence requirements. Given the absence of long dsRNA under physiological conditions, TLR3 should be inactive in the absence of an infection. Still, a number of studies proposed recognition of endogenous dsRNA by TLR3 in situations of sterile tissue damage, but the specific ligand is not well defined.

9.2 TLR7 and TLR8 The identification of ligand specificities of TLR7 and TLR8 has been hampered by their mutually exclusive expression in different cell types and by considerable differences between mouse and human. TLR7 and TLR8 are examples where distinct function of nucleic acid-sensing TLRs is determined by their differential expression in immune cell subsets. While the expression of TLR7 in the human immune system is almost restricted to B cells and PDC, TLR8 is preferentially expressed in myeloid immune cells. Consequently, TLR7 ligands drive B cell activation and the production of large amounts of IFN-alpha in PDC, while TLR8 induces the secretion of high amounts of IL-12p70 in myeloid immune cells.

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TLR3 Cell membrane

Endolysosome TLR3 TLR7 TLR8

3′–

TLR9 CG

RIG-I 5¢– –5′

Type I interferon Interferon-induced genes

cGAS

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–3′ –5′ G

3′– G G 5′–

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Type I interferon Interferon-induced genes Inflammasome

G

cGAS GTP

via gap junctions to neighboring cells

MDA5 G

ATP

2′–5′ cGAMP Sting

AIM2

3′– 5′–

–3′ –5′

Inflammasome

Type I interferon NF-κB-induced inflammatory cytokines

Nucleus

Fig. 6 Immune-sensing receptors-detecting foreign nucleic acids and inducing indirect effector responses. This graph provides an overview of immune-sensing receptors of nucleic acids. TLR3 is the only one which besides its endosomal localization is also reported to be expressed on the cell membrane. TLR3 binds long double-stranded RNA which is not present in the cytosol of normal cells and is an indicator of foreign. TLR3 is expressed in myeloid immune cells and in a number of somatic cells including fibroblasts and endothelial cells. The other three TLRs expressed in the endolysosomal compartment of distinct immune cell subsets are TLR7, TLR8, and TLR9. TLR7 detects even short RNA, preferentially double-stranded and containing G and U. TLR8 detects single-stranded RNA. While TLR8 is expressed in human myeloid immune cells, TLR7 and TLR9 are predominantly expressed in human B cells and plasmacytoid dendritic cells. TLR9 detects single-stranded DNA containing unmethylated CpG dinucleotides. In the cytoplasm, RIG-I specifically detects RNA if it contains at least a short double strand with a blunt end and a 50 -triphosphate. The RIG-I-like receptor MDA5 detects long irregular forms of double-stranded RNA, but the exact definition of the ligand structure is unclear. Both RIG-I and MDA5 are widely expressed in immune cells and nonimmune cells, and induce a broad array of cell autonomous and extracellular antiviral responses including the production of type I interferon. MDA5 ligands also activate multiple other receptor pathways that depend on the detection of long double-stranded RNA, including PKR, ADAR1, and TLR3. The cytosolic receptor AIM2 detects long double-stranded DNA and activates the inflammasome. The other key receptor for the detection of DNA in the cytoplasm is cGAS. cGAS is activated by long double-stranded DNA and short forms of double-stranded DNA with single-stranded overhangs containing Gs, a structure which was termed Y-form DNA and which is presented during retroviral infection or by endogenous retroelements. Upon activation, cGAS catalyzes the formation of 20 –50 -cGAMP from GTP and ATP. 20 –50 -cGAMP acts as a second messenger which binds to the downstream signaling protein Sting which induces type I interferon via TBK1 and IRF3. 20 –50 -cGAMP can travel to and alarm neighboring cells via gap junctions. Sting also activates NF-κB activation and inflammatory cytokines.

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TLR7 and TLR8 are preferentially activated by polyU or by G- and U-rich sequences (Diebold et al., 2004; Heil et al., 2004; Hornung et al., 2005; Judge et al., 2005). However, confounding factors need to be considered while interpreting these results (Forsbach et al., 2008). Furthermore, it has been demonstrated that TLR8 selectively detects ssRNA, while TLR7 primarily detects short stretches of dsRNA but can also accommodate certain ssRNA oligonucleotides (Ablasser et al., 2009; Sioud, 2006). However, since neither polyU or G- and U-rich sequences nor ssRNA or short dsRNA structures are overrepresented in microbial or viral RNA, the distinction of self vs nonself by TLR7 and TLR8 requires additional information. In fact, endogenous RNAs are posttranscriptionally modified at their nucleobases and backbone. The current concept is that the addition of certain modifications to self RNA inside the nucleus provides a signature for self. One example is 20 -O-methylation which is a common nuclear modification of RNA performed by a specific MTase located in the nucleolus. The MTase adds a methyl group to the 20 -hydroxyl group of the ribose. This modification represents a marker of self in higher eukaryotic cells and prevents the recognition of endogenous RNA by TLR7 and TLR8 (Hornung et al., 2005; Judge et al., 2005; Kariko, Buckstein, Ni, & Weissman, 2005). Other modifications of RNA molecules which potently inhibit the detection of transfer RNA and ribosomal RNA by TLR7 and TLR8 are the incorporation of pseudouridine (Ψ), 5-methylcytidine (m5C), 2-thio-uridine (s2U), or N6-methyladenosine (m6A) (Kariko et al., 2005). The presence of such modifications in part explains the lack of immunostimulation of host-derived RNA vs microbial RNA (Gehrig et al., 2012; Jockel et al., 2012). However, endogenous RNA from apoptotic or dying cells still activates TLR7 and TLR8 once entering the endolysosomal compartment (Busconi et al., 2006; Ganguly et al., 2009; Vollmer et al., 2005). Thus, additional factors such as intracellular localization and degradation by nucleases likely support a faithful discrimination of self from nonself by TLR7 and TLR8. It is interesting to note that there is an obvious need to sense and eliminate certain endogenous RNAs as well. In this context, it has been reported that the loss of TLR7 function causes retroviral viremia (Yu et al., 2012) indicating that endogenous RNAs transcribed from RNA polymerase II promoters are not generally excluded from TLR-mediated recognition.

9.3 TLR9 TLR9 senses DNA in the endolysosomal compartment of certain immune cells (Hemmi et al., 2000). Like TLR7 and TLR8, TLR9 travels to the

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endolysosomal compartment via the chaperone protein UNC93B1 (protein unc-93 homolog B1) (Latz et al., 2004; Pelka, Shibata, Miyake, & Latz, 2016). There, TLR9 is proteolytically processed at a defined protruding loop structure without disrupting the horseshoe shape of the protomer (Bauer, 2013; Onji et al., 2013; Peter, Kubarenko, Weber, & Dalpke, 2009). Cleavage is necessary for the activation of TLR signaling (Ewald et al., 2008; Park et al., 2008). TLR9 preferentially detects DNA with unmethylated (no methylation at the C5 carbon of cytosine) CpG dinucleotides (CpG DNA) with a preference for certain sequence contexts (hexamer CpG motifs, in humans 50 -GTCGTT-3) which vary between species and which altogether are less frequent in eukaryotic self DNA (Hartmann & Krieg, 1999, 2000; Hartmann et al., 2000; Hartmann, Weiner, & Krieg, 1999; Hemmi et al., 2000; Krieg et al., 1995). Activation of TLR9 signaling is preceded by dimer formation where two CpG DNA molecules symmetrically bind two TLR9 molecules (Ohto et al., 2015). Both CpG DNA molecules bind to the C-terminal fragment of one protomer and the CpG-binding groove in the N-terminal fragment of the other. Inhibitory DNA oligonucleotides only bind to the N-terminal fragment. Methylated single-stranded CpG DNA and double-stranded DNA exhibit lower binding to TLR9 and are less potent to induce TLR9 dimer formation. Of note, digestion of DNA molecules by the lysosomal endonuclease DNase II creates short TLR9stimulatory DNA fragments (Chan et al., 2015; Pawaria et al., 2015). Notably, specificity for unmethylated CpG motifs is reduced if the CpG motif is within a phosphorothioate-modified DNA often used to stabilize oligodeoxynucleotides against DNases. Nevertheless, a high degree of specificity is well established for unmethylated CpG motif containing DNA within a natural phosphodiester backbone including microbial DNA (Coch et al., 2009; Hartmann & Krieg, 2000). It is specifically noteworthy that genomic microbial DNA displays a much stronger activity to stimulate TLR9 as compared to genomic DNA of vertebrates. Although eukaryotic DNA presents with a lower frequency of nonmethylated CpG motifs than microbial DNA, this difference in frequency of unmethylated CpG motifs does not allow a clear cut distinction of self from nonself on a structural basis. Endogenous DNA at high concentrations can activate TLR9 once delivered into the endolysosome (Marshak-Rothstein, 2006). It is also of great importance to be aware of the differences of TLR9mediated DNA recognition between species. In humans, TLR9 is almost exclusively expressed in B cells and PDC (Hornung et al., 2002), while in mice, TLR9 is expressed more widely including myeloid immune cells.

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In humans, TLR9 predominantly induces type IFN production in PDCs and polyclonal activation in B cells via MyD88/IRF7-dependent signaling. Species-specific expression patterns of TLR9 are responsible for the fundamental functional differences of TLR9 in mouse and man. Another important issue is that in preparations of human immune cell subsets, minute amounts of PDC indirectly activate other immune cell subsets such as monocytes, myeloid dendritic cells, or NK cells. This needs to be carefully considered when direct effects of TLR9 activation in immune cells other than B cells and PDC are claimed, such as direct TLR9 effects in human myeloid immune cells and NK cells. Three different classes of CpG oligonucleotides have been described, CpG-A, CpG-B, and CpG-C (Avalos et al., 2009; Hartmann et al., 2003; Kerkmann et al., 2003; Krug et al., 2003; Rothenfusser et al., 2004). Based on the palindromic structure, CpG-A spontaneously forms nanoparticle-like complexes (Kerkmann et al., 2005) that explain much higher type I IFN-inducing capacity in PDC as compared to CpG-B which are monomeric. Monomeric CpG-B potently activates B cells which do not internalize larger particles of DNA as with CpG-A complexes. CpG-C potently stimulates both B cells and PDCs. In cell culture, delayed TLR9 activation due to slower uptake of CpG-A nanoparticles allows a longer self-priming of PDC by minute amounts of spontaneously released type I IFN. Priming of PDC results in higher IFN-inducing activity of CpG-A seen in cell culture (Kim et al., 2014).

9.4 RIG-I RIG-I belongs to the cytosolic DExD/H box RNA helicases and is one of three members of the so-called family of RIG-I-like helicases (others: MDA5 and LGP2). RIG-I is closely related to the Dicer family of helicases of the RNAi pathway. RIG-I contains a RNA helicase domain and a two N-terminal CARD domains which relay the signal to the downstream signaling adaptor MAVS (mitochondrial antiviral-signaling protein). RIG-I signaling via MAVS not only leads to the induction of type I IFN responses via TBK1 and IRF7/8, it also activates caspase-8-dependent apoptosis, preferentially in tumor cells (Besch et al., 2009; El Maadidi et al., 2014; Glas et al., 2013; Kumar et al., 2015). Furthermore, RIG-I was also found to mediate MAVS-independent inflammasome activation (Poeck et al., 2010), specifically in the context of viral infection (Poeck et al., 2010; Pothlichet et al., 2013).

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RIG-I detects blunt ends of double-stranded RNA containing a 5 -triphosphate or a 50 -diphosphate (Goubau et al., 2014; Hornung et al., 2006; Marq, Hausmann, Veillard, Kolakofsky, & Garcin, 2010; Marq, Kolakofsky, & Garcin, 2010; Pichlmair et al., 2006; Schlee, Roth, et al., 2009). Such RNA ligands are presented, for example, by negative-strand RNA viruses which form panhandle structures with their matching 50 - and 30 -ends of the single-stranded genomic RNA (Rehwinkel et al., 2010; Schlee, Roth, et al., 2009). While crystal structures confirmed the structural requirements as determined in functional studies (Civril et al., 2011; Jiang et al., 2011; Kowalinski et al., 2011; Luo et al., 2011; Wang et al., 2010), the minimum length of the double-strand required for RIG-I activation is still controversial. While approaches with synthetic or highly purified enzymatic double-stranded 50 -triphosphate RNA revealed a minimum length of 18–19 bp (Marq, Hausmann, et al., 2010; Schlee, Hartmann, et al., 2009), 10 bp were demonstrated to be sufficient for a hairpin forming oligonucleotide (Kohlway, Luo, Rawling, Ding, & Pyle, 2013). However, alternatively to the predicted hairpin, these oligonucleotides may form 20mer duplexes when entering the cell. Although oligomerization of 2CARD modules of each RIG-I protein along the RIG-I filament bound to a longer double-stranded RNA molecule induces robust RIG-I signaling, the minimal signaling unit is sufficient for RIG-I to trigger signal transduction. In the latter case, a 2CARD tetramer is stabilized by ubiquitin chains (Wu & Hur, 2015). Furthermore, it is interesting to note that RIG-I mutants deficient in ATP hydrolysis of their helicase domain cannot detach from suboptimal endogenous RNA ligands leading to erroneous signaling which can cause autoimmunity (Lassig et al., 2015). Importantly, N1-methylation (20 -O-methylation at the first nucleotide of capped RNA) serves as a signature of self RNA and completely abrogates RIG-I sensing of RNA, while in the absence of N1 methylation, RIG-I binding is hardly impaired by the 50 ppp50 -linked m7G cap structure itself (Schuberth-Wagner et al., 2015). RIG-I is ubiquitously expressed in all cell types including tumor cells. However, the type of RIG-I induced responses differs between cells. While normal healthy cells such as melanocytes and fibroblasts are quite resistant to RIG-I-induced apoptosis, tumor cells are highly susceptible to RIG-Iinduced cell death (Besch et al., 2009; Kubler et al., 2010). Based on this tumor selective activity and a favorable toxicity profile, RIG-I-specific ligands are currently being developed for immunotherapy of cancer (Duewell et al., 2014, 2015; Ellermeier et al., 2013; Schnurr & Duewell, 0

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2013, 2014). Part of the potent antitumor activity of RIG-I ligands is its ability to promote crosspresentation of antigens to CD8 T cells and to induce cytotoxic activity (Hochheiser et al., 2016). RIG-I ligands show strong therapeutic activity in viral infection models such as influenza (WeberGerlach & Weber, 2016). Notably, RIG-I has also been shown to be involved in the detection of intracellular bacteria (Abdullah et al., 2012). Rare genetic gain-of-function variants of RIG-I have been associated with an atypical form of Singleton Merten syndrome (Jang et al., 2015).

9.5 MDA5 and LGP2 Like RIG-I, melanoma differentiation associated gene 5 (MDA5) is a cytosolic DExD/H box RNA helicase which signals through MAVS and IRF3/ IRF7 (Yoneyama et al., 2005). Despite its similar structure, MDA5 senses a different type of ligand which has been described as higher order RNA structures (Pichlmair et al., 2009). So far, a MDA5-specific ligand has not been described. Double-stranded RNA ligands activating MDA5 are typically promiscuous ligands, such as poly(I:C) which also activates TLR3 and antiviral effector proteins which inhibit translation upon binding to doublestranded RNA, such as PKR and OAS. Multiple effects of MDA5 ligands cause a high degree of toxicity in vivo strictly limiting the clinical application of MDA5 ligands. Unlike RIG-I which primarily binds to the ends of RNA, MDA5 proteins bind double-stranded RNA internally, independently of its terminal structures. Additional MDA5 molecules then closely stack in a helical headto-tail arrangement around dsRNA resulting in the formation of long MDA5 filaments which initiate signaling toward activation of MAVS (del Toro Duany, Wu, & Hur, 2015). LGP2 (laboratory of genetics and physiology 2) is a third cytosolic RIG-I-like helicase lacking CARD domains for signaling. LGP2 appears to contribute to the fine tuning of immune responses by inhibition of RIG-I and supporting MDA5 signaling (Rothenfusser et al., 2005; Venkataraman et al., 2007). While MDA5 contains the signaling CARD domains but has relatively weak binding to double-stranded RNA, LGP2 readily detects diverse double-stranded RNA species but lacks a signaling domain. The current concept is that LGP2 assists the interaction of MDA5 with double-stranded RNA and filament formation, thereby enhancing MDA5-mediated antiviral signaling (Bruns & Horvath, 2015; Bruns, Leser, Lamb, & Horvath, 2014). Notably, genetic gain-of-function variants of MDA5 have been associated with autoimmune disorders (Junt & Barchet, 2015; Kato & Fujita, 2015).

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9.6 AIM2 The HIN-200 (hematopoietic interferon-inducible nuclear proteins with a 200-amino acid repeat) family member AIM2 (absent in melanoma 2) binds and oligomerizes on cytoplasmic double-stranded DNA through its C-terminal HIN domain in a sequence-independent manner (FernandesAlnemri, Yu, Datta, Wu, & Alnemri, 2009; Hornung et al., 2009; Roberts et al., 2009). DNA binding of the HIN domain relieves the autoinhibitory conformation of AIM2 and allows the N-terminal pyrin domain of multiple AIM2 proteins to form a helical structure which nucleates the helical assembly of ASC (apoptosis-associated speck-like protein containing a CARD) filaments (Lu, Kabaleeswaran, Fu, Magupalli, & Wu, 2014; Lu, Magupalli, et al., 2014; Lu et al., 2015) thereby forming an inflammasome that results in the release of IL-1beta. The formation of an AIM2 inflammasome requires a minimal length of double-stranded DNA of 50–80 bp (Jin et al., 2012).

9.7 cGAS/Sting The cytosolic immune-sensing receptor cGAS (Cai, Chiu, & Chen, 2014; Wu et al., 2012) detects long double-stranded DNA (dsDNA) or short dsDNA with unpaired open ends containing guanosines (Y-form DNA) as, for example, presented in highly structured single-stranded DNA of retroviruses or certain endogenous retroelements (Herzner et al., 2015). It is important to note that Trex1 has a gate keeper function for cGAS. Usually, cytosolic DNA is efficiently degraded by TREX1. Only in the case of excess cytosolic DNA, or DNA modifications rendering DNA resistant to Trex1mediated degradation, DNA gains access to cGAS resulting in downstream signaling. Along these lines, it has been reported that oxidized DNA (e.g., 8-hydroxyguanosine, 8-OHG) as occurring in the context of UV radiation or upon exposure to reactive oxygen species resists Trex1-mediated degradation (Gehrke et al., 2013). This results in an accumulation of DNA in the cytosol. Oxidized DNA has the same affinity to cGAS than nonoxidized DNA. cGAS is monomeric in its unligated state. However, two cGAS molecules bind to two dsDNA molecules in a way that each cGAS protomer presents an additional interaction site with the DNA bound to the other cGAS protein. Upon activation by cytosolic DNA, cGAS catalyzes the formation of 20 –50 -cGAMP from GTP and ATP (Ablasser, Goldeck, et al., 2013; Gao, Ascano, Wu, et al., 2013). 20 –50 -cGAMP acts as a second messenger which binds to the downstream signaling protein Sting (Gao, Ascano, Zillinger,

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et al., 2013) which induces type I interferon via TBK1 and IRF3. 20 –50 cGAMP can travel to and alarm neighboring cells via gap junctions (Ablasser, Schmid-Burgk, et al., 2013). Sting activation is also associated with NF-κB activation and prominent induction of inflammatory cytokines such as IL-6 and TNF-a.

10. CONCLUSIONS The general view is that most of the relevant immune receptors, effector proteins, and pathways participating in nucleic acid immunity have now been identified. These different players have in common that they all serve the function to detect and to disable foreign nucleic acids. Altogether they constitute the system of nucleic acid immunity. All of these pathways are relevant for human disease, either as part of the human antiviral defense system, or indirectly by being active in pathogens or pathogen-transmitting organisms. Examples are arboviruses (Zika, Dengue, Yellow-fever, West Nile) which are transmitted by arthropod vectors. Successful arboviruses need to escape both RNAi in insects and immunoreceptors such as RIG-I in humans. Only if they manage to inhibit both pathways, they can establish as pathogens. Therefore, it will be interesting to understand the molecular evolution of escape strategies in emerging viruses such as Zika, which may lead to the identification of the molecular step that allowed the virus to spread more efficiently (Rasmussen & Katze, 2016). Another example is the important role of RNAi in pathogens such as filariae and other human pathogenic helminths. There, RNAi may be useful as a therapeutic target. Another example is the presence of endosymbionts such as Wolbachia, a genus of bacteria, in filarial nematodes (Taylor, Bandi, & Hoerauf, 2005). The release of Wolbachia nucleic acids may contribute to the pathogenesis of filarial infection. Furthermore, CRISPR/Cas is involved in the evolution of pathogenic bacteria. Nucleic acid sensing in vertebrates is required for antimicrobial immunity and is involved in the pathogenesis of many inflammatory diseases. Although much is known about the structure and the function of the single pathways, the functional interaction of the different pathways is far from being understood. Distinct expression patterns of the receptors in different cell types and cell-type-dependent differences in the expression of downstream signaling components and transcription factors contribute to the complexity of nucleic acid immunity. As a result, the same type of ligand can have different functional outcomes in different cell types. Furthermore,

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since different receptor pathways are activated by the same type of ligand, it is currently unclear whether competition of receptor binding or distinct molecular trafficking to the corresponding receptors or both impact on the functional outcome of ligand exposure. For example, long doublestranded DNA in principle binds to both AIM2 and cGAS, and functional cooperation or inhibition of the respective pathways are unclear. Now since most of the individual molecular pathways of nucleic acid immunity are on the table, we see ourselves just at the dawn of an exciting new research field which is expected to advance medicine specifically in the areas of infection and inflammation and with broad implication for human diseases.

ACKNOWLEDGMENTS This work was supported by the DFG-funded excellence cluster ImmunoSensation, and the Helmholtz-funded German Center for Infection Research (Deutsches Zentrum f€ ur Infektionsforschung, DZIF).

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CHAPTER FIVE

About Training and Memory: NK-Cell Adaptation to Viral Infections Q. Hammer, C. Romagnani1 Deutsches Rheuma Forschungszentrum, a Leibniz Institute, Berlin, Germany 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 NK-Cell Recognition Repertoire 1.2 Role of NK Cells in the Defense Against Viral Infections 2. NK-Cell Adaptations to Viral Infections 2.1 Skewing and Adaptation of NK-Cell Subsets After MCMV Infection 2.2 Skewing and Adaptation of NK-Cell Subsets After HCMV Infection 3. Functional Imprinting of Adaptive NK Cells 3.1 Functional Properties of MCMV-Induced Memory Ly49H+ NK Cells 3.2 Functional Properties of HCMV-Induced Adaptive NKG2C+ NK Cells 4. Epigenetic Remodeling as Hallmark of Immune Training and Memory 4.1 Epigenetic Imprinting of Human Adaptive NK Cells 4.2 Possible Role of Epigenetic Remodeling for NK-Cell Memory in MCMV 5. Training and Cross-Reactive Memory: Consequences for Heterologous Immunity 5.1 NK-Cell Training and Responses to Heterologous Pathogens 5.2 NK-Cell Cross-Reactive Memory and Heterologous Immunity 6. Outlook and Outstanding Questions Acknowledgments References

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Abstract Viral infections continuously challenge and shape our immune system. Due to their fine antigen recognition ability, adaptive lymphocytes protect against pathogen reencounter by generating specific immunological memory. Innate cells such as macrophages also adapt to pathogen challenge and mount resistance to reinfection, a phenomenon termed trained immunity. As part of the innate immunity, natural killer (NK) cells can display rapid effector functions and play a crucial role in the control of viral infections, especially by the β-herpesvirus cytomegalovirus (CMV). CMV activates the NK-cell pool by inducing proinflammatory signals, which prime NK cells, paralleling macrophage training. In addition, CMV dramatically shapes the NK-cell repertoire due to its ability to trigger specific NK

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cell-activating receptors, and enables the expansion and persistence of a specific NK-cell subset displaying adaptive and memory features. In this chapter, we will discuss how different signals during CMV infection contribute to NK-cell training and acquisition of classical memory properties and how these events can impact on reinfection and crossresistance.

1. INTRODUCTION Natural killer (NK) cells are lymphocytes belonging to the family of innate lymphoid cells (Annunziato, Romagnani, & Romagnani, 2015), and since their discovery as cytotoxic effector cells of the innate immune system in 1975 (Kiessling, Klein, & Wigzell, 1975), our knowledge of NK cells has progressed abundantly. Initially, their functional characterization was focused on the killing of malignantly transformed and virally infected cells and has over time been extended to their contribution to and regulation of host immune responses (Vivier, Tomasello, Baratin, Walzer, & Ugolini, 2008). The key functions of NK cells include direct killing of target cells by release of granules containing the cytotoxic proteins perforin and granzymes and secretion of their signature cytokine interferon (IFN)-γ as well as of tumor necrosis factor (TNF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and chemokine (C–C motif ) ligands CCL3 and CCL4. As such, NK cells contribute to host defense as direct effectors and immunoregulators.

1.1 NK-Cell Recognition Repertoire The functionality of NK cells is regulated by the integration of signals from a broad range of activating and inhibitory receptors, which, unlike the T-cell receptor (TCR), do not undergo rearrangement. Activating receptors such as NKG2D, DNAM-1, and the natural cytotoxicity receptors (NCRs, namely NKp46, NKp30, and NKp44) sense ligands, which are upregulated during cellular stress, tumor transformation, or viral infection (Moretta et al., 2001). CD16, the low affinity receptor for the Fc part of IgG, enables NK cells to recognize and be activated by antibody-coated target cells, a phenomenon known as antibody-dependent cell cytotoxicity (ADCC). The majority of NK-cell activating receptors contain short intracellular domains, which do not possess signaling capacity. Instead, the cytoplasmic regions associate with signaling chains such as CD3ζ, FcεRγ, and DAP12, which in turn contain immunoreceptor tyrosine-based activation motifs

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(ITAMs), and thus transmit their signals similar to the TCR (Lanier, 2008). However, some exceptions with different signaling properties exist. Human NKG2D selectively couples to DAP10 containing a YINM motif (Wu et al., 1999), the family of SLAM-related receptors contains immunoreceptor tyrosine-based switch motifs (ITSMs) (Watzl, 2014), and DNAM-1 can directly recruit the tyrosine kinase Fyn and the serine-threonine kinase PKC via signal-transducing motifs in its cytoplasmic domain (Shibuya, Lanier, & Phillips, 1998; Shirakawa, Shibuya, & Shibuya, 2005). Engagement of activating receptors or CD16 by their respective ligands on target cells induces signaling, leading to the release of cytotoxic mediators and the secretion of cytokines and chemokines by the activated NK cells. In addition, NK cells express the receptors for different cytokines, among which are the receptors for type I IFNs, interleukin (IL)-12, IL-15, and IL-18. Stimulation via cytokines induces phosphorylation of signaling molecules of the JAK/STAT families downstream of most cytokine receptors (O’Shea & Plenge, 2012) or mediates signaling through the adaptor MyD88 for the IL-1R/IL-18R (Fitzgerald & O’Neill, 2000). Ultimately, cytokine receptor signaling yields in secretion of chemokines and cytokines by NK cells and enhances their functionality against cellular targets (Fauriat, Long, Ljunggren, & Bryceson, 2010). The majority of inhibitory receptors such as those belonging to the killer cell immunoglobulin-like receptor (KIR) family in humans, to the C-type lectin-like superfamily of Ly49 receptors in mice and CD94/NKG2A in both species bind to major histocompatibility complex (MHC) class I molecules to prevent autoreactivity against host cells, while allowing the detection of cells lacking MHC class I expression (missing self ), which often occurs as an immune evasion strategy during viral infection or malignant transformation. In contrast to activating receptors, engagement of inhibitory receptors induces phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs), directly and indirectly counterbalancing stimulation through activating signals (Lanier, 2008). Importantly, these inhibitory receptors confer education, that is, functional competence, and NK cells lacking the expression of inhibitory receptors remain in a hyporesponsive state toward target cells, thus ensuring tolerance (Goodridge, Onfelt, & Malmberg, 2015). The majority of activating receptors including NKp30, NKp46, DNAM-1, and NKG2D are uniformly expressed on NK cells in the periphery. Conversely, other inhibitory or activating receptors belonging to the KIR, Ly49, or CD94/NKG2 families are stochastically distributed on different NK-cell subsets. This combinatorial coexpression pattern of

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activating and inhibitory receptors determines the NK-cell receptor repertoire. State-of-the-art mass cytometry estimated the presence of up to 30,000 phenotypically distinct subpopulations of NK cells within single human individuals (Horowitz et al., 2013). Monitoring shifts and deviations in the NK-cell repertoire under varying pathogenic, environmental, or experimental conditions has enabled to recognize how the NK-cell pool adapts in response to viral infections.

1.2 Role of NK Cells in the Defense Against Viral Infections The role of NK cells in the defense against viral infections has been extensively studied in animal models. In particular, the analysis of the NK-cell response to the herpesvirus murine cytomegalovirus (MCMV) has provided important insights as to how NK cells can specifically recognize virusinfected cells and contribute to the control of viral infections (Scalzo & Yokoyama, 2008). Infection of inbred mouse lines enabled the identification of strains with differing resistance to MCMV infection due to variations of a host locus, named Cmv1, which confers protection toward MCMV infection in an NK cell-dependent fashion (Scalzo, Fitzgerald, Simmons, La Vista, & Shellam, 1990). Genetic analyses revealed that Cmv1 maps to the distal region of mouse chromosome 6 in the NK-cell gene complex (NKC) (Scalzo et al., 1992). Further studies demonstrated that the NK-cell receptor Ly49H, encoded within the Cmv1 locus, is responsible for MCMV resistance in C57BL/6 mice (Depatie, Muise, Lepage, Gros, & Vidal, 1997; Forbes et al., 1997). Ly49H is an activating receptor of the C-type lectin-like superfamily, which associates with the signaling adaptor DAP12 (Smith, Wu, Bakker, Phillips, & Lanier, 1998) and recognizes the MHC class I-like glycoprotein m157 derived from the Smith and K181 MCMV laboratory strains. Introduction of Ly49H in susceptible mice is sufficient to confer resistance to MCMV infection (Arase, Mocarski, Campbell, Hill, & Lanier, 2002; Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001, 2003; Smith et al., 2002). However, analysis of MCMV isolates from wildtype mice revealed a large variety of m157 sequence variants displaying low binding to Ly49H and maintaining the ability to efficiently replicate in C57BL/6 mice, indicating that MCMV resistance displayed by different mouse lines is also dependent on the virus strain (Corbett, Coudert, Forbes, & Scalzo, 2011; Voigt et al., 2003). m157 from Smith and K181 MCMV strains can also bind to the inhibitory receptor Ly49I in 129/J mice, while the MCMV G1F strain binds to both the activating receptor

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Ly49H and the inhibitory receptor Ly49C from C57BL/6 mice, suggesting that m157 may have evolved as an immune evasion protein engaging NK-cell inhibitory receptors (Arase et al., 2002). Rare cases of human genetic diseases have allowed investigating the clinical importance of NK cells during viral infections, since patients suffering from combined NK-cell deficiencies present with severe and disseminated infections, especially caused by β-Herpesviridae such as varicella zoster virus (VZV), human (H)CMV, Epstein–Barr virus (EBV), and herpes simplex virus (HSV) (Biron, Byron, & Sullivan, 1989; Orange, 2013). Further supporting the role of human NK cells in the defense against viruses, control of acute HCMV infection, which coincided with expansion of a distinct NK-cell subset, was observed in a pediatric SCID patient in the absence of T cells (Kuijpers et al., 2008). Altogether, human and mouse studies have highlighted the key role of NK cells in controlling infections by herpesviruses, in particular by CMV.

2. NK-CELL ADAPTATIONS TO VIRAL INFECTIONS It is well established that viral infection can activate T-cell responses and generate immunological memory, which is able to protect the host against subsequent infections. The initial antigen encounter triggers activation of selected T-cell clones, which receive signal 1 by interaction of their TCR with its cognate peptide presented by MHC class I and signal 2 by costimulatory signals mediated through CD28 (Williams & Bevan, 2007). Signaling delivered by exposure to proinflammatory cytokines, referred to as signal 3, dictates the type of T-cell effector response elicited, and the amount of inflammation during T-cell priming can even influence the generation of effector and memory CD8+ T cells (Joshi et al., 2007; Pearce & Shen, 2007; Pipkin et al., 2010). Pathogen-specific T cells, which have coordinately received all three signals, undergo sustained clonal proliferation and acquire effector programs tailored to optimally clear the infection. The expansion phase is followed by a regulated contraction of the T-cell clones, which finally yields in the generation of a small pool of antigen-specific memory cells persisting throughout the life span of the host and maintaining the capacity to vigorously respond to pathogen reencounter (Althaus, Ganusov, & De Boer, 2007). In this paragraph, it will be discussed how human and mouse CMV can induce preferential expansion of a defined subset of NK cells and drive the acquisition of adaptive and even memory features.

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2.1 Skewing and Adaptation of NK-Cell Subsets After MCMV Infection Despite evident ontogenic and functional differences, the dynamics of the NK-cell response to CMV infection strikingly parallels the triphasic T-cell response described above. In-depth analyses of the NK-cell response to MCMV infection in C57BL/6 mice revealed that, after an initial stage characterized by a global activation of the entire NK-cell compartment driven by systemic proinflammatory cytokines, a subset of NK cells expressing the activating receptor Ly49H undergoes preferential expansion, followed by constant contraction, and then persistence as a pool of memory cells up to 2 months after infection (Dokun et al., 2001; Robbins, Tessmer, Mikayama, & Brossay, 2004; Sun, Beilke, & Lanier, 2009). Although the frequency of naive Ly49H+ NK cells (approximately 40–50% of the initial NK-cell repertoire in uninfected C57BL/6 mice) is much higher compared to antigen-specific T-cell precursors within the naive pool, Ly49H+ NK cells undergo a 2- to 3-fold expansion in the spleen and a roughly 10-fold increase in the liver 7 days after MCMV infection (Dokun et al., 2001). The dramatic proliferative potential of Ly49H+ NK cells has been highlighted by adoptive transfer of naive NK cells into DAP12- or Ly49H-deficient mice followed by MCMV infection. In this setting, Ly49H+ NK-cell numbers are increased 100-fold in the spleen and up to 1000-fold in the liver (Sun et al., 2009), demonstrating that MCMV infection can induce a dramatic skew of the NK-cell repertoire. The expansion phase of Ly49H+ NK cells is primarily dependent on the specific interaction between m157 and Ly49H, as both blockade of Ly49H by antibody treatment and infection with a MCMV mutant strain lacking the m157 protein abolish this response (Dokun et al., 2001). Paralleling the interaction of a TCR with its cognate antigen, the exquisitely specific recognition of infected cells by Ly49H and the resulting activation of a selected population of NK cells can be regarded as signal 1 for MCMV-induced memory NK cells (Fig. 1). Utilizing the above-mentioned adoptive transfer system, costimulatory signals and inflammatory cues required by Ly49H+ NK cells during the expansion phase after MCMV infection have been identified. Engagement of the activating receptor DNAM-1 on Ly49H+ NK cells cooperates together with Ly49H to trigger the initial proliferative burst, enabling sustained expansion of the Ly49H+ NK-cell subset and efficient differentiation into memory NK cells (Nabekura et al., 2014). Importantly, the DNAM-1 ligands poliovirus receptor (PVR, CD155) and Nectin-2 (CD122), which are constitutively expressed on hematopoietic

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CD11b+/– CD27+/– KLRG1+/– Ly6C+/– DNAM-1+/–

MCMV infection Signal 1 : m157 Signal 2 : DNAM-1L Signal 3 : IL-12 Conventional NK cell

CD57+/– NKG2A+/– sKIR+/– CD2+/– ILT2+/– NCR+ Siglec-7+ CD161+ Conventional CD7high NK cell

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CD11b+ CD27– KLRG1+ Ly6C+ DNAM-1–

Memory NK cell

HCMV infection Signal 1 : HLA-E? Antibodies? Signal 2 : CD58? Signal 3 : IL-12? Adaptive NK cell

CD57+ NKG2A– sKIR+ CD2+ ILT2+ NCR– Siglec-7– CD161– CD7low

Epigenetic remodeling

Fig. 1 Signals inducing the phenotype of adaptive NK cells after CMV infection. MCMV infection (first row) provides the viral antigen m157, ligands for costimulatory DNAM-1, as well as proinflammatory cytokines such as IL-12, which coordinately induces adaptive NK cells with an altered surface receptor phenotype. The contribution of epigenetic remodeling in driving the phenotype of murine adaptive NK cells after MCMV infection is still unclear. In vitro studies have recapitulated the potential ligands for activating and costimulatory receptors, which, in combination with IL-12, might contribute to induce the surface receptor signature and epigenetic remodeling displayed by adaptive NK cells after HCMV infection (second row).

and other host cells (Aoki et al., 1997; Nabekura et al., 2014; Ravens, Seth, Forster, & Bernhardt, 2003), are upregulated on dendritic cells and macrophages early after MCMV infection, implying synergistic recognition of virally infected cells by Ly49H+ NK cells (Nabekura et al., 2014). The requirement of DNAM-1 costimulation for proficient proliferation of Ly49H+ NK cells closely resembles the initiation of activation of T cells by signal 2 (Fig. 1). Whether ligands for Ly49H and DNAM-1 need to be provided simultaneously by the same infected cell and whether additional costimulatory molecules can similarly influence the primary and memory responses of Ly49H+ NK cells requires further investigation. Along this line, cognate ligand recognition of the activating receptor Ly49D can also enhance Ly49H+ NK-cell responses and memory formation in MCMV-infected mice, as it will be discussed in more detail in Section 5.2 (Nabekura & Lanier, 2016).

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In addition to providing cellular ligands for recognition by NK cells, MCMV infection creates an intense inflammatory milieu (Biron & Tarrio, 2015) through activation of different pattern-recognition receptors (Krug et al., 2004; Rathinam et al., 2010; Tabeta et al., 2004). Proinflammatory cytokines are crucial to control the viral infection as well as to expand and activate IFN-γ production and cytotoxicity in the entire NK-cell compartment in an unspecific fashion (Dokun et al., 2001; Nguyen et al., 2002). However, they also contribute to shape the specific Ly49H+ NK-cell response to MCMV. This concept is supported by the observation that IL-12 is essential for both the initial expansion and subsequent formation of memory NK cells (Sun et al., 2012). In addition, IL-12 and type I IFNs reshape the granule content by induction of granzymes and perforin, and thus enable Ly49H+ NK cells to efficiently mediate cytotoxicity against m157-expressing target cells (Nguyen et al., 2002; Parikh et al., 2015). Further cytokines including IL-18 and IL-33 sustain the primary response against MCMV, but do not impact on generation of memory (Madera & Sun, 2015; Nabekura, Girard, & Lanier, 2015). The molecular mechanisms underlying IL-12-dependent effects on expansion and generation of memory Ly49H+ NK cells after MCMV infection have started to be elucidated. Proinflammatory cytokine signaling induces early expression of the transcription factor Zbtb32 and of miR-155 in NK cells, which antagonize the repressor of proliferation Blimp-1 and the proapoptotic molecule Noxa, respectively (Beaulieu, Zawislak, Nakayama, & Sun, 2014; Zawislak et al., 2013). However, both Zbtb32 and miR-155 are equally upregulated in both Ly49H+ and Ly49H NK cells, indicating that in Ly49H+ NK cells, additional signals might drive preferential expansion or survival. These data delineate a complex host– pathogen interaction, which is not confined to a specific receptor–ligand interaction, but is markedly tuned by nonspecific global cytokine signals, which can be envisaged as signal 3 (Fig. 1). After the expansion phase, transferred Ly49H+ NK cells undergo a slow and constant contraction phase, returning to baseline or even below endogenous preinfection levels (Robbins et al., 2004; Schlub et al., 2011; Sun et al., 2009). However, unlike T cells, MCMV-experienced NK cells do not undergo biphasic decay after the response peak, a trait associated with T-cell memory formation (Schlub et al., 2011). Paralleling the generation of memory T cells, the contraction phase of Ly49H+ NK cells is regulated by the proapoptotic molecule Bim, which is also required for efficient differentiation into functionally competent memory NK cells (Min-Oo, Bezman, Madera, Sun, & Lanier, 2014). Moreover, autophagy has been

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implied as a mechanism enabling NK-cell memory generation, similar to observations in CD8+ T cells (Puleston et al., 2014; Xu et al., 2014). Activation of autophagy during the contraction phase after MCMV infection enables a small population of Ly49H+ NK cells to be rescued from cell death provoked by dysfunctional mitochondria accumulation during the expansion phase and to subsequently differentiate into memory NK cells (O’Sullivan, Johnson, Kang, & Sun, 2015). These findings point toward a potential selection for memory cells from the Ly49H+ NK-cell pool during the contraction phase, as reported for T cells. Two months after MCMV infection, Ly49H+ NK cells display an altered signature of surface receptors as shown by high KLRG1 and CD11b and by low CD27 and DNAM-1 expression. This phenotype is fully acquired already during the effector phase, 7 days after MCMV infection, and is shared by a large fraction of mature NK cells in noninfected mice, thus likely reflecting their terminal differentiation and proliferative history (Huntington et al., 2007; Kim et al., 2002). Additionally, supported by data from genome-wide transcriptional analysis, Ly6C and CD49a have been identified as differentially expressed between naive and memory Ly49H+ NK cells emerging 27 days after MCMV infection (Bezman et al., 2012). Interestingly, these markers are also preferentially expressed by CD8+ memory T cells (Bezman et al., 2012; Wherry et al., 2007), pointing toward a molecular response pattern possibly shared by pathogen-specific memory cells of the NK- and T-cell lineage. The phenotypic markers of memory Ly49H+ NK cells are summarized in Fig. 1. Altogether, these findings illustrate that the immune response against MCMV in C57BL/6 mice elicits the selective expansion of a defined NK-cell subset. The skewing of the NK-cell repertoire is directly driven by recognition of the pathogen in concert with substantial inflammatory signals and intrinsically regulated during the contraction phase, resulting in a dynamic adaptation to the viral infection.

2.2 Skewing and Adaptation of NK-Cell Subsets After HCMV Infection Similar to the remarkable interaction of MCMV with mouse NK cells, HCMV exhibits a pronounced impact on the NK-cell compartment in humans. Pioneering studies indicated a skew of the repertoire in healthy HCMV-seropositive compared to seronegative individuals, illustrated by an increased average frequency of NK cells expressing the activating receptor NKG2C (Guma et al., 2004). Similar to Ly49H in mouse, NKG2C

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is a member of the C-type lectin superfamily and associates to the adaptor protein DAP12 (Lanier, Corliss, Wu, & Phillips, 1998). NKG2C forms heterodimers with CD94 (Lazetic, Chang, Houchins, Lanier, & Phillips, 1996) and binds to the nonclassical MHC class I molecules HLA-E bound to HLA-E-stabilizing peptides (Braud et al., 1998). Along with the detection of elevated frequencies of NKG2C+ NK cells in HCMV-seropositive individuals, Guma and colleagues also described an altered receptor expression pattern on this NK-cell population consisting of reduced expression of NKG2A (the inhibitory receptor counterpart of NKG2C), NKp30, and NKp46 as well as increased expression of the inhibitory ILT2 (LIR-1a). Conversely, other receptors such as NKG2D were expressed at comparable levels (Guma et al., 2004). More recently, extensive characterization of NKG2C+ NK cells from HCMV-seropositive individuals revealed further phenotypic hallmarks of this subset, such as preferential expression of the activating receptor CD2 together with reduced expression of the inhibitory receptor Siglec-7 as well as low expression of CD161 and CD7 (Beziat et al., 2012, 2013). Importantly, NKG2C+ NK cells from HCMV-seropositive individuals display a strikingly dominant expression of the terminal differentiation marker CD57 and of KIRs specific for self-MHC class I molecules (sKIRs), suggestive of clonal or oligoclonal expansion (Beziat et al., 2012, 2013) and due to their extensive adaptation to HCMV infection are referred to as adaptive NK cells. The phenotypic hallmarks of human adaptive NK cells are illustrated in Fig. 1. The direct link between HCMV infection and expansion of NKG2C+ NK cells was obtained from consecutive studies delineating a coordinated response consisting of preferential proliferation of NKG2C+ NK cells during acute HCMV infection in patients undergoing transplantation (Foley et al., 2012; Lopez-Verges et al., 2011). Expression of CD57 and sKIRs appears to represent typical phenotypic adaptations of NKG2C+ NK cells induced by HCMV since NKG2C+ CD57+ sKIR+ NK cells accumulate during acute infection in vivo (Foley et al., 2012; Lopez-Verges et al., 2011). Expansion of human NKG2C+ NK cells is followed by a stable persistence phase in the absence of detectable viremia (Foley et al., 2012; Lopez-Verges et al., 2011), which does not resemble the contraction phase observed in Ly49H+ NK cells after MCMV infection. In some healthy HCMV-seropositive individuals, the adaptive NKG2C+ NK-cell compartment can persist in high frequencies for years (Beziat et al., 2013; Luetke-Eversloh, Hammer, et al., 2014). Potential subclinical or

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tissue-specific reactivation events of HCMV during its life-long latent infection might contribute to maintain the NKG2C+ NK-cell pool. A more detailed follow-up of the ex vivo dynamics of NKG2C+ NK-cell response during and after HCMV acute infection will help to clarify the in vivo dynamics and behavior of human adaptive NK cells. Exploiting the preferential expression of sKIR as a strategy to identify HCMV-induced subsets of NK cells led to the major finding that HCMV can drive the expansion of NK cells in the absence of NKG2C expression (Beziat et al., 2013, 2014; Della Chiesa et al., 2014). As such, NK-cell subsets which do not express NKG2C, but share the functional and surface receptor signature (NKG2A NCR CD161 CD57+ LIR-1a+ CD2+ Siglec-7 CD7low sKIR+) with adaptive NKG2C+ NK cells, have been detected in HCMV-seropositive donors (Beziat et al., 2013). Intriguingly, some of these NKG2C HCMV-induced NK-cell populations express activating KIRs, implying a potential role of other receptors than NKG2C in the recognition of and response to HCMV infection (Beziat et al., 2013). Analysis of patients undergoing umbilical cord blood transplantation with grafts carrying a homozygous deletion for the NKG2C gene (KLRC2) revealed that adaptive NK cells expressing activating KIRs emerge and mature during acute HCMV infection (Della Chiesa et al., 2014). Additionally, a study focusing on HCMV-seropositive KLRC2-deficient healthy individuals conclusively demonstrated that adaptive NK cells can be generated in the absence of NKG2C and are phenotypically and functionally similar to their NKG2Cexpressing counterparts, indicating alternative generation pathways, which result in identical cellular programs (Liu et al., 2016). The detection of adaptive NK-cell responses independent of NKG2C, and even in the absence of the gene encoding NKG2C, is in line with previous observation of mouse strain-dependent differences in Ly49H expression. Ly49H-deficient mouse strains displaying NK cell-mediated MCMV resistance have also been described (Adam et al., 2006; Desrosiers et al., 2005; Kielczewska et al., 2009; Pyzik et al., 2011). In these strains, other Ly49 activating receptors such as Ly49P or Ly49L can recognize MCMV-infected cells in an MHC class I (H-2) and MCMV-m04-dependent fashion. Interestingly, specific expansion of Ly49L+ NK cells during MCMV infection was reported in BALB.K mice, in which the frequency of this fraction increased from 15% in uninfected mice up to 40% 6 days postinfection (Pyzik et al., 2011). Whether Ly49P+ or Ly49L+ NK cells expanded during MCMV infection display similar phenotypic and functional memory properties as the Ly49H+ NK cells still needs to be established.

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In addition to data from patients with acute viremia, mechanistic studies analyzing the generation and maintenance of adaptive NKG2C+ NK cells mainly employed reductionist in vitro models to enable modification of experimental parameters. Such studies have shown the preferential proliferation of NKG2C+ NK cells in coculture systems with HCMV-infected fibroblasts (Guma, Budt, et al., 2006; Rolle et al., 2014). Importantly, NKG2C and its ligand HLA-E are involved in driving this proliferation, as blockade of both CD94/NKG2C and HLA-E as well as shRNAmediated knockdown of HLA-E impaired the relative accumulation of NKG2C+ NK cells (Guma, Budt, et al., 2006; Rolle et al., 2014). Although it has been demonstrated that both NKG2A and NKG2C can recognize HLA-E bound to peptides derived from HCMV-gpUL40 (Tomasec et al., 2000), a role for gpUL40 in driving expansion of NKG2C+ NK cells after in vitro infection has not been recognized (Guma, Budt, et al., 2006; Rolle et al., 2014). Due to the undefined recognition of HCMV-infected cells by NKG2C, it still remains unclear whether it may function as signal 1 or signal 2 for the expansion of adaptive NK cells. Paralleling the data in the murine system, monocyte-derived IL-12 was required for efficient expansion of NKG2C+ NK cells in response to HCMV-infected fibroblasts (Rolle et al., 2014). The importance of monocytes in the expansion of adaptive NK cells is supported by data obtained from SCT patients, in which the number of monocytes at the time of HCMV reactivation correlated positively with the frequency of NKG2C+ NK cells 6–12 months after infection (Cichocki et al., 2016). The putative signals driving in vitro expansion of human adaptive NK cells are depicted in Fig. 1. Overall, identification of a phenotypic imprint in the NK-cell compartment induced by acute HCMV infection represents a central finding in human NK-cell biology and serves as the foundation to study the intense interaction of HCMV with human NK cells, which profoundly reshapes the NK-cell receptor repertoire.

3. FUNCTIONAL IMPRINTING OF ADAPTIVE NK CELLS The critical role of adaptive NK cells for viral control is highlighted by the seminal observation that Ly49H is responsible for MCMV resistance in C57BL/6 mice and following studies, as discussed in Section 1.2. In humans, although control of acute HCMV infection in a pediatric SCID patient was associated with a typical expansion of NKG2C+ adaptive NK cells (Kuijpers et al., 2008), the protective role of such adaptive NK cells in humans remains

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elusive. Even analysis of NKG2C-deficient individuals as control group cannot be helpful since expansions of NKG2C adaptive NK cells emerge (Liu et al., 2016). Independent of the clinical outcome, expansion and persistence of adaptive NK cells during CMV infection results in discrete functional consequences, both at the single cell and at the compartment level, which will be discussed in detail in this paragraph.

3.1 Functional Properties of MCMV-Induced Memory Ly49H+ NK Cells Two months after MCMV infection, memory Ly49H+ NK cells maintain enhanced functional properties: they display elevated degranulation and IFN-γ production upon cross-linking of the activating receptor NK1.1 or of Ly49H itself compared to Ly49H+ NK cells from noninfected hosts (Sun et al., 2009), which parallels the increased functionality of pathogenspecific memory cells of the adaptive immune system (Lohning, Richter, & Radbruch, 2002). In line with their potent effector functions, transfer of memory Ly49H+ NK cells from MCMV-infected animals mediates protection against a subsequent infection with MCMV at lower cell number than transfer of naive Ly49H+ NK cells from uninfected mice (Sun et al., 2009). By performing a series of comparative experiments using mouse models of cytokine-driven inflammation, Min-Oo and Lanier demonstrated that MCMV-induced memory Ly49H+ NK cells are less responsive to the proinflammatory cytokines IL-12 and type I IFNs in vitro: compared to naive Ly49H+ NK cells, treatment with proinflammatory cytokines results in reduced phosphorylation of signaling molecules downstream of the cytokine receptors as well as decreased IFN-γ production, possibly reflecting the extensive pathogen-driven terminal differentiation of memory Ly49H+ NK cells (Min-Oo & Lanier, 2014). However, in the presence of m157 on target cells, IL-12 is still capable of synergizing with cross-linking of Ly49H by costimulating IFN-γ production, suggesting that mouse memory NK cells have not completely lost responsiveness to proinflammatory signals (Min-Oo & Lanier, 2014).

3.2 Functional Properties of HCMV-Induced Adaptive NKG2C+ NK Cells In humans, the activation properties and effector functions of adaptive NKG2C+ NK cells have been thoroughly studied ex vivo. In line with murine data, a key feature of their functional responsiveness is a shift away

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from proinflammatory cytokine cues and toward vigorous activation by engagement of selected receptors by target cells. Likewise, when stimulated with the classical combination of dendritic cell-derived IL-12 and IL-18, adaptive NK cells produce IFN-γ at very low frequencies compared to conventional NK cells (Beziat et al., 2012). Further studies demonstrated an association between expression of PLZF and responsiveness to IL-12 and IL-18 in adaptive NK cells and proposed that loss of PLZF expression, which controls expression of IL-12R and IL-18R in murine NKT cells (Gleimer, von Boehmer, & Kreslavsky, 2012), might underlie the altered activation strategy of these cells (Schlums et al., 2015). However, loss of IL12RB2 transcripts or of STAT4 phosphorylation is also a typical feature of terminally differentiated human conventional NK cells (Bjorkstrom et al., 2010; Luetke-Eversloh, Cicek, et al., 2014; Yu et al., 2010), which generally express PLZF (Schlums et al., 2015). Initial studies of HCMV reactivation in patients undergoing hematopoietic stem cell (HSC) transplantation included the description of robust IFN-γ production after stimulation with MHC class I-deficient leukemia target cells (Foley et al., 2012). However, education via expression of self MHC-specific KIR is a prerequisite for preferential activation in this setting (Foley et al., 2012). Direct comparison of equally differentiated (NKG2A and CD57+) and educated NKG2C+ NK cells from HCMVseronegative and HCMV-seropositive healthy individuals did not reveal an elevated capacity for IFN-γ production in adaptive NKG2C+ NK cells in response to classical MHC class I-negative targets (Luetke-Eversloh, Hammer, et al., 2014). As previously discussed, one peculiarity of adaptive NKG2C+ NK cells from HCMV-seropositive individuals is the downregulation of major activating receptors, namely NKp30 and NKp46 (Guma et al., 2004), which generally contribute to triggering cytotoxicity and cytokine production in response to several MHC class I-negative target cells (Moretta et al., 2001). Despite the lack of major differences in functionality against HLA-deficient target cells, engagement of their signature activating receptor NKG2C by HLA-E-expressing target cells potently activates adaptive NKG2C+ NK cells and leads to polyfunctional responses characterized by degranulation as well as TNF and IFN-γ production (Beziat et al., 2012). Importantly, cross-linking of NKG2C alone is even sufficient to drive IFN-γ production in adaptive NKG2C+ NK cells (Liu et al., 2016; Luetke-Eversloh, Hammer, et al., 2014), thereby violating the previously described minimal requirements of NK-cell activation (Bryceson,

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Ljunggren, & Long, 2009) and suggesting a restricted specificity toward selected stimuli. Apart from NKG2C as a key activation pathway, adaptive NK cells can be efficiently stimulated by cross-linking of CD16, for instance by recognition of antibody-coated target cells, to drive ADCC (Beziat et al., 2012). By exploiting the specificity of the antibody, adaptive NKG2C+ NK cells can react against any immunogenic epitope. This notion is supported by the CD16-mediated activation of adaptive NK cells against HCMV-, HSV-, and Influenza-infected cells in the presence of antibodies specific for the corresponding pathogens (Costa-Garcia et al., 2015; Hwang et al., 2012; Lee et al., 2015; Zhang, Scott, Hwang, & Kim, 2013). Since CD16 is equally expressed by conventional and adaptive NK cells, the higher functional response of adaptive NK cells seems to underlie intrinsic differences. Associated signaling molecules downstream of the receptor were implicated to function as molecular switches for elevated activating signals. Human CD16 can pair with ITAM-bearing homodimers of FcεRγ or CD3ζ or with heterodimers of FcεRγ and CD3ζ. Notably, the number of activation motifs differs between signaling adaptors: FcεRγ contains one ITAM domain, whereas CD3ζ contains three. As a direct consequence, engagement of CD16 might induce distinct quantitative levels of ITAM-mediated signaling and result in discrete activation levels dependent on the signaling adapters associated to CD16. CD3ζ is expressed by all human CD56dim NK cells, while FcεRγ is partially downregulated in adaptive NK cells (Hwang et al., 2012; Lee et al., 2015; Schlums et al., 2015; Zhang et al., 2013). It is perceivable that in FcεRγ adaptive NK cells, CD16 exclusively associates with CD3ζ homodimers containing a total of six ITAMs, which could mediate increased downstream signaling compared to FcεRγ homodimers with only two ITAMs. As such, expression of signaling adaptor molecules rather than expression of receptors might fine-tune the preferential activation of adaptive NK cells. However, not all adaptive NK cells display deficient expression of FcεRγ (Schlums et al., 2015). Moreover, only IFN-γ and TNF production, but not degranulation, is more pronounced in adaptive compared to conventional NK cells after CD16 triggering, suggesting additional regulatory mechanisms (Liu et al., 2016; Schlums et al., 2015). Another functional feature associated to the loss of signaling adapters is diminished degranulation of adaptive NK cells in response to activated CD4+ T cells, which might impact

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on the regulation of immune responses by NK cells (Schlums et al., 2015). In quest of synergistic activation pathways in driving effector functions of adaptive NK cells, Liu and colleagues identified CD2, which is abundantly expressed on NKG2C+ and NKG2C adaptive NK cells, as crucial mediator of costimulation (Liu et al., 2016). Concomitant engagement of CD16 and CD2 yields in enhanced phosphorylation of signaling molecules in the cascade downstream of CD16 and, consequently, results in increased frequencies of IFN-γ- and TNF-producers among adaptive NK cells (Liu et al., 2016). The synergy for cytokine production is also present in conventional NK cells, but to a considerably lesser extent (Liu et al., 2016). Cross-linking of CD2 also potently costimulates activation through NKG2C, thus bridging the two major activation pathways of adaptive NK cells (Liu et al., 2016). The ligand of CD2, CD58 (also termed LFA-3a), is abundantly expressed by various tissues (Smith & Thomas, 1990) and upregulated by HCMV-infected fibroblasts (Rolle et al., 2016), further emphasizing how adaptive NKG2C+ NK cells rely on selected signals to orchestrate their activation, which might be predominantly directed against HCMV-infected host cells. In summary, adaptive NK cells are not necessarily more functional compared to equally differentiated and educated NK cells in response to targetcell recognition. Their peculiar functionality lies in a more selective recognition repertoire compared to conventional NK cells, with a reduced ability to respond to bystander activation by proinflammatory cytokines or engagement of multiple activating receptors. Adaptive NK cells rather rely on single receptors such as Ly49H, in case of adaptive NK cells from B6 mice infected by MCMV strains expressing m157, or such as NKG2C and CD16, as in the case of human adaptive NK cells. Although clearly utilizing distinct recognition tools, the increased specificity together with the partial loss of B-cell/ myeloid signaling adaptors (at least observed in humans) parallels memory T cells, and points toward an adaptation of a specific subset of these innate effectors to counterbalance HCMV infection. Moreover, enhanced functional competence of adaptive NK cells in response to these selected stimuli is, at least in humans, mostly confined to cytokine production rather than to cytotoxicity. The potential mechanisms explaining this phenomenon are more extensively discussed in the next paragraph. The different recognition strategies and consequent effector functions displayed by conventional and adaptive human NK cells are depicted in Fig. 2.

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Fig. 2 Functional properties of human adaptive NK cells derived from HCMVseropositive individuals. Human conventional and adaptive NK cells can be activated to produce effector molecules by various stimuli including DC-derived proinflammatory cytokines, MHC class I-deficient target cells, HLA-E-expressing target cells, or antibodycoated target cells. Differential expression of receptors and epigenetic modifications underlie the varying responsiveness of NK-cell subpopulations to selected stimuli. “ ” indicates low or no production; “+” intermediate production; “++” high production; and “+++” very high production. CD107 detects degranulation and serves as an indirect measurement of cytotoxicity.

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4. EPIGENETIC REMODELING AS HALLMARK OF IMMUNE TRAINING AND MEMORY Since the first description of the “epigenotype” as a complex connection of genotype and phenotype in Drosophila more than 70 years ago (Waddington, 1942), regulation of gene expression beyond the DNA sequence level has emerged as the pioneering field of epigenetics. In cellular biology, global changes in epigenetic landscapes are most recognized during programmed differentiation from progenitor/precursor cells into their respective progeny. Epigenetic reprogramming is also an adaptive mechanism employed by T cells to switch different effector programs on or off and to differentiate into memory cells (Wilson, Rowell, & Sekimata, 2009). By stable imprinting of these programs into their progeny, memory T cells are able to “remember” pathogens previously encountered and display a faster and more robust response upon rechallenge (Balasubramani, Mukasa, Hatton, & Weaver, 2010). More recently, it became clear that primitive organisms, which lack adaptive immunity, as well as cells of the innate immune system in mammals, can specifically sense and adapt to the environment by displaying a qualitatively and quantitatively different secondary response (Hamon & Quintin, 2016; Milutinovic & Kurtz, 2016; Reimer-Michalski & Conrath, 2016). This phenomenon, also termed trained immunity, has been studied especially in monocytes/macrophages and is largely mediated by epigenetic modifications induced by recognition of pathogens or of pathogenassociated molecular patterns (PAMPs) during the primary response (Netea et al., 2016). In a seminal study using a model of LPS-induced tolerance, Foster et al. showed that macrophages undergo epigenetic modifications associated with silencing of genes coding for inflammatory molecules, while priming other genes coding for antimicrobial molecules (Foster, Hargreaves, & Medzhitov, 2007). This report was followed by further studies demonstrating that exposure of monocytes and macrophages to C. albicans, β-glucan, or Bacille Calmette-Guerin (BCG) induces reprogramming of chromatin marks and a more robust responses after challenge with unrelated pathogens or PAMPs (Kleinnijenhuis et al., 2012; Quintin et al., 2012; Saeed et al., 2014). Monocyte and macrophage training has also been observed in response to parasites (Chen et al., 2014) and viruses (Barton et al., 2007), although it remains to be elucidated whether these phenomena are also epigenetically regulated.

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4.1 Epigenetic Imprinting of Human Adaptive NK Cells The thorough analysis of the epigenome of human adaptive NK cells has recently revolutionized our understanding of how adaptive features are controlled in innate NK cells. Different groups independently reported that adaptive NK cells derived from HCMV-seropositive individuals undergo a global epigenetic reprogramming as shown by extensive changes in DNA methylation, similar to differentiation from naive to memory CD8+ and CD4+ T cells (Luetke-Eversloh, Hammer, et al., 2014; Schlums et al., 2015). The surface receptor signature of adaptive NK cells is not only reflected at the transcriptome but also at the DNA methylome level: the loci encoding for CD2 and ILT2 are hypomethylated in adaptive NK cells, whereas the transcriptional start sites of the genes encoding NKG2A, CD7, and CD161 display increased DNA methylation compared to conventional NK cells (Schlums et al., 2015). Likewise, the expression of the signaling adaptor proteins SYK, FcεRγ, and EAT-2 as well as of the transcription factor PLZF, which are downregulated in subsets of adaptive NK cells, is silenced by DNA methylation of the corresponding promoter regions (Lee et al., 2015; Schlums et al., 2015). Notably, PLZF can modulate transcriptional activity by recruiting histone deacetylases (Sadler et al., 2015), and thus potentially contributs to a regulatory network orchestrating epigenetic alterations in adaptive NK cells (Schlums et al., 2015). Therefore, the previously recognized stable imprint of HCMV infection on the NK-cell repertoire is epigenetically controlled, which can explain the long-term stability of the phenotype of adaptive NK cells. One of the major epigenetic hallmarks of adaptive NK cells is at the level of cytokine genes, as shown by significant hypomethylation at both IFNG and TNF loci (Luetke-Eversloh, Hammer, et al., 2014; Schlums et al., 2015). In the adaptive immune system, CD4+ T helper and CD8+ cytotoxic T cells possess memory for key cytokines, and this phenomenon is strictly controlled by epigenetic mechanisms (Balasubramani et al., 2010). To enable rapid and robust expression, IFN-γ-competent memory CD4+ Th1 and CD8+ T cells display an open configuration at the IFNG gene, both at the promoter and at conserved noncoding sequences (CNSs), which act as transcriptional enhancers. CNS1, located 4 and 6 kb upstream of the transcriptional start site in humans and mice, respectively, contains NFATbinding sites and, thus, is particularly functional in enhancing IFNG transcription upon engagement of the TCR, which drives NFAT

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dephosphorylation in T cells (Agarwal, Avni, & Rao, 2000; Balasubramani et al., 2010; Kiani et al., 2001; Lee, Avni, Chen, & Rao, 2004; Shnyreva et al., 2004). High-resolution analysis of DNA methylation revealed that human adaptive NK cells have lost repressive DNA methylation marks at the six CpG residues within the IFNG CNS1, while conventional NK cells display complete methylation of the enhancer region independent of their differentiation state (Liu et al., 2016; Luetke-Eversloh, Hammer, et al., 2014). Accordingly, hypomethylated CNS1 enhances IFNG transcriptional activity in NK cells, in response to stimulation with NKG2C (LuetkeEversloh, Cicek, et al., 2014). Consequently, IFNG CNS1 accessibility in adaptive NK cells could provide a molecular mechanism underlying potent IFN-γ production upon single engagement of NKG2C or CD16. Due to HCMV-induced downregulation of NCRs, CD16 and the DAP12-associated NKG2C receptor might represent two major stimuli, which can induce NFAT dephosphorylation in adaptive NKG2C+ NK cells, thus enabling CNS1 to enhance IFNG transcription. Similar mechanisms might apply for the TNF locus, which has also been shown to display a more open configuration in adaptive NK cells (Luetke-Eversloh, Hammer, et al., 2014; Schlums et al., 2015). Comparable to phenotypic features, the cytokine memory of adaptive NK cells is stably imprinted in adaptive NKG2C+ NK cells, as IFNG CNS1 accessibility is invariable over time (LuetkeEversloh, Hammer, et al., 2014). The signals driving the epigenetic reprogramming in adaptive NK cells have only been started to be elucidated. In vitro, priming by inflammatory cytokines IL-12, IL-18 (or IL-1), and IL-15 is able to induce demethylation of the IFNG CNS1 in human naive NK cells to a similar extent displayed by HCMV-induced adaptive NK cells (Luetke-Eversloh, Hammer, et al., 2014). Interestingly, it was previously proposed that cytokine priming can induce memory-like properties in NK cells in vitro and in vivo, as illustrated by enhanced and stable IFN-γ production after cytokine or activating receptor restimulation (Cooper et al., 2009; Romee et al., 2012). Epigenetic remodeling of the IFNG and possibly of other loci represents a potential molecular mechanism underlying this phenomenon. Thus, it is possible that IL-12 and other proinflammatory cytokines play an important role in the establishment of epigenetic reprogramming in adaptive NK cells after HCMV infection. This would suggest that molecular regulation of CMV- and cytokine-induced NK-cell memory might be overlapping. However, although inducible in vitro, epigenetic modifications of the IFNG locus are detectable ex vivo selectively in adaptive NK cells from

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HCMV-seropositive donors. This finding suggests that additional signals such as engagement of NKG2C or CD16 might be required in order to gain stable epigenetic modifications or to enable the survival and persistence of the adaptive NK-cell pool. Overall, the vast majority of properties defining the identity of human adaptive NK cells are epigenetically imprinted. DNA methylation likely underlies the stable phenotype of highly specialized adaptive NK cells, but key regulators of the acquisition and maintenance of the alterations in the epigenome warrant further investigation.

4.2 Possible Role of Epigenetic Remodeling for NK-Cell Memory in MCMV The definitive role of epigenetic mechanisms in the control of murine NK-cell responses against MCMV remains to be elucidated, but the phenotypic and functional signature of MCMV-induced memory Ly49H+ NK cells as well as their persistence and stable phenotype in the absence of antigenic signals implies long-term alterations in regulatory networks. Such features are indicative of regulation beyond the proteome and transcriptome levels and likely reflect stably imprinted epigenetic programs specifically tailored to combat MCMV throughout the host’s life span. Interestingly, Tarrio et al. have recently shown that during MCMV infection, NK cells undergo histone modifications for opening up the IL-10 (Il10) gene, while no major differences could be observed within the Ifng locus regions analyzed (Tarrio et al., 2014). However, changes in DNA methylation at Ifng enhancer regions have not been analyzed in this study. Recent data demonstrate that Ifng CNS1 is largely methylated in naive NK cells and that cytokine priming induces extensive CNS1 demethylation (Ni et al., 2016), as it has been shown for human NK cells (Luetke-Eversloh, Hammer, et al., 2014). In light of these findings, it would be of great interest to study whether similar epigenetic reprogramming is also occurring after MCMV infection in vivo and to clarify the relative contribution of proinflammatory cytokines and of Ly49H engagement in this phenomenon.

5. TRAINING AND CROSS-REACTIVE MEMORY: CONSEQUENCES FOR HETEROLOGOUS IMMUNITY In this paragraph, we would like to discuss whether and how priming and imprinting of NK cells during a primary infection can drive heterologous immunity. The term heterologous immunity refers to immunity that

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can develop to one pathogen after a host has had exposure to nonidentical pathogens (Welsh, Che, Brehm, & Selin, 2010). This concept explains previous seminal data of acquired resistance to heterologous pathogens (Mackaness, 1964) and is supported by epidemiologic and immunological studies on heterologous effects of vaccines, especially BCG, toward unrelated pathogens (Jensen, Benn, & van Crevel, 2016). With this broad definition, heterologous immunity includes both the epigenetically driven training of the innate immune system discussed in the previous paragraph and effects mediated by adaptive lymphocytes. In trained immunity, the increased responsiveness to secondary stimuli achieved by macrophages after pathogen encounter is not specific for a particular pathogen (Netea et al., 2016); it is therefore implied in its own definition that innate training leads to heterologous immunity. Unlike macrophages, immunological memory of T cells is antigen-specific and preferentially reactivated after rechallenge with the same pathogen. Nonetheless, several lines of evidence support the concept that T cells directly contribute to heterologous immunity. The underlying molecular mechanisms can be various: (1) pathogen-induced cytokines such as IL-12 and IL-18 may activate memory T cells independent of TCR engagement; (2) the TCR of antigen-specific T cells may directly cross-react with a different pathogen; (3) induction of type I IFNs during infection may upregulate host MHC and self-antigens and thereby provide signals sensitizing self-reactive T cells to participate in immune responses (Welsh et al., 2010).

5.1 NK-Cell Training and Responses to Heterologous Pathogens Can NK cells, which have experienced previous pathogen encounter, mediate heterologous immunity? In a general term, several studies support this concept as it has been extensively shown in vivo and in vitro that NK cells require priming to acquire functional competence. Resting NK cells from specific pathogen-free (SPF) laboratory mice display poor effector functions directly ex vivo. Efficient cytotoxicity by these cells requires priming from APC-derived cytokines, such as type I IFNs, IL-15 trans-presentation, IL-12, and IL-18, which among other potential mechanisms, induce the translation of perforin and granzyme B mRNA (Biron & Tarrio, 2015; Chaix et al., 2008; Fehniger et al., 2007; Lucas, Schachterle, Oberle, Aichele, & Diefenbach, 2007). The duration and the intensity of exposure to proinflammatory stimuli required for priming NK cells at steady state or during infections have not been elucidated in detail but might differentially

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impact on the differentiation and/or adaptation of the NK-cell pool. In steady state, priming of NK cells is severely compromised in germ-free mice, in which intestinal mononuclear phagocytes fail to induce expression of inflammatory response genes, including type I IFNs, due to the absence of epigenetic remodeling mediated by postnatally colonizing microbiota (Ganal et al., 2012). Furthermore, germ-free mice display diminished immunity against MCMV infection (Ganal et al., 2012), suggesting that reduced innate training can collectively impact on the NK-cell activation status and antiviral immunity. Experimental models of persistent viral infections have shown that low but chronic levels of proinflammatory cytokines induce durable activation of NK cells. NK cells derived from mice infected with the lymphocytic choriomeningitis virus Armstrong strain display prolonged enhanced cytotoxic activity, lasting for at least 6 months (Bukowski, Biron, & Welsh, 1983). Similarly, NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) display higher expression of granzyme B as well as enhanced cytotoxicity and IFN-γ production (Barton et al., 2007). Latently infected mice from both studies show increased resistance to tumor-cell implants, and this resistance was proven to be NK cell-mediated, at least in MuHV-4 infection, providing a proof of principle for NK cell-mediated heterologous immunity (Barton et al., 2007; Bukowski et al., 1983). Exposure of NK cells to proinflammatory stimuli does not need to be persistent in order to induce detectable effects on their effector functions. Short-term in vitro priming of murine and human NK cells by proinflammatory cytokines can induce longlasting effects on cytotoxic as well as cytokine production capacity, which are detectable up to 1 month after in vivo transfer into immunodeficient mice (Cooper et al., 2009; Romee et al., 2012). In line with these observations, vaccination of healthy volunteers with BCG (Kleinnijenhuis et al., 2014), yellow fever virus (YFV) (Marquardt et al., 2015), or Influenza virus (Goodier et al., 2016) primes human NK cells in vivo, which translates into increased cytokine production following ex vivo restimulation with either the challenging or other unrelated stimuli. In the case of BCG and Influenza virus vaccination, the effect is evident up to 4 months after priming. Exposure to commensals, pathogens, or vaccines might also contribute to the heterogeneity of maturity stages observed in the conventional NK-cell pool of healthy individuals (Bjorkstrom et al., 2010; Juelke et al., 2010; Yu et al., 2010). Since IL-15, which can prime NK cells in vivo, is produced in steady state as well as during inflammation, it is challenging to establish to what extent the adaptations observed in the peripheral NK-cell pool are part of

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a programmed differentiation process or rather occur in response to environmental stimuli. These events might not be mutually exclusive and cytokines produced under homeostatic conditions can concur with those produced in response to external stimuli to shape maturation and functional competence of the NK-cell pool. In this broad view, delivery of tolerogenic signals by repetitive stimulation via activating receptors in the absence of counterbalanced inhibition, as postulated by the disarming model (Raulet & Vance, 2006), can also be considered as an innate adaptation or training. Whether this mechanism is regulated by transcriptional and epigenetic reprogramming of hyporesponsive disarmed NK cells is still unclear. Altogether, the alterations induced in NK cells by exposure to various pathogens or commensals or after vaccination are likely triggered by proinflammatory cytokines produced by APC or other accessory cells. The acquired functional changes can last several months and are possibly associated with in part transient, in part stable adaptations. In this sense, priming of NK cells by cytokines closely resembles training of macrophages and, due to NK cell–macrophage cross-talk, these two phenomena are possibly linked and synergize to mediate innate heterologous immunity. Indeed, the role of IFN-γ produced by NK cells during infections in priming and shaping monocyte differentiation has been extensively documented (Askenase et al., 2015; Coombes, Han, van Rooijen, Raulet, & Robey, 2012; Goldszmid et al., 2012; Kang, Liang, Reizis, & Locksley, 2008).

5.2 NK-Cell Cross-Reactive Memory and Heterologous Immunity Adaptations of NK cells in response to CMV infection are more complex than training induced by cytokine priming only. As Ly49H+ NK cells display fine antigen specificity, it would be reasonable to predict that NK cellmediated heterologous immunity might also rely on mechanisms of direct or indirect cross-reactivity, as enumerated for T cells. Are there evidences of heterologous immunity mediated by CMV-specific adaptive NK cells? Barton et al. have demonstrated that persistent infection with MCMV confers protection toward following bacterial infection with Listeria and Yersinia pestis. Importantly, protection is associated with production of IFN-γ from an unclear cellular source and systemic activation of macrophages (Barton et al., 2007). However, MCMV-induced memory Ly49H+ NK cells are unlikely to mediate this effect, as they do not display enhanced activation or proliferation after infection with Listeria, as compared to naive Ly49H+ or Ly49H NK cells (Min-Oo & Lanier, 2014). As discussed in Section 3.1, this phenomenon is likely due to low expression of cytokine receptors

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and therefore reduced susceptibility to cytokine stimulation of adaptive Ly49H+ compared to naive NK cells (Min-Oo & Lanier, 2014). If NK cells played any role in MCMV-induced resistance to Listeria infection (Barton et al., 2007), conventional rather than adaptive NK cells might be preferentially responsible for this effect. Human data indirectly confirmed this observation by showing that the increased ex vivo NK-cell baseline response induced after Influenza virus vaccination is higher in HCMV-seronegative individuals than in HCMV-seropositive ones, in which adaptive NK cells are accumulating (Goodier et al., 2016). These data are in line with conserved downregulation of cytokine receptors (and therefore lower activation) between murine and human adaptive NK cells. Downregulation of cytokine receptors by adaptive NK cells can represent a mechanism to minimize bystander activation or heterologous immunity by adaptive NK cells. However, this could decrease the ability of the NK-cell pool to mediate cross-resistance to subsequent infections. To date, m157 is the only pathogen-derived antigen recognized by Ly49H. Thus, there is no evidence of direct Ly49H cross-reactivity between different pathogens, although such mechanisms can in principle not be excluded. Unlike other activating Ly49 receptors, Ly49H does not efficiently bind to self H-2 alleles in B6 mice (Nakamura & Seaman, 2001), suggesting that in this case cross-reactivity between viral and self-antigens might be an unlikely event. In line with this concept, engagement of self H-2 molecules even in the presence of proinflammatory cytokines provided by a subsequent heterologous infection is not sufficient to trigger expansion and activation of memory Ly49H+ NK cells (Min-Oo & Lanier, 2014). The situation might be different for the activating receptor Ly49D which recognizes the MHC class I molecule H-2Dd (George, Mason, Ortaldo, Kumar, & Bennett, 1999). Although Ly49D+ NK cells in H-2Dd mice are hyporesponsive when assayed ex vivo (Nabekura & Lanier, 2016) and do not mediate autoimmunity (George et al., 1999), Ly49D recognition of H-2Dd (as self-antigen) can augment IFN-γ production by Ly49H+ NK cells in an acute phase of MCMV infection and thereafter mediate preferential differentiation of memory Ly49H+ Ly49D+ NK cells, with an impact on host defense against MCMV (Nabekura & Lanier, 2016). However, the pathogen-derived ligand of Ly49D is not known. Thus, NK cellactivating receptors for self-MHC class I can contribute to the immune response in the presence of high inflammatory signals and could potentially mediate cross-reactive recognition between viral and self-antigens. While Influenza, YFV vaccination, as well as HSV-2- and EBVpersistent infections fail to do so (Bjorkstrom, Svensson, Malmberg,

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Eriksson, & Ljunggren, 2011; Goodier et al., 2016; Hendricks et al., 2014; Marquardt et al., 2015), selected infections by hantavirus (Bjorkstrom, Lindgren, et al., 2011), chikungunya virus (Petitdemange et al., 2011), chronic Hepatitis virus B and C (Beziat et al., 2012), and human immunodeficiency virus (Guma, Cabrera, et al., 2006) preferentially activate or expand the human adaptive NKG2C+ NK-cell pool generated after HCMV infection. The molecular mechanisms underlying this phenomenon are poorly understood. One possible explanation is that these infections provide a profound inflammatory milieu sufficient to overcome the reduced sensitivity of adaptive NKG2C+ NK cells to cytokine stimulation. Alternatively, NKG2C could directly recognize cross-reactive antigens derived from HCMV and one of these viruses. In hantavirus infection, it was suggested that activation and preferential proliferation of NKG2C+ NK cells could be triggered by elevated HLA-E expression (Bjorkstrom, Lindgren, et al., 2011). In this scenario, inflammatory cytokines and cross-reactivity to selfantigens could both contribute to mediate activation of adaptive NKG2C+ NK cells and thereby possibly induce heterologous immunity. Alternatively, as HCMV persists in the host, it cannot be excluded that subclinical locally confined HCMV reactivation contributes to the expansion of adaptive NKG2C+ NK cells during this heterologous infection. Whether the expansion of adaptive NKG2C+ NK cells is beneficial or detrimental for control of these heterologous infections still remains to be determined. However, potential cross-reactivity of adaptive NK cells could be advantageous in a tumor setting: patients undergoing HSC transplantation for the treatment of acute myeloid leukemia have a reduced relapse risk when the donor and/or recipient are HCMV seropositive before the transplantation (Behrendt et al., 2009; Elmaagacli et al., 2011). Cross-reactive recognition of HLA-E+ leukemic blasts (Nguyen et al., 2009) by adaptive NKG2C+ NK cells expanded in response to HCMV infection might contribute to the eradication of minimal residual disease, as expansion of adaptive NKG2C+ NK cells was associated with reduced relapse risk in leukemia patients after HSC transplantation (Cichocki et al., 2016). Cross-reactivity of HCMV-induced adaptive NK cells bearing an activating KIR capable of recognizing allogeneic HLA class I on leukemic blasts might have similar effects. In support of this concept, patients who undergo allogeneic HSC grafts with KIR2DS1, an activating KIR specific for group 2 HLA-C ligands, have a lower rate of relapse (Venstrom et al., 2012). Different mechanisms potentially underlying NK cell-mediated heterologous immunity are depicted in Fig. 3.

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Virus 2

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NK cell

NK cell

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to self and inflammation

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Fig. 3 Possible mechanisms driving heterologous immunity of NK cells as consequence of cytokine training or cross-reactive memory after viral infections. Exposure to proinflammatory cytokines during a primary viral infection can prime NK cells for elevated responses to heterologous viral infections (first row). Heterologous immunity can be potentially mediated by NK cells through cross-reactivity of one activating receptor recognizing two unrelated viral antigens (second row). NK cells primed with a first virus can cross-react with self-antigens in the presence of proinflammatory signals provided during a subsequent infection with a heterologous pathogen (or by leukemic blasts in HSC-transplanted patients, as discussed in Section 5.2). Proinflammatory signals can overcome tolerance and the low avidity recognition of self-antigens by providing direct costimulation to NK cells and upregulating MHC class I and self-antigens on uninfected host cells (third row).

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Altogether, adaptations induced in NK cells and other innate cells by viral infections or vaccinations can mediate heterologous immunity mainly by two potential mechanisms, namely priming and imprinting by proinflammatory cytokines as well as cross-reactivity toward heterologous pathogens or selfantigens. Although there are no evidences of cross-reactive recognition of Ly49H in mice so far, cross-reactivity between viral and self-antigens under inflammatory conditions might mediate NK-cell heterologous immunity.

6. OUTLOOK AND OUTSTANDING QUESTIONS In the last years our understanding of how NK cells adapt to CMV infection has dramatically changed our way of thinking, further blurring the schematic differences between innate and adaptive immunity. These findings paved the way for new fields of research, which will hopefully help to answer many open questions: what is the relative contribution of inflammatory signals vs specific recognition of viral antigens in driving phenotypic, functional, and epigenetic imprinting of adaptive NK cells during CMV infection and which are the key molecular events underlying this phenomenon? Which are the signals and niches required for NK-cell subset persistence during CMV infection? Which are the relevant NK-cell receptors and specific viral ligands driving CMV recognition and expansion of adaptive NK cells in humans and in different inbred and outbred mouse strains? Can other infections drive similar features, and by which signals? The fight and coexistence between CMV and NK cells can still teach us a lot about immune resistance and guide us to develop new strategies to enhance vaccine efficacy and protective memory.

ACKNOWLEDGMENTS We thank Marina Babic, Kerstin Juelke, Timo Rueckert, and Josefine Dunst for insightful comments and helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants SFB 650 and RO 3565 to C.R. C.R. is supported by the DFG Heisenberg grant RO 3565/1, and Q.H. is supported by the Leibniz Association (Leibniz Graduate School for Rheumatology).

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INDEX Note: Page numbers followed by “f ” indicate figures, and “t” indicate tables.

A Activation induced deaminase (AID), 38–42 activity biochemical, 42–44 Ig loci contribution, 49–52 to Ig locus, phosphorylation, 56–57 recruiting, 52–54 in B cells, 48–49 initiates DNA repair cascade, 57 UNG and base excision repair arm, 58–61 uracil detection, 57–58 limiting the levels, in nucleus, 44–47 localization, 46 oncogenic activity of, 44 piggybacks on transcription to access DNA, 47–49 postdeamination roles of, 69–70 regulation of, 43f targeting, RNA processing to, 54–56 topological and functional factors promoting, 48f Adaptive immunity, 122–123, 133–134 in prokaryotes, 134 ADAR. See Adenosine deaminase acting on RNA (ADAR) ADCC. See Antibody-dependent cell cytotoxicity (ADCC) Adenosine deaminase acting on RNA (ADAR), 136–137 Aicardi–Goutieres syndrome (AGS), 130 AID. See Activation induced deaminase (AID) AIM2, 153 Alternative end joining (A-EJ) pathway, 68 Antibody-dependent cell cytotoxicity (ADCC), 172–173 Antibody diversification in B cells, 40f during humoral response, 38–39 interactions between BER and MMR during, 63–65 Antibody response, 38–40, 70

Antigen-presenting cells (APCs), NLRC5 role in, 106–108 Antigen-processing machinery (APM), 96 Antiinflammatory macrophages, 10 Antiviral immunity, 126–127f, 130, 144–145 mitochondrial dynamics regulate, 20–22 mitochondrial-mediated, 14–22 Antiviral response, mitochondria signaling platforms for, 18f Antiviral RNAi, 130 Antiviral signaling mitochondria, 16–17 ROS regulation of, 22 APE1. See Apurinic/apyrimidinic endonuclease 1 (APE1) APM. See Antigen-processing machinery (APM) APOBEC enzyme, 42 Apurinic/apyrimidinic endonuclease 1 (APE1), 56 Autosomal dominant hyper IgM syndrome 2, 69–70

B Bacille Calmette-Guerin (BCG), 188 Baculovirus inhibitor repeat (BIR), 90–91 Base excision repair (BER) arm, 58–61 and MMR interactions, 63–65 B cell, 122–123 antibody diversification in, 40f germinal center, 65–66, 70 lymphoma, 44 B cell receptor (BCR), 40f BIR. See Baculovirus inhibitor repeat (BIR) Bone marrow derived macrophages (BMDMs), 106–107, 107f

C Cancer, NLRC5 and, 110–112 Canonical MMR, 61–62 Carbohydrate kinase-like protein (CARKL), 7 209

210 CARD. See Caspase recruitment domain (CARD) CARKL. See Carbohydrate kinase-like protein (CARKL) Caspase recruitment domain (CARD), 90–91 CD16, 172–173 CDRs. See Complementary determining regions (CDRs) cGAMP, 125–128, 147f, 153–154 cGAS. See Cyclic GMP-AMP synthase (cGAS) Chromatin immunoprecipitation, 69–70 Cis-elements, 51–52 Classical MHC class I, 96–97 Class II major histocompatibility complex (MHC) transactivator (CIITA), 94f master transcriptional regulator of MHC class II genes, 91–92 vs. NLRC5, 91f, 98–99 transcriptional regulation by, 93–95 transcriptional regulation of, 92–93 transcriptional targets of, 95–96 Class switch recombination (CSR), 38–40, 40f, 44–45, 68 AID activity during, 48f AID for, 69 biochemical pathways of, 59–60f epigenetics of, 54 CNSs. See Conserved noncoding sequences (CNSs) Complementary determining regions (CDRs), 38–40 relevance for humoral immunity, 41 Conserved noncoding sequences (CNSs), 189–190 CRISPR/Cas system, 122–125 CRISPR RNAs (CrRNAs), 134 CSR. See Class switch recombination (CSR) CTL. See Cytotoxic T lymphocytes (CTL) CTNNBL1 ablation, 45 Cyclic GMP-AMP synthase (cGAS), 125–128, 153–154 cytosolic immune-sensing receptor, 153 Cytochrome c oxidase complex subunit 5B (COX5B), 22 Cytokines, proinflammatory, 178

Index

Cytosolic immune-sensing receptor cGAS, 153 Cytotoxic T lymphocytes (CTL), 111f

D Danger-associated molecular patterns (DAMPs), 4–5, 90 Dendritic cells (DCs), NLRC5 role in, 108 Deoxyribonuclease (DNase) DNase I, 141–142 DNase II, 142–143 DNase III/Trex1, 143–144 DIVAC, 51–52 DNA deamination at IGS, activation-induced deaminase, 41–42 DSBs, 68 nicks and breaks, 65–67 repairing the breaks, 67–69 Double strand breaks (DSBs), 38, 67–68

E Electron transport chain (ETC), 6 Endogenous peptides, 96 Enhanceosome, 93 MHC, 94f Enzyme, APOBEC, 42 Epigenetic mechanism, 191 Epigenetic remodeling as hallmark of immune training and memory, 188–191 for NK-cell memory in MCMV, 191 Epigenetic reprogramming, 188 Error-free repair, biochemical pathways of, 59–60f ETC. See Electron transport chain (ETC)

G GANP, 45, 56 Germinal center, 38–39, 51, 54–55 B cells, 65–66, 70 response, 70 Germline-encoded receptors, 122–123 innate immune, 122–123 Globular domain of complement component 1q (gC1qR), 19

211

Index

H HATs. See Histone acetylation (HATs) HCMV-induced Adaptive NKG2C+ NK cells, 183–187 Heat-shock protein 90 (HSP90), 18–19 Hematopoietic stem cell (HSC) transplantation, 184–185 Heterologous immunity, NK cell, 191–198, 197f Heterologous pathogens, 192–194 HIF-1α. See Hypoxia-inducible factor-1α (HIF-1α) Histone acetylation (HATs), 93–95 Human adaptive NK cells, epigenetic imprinting of, 189–191 Human NLRC5 targets, 103t Humoral immunity, 41 Humoral response, antibody diversification during, 38–39 Hypoxia-inducible factor-1α (HIF-1α), 7

I IFIT. See Interferon-induced proteins with tetratricopeptide repeat (IFIT) IGS, DNA deamination at, 41–57 Immune function, 134–144 Immune receptor germline-encoded innate, 122–123 nucleic acid-detecting, 122–123 nucleic acid-sensing, 122–123 Immune-responsive 1 homolog (Irg1), 7–8 Immune sensing of nucleic acid, 125–128, 126–127f, 130–133 Immune-sensing receptor, 145–146, 147f AIM2, 153 cGAS/sting, 153–154 LGP2, 152 MDA5, 152 RIG-I, 150–152 TLR3, 146, 147f TLR7, 146–148, 147f TLR8, 146–148, 147f TLR9, 148–150 Immune signaling pathway, 5 Immune system, prokaryotic, 130 Immunity, humoral, 41 Immunological memory, 175

Immunoreceptor tyrosine-based switch motifs (ITSMs), 172–173 Inflammasome activation, 23 mitochondrial signals in, 24–27 mitochondrion as scaffold for, 23–24, 25f mitophagy restrains, 27 Inflammatory macrophages, 5–6 Inflammatory signaling pathway, mROS, 12–14 Innate immunity, 133–134 in prokaryotes, 134 response, mROS in, 10–14, 11f SAMHD1 in, 137–138 Interferon-induced proteins with tetratricopeptide repeat (IFIT), 139–140 Interferon-stimulated genes (ISGs), 139–140 ITSMs. See Immunoreceptor tyrosine-based switch motifs (ITSMs)

K Killer cell immunoglobulin-like receptor (KIR), 172–173

L Laboratory of genetics and physiology 2 (LGP2), 152 LCMV. See Lymphocytic choriomeningitis virus (LCMV) Leucine-rich repeats (LRRs), 90–91 Leukemia, 44 LGP2. See Laboratory of genetics and physiology 2 (LGP2) Lipopolysaccharide (LPS), 7 LRRs. See Leucine-rich repeats (LRRs) Ly49H, 174–175, 182–183, 195 ligands for, 176–177 in mouse, 179–181 Ly49H+ NK cells, 176–179, 191, 194–195 MCMV-induced memory, 183 Lymphocyte, 172 Lymphocytic choriomeningitis virus (LCMV), 109

M Macrophage, 4 antiinflammatory, 10 inflammatory, 5–6

212 Macrophage (Continued ) metabolic profile in IL-4-activated, 8–10, 9f proinflammatory, 6 metabolic reprogramming in, 7–8 Major histocompatibility complex (MHC), 172–173 class Ia, 96–97 class I genes complex transcriptional regulation of, 96–97 transcriptional regulator of, 97–98 class II molecules, 91–92 enhanceosome, 94f MAPK phosphatases (MKPs), 12 MAPKs. See Mitogen-activated protein kinases (MAPKs) MAVS. See Mitochondrial antiviral signaling (MAVS) MCMV. See Murine cytomegalovirus (MCMV) Melanoma differentiation-associated protein 5 (MDA5), 152 Memory epigenetic remodeling as hallmark of immune training and, 188–191 NK-cell cross-reactive, 194–198 training and cross-reactive, 191–198, 197f Memory Ly49H+ NK cells, MCMV-induced, 183 Memory NK cells, 178–179 MCMV-induced, 176–177 Metabolic profile, in IL-4-activated macrophages, 8–10 Metabolic reprogramming, in proinflammatory macrophages, 7–8 Metabolism, mitochondria, 5–10 MHC. See Major histocompatibility complex (MHC) Microglia, 4 Mismatch repair (MMR), 61, 70 and BER interaction, 63–65 mutagenic noncanonical pathway, 64 Mitochondria, 5 antiviral immunity, 14, 16–17 antiviral signaling, 16–17 metabolism, 5–10

Index

signaling platforms for antiviral responses, 18f Mitochondrial antiviral signaling (MAVS) proteins regulation, 17–19 Mitochondrial dynamics, regulate antiviral immunity, 20–22 Mitochondrial fusion, 20 Mitochondrial ROS (mROS) inflammatory signaling pathways, 12–14 in innate immune responses, 10–14, 11f production, 14 Mitochondrial signal, in inflammasome activation, 24–27 Mitogen-activated protein kinases (MAPKs), 90 Mitophagy inhibition of, 27 restrains inflammasome activation, 27 Murid herpesvirus 4 (MuHV-4), 193–194 Murine cytomegalovirus (MCMV), 174–175 induced memory Ly49H+ NK cells, 183 infection, 176–179, 177f NK-cell memory in, role of epigenetic remodeling for, 191 Murine NLRC5 targets, 100, 101–102t Mutagenic noncanonical pathway of MMR, 64 Mutation, in SAMHD1, 137–138 MutSα-initiated arm, 61–63

N Natural killer (NK) cells, 109, 172 adaptations to viral infections, 175–182 cross-reactive memory, 194–198 epigenetic imprinting of human adaptive, 189–191 functional imprinting of adaptive, 182–187 functions of, 172 HCMV-induced adaptive NKG2C+, 183–187 heterologous immunity, 191–198, 197f human adaptive, functional properties of, 187f Ly49H+, 176–179, 191, 194–195 MCMV-induced memory Ly49H+, 183

213

Index

memory in MCMV, 191 NKG2C+, 179–182, 195–196 NLRC5 protects T cells from, 109f recognition repertoire, 172–174 responses to heterologous pathogens, 192–194 role in defense against viral infections, 174–175 skewing and adaptation of, 176–182 subsets after HCMV infection, 179–182 subsets after MCMV infection, 176–179 NBD. See Nucleotide binding domain (NBD) NES. See Nuclear export signal (NES) NHEJ pathway. See Nonhomologous end joining (NHEJ) pathway NK cells. See Natural killer (NK) cells NKG2C, 179–181 expression, 181 NKG2C+ NK cells, 179–182, 195–196 HCMV-induced adaptive, 183–187 NLR family CARD domain containing 5 (NLRC5), 94f, 97–98 and cancer, 110–112 vs. CIITA, 91f, 98–99 protects T cells from NK cell, 109f role in APCs, 106–108 CD8+ T cell selection and maintenance, 105–106 dendritic cells, 108 infections, 108–110 tumors, 112–114 transcriptional activity, 99–100 transcriptional targets of, 100–103, 101–103t tunes MHC class I gene transcription, 104–105 NLRs. See Nucleotide-binding oligomerization domain-like receptors (NLRs) NLS. See Nuclear localization sequence (NLS) Nodamura virus (NoV), 144–145 Non-classical MHC class I, 97 Non-Hodgkin lymphoma, 44 Nonhomologous end joining (NHEJ) pathway, 68

NoV. See Nodamura virus (NoV) Nuclear export signal (NES), 45–46 Nuclear localization sequence (NLS), 98 Nucleic acid detecting immune receptors, 122–123 immune sensing of, 125–128 proteins targeting foreign, 134–135 sensing immune receptor, 122–123 Nucleic acid immunity functional components of, 130–133, 132f historic overview of different fields merging into, 125–130, 126–127f innate and adaptive components in, 133–134, 133f mechanism, 123–125, 124f Nucleotide binding domain (NBD), 90–91 Nucleotide-binding oligomerization domain-like receptors (NLRs), 90–91 Nucleus, limiting levels of AID in, 44–47

O

20 –50 -Oligoadenylate synthetase (OAS) system, 140 Oxidative phosphorylation (OXPHOS), 6

P Pathogen heterologous, 192–194 specific T cells, 175 Pathogen associated molecular patterns (PAMPs), 4–5, 90, 188 Pattern-recognition receptors (PRRs), 4–5 Peripheral blood mononuclear cells (PBMCs), 12 Phosphorylation, 56–57 PKA. See Protein kinase A (PKA) PKR, 138–139 Polypyrimidine tract-binding protein 2 (PTBP2), 54–55 Proinflammatory cytokines, 178 Proinflammatory macrophages, 6 metabolic reprogramming in, 7–8 Prokaryotes DNA R-M system, 134 immune system, 130 innate and adaptive nucleic acid immunity in, 134 Proteasome, 96

214 Protein RNA-binding, 56 targeting foreign nucleic acids, 134–135 Protein kinase A (PKA), 48–49 PRRs. See Pattern-recognition receptors (PRRs) PTBP2. See Polypyrimidine tract-binding protein 2 (PTBP2) Pyrin domain (PYD), 90–91

R Reactive oxygen species (ROS), 6 regulation of antiviral signaling, 22 Receptor, immune-sensing, 145–154 Replication protein A (RPA), 48–49 Retinoic acid-inducible gene I (RIG-I), 150–152 Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), 14–16 RIG-I. See Retinoic acid-inducible gene I (RIG-I) RLRs. See Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RNA-binding protein, 56 RNA exosome, 48–49 RNA interference (RNAi), 130, 144–145 RNApolymerase II (RNAPII) complex, 48–49, 52 RNase H, 124–125, 130, 135f, 141 RNase L, 129–133, 135f, 140 ROS. See Reactive oxygen species (ROS) RPA. See Replication protein A (RPA)

S SAMHD1, 137–138 Self-MHC class I molecules (sKIRs), 179–180 SHM. See Somatic hypermutation (SHM) Single-stranded DNA (ssDNA), 42–44 SMUG1, 64 Somatic hypermutation (SHM), 38–40, 40f, 44–45 AID activity during, 48f AID for, 69 biochemical pathways of, 59–60f epigenetics of, 54 function of GANP in, 56 intrinsic features of, 51

Index

mutagenic BER for, 58–60 relevance for humoral immunity, 41 ssDNA. See Single-stranded DNA (ssDNA) STING, 17–18, 153–154

T T-cell, 122–123 clone, 175 immunological memory of, 191–192 memory formation, 178–179 pathogen-specific, 175 T-cell receptor (TCR), 172–173 TCR. See T-cell receptor (TCR) TECs. See Thymic epithelial cells (TECs) Thymic epithelial cells (TECs), 105 TLR. See Toll-like receptor (TLR) TNF receptor-associated factor 6 (TRAF6), 14 TNF receptor associated periodic syndrome (TRAPS), 12 Toll-like receptor (TLR), 122–123 signaling upregulates mROS production, 14 TLR3, 146, 147f TLR7, 146–148, 147f TLR8, 146–148, 147f TLR9, 148–150 TOM70, 18–19 TRAF6. See TNF receptor-associated factor 6 (TRAF6) Trained immunity, 188, 191–192 Transcriptional activity, NLCR5, 99–100 Transcriptional regulation by CIITA, 93–95 of CIITA, 92–93 Transcriptional regulator, of MHC class I genes, 97–98 Transcriptional targets of CIITA, 95–96 of NLRC5, 100–103 TRAPS. See TNF receptor associated periodic syndrome (TRAPS) Trex1, 143–144

U Uncoupling protein 2 (UCP2), 13 Unmethylated CpG motifs, 125–128 Uracil detection, 57–58

215

Index

Uracil-DNA glycosylase (UNG), 57–58, 63–67, 69–70 activity, 58–60 and base excision repair arm, 58–61 inhibitor, 60–61 promutagenic activity of, 60–61

V Viral dsRNA, 136 Viral infections

NK cells adaptations to, 175–182 role of NK cells in defense against, 174–175 Virus-derived small RNAs (vsRNAs), 144–145 Voltage-dependent anion-selective channels (VDACs), 10–11

W Warburg effect, 6–7

CONTENTS OF RECENT VOLUMES Volume 85

Volume 87

Cumulative Subject Index Volumes 66–82

Role of the LAT Adaptor in T-Cell Development and Th2 Differentiation Bernard Malissen, Enrique Aguado, and Marie Malissen

Volume 86 Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation Michael R. Blackburn and Rodney E. Kellems Mechanism and Control of V(D)J Recombination Versus Class Switch Recombination: Similarities and Differences Darryll D. Dudley, Jayanta Chaudhuri, Craig H. Bassing, and Frederick W. Alt Isoforms of Terminal Deoxynucleotidyltransferase: Developmental Aspects and Function To-Ha Thai and John F. Kearney Innate Autoimmunity Michael C. Carroll and V. Michael Holers Formation of Bradykinin: A Major Contributor to the Innate Inflammatory Response Kusumam Joseph and Allen P. Kaplan Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells Brian Becknell and Michael A. Caligiuri Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Cell Network: Facts, Hypothesis, and Immunological Implications Nicholas S. Wilson and Jose A. Villadangos Index

The Integration of Conventional and Unconventional T Cells that Characterizes Cell-Mediated Responses Daniel J. Pennington, David Vermijlen, Emma L. Wise, Sarah L. Clarke, Robert E. Tigelaar, and Adrian C. Hayday Negative Regulation of Cytokine and TLR Signalings by SOCS and Others Tetsuji Naka, Minoru Fujimoto, Hiroko Tsutsui, and Akihiko Yoshimura Pathogenic T-Cell Clones in Autoimmune Diabetes: More Lessons from the NOD Mouse Kathryn Haskins The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling Louis M. Staudt and Sandeep Dave New Insights into Alternative Mechanisms of Immune Receptor Diversification Gary W. Litman, John P. Cannon, and Jonathan P. Rast The Repair of DNA Damages/ Modifications During the Maturation of the Immune System: Lessons from Human Primary Immunodeficiency Disorders and Animal Models Patrick Revy, Dietke Buck, Franc¸oise le Deist, and Jean-Pierre de Villartay Antibody Class Switch Recombination: Roles for Switch Sequences and Mismatch Repair Proteins Irene M. Min and Erik Selsing Index 217

218

Volume 88 CD22: A Multifunctional Receptor That Regulates B Lymphocyte Survival and Signal Transduction Thomas F. Tedder, Jonathan C. Poe, and Karen M. Haas Tetramer Analysis of Human Autoreactive CD4-Positive T Cells Gerald T. Nepom Regulation of Phospholipase C-γ2 Networks in B Lymphocytes Masaki Hikida and Tomohiro Kurosaki Role of Human Mast Cells and Basophils in Bronchial Asthma Gianni Marone, Massimo Triggiani, Arturo Genovese, and Amato De Paulis A Novel Recognition System for MHC Class I Molecules Constituted by PIR Toshiyuki Takai Dendritic Cell Biology Francesca Granucci, Maria Foti, and Paola Ricciardi-Castagnoli The Murine Diabetogenic Class II Histocompatibility Molecule I-Ag7: Structural and Functional Properties and Specificity of Peptide Selection Anish Suri and Emil R. Unanue RNAi and RNA-Based Regulation of Immune System Function Dipanjan Chowdhury and Carl D. Novina Index

Volume 89 Posttranscriptional Mechanisms Regulating the Inflammatory Response Georg Stoecklin Paul Anderson Negative Signaling in Fc Receptor Complexes Marc Dae¨ron and Renaud Lesourne

Contents of Recent Volumes

The Surprising Diversity of Lipid Antigens for CD1-Restricted T Cells D. Branch Moody Lysophospholipids as Mediators of Immunity Debby A. Lin and Joshua A. Boyce Systemic Mastocytosis Jamie Robyn and Dean D. Metcalfe Regulation of Fibrosis by the Immune System Mark L. Lupher, Jr. and W. Michael Gallatin Immunity and Acquired Alterations in Cognition and Emotion: Lessons from SLE Betty Diamond, Czeslawa Kowal, Patricio T. Huerta, Cynthia Aranow, Meggan Mackay, Lorraine A. DeGiorgio, Ji Lee, Antigone Triantafyllopoulou, Joel Cohen-Solal Bruce, and T. Volpe Immunodeficiencies with Autoimmune Consequences Luigi D. Notarangelo, Eleonora Gambineri, and Raffaele Badolato Index

Volume 90 Cancer Immunosurveillance and Immunoediting: The Roles of Immunity in Suppressing Tumor Development and Shaping Tumor Immunogenicity Mark J. Smyth, Gavin P. Dunn, and Robert D. Schreiber Mechanisms of Immune Evasion by Tumors Charles G. Drake, Elizabeth Jaffee, and Drew M. Pardoll Development of Antibodies and Chimeric Molecules for Cancer Immunotherapy Thomas A. Waldmann and John C. Morris

219

Contents of Recent Volumes

Induction of Tumor Immunity Following Allogeneic Stem Cell Transplantation Catherine J. Wu and Jerome Ritz Vaccination for Treatment and Prevention of Cancer in Animal Models Federica Cavallo, Rienk Offringa, Sjoerd H. van der Burg, Guido Forni, and Cornelis J. M. Melief Unraveling the Complex Relationship Between Cancer Immunity and Autoimmunity: Lessons from Melanoma and Vitiligo Hiroshi Uchi, Rodica Stan, Mary Jo Turk, Manuel E. Engelhorn, Gabrielle A. Rizzuto, Stacie M. Goldberg, Jedd D. Wolchok, and Alan N. Houghton Immunity to Melanoma Antigens: From Self-Tolerance to Immunotherapy Craig L. Slingluff, Jr., Kimberly A. Chianese-Bullock, Timothy N. J. Bullock, William W. Grosh, David W. Mullins, Lisa Nichols, Walter Olson, Gina Petroni, Mark Smolkin, and Victor H. Engelhard Checkpoint Blockade in Cancer Immunotherapy Alan J. Korman, Karl S. Peggs, and James P. Allison Combinatorial Cancer Immunotherapy F. Stephen Hodi and Glenn Dranoff

Accessibility Control of V(D)J Recombination Robin Milley Cobb, Kenneth J. Oestreich, Oleg A. Osipovich, and Eugene M. Oltz Targeting Integrin Structure and Function in Disease Donald E. Staunton, Mark L. Lupher, Robert Liddington, and W. Michael Gallatin Endogenous TLR Ligands and Autoimmunity Hermann Wagner Genetic Analysis of Innate Immunity Kasper Hoebe, Zhengfan Jiang, Koichi Tabeta, Xin Du, Philippe Georgel, Karine Crozat, and Bruce Beutler TIM Family of Genes in Immunity and Tolerance Vijay K. Kuchroo, Jennifer Hartt Meyers, Dale T. Umetsu, and Rosemarie H. DeKruyff Inhibition of Inflammatory Responses by Leukocyte Ig-Like Receptors Howard R. Katz Index

Volume 92

Volume 91

Systemic Lupus Erythematosus: Multiple Immunological Phenotypes in a Complex Genetic Disease Anna-Marie Fairhurst, Amy E. Wandstrat, and Edward K. Wakeland

A Reappraisal of Humoral Immunity Based on Mechanisms of Antibody-Mediated Protection Against Intracellular Pathogens Arturo Casadevall and Liise-anne Pirofski

Avian Models with Spontaneous Autoimmune Diseases Georg Wick, Leif Andersson, Karel Hala, M. Eric Gershwin,Carlo Selmi, Gisela F. Erf, Susan J. Lamont, and Roswitha Sgonc

Index

220 Functional Dynamics of Naturally Occurring Regulatory T Cells in Health and Autoimmunity Megan K. Levings, Sarah Allan, Eva d’Hennezel, and Ciriaco A. Piccirillo BTLA and HVEM Cross Talk Regulates Inhibition and Costimulation Maya Gavrieli, John Sedy, Christopher A. Nelson, and Kenneth M. Murphy The Human T Cell Response to Melanoma Antigens Pedro Romero, Jean-Charles Cerottini, and Daniel E. Speiser Antigen Presentation and the Ubiquitin-Proteasome System in Host–Pathogen Interactions Joana Loureiro and Hidde L. Ploegh Index

Volume 93 Class Switch Recombination: A Comparison Between Mouse and Human Qiang Pan-Hammarstr€ om, Yaofeng Zhao, and Lennart Hammarstr€ om Anti-IgE Antibodies for the Treatment of IgE-Mediated Allergic Diseases Tse Wen Chang, Pheidias C. Wu, C. Long Hsu, and Alfur F. Hung Immune Semaphorins: Increasing Members and Their Diverse Roles Hitoshi Kikutani, Kazuhiro Suzuki, and Atsushi Kumanogoh Tec Kinases in T Cell and Mast Cell Signaling Martin Felices, Markus Falk, Yoko Kosaka, and Leslie J. Berg Integrin Regulation of Lymphocyte Trafficking: Lessons from Structural and Signaling Studies Tatsuo Kinashi

Contents of Recent Volumes

Regulation of Immune Responses and Hematopoiesis by the Rap1 Signal Nagahiro Minato, Kohei Kometani, and Masakazu Hattori Lung Dendritic Cell Migration Hamida Hammad and Bart N. Lambrecht Index

Volume 94 Discovery of Activation-Induced Cytidine Deaminase, the Engraver of Antibody Memory Masamichi Muramatsu, Hitoshi Nagaoka, Reiko Shinkura, Nasim A. Begum, and Tasuku Honjo DNA Deamination in Immunity: AID in the Context of Its APOBEC Relatives Silvestro G. Conticello, Marc-Andre Langlois, Zizhen Yang, and Michael S. Neuberger The Role of Activation-Induced Deaminase in Antibody Diversification and Chromosome Translocations Almudena Ramiro, Bernardo Reina San-Martin, Kevin McBride, Mila Jankovic, Vasco Barreto, Andre Nussenzweig, and Michel C. Nussenzweig Targeting of AID-Mediated Sequence Diversification by cis-Acting Determinants Shu Yuan Yang and David G. Schatz AID-Initiated Purposeful Mutations in Immunoglobulin Genes Myron F. Goodman, Matthew D. Scharff, and Floyd E. Romesberg Evolution of the Immunoglobulin Heavy Chain Class Switch Recombination Mechanism Jayanta Chaudhuri, Uttiya Basu, Ali Zarrin, Catherine Yan, Sonia Franco, Thomas Perlot, Bao Vuong, Jing Wang, Ryan T. Phan, Abhishek Datta, John Manis, and Frederick W. Alt

221

Contents of Recent Volumes

Beyond SHM and CSR: AID and Related Cytidine Deaminases in the Host Response to Viral Infection Brad R. Rosenberg and F. Nina Papavasiliou Role of AID in Tumorigenesis Il-mi Okazaki, Ai Kotani, and Tasuku Honjo Pathophysiology of B-Cell Intrinsic Immunoglobulin Class Switch Recombination Deficiencies Anne Durandy, Nadine Taubenheim, Sophie Peron, and Alain Fischer Index

Volume 95 Fate Decisions Regulating Bone Marrow and Peripheral B Lymphocyte Development John G. Monroe and Kenneth Dorshkind Tolerance and Autoimmunity: Lessons at the Bedside of Primary Immunodeficiencies Magda Carneiro-Sampaio and Antonio Coutinho B-Cell Self-Tolerance in Humans Hedda Wardemann and Michel C. Nussenzweig Manipulation of Regulatory T-Cell Number and Function with CD28-Specific Monoclonal Antibodies Thomas H€ unig Osteoimmunology: A View from the Bone Jean-Pierre David Mast Cell Proteases Gunnar Pejler, Magnus A˚brink, Maria Ringvall, and Sara Wernersson Index

Volume 96 New Insights into Adaptive Immunity in Chronic Neuroinflammation Volker Siffrin, Alexander U. Brandt, Josephine Herz, and Frauke Zipp Regulation of Interferon-γ During Innate and Adaptive Immune Responses Jamie R. Schoenborn and Christopher B. Wilson The Expansion and Maintenance of Antigen-Selected CD8+ T Cell Clones Douglas T. Fearon Inherited Complement Regulatory Protein Deficiency Predisposes to Human Disease in Acute Injury and Chronic Inflammatory States Anna Richards, David Kavanagh, and John P. Atkinson Fc-Receptors as Regulators of Immunity Falk Nimmerjahn and Jeffrey V. Ravetch Index

Volume 97 T Cell Activation and the Cytoskeleton: You Can’t Have One Without the Other Timothy S. Gomez and Daniel D. Billadeau HLA Class II Transgenic Mice Mimic Human Inflammatory Diseases Ashutosh K. Mangalam, Govindarajan Rajagopalan, Veena Taneja, and Chella S. David Roles of Zinc and Zinc Signaling in Immunity: Zinc as an Intracellular Signaling Molecule Toshio Hirano, Masaaki Murakami, Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, and Tomoyuki Suzuki

222

Contents of Recent Volumes

The SLAM and SAP Gene Families Control Innate and Adaptive Immune Responses Silvia Calpe, Ninghai Wang, Xavier Romero, Scott B. Berger, Arpad Lanyi, Pablo Engel, and Cox Terhorst

Volume 99

Conformational Plasticity and Navigation of Signaling Proteins in Antigen-Activated B Lymphocytes Niklas Engels, Michael Engelke, and J€ urgen Wienands

DNA-PK: The Means to Justify the Ends? Katheryn Meek, Van Dang, and Susan P. Lees-Miller

Index

Volume 98 Immune Regulation by B Cells and Antibodies: A View Towards the Clinic Kai Hoehlig, Vicky Lampropoulou, Toralf Roch, Patricia Neves, Elisabeth Calderon-Gomez, Stephen M. Anderton, Ulrich Steinhoff, and Simon Fillatreau Cumulative Environmental Changes, Skewed Antigen Exposure, and the Increase of Allergy Tse Wen Chang and Ariel Y. Pan New Insights on Mast Cell Activation via the High Affinity Receptor for IgE Juan Rivera, Nora A. Fierro, Ana Olivera, and Ryo Suzuki B Cells and Autoantibodies in the Pathogenesis of Multiple Sclerosis and Related Inflammatory Demyelinating Diseases Katherine A. McLaughlin and Kai W. Wucherpfennig Human B Cell Subsets Stephen M. Jackson, Patrick C. Wilson, Judith A. James, and J. Donald Capra Index

Cis-Regulatory Elements and Epigenetic Changes Control Genomic Rearrangements of the IgH Locus Thomas Perlot and Frederick W. Alt

Thymic Microenvironments for T-Cell Repertoire Formation Takeshi Nitta, Shigeo Murata, Tomoo Ueno, Keiji Tanaka, and Yousuke Takahama Pathogenesis of Myocarditis and Dilated Cardiomyopathy Daniela Cihakova and Noel R. Rose Emergence of the Th17 Pathway and Its Role in Host Defense Darrell B. O’Quinn, Matthew T. Palmer, Yun Kyung Lee, and Casey T. Weaver Peptides Presented In Vivo by HLA-DR in Thyroid Autoimmunity Laia Muixı´, In˜aki Alvarez, and Dolores Jaraquemada Index

Volume 100 Autoimmune Diabetes Mellitus—Much Progress, but Many Challenges Hugh O. McDevitt and Emil R. Unanue CD3 Antibodies as Unique Tools to Restore Self-Tolerance in Established Autoimmunity: Their Mode of Action and Clinical Application in Type 1 Diabetes Sylvaine You, Sophie Candon, Chantal Kuhn, Jean-Franc¸ois Bach, and Lucienne Chatenoud GAD65 Autoimmunity—Clinical Studies Raivo Uibo and A˚ke Lernmark

223

Contents of Recent Volumes

CD8+ T Cells in Type 1 Diabetes Sue Tsai, Afshin Shameli, and Pere Santamaria Dysregulation of T Cell Peripheral Tolerance in Type 1 Diabetes R. Tisch and B. Wang Gene–Gene Interactions in the NOD Mouse Model of Type 1 Diabetes William M. Ridgway, Laurence B. Peterson, John A. Todd, Dan B. Rainbow, Barry Healy, and Linda S. Wicker

Volume 102 Antigen Presentation by CD1: Lipids, T Cells, and NKT Cells in Microbial Immunity Nadia R. Cohen, Salil Garg, and Michael B. Brenner How the Immune System Achieves Self–Nonself Discrimination During Adaptive Immunity Hong Jiang and Leonard Chess

Index

Cellular and Molecular Mechanisms in Atopic Dermatitis Michiko K. Oyoshi, Rui He, Lalit Kumar, Juhan Yoon, and Raif S. Geha

Volume 101

Micromanagers of Immune Cell Fate and Function Fabio Petrocca and Judy Lieberman

TSLP in Epithelial Cell and Dendritic Cell Cross Talk Yong-Jun Liu Natural Killer Cell Tolerance: Licensing and Other Mechanisms A. Helena Jonsson and Wayne M. Yokoyama Biology of the Eosinophil Carine Blanchard and Marc E. Rothenberg Basophils: Beyond Effector Cells of Allergic Inflammation John T. Schroeder DNA Targets of AID: Evolutionary Link Between Antibody Somatic Hypermutation and Class Switch Recombination Jason A. Hackney, Shahram Misaghi, Kate Senger, Christopher Garris, Yonglian Sun, Maria N. Lorenzo, and Ali A. Zarrin Interleukin 5 in the Link Between the Innate and Acquired Immune Response Kiyoshi Takatsu, Taku Kouro, and Yoshinori Nagai Index

Immune Pathways for Translating Viral Infection into Chronic Airway Disease Michael J. Holtzman, Derek E. Byers, Loralyn A. Benoit, John T. Battaile, Yingjian You, Eugene Agapov, Chaeho Park, Mitchell H. Grayson, Edy Y. Kim, and Anand C. Patel Index

Volume 103 The Physiological Role of Lysyl tRNA Synthetase in the Immune System Hovav Nechushtan, Sunghoon Kim, Gillian Kay, and Ehud Razin Kill the Bacteria … and Also Their Messengers? Robert Munford, Mingfang Lu, and Alan Varley Role of SOCS in Allergic and Innate Immune Responses Suzanne L. Cassel and Paul B. Rothman

224

Contents of Recent Volumes

Multitasking by Exploitation of Intracellular Transport Functions: The Many Faces of FcRn E. Sally Ward and Raimund J. Ober

The Family of IL-10-Secreting CD4+ T Cells Keishi Fujio, Tomohisa Okamura, and Kazuhiko Yamamoto

Index

Artificial Engineering of Secondary Lymphoid Organs Jonathan K. H. Tan and Takeshi Watanabe

Volume 104 Regulation of Gene Expression in Peripheral T Cells by Runx Transcription Factors Ivana M. Djuretic, Fernando Cruz-Guilloty, and Anjana Rao Long Noncoding RNAs: Implications for Antigen Receptor Diversification Grace Teng and F. Nina Papavasiliou

AID and Somatic Hypermutation Robert W. Maul and Patricia J. Gearhart BCL6: Master Regulator of the Germinal Center Reaction and Key Oncogene in B Cell Lymphomagenesis Katia Basso and Riccardo Dalla-Favera

Pathogenic Mechanisms of Allergic Inflammation: Atopic Asthma as a Paradigm Patrick G. Holt, Deborah H. Strickland, Anthony Bosco, and Frode L. Jahnsen

Index

The Amplification Loop of the Complement Pathways Peter J. Lachmann

Volume 106

Index

Volume 105 Learning from Leprosy: Insight into the Human Innate Immune Response Dennis Montoya and Robert L. Modlin The Immunological Functions of Saposins Alexandre Darmoise, Patrick Maschmeyer, and Florian Winau OX40–OX40 Ligand Interaction in T-Cell-Mediated Immunity and Immunopathology Naoto Ishii, Takeshi Takahashi, Pejman Soroosh, and Kazuo Sugamura

The Role of Innate Immunity in B Cell Acquisition of Antigen Within LNs Santiago F. Gonzalez, Michael P. Kuligowski, Lisa A. Pitcher, Ramon Roozendaal, and Michael C. Carroll Nuclear Receptors, Inflammation, and Neurodegenerative Diseases Kaoru Saijo, Andrea Crotti, and Christopher K. Glass Novel Tools for Modulating Immune Responses in the Host— Polysaccharides from the Capsule of Commensal Bacteria Suryasarathi Dasgupta and Dennis L. Kasper The Role of Mechanistic Factors in Promoting Chromosomal

225

Contents of Recent Volumes

Translocations Found in Lymphoid and Other Cancers Yu Zhang, Monica Gostissa, Dominic G. Hildebrand, Michael S. Becker, Cristian Boboila, Roberto Chiarle, Susanna Lewis, and Frederick W. Alt Index

Volume 107 Functional Biology of the IL-22-IL-22R Pathway in Regulating Immunity and Inflammation at Barrier Surfaces Gregory F. Sonnenberg, Lynette A. Fouser, David Artis Innate Signaling Networks in Mucosal IgA Class Switching Alejo Chorny, Irene Puga, and Andrea Cerutti Specificity of the Adaptive Immune Response to the Gut Microbiota Daniel A. Peterson and Roberto A. Jimenez Cardona

Volume 108 Macrophage Proinflammatory Activation and Deactivation: A Question of Balance Annabel F. Valledor, Monica Comalada, Luis Santamarı´a-Babi, Jorge Lloberas, and Antonio Celada Natural Helper Cells: A New Player in the Innate Immune Response against Helminth Infection Shigeo Koyasu, Kazuyo Moro, Masanobu Tanabe, and Tsutomu Takeuchi Mapping of Switch Recombination Junctions, a Tool for Studying DNA Repair Pathways during Immunoglobulin Class Switching Janet Stavnezer, Andrea Bj€ orkman, Likun Du, Alberto Cagigi, and Qiang Pan-Hammarstr€ om How Tolerogenic Dendritic Cells Induce Regulatory T Cells Roberto A. Maldonado and Ulrich H. von Andrian Index

Intestinal Dendritic Cells Maria Rescigno The Many Face-Lifts of CD4 T Helper Cells Daniel Mucida and Hilde Cheroutre GALT: Organization and Dynamics Leading to IgA Synthesis Keiichiro Suzuki, Shimpei Kawamoto, Mikako Maruya, and Sidonia Fagarasan Bronchus-Associated Lymphoid Tissue (BALT): Structure and Function Troy D. Randall Host–Bacterial Symbiosis in Health and Disease Janet Chow, S. Melanie Lee, Yue Shen, Arya Khosravi, and Sarkis K. Mazmanian Index

Volume 109 Dynamic Palmitoylation and the Role of DHHC Proteins in T Cell Activation and Anergy Nadejda Ladygina, Brent R. Martin, and Amnon Altman Transcriptional Control of Natural Killer Cell Development and Function David G. T. Hesslein and Lewis. L. Lanier The Control of Adaptive Immune Responses by the Innate Immune System Dominik Schenten and Ruslan Medzhitov The Evolution of Adaptive Immunity in Vertebrates Masayuki Hirano, Sabyasachi Das, Peng Guo, and Max D. Cooper

226 T Helper Cell Differentiation: More than Just Cytokines Beata Zygmunt and Marc Veldhoen Index

Volume 110 AID Targeting in Antibody Diversity Rushad Pavri and Michel C. Nussenzweig The IgH Locus 30 Regulatory Region: Pulling the Strings from Behind Eric Pinaud, Marie Marquet, Remi Fiancette, Sophie Peron, Christelle Vincent-Fabert, Yves Denizot, and Michel Cogne Transcriptional and Epigenetic Regulation of CD4/CD8 Lineage Choice Ichiro Taniuchi and Wilfried Ellmeier Modeling a Complex Disease: Multiple Sclerosis Florian C. Kurschus, Simone W€ ortge, and Ari Waisman Autoinflammation by Endogenous DNA Shigekazu Nagata and Kohki Kawane Index

Volume 111 Early Steps of Follicular Lymphoma Pathogenesis Sandrine Roulland, Mustapha Faroudi, Emilie Mamessier, Stephanie Sungalee, Gilles Salles, and Bertrand Nadel “A Rose is a Rose is a Rose,” but CVID is Not CVID: Common Variable Immune Deficiency (CVID), What do we Know in 2011? Patrick F. K. Yong, James E. D. Thaventhiran, and Bodo Grimbacher Role of Activation-Induced Cytidine Deaminase in Inflammation-Associated Cancer Development Hiroyuki Marusawa, Atsushi Takai, and Tsutomu Chiba

Contents of Recent Volumes

Comparative Genomics and Evolution of Immunoglobulin-Encoding Loci in Tetrapods Sabyasachi Das, Masayuki Hirano, Chelsea McCallister, Rea Tako, and Nikolas Nikolaidis Pax5: A Master Regulator of B Cell Development and Leukemogenesis Jasna Medvedovic, Anja Ebert, Hiromi Tagoh, and Meinrad Busslinger Index

Volume 112 Stability of Regulatory T-cell Lineage Shohei Hori Thymic and Peripheral Differentiation of Regulatory T Cells Hyang-Mi Lee, Jhoanne Lynne Bautista, and Chyi-Song Hsieh Regulatory T Cells in Infection Rick M. Maizels and Katherine A. Smith Biological Functions of Regulatory T Cells Ethan M. Shevach Extrathymic Generation of Regulatory T Cells—Chances and Challenges for Prevention of Autoimmune Disease Carolin Daniel, and Harald von Boehmer Index

Volume 113 Studies with Listeria monocytogenes Lead the Way Emil R. Unanue and Javier A. Carrero Interactions of Listeria monocytogenes with the Autophagy System of Host Cells Grace Y. Lam, Mark A. Czuczman, Darren E. Higgins and John H. Brumell

227

Contents of Recent Volumes

Virulence Factors That Modulate the Cell Biology of Listeria Infection and the Host Response Serge Mostowy and Pascale Cossart

Structure-Based Design for High-Hanging Vaccine Fruits Jaap W. Back and Johannes P. M. Langedijk

Dendritic Cells in Listeria monocytogenes Infection Brian T. Edelson

Mechanisms of Peptide Vaccination in Mouse Models: Tolerance, Immunity, and Hyperreactivity Thorbald van Hall and Sjoerd H. van der Burg

Probing CD8 T Cell Responses with Listeria monocytogenes Infection Stephanie A. Condotta, Martin J. Richer, Vladimir P. Badovinac and John T. Harty

Experience with Synthetic Vaccines for Cancer and Persistent Virus Infections in Nonhuman Primates and Patients Esther D. Quakkelaar and Cornelis J. M. Melief

Listeria monocytogenes and Its Products as Agents for Cancer Immunotherapy Patrick Guirnalda, Laurence Wood and Yvonne Paterson

Malaria Vaccine Development Using Synthetic Peptides as a Technical Platform Giampietro Corradin, Nora Cespedes, Antonio Verdini, Andrey V. Kajava, Myriam Arevalo-Herrera, and So´crates Herrera

Monocyte-Mediated Immune Defense Against Murine Listeria monocytogenes Infection Natalya V. Serbina, Chao Shi and Eric G. Pamer Innate Immune Pathways Triggered by Listeria monocytogenes and Their Role in the Induction of Cell-Mediated Immunity Chelsea E. Witte, Kristina A. Archer, Chris S. Rae, John-Demian Sauer, Josh J. Woodward and Daniel A. Portnoy Mechanisms and Immunological Effects of Lymphocyte Apoptosis Caused by Listeria monocytogenes Javier A. Carrero, and Emil R. Unanue Index

Enhancing Cancer Immunotherapy by Intracellular Delivery of Cell-Penetrating Peptides and Stimulation of PatternRecognition Receptor Signaling Helen Y. Wang and Rong-Fu Wang TLR Ligand–Peptide Conjugate Vaccines: Toward Clinical Application Gijs G. P. Zom, Selina Khan, Dmitri V. Filippov, and Ferry Ossendorp Behavior and Function of Tissue-Resident Memory T cells Silvia Ariotti, John B. Haanen, and Ton N. Schumacher Rational Design of Vaccines: Learning from Immune Evasion Mechanisms of Persistent Viruses and Tumors Ramon Arens Index

Volume 114 Nucleic Acid Adjuvants: Toward an Educated Vaccine Jasper G. van den Boorn, Winfried Barchet, and Gunther Hartmann

Volume 115 The Immunobiology of IL-27 Aisling O’Hara Hall, Jonathan S. Silver, and Christopher A. Hunter

228

Contents of Recent Volumes

Autoimmune Arthritis: The Interface Between the Immune System and Joints Noriko Komatsu and Hiroshi Takayanagi

What is Unique About the IgE Response? Huizhong Xiong, Maria A. Curotto de Lafaille, and Juan J. Lafaille

Immunological Tolerance During Fetal Development: From Mouse to Man Jeff E. Mold and Joseph M. McCune

Prostanoids as Regulators of Innate and Adaptive Immunity Takako Hirata and Shuh Narumiya

Mapping Lupus Susceptibility Genes in the NZM2410 Mouse Model Laurence Morel

Lymphocyte Development: Integration of DNA Damage Response Signaling Jeffrey J. Bednarski and Barry P. Sleckman

Functional Heterogeneity in the Basophil Cell Lineage Mark C. Siracusa, Elia D. Tait Wojno, and David Artis

Index

An Emerging Role of RNA-Binding Proteins as Multifunctional Regulators of Lymphocyte Development and Function Martin Turner and Daniel J. Hodson

Volume 117

Active and Passive Anticytokine Immune Therapies: Current Status and Development Hele`ne Le Buanec, Armand Bensussan, Martine Bagot, Robert C. Gallo, and Daniel Zagury Index

Volume 116 Classical and Alternative End-Joining Pathways for Repair of Lymphocyte-Specific and General DNA Double-Strand Breaks Cristian Boboila, Frederick W. Alt, and Bjoern Schwer The Leukotrienes: Immune-Modulating Lipid Mediators of Disease Antonio Di Gennaro and Jesper Z. Haeggstr€ om Gut Microbiota Drives Metabolic Disease in Immunologically Altered Mice Benoit Chassaing, Jesse D. Aitken, Andrew T. Gewirtz, and Matam Vijay-Kumar

Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression Panagiotis Ntziachristos, Jasper Mullenders, Thomas Trimarchi, and Iannis Aifantis Translocations in Normal B Cells and Cancers: Insights from New Technical Approaches Roberto Chiarle The Intestinal Microbiota in Chronic Liver Disease Jorge Henao-Mejia, Eran Elinav, Christoph A. Thaiss, and Richard A. Flavell Intracellular Pathogen Detection by RIG-ILike Receptors Evelyn Dixit and Jonathan C. Kagan Index

Volume 118 Fate Determination of Mature Autoreactive B Cells Yong-Rui Zou and Betty Diamond Epigenetic Control of Cytokine Gene Expression: Regulation of the TNF/LT Locus and T Helper Cell Differentiation James V. Falvo, Luke D. Jasenosky, Laurens Kruidenier, and Anne E. Goldfeld

229

Contents of Recent Volumes

Autoimmunity in Rheumatoid Arthritis: Citrulline Immunity and Beyond Lars Klareskog, Karin Lundberg, and Vivianne Malmstr€ om

Plasmacytoid Dendritic Cell Development Ken Shortman, Priyanka Sathe, David Vremec, Shalin Naik, and Meredith O’Keeffe

Index

Pathological Consequence of Misguided Dendritic Cell Differentiation in Histiocytic Diseases Marie-Luise Berres, Carl E. Allen, and Miriam Merad

Volume 119 The Interdisciplinary Science of T-cell Recognition Johannes B. Huppa and Mark M. Davis Residual Immune Dysregulation Syndrome in Treated HIV infection Michael M. Lederman, Nicholas T. Funderburg, Rafick P. Sekaly, Nichole R. Klatt, and Peter W. Hunt Developmental Plasticity of Murine and Human Foxp3+ Regulatory T Cells Adrian Liston and Ciriaco A. Piccirillo Logic of the Inflammation-Associated Transcriptional Response Alexander Tarakhovsky Structural Basis of Signal Transduction in the TNF Receptor Superfamily Jixi Li, Qian Yin, and Hao Wu Index

Macrophage Activation and Polarization as an Adaptive Component of Innate Immunity Massimo Locati, Alberto Mantovani, and Antonio Sica Terminal Differentiation of Dendritic Cells Cyril Seillet and Gabrielle T. Belz Diversity of Pathogen Sensors in Dendritic Cells Silvia Cerboni, Matteo Gentili, and Nicolas Manel Transcriptional Control of Dendritic Cell Development Kenneth M. Murphy Transcriptional Control of Macrophage Identity, Self-Renewal, and Function Kaaweh Molawi and Michael H. Sieweke Index

Volume 120

Volume 121

Ontogeny and Functional Specialization of Dendritic Cells in Human and Mouse Muzlifah Haniffa, Matthew Collin, and Florent Ginhoux

Multifarious Determinants of Cytokine Receptor Signaling Specificity Ignacio Moraga, Jamie Spangler, Juan L. Mendoza, and K. Christopher Garcia

Dendritic Cell Migration Through the Lymphatic Vasculature to Lymph Nodes Andrew M. Platt and Gwendalyn J. Randolph

Pathogenic Mechanisms of Bradykinin Mediated Diseases: Dysregulation of an Innate Inflammatory Pathway Allen P. Kaplan and Kusumam Joseph

A Close Encounter of the Third Kind: Monocyte-Derived Cells Alexander Mildner, Simon Yona, and Steffen Jung

The Role of Short-Chain Fatty Acids in Health and Disease Jian Tan, Craig McKenzie, Maria Potamitis, Alison N. Thorburn, Charles R. Mackay, and Laurence Macia

230 Combined Immunodeficiencies with Nonfunctional T Lymphocytes Luigi D. Notarangelo The CD200–CD200R1 Inhibitory Signaling Pathway: Immune Regulation and Host–Pathogen Interactions Christine A. Vaine and Roy J. Soberman Immunopathogenesis of Neuromyelitis Optica Michael Levy, Brigitte Wildemann, Sven Jarius, Benjamine Orellano, Saranya Sasidharan, Martin S. Weber, and Olaf Stuve

Contents of Recent Volumes

Development of Mast Cells and Importance of Their Tryptase and Chymase Serine Proteases in Inflammation and Wound Healing Jeffrey Douaiher, Julien Succar, Luca Lancerotto, Michael F. Gurish, Dennis P. Orgill, Matthew J. Hamilton, Steven A. Krilis, and Richard L. Stevens Why Does Somatic Hypermutation by Aid Require Transcription of Its Target Genes? Ursula Storb Index

Index

Volume 123 Volume 122 Regulation of Immunoglobulin Class-Switch Recombination: Choreography of Noncoding Transcription, Targeted DNA Deamination, and Long-Range DNA Repair Allysia J. Matthews, Simin Zheng, Lauren J. DiMenna, and Jayanta Chaudhuri Two Forms of Adaptive Immunity in Vertebrates: Similarities and Differences Masanori Kasahara and Yoichi Sutoh Recognition of Tumors by the Innate Immune System and Natural Killer Cells Assaf Marcus, Benjamin G. Gowen, Thornton W. Thompson, Alexandre Iannello, Michele Ardolino, Weiwen Deng, Lin Wang, Nataliya Shifrin, and David H. Raulet Signaling Circuits in Early B-Cell Development Michael Reth and Peter Nielsen Interleukin 10 Receptor Signaling: Master Regulator of Intestinal Mucosal Homeostasis in Mice and Humans Dror S. Shouval, Jodie Ouahed, Amlan Biswas, Jeremy A. Goettel, Bruce H. Horwitz, Christoph Klein, Aleixo M. Muise, and Scott B. Snapper

B-Cell Receptor Signaling in Lymphoid Malignancies and Autoimmunity Ana M. Avalos, Friederike Meyer-Wentrup, and Hidde L. Ploegh A Critical Role for Cell Polarity in Antigen Extraction, Processing, and Presentation by B Lymphocytes Dorian Obino and Ana-Maria Lennon-Dumenil Force Generation in B-Cell Synapses: Mechanisms Coupling B-Cell Receptor Binding to Antigen Internalization and Affinity Discrimination Pavel Tolar and Katelyn M. Spillane The Role of BCR Isotype in B-Cell Development and Activation Elena Surova and Hassan Jumaa Index

Volume 124 Group 2 Innate Lymphoid Cells in the Lung Li Yin Drake and Hirohito Kita The Ubiquitin System in Immune Regulation Yoon Park, Hyung-seung Jin, Daisuke Aki, Jeeho Lee, and Yun-Cai Liu

231

Contents of Recent Volumes

How Immunoglobulin G Antibodies Kill Target Cells: Revisiting an Old Paradigm Markus Biburger, Anja Lux, and Falk Nimmerjahn A Transendocytosis Perspective on the CD28/CTLA-4 Pathway Blagoje Soskic, Omar S. Qureshi, Tiezheng Hou, and David M. Sansom How to Trigger a Killer: Modulation of Natural Killer Cell Reactivity on Many Levels Carsten Watzl Roles for Helper T Cell Lineage-Specifying Transcription Factors in Cellular Specialization Amy S. Weinmann MHC Class I Recognition by Monocyte-/ Macrophage-Specific Receptors Ryotaro Yoshida Regulation of Regulatory T Cells: Epigenetics and Plasticity Masahiro Okada, Sana Hibino, Kazue Someya, and Akihiko Yoshmura

Microbes and B Cell Development Duane R. Wesemann Index

Volume 126 NOD.H-2h4 Mice: An Important and Underutilized Animal Model of Autoimmune Thyroiditis and Sjogren’s Syndrome Helen Braley-Mullen and Shiguang Yu Approaches for Analyzing the Roles of Mast Cells and Their Proteases In Vivo Stephen J. Galli, Mindy Tsai, Thomas Marichal, Elena Tchougounova, Laurent L. Reber, and Gunnar Pejler Epithelial Cell Contributions to Intestinal Immunity Lora V. Hooper Innate Memory T cells Stephen C. Jameson, You Jeong Lee, and Kristin A. Hogquist Index

Index

Volume 127

Volume 125

Cross-Presentation in Mouse and Human Dendritic Cells Elodie Segura and Sebastian Amigorena

Regulation of CD4 and CD8 Coreceptor Expression and CD4 Versus CD8 Lineage Decisions Takeshi Egawa Mast Cells’ Integrated Actions with Eosinophils and Fibroblasts in Allergic Inflammation: Implications for Therapy Nadine Landolina, Roopesh Singh Gangwar, and Francesca Levi-Schaffer Positive-Selection-Inducing Self-Peptides Displayed by Cortical Thymic Epithelial Cells Kensuke Takada and Yousuke Takahama Group 2 Innate Lymphoid Cells in the Regulation of Immune Responses Ben Roediger and Wolfgang Weninger

HLA-G: An Immune Checkpoint Molecule Edgardo D. Carosella, Nathalie RouasFreiss, Diana Tronik-Le Roux, Philippe Moreau, and Joel LeMaoult Activation and Function of iNKT and MAIT Cells Shilpi Chandra and Mitchell Kronenberg IgE and Mast Cells: The Endogenous Adjuvant Hans C. Oettgen and Oliver T. Burton RNA Exosome Regulates AID DNA Mutator Activity in the B Cell Genome Evangelos Pefanis and Uttiya Basu Index

232

Volume 128 Regulation and Evolution of the RAG Recombinase Grace Teng and David G. Schatz Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly Gita Kumari and Ranjan Sen Spatial Regulation of V–(D)J Recombination at Antigen Receptor Loci Anja Ebert, Louisa Hill, and Meinrad Busslinger Long-Range Regulation of V(D)J Recombination Charlotte Proudhon, Bingtao Hao, Ramya Raviram, Julie Chaumeil, and Jane A. Skok Dynamic Control of Long-Range Genomic Interactions at the Immunoglobulin κ Light-Chain Locus Claudia Ribeiro de Almeida, Rudi W. Hendriks, and Ralph Stadhouders Regulation of Tcrb Gene Assembly by Genetic, Epigenetic, and Topological Mechanisms Kinjal Majumder, Craig H. Bassing, and Eugene M. Oltz Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires Zachary Carico and Michael S. Krangel Long-Range Control of V(D)J Recombination & Allelic Exclusion: Modeling Views Pernelle Outters, Sebastien Jaeger, Nancy Zaarour, and Pierre Ferrier Index

Volume 129 Rheumatoid Rescue of Misfolded Cellular Proteins by MHC Class II Molecules:

Contents of Recent Volumes

A New Hypothesis for Autoimmune Diseases Hisashi Arase Mechanism of Diapedesis: Importance of the Transcellular Route Marie-Dominique Filippi Evolution of the Humoral Response during HCV Infection: Theories on the Origin of Broadly Neutralizing Antibodies and Implications for Vaccine Design Armstrong Murira, Pascal Lapierre, and Alain Lamarre Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control Ellen V. Rothenberg, Jonas Ungerb€ ack, and Ameya Champhekar Gene Map of the HLA Region, Graves’ Disease and Hashimoto Thyroiditis, and Hematopoietic Stem Cell Transplantation Takehiko Sasazuki, Hidetoshi Inoko, Satoko Morishima, and Yasuo Morishima The Pathogenesis and Immunobiology of Mousepox Luis J. Sigal MAP4K Family Kinases in Immunity and Inflammation Huai-Chia Chuang, Xiaohong Wang, and Tse-Hua Tan Index

Volume 130 Mouse Models of Tumor Immunotherapy Shin Foong Ngiow, Sherene Loi, David Thomas, and Mark J. Smyth The Role of Neoantigens in Naturally Occurring and Therapeutically Induced Immune Responses to Cancer Jeffrey P. Ward, Matthew M. Gubin, and Robert D. Schreiber

233

Contents of Recent Volumes

Tumor and Host Factors Controlling Antitumor Immunity and Efficacy of Cancer Immunotherapy Stefani Spranger, Ayelet Sivan, Leticia Corrales, and Thomas F. Gajewski Immune Contexture, Immunoscore, and Malignant Cell Molecular Subgroups for Prognostic and Theranostic Classifications of Cancers Etienne Becht, Nicolas A. Giraldo, Claire Germain, Aurelien de Reynie`s, Pierre Laurent-Puig, Jessica Zucman-Rossi, Marie-Caroline Dieu-Nosjean, Catherine Saute`s-Fridman, and Wolf H. Fridman Advances in Therapeutic Cancer Vaccines Karrie K. Wong, WeiWei Aileen Li, David J. Mooney, and Glenn Dranoff Combinatorial Cancer Immunotherapies Matthew D. Hellmann, Claire F. Friedman, and Jedd D. Wolchok Adoptive T-Cell Therapy for Cancer James C. Yang and Steven A. Rosenberg Index

Volume 131 Malondialdehyde Epitopes as Targets of Immunity and the Implications for Atherosclerosis N. Papac-Milicevic, C.J.-L. Busch, and C.J. Binder Factors That Regulate the Generation of Antibody-Secreting Plasma Cells Y.-H. Yu and K.-I. Lin

Deep Profiling Human T Cell Heterogeneity by Mass Cytometry Y. Cheng and E.W. Newell Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation N. Sakaguchi and K. Maeda Advances in PET Detection of the Antitumor T Cell Response M.N. McCracken, R. Tavare, O.N. Witte, and A.M. Wu Index

Volume 132 Context- and Tissue-Specific Regulation of Immunity and Tolerance by Regulatory T Cells A. Ulges, E. Schmitt, C. Becker, and T. Bopp Endogenous Retroelements and the Host Innate Immune Sensors X. Mu, S. Ahmad, and S. Hur B-Lymphopoiesis in Fetal Liver, Guided by Chemokines K. Kajikhina, M. Tsuneto, and F. Melchers The Roles of the Secreted Phospholipase A2 Gene Family in Immunology M. Murakami, K. Yamamoto, Y. Miki, R. Murase, H. Sato, and Y. Taketomi Pleiotropic Roles of Type 1 Interferons in Antiviral Immune Responses J.R. Teijaro Index