Genetic and Epigenetic Regulation of PD-1 Expression Alexander P. R. Bally, James W. Austin and Jeremy M. Boss This information is current as of September 27, 2021. J Immunol 2016; 196:2431-2437; ; doi: 10.4049/jimmunol.1502643 http://www.jimmunol.org/content/196/6/2431 Downloaded from

References This article cites 98 articles, 44 of which you can access for free at: http://www.jimmunol.org/content/196/6/2431.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 27, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

Genetic and Epigenetic Regulation of PD-1 Expression Alexander P. R. Bally, James W. Austin,1 and Jeremy M. Boss The inhibitory immune programmed cell functional tumor-infiltrating cells during multiple types of death-1 (PD-1) is intricately regulated. In T cells, cancers (25–29). Thus, the expression of PD-1 is critical to PD-1 is expressed in response to most immune chal- the development and maintenance of a healthy and robust lenges, but it is rapidly downregulated in acute settings, immune response. allowing for normal immune responses. On chronically PD-1 expression is tightly and dynamically regulated. On stimulated Ag-specific T cells, PD-1 expression remains resting naive T cells, as well as in certain populations of de- high, leading to an impaired response to stimuli. Ab veloping thymocytes, PD-1 is expressed at low basal levels blockade of PD-1 interactions during chronic Ag set- (30, 31). This low level has been linked to immune tolerance (3, 30, 31). Following an initial immune stimulus, PD-1 is tings partially restores immune function and is now Downloaded from used clinically to treat a variety of devastating cancers. transiently expressed on multiple immune cells types, in- Understanding the regulation of PD-1 expression may cluding CD4 and CD8 T cells, B cells, macrophages, and be useful for developing novel immune-based therapies. dendritic cells (30, 32–38). In the case of acute Ag exposure, In this review, the molecular mechanisms that drive PD-1 is downregulated in a time course that is concurrent dynamic PD-1 expression during acute and chronic with, or prior to, Ag clearance. During chronic immune

antigenic stimuli are discussed. An array of cis-DNA stimulation, PD-1 remains highly expressed, which can lead http://www.jimmunol.org/ elements, transcription factors, and epigenetic compo- to functional exhaustion (5, 6, 12–18). Functional exhaustion nents, including DNA methylation and histone mod- can be partially reversed by blocking PD-1/PD ligand-1 in- ifications, control PD-1 expression. The interplay teractions with Abs. These Ab-based immune checkpoint between these regulators fine-tunes PD-1 expression blockade therapies (nivolumab [under the trade name in different inflammatory environments and across nu- Opdivo] or pembrolizumab [Keytruda]) have been shown to merous cell types to modulate immune responses. be highly efficacious in reinvigorating the antitumor immune The Journal of Immunology, 2016, 196: 2431–2437. response in patients with advanced cancers, including mela-

nomas, non–small cell lung cancer, colorectal cancer, and by guest on September 27, 2021 renal cell cancer (39–45). Additionally, high PD-1 expression he inhibitory receptor programmed cell death-1 is necessary for regulatory development, and follicular (PD-1) is a mediator of central and peripheral im- helper T (TFH) cells also constitutively express high PD-1 T mune tolerance and immune exhaustion (1–6). The (1, 2, 46, 47), although PD-1 regulation has not been stud- immune inhibitory function of PD-1 was demonstrated in ied in this population. PD-1 and PD ligand-1 knockout mice, which presented hy- In this review, the mechanisms that drive the initial PD-1 peractive immune phenotypes (4, 7, 8), and mutations in PD-1 induction and its continued expression during chronic Ag have been associated with disease progression in multiple hu- exposure will be discussed. In addition to providing an un- man autoimmune disorders (9–11). High levels of PD-1 were derstanding of which active immune environments may in- linked to CD8 T cell exhaustion during chronic viral infections duce and be affected by PD-1’s function, knowledge of the (5, 6, 12–18). Exhausted CD8 T cells are unable to secrete molecular mechanisms regulating PD-1 may help tailor fu- normal amounts of , proliferate, or perform immune ture immunotherapies for fighting cancer and chronic HIV functions such as initiating cellular cytotoxicity (12, 19–22). and hepatitis C virus infections, or for prevention of allergic Although the PD-1 pathway represents a major contributing responses, transplant rejection, and autoimmunity (48), as factor to cellular exhaustion, other inhibitory receptors also well as boosting long-lived memory responses to vaccines contribute (23, 24). PD-1 expression was also found on poorly (49).

Department of Microbiology and Immunology and Emory Vaccine Center, Emory Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of University School of Medicine, Atlanta, GA 30322 Microbiology and Immunology, Emory University, 1510 Clifton Road NE, Room 3001, Atlanta, GA 30322. E-mail address: [email protected] 1Current address: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. Abbreviations used in this article: Blimp-1, B lymphocyte–induced maturation protein- 1; CR-B/C, conserved region B/C; CTCF, CCCTC binding factor; H3, histone H3; ORCIDs: 0000-0003-4494-5033 (A.P.R.B.); 0000-0002-2432-1840 (J.M.B.). 5hmC, 5-hydroxymethylcytosine; Io, ionomycin; IRF, IFN regulatory factor; ISGF, Received for publication December 21, 2015. Accepted for publication January 16, IFN-stimulated gene factor; ISRE, IFN-stimulated response element; K27ac, lysine 27 2016. acetylation; K4me1, lysine 4 monomethylation; K4me3, lysine 4 trimethylation; LCMV, lymphocytic choriomeningitis virus; 5mC, 5-methylcytosine; PD-1, programmed cell This work was supported by National Institutes of Health Grant R01 AI113021 (to J.M.B.). death-1; T-bet, T-box expressed in T cells; T , follicular helper T; TSS, transcription A.P.R.B. was supported in part by National Institutes of Health Grant T32AI007610-12. FH start site. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502643 2432 BRIEF REVIEWS: REGULATION OF PD-1

Models for studying PD-1 regulation. The expression and regu- actions of distant enhancers from controlling a downstream lation of PD-1 have been studied in a variety of model systems. gene (67), these CTCF sites likely define the extreme ends of In culture, PD-1 has been studied using both the EL4 mouse the Pdcd1 regulatory locus. T cell line and isolated mouse and human primary immune Transcription factors inducing acute PD-1 expression. PD-1 cells. A variety of stimuli can activate these cells, including expression on CD8 T cells correlates with the strength of the small molecule drugs PMA and ionomycin (Io), Con A, TCR signaling (12, 54, 68). TCR stimulation initiates a or anti-CD3/CD28 Abs, which acutely activate immune signaling cascade through the calcineurin pathway, resulting cells and transiently induce PD-1 expression (30, 50–53). in activation and translocation of the transcription factor In vivo, PD-1 expression is probably most studied and best NFATc1 (also known as NFAT2) (69). NFATc1 binds understood during infection with lymphocytic choriomenin- strongly to the CR-C region (Fig. 1B) and is likely one of gitis virus (LCMV) (12, 19, 20, 54–57). The LCMV the first steps in activating Pdcd1 gene expression (52, 53). infection model includes two strains that differ by 2 aa Blocking this pathway using the calcineurin inhibitor cyclo- (22). Infection with the Armstrong strain results in an sporine A or the NFATc1-specific inhibitor peptide VIVIT acute infection, correlating with transient upregulation and abrogated PD-1 expression (52). Thus, NFATc1 is necessary subsequent loss of PD-1 that coincides with viral clearance. for initial activation-induced expression of PD-1 in CD4 In contrast, establishing a chronic infection with the clone-13 and CD8 T cells. PMA/Io-mediated NFATc1 binding and strain results in durable PD-1 expression and concurrent transcriptional activities were also found at the 23.7 and immune cell dysfunction and exhaustion. Side-by-side analysis 117.1 sites, suggesting that multiple NFATc1 elements are Downloaded from of these two infections has provided direct comparisons required for driving Pdcd1 expression or that there is genetic between the transient/acute versus the chronic/prolonged redundancy in the mechanism by which Pdcd1 is induced modalities of PD-1 expression in CD8 T cells. Additionally, (62). Intriguingly, NFATc1 expression is repressed at extended prolonged PD-1 expression during an antitumor response time points after induction, and this is likely part of the in mice has been studied using the B16.f10 melanoma cell mechanism for restricting PD-1 expression during acute Ag line or Lewis lung carcinoma cell line, both of which are exposure (53). During chronic LCMV infection, NFATc1 http://www.jimmunol.org/ terminally tumorigenic but nonetheless immunogenic in translocation is also impaired (70). These observations suggest C57BL/6 mice (25, 58, 59). Although these different models that although NFATc1 may be necessary to initially induce have largely been used to study separate aspects of PD-1 PD-1, another mechanism is required to maintain or augment regulation, in vivo infection models have recapitulated expression during chronic Ag exposure. findings from in vitro–activated cell lines. Although most AP-1 frequently couples with NFAT activity during T cell of the studies to date have been performed on murine cells activation (71). TCR-mediated stimulation of the protein and cell lines (36, 52–54, 59–64), those performed with kinase C/Raf pathway activates the MAPK cascade, ultimately

human cells have yielded similar findings (65, 66). As described leading to AP-1 activity (71). Notably, PMA/Io stimulation, by guest on September 27, 2021 below, this is likely due to conservation of important regulatory originally used to identify NFATc1 binding and activity at elements at the PD-1 locus. CR-C, concurrently triggers this same pathway. In a Lewis Identifying cis-regulatory elements of the Pdcd1 gene. PD-1 is lung carcinoma tumor model, overexpression of the AP-1 encoded in the Pdcd1 gene. At least eight cis-elements subunit c-Fos was found to inhibit antitumor T cell re- regulate Pdcd1 expression. Strong mammalian DNA sequence sponses by direct binding to the AP-1 site in the CR-B region conservation coupled with DNAse I hypersensitivity assays in of Pdcd1 (Fig. 1B). This resulted in induction of PD-1 ex- EL4 cells and CD8 T cells initially identified two conserved pression in CD4 and CD8 T cells (59). A mouse containing a regions (CR-B and CR-C)associatedwithPdcd1 activation mutation of this AP-1 site had less PD-1 expression on (52). These elements, which are located 100 bp and 1.1 kb tumor-infiltrating T cells and demonstrated increased anti- upstream of the transcription start site (TSS), contain multiple tumor immunity (59). Unlike other NFAT-coupled AP-1 transcription factor binding sites (Fig. 1A). An AP-1 binding sites, which are typically adjacent (71), the Pdcd1 AP-1 site site is encoded within CR-B (59). CR-C contains an IFN- was located .1 kb away from the NFATc1 binding site (52). stimulated response element (ISRE) (60), an NFATc1 binding Additionally, the Notch pathway, which regulates T cell site (52), a FoxO1 binding site (61), and an NF-kBbindingsite effector functions (72, 73), also regulates PD-1 expression (36).Notably,reporterconstructsthatusethePD-1promoter in acute Ag settings (64). In an in vitro experiment, blockade but do not contain the CR-C region failed to induce PD-1 of the Notch pathway showed a moderate, dose-dependent expression in response to a variety of stimuli (36, 52). reduction of PD-1 expression, without affecting the overall Collectively, this suggests that CR-C is of critical importance activation of the T cell (64). Corroborating this, the Notch to the observed expression patterns of the gene. intracellular domain and the recombination signal binding Two conserved DNase I hypersensitive elements located protein for Igk J (RBPJk), two critical components of the 23.7 and 117.1 kb from the TSS enhanced transcriptional Notch signaling pathway, were bound to the Pdcd1 locus activity following TCR and IL-6 or IL-12 stimula- within 6 h of in vitro peptide–mediated TCR stimulation tion (Fig. 1A, 1E). These elements encode STAT binding sites (Fig. 1B). Similarly, in T cells of patients experiencing sepsis- and were found to interact directly with the Pdcd1 promoter induced immunosuppression, Notch pathway activation also following ex vivo cell activation and cytokine treatment (62). correlated with PD-1 expression (74). In a model using IL-10 Additional elements at 226.7 and 117.5 kb bind the and LPS to cause sepsis-induced immunosuppression in CD4 mammalian transcriptional insulator CCCTC-binding factor T cells and macrophages, inhibition of Notch signaling again (CTCF) and form constitutively interacting chromatin loops reduced PD-1 expression. Thus, the Notch pathway appears to (Fig. 1) (62). Because transcriptional insulators prevent the directly augment PD-1 expression during T cell activation settings. The Journal of Immunology 2433

FIGURE 1. Schematic models of Pdcd1 gene regula- tion. The Pdcd1 gene is represented in the various acti- vation states (ON, OFF) with (A) major cis-regulatory elements identified relative to the TSS. The positions of two transcriptional insulators (In) that bind CTCF and encompass the locus are represented. Three in vivo transcriptional states representing the activity of the locus in the (A) naive state and then following (B and C) acute (early and late) and chronic (D) LCMV infections are shown. (E) Ex vivo stimulation of CD8 T cells with cytokines IFN-a, IL-6, or IL-12. (F) Ex vivo and/or in vitro stimulation of macrophages with TLR ligands or IFN-a. In each, transcriptional activators/repressors, ac- tivating or repressive nucleosomes, and DNA methyl- Downloaded from ation states are depicted as indicated. http://www.jimmunol.org/

Inhibitors of PD-1 expression. T-box expressed in T cells lowing an acute infection. Furthermore, TFH cells, which (T-bet) was the first identified inhibitor of PD-1 (63). T-bet express high levels of PD-1, are also noted for their lack expression influences CD8 T cell differentiation, with high of Blimp-1 expression (46). In germinal centers, retrovirus- T-bet levels associated with short-lived effector cells and mediated exogenous expression of Blimp-1 resulted in a hi intermediate levels with memory precursor cells (75). Fol- failure to generate PD-1 TFH cell subsets, again showing an lowing TCR stimulation or in PD-1lo memory CD8 T cells inverse correlation between Blimp-1 and PD-1 (46). Notably, from acutely infected mice, T-bet binds 500 bp upstream Bcl6, an antagonist of Blimp-1 (78), was necessary for gen- hi by guest on September 27, 2021 of the Pdcd1 TSS (Fig. 1C) (63). In a chronic LCMV eration of a PD-1 TFH population (46). However, a direct infection, T-bet overexpression (normally only expressed role for Bcl6 inducing PD-1 expression has not been reported. at intermediate levels) was inverselycorrelatedwithPD-1 Surprisingly, Blimp-1 is expressed in exhausted CD8 T cells, expression, resulting in a PD-1int phenotype (63). Furthermore, where PD-1 levels are at their highest (79). In exhausted CD8 PD-1 expression was further increased in this infection model T cells of Blimp-1 knockout mice, the high levels of PD-1 in a T-bet knockout mouse. In acute LCMV infections, CD8 expression were slightly reduced (79), suggesting a different T cells from T-bet knockout mice only showed moderately role for Blimp-1 in regulating Pdcd1 in these cells. The higher expression of PD-1 compared with controls, rather molecular mechanism for how Blimp-1 could function than the PD-1hi phenotype seen during chronic or peak differentially as a repressor or activator of gene expression is acute infection. Collectively, these data suggest that whereas unclear. As a repressor, Blimp-1 is known to recruit additional T-bet does repress PD-1, T-bet alone is neither sufficient nor transcriptional repressors that result in silencing the local necessary to completely downregulate PD-1, and other factors chromatin environment (80–82). In the case where expression may synergize with T-bet following an acute infection to silence is increased in the presence of high levels of Blimp-1 in the gene expression. cell, several possibilities exist. First, Blimp-1 may not bind to In acute LCMV viral infection, expression of the inhibitory its site in the Pdcd1 gene and PD-1 expression continues to be factor B lymphocyte–induced maturation protein-1 (Blimp-1, active. Second, Blimp-1 could bind but fail to recruit the encoded by Prdm1) is induced by day 8 after T cell activation, repressive cofactors necessary to silence the gene. This could and similar to T-bet is necessary for terminal T cell differ- be due to novel posttranslational modifications that control entiation and functional memory formation (76, 77). At day its function. Third, Blimp-1 could function indirectly by 8, Blimp-1 was bound between CR-C and CR-B (Fig. 1C) repressing a corepressor necessary for silencing. Lastly, under (53). In this system, Blimp-1 binding resulted in the direct the exhausted conditions, an additional activating factor may loss of NFATc1 occupancy at CR-C. Blimp-1 also directly override the Blimp-1 repressor pathway. downregulated PD-1 expression in tissue culture cells. Blimp- Chronic regulators of PD-1 and factors in other cell types. The 1–mediated repression of PD-1 occurred irrespective of transcription factor FoxO1 promotes transcription of a whether NFATc1 was overexpressed from an exogenous vec- number of genes necessary for homeostatic maintenance of tor. Moreover, acute LCMV infection of a Blimp-1 knockout naive and memory T cells (83). FoxO1 antagonizes the mouse resulted in prolonged PD-1 expression on virus- transcription factor T-bet (84), and as such is a candidate specific CD8 T cells (53). These experiments indicate that for preventing PD-1 downregulation. In chronic LCMV Blimp-1 is necessary for normal loss of PD-1 expression fol- infections, FoxO1 protein is highly expressed and retained 2434 BRIEF REVIEWS: REGULATION OF PD-1 within the nucleus (61). Despite FoxO1 promoting homeostatic enhanced TCR-mediated PD-1 expression (66). Much like division and relative viral control by supporting high T cell the cytokine-induced STAT3 and STAT4 activity, IFN- numbers, it was also necessary to generate PD-1hi T cells in stimulated STAT1, STAT2, and IRF9 seem to have supple- the chronic infection model, as FoxO1 knockout mice have mentary roles in increasing PD-1 expression that require ad- lower PD-1 expression. FoxO1 acted directly on the Pdcd1 ditional TCR-mediated signals. locus by binding to a region in CR-C, and therein induced Thus, collectively, to date 10 transcription factor complexes promoter activity and PD-1 expression (Fig. 1D). Interestingly, are known to modulate PD-1, including 8 activators (NFATc1, theputativeFoxO1bindingsiteoverlapswithoneofthe c-fos/AP-1,Notch,FoxO1,STAT3,STAT4,ISGF3,and NFATc1 binding sites in CR-C. This may indicate a molecular NF-kB) and 2 inhibitory molecules (Blimp-1 and T-bet), system in which FoxO1 replaces NFATc1 and can override which all interact with the locus in response to different Blimp-1’s repressive activity. stimuli. The complexity and variability of the Pdcd1 regulome, To partially replicate in vitro some of the immune micro- the entire set of transcription factors and genetic elements that environments seen in chronic infection, CD8 T cells were affect this one gene, may contribute not only to a differential activated with anti-CD3/CD28 beads and treated with IL-6 or expression in response to different inflammatory stimuli, in- IL-12, resulting in the activation of STAT3 and STAT4 (62). cluding the difference between acute and chronic infection, Under these conditions, STAT3 and STAT4 bound the 23.7 but may also account for differential patterns seen across 1 and 17.1 regulatory regions and were able to increase PD-1 multiple cell types in response to the same infection. expression in conjunction with TCR signaling (Fig. 1E). The Epigenetic regulation of Pdcd1. Epigenetics refers to a stable, Downloaded from STAT proteins alone were not sufficient, as cytokine stimu- heritable mechanism by which cells may maintain transcrip- lation of naive T cells without concurrent TCR stimulation tional profiles and corresponding states of differentiation across did not induce PD-1 expression (62). However, this could be cell generations without modifying the underlying genetic due in part to the low levels of IL-12 receptor on naive CD8 code. Such epigenetic mechanisms include DNA methylation, T cells (85). Intriguingly, preconditioning CD8 T cells with histone protein modifications, and overall chromatin looping a http://www.jimmunol.org/ IL-12 and/or IFN- (which induces STAT1/STAT2/IFN and organization, which individually or together alter the chro- regulatory factor [IRF]9 activity) resulted in higher expres- matin state/accessibility and control transcriptional activity of a sion of the repressor T-bet and subsequently led to lower gene (89). These processes are widely used by the immune expression of PD-1 upon later Ag encounter (86). Thus, cy- system to control immune cell differentiation, fate, and gene tokine exposure prior to Ag-mediated TCR signaling may expression (90–93). have profound effects on the level of PD-1 expressed. Exhausted T cells, even upon total removal of Ag, stably Owing to the immune reinvigoration potential related to maintain their exhausted phenotype during subsequent divi- PD-1 expression on exhausted CD8 T cells, most of the above sions (94). This has been demonstrated in exhausted CD8 transcription factors have been studied in CD8 T cells alone by guest on September 27, 2021 T cells adoptively transferred from chronic LCMV-infected or in a combination of CD4 and CD8 T cells. When queried mice into naive mice, in which the cells proliferated yet in B cells, a similar NFATc1-based regulatory system was remained functionally exhausted and PD-1hi upon rechallenge implicated for PMA/Io- or BCR-mediated induction of PD-1 with an acute virus (LCMV Armstrong), which otherwise (36). However, in macrophages the calcineurin and MAPK would not induce exhaustion (94). Similarly, PD-1 levels pathways stimulated by PMA/Io did not induce PD-1, and blockade of these pathways had no effect on PD-1 expression remain high on the CD8 T cells of human patients infected induced in macrophages by TLR ligands (36, 87). It was with HIV but undergoing antiretroviral therapy, which instead found that NF-kB is necessary to induce PD-1 in drastically decreases viral loads (65). These data indicate that a response to TLR stimulation (36). Inhibition of NF-kB heritable epigenetic mechanism establishes and maintains binding to DNA using a small molecule inhibitor resulted in a both the exhausted phenotype and expression of PD-1 itself total loss of PD-1 expression. Furthermore, the NF-kB p65 after the exhaustion-inducing stimulus has been removed. subunit was bound to a site in CR-C in response to TLR DNA methylation of Pdcd1. The DNA modification 5-meth- stimulation of macrophages (Fig. 1F), and a mutation of this ylcytosine (5mC) at CpG sites in transcriptional enhancers or site abrogated transcriptional activity of the region in reporter at gene promoters is associated with silencing gene expression assays (36). The ability of NF-kB to induce PD-1 directly in (95). In mice, two CpG-rich regions upstream of the Pdcd1 cell types other than macrophages has not been studied. TSS (CR-C itself and a regulatory region starting ∼300 bp PD-1 regulation in macrophages in response to cytokine upstream of the promoter and labeled as CR-B) were dy- stimuli has also been examined. IFN-a stimulation of macro- namically methylated in CD8 T cells responding to an acute phages resulted in IFN-stimulated gene factor (ISGF)3 LCMV infection (54). Both of these regions were fully complex binding to the ISRE in the CR-C region (Fig. 1F) methylated in resting, naive CD8 T cells that do not express and induction of PD-1 expression (60). ISGF3 is a complex PD-1. At day 4 following acute LCMV infection, both regions composed of STAT1, STAT2, and IRF9 (88), again impli- showed a profound loss of methylation that was restored at day cating the STAT family of transcription factors as key mem- 8. DNA methylation was inversely correlated to both PD-1 bers of PD-1 regulation in addition to IRF9. Type I IFN expression and viral load. In contrast, during chronic LCMV activity was subsequently examined for its effects on cultured infection, the regions remained unmethylated at day 8 and T cells (66). In T cells, IFN-a alone had no effect on PD-1 later time points (Fig. 1B). Similarly, in a model of Ag tolerance expression. However, although it could not autonomously in- induced by peptide immunotherapy, it was found that duce expression, IFN-a administered concurrently with TCR demethylation of the Pdcd1 locus and corresponding induction stimulation induced IRF9 binding to the ISRE (Fig. 1E) and of PD-1 was necessary for tolerizing T cells (48). This inverse The Journal of Immunology 2435 correlation between DNA methylation and PD-1 expression effect “licensing” the elements for activity. When cytokine treat- strongly indicates a role for DNA methylation in regulating ment and TCR stimulation were combined, both H3K4me1 and Pdcd1 gene expression. H3K27ac were enriched at these elements, indicating the In patients with chronic HIV, the PD-1 regulatory region formation of an active chromatin state at these elements was similarly demethylated in PD-1hi virus-specific cells, and that contributed to increased PD-1 expression (62). Thus, unmethylated in naive, nonexhausted PD-1lo cells from the distinct cytokine profiles elicited during infections are capable same donors (65). Intriguingly, patients who were on anti- of manipulating the epigenetic program governing Pdcd1 gene retroviral therapy at the time showed no remethylation of expression. DNA at the PD-1 locus, despite significantly lower viral loads. Following resolution of an acute CD8 T cell activation, This indicates that there is no mechanism to reverse DNA the CR-C region of Pdcd1 wasenrichedfortherepressivehis- methylation changes at the Pdcd1 locus in exhausted cells, tone modifications H3 lysine 4 trimethylation (H3K9me3), regardless of changes in viral burden. Notably, one instance H3K27me3, and H4K20me3, as summarized in Fig. 1 (53). has been found in which DNA methylation is not linked to However, H3K9me3 and H3K27me3 were absent from the PD-1 expression. In macrophages, transient induction of PD- locus at the same time point in chronic infection or in 1 through TLR signaling and NF-kB was not accompanied tolerized cells expressing high levels of PD-1, showing a by a corresponding demethylation of the Pdcd1 locus (36). correlation between appearance of these inhibitory histone However, TLR-mediated PD-1 induction in macrophages profiles and loss of PD-1 expression (48, 54). Exogenous was much shorter in duration and not as robust as that in expression of Blimp-1 in acutely activated T cells was capable Downloaded from CD8 T cells during acute infection. Collectively, these data of inducing these repressive modifications and driving down suggest that demethylation may only be necessary for high or PD-1 expression (53). Although Blimp-1 itself is not known prolonged PD-1 expression. to directly modify histone proteins, it does recruit other re- There are two general models for how a gene loses DNA pressive histone-modifying enzymes, including the histone methylation. In the passive model, following DNA replication deacetylases HDAC1 and HDAC2, the histone methyl- me2/me1 a locus fails to remethylate hemimethylated CpG-containing transferase G9a, and the H3K4 lysine–specific de- http://www.jimmunol.org/ DNA, resulting in dilution of the methylation mark during methylase LSD1 (81, 82, 99). Importantly, note that both successive divisions. In an active model, methylated CpG loci repressive and activating marks are absent from the locus in are targeted by members of the ten-eleven translocase family, naive cells (53, 54), indicating that active repression is employed resulting in an initial catalytic oxidation of the 5mC to 5- only after PD-1 induction, and that the low PD-1 expression in hydroxymethylcytosine (5hmC) as an intermediate (96, 97). naive cells may be maintained exclusively by the absence of Continued oxidation and base removal result in replacement of transcriptional activators and the presence of DNA methylation. the cytosine with a nonmethylated cytosine. Note that the data

discussed above (54, 65) used bisulfite sequencing, which does Conclusions by guest on September 27, 2021 not distinguish between 5mC and the 5hmC modification. The reviewed data suggest that cell-specific and cytokine Intriguingly, 5hmC was enriched at the Pdcd1 locus (albeit at microenvironmental regulation of PD-1 allows for multiple low levels compared with 5mC) in naive mouse CD4 T cells expression paradigms. Exhaustion and high levels of PD-1 are (48). Both 5hmC and 5mC were lost upon CD4 T cell acti- correlated with Ag exposure and prolonged TCR stimulation. vation and induction of PD-1 (48). This result would argue How prolonged TCR stimulation and cytokine exposure that demethylation of the Pdcd1 locus upon gene induction drive the distinct epigenetic states of the locus remains to be occurs through an active and targeted mechanism. The occur- discovered. Because these induction pathways are common rence of this transitional mark in naive cells may indicate that to other genes, careful consideration of the the Pdcd1 locus rests in a state capable of rapid demethylation. transcription factors and epigenetic parameters governing The histone landscape of Pdcd1. Modifications to histone proteins Pdcd1 expression is necessary to be able to control the regu- within a chromatin region can change chromatin accessibility lation of PD-1 expression in therapeutic settings. Nonetheless, and affect promoter and enhancer activity, leading to changes such pathways offer the prospect of novel targets to intervene in gene transcription. On a genomic level, enhancers are with or augment PD-1–controlled immune responses. marked by histones enriched for histone H3 (H3) lysine 4 monomethylation (H3K4me1), and when considered “active” Acknowledgments also contain H3 lysine 27 acetylation (H3K27ac) (98). When We thank the members of the Boss laboratory for critical input and feedback for this work. PD-1 expression was induced on CD8 T cells in vitro, H3K9ac (another activation-specific mark) and H3K27ac were present at CR-C and the promoter (48, 53). The other Disclosures cis-elements 23.7 and 117.1 did not contain active histone The authors have no financial conflicts of interest. marks following TCR stimulation alone even though Pdcd1 expression was induced (62), suggesting that these elements References do not play a role unless cytokine stimulation occurs. In 1. Francisco, L. M., P. T. Sage, and A. H. Sharpe. 2010. The PD-1 pathway in tol- erance and autoimmunity. Immunol. Rev. 236: 219–242. agreement, treatment of CD8 T cells ex vivo with the 2. Boussiotis, V. A., P. Chatterjee, and L. Li. 2014. Biochemical signaling of PD-1 on cytokines IL-6 or IL-12 alone resulted in the enrichment of T cells and its functional implications. Cancer J. 20: 265–271. me1 2 1 3. Nishimura, H., T. Honjo, and N. Minato. 2000. Facilitation of b selection and the histone mark H3K4 at both the 3.7 and 17.1 modification of positive selection in the thymus of PD-1-deficient mice. J. Exp. regulatory sites (62). Cytokine stimulation did not result in Med. 191: 891–898. ac 4. Nishimura, H., T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori, the appearance of H3K27 at these sites or in induction of S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, and T. Honjo. 2001. Autoimmune Pdcd1 expression, suggesting that cytokine treatment was in dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291: 319–322. 2436 BRIEF REVIEWS: REGULATION OF PD-1

5. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy, 30. Agata, Y., A. Kawasaki, H. Nishimura, Y. Ishida, T. Tsubata, H. Yagita, and E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al. 2006. PD-1 expression T. Honjo. 1996. Expression of the PD-1 on the surface of stimulated mouse on HIV-specific T cells is associated with T-cell exhaustion and disease progression. T and B lymphocytes. Int. Immunol. 8: 765–772. Nature 443: 350–354. 31. Nishimura, H., Y. Agata, A. Kawasaki, M. Sato, S. Imamura, N. Minato, H. Yagita, 6. Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette, T. Nakano, and T. Honjo. 1996. Developmentally regulated expression of the PD-1 2 2 M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al. 2006. Upregulation protein on the surface of double-negative (CD4 CD8 ) thymocytes. Int. Immunol. of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune 8: 773–780. dysfunction. Nat. Med. 12: 1198–1202. 32. Haynes, N. M., C. D. Allen, R. Lesley, K. M. Ansel, N. Killeen, and J. G. Cyster. 7. Nishimura, H., M. Nose, H. Hiai, N. Minato, and T. Honjo. 1999. Development 2007. Role of CXCR5 and CCR7 in follicular Th cell positioning and appearance of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an of a programmed cell death gene-1high germinal center-associated subpopulation. ITIM motif-carrying immunoreceptor. Immunity 11: 141–151. J. Immunol. 179: 5099–5108. 8. Rui, Y., T. Honjo, and S. Chikuma. 2013. Programmed cell death 1 inhibits in- 33. Yao, S., S. Wang, Y. Zhu, L. Luo, G. Zhu, S. Flies, H. Xu, W. Ruff, M. Broadwater, flammatory helper T-cell development through controlling the innate immune re- I. H. Choi, et al. 2009. PD-1 on dendritic cells impedes innate immunity against sponse. Proc. Natl. Acad. Sci. USA 110: 16073–16078. bacterial infection. Blood 113: 5811–5818. 9. Kroner, A., M. Mehling, B. Hemmer, P. Rieckmann, K. V. Toyka, M. Ma¨urer, and 34. Zhang, Y., C. J. Ma, L. Ni, C. L. Zhang, X. Y. Wu, U. Kumaraguru, C. F. Li, H. Wiendl. 2005. A PD-1 polymorphism is associated with disease progression in J. P. Moorman, and Z. Q. Yao. 2011. Cross-talk between programmed death-1 and multiple sclerosis. Ann. Neurol. 58: 50–57. suppressor of cytokine signaling-1 in inhibition of IL-12 production by monocytes/ 10. Prokunina, L., C. Castillejo-Lo´pez, F. Oberg, I. Gunnarsson, L. Berg, macrophages in hepatitis C virus infection. J. Immunol. 186: 3093–3103. V. Magnusson, A. J. Brookes, D. Tentler, H. Kristjansdo´ttir, G. Gro¨ndal, et al. 35. Okazaki, T., A. Maeda, H. Nishimura, T. Kurosaki, and T. Honjo. 2001. PD-1 2002. A regulatory polymorphism in PDCD1 is associated with susceptibility to immunoreceptor inhibits receptor-mediated signaling by recruiting src ho- systemic lupus erythematosus in humans. Nat. Genet. 32: 666–669. mology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc. 11. Nielsen, C., D. Hansen, S. Husby, B. B. Jacobsen, and S. T. Lillevang. 2003. Natl. Acad. Sci. USA 98: 13866–13871. Association of a putative regulatory polymorphism in the PD-1 gene with suscep- 36. Bally, A. P., P. Lu, Y. Tang, J. W. Austin, C. D. Scharer, R. Ahmed, and J. M. Boss. tibility to type 1 diabetes. Tissue 62: 492–497. 2015. NF-kB regulates PD-1 expression in macrophages. J. Immunol. 194: 4545– 12. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, 4554. G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells 37. Honda, T., J. G. Egen, T. La¨mmermann, W. Kastenmuller,€ P. Torabi-Parizi, and Downloaded from during chronic viral infection. Nature 439: 682–687. R. N. Germain. 2014. Tuning of antigen sensitivity by T cell receptor-dependent 13. Urbani, S., B. Amadei, D. Tola, M. Massari, S. Schivazappa, G. Missale, and negative feedback controls T cell effector function in inflamed tissues. Immunity 40: C. Ferrari. 2006. PD-1 expression in acute hepatitis C virus (HCV) infection is 235–247. associated with HCV-specific CD8 exhaustion. J. Virol. 80: 11398–11403. 38. Costa, P. A., F. M. Leoratti, M. M. Figueiredo, M. S. Tada, D. B. Pereira, 14. Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick, W. C. Adams, C. Junqueira, I. S. Soares, D. L. Barber, R. T. Gazzinelli, and L. R. Antonelli. 2015. M. L. Precopio, T. Schacker, M. Roederer, D. C. Douek, and R. A. Koup. 2006. Induction of inhibitory receptors on T cells during Plasmodium vivax malaria im- PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J. Exp. pairs cytokine production. J. Infect. Dis. 212: 1999–2010.

Med. 203: 2281–2292. 39. Topalian, S. L., F. S. Hodi, J. R. Brahmer, S. N. Gettinger, D. C. Smith, http://www.jimmunol.org/ 15. Grakoui, A., E. John Wherry, H. L. Hanson, C. Walker, and R. Ahmed. 2006. D. F. McDermott, J. D. Powderly, R. D. Carvajal, J. A. Sosman, M. B. Atkins, et al. Turning on the off switch: regulation of anti-viral T cell responses in the liver by the 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. PD-1/PD-L1 pathway. J. Hepatol. 45: 468–472. Engl. J. Med. 366: 2443–2454. 16. Golden-Mason, L., B. Palmer, J. Klarquist, J. A. Mengshol, N. Castelblanco, and 40. Brahmer, J. R., S. S. Tykodi, L. Q. Chow, W. J. Hwu, S. L. Topalian, P. Hwu, H. R. Rosen. 2007. Upregulation of PD-1 expression on circulating and intra- C. G. Drake, L. H. Camacho, J. Kauh, K. Odunsi, et al. 2012. Safety and activity of hepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366: 2455– dysfunction. J. Virol. 81: 9249–9258. 2465. 17. Peng, G., S. Li, W. Wu, X. Tan, Y. Chen, and Z. Chen. 2008. PD-1 upregulation is 41. Hamid, O., C. Robert, A. Daud, F. S. Hodi, W. J. Hwu, R. Kefford, associated with HBV-specific T cell dysfunction in chronic hepatitis B patients. Mol. J. D. Wolchok, P. Hersey, R. W. Joseph, J. S. Weber, et al. 2013. Safety and tumor Immunol. 45: 963–970. responses with lambrolizumab (anti-PD-1) in melanoma. N. Engl. J. Med. 369: 18. Fuller, M. J., B. Callendret, B. Zhu, G. J. Freeman, D. L. Hasselschwert, 134–144.

W. Satterfield, A. H. Sharpe, L. B. Dustin, C. M. Rice, A. Grakoui, et al. 2013. 42. Topalian, S. L., M. Sznol, D. F. McDermott, H. M. Kluger, R. D. Carvajal, by guest on September 27, 2021 Immunotherapy of chronic hepatitis C virus infection with antibodies against W. H. Sharfman, J. R. Brahmer, D. P. Lawrence, M. B. Atkins, J. D. Powderly, programmed cell death-1 (PD-1). Proc. Natl. Acad. Sci. USA 110: 15001–15006. et al. 2014. Survival, durable tumor remission, and long-term safety in patients with 19. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, advanced melanoma receiving nivolumab. J. Clin. Oncol. 32: 1020–1030. J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of 43. Goding, S. R., K. A. Wilson, Y. Xie, K. M. Harris, A. Baxi, A. Akpinarli, A. Fulton, activated T cells without effector function. J. Exp. Med. 188: 2205–2213. K. Tamada, S. E. Strome, and P. A. Antony. 2013. Restoring immune function of 20. Gallimore, A., A. Glithero, A. Godkin, A. C. Tissot, A. Pluckthun,€ T. Elliott, tumor-specific CD4+ T cells during recurrence of melanoma. J. Immunol. 190: H. Hengartner, and R. Zinkernagel. 1998. Induction and exhaustion of lympho- 4899–4909. cytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using sol- 44. Jin, H. T., A. C. Anderson, W. G. Tan, E. E. West, S. J. Ha, K. Araki, uble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. G. J. Freeman, V. K. Kuchroo, and R. Ahmed. 2010. Cooperation of Tim-3 and Med. 187: 1383–1393. PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc. Natl. Acad. Sci. 21.Chen,L.2004.Co-inhibitorymoleculesoftheB7-CD28familyinthecontrolof USA 107: 14733–14738. T-cell immunity. Nat. Rev. Immunol. 4: 336–347. 45. Duraiswamy, J., K. M. Kaluza, G. J. Freeman, and G. Coukos. 2013. Dual 22.Wherry,E.J.,S.J.Ha,S.M.Kaech,W.N.Haining,S.Sarkar,V.Kalia, blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores S. Subramaniam, J. N. Blattman, D. L. Barber, and R. Ahmed. 2007. Molecular T-cell rejection function in tumors. Cancer Res. 73: 3591–3603. signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 46. Johnston, R. J., A. C. Poholek, D. DiToro, I. Yusuf, D. Eto, B. Barnett, A. L. Dent, 27: 670–684. J. Craft, and S. Crotty. 2009. Bcl6 and Blimp-1 are reciprocal and antagonistic 23. Odorizzi, P. M., K. E. Pauken, M. A. Paley, A. Sharpe, and E. J. Wherry. 2015. regulators of T follicular helper cell differentiation. Science 325: 1006–1010. Genetic absence of PD-1 promotes accumulation of terminally differentiated 47. Crotty, S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29: exhausted CD8+ T cells. J. Exp. Med. 212: 1125–1137. 621–663. 24. Turnis, M. E., L. P. Andrews, and D. A. Vignali. 2015. Inhibitory receptors as 48. McPherson, R. C., J. E. Konkel, C. T. Prendergast, J. P. Thomson, R. Ottaviano, targets for cancer immunotherapy. Eur. J. Immunol. 45: 1892–1905. M. D. Leech, O. Kay, S. E. Zandee, C. H. Sweenie, D. C. Wraith, et al. 2014. 25. Blank, C., I. Brown, A. C. Peterson, M. Spiotto, Y. Iwai, T. Honjo, and Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells T. F. Gajewski. 2004. PD-L1/B7H-1 inhibits the effector phase of tumor rejection tolerized by peptide immunotherapy. eLife 3: 4. by T cell receptor (TCR) transgenic CD8+ T cells. Cancer Res. 64: 1140–1145. 49. Allie, S. R., W. Zhang, S. Fuse, and E. J. Usherwood. 2011. Programmed death 1 26.Curiel,T.J.,S.Wei,H.Dong,X.Alvarez,P.Cheng,P.Mottram,R.Krzysiek, regulates development of central memory CD8 T cells after acute viral infection. K. L. Knutson, B. Daniel, M. C. Zimmermann, et al. 2003. Blockade of B7-H1 J. Immunol. 186: 6280–6286. improves myeloid dendritic cell-mediated antitumor immunity. Nat. Med. 9: 50. Chemnitz, J. M., R. V. Parry, K. E. Nichols, C. H. June, and J. L. Riley. 2004. 562–567. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of 27. Sierro, S. R., A. Donda, R. Perret, P. Guillaume, H. Yagita, F. Levy, and programmed death 1 upon primary human T cell stimulation, but only receptor P. Romero. 2011. Combination of lentivector immunization and low-dose che- ligation prevents T cell activation. J. Immunol. 173: 945–954. motherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti- 51. Vibhakar, R., G. Juan, F. Traganos, Z. Darzynkiewicz, and L. R. Finger. 1997. tumor immunity. Eur. J. Immunol. 41: 2217–2228. Activation-induced expression of human programmed death-1 gene in T-lympho- 28. Winograd, R., K. T. Byrne, R. A. Evans, P. M. Odorizzi, A. R. Meyer, D. L. Bajor, cytes. Exp. Cell Res. 232: 25–28. C. Clendenin, B. Z. Stanger, E. E. Furth, E. J. Wherry, and R. H. Vonderheide. 52. Oestreich, K. J., H. Yoon, R. Ahmed, and J. M. Boss. 2008. NFATc1 regulates PD- 2015. Induction of T-cell immunity overcomes complete resistance to PD-1 and 1 expression upon T cell activation. J. Immunol. 181: 4832–4839. CTLA-4 blockade and improves survival in pancreatic carcinoma. Cancer Immunol. 53. Lu, P., B. A. Youngblood, J. W. Austin, A. U. Mohammed, R. Butler, R. Ahmed, Res. 3: 399–411. and J. M. Boss. 2014. Blimp-1 represses CD8 T cell expression of PD-1 using a 29. Taube, J. M., A. Klein, J. R. Brahmer, H. Xu, X. Pan, J. H. Kim, L. Chen, feed-forward transcriptional circuit during acute viral infection. J. Exp. Med. 211: D. M. Pardoll, S. L. Topalian, and R. A. Anders. 2014. Association of PD-1, PD-1 515–527. ligands, and other features of the tumor immune microenvironment with response 54. Youngblood, B., K. J. Oestreich, S. J. Ha, J. Duraiswamy, R. S. Akondy, E. E. West, to anti-PD-1 therapy. Clin. Cancer Res. 20: 5064–5074. Z. Wei, P. Lu, J. W. Austin, J. L. Riley, et al. 2011. Chronic virus infection enforces The Journal of Immunology 2437

demethylation of the locus that encodes PD-1 in antigen-specific CD8+ T cells. Blimp-1 promotes CD8+ T cell terminal differentiation and represses the acquisition Immunity 35: 400–412. of central memory T cell properties. Immunity 31: 296–308. 55. Mueller, S. N., W. A. Langley, G. Li, A. Garcı´a-Sastre, R. J. Webby, and R. Ahmed. 78. Shaffer, A. L., X. Yu, Y. He, J. Boldrick, E. P. Chan, and L. M. Staudt. 2000. BCL- 2010. Qualitatively different memory CD8+ T cells are generated after lymphocytic 6 represses genes that function in lymphocyte differentiation, inflammation, and cell choriomeningitis virus and influenza virus infections. J. Immunol. 185: 2182–2190. cycle control. Immunity 13: 199–212. 56. West, E. E., H. T. Jin, A. U. Rasheed, P. Penaloza-Macmaster, S. J. Ha, W. G. Tan, 79. Shin, H., S. D. Blackburn, A. M. Intlekofer, C. Kao, J. M. Angelosanto, B. Youngblood, G. J. Freeman, K. A. Smith, and R. Ahmed. 2013. PD-L1 blockade S. L. Reiner, and E. J. Wherry. 2009. A role for the transcriptional repressor Blimp- synergizes with IL-2 therapy in reinvigorating exhausted T cells. J. Clin. Invest. 123: 1 in CD8+ T cell exhaustion during chronic viral infection. Immunity 31: 309–320. 2604–2615. 80. Shin, H. M., V. N. Kapoor, T. Guan, S. M. Kaech, R. M. Welsh, and L. J. Berg. 57. Blackburn, S. D., H. Shin, W. N. Haining, T. Zou, C. J. Workman, A. Polley, 2013. Epigenetic modifications induced by Blimp-1 regulate CD8+ T cell memory M. R. Betts, G. J. Freeman, D. A. Vignali, and E. J. Wherry. 2009. Coregulation of progression during acute virus infection. Immunity 39: 661–675. CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral in- 81. Su, S. T., H. Y. Ying, Y. K. Chiu, F. R. Lin, M. Y. Chen, and K. I. Lin. 2009. fection. Nat. Immunol. 10: 29–37. Involvement of histone demethylase LSD1 in Blimp-1-mediated gene repression 58. Overwijk, W. W., and N. P. Restifo. 2001. B16 as a mouse model for human melanoma. during plasma cell differentiation. Mol. Cell. Biol. 29: 1421–1431. Curr. Protoc. Immunol. Chapter 20: Unit 20.21. doi:10.1002/0471142735.im2001s39 82. Yu, J., C. Angelin-Duclos, J. Greenwood, J. Liao, and K. Calame. 2000. Tran- 59. Xiao, G., A. Deng, H. Liu, G. Ge, and X. Liu. 2012. Activator protein 1 suppresses scriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone antitumor T-cell function via the induction of programmed death 1. Proc. Natl. deacetylase. Mol. Cell. Biol. 20: 2592–2603. Acad. Sci. USA 109: 15419–15424. 83. Kerdiles, Y. M., D. R. Beisner, R. Tinoco, A. S. Dejean, D. H. Castrillon, 60. Cho, H. Y., S. W. Lee, S. K. Seo, I. W. Choi, I. Choi, and S. W. Lee. 2008. R. A. DePinho, and S. M. Hedrick. 2009. Foxo1 links homing and survival of naive -sensitive response element (ISRE) is mainly responsible for IFN-a-in- T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. Nat. Immunol. duced upregulation of programmed death-1 (PD-1) in macrophages. Biochim. 10: 176–184. Biophys. Acta 1779: 811–819. 84. Rao, R. R., Q. Li, M. R. Gubbels Bupp, and P. A. Shrikant. 2012. Transcription 61. Staron, M. M., S. M. Gray, H. D. Marshall, I. A. Parish, J. H. Chen, C. J. Perry, factor Foxo1 represses T-bet-mediated effector functions and promotes memory G. Cui, M. O. Li, and S. M. Kaech. 2014. The transcription factor FoxO1 sustains CD8+ T cell differentiation. Immunity 36: 374–387. expression of the inhibitory receptor PD-1 and survival of antiviral CD8+ T cells 85. Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, and during chronic infection. Immunity 41: 802–814. F. Sinigaglia. 1997. Selective expression of an interleukin-12 receptor component by Downloaded from 62. Austin, J. W., P. Lu, P. Majumder, R. Ahmed, and J. M. Boss. 2014. STAT3, human T helper 1 cells. J. Exp. Med. 185: 825–831. STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory 86. Schurich, A., L. J. Pallett, M. Lubowiecki, H. D. Singh, U. S. Gill, P. T. Kennedy, regions in murine T cells. J. Immunol. 192: 4876–4886. E. Nastouli, S. Tanwar, W. Rosenberg, and M. K. Maini. 2013. The third signal 63. Kao, C., K. J. Oestreich, M. A. Paley, A. Crawford, J. M. Angelosanto, M. A. Ali, cytokine IL-12 rescues the anti-viral function of exhausted HBV-specific CD8 A. M. Intlekofer, J. M. Boss, S. L. Reiner, A. S. Weinmann, and E. J. Wherry. 2011. T cells. PLoS Pathog. 9: e1003208. Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and 87. Said, E. A., F. P. Dupuy, L. Trautmann, Y. Zhang, Y. Shi, M. El-Far, B. J. Hill, sustains virus-specific CD8+ T cell responses during chronic infection. Nat. A. Noto, P. Ancuta, Y. Peretz, et al. 2010. Programmed death-1-induced +

Immunol. 12: 663–671. interleukin-10 production by monocytes impairs CD4 T cell activation during http://www.jimmunol.org/ 64. Mathieu, M., N. Cotta-Grand, J. F. Daudelin, P. The´bault, and N. Labrecque. HIV infection. Nat. Med. 16: 452–459. 2013. Notch signaling regulates PD-1 expression during CD8+ T-cell activation. 88. Horvath, C. M., G. R. Stark, I. M. Kerr, and J. E. Darnell, Jr. 1996. Interactions Immunol. Cell Biol. 91: 82–88. between STAT and non-STAT proteins in the interferon-stimulated gene factor 3 65. Youngblood, B., A. Noto, F. Porichis, R. S. Akondy, Z. M. Ndhlovu, J. W. Austin, transcription complex. Mol. Cell. Biol. 16: 6957–6964. R. Bordi, F. A. Procopio, T. Miura, T. M. Allen, et al. 2013. Cutting edge: pro- 89. Li, E. 2002. Chromatin modification and epigenetic reprogramming in mammalian longed exposure to HIV reinforces a poised epigenetic program for PD-1 expression development. Nat. Rev. Genet. 3: 662–673. in virus-specific CD8 T cells. J. Immunol. 191: 540–544. 90. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar, 66. Terawaki, S., S. Chikuma, S. Shibayama, T. Hayashi, T. Yoshida, T. Okazaki, and M. Pe´rez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al. 2001. A critical T. Honjo. 2011. IFN-a directly promotes programmed cell death-1 transcription role for Dnmt1 and DNA methylation in T cell development, function, and sur- and limits the duration of T cell-mediated immunity. J. Immunol. 186: 2772–2779. vival. Immunity 15: 763–774. 67. Hou, C., H. Zhao, K. Tanimoto, and A. Dean. 2008. CTCF-dependent enhancer- 91. Araki, Y., Z. Wang, C. Zang, W. H. Wood, III, D. Schones, K. Cui, T. Y. Roh,

blocking by alternative chromatin loop formation. Proc. Natl. Acad. Sci. USA 105: B. Lhotsky, R. P. Wersto, W. Peng, et al. 2009. Genome-wide analysis of histone by guest on September 27, 2021 20398–20403. methylation reveals chromatin state-based regulation of gene transcription and 68. Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, and R. Ahmed. function of memory CD8+ T cells. Immunity 30: 912–925. 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution 92. Scharer, C. D., B. G. Barwick, B. A. Youngblood, R. Ahmed, and J. M. Boss. 2013. and results in distinct stages of functional impairment. J. Virol. 77: 4911–4927. Global DNA methylation remodeling accompanies CD8 T cell effector function. 69. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT family: J. Immunol. 191: 3419–3429. regulation and function. Annu. Rev. Immunol. 15: 707–747. 93. Shih, H. Y., G. Sciume`, A. C. Poholek, G. Vahedi, K. Hirahara, A. V. Villarino, 70. Agnellini, P., P. Wolint, M. Rehr, J. Cahenzli, U. Karrer, and A. Oxenius. 2007. M. Bonelli, R. Bosselut, Y. Kanno, S. A. Muljo, and J. J. O’Shea. 2014. Tran- Impaired NFAT nuclear translocation results in split exhaustion of virus-specific scriptional and epigenetic networks of helper T and innate lymphoid cells. Immunol. CD8+ T cell functions during chronic viral infection. Proc. Natl. Acad. Sci. USA Rev. 261: 23–49. 104: 4565–4570. 94. Utzschneider, D. T., A. Legat, S. A. Fuertes Marraco, L. Carrie´, I. Luescher, 71. Macia´n, F., C. Lo´pez-Rodrı´guez, and A. Rao. 2001. Partners in transcription: D. E. Speiser, and D. Zehn. 2013. T cells maintain an exhausted phenotype after NFAT and AP-1. Oncogene 20: 2476–2489. antigen withdrawal and population reexpansion. Nat. Immunol. 14: 603–610. 72. Cho, O. H., H. M. Shin, L. Miele, T. E. Golde, A. Fauq, L. M. Minter, and 95. Xu, J., S. D. Pope, A. R. Jazirehi, J. L. Attema, P. Papathanasiou, J. A. Watts, B. A. Osborne. 2009. Notch regulates cytolytic effector function in CD8+ T cells. K. S. Zaret, I. L. Weissman, and S. T. Smale. 2007. Pioneer factor interactions and J. Immunol. 182: 3380–3389. unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic 73. Maekawa, Y., Y. Minato, C. Ishifune, T. Kurihara, A. Kitamura, H. Kojima, stem cells. Proc. Natl. Acad. Sci. USA 104: 12377–12382. H. Yagita, M. Sakata-Yanagimoto, T. Saito, I. Taniuchi, et al. 2008. Notch2 in- 96. Hashimoto, H., Y. Liu, A. K. Upadhyay, Y. Chang, S. B. Howerton, P. M. Vertino, tegrates signaling by the transcription factors RBP-J and CREB1 to promote T cell X. Zhang, and X. Cheng. 2012. Recognition and potential mechanisms for repli- cytotoxicity. Nat. Immunol. 9: 1140–1147. cation and erasure of cytosine hydroxymethylation. Nucleic Acids Res. 40: 4841– 74. Pan, T., Z. Liu, J. Yin, T. Zhou, J. Liu, and H. Qu. 2015. Notch signaling pathway 4849. was involved in regulating programmed cell death 1 expression during sepsis- 97. Tahiliani, M., K. P. Koh, Y. Shen, W. A. Pastor, H. Bandukwala, Y. Brudno, induced immunosuppression. Mediators Inflamm. 2015: 539841. S. Agarwal, L. M. Iyer, D. R. Liu, L. Aravind, and A. Rao. 2009. Conversion of 5- 75. Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin, methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner and S. M. Kaech. 2007. Inflammation directs memory precursor and short-lived TET1. Science 324: 930–935. effector CD8+ T cell fates via the graded expression of T-bet transcription factor. 98. Creyghton, M. P., A. W. Cheng, G. G. Welstead, T. Kooistra, B. W. Carey, Immunity 27: 281–295. E. J. Steine, J. Hanna, M. A. Lodato, G. M. Frampton, P. A. Sharp, et al. 2010. 76. Kallies, A., A. Xin, G. T. Belz, and S. L. Nutt. 2009. Blimp-1 transcription factor is Histone H3K27ac separates active from poised enhancers and predicts develop- required for the differentiation of effector CD8+ T cells and memory responses. mental state. Proc. Natl. Acad. Sci. USA 107: 21931–21936. Immunity 31: 283–295. 99. Gyory, I., J. Wu, G. Feje´r, E. Seto, and K. L. Wright. 2004. PRDI-BF1 recruits the 77. Rutishauser, R. L., G. A. Martins, S. Kalachikov, A. Chandele, I. A. Parish, histone H3 methyltransferase G9a in transcriptional silencing. Nat. Immunol. 5: E. Meffre, J. Jacob, K. Calame, and S. M. Kaech. 2009. Transcriptional repressor 299–308.