Epigenetic Regulation of T Cell Memory: Recalling Therapeutic Implications

Please cite this article in press as: Tough et al., Epigenetic Regulation of T Cell Memory: Recalling Therapeutic Implications, Trends in Immunology (2019), https://doi.org/10.1016/j.it.2019.11.008

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Feature Review

Epigenetic Regulation of T Cell Memory: Recalling Therapeutic Implications David F. Tough,1 Inma Rioja,1 Louise K. Modis,2 and Rab K. Prinjha1,2,* Memory T cells possess functional differences from naı¨ve T cells that powerfully contribute to the efficiency of secondary immune responses. These abilities are imprinted during the primary response, linked to the acquisition of novel patterns of gene expression. Underlying this are alterations at the chromatin level (epigenetic modifications) that regulate constitutive and inducible gene transcription. T cell epigenetic memory can persist long-term, contributing to long-lasting immunity after infection or vaccination. However, acquired epigenetic states can also hinder effective tumor immunity or contribute to autoimmunity. The growing understanding of epigenetic gene regulation as it relates to both the stability and malleability of T cell memory may offer the potential to selectively modify T cell memory in disease by targeting epigenetic mechanisms.

T Cell Memory: An Imprinting Process Memory is a fundamental and essential feature of the immune system, allowing the long-term pathogen-specific protection that is provided by initial infection or vaccination. While memory T cells (see Glossary) are well known to have altered functional properties compared with naı¨ve T cells, the cellintrinsic mechanisms underlying memory T cell functionality are only now beginning to be elucidated. Recent advances in the field of epigenetics are providing key insights into the molecular processes controlling T cell memory. An immensely diverse T cell repertoire is generated through genetic recombination of T cell receptor (TCR) gene segments during T cell development in the thymus. Consequently, the frequency and absolute number of T cells with reactivity to individual antigenic peptides to which the host has not been previously exposed, is extremely low. In humans, the frequency of epitope-specific naive CD8+ T cells has been found to be in the range of 1/200 000 to less than 1/1 000 000 cells, depending on the antigen [1]. Upon initial exposure to an infectious agent, rare cells present in the naı¨ve T cell pool, with specificity for antigens in the pathogen selectively undergo massive expansion, and concurrent differentiation to generate a large population of effector CD4+ and CD8+ T cells, possessing functional activities that can directly or indirectly kill the pathogen and/or pathogen-infected cells [2]. In an infection where the pathogen is cleared or contained by the immune response, the great majority of effector T cells subsequently die [3]. The surviving cells return to a more resting state as the antigenic stimulus disappears and they persist as memory T cells. Memory T cells with specificity for the pathogen are maintained at a much higher frequency than amongst T cells that existed in the naı¨ve state, which is in turn critical for the host’s ability to mount a faster response upon re-infection with the same pathogen. T cell memory is extremely long-lived and this is exemplified by the elevated frequency of circulating memory T cells reactive with smallpox antigens detected 83 years postinfection in humans [4]. In addition to surviving with expanded clone sizes, memory T cells also possess important differences from naı¨ve cells at the individual cell level that allow them to function more efficiently during a secondary response [5]. This includes distinct migratory properties such that memory T cells localize quickly to the sites of pathogen entry, with the ability to rapidly generate effector functions. However, memory T cells are well-recognized to be phenotypically and functionally heterogeneous. Seminal early insights into this heterogeneity resulted in an initial subdivision of human circulating memory T cells into central memory (TCM) cells that recirculate, like naı¨ve T cells between blood and lymph, and effector memory (TEM) cells, which have a migration pattern that takes them between blood and inflamed tissues and are capable of immediate expression of effector cytokines [6]. Subsequently,

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Highlights Precise control of gene expression is achieved by epigenetic mechanisms involving covalent modifications to DNA and histones that alter chromatin structure. T cell activation and differentiation into effector cells is accompanied by widespread epigenetic modifications and marked changes in gene expression. Memory T cells retain a long-term epigenetic imprint that confers constitutive and inducible gene expression associated with a rapid recall response capacity. Exhausted T cells generated in response to chronic antigen exposure are functionally defective and epigenetically distinct from both effector and memory T cells. Blocking inhibitory receptors on Tex cells transiently restores function but has little effect on the epigenetic memory of these cells. Epigenetic mediators have been identified that can modulate T cell differentiation into effector, memory, and exhausted cells.

1Epigenetics Research Unit, Oncology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK 2Adaptive Immunity Research Unit, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK

*Correspondence: [email protected]

https://doi.org/10.1016/j.it.2019.11.008 ª 2019 Published by Elsevier Ltd.

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further subpopulations of memory T cells have been described, including resident memory T (TRM) cells that do not circulate, but rather persist as a self-renewing population in peripheral sites such as the epithelium of the skin or mucosal tissues in mice and humans [7]. TRM cells can respond immediately to re-infection at the site of pathogen entry with the production of effector molecules and have been shown to play a key role in infection- or vaccine-induced protection against certain viruses, bacteria, and parasites [8–11]; mediate surveillance against epithelial tumors [12]; and contribute to chronic inflammation and autoimmunity [13–15]. The modified functional properties of memory T cells are intrinsically linked to changes in gene expression patterns compared with naı¨ve T cells. This includes altered constitutive gene expression; for example, TEM cells turn off expression of genes encoding receptors required for homing to lymph nodes, including CCR7 and CD62L, while turning on expression of genes encoding receptors for inflammatory chemokines such as CCR1, CCR3m and CCR5, across various mammalian species [6]. Additionally, genes in effector CD4+ and CD8+ memory T cells can be ‘primed’ for rapid expression upon reactivation; this is the case for effector cytokines such as interferon (IFN)-g in TEM cells in contrast to naı¨ve CD4+ and CD8+ T cells, which require several rounds of cell division before acquiring the ability to express cytokines other than IL-2. The imprinting of gene expression patterns is mediated by epigenetic mechanisms that act during T cell activation and differentiation and are maintained in memory T cells, even following cell division [16]. Here, we provide a brief overview of epigenetic mechanisms that regulate gene expression and review recent findings showing how these processes contribute to CD4+ and CD8+ effector T cell differentiation, the generation and maintenance of memory CD4+ and CD8+ T cells, and the development of ‘dysfunctional’ (exhausted) CD8+ T cells. Finally, we discuss the exciting emerging potential to manipulate epigenetic mechanisms to modulate T cell memory for benefit in human disease.

Epigenetic Mechanisms Regulating Gene Expression Gene expression is constrained and controlled to a significant extent by DNA packing, in the form of chromatin, into the cell nucleus (Figure 1). The basic unit of chromatin is the nucleosome, comprised of 147 base pairs of DNA wrapped around a core of eight histone proteins. Nucleosomes can be further packaged, together with additional histones, into highly ordered and compact structures. Gene expression is linked to the extent and density of DNA packing into chromatin. Thus, in regions of dense nucleosome packing, referred to as heterochromatin, DNA is inaccessible to the transcriptional machinery and genes in these regions are generally silent. Conversely, expressed genes are found in areas where chromatin is less condensed, referred to as euchromatin, with much more subtle differences in chromatin structure providing fine control of gene expression. In addition, long-range interactions between distantly separated regions of DNA, such as those between gene enhancers and promoters, provide an essential level of transcriptional regulation. Of relevance, chromatin structure is not static and can be markedly, and specifically, modified in response to signals received by a cell. Processes that regulate gene expression by modulating chromatin accessibility are referred to as epigenetic mechanisms, based around two broad classes of covalent modifications. First, DNA itself can be modified, which in mammals occurs primarily through methylation of cytosine bases by DNA methyltransferases (DNMTs) to produce 5-methylcytosine (5mC). DNMT3a and DNMT3b are termed de novo DNMTs, responsible for depositing new 5mC modifications, while DNMT1 functions mainly in propagating existing 5mC marks through cell division and DNA replication based on its preferential activity on hemi-methylated DNA [17]. Cytosine methylation occurs mainly within cytosine-guanine (CpG) di-nucleotides; 5-hydroxymethylcytosine (5hMC), formed as an intermediate during cytosine demethylation through the activity of ten-eleven translocation (TET) enzymes, also acts as an epigenetic mark. Second, histone tails are subject to a wide range of post-translational modifications, including acetylation, methylation, and phosphorylation amongst many others, which can occur on several different amino acids [18]. Some histone modifications can be of different valencies (e.g., lysine residues may be mono-, di-, or trimethylated), producing even further diversity on the

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Glossary Bivalent modifications: distinctive histone modification signature characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Central memory (TCM) cells: CD45R0+ memory T lymphocyte subset constitutively expressing CCR7 and CD62L. The cells home to T cell areas of secondary lymphoid organs, have little or no effector function, but readily proliferate and differentiate into effector cells in response to antigenic stimulation, providing reactive immunological memory. Chimeric antigen receptor (CAR)T cell: T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. CARs are chimeric because they combine both antigen-binding and TCR. Chromatin: form in which DNA is found in eukaryotic cell nuclei in combination with histone proteins. DNA methyltransferase (DNMT): enzyme capable of catalyzing the transfer of a methyl group to DNA. Epigenetic erasers: enzymes that catalyze the removal of epigenetic marks onto either histone tails or the DNA itself. Effector memory (TEM) cells: subset of memory T lymphocytes (CCR7–) that migrate to inflamed peripheral tissues and display immediate effector function in response to antigenic stimulation, providing protective immunological memory. Effector T cells: activated T cells with functions such as cytotoxic activity or cytokine production. Epigenetic writers: enzymes that catalyze the addition of chemical groups (acetylation, methylation, phosphorylation, and ubiquitination amongst others) onto either histone tails or the DNA itself. These modifications are known as epigenetic marks. Exhausted T cells (TEX): T cells with defective effector function due to chronic antigen exposure. Gene enhancer: region of DNA located away from the transcription start site or gene promoter that can enhance expression of a gene. Enhancers can be located upstream or downstream for the

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epigenetic landscape. Modifications to histones are highly dynamic and reversible, with families of enzymes mediating the addition (epigenetic writers) or removal (epigenetic erasers) of these marks in a highly specific way [19,20]. Due to the multiplicity of different possible covalent modifications, a large number of combinations of epigenetic marks can be generated, enabling an incredible level of specificity, control, and memory of any given gene’s function, status, and history. Studies investigating the relationship between the occurrence of post-translational chromatin modifications and gene expression at a genome-wide level have identified distinct patterns of modifications, or epigenetic signatures, that are associated with groups of genes showing similar expression characteristics. For example, genes that are constitutively expressed, silenced, or inducibly expressed, each have distinct combinations of DNA and histone modifications at their promoters. Specific epigenetic features of gene enhancers have also been described and are often used to identify these regions (Figure 2). Epigenetic signatures translate into different states of gene expression, mainly by serving as recognition sites for chromatin-binding ‘reader’ proteins. Thus, depending on the presence or absence of different epigenetic modifications, distinct sets of proteins will be recruited to genetic loci. These readers, which often form large protein multicomplexes, can bring with them enzymatic activities that create a more open or closed chromatin structure, promote/inhibit long-range chromatin interactions, or recruit proteins required for transcriptional initiation or elongation. Additionally, some histone modifications can modify chromatin structure directly by affecting nucleosome stability [18], while DNA methylation can alter the binding of transcription factors (TFs). While epigenetic writers, erasers, and readers do not typically possess intrinsic DNA sequence specificity, they impose patterns of chromatin structure and gene expression in a cell- and stimulus-specific way by functioning together with TFs and noncoding RNAs that direct these proteins to precise genomic locations. So-called ‘pioneer’ factors are thought to be particularly important in establishing new regions of open chromatin because of their ability to bind to and further expose specific DNA sequences within closed chromatin, where genomic DNA is not readily accessible [21]. However, the ability of many TFs to bind DNA is also determined by the pre-existing chromatin landscape, so there is an important interdependency between these two mechanisms. TFs induced by TCR or cytokine signaling have been long known to guide the generation of widespread chromatin modifications in activated T cells [22]. Whereas TFs largely dictate where chromatin modifications occur, the types of modifications generated during memory formation are influenced by other factors regulated following T cell activation, including changes in the expression of epigenetic writers, erasers, and readers and post-translational modifications that modulate their activity. In addition, alterations in cellular metabolism make important contributions to epigenetic modulation through metabolites or metabolic enzymes, which can modify chromatin directly or indirectly [23]. TCR and cytokine signaling induce rapid metabolic changes; in mice, activation and differentiation of naı¨ve CD4+ and CD8+ T cells into various effector T cell subsets requires metabolic reprogramming and a switch to aerobic glycolysis [24,25]. In turn, a switch to mitochondrial oxidative phosphorylation is associated with memory T cell formation, with this switch being more prominent amongst TCM and TRM than TEM cells [26–28]. There is evidence that metabolic intermediates also serve as signaling molecules (immunometabolism) and cells can use them differentially (reviewed in [23]). How these changes in cellular metabolism influence epigenetic imprinting to influence T cell memory is an emerging area [28,29], with examples such as acetylCoA generated from pyruvate contributing to acetylation of proteins and histones [30], and acetate enhancing histone acetylation, chromatin accessibility, and effector function in glucose-restricted CD8+ T cells [31].

Epigenetic Regulation of Effector T Cell Differentiation Following stimulation by cognate antigen presented on the surface of antigen presenting cells, T cells differentiate into specialized effector cells, with the resultant phenotype dependent on the

start site and up to 1 million base pairs away from a regulated gene. Gene promoter: region of DNA located near the transcription start site of a gene, which initiates transcription of that gene via the binding of TFs and proteins involved in transcribing DNA into RNA. Histones: family of proteins found in complex with DNA as a central component of chromatin. Homeostatic proliferation: normal physiological process triggered by lymphopenia to maintain a constant number of T cells; predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire a memory phenotype, can contribute to autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. Immune-mediated adverse events (imAEs): set of possible side effects believed to arise from increased activity in the immune system after immune checkpoint blockade. Inducible Treg (iTreg): adaptive Foxp3+CD4+ regulatory T cells that develop outside the thymus under subimmunogenic antigen presentation, during chronic inflammation, and during normal homeostasis of the gut. iTreg cells are essential in mucosal immune tolerance and in the control of severe chronic allergic inflammation; they are, most likely, one of the main barriers to the eradication of tumors. Inhibitory receptor: (here) expressed on the surface of T cells (and other lymphocytes) that, when bound to its ligand, delivers a signal that negatively regulates T cell activation. Memory T cells: antigen-activated T cells that persist following the resolution of a primary immune response and mediate a specific and enhanced secondary response to the same antigen. Naı¨ve T cells: T cells that have not yet been activated by specific antigen. Noncoding RNAs: ncRNAs; functional RNA molecules transcribed from DNA but not translated into proteins. Epigenetic related

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conditions (particularly the cytokine milieu) present during activation. CD4+ effector T cells are especially diverse, with many well-characterized functional subtypes described (helper T cells : Th1, Th2, Th17, T follicular-helper, etc.), and likely much further heterogeneity yet to be revealed as the use of single cell profiling technologies becomes increasingly prevalent [32]. This Th cell specialization is in accordance with the broad range of ‘helper’ activities mediated by these cells, such as assisting B cells to make antibody, enhancing and maintaining CD8+ T cell responses, regulating macrophage functions, as well as the requirements for tailored suites of effector mechanisms in defending against different classes of pathogens. Acquisition of cytotoxic activity and IFN-g production are the most characteristic features of CD8+ effector T cells, but these cells also demonstrate functional heterogeneity. Links between epigenetic mechanisms and effector T cell differentiation have been studied by analyzing changes in gene expression in the context of three main types of chromatin modifications: (i) specific histone modifications associated with gene expression or repression or with gene enhancer regions; (ii) cytosine modification within DNA, particularly within gene promoters; and (iii) the presence of accessible DNA in nonexonic regions of the genome, indicative of regulatory regions, including gene enhancers. Due to their well-characterized and distinct phenotypes, Th1 and Th2 cells have served as informative comparators for such studies (Figure 3). Early work analyzing mouse T cells differentiated under Th1 or Th2 conditions in vitro reported hyperacetylation of histones H3 and H4 (two of the core histones) in the enhancer and promoter regions of the Il4 gene, but not Ifng in Th2 cells, whereas the opposite was true in Th1 cells [33,34]. This is in accordance with a consistently observed association between histone lysine acetylation and active gene expression. The distinct patterns of histone acetylation in polarized Th cells observed in these studies was shown to be dependent on key TFs, T-bet (Tbx21) in Th1 cells and Gata3 and Stat6 in Th2 cells, and largely coincided with accessible regions of DNA present selectively in the two effector subtypes [33,34]. Reciprocally, another study reported that silenced Th1-related genes were marked with the repressive histone mark H3K9me3 (trimethylation of lysine 9 on histone 3) after Th2 differentiation in vitro, and loss of the enzyme responsible for deposition of this mark, SUV39H1, led to lack of silencing of these genes (including Ifng expression in Th2 cells) after in vitro Th2 differentiation and in an in vivo mouse model of ovalbumin (OVA)-induced, Th2-associated asthma [35]. Indeed, the epigenetic mark H3K9me3 (trimethylation) serves as a recognition mark for the epigenetic reader heterochromatin protein 1a (HP1a), a central component of transcriptional repressor complexes, silencing Th1 genes in mice [36,37]. Similar studies have implicated DNA modifications in guiding Th cell differentiation. For instance, CpG residues within the Ifng promoter were reported to become hypermethylated during the in vitro differentiation of mouse naı¨ve T cells into Th2, but not Th1 cells [38]. CpG methylation within promoters is normally associated with gene repression, mediated by specific methyl CpG-binding reader proteins able to recognize this mark. Subsequent studies showed that expression of Dnmt3a was increased following TCR stimulation of mouse CD4+ T cells and that selective knockout of Dnmt3a in T cells resulted in the absence of Ifng promoter methylation and in significant production of IFN-g by T cells differentiated under Th2, Th17, or inducible Treg (iTreg) conditions [39,40]. Patterns of DNA methylation at the Ifng promoter in mouse CD8+ T cells have been shown to be faithfully inherited following extensive cell proliferation (16 cell divisions), illustrating how effector function can be stably programmed by differentiation events following T cell activation [16]. Recently, heritable stability of DNA methylation patterns was demonstrated as well for human CD8+ T cells, driven to divide in vitro by exposure to IL-7/IL-15 for 7 days, or after homeostatic proliferation in vivo for 2 months after transfer of TCM and TEM cells into haploidentical bone marrow transplant recipients [41]. An interplay between epigenetic modifications and metabolic activity is illustrated by the requirement for lactate dehydrogenase (LDH) and aerobic glycolysis in mouse CD4+ effector T cells for acetylation of the Ifng promoter and enhancer regions [42]. Similarly, an immediate-early glycolytic switch was shown to be necessary for the rapid production of IFN-g by reactivated human CD8+ TEM cells, based on blockade of these responses with an inhibitor of the serine-threonine kinase Akt; this was linked to mTOR signals and also to epigenetic modifications of the Ifng locus [43].

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ncRNAs include miRNA, siRNA, Piwi-interacting RNA, and long noncoding RNA. In general, ncRNAs function to regulate gene expression at transcriptional and post-transcriptional levels. Pioneer factors: transcription factors (TFs) that can directly bind condensed chromatin. They can have positive and negative effects on transcription and are important in recruiting other TFs and histone modification enzymes, as well as in controlling DNA methylation. Progenitor exhausted cells: subpopulation of exhausted CD8+ tumor-infiltrating T lymphocytes (TILs) that retain polyfunctionality, persist long term, and differentiate into ‘terminally exhausted’ TILs. Progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Resident memory T (TRM) cells: subset that occupies tissues without recirculating. They provide a first response against infections reencountered at body surfaces, where they accelerate pathogen clearance. Epigenetic reader proteins: diverse range of proteins that possess specialized domains capable of recognizing specific epigenetic marks in a locus. Proteins that contain reader domains can be broadly classified into four groups: chromatin architectural proteins, chromatin remodeling enzymes, chromatin modifiers, and adaptor proteins that recruit other machinery involved in gene expression. Th cells: CD4+ T helper cells that have differentiated to acquire specific effector functions, typically defined by the profile of cytokines and TFs that they express. Transcription factor (TF): protein that binds DNA, typically in a sequence-specific manner, and controls the rate of gene transcription.

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Methyla on Acetyla on Phosphoryla on Crotonyla on

Chromosome Chroma n

Methylated cytosine (5mC)

Histone tail Histone DNA accessible Gene transcrip on ON H2A H3

H2B

Nucleosome

H4

Euchroma n

DNA inaccessible Gene transcrip on OFF Heterochroma n

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Figure 1. Basic Chromatin Structure and Categories of Epigenetic Modifications. Chromatin is a complex, highly ordered, and heterogeneous structure in which DNA is packaged in the nucleus together with histone proteins. Nucleosomes, comprised of DNA wrapped around a core of eight histone proteins (two each of histones H2A, H2B, H3, and H4), are combined in higher-order structures that lead to greater or lesser compaction and less or more accessible DNA, respectively. DNA found in less compacted chromatin is amenable to binding by transcription factors and other proteins involved in mediating gene transcription. Chromatin can be modified on DNA, through cytosine methylation, and by various post-translational modifications to histones (of which methylation, acetylation, phosphorylation, and crotonylation are four examples). These can directly or indirectly alter the accessibility of DNA within chromatin.

In addition to studies focusing on specific gene loci, work investigating epigenetic features at a genome-wide level have highlighted the extensive chromatin modulation that occurs upon effector T cell differentiation. Global analysis of histone marks at a relatively early time-point (72 h) during the in vitro differentiation of human naı¨ve T cells into Th1 or Th2 phenotypes identified >2000 unique regions in each cell type possessing chromatin features characteristic of gene enhancers [44]. Extensive remodeling of the enhancer landscape was also demonstrated in mouse CD8+ T cells responding to lymphocytic choriomeningitis viral (LCMV) or bacterial (Listeria monocytogenes) infection in vivo, with thousands of unique enhancers being identified selectively in effector versus naı¨ve T cells (based on the presence of characteristic histone modifications and, in some cases, via identification of accessible DNA regions) [45–47]. This was due to both the loss or ‘decommissioning’ of naı¨ve T cell enhancers as well as the generation of new enhancers in effector cells [45]. In a similar setting, genome-wide analysis of permissive (H3K4me3) and repressive (H3K27me3) marks in influenza A virus-specific mouse CD8+ T cells revealed extensive and varied changes in these histone modifications that correlated with changes in gene expression. Of note, different combinations of these histone marks were observed, including the occurrence of ‘bivalent’ modifications, with both H3K4me3 and H3K27me3 at some promoters (e.g., genes encoding TFs known to be important regulators of CTL differentiation, such as Tbx21, Irf4, BmiI, Gata3, and Eomes) [48]. This signature identified genes in a poised state, which, following cell differentiation, could resolve into either expressed (losing H3K27me3) or repressed (losing H3K4me3) states [48].

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Histone modifica ons at promoters: Gene transcrip on ON/ OFF Promoter

TF1 TF2

Enhancer 1 Enh

Pol2

Ac ve gene • ↑ H3K9ac • ↑ H3K4me2/3 • ↑ H3K9me1, H3K79me2 • ↑ H4K20me1 Repressed gene • ↑ H3K9me2/3 • ↑ H3K27me3 • ↑ H4K20me3 • ↑ 5mC Poised/bivalent gene • ↑ H3K4me2/3 • ↑ H3K27me3 • ↑ 5hMC

Enhancer 2

Histone modifica ons at enhancers: Ac ve gene • ↑ H3K27ac • ↑ H3K4me1 • ↑ 5hMC Repressed gene • ↑ H3K9me2/3 • ↑ H3K27me3 • ↑ 5mC Poised/bivalent gene • ↑ H3K4me1 • ↑ H3K27me3 • ↑ 5hMC

Super-enhancer • Clustered enhancers • Cover large stretches of DNA

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Figure 2. Epigenetic Features of Gene Regulatory Regions. Gene promoters and enhancers are found in open, ‘nucleosome-poor’ regions of chromatin. The status of the gene under regulation (e.g., actively transcribed, repressed, or primed for expression) is reflected by the combination of chromatin modifications present in these regulatory regions (as indicated in the boxes). In some cases, multiple or stretch enhancers regulating the same gene can be found in large clusters and have sometimes been termed ‘super-enhancers’. Abbreviations: 5hMC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; Pol2, RNA polymerase 2; TF, transcription factor.

Genome-wide analysis of DNA methylation has also demonstrated extensive changes in the distribution of 5mC during mouse and human CD4+ Th cell differentiation [49,50]. Consistent with the repressive function of 5mC, loss of this mark was observed at the promoters and enhancers of lineage-specific TFs and cytokines during differentiation to Th1, Th2, or Th17 cells [49,50]. Studies of LCMVspecific mouse CD8+ T cells have shown similarly widespread alterations in DNA methylation patterns upon CD8+ effector T cell differentiation [51]. Both gain and loss of 5mC were observed, with the presence of DNA CpG methylation at promoter and enhancer regions correlating negatively with gene expression [51]. In contrast to 5mC, genome-wide investigations have shown that 5hmC is associated with expressed genes in mouse and human CD4+ and CD8+ effector T cells [49,52,53]. Conversion of 5mC to 5hmC eliminates binding sites for methyl-binding proteins involved in gene repression and, hence, can facilitate nucleosome remodeling and binding of TFs. A direct role for one TET family member, TET2, in this process was shown in studies using TET2-deficient mouse CD4+ T cells [49]. Here, mouse CD4+ TET2–/– T cells, differentiated under Th1 and Th17 conditions in vitro showed reduced prevalence of 5hmC at Ifng and Il17a gene loci, respectively, associated with reduced expression of these genes, relative to TET2+/+ T cells [49]. Overall, however, TET2-deficiency led to only a partial loss of 5hmC, suggesting a role for other TET family members in the conversion of 5mC to 5hmC (TET1, 3). Taken together, these and other studies have demonstrated that T cell activation and differentiation into effector cells is accompanied by extensive alterations in the chromatin landscape, with widespread changes in the pattern of gene enhancers, which mediate globally altered patterns of gene expression. Such changes are driven by stimulation through the TCR, co-stimulatory molecules and

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Figure 3. Epigenetic Changes Associated with Effector T Cell Differentiation. Differentiation of naı¨ve T cells into effector cells is accompanied by epigenetic modifications that establish new patterns of gene expression. In the simplified example shown, T helper (Th)1 and Th2 cells acquire reciprocal chromatin changes at canonical cytokine loci. In Th1 cells, the IFNG locus is marked with histone acetylation and becomes accessible to transcription factors and Pol2, while CpG residues within the locus encoding Th2 cytokines [interleukin (IL)-13, IL-4] become methylated, leading to closed chromatin and gene repression. The opposite changes occur upon Th2 differentiation. Abbreviations: Pol2, RNA polymerase 2; TN, naı¨ve T cells.

cytokine receptors, as well as by the integration of these signaling pathways and metabolism by TFguided epigenetic modifications.

Epigenetic Regulation of T Cell Memory Generation and Maintenance As discussed above, memory T cells represent a small subset of the activated T cell population that persists following the disappearance of short-lived effector cells. Epigenetic mechanisms, guided by TFs, have been shown to play an important role in this fate decision. For example, Blimp-1 (encoded by Prdm1), a TF and transcriptional repressor that drives CD8+ T cells towards an effector cell fate, was shown to direct the histone-modifying enzymes G9a and HDAC2 to the Il2ra and Cd27 loci at the peak of an anti-LCMV mouse CD8+ T cell response; Blimp1 protein colocalized with G9a and HDAC2 at these gene loci in activated Prdm1+/+ CD8+ T cells, while G9a and HDAC2 failed to bind to Il2ra and Cd27 loci in Prdm1–/– cells [54]. Generation of the resultant repressive histone modifications (H3K9 methylation and histone deacetylation, respectively) at these loci in Prdm1+/+ cells led to reduced expression of two receptors that contributed to the survival of activated T cells relative to Prdm1–/– cells , hence contributing to the short lifespan of effector CD8+ T cells [54]. At a genomewide level, TFs YY1 and Nr3c1 were shown to have critical roles in establishing the enhancer landscape in mouse CD8+ effector versus memory precursor CD8+ T cells, respectively [47]. Whether memory T cells arise from fully differentiated effector T cells or at an earlier stage of activation has been a subject of debate for decades, with published evidence supporting both views. Unsurprisingly, some epigenetic mechanisms are required for the generation of both effector and

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memory T cells. For instance, CD8+ T cells deficient in the histone acetyltransferase CREB-binding protein [CBP; (CBP –/–)], which exhibits enhanced binding to chromatin upon TCR-triggered phosphorylation, were unable to differentiate into either effector or memory CD8+ T cells in response to infection with L. monocytogenes in mice [55]. However, this shared requirement for an epigenetic mechanism at an early stage post-T cell activation did not preclude divergence of cell fates prior to effector cell differentiation [55]. Conversely, results from a recent study analyzing genome-wide DNA methylation amongst virus-specific CD8+ T cells during acute LCMV infection (Armstrong strain) in mice have been used to argue for a linear model of differentiation (naı¨ve / effector / memory) [56]. The authors observed that cells destined to become memory cells initially acquired de novo methylation and repression of naı¨ve T cell associated genes, as well as demethylation and expression of effector genes (such as Prf1, granzyme genes, and Ifng). Notably, a subset of the methylated naı¨ve genes subsequently became demethylated and re-expressed in memory cells, a process that was accelerated by loss of DNMT3a; similar potentiation of memory CD8+ T cell generation was observed upon conditional depletion of DNMT3a ( DNMT3a2loxp/2loxp under the control of the Cd4 promoter or the dLck promoter) in mouse CD8+ T cells during the effector phase of the anti-viral (LCMV) response in a previous study [57]. Of note, enhanced differentiation of CD8+ T cells into memory cells after LCMV infection has also been reported in TET2-deficient mice, but in this case, it was associated with increased DNA methylation at gene loci encoding TFs that favored CD8+ effector T cell differentiation, including Tbx21, Prdm1, Irf4, and Runx3 [58]. While these observations provide persuasive evidence that an effector-to-memory T cell transition can take place, memory T cells may also be generated at other stages of T cell activation/differentiation, perhaps contributing to the heterogeneity of memory T cell phenotypes. In addition to directing activated T cells towards short-lived effector versus long-lived memory fates, epigenetic mechanisms play a central role in maintaining gene expression patterns in established memory T cells (Figure 4). This idea is supported by numerous studies focusing on chromatin modifications at specific gene loci. Examples, amongst many others, include histone H3K9/14 hyperacetylation and priming at the Il4 locus in mouse memory Th2 cells [59], hyperacetylation at the IL4 or IFNG loci in human TEM Th2 or Th1 cells, respectively [60], elevated deposition of H3K9Ac at the promoters of constitutively expressed EOMES and PRF1 and inducibly-expressed GZMB genes in human memory CD8+ T cells [29], reduced promoter DNA methylation and constitutive expression of the chemokine receptor CX3CR1 on human CD8+ TEM [61], demethylation of DNA within a CCR6 enhancer in human CCR6+ memory CD4+ and CD8+ T cells [62], and demethylation of an enhancer element in the mouse Itga4 locus and its human counterpart (ITGA4) encoding a component of the a4b7 integrin on a4b7+ gut-homing memory CD4+ T cells [63]. Furthermore, in addition to imparting constitutive gene expression/repression or priming for a rapid response upon T cell reactivation, genes may also be primed to respond to alternative stimuli. For example, the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis, was found to have an open chromatin configuration in LCMV-specific mouse memory but not naı¨ve CD8+ T cells, which allowed the former to upregulate expression of Gcnt1 in response to the cytokine IL-15 [64]. Since 2 O-glycans interact with P- and Eselectins, this example is interesting, as such an epigenetic modification allows for memory T cells to migrate into sites of inflammation, where IL-15 is produced [64]. In accordance with this, when LCMVspecific memory CD8+ T cells were transferred into Il15–/– mice, these cells were much less able to migrate into the lungs of mice challenged intranasally with an inflammatory stimulus (CpG oligonucleotides) than when the cells were transferred into Il15+/+ mice [64]. Moreover, genome-wide analysis of permissive (H3K4me3) and repressive (H3K27me3) histone modifications in human and mouse naı¨ve, TCM and TEM CD8+ T cells has shown that a markedly altered epigenetic landscape can persist into memory subsets [65–67]. Indeed, the presence of these histone marks, and combinations thereof at gene regulatory loci, were shown to correlate with gene expression status in these studies (i.e., expressed, repressed, or primed genes). Similarly, analysis of DNA methylation in mouse antigen-specific CD4+ T cells and human TCM and TEM subsets have shown extensive differences in the genome-wide distribution of 5mC between naı¨ve and memory cell subsets associated with differential gene expression [68,69]. Additionally, genome-wide analysis of

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Figure 4. Epigenetic Changes Associated with Memory T Cell Differentiation. When naı¨ve T cells differentiate into effector cells, large numbers of genes are turned on and off, generating highly distinct gene expression patterns. These changes in gene expression are guided by repressive and activating epigenetic modifications, highly simplified and depicted here with representative examples of H3K27me3 (repressive) and H3K4me3 (activating). The gene expression and epigenetic landscape of memory cells share features of both effector and naı¨ve cells. Some genes that are turned on in effector cells are found in a poised state in memory cells: not expressed but able to be induced rapidly upon T cell reactivation. Note that while there is strong evidence that memory T cells can arise from effector T cells, there is also support for a pathway in which naı¨ve T cell activation generates memory cells in the absence of an intermediate effector cell stage (indicated by broken arrow). Abbreviations: APC, antigen-presenting cells; TM, memory T cells; TEFF, effector T cells; TN, naı¨ve T cells.

epigenetic modifications has extended the understanding of how memory T cells maintain gene expression patterns that are distinct from those in either naı¨ve or effector T cells (Figure 4). Specifically, genome-wide analysis of accessible regions of chromatin in mouse LCMV-specific CD8+ T cells 8d (effector) or 30d (memory) after infection demonstrated unique widespread patterns of open chromatin in memory compared with naı¨ve or effector CD4+ T cells, which correlated with both constitutive gene expression and expression in response to re-stimulation (primed genes) [46]. The implications of such epigenetic memory in the context of autoimmunity/inflammation was demonstrated in a recent example investigating chromatin patterns amongst a population of granulocyte-macrophage-colony stimulating factor (GM-CSF) producing CD4+ T cells; these were identified as critical for contributing to pathology in a mouse model of neuroinflammation (experimental autoimmune encephalomyelitis) [70]. Specifically, analysis of open chromatin regions in reactivated CD4+ Th cells that had been actively producing GM-CSF versus those that had previously produced GM-CSF at the point of isolation demonstrated that they had closely related epigenetic modifications (including enrichment of accessible binding sites for key TFs Runx1 and Runx2, Erg, Etv4, Fev, Batf, and Fos-Jun motifs) compared with non-GM-CSF producing reactivated CD4+ T cells. Since the memory cells possessing these epigenetic modifications produced GM-CSF upon re-stimulation, this

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suggested the presence of an epigenetically stable and specific population of pathogenic T cells with memory and effector capabilities that contributed to maintaining chronic inflammation [70]. Overall, it is clear that the properties of memory cells that mediate efficient secondary responses are both established and maintained by epigenetic modification of gene expression. These epigenetic changes can persist long-term in memory T cells, as demonstrated in a study of human yellow fever virus-specific CD8+ T cells, where T cell effector genes were found to be associated with open chromatin a decade after vaccination [71]. In fact, rapid expression of effector cytokines in smallpox-specific T cells up to 83 years postinfection also suggests that there can be life-long maintenance of epigenetic priming in memory CD4+ and CD8+ T cells [4].

Epigenetics of T Cell Exhaustion Whereas ‘typical’ memory T cells are generated in situations in which pathogens are cleared by the primary immune response, antigen-specific T cells that survive long-term during chronic viral infection exhibit markedly different properties. In this setting, first characterized in mice administered variants of LCMV that cause persistent infection (Clone 13 strain), virus-specific CD8+ T cells show significantly reduced effector functions as well as sustained expression of inhibitory receptors [(IRs); negative regulators of T cell activation], including programmed cell death protein 1 (PD-1) [72–74]. Linked to chronic antigen exposure, this dysfunctional state has been termed T cell ‘exhaustion’, and cells with these characteristics [exhausted T cells (Tex)] have also been observed in the context of other viruses that cause chronic infections in mice and humans, including HIV, hepatitis C, and hepatitis B viruses [75]. Although predominantly reported for CD8+ T cells, CD4+ T cells with characteristics of exhaustion have also been observed in chronic LCMV infection in mice [76]. While ineffective in clearing the pathogen, conversion to an exhausted state is likely an adaptation to prevent immunopathology in the host that would otherwise occur with widespread and long-term production of inflammatory mediators and the killing of infected cells. The observation that Tex gene signatures are associated with a better prognosis in a variety of autoimmune diseases (e.g., type 1 diabetes, anti-neutrophil cytoplasmic antibody-associated vasculitis, systemic lupus erythematosus, idiopathic pulmonary fibrosis) suggests that conversion to an exhausted state may also provide protection in the context of self-antigen-reactive T cells [77]. Notably, rather than representing a terminally differentiated state, the dysfunction of Tex cells appears to be actively maintained via the IRs expressed on these cells and may be at least partially reversible. This was first shown in chronic LCMV infection in mice, where treatment with antibodies that blocked PD-1 interaction with its ligand PD-L1 [immune checkpoint blockade (ICB)] restored the ability of CD8+ Tex cells to undergo proliferation, secrete cytokines, kill virally infected cells, and to reduce viral load in vivo [74]. Later studies showed this response to be heterogeneous, with proliferation being restricted to one subpopulation of PD-1+ cells expressing lower amounts of other IRs and characterized by the expression of TF TCF1; however, nonproliferating Tex cells also appeared to be required for the PD-1-blockade-induced control of chronic viral infection, since depletion of either subpopulation resulted in failure to control chronic LCMV infection [78,79]. TCF1+ PD-1+ Tex exhibit stem cell-like properties, being capable of self-renewal and also giving rise to more terminally differentiated Tex cells, and hence are crucial for maintaining the Tex population [79]. The concept of ‘re-invigorating’ Tex cells by blocking IRs was subsequently extended into the setting of oncology. This was based on the finding that features of exhaustion are observed amongst CD8+ T cells infiltrating a range of human tumors, suggesting that such T cells recognize tumor antigens but are functionally defective and unable to kill the malignant cells [75,80–82]. As in chronic viral infections, tumor-infiltrating Tex cells are heterogeneous, with preclinical studies in mice identifying a subpopulation of ‘progenitor exhausted cells’ that respond to anti-PD-1 antibody treatment by differentiating into more cytotoxic ‘terminally exhausted’ cells [83]. Clinically, approaches to block IR signaling (i.e., checkpoint-inhibitor therapy or ICB) has yielded striking results in certain tumor types, including melanoma and non-small cell lung cancer [84]. However, only a fraction of cancer patients currently benefit, potentially because sufficient numbers of tumor-specific Tex cells are often not

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present. The nature of the Tex might also play a role, given that the relative frequency of progenitor exhausted CD8+ T cells has been found to correlate with improved duration of response to ICB in patients with advanced melanoma [83]. The clinical experience with ICB has also highlighted the role that IRs might play in maintaining tolerance to self-tissues; indeed severe immune-mediated adverse events (imAEs), such as colitis, hypophysitis (pituitary inflammation), and neurological toxicities (amongst many others) are common [85]. Most imAEs are reversible upon corticosteroid treatment and cessation of checkpoint-inhibitor therapy, implying that transiently blocking the IR alone does not cause long-term alterations in T cell function (see further below). Studies in the setting of chronic LCMV infection have shown that the gene expression patterns of CD8+ and CD4+ Tex cells are distinct from those of both effector and memory T cells [76,86,87]. Linked to this, Tex cells are also epigenetically unique; analysis of accessible chromatin regions in mouse CD8+ T cells responding to acute or chronic LCMV infection identified thousands of differentially accessible regions in cells with an exhausted phenotype compared with memory and effector T cells [88–90]. One mechanism that has been shown to shape the epigenetic landscape of Tex cells is de novo DNA methylation [91]. In this study, new patterns of DNA methylation were found to accompany the generation of mouse CD8+ Tex cells at the post-effector cell stage during chronic LCMV infection and amongst tumor-infiltrating Tex cells in a mouse prostate cancer model [91]. Conditional knockout of the de novo DNA methyltransferase DNMT3a in responding CD8+ T cells (Dnmt3a-cKO; Cre; under the control of the granzyme b promoter) abrogated the acquisition of these methylation patterns and inhibited the development of functional exhaustion relative to wild type CD8+ T cells [91]. Moreover, accessible chromatin regions in antigen-specific CD8+ T cells in human chronic viral infections (HIV-1, hepatitis C virus) show significant overlap with those found in mouse Tex, supporting the view that similar mechanisms might operate in human Tex cells [89]. Consistent with the functional heterogeneity observed amongst Tex cells, analysis of accessible chromatin regions in CD8+ T cells in the context of chronic LCMV infection demonstrated that progenitor (stem cell-like) Tex cells were epigenetically distinct from more terminally differentiated Tex cells, as shown by their distinct patterns of accessible/inaccessible chromatin across the genome [92]. In addition, the epigenetic landscapes of both populations of Tex cells were shown to be different from those of either effector and memory T cells generated during acute LCMV infection (Armstrong strain). The data imply that the differing properties of these T cell subpopulations may be largely driven by epigenetic mechanisms. Furthermore, the number of distinct accessible chromatin regions with features of enhancers in Tex versus effector CD8+ T cells generated during LCMV infection in mice has been shown to substantially exceed (four to five times) the number of differentially expressed genes, perhaps reflective of the fact that genes are often under the control of multiple enhancers [88]. Detailed analysis of enhancer regions upstream of the Pdc1 locus (encoding mouse PD-1) was informative in this context [89]. Specifically, several accessible enhancers were present in this region of DNA in both Tex versus effector CD8+ T cells, consistent with the fact that PD-1 is also transiently expressed by effector CD8+ T cells during acute LCMV infection. However, a further unique enhancer was identified in Tex cells, shown to enable binding of specific TFs to this site, only in Tex cells [89]. Indeed, the presence of unique accessible Pdc1 regulatory regions in Tex cells is consistent with a previous study identifying a CpG-rich regulatory region upstream of Pdc1 that underwent transient demethylation followed by remethylation in CD8+ T cells responding to acute LCMV infection in mice, but which remained demethylated in Tex CD8+ T cells independently of continued exposure to virus [93,94]. Tex cells identified in mouse and human tumors also exhibit chromatin signatures that are distinct from memory or naı¨ve T cells [95,96]. As in models of viral infection, there is heterogeneity amongst cells categorized as Tex (primarily on the basis of PD-1hi expression), which is apparent at the epigenetic level [95,96]. In one study, transfer of CD8+ TCR transgenic T cells specific for SV40 large T antigen epitope I (TAG) into mice bearing an autochthonous TAG-expressing liver tumor showed that tumor-infiltrating T cells underwent two waves of chromatin remodeling, as indicated by measurement of global chromatin accessibility [95]. Widespread changes in chromatin accessibility occurred

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Key Figure

Potential for Modulating T Cell Memory in Disease by Targeting Epigenetic Mechanisms

Figure 5. Given the role of epigenetic mechanisms in generating and maintaining gene expression programs in T cells, chromatin writers, erasers, and readers may represent promising therapeutic targets for modulating T cell memory in disease. In autoimmunity, diverting self-reactive T cells towards a less functional, nonpathogenic state would be desirable, while converting exhausted tumor-reactive T cells into functional memory cells would be the objective in cancer. Although our understanding in this area is at an early stage, examples of epigenetic regulators that influence the generation or maintenance of exhausted and memory T cells have been described (main text). Abbreviations: TEFF, effector T cells; Tex, exhausted T cells.

by day 5, followed by a second wave of remodeling between days 7 and 14, after which few accessibility changes occurred. While tumor-infiltrating CD8+ T cells were dysfunctional at both the early and later time-points, only cells isolated at the early time-point were able to regain functional properties (cytokine secretion, proliferation) when cultured ex vivo with IL-15. Since the change in chromatin state and conversion to a more refractory exhausted state occurred with increasing time after tumor infiltration, the data imply that this included a process of progressive differentiation towards terminal exhaustion [95]. Recently, several research groups independently identified a TF, thymic selectionassociated high mobility group box protein (TOX), that is critically required for the development and maintenance of CD8+ Tex in tumors and chronic viral infections (LCMV in mice, hepatitis C virus in humans), but which is dispensable for the formation of effector and memory CD8+ T cells [97–101]. TOX appears to be required for maintaining high expression of IRs, linked with increased chromatin accessibility of the genes encoding these receptors, since deletion of TOX, or removal of its DNAbinding domain in CD8+ T cells led to downregulation of IRs and increased effector function; this was shown both in a TAG-specific TCR transgenic adoptive transfer model (see above) and in the context of LCMV infection [98,99].

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Notably, some intratumoral PD-1hi T cells exhibit neither functional nor epigenetic hallmarks of exhaustion. For example, in a study of infiltrating CD8+ T cells in human urinary bladder cancer, epigenetic analysis of the PRF1 locus (encoding the pore-forming protein perforin) demonstrated reduced levels of methylation at the enhancer region in tumor-infiltrating cells compared with those in peripheral blood [102]. Associated with this, CD8+ T cells from the tumor expressed greater quantities of perforin protein than peripheral blood CD8+ cells after stimulation, indicating that they functioned more like TRM than Tex cells. As discussed earlier, therapeutic blocking of IR/pathways can re-invigorate Tex cells and is well known as a promising new approach in the treatment of certain cancers. However, the majority of patients given such treatments fail to develop durable responses and many eventually progress [103,104]. One factor contributing to the lack of sustained efficacy may be the failure of checkpoint blockade to alter the epigenetic landscape of Tex cells. In support of this idea, mouse CD8+ Tex cells generated during chronic LCMV infection have shown only minor changes in accessible chromatin regions in response to PD-L1 blockade-induced re-invigoration [90]. Notably, these cells became re-exhausted if antigen concentration remained high and failed to become memory cells upon antigen clearance [90]. Similarly, limited effects of PD-L1 blockade on chromatin accessibility were observed amongst antigen-specific infiltrating CD8+ T cells in a mouse tumor model in which OVA-specific CD8+ T cells were injected into mice bearing B16-OVA, a melanoma cell line engineered to express OVA [105]. These data suggest that approaches combining epigenetic modulation with checkpoint inhibitors might be able to generate tumor-targeting CD8+ T cells with longer-term potential and, presumably, greater efficacy, although this remains to be further assessed (Figure 5, Key Figure). Nevertheless, the dramatic progress in the identification of small molecules able to inhibit the function of epigenetic writers, erasers, and readers might contribute to making this approach more feasible [19,106]. Although this specific strategy remains to be proven, there is some evidence that T cell functional exhaustion can be alleviated by targeting epigenetic pathways. For example, treatment of splenocytes from chronically LCMV-infected mice with a histone deacetylase inhibitor (HDACi) resulted in increased anti-CD3/CD28-induced IFN-g production, associated with increased histone acetylation at the Ifng enhancer and promoter in CD8+ T cells relative to control cells [96]. Moreover, HDACitreated CD8+ T cells were able to differentiate into functional memory T cells after adoptive transfer into naı¨ve recipient mice, indicating that these cells had undergone long-term reprogramming [96]. Recently, treatment with DNA-demethylating agent 5-aza-2-deoxycytidine prior to PD-L1 blockade was shown to enhance the number of proliferating antigen-specific CD8+ T cells, as well as the ability of these cells to produce cytokines, in mouse models of chronic viral (LCMV) infection and cancer (mouse prostate cancer) [91]. Thus, the effects of 5-aza-2-deoxycytidine treatment on CD8+ T cells support the findings that de novo DNA methylation can contribute to the epigenetic programming of Tex cells and further suggests that it may be possible to reverse this program for therapeutic gain [91]. A striking recently published example of how epigenetic manipulation might be used to prevent T cell exhaustion in humans came from an accidental gene ‘knockout’ in a chimeric antigen receptor (CAR)T cell trial in one characterized patient achieving remission [107]. Transfer of CD8+ T cells genetically modified to express tumor-specific CARs has been used successfully to treat certain B cell lymphomas, but the expansion and persistence of CAR-T cells is often limited [108]. In a recent study, complete remission in a CAR-T cell recipient with advanced/relapsed chronic lymphocytic leukemia was associated with expansion and persistence of a single CD8+ T cell clone in which the transgene had inserted into the TET2 locus [107]. Because the patient harbored a hypomorphic mutation in the second TET2 allele, this led to a near knockout of this epigenetic enzyme. Accordingly, the CAR-T cells exhibited lower amounts of 5hmC and altered chromatin accessibility at gene loci for several regulators of T cell effector differentiation or exhaustion. Phenotypically, the cells resembled TCM at the peak of the response, perhaps accounting for their persistence and the antitumor efficacy that was observed [107]. This finding suggests that specific epigenetic proteins such as TET2 can play a decisive role in mediating fate decisions between memory and exhausted T cell fates.

Outstanding Questions Is it possible to target epigenetic mechanisms for conversion of exhausted T cells into functional memory T cells? This approach, in the presence or absence of checkpoint inhibitors, could lead to enhanced and prolonged efficacy in cancer immunotherapy. However, strategies would need to be developed that do not have systemic effects on self-reactive exhausted T cells to avoid fulminant autoimmunity. Can memory T cells that contribute to autoimmunity/inflammation be converted to an exhausted phenotype by targeting epigenetic mechanisms? Consideration would need to be given to avoid broader effects on T cells that could eliminate protective memory against pathogens. It will also be important to understand the potential to manipulate the epigenetic state of memory cells at rest versus under conditions of antigen-induced activation. Can epigenetic manipulation be used to enhance vaccine efficacy, for instance, by promoting the generation of memory cells rather than short-lived effector cells? Can epigenetic mechanisms be targeted to selectively alter the effector function of memory T cells? Such an approach could be considered, for example, in allergy, where Th2 memory to allergens drives the pathology and maintains the allergic state. An ability to modify the function of TRM cells might be particularly valuable in autoimmune and inflammatory diseases, in which polarized tissue-resident memory cells are thought to make a major contribution; in an ideal scenario, the function of these cells could be changed from that of an effector to a regulatory T cell. Can selective modulation of metabolic pathways, during T cell activation or memory, be used to modify the epigenetic landscape and functional properties of T cells? Such an approach might offer an opportunity to target T cells in a particular

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Approaches that target such mechanisms selectively in T cells may hold promise for reversal of T cell exhaustion in cancer immunotherapy.

Concluding Remarks Epigenetic imprinting is central to the formation and maintenance of memory in T cells and reflects the conditions of primary T cell activation: exposure to antigen, inflammatory signals, and the microenvironment, including nutrient availability. T cell memory can be maintained for the lifetime of the organism and is key for long-term responses to pathogens. However, it is likely also involved in chronic autoimmune disorders, including relapsing remitting immune diseases such as multiple sclerosis, inflammatory bowel disease, and psoriasis. Moreover, tumor-specific exhausted CD8+ T cells retain a memory that limits their ability to maintain effective antitumor activity. The central themes of epigenetics and T cell memory formation are that the specific epigenetic drivers and pathways are highly context-dependent and vary with each subpopulation and location; moreover, the epigenetic signature, while stable, has the potential to be modified, which is relevant from a therapeutic perspective. The contextual rules governing epigenetic control in some populations are becoming clearer, as in the case of the priming of the Ifng locus in memory compared with naı¨ve CD4+ T cells [60]. While the possibility of selectively modulating T cell memory by targeting epigenetic mechanisms holds significant therapeutic potential, many questions still remain concerning how this can be achieved and extensive and robust work is still required (see Outstanding Questions). The emerging themes and areas for additional investigation include understanding the specific drivers for local and specific memory responses in different subpopulations and defining when, in the differentiation process, does memory form. The application of genome-wide functional genomics approaches, lineage tracing technologies in model systems, all combined with major advances in our ability to apply single cell sequencing of disease samples, should collectively inform greater understanding of this key question. Concomitant translation of any epigenetic signatures observed in mouse, to human, will continue to be important. Understanding the molecular basis of epigenetic imprinting, the drivers, and the differences in different subpopulations of memory cells (CD4 vs CD8) and in memory cell-specific locations (TCM vs TRM) will provide opportunities for novel therapeutic interventions both to accentuate memory and to block inappropriate memory. This may include discrete therapy directly with epigenetic modulators or with upstream stimuli to boost memory formation in T cells during vaccination regimes. Alternatively, treatment of cells ex vivo to induce a robust memory response could be used in the case of adoptively transferred T cells during cancer therapy to enable their long-term survival and ideally increase their therapeutic efficacy. Conversely, the treatment of patients with autoimmune disorders to reduce the pathogenic memory cell population may be highly desirable (Figure 5). Current therapies that reduce T effector cell function and trafficking, such as abatacept (which blocks CD28mediated co-stimulation) and vedolizumab (which blocks T cell migration to intestinal tissues) are offering some efficacy [109,110]. Nevertheless, there is an unmet need for increased efficacy in these approaches and this may rest with targeting different memory populations, including resident memory cells, to break cycles of autoimmunity.

Acknowledgments We thank colleagues at GSK and many collaborators for helpful discussions and clarifications during the writing of this review.

Disclaimer Statement D.F.T., I.R., L.K.M., and R.K.P. are employees and shareholders of GSK.

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metabolic state, reducing wider effects that might be observed when modulating epigenetic mechanisms occurring more broadly across different cell types.

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