Adaptive immunity after cell death

Review

Adaptive immunity after cell death Santiago Zelenay and Caetano Reis e Sousa Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

We understand much about the agents, receptors, and signalling pathways that lead to immunity to pathogens. Less is known about how the process is initiated in apparently sterile conditions such as spontaneous immunity to certain tumours, tissue grafts, or autoimmune disorders. Proinflammatory molecules released by dying cells, termed damage-associated molecular patterns (DAMPs), have been proposed to activate dendritic cells (DCs) to promote T cell responses to antigens present in cell corpses. Surprisingly, rather than affecting activation, some recently identified DAMP receptors control specialised DC functions such as antigen acquisition and presentation. This selectivity reveals a new point of control in the regulation of adaptive immunity and, potentially, tolerance that renders DAMPs nonredundant players in responses to both sterile and nonsterile insults. The response to infection and injury Injury or infection in multicellular organisms triggers a sophisticated sequence of reactions, known as the inflammatory response, that plays a crucial role in minimising the insult and repairing damaged tissue. In the case of infection, inflammation helps to contain and subsequently eliminate the invading organism. In sterile injury, the inflammatory response clears dead tissue and promotes wound healing. Although they share common features, sterile and nonsterile inflammation differ fundamentally in terms of their initiation, regulation, and function. The past two decades have witnessed an explosion of knowledge on the receptors and signalling pathways that initiate inflammatory responses to pathogens. By contrast, the mechanisms underlying sterile inflammation in response to damage remain somewhat obscure despite the growing realisation that this process may be a major component of tissue homeostasis and, when dysregulated, a contributing factor to chronic diseases ranging from atherosclerosis to neurodegeneration [1]. Furthermore, injury and infection often overlap and our understanding of immunity therefore necessitates consideration of the interplay between the processes that detect pathogen invasion and those that sense host damage. Finally, in vertebrates, the pathways involved in initiating both sterile and nonsterile inflammation can additionally lead to T and B cell priming. This forms the basis of acquired immunity to infection, but may also underlie antigen-specific responses to tumours and allografts, as well as cause autoimmunity. Here, we discuss Corresponding author: Reis e Sousa, C. ([email protected]) Keywords: inflammation; infection; dead cell; adjuvant; injury. 1471-4906/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2013.03.005

recent progress on understanding the sterile inflammatory response, with a particular focus on cell death as the initiating factor, and the subsequent generation of adaptive immune responses to dead-cell-associated antigens. Sterile inflammatory response Inflammation is an umbrella term that denotes the functional consequences of innate immune responses. The concept that inflammation can result from tissue injury dates back to the time of the Romans. Far more recent, but still long known, is the notion that the innate immune system mediates this process and that cell death is a key initiating factor. The work of Metchnikoff at the beginning of the 20th century already highlighted the significance of phagocytosis as an evolutionarily conserved process, not only in host defence but also in the removal of damaged and dead cells [2,3]. However, not all types of cell death promote an inflammatory reaction. In multicellular organisms, cells die continuously during development and tissue regeneration and this type of programmed cell death does not normally function as a proinflammatory event. In fact, engulfment of apoptotic bodies often causes phagocytes and other cells to secrete transforming growth factor (TGF)-b, interleukin (IL)-10, and prostaglandin E2; all of which dampen inflammation [4–6]. By contrast, aberrant or nonhomeostatic cell death, such as occurring upon injury or during infection, often triggers an inflammatory reaction. According to current views, this arises from loss of cell membrane integrity in dead cells, which allows the release and/or exposure of intracellular molecules that recruit and stimulate cells of the innate immune system [7]. These proinflammatory stimuli of self-origin have been termed DAMPs and the identity of some of them has been uncovered during the past decade (Box 1). They add up to a diverse list of compounds including uric acid, DNA and RNA, spliceosome-associated protein 130 (SAP130), high mobility group box 1 (HMGB1), high mobility group nucleosome binding domain 1 (HMGN1), H2O2, and ATP (Table 1). Although it is often thought that DAMP release equates to necrotic cell death, it is important to note that it can in some cases follow apoptosis; in particular if apoptotic bodies are not rapidly cleared by scavenger cells and allowed to undergo secondary disintegration [8–10]. Moreover, in recent years, other types of nonapoptotic programmed cell death have been uncovered, including pyroptosis and necroptosis, which also can cause DAMP release [11,12]. Coupling innate to adaptive immunity The inflammatory response triggered by the detection of DAMPs is an evolutionarily conserved mechanism present Trends in Immunology, July 2013, Vol. 34, No. 7

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Review Box 1. What is a DAMP? A DAMP is generally defined as a mediator released by damaged cells that acts to stimulate inflammation or immunity. By this definition, eat-me signals exposed by dying cells in most cases are not DAMPs, because they are generally associated with silent, antiinflammatory, or tolerogenic responses. However, it is becoming clear that immunogenic dead cells also emit signals that contribute to immunity by engaging receptors that facilitate corpse uptake or processing of dead-cell-associated antigens (see main text). These findings suggest a broader use for the term DAMP, to include molecules released by dead cells that contribute to immunity even if they are not stimulatory for DCs. By analogy, some PAMPs also act to promote pathogen uptake without signalling for DC activation [13]. The role of some DAMPs in regulating antigenicity rather than adjuvanticity (Box 2) raises the theoretical possibility that, in some instances, they could potentially contribute to antigen-specific tolerance rather than immunity to dead cells (see main text), in which case the definition of the term DAMP may need to be revised.

in both invertebrates and vertebrates. It is thought to help promote wound healing and tissue repair, in addition to maintaining wound sterility. However, in vertebrates, the existence of an adaptive immune system raises an important consideration, namely, whether the recognition of DAMPs also plays a role in the initiation of T or B cell responses against antigens present within damaged cells. In fact, at the origin of the term DAMP is the proposal that cell death triggers adaptive immunity against dead-cellassociated antigens. A major function of adaptive immunity is host defence against infectious organisms. Host protective B and T cell responses to infection are induced by antigen-presenting cells (APCs), which become activated upon pathogen encounter. As predicted by Janeway [13], activation of APCs can result from signals by pattern-recognition receptors (PRRs) that bind to molecular structures present in the invading microorganism. These structures are termed pathogen-associated molecular patterns (PAMPs) and include lipopolysaccharide (LPS), peptidoglycan, b-glucans, flagellin, double-stranded RNA, and RNA bearing 50 triphosphates. Such PAMPs can be variably detected by PRRs belonging to the family of Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), among others. DCs are widely believed to constitute the primary APCs regulating adaptive immunity. PRR signalling greatly augments the T cell stimulatory potential of DCs by potentiating antigen processing and presentation, inducing the expression of chemokine receptors that allow DC migration to T cell areas of secondary lymphoid tissues and promoting synthesis of co-stimulatory molecules and cytokines that act on T cells to promote their expansion and effector differentiation [14,15]. Inducing these changes in DCs is the purpose of adding adjuvants to antigens in vaccination protocols and many microbial products are powerful adjuvants, consistent with their identity as PAMPs. However, some adaptive immune responses occur in the apparent absence of infection or PAMP recognition. This is the case of spontaneous and therapy-induced T cell responses against certain tumours, rejection of histoincompatible tissue transplants and several autoimmune disorders. In addition, some widely used adjuvants, such as alum, are of nonmicrobial origin. These observations led 330

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Matzinger to propose that detection of stressed or damaged cells by DCs is the primary force driving adaptive immune responses [16,17]. According to this so-called ‘danger model’, the key event in the initiation of adaptive immunity is the release or exposure of immunostimulatory intracellular molecules, that is, DAMPs [18], regardless of the presence of PAMPs. The terms PAMP and DAMP have stuck and are used in a wider context nowadays to denote molecules of microbial or self-origin that induce immune responses, even in organisms such as insects that do not possess an adaptive immune system (Box 1). Much evidence supports the concept that DAMPs contribute to adaptive immunity in vertebrates. For example, in a mouse model, the adjuvanticity of alum appears to be due to its toxicity and consequent release of DNA from dead cells at the site of vaccination [19]. Furthermore, deliberate immunisation with dead cells bearing a foreign antigen often provokes an immune response such as, for example, the generation of antitumour CTLs by vaccination with dead tumour cells [20]. In the same vein, some of the beneficial effects of radio- or chemotherapy in cancer appear to be due to their ability to incite ‘immunogenic tumour cell death’ and prime or boost the generation of specific T cell responses against tumour antigens [21–23]. The features of immunogenic tumour cell death elicited by administration of chemotherapeutic agents have been extensively characterised in recent years and involve calreticulin exposure, release of HMGB1 and ATP discharge [9]. These molecules act on DCs to promote phagocytosis of corpses, antigen processing, and inflammasome activation, collectively promoting T cell priming against tumour antigens [9]. Additional DAMPs are implicated in immunity to dead-cell-associated antigens such as, for example, uric acid, heat shock proteins (HSPs), or granulysin [7,24,25]. Finally, the importance of DAMPs is emphasised by the fact that they are actively regulated in nonimmunogenic situations. For instance, in cells undergoing nonimmunogenic apoptosis, the proinflammatory effects of HMGB1 and the cytokine IL-33 are limited by oxidation [26,27] and caspase-mediated cleavage [28], respectively. Is DAMP recognition sufficient to initiate adaptive immunity? Even if DAMP recognition contributes to adaptive immunity, a key question is whether it is sufficient. This is important because even if individual DAMPs can be shown to have proinflammatory properties, it does not mean that they necessarily possess the ability to promote DC activation and adaptive immunity to associated foreign antigens (i.e., adjuvanticity – Box 2). For example, the original discovery of uric acid as a DAMP focused on its role as an endogenous adjuvant released from dead cells that could promote CTL responses to nonimmunogenic antigen-coated beads [24]. However, most of the subsequent studies have focussed on the ability of uric acid to induce inflammasome activation and promote inflammation (e.g., neutrophilia), including precipitating the autoinflammatory disease, gout. Importantly, none of these processes involve antigen-specific lymphocyte responses. In fact, many of the DAMPs in Table 1 have proinflammatory properties but remarkably few have been conclusively

Review

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Table 1. DAMPs and DAMP receptors DAMP HMGB1 Uric acid DNA RNA HSPs SAP130 Tenascin-C HMGN1 ATP IL-1a IL-33 S100 proteins b-Amyloid Oxidised low-density lipoprotein Granulysin H2O2 Hyaluronan Heparan sulfate Biglycan Versican Mitochondrial formyl peptides Calreticulin Filamentous actin

Receptor TLR4, TLR2, RAGE, CD24, and others NLRP3 and others TLR9, AIM2, and others TLR3, TLR7, and TLR8 TLR2, TLR4, CD14, CD24, CD91, and others Mincle TLR4 TLR4 P2X7 IL-1R ST2 RAGE NLRP3, RAGE, TLR4, TLR6, and CD36 TLR4, TLR6, and CD36

Refs [31,70] [7,71] [36,41,72,73] [36,40,42] [7,74] [32] [45] [65] [9,37] [75,76] [63] [77] [78–80] [80]

TLR4 Lyn TLR2, TLR4, and CD44 TLR4 TLR2 and TLR4 TLR2 FPR1

[25] [81,82] [83,84] [85] [86] [87] [88]

CD91 DNGR-1

[9,21] [52,53]

proven to act as adjuvants in the absence of PAMP contamination. As such, it is important to establish whether DAMPs can fully substitute for PAMPs in initiating adaptive immunity. Evidence that this may be the case is the aforementioned observation that T cell responses can be generated against antigens associated with dead cell corpses in the apparent absence of microbial infection. However, all cells have endogenous retroviruses and retroelements that naturally generate viral PAMPs [29]. In addition, commensal microbes and their constituents are a factor in most animal experiments [30] and therefore PAMPs present in the animals or the environment can never be excluded. Thus, whether there truly is something as sterile adaptive immunity to foreign antigens remains to be conclusively demonstrated. This possibility needs to be explored more rigorously, not only from its basic research implications but also because it could help mitigate the current dearth of adjuvants available for use in human vaccination. DAMP receptors Several receptors that bind DAMPs have been identified (Table 1), although not all have been shown to mediate DC activation and the adjuvant effects of DAMPs. Nevertheless, polymorphisms in some of these receptors have been reported to associate with prognosis in breast cancer patients treated with chemotherapeutic drugs that induce immunogenic cell death [22,23]. Interestingly, many DAMP receptors also function as PRRs for microbial components. For instance, TLR4 can act as a sensor for

self-molecules such as HMGB1, HMGN-1, HSPs, as well as degradation products of the extracellular matrix (Table 1), in addition to its better-known function as an LPS receptor [31]. The CLR, Mincle, is involved in the recognition of SAP130, a small nuclear ribonucleoprotein component released from dead cells, but also of a-mannose in Malassezia and some Candida fungal species, as well as trehalose-6,60 -dimycolate, a mycobacterial cell wall glycolipid [32–35]. The nucleic acid sensors TLR7 and TLR9 can be triggered by both foreign and self-nucleic acids [36]. Additionally, the NLRP3-inflammasome triggers the secretion of bioactive IL-1b in response to DAMPs such as ATP or uric acid or after infections with viruses, fungi, and bacteria [37,38]. The finding that DAMPs can be detected by the same receptors that recognise PAMPs could be seen as support for the notion that DAMPs and PAMPs act similarly and have equivalent properties; most notably when it comes to coupling innate to adaptive immunity. Consistent with that notion, some DAMPs induce changes in DCs that are reminiscent of those observed in response to PAMPs, including the upregulation of MHC and co-stimulatory molecules and production of some cytokines. This has been extensively documented, for example, for DC stimulation by self-DNA and RNA [36,39–42]. However, it need not always be the case. For example, it is possible that the immunogenicity of dead cells involves a type of DC activation distinct from that seen in response to pathogen encounter. As such, assays for DC activation developed on the basis of the response to PAMPs may fail to detect DAMPinduced changes that might be relevant for T cell priming. This hypothesis is predicated on the concept that innate immune receptors are able to respond differently to DAMP and PAMP ligands. This has been argued, for example, for TLR4 based on the notion that HMGB1, Tenascin C, HSP60, and hyaluronan fragments induce distinct patterns of gene expression that do not always overlap with those induced by LPS [43–47]. It is presently unclear how ligand discrimination might be achieved but a possibility may be that some DAMP ligands act as partial agonists for PRRs, eliciting only a subset of signalling responses. Such partial agonism has been observed for other immune receptors that can recognise self and foreign ligands such as the TCR [48]. It can be facilitated by co-receptors that specifically recognise the DAMP or the PAMP ligand and modulate signalling from the shared receptor. For example, HMGB1 but not LPS binds CD24, which, through association with Siglec-G in humans or Siglec-10 in mice, specifically inhibits the TLR4-dependent activation of nuclear factor (NF)-kB [49]. An important area for future research will be the systematic characterisation of DAMP versus PAMP responses induced by individual innate immune receptors. This may allow the design of treatments that specifically dampen the inflammatory and/or immunogenic response to tissue damage but not to microbial components. Decoding of dead cell antigenicity by DAMP receptors The original Danger model predicted the existence of dedicated DAMP receptors in DCs that specifically regulate adaptive immunity. Our own research has led to the identification of one putative such receptor, DNGR-1 (also known as CLEC9A), which binds dead cells and 331

Review Box 2. Antigenicity and adjuvanticity The immunogenicity of a given stimulus, that is the ability to generate an antigen-specific response, can be considered as the combination of two basic properties: antigenicity and adjuvanticity. Antigenicity denotes the ability of a given immunogen to be seen by lymphocytes. For T cells, this implies that the material is accessible to APCs, can be engulfed and processed into peptides suitable for loading onto MHC molecules, and is subsequently displayed. Antigenicity requires, in addition, the existence of a repertoire of lymphocytes specific for the antigen in question. Adjuvanticity, by contrast, refers to the property of a substance to promote the priming of lymphocytes against an associated antigen. In the case of T cells, this is thought to require that the substance in question induces the activation of DCs (e.g., upregulation of co-stimulatory molecules, or production of cytokines) such that they become stimulatory for T cells. Distinguishing antigenicity from adjuvanticity is often difficult because most DC activators additionally potentiate the ability of the cells to acquire, process, and present antigens. The study of responses to DAMPs has revealed that, in some cases, DCs regulate antigen processing independently of other activation parameters. This can selectively affect antigenicity and act as an important point of control of adaptive immunity even in situations when DCs are fully activated.

contributes to crosspriming of cytotoxic T cell responses against dead-cell-associated antigens [50]. DNGR-1 is a CLR selectively expressed at highest levels by a subset of mouse and human DCs, often known as the ‘CD8a+ DC family’. These DCs stand out for their ability to capture and process dead-cell-derived material for crosspresentation to CD8+ T cells [51]. The DAMP recognised by DNGR-1 has been recently identified by us and others as F-actin; a universal and abundant component of the cytoskeleton that is exposed in cells that have lost membrane integrity [52,53]. Thus, cytoskeletal recognition by innate immune cells can serve as a means of detecting damage and initiating suitable immune responses. DNGR-1 bears an intracellular tail with a modified immunoreceptor tyrosine-based activation motif (termed hemITAM) that allows signalling via spleen tyrosine kinase (Syk) [50,54]. The structurally related CLR, Dectin-1, functions as a PRR for b-glucans from fungi and bacteria and contains an analogous hemITAM motif that allows it to function as an activating receptor for myeloid cells, promoting NF-kB activation and proinflammatory cytokine production [55]. Based on the similarity with Dectin1, we hypothesised that DNGR-1 signals via Syk to promote DC activation in response to F-actin exposed or released by dead cells. Indeed, dead cells weakly activate DCs, for example, inducing upregulation of co-stimulatory molecules and production of low levels of IL-12/23 p40 [56]. However, DNGR-1 is dispensable for such activation, which is therefore likely to be attributable to DAMPs other than F-actin [56]. Rather, DNGR-1 plays a nonredundant role in DC handling of dead cell cargo, diverting the latter into a subcellular nondegradative recycling endosome that favours antigen extraction for crosspresentation [56] (Figure 1). This alone appears to account for the functions of the receptor in immunity as the defect of DNGR-1-deficient DCs in crosspriming CD8+ T cells against dead-cell-associated antigens can be rescued by inhibition of lysosomal degradation [57]. Uptake and diversion of soluble ovalbumin into nondegradative 332

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endosomes for crosspresentation has similarly been reported for another CLR, the mannose receptor [58]. It is presently unclear by which mechanisms DNGR-1 diverts dead cell cargo into nonlysosomal compartments and how Syk-dependent signalling favours crosspresentation of antigens contained in the corpses (Figure 1). Nevertheless, these findings demonstrate that a DAMP receptor can affect adaptive immunity, not by controlling DC activation, as predicted by the Danger model, but by selectively affecting antigen processing. Of note, a similar phenomenon has been observed during the study of tumour-specific immune responses induced by chemotherapy in mice [22]. These responses depend on TLR4 engagement on DCs by HMGB1 released from dying tumour cells (Figure 1). However, TLR4 signalling in this context does not affect the activation status of the DCs but is selectively required for the efficient processing and crosspresentation of antigens from dead tumour cells [22]; another example of partial agonism. Finally, ‘eat-me’ signals such as calreticulin on immunogenic dead cells [21] can be seen as additional DAMPs that primarily contribute to antigen presentation rather than DC activation, by promoting dead cell (i.e., antigen) uptake by DCs (Figure 1, Box 1). Thus, PAMPs and DAMPs can, in some instances, control distinct aspects of T cell priming by differentially impacting antigenicity and adjuvanticity (Box 2). If some DAMP receptors, such as DNGR-1, selectively affect antigenicity, they might be expected also to play a role in the induction of T cell tolerance to antigens from dead cells. This might be the case if such receptors are to be selectively engaged. However, in normal circumstances, engagement of the receptors does not occur in isolation. For example, the DNGR-1 ligand is only revealed when dead cells lose membrane integrity [50,52,53]. This exposes additional DAMPs that likely serve to activate DCs and bias the subsequent T cell response towards immunity rather than tolerance. Conversely, tolerogenic responses to dead cells are often associated with nonimmunogenic apoptotic cells, which retain membrane integrity and therefore do not expose F-actin. Consistent with that notion, DNGR-1 deficiency does not have an impact on a mouse model of crosstolerance to pancreatic antigens that is likely driven by antigen contained within apoptotic b cells [56]. Thus, despite acting as a nonactivating receptor for DCs, DNGR-1 primarily functions to regulate immunity rather than tolerance to dead-cell-associated antigens by virtue of its function as a DAMP receptor. A final point of regulation of dead cell antigenicity is the antigen donor cell itself. Several reports have emphasised that the mode of cell death can profoundly influence the subsequent availability of antigens for crosspriming. For example, induction of autophagy prior to cell death can facilitate antigen delivery to DCs [59]. In addition, the pool of antigenic substrates for crosspresentation can be altered by the process of cell death itself. Indeed, caspase-mediated cleavage of cellular proteins during apoptosis has been shown to generate neoantigens that act as efficient substrates for DC crosspresentation and can lead to priming of autoreactive CD8+ T cells [60]. Thus, the ability of dead cell antigens to lead to T cell priming via DCs will depend

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Dead cell

Calreculin

Acn filament

DNA HMGB1

Dendric cell Uptake receptor

TLR4

L X X Y P

L X X P Y

DNGR-1

S Y K

Nucleus

Early/recycling endosomes Gene transcripon leading to DC acvaon Crosspresentaon of dead cell-associated angens TRENDS in Immunology

Figure 1. Damage-associated molecular pattern (DAMP) exposure promotes adaptive immunity to dead-cell-associated antigens. Damaged cells expose or release DAMPs such as high mobility box group 1 (HMGB1), DNA, calreticulin, or F-actin. Some DAMPs engage receptors expressed by dendritic cells (DCs) and contribute to specific immunity to antigens present in dead cells by inducing DC activation. Other DAMP receptors facilitate the engulfment or the trafficking of dead cell material to early/ recycling endocytic compartments favouring antigen extraction for crosspresentation.

dually on the antigenic status of the corpse and the DAMP receptors that it encounters. Cell death sensing in immunity to infection The notion that DAMP and PAMP sensing can control different aspects of DC function is also apparent in situations when both are present. This is the case, for example, when DCs encounter dying cells infected with cytopathic viruses or bacteria. Interestingly, and contrary to expectations, redundancy between the two types of sensors is not observed. For example, phagocytosis of dying virusinfected cells leads to CTL responses that are highly dependent on TLR3, which senses the viral PAMP (double-stranded RNA) content of the corpse [61]. Similarly, uptake of dead cells by DCs is accompanied by TLR-dependent production of IL-6 if the corpse contains bacterial PAMPs, which can lead to priming of IL-17-producing T cells [62]. Recent data indicate that the converse is also true; namely that DAMP recognition is likewise nonredundant for effective immunity to cytopathic infection. For example, IL-33, a DAMP released by necrotic cells has been recently shown to play a major role in the generation of an effective CTL response to viral infections, by acting directly to promote expansion and effector differentiation of newlyprimed CD8+ T cells [63]. Finally, our own work has revealed a nonredundant role for DNGR-1 in the generation of an efficient CTL response after infection with herpes simplex or vaccinia viruses [56,57].

Independently of redundancy or lack thereof, there is interdependence between DAMP and PAMP recognition that has only recently come to be widely appreciated. For example, the ability of the PAMP LPS to induce toxic shock in mice is greatly potentiated by the DAMP HMGB1 [64]. Similarly, the ability of LPS to act as an adjuvant for priming of antigen-specific responses appears to depend on a distinct DAMP, HMGN1 [65]. Conversely, the adjuvant properties of many DAMPs may themselves depend on LPS contamination, as already discussed earlier [66,67]. Finally, microbes subvert the immune response in part by manipulating cell death pathways, which shows that they have understood the interrelatedness of PAMP and DAMP sensing long before researchers in the field [68,69]. The study of synergistic and/or antagonistic effect of PAMPs and DAMPs on innate and adaptive immune responses is therefore an important area for further investigation. Concluding remarks DAMP recognition is an evolutionarily ancient mechanism for induction of inflammation that pre-dates adaptive immunity. However, there is accumulating evidence that the release or exposure of DAMPs also contributes to immunogenicity in vertebrates. Some DAMPs engage PRRs commonly involved in the direct recognition of microbial components. These DAMPs may in some instances promote specific immunity against dead-cell-associated antigens by inducing the activation of DCs, effectively acting as 333

Review mediators of the adjuvanticity of dead cells. Other DAMPs fail to induce DC activation but contribute to the adaptive immune response by affecting the presentation of dead-cellassociated antigens, either by facilitating engulfment of corpses (eat-me signals such as calreticulin) or by contributing to antigen extraction and processing (‘present-me signals’ such as F-actin or HMGB1). Finally, PAMPs and DAMPs are not redundant in the induction of adaptive immunity and their interdependence becomes most apparent in immunity to cytopathic pathogens, which relies on both PAMP and DAMP sensing. Most of what we know about adaptive immune responses and DC activation comes from the study of responses to PAMPs. The next decade is likely to witness a rapid increase in our knowledge of the receptors and pathways underlying sterile inflammation. It will be interesting to find out how many will influence the function of DCs and impact adaptive immunity. Acknowledgements We are grateful to David Sancho and members of the Immunobiology laboratory for critical review of the manuscript. Work in the CRS laboratory is funded by Cancer Research UK, a prize from Fondation Bettencourt-Schueller and a European Research Council Advanced Researcher Grant.

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