Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Sep 23.
Published in final edited form as: Immunity. 2011 Sep 23;35(3):400–412. doi: 10.1016/j.immuni.2011.06.015

Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8+ T cells

Ben Youngblood 1,2,‡, Kenneth J Oestreich 2,4,‡, Sang-Jun Ha 1,2,5, Jaikumar Duraiswamy 1,2, Rama S Akondy 1,2, Erin E West 2, Zhengyu Wei 3, Peiyuan Lu 2, James W Austin 2, James L Riley 3, Jeremy M Boss 2,*, Rafi Ahmed 1,2,*
PMCID: PMC3183460  NIHMSID: NIHMS324340  PMID: 21943489

SUMMARY

Functionally exhausted T cells express high levels of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. Using human and murine systems of acute and chronic viral infections we analyzed epigenetic regulation of PD-1 expression during CD8 T cell differentiation. During acute infection, naïve to effector CD8 T cell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of TCR signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8 T cells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.

INTRODUCTION

Heritable changes in gene regulation that occur via modification of the DNA without changing the DNA sequence are often referred to as epigenetic programming. The major eukaryotic mechanisms for epigenetic programming include DNA methylation, histone modifications, non-coding RNA-mediated transcriptional, and post-transcriptional regulation (Li, 2002; Ting et al., 2006). The best understood and most extensively studied epigenetic mechanism is transcriptional repression via DNA methylation (Ballestar and Wolffe, 2001; Baylin, 2005; Bestor et al., 1988; Hendrich and Bird, 1998; Holliday and Pugh, 1975; Johnson and Coghill, 1925; Jones, 2003; Klose and Bird, 2006; Meehan et al., 1989; Watt and Molloy, 1988; Wilson et al., 2009). Heritable transcriptional programming through DNA methylation in conjunction with other epigenetic mechanisms, orchestrate tissue and locus specific chromatin access.

Epigenetic modifications are coupled to a vast array of mammalian cell fate decisions (Jaenisch and Bird, 2003; Li, 2002), including T cell differentiation (Ansel et al., 2006; Hutchins et al., 2002; Lee et al., 2001; Wilson et al., 2009). After vaccination or infection, antigen-specific naïve CD8 T cells rapidly expand and differentiate into cytotoxic effector cells that produce cytokines to facilitate the control and clearance of the pathogen. Following clearance of the pathogen, the antigen-specific cell population shifts to a state of preparedness with the emergence of memory cells (Ahmed and Gray, 1996; Bevan and Goldrath, 2000; Doherty et al., 1996; Lefrancois and Masopust, 2002; McKinstry et al., 2008; Parish and Kaech, 2009; Sallusto and Lanzavecchia, 2009; Seder and Ahmed, 2003; Wherry and Ahmed, 2004). Epigenetic reprogramming has emerged as a mechanism to explain the cell-transmissible nature of the acquired functional properties in self-renewing memory CD8 T cells (Ansel et al., 2006; Kersh et al., 2006; Reiner, 2005; Youngblood et al., 2010).

In contrast to acute viral infection, the sustained interaction of the virus-specific CD8 T cells with antigen during chronic viral infection results in a functional impairment referred to as T cell exhaustion. CD8 T cell exhaustion is characterized by the diminished ability of the cell to express the cytokines IFNγ, TNFγ, and IL-2, reduced cytotoxicity, and an impaired ability to proliferate (Wherry et al., 2003; Zajac et al., 1998). Recent studies have reported that naïve, effector, functional memory, and exhausted CD8 T cells each have a distinct gene expression profile that reflects their status of differentiation, indicating that many of the functional differences between these T cell populations are manifest through changes in transcriptional programming (Ansel et al., 2003; Kaech et al., 2002; Reiner, 2005; Sarkar et al., 2008; Wherry, 2007). Specifically, expression of the inhibitory receptor PD-1 exemplifies the dynamic and differential gene regulation that occurs during memory CD8 T cell differentiation and directly influences the functional capacity of these cells.

PD-1 is a cell surface receptor in the immunomodulatory family of CD28 receptors. Expression of PD-1 is mainly restricted to activated lymphocytes and provides a negative signal that counters the activation signal provided by T cell receptor ligation (Greenwald et al., 2005; Okazaki and Honjo, 2007). Signaling through PD-1 is initiated by binding of its Ig surface domain to its ligands (PD-L1 and PD-L2) on infected (or antigen presenting) cells ultimately blocking many effector functions of the activated T cell (Blank and Mackensen, 2007; Greenwald et al., 2005; Hirano et al., 2005; Kaufmann and Walker, 2009; Nishimura et al., 1998; Okazaki and Honjo, 2007; Riley, 2009; Sharpe et al., 2007; Thompson et al., 2007; Yamamoto et al., 2008). Recently, using the mouse model of infection with lymphocytic choriomeningitis virus (LCMV), it was shown that exhausted antigen-specific CD8 T cells generated in chronically infected mice express high amount of PD-1 while functional antigen-specific memory T cells in acutely infected mice have reduced expression of PD-1. Treatment of chronically infected mice with a PD-1 blocking antibody drastically improved T cell function and reduced viral load (Barber et al., 2006; Blackburn et al., 2008). PD-1's causal role in T cell exhaustion and the therapeutic potential by blocking PD-1 signaling has been expanded to nonhuman primate and human studies (Boettler et al., 2006; Day et al., 2006; Golden-Mason et al., 2007; Kaufmann and Walker, 2009; Radziewicz et al., 2007; Trautmann et al., 2006; Urbani et al., 2006; Velu et al., 2009; Zhang et al., 2009).

In addition to blocking PD-1 signaling, modulating PD-1 expression may also serve to rejuvenate exhausted T cells. To better define the manner by which Pdcd1 is regulated in antigen-specific CD8 T cells during acute and chronic infection we have examined DNA methylation of PD-1 transcriptional regulatory regions in human and murine virus-specific CD8 T cells during acute and chronic infection. We present data that reveals an antigen driven transcriptional program that is distinct between exhausted and functional memory CD8 T cells.

RESULTS

Identification of Epigenetic Regulatory Regions in the PDCD1 Locus

Due to the important impact PD-1 has on CD8 T cell function we wished to determine the transcriptional mechanisms involved in regulating expression of the gene encoding PD-1 (PDCD1). Bioinformatic analysis of the upstream DNA sequence of the transcriptional start site of the human PDCD1 gene revealed a putative CpG Island, a CpG rich genomic region associated with DNA methylation-mediated transcriptional repression, that overlaps with previously identified conserved regions C and B (CR-C & CR-B) (Figure 1a). A major epigenetic mechanism is the repression of transcriptional activation via methylation of CpG dinucleotides. To test if CpG demethylation correlated with PD-1 expression, we examined the methylation status of CpGs at CR-C and -B in the mouse T cell line EL4 which express high amounts of PD-1 transcript and protein and the mouse B cell line A20 which produce very little PD-1 transcript and protein (Figure 1b) (Oestreich et al., 2008). Using a restriction digest analysis relying on enzymes that are sensitive to CpG methylation, we determined that the PD-1 conserved regions in A20 cells (PD-1negative) contained a greater degree of CpG methylation compared to EL4 cells (PD-1hi) (Figure 1b). To obtain nucleotide resolution of DNA methylation we performed bisulfite sequencing of CR-C and -B on genomic DNA from EL4 and A20 cells. Using this technique, EL4 cells were found to be void of CpG methylation while A20 cells displayed near complete methylation in the Pdcd1 conserved regions (Figure 1c). The observed inverse correlation of PD-1 expression with CpG methylation of CR-C and B in cell lines provides evidence that PD-1 transcription regulation is in part mediated through site-specific DNA methylation.

Figure 1.

Figure 1

Open in a new tab

Conserved regions of the PDCD1 promoter contain differentially methylated sites that correlate with PD-1 expression. A) The PDCD1 gene contains several highly conserved regions as determined by the UCSC Genome Browser http://genome.ucsc.edu/ (Karolchik et al., 2003). This genomic region contains a CpG island in the human genome as defined by CpG Plot from the European Bioinformatics Institute. The location of the CpG island overlaps with a DNase I hypersensitive region identified by Oestreich et al.(Oestreich et al., 2008). B) Genomic DNA from EL4 (PD-1hi) and A20 (PD-1lo) was digested with either HpaII (H, Methylation sensitive) or MspI (M, non-sensitive) along with HindIII (HIII, non-sensitive). A southern blot of the methylation sensitive restriction endonuclease challenge reveals that A20 cells have a methylated promoter. The undigested Hind III fragment is indicated by the arrowhead. The cartoon of the promoter region displays the approximate location of the restriction cut sites. C) Bisulfite sequencing of Conserved Region C (CR-C) and CR-B was performed on genomic DNA from A20 and EL4 cells. Each line represents an individual clone picked for sequencing. Filled circles = methylated cytosine. Open circles = non-methylated cytosine. D) Peripheral blood CD8+ T cells were column purified to >95% purity and stimulated with anti-CD3/CD28 beads for 3 days. Stimulated cells were cultured for an additional 5 days with and without 5-aza-2′-deoxycytidine. Cells were harvested and analyzed by FACS for PD-1 and CD8 expression. E) PD-1 mRNA from the indicated CD8 T cell population were analyzed using qRT-PCR . The values obtained were normalized to 18s rRNA and expressed as fold over the amount of transcript from Day 0 cells.

To determine if DNA methylation is causal in PD-1 repression, we measured PD-1 expression on in vitro activated primary CD8 T cells cultured in presence and absence of the DNA demethylating agent 5-aza-2′-deoxycytidine (5′AzaC). Polyclonal human naïve CD8 T cells rapidly produce PD-1 when cultured in the presence of anti-CD3 and anti-CD28 (Figure 1d & e). In vitro activated CD8 T cells were cultured for 8 days with and without 5′AzaC. Indeed, treatment of the activated CD8 T cells with 5′AzaC resulted in retained expression of PD-1 protein and transcript (Figure 1d & e). Taken together these data suggest that the DNA methylation status is inversely correlated with expression of PD-1. Further these data indicate that DNA methylation serves to repress PD-1 expression following TCR activation.

Dynamic Regulation of the Pdcd1 Locus During Memory CD8 T cell Differentiation

Our data using cell lines has established that changes in DNA methylation are coupled to transcriptional activation of PD-1 expression. Further these data demonstrate that in vitro activation of primary CD8 T cells through TCR signaling results in PD-1 expression and DNA methylation is causal in repression of transcription post-induction. We next sought to determine if alterations in DNA methylation programming is a mechanism coupled to the dynamic PD-1 expression that occurs during the differentiation of antigen-specific CD8 T cell in response to in vivo viral infection.

To investigate the role of DNA methylation in the transcriptional regulation of PD-1 expression in the differentiation of virus-specific T cells we used the mouse model of acute viral infection with LCMV Armstrong. LCMV Armstrong infection peaks ~3-4 days post infection and generates a robust effector CD8 T cell response (Ahmed et al., 1984; Wherry et al., 2003). The virus is cleared by 8 days post infection, and parallel to the virus titer, PD-1 expression is upregulated in antigen-specific CD8 T cells by 4 days post-infection and downregulated between day 4 and day 8 (Figure 2a) (Ahmed et al., 1984; Barber et al., 2006; Wherry et al., 2003; Wherry, 2007). The transient increase in PD-1 protein expression is coupled to an increase in PD-1 transcriptional activity during early effector stages of differentiation and ensuing downregulation of PD-1 transcriptional activity in day 8 effector CD8 T cells and throughout the maintenance stage of memory differentiation (Figure 2b). Genomic DNA was isolated from purified LCMV gp33-specific CD8 T cells harvested at varying time points post acute LCMV infection. Naïve, effector, and memory CD8 T cells were sorted to > 95% purity based on gp33 class I tetramer staining (Figure S1) and the percent of DNA methylation at the Pdcd1 locus was analyzed by bisulfite sequencing. We found naïve cells to be nearly 100% methylated at CpG sites 5 through 14 (Figure 2c). As a consequence of the infection induced differentiation of LCMV-specific naïve T cells, CpG sites 5 through 14 in antigen-specific day 4 effector T cells are 75 – 100 % unmethylated. Interestingly antigen-specific day 8 effector CD8 T cells have already remethylated some of the CpG sites in CR-C and CR-B compared to day 4 effector cells (Figure 2c). Moreover, the reinstated DNA methylation program becomes much more distinct in memory cells with the majority of the CpG sites in CR-B > 70% remethylated. Interestingly CpG sites 5, 11, and 14 were observed to be unmethylated on ~30% or more of the alleles from the memory CD8 T cells. These results indicate that the transient upregulation in expression of PD-1 protein and transcript in virus-specific CD8 T cells during their differentiation from a naïve CD8 T cell to a day 8 effector CD8 T cell is associated with a transient demethylation of the Pdcd1 locus (Figure 2). The reinstatement of the methylation program in day 8 effector cells before the T cell contraction phase suggests that the DNA methylation observed in memory CD8 T cells occurs due to an enzyme catalyzed process rather than selective death of terminal effector cells.

Figure 2.

Figure 2

Open in a new tab

DNA methylation of Pdcd1 CR-C and CR-B is inversely correlated with PD-1 expression in antigen-specific cells from mice acutely infected with LCMV. A) Histogram analysis of PD-1 protein expression on antigen-specific CD8 T cells at 4, 8, and >30 dpi during acute infection with 2*105 pfu of LCMV Armstrong. The red line = LCMV-specific CD8 T cells. The filled gray histogram = naïve CD8 T cells. B) Real-time PCR analysis of PD-1 transcript from purified naïve, day 4 effector, day 8 effector, and memory antigen-specific CD8 T cells. C) Bisulfite sequencing analysis and graphical summary of Pdcd1 CR-C and CR-B from FACS purified naïve, day 4 effector, day 8 effector, and memory cells. D) Bisulfite sequencing of Pdcd1 CR-C and CR-B was performed on genomic DNA from terminal effector and memory precursor CD8 T cell subsets purified from mice at 8 days post infection with 2*105 LCMV Armstrong. Filled circles = methylated cytosine. Each line represents a sequenced clone. Open circles = nonmethylated cytosine. CpG sites 1 – 14 correspond to sites -1280, -1158, -1099, -1069, -986, -778, -672, -667, -636, -612, -535, -496, -491, and -465.

To further test that recovery of the methylation profile in memory CD8 T cells is due to remethylation rather than selective death of terminal effector cells we measured the methylation status in day 8 terminal effector (Klrg1hi IL7Rαlo) and memory precursor (Klrg1lo IL7Rαhi) subsets (Kaech et al., 2003; Sarkar et al., 2008) (Figure 2d). We found that the percent of methylation at CR-C and CR-B of PD-1 was strikingly similar between the two effector subsets (Figure 2d). Thus, day 8-terminal effector and memory precursor CD8 T cells retain the same capacity to remethylate the Pdcd1 locus. These data suggest that the reprogramming of PD-1 expression is not a unique property of cells committed to a memory fate, but a consequence of cessation of TCR signaling. Furthermore, the site-specific nature of the CpG methylation observed in memory CD8 T cells is consistent with previous studies that have shown that the antigen-specific memory CD8 T cells arise from an effector population (Jacob and Baltimore, 1999; Kaech et al., 2003; Sarkar et al., 2008).

Chronic Viral Infection Inhibits DNA Remethylation of the Pdcd1 Locus

The methylation data from antigen-specific CD8 T cells generated during an acute infection suggest that remethylation of DNA in antigen-experienced cells occurs after cessation of TCR stimulation (Figure 2). In contrast to an acute viral infection, upregulation of PD-1 protein and transcriptional expression is maintained in antigen-specific CD8 T cells in the presence of persistent viral infection (Figure 3a & b). To investigate the effect of prolonged TCR stimulation on the PD-1 methylation program we measured the percent of Pdcd1 CR-C and CR-B methylation in LCMV-specific CD8 T cells during chronic LCMV infection. LCMV-specific CD8 T cells were obtained at 8 and >30 days post infection. Antigen-specific T cells were sorted to greater than 95% purity (Figure S1). We found that the CpG sites in CR-C and CR-B were demethylated in antigen experienced cells at day 8 post LCMV clone-13 infection and remained demethylated > 30 days post LCMV clone-13 infection following the contraction of the virus-specific CD8 T cell population (Figure 3c).

Figure 3.

Figure 3

Open in a new tab

Chronic antigen exposure results in prolonged demethylation of Pdcd1 CR-C and CR-B. A) Histogram analysis of PD-1 protein expression on antigen-specific CD8 T cells during acute infection at 4, 8, and 60 dpi with 2*106 pfu of LCMV clone 13. The red line = LCMV-specific CD8 T cells. The filled gray histogram = naïve CD8 T cells. B) Real-time PCR analysis of PD-1 transcript from purified naïve and exhausted antigen-specific CD8 T cells. C) Bisulfite sequencing analysis and graphical summary of Pdcd1 CR-C and CR-B from FACS purified antigen-specific cells at 8 and >30 dpi from mice infected with LCMV clone 13. D) P14 chimeric mice were infected with 2*105 pfu of LCMV Armstrong, 2*106 LCMV Armstrong, and 2*106 LCMV clone-13. PD-1 expression was measured on the LCMV-specific CD8 T cells harvested at day 8 post infection from the acutely and chronically infected mice. Adoptively transferred LCMV-specific CD8 T cells were purified at 8 days post infection. E) Bisulfite sequencing and collective summary of CpG methylation at all sites in Pdcd1 CR-B was performed on purified Day 8-effector cells. F) Day 40 LCMV-specific cells isolated from LCMV clone 13 infected mice that were treated with GK1.5 to deplete CD4 T cells to achieve full exhaustion. Antigen-specific cells were harvested from the spleen and purified by FACS using. Bisulfite sequencing analysis and graphical summary of CR-C and CR-B from the fully exhausted antigen-specific cells.

We next performed experiments to determine if duration of TCR stimulation provides a real-time signal to maintain the demethylated state of the Pdcd1 regulatory regions. To test this we varied the amount of TCR stimulation that virus-specific CD8 T cells would receive by infecting mice with 2*105 pfu of LCMV Armstrong, 2*106 LCMV Armstrong, or 2*106 LCMV clone-13. Mice infected with 2*105 LCMV Armstrong clear the virus ~ 4-5 dpi, while an acute infection initiated with 2*106 LCMV Armstrong clears ~ 6-7 dpi. Infection with 2*106 LCMV clone-13 results in high viremia at 8 dpi and persists for several months. Splenocytes were harvested at 8 days post infection and LCMV-specific CD8 T cells were sorted to greater than 95 % purity using FACS. We observed that the loss of DNA methylation of Pdcd1 CR-B in LCMV-specific CD8 T cells was dependent upon the duration of TCR stimulation (Figure 3e), with the most demethylation observed in CD8 T cells from animals infected with 2*106 LCMV clone-13 and the least demethylation in CD8 T cells from animals infected with 2*105 LCMV Armstrong.

It is well established that the loss of CD8 T cell function during chronic infection is exacerbated in the absence of CD4 help. To determine if the absence of CD4 help influences the demethylation of the Pdcd1 locus we measured the percent of DNA methylation in CD8 T cells from mice that had been treated with the CD4 depleting antibody GK1.5 then chronically infected. Both CR-C and CR-B remained demethylated in CD8 T cell isolated from mice at 40 days post LCMV clone 13 infection without CD4 help (Figure 3f). These data further supports the finding that the lack of DNA remethylation of Pdcd1 in exhausted CD8 T cells (Figure 3c & f) is a consequence of TCR signaling alone and not due to extrinsic effects to CD4 T cells induced by a chronic infection.

PD-1 Expression In Virus-specific CD8 T cells is Coupled to Chromatin Accessibility

Changes in DNA methylation are often accompanied with, and possibly directly code for, changes in chromatin accessibility by transcription factors and RNA polymerase. The “off-on-off” expression of PD-1 that occurs during an acute antiviral CD8 T cell response along with the observed dynamic methylation of Pdcd1 CR-C and CR-B (Figure 2) prompted us to determine if TCR signaling actively signals for chromatin accessibility at the Pdcd1 regulatory regions. Accessible vs. inaccessible chromatin is traditionally assayed by measuring the ability of DNase I to digest the target chromatin and was previously employed in the initial mapping of conserved regulatory regions upstream of the Pdcd1 translational start site (Oestreich et al., 2008). To determine if changes in Pdcd1 chromatin architecture correlate with antigen presentation, DNase I hypersensitivity of the Pdcd1 conserved regions was assayed in antigen-specific CD8 T cells from acutely infected mice before and after viral clearance (Figure 4). Antigen-specific CD8 T cells from mice acutely infected with LCMV Armstrong only exhibit a significant increase in sensitivity to DNase I treatment at CR-C in PD-1hi effector cells at 4 days post infection relative to naïve, PD-1lo day 9 effector, and memory antigen-specific CD8 T cells (Figure 4b & c). In contrast to virus-specific CD8 T cells obtained from mice infected with the acute strain of LCMV, virus-specific CD8 T cells from mice infected with the chronic strain of LCMV were more sensitive to DNase I digestion at both CR-C and CR-B, indicating that the transcriptional regulatory regions are more accessible (Figure 4b & c). These data reveals that changes in chromatin architecture at the Pdcd1 locus are mediated through T-cell receptor stimulation. Further, our results indicate that PD-1 expression in early effector cells is coupled to restructuring the chromatin into a condition that is poised for transcriptional activation, and that viral clearance ultimately results in restricting transcription factors from accessing the Pdcd1 locus.

Figure 4.

Figure 4

Open in a new tab

CR-C chromatin is hypersensitive to digestion by DNase I in PD-1hi virus-specific CD8 T cells. A) Chimeric mice were generated by adoptive transfer of P14 transgenic cells. Antigen-specific transgenic cells were harvested from chimeric mice following at 4, 9, and >40 dpi with LCMV clone-13 or Armstrong. Cells were then column purified using a biotinylated antibody to the congenic marker Thy1.1 on the P14 cells and assayed for DNase I hypersensitive regions in the Pdcd1 promoter. Viremia is detectable only in mice infected with clone 13 at 9 dpi vs. mice infected with Armstrong at 9 dpi (Data not shown). B) Histogram panels show PD-1 expression on naïve (shaded gray line), LCMV Armstrong differentiated (open blue line), and LCMV clone-13 differentiated (open red line) P14 LCMV-specific CD8 T cells. C) Relative DNase I hypersensitivity for CR-C and CR-B in the Pdcd1 regulatory region of naïve, day 4-effector, day 9-effector and memory CD8 T cells is plotted. P values were determined using the student T test. Not shown, the P value for the difference in DNase I hypersensitivity of CR-C between naïve and Day 4 effector cells is <0.005. D) Chromatin immunoprecipitation analysis for histone 3 lysine 9 tri-methylation (H3K9me3) and histone 3 lysine 27 tri-methylation (H3K27me3) at Pdcd1 CR-C was performed on chromatin extracted from the purified naïve, D8 effector-armstrong, and D8 effector-clone13 CD8 T cells. The bar graphs were generated from an average of three independent experiments.

DNA methylation is utilized in concert with other epigenetic mechanisms, including histone modifications, to program cell-specific restriction of transcription factors to select regions of chromatin. To further determine if the chromatin at Pdcd1 regulatory regions acquires epigenetic programs for transcriptional repression in PD-1lo cells, we measured histone tri-methylation at histone 3 lysine 9 and 27 (H3K9me3 & H3K27me3) using chromatin immunoprecipitation. Naïve CD8 T cells were observed to have very little repressive histone marks despite their low amount of PD-1 expression. Interestingly, we observed that PD-1lo antigen-specific effector CD8 T cells generated from an acute viral infection are enriched for these repressive marks relative to both naïve CD8 T cells and PD-1hi effector CD8 T cells obtained from chronically infected mice (Figure 4d and S2). These data further suggest that effector differentiation and the cessation of TCR signaling is required for the acquisition of new epigenetic programs for gene expression.

Exhausted CD8 T Cells From Chronic Infection are Poised For Rapid PD-1 Re-expression

Based on the previous observation that DNA remethylation is coupled with reduction of virus and PD-1 expression in the generation of functional memory CD8 T cells from acute LCMV infection (Figure 2), we sought to determine if reduction in antigen presentation to antigen-specific CD8 T cells from chronically infected animals would also result in remethylation of the Pdcd1 locus. LCMV clone-13 viremia is undetectable at ~3-4 months post infection and virus titers are reduced in most tissues except for the kidney and brain (Shin et al., 2007; Wherry et al., 2003). Following the waning virus titer, PD-1 expression is reduced on the LCMV-specific T cells (Figure S3). PD-1lo LCMV-specific CD8 T cells from chronically infected mice with non-detectable titers of virus in the serum at 135 days post-infection with LCMV clone-13 were sorted to greater than 95 % purity (Figure 5a) and bisulfite sequencing of Pdcd1 CR-C and CR-B was performed on the purified genomic DNA (Figure 5b). Surprisingly we found that Pdcd1 CR-C and CR-B were still fully unmethylated (Figure 5b).

Figure 5.

Figure 5

Open in a new tab

Prolonged antigen exposure results in an inability for antigen-specific CD8 T cells to reinstate the epigenetic program. A) PD-1 expression at day 8, 30 and 135 post LCMV clone-13 infection reveals that PD-1 expression decreases following the clearance of virus. Day 135 pi antigen-specific cells were sorted for methylation analysis. Serum virus titers (PFU/ml) for days 8, 30, and 135 post-infection were 6*104, 6*103, and undetectable respectively. B) Methylation analysis was performed on DNA from antigen-specific cells from naïve and virus-specific cells isolated from clone-13 infected mice at >135 dpi. Bisulfite sequencing analysis and graphical summary of CR-C & B reveals that the promoter is not remethylated in endogenous LCMV-specific CD8 T cells C) Endogenous levels (~200) of transgenic P14 CD8 T cells specific for the LCMV antigen gp33-41 were adoptively transferred into WT B6 mice. Chimeric mice were infected the next day with 2*106 pfu of LCMV clone 13. Bisulfite sequencing was performed on purified adoptively transferred LCMV-specific CD8 T cells 180 dpi. D) P14 Chimeric mice were infected with either LCMV Armstrong or LCMV clone-13. Splenocytes were isolated from infected mice at 6 months pi when PD-1 expression was significantly reduced on antigen-specific cells from clone-13 infected mice. Antigen-specific cells from either immune mice or chronically infected mice were cultured for 0, 6, and 12 hours in media containing the gp33 peptide. The amount of PD-1 expression on the virus-specific CD8 T cells (gated on Thy1.1+ cells) was determined by flow cytometric analysis. LCMV antigen-specific cells from the long-term chronic environment (blue line) produce more PD-1 relative to antigen-specific cells from the acute environment (red line) upon restimulation.

We next assessed whether adoptively transferred LCMV-specific TCR transgenic CD8 T cells (P14) would also maintain an unmethylated Pdcd1 CR-C and CR-B as a result of prolonged TCR stimulation. The analysis of adoptively transferred P14 CD8 T cells has the advantage that analysis remains restricted to cells that were present prior to the infection, as opposed to measurements being performed on heterogeneous antigen-specific populations that arise from the continuous thymic supply of naïve cells in the endogenous pool of cells. Prior to LCMV clone 13 infection we transferred endogenous levels of congenically labeled P14s into mice. After 180 days post-infection we analyzed the methylation status in the adoptively transferred LCMV-specific CD8 T cells. Indeed the continuous maintenance of an unmethylated regulatory region that occurs during the chronic infection establishes a heritably demethylated state in the adoptively transferred virus-specific CD8 T cells. Thus, naïve CD8 T cells start with the potential to obtain either a functional memory CD8 T cell epigenetic program or an exhausted epigenetic program and the commitment to these programs is dictated by the duration of antigen persistence (Figure 5c).

The observed inability to remethylate the regulatory regions of Pdcd1 in exhausted CD8 T cells implies that the locus is poised for early transcriptional activation during a secondary immune response. Thus, the effector CD8 T cell response from antigen-specific PD-1lo cells generated from a prolonged infection may be prematurely terminated as a result of inappropriately timed inhibitory signaling through PD-1. This prompted us to determine if DNA remethylation is utilized to the control PD-1 re-expression during a secondary encounter with antigen. Therefore we measured the kinetics of PD-1 expression from memory PD-1lo cells with or without DNA methylation of the Pdcd1 CR-C and CR-B. Highly functional LCMV-specific memory CD8 T cells from acutely infected animals or less functional exhausted PD-1lo LCMV-specific CD8 T cells from chronically infected animals were ex vivo stimulated for 6 and 12 hours with the LCMV peptide gp33-41 (Figure 5d). We observed that TCR-mediated stimulation of both populations of cells resulted in increased PD-1 expression, but maximal PD-1 expression was achieved from LCMV-specific cells from the chronically infected mice much faster relative to the LCMV-specific CD8 T cells from immune mice (Figure 5d). The greater rate of PD-1 protein re-expression on the LCMV-specific cells from the chronically infected mice relative to the LCMV-specific cells from acutely infected mice is consistent with the hypothesis that the remethylation of fully functional memory cells serves as a repressive mark for expression. Furthermore, the data suggest that chronic antigen exposure can heritably modify the ability to re-express PD-1.

DNA Methyltransferase 3a Isoform 2 Expression is Decreased in Exhausted CD8 T Cells

The differentiation of stem cells into cells with specialized functions is coupled to the restricted capacity to perform de novo DNA methylation mediated in part by the downregulated expression of different isoforms of de novo DNA methyltransferases (Chen et al., 2002; La Salle and Trasler, 2006). It has previously been noted that isoform 2 of the de novo DNA methyltransferase Dnmt3a is expressed in embryonic stem cells but downregulated in cells from various tissues. Interestingly, a significant amount of Dnmt3a isoform 2 expression was also detected in cells from the spleen and thymus (Chen et al., 2002). To determine if the antigen driven commitment of naïve CD8 T cells towards a functional vs. nonfunctional memory fate is associated with restriction of de novo DNA methyltransferase expression we measured the relative amount of transcript of both de novo and maintenance DNA methyltransferases in antigen-specific CD8 T cells generated from acute or chronic viral infection. Interestingly, Dnmt3a isoform 2 (Dnmt3a2) expression was downregulated in exhausted cells ~18 and ~7 fold relative to the expression in day 4 effector and functional memory CD8 T cells, respectively. However, isoform 1 of Dnmt3a (Dnmt3a1) was not downregulated in exhausted CD8 T cells (Figure 6). Furthermore, the maintenance methyltransferase Dnmt1 was upregulated ~6 fold in day 4 effector cells during acute LCMV infection relative to naïve cells, but was not significantly different in expression between functional memory and exhausted virus-specific CD8 T cells (Figure 6).

Figure 6.

Figure 6

Open in a new tab

Persistent TCR stimulation results in downregulation of the stem cell isoform of the de novo methyltransferase Dnmt3a. The relative expression of maintenance and de novo methyltransferase mRNA was measured using real-time RT-PCR. Analysis was performed on cDNA generated from total RNA from naïve, day 4, day 8, memory and exhausted LCMV-specific CD8 T cells. The difference in expression of isoform 1 and isoform 2 of the de novo methyltransferase Dnmt3a was determined using two different sets of primers that distinguish between the two isoforms. Only isoform 2 of Dnmt3a is differentially expressed between LCMV-specific CD8 T cells generated from acute and chronic infections. Transcript expression values were normalized to 18s ribosomal RNA.

To validate our expression data, and to better understand the mechanism for Dnmt3a2 downregulation, we measured the methylation status of the CpG sites near the CpG island upstream of the transcriptional start site of Dnmt3a2. We found that naïve CD8 T cells have several CpGs that are methylated. Furthermore, exhausted CD8 T cells acquired additional DNA methylation at several CpG sites (Figure S4). The increased DNA methylation at the CpGs upstream of the transcriptional start site of Dnmt3a2 in exhausted CD8 T cells is consistent with reduced expression of this Dnmt isoform. Our results showing specific downregulation of Dnmt3a2 expression in exhausted cells is consistent with the broader observation that virus-specific CD8 T cells become progressively restricted in function during chronic infection.

Epigenetic Regulation of PD-1 Expression in Human Virus-Specific CD8 T Cells

Results obtained from in vitro experiments on cell lines and primary CD8 T cells demonstrate that regulation of PD-1 expression is coupled to the ability to methylate the conserved mammalian regulatory regions (Figure 1). Further, our in vivo studies focused on the DNA methylation at the conserved regulatory regions in CD8 T cells differentiating during acute viral infections in mice demonstrated that the site-specific remethylation of the PD-1 locus is coupled with development of fully functional memory CD8 T cells (Figure 2). Additionally, development of exhausted CD8 T cells during chronic viral infection is coupled to maintenance of PD-1 locus demethylation, leaving the transcriptional regulatory regions poised for premature activation and the subsequent dampening of the effector response (Figure 3 & 5). To determine if this mechanism is utilized in the context of regulating human antigen-specific CD8 T cell functions, we proceeded to perform the methylation analysis on effector and memory CD8 T cells generated in response to both acute and chronic human infections.

We have previously shown that a primary immune response to the live virus yellow fever vaccine results in expansion of an MHC class I tetramer+ effector CD8 T cell population specific for the NS4B dominate epitope between 15 to day 30 post vaccination. The expansion of the YF-17D specific effector CD8 T cells is coupled to the transient expression of PD-1. Following contraction of the YF-17D-specific CD8 T cells and downregulation of PD-1 expression, a highly functional memory population emerges (>90 days post vaccination) (Akondy et al., 2009) (Figure 7). The yellow fever vaccine model allows for longitudinal studies of human virus-specific CD8 T cells as they progress through memory differentiation (Akondy et al., 2009; Miller et al., 2008). Further, it provides a unique opportunity to measure the temporal associations of PD-1 expression and locus programming during primary human memory differentiation of virus-specific CD8 T cells.

Figure 7.

Figure 7

Open in a new tab

PDCD1 CR-C is remethylated during effector to memory differentiation but remains demethylated in CD8 T cells specific to human viruses that cause chronic infections. A) Histogram analysis of PD-1 expression on naïve and virus-specific CD8 T cells. Bisulfite sequencing and graphical summary of CR-C of the PDCD1 promoter in human naïve (CCR7hi, CD45RAhi, PD-1lo), YF-17D-specific effector, and YF-17D-specific memory CD8 T cells. B) Histogram analysis of PD-1 expression on virus-specific CD8 T cells. Bisulfite sequencing and graphical summary of CR-C of the PDCD1 promoter in CMV and EBV-specific CD8 T cells (Cytomegalovirus & Epstein Barr virus). Class-I tetramers were used to sort antigen-specific cells from immune and chronically infected individuals. Cell purity post-sorting was ≥ 94%. Bisulfite sequencing and graphical summary of DNA from. Only CpG sites 18-26 are shown for clarity. CpG sites 1-17 reside inside the predicted CpG island which remains predominantly demethylated (Figure S5). Histogram analysis of PD-1 expression is above the corresponding methylation summary. PD-1 expression of the naïve cells is shown with the shaded gray line and PD-1 expression on the antigen-specific CD8 T cells is indicated by the open black line.

To first determine if changes in DNA methylation of the PD-1 regulatory regions correlated with PD-1 expression in human CD8 T cells we measured the methylation status of the conserved regions in naïve (CCR7+ CD45RA+) CD8 T cells. Human CD8 T cells were obtained from PBMCs of healthy human donors and sorted to a purity of >94 % (Supplemental Figure 1)(Day et al., 2006; Sallusto et al., 1999) (Duraiswamy and Ahmed unpublished results). We found that CpGs in the 5’ region of CR-C in polyclonal human naïve cells were more than 70% methylated (Figure 7a), similar to the observed level of methylation in mouse naïve CD8 T cells (Figure 2). We next measured the methylation of PD-1 CR-C in polyclonal antigen experienced (CCR7-) PD-1hi CD8 T cells and polyclonal antigen experienced PD-1lo CD8 T cells. PD-1 was CR-C was fully demethylated in the antigen experienced PD-1hi CD8 T cells, whereas it was intermediately methylated in antigen experienced PD-1lo CD8 T cells relative to the naïve cells (Supplemental Figure 5).

We next proceeded to obtain virus-specific effector and memory CD8 T cells generated from a primary response to an acute infection of healthy individuals with the yellow fever virus vaccine (YF-17D) (Figure 7a, Supplemental Figure 1 & 5). Using the MHC class I tetramer for the YFV dominate epitope we were able to obtain YF-17D-specific effector and memory CD8 T cells sorted to > 94% purity (Supplemental Figure 1c). Yellow fever virus-specific effector CD8 T cells were nearly completely demethylated relative to naïve CD8 T cells, which were more than 70% methylated (Figure 7a). Following the contraction phase of the CD8 T cell response to the infection, yellow fever virus-specific memory CD8 T cells recovered a substantial level of DNA methylation at PD-1 CR-C. Interestingly, the site-specific lack of methylation at CpG site 21 in yellow fever virus-specific memory CD8 T cells relative to naïve cells is strikingly similar to the reduced level of methylation in yellow fever virus-specific effector CD8 T cells (Figure 7a). This is consistent with the idea that human memory CD8 T cells transitioned through an effector stage of differentiation.

Similar to the mouse model of acute viral infection, acute TCR ligation of human CD8 T cells results in transient demethylation of the PD-1 regulatory regions followed by the reacquisition of a methylation program in highly functional memory CD8 T cells (Figure 2c vs. 7a). Therefore we proceeded to determine if chronic infections in humans would result in a retained demethylated state of the PD-1 locus. To gain better insight into the DNA remethylation capacity of human CD8 T cells during prolonged TCR stimulation we measured PD-1 CpG methylation in CD8 T cells specific to chronic virus infections. Antigen experienced PD-1hi CD8 T cells generated in response to Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infection were obtained from PBMCs of human donors. The antigen-specific CD8 T cells were sorted to > 94% purity using class I tetramers (Supplemental Figure 1c). We found that CMV- and EBV-specific CD8 T cells have no methylation of the PD-1 conserved region (Figure 7b). Taken together, these results suggest that persistent TCR signaling to CD8 T cells results in the maintenance of PD-1 regulatory region demethylation, leaving the locus poised for rapid transcriptional activation and a premature dampening of the effector response due to PD-1 signaling (Figure 7a & b).

DISCUSSION

Phenotypic differences between functional vs. non-functional antigen-specific memory CD8 T cells are largely manifested through changes in transcriptional regulation (Kaech et al., 2002; Slifka and Whitton, 2001; Wherry, 2007). The mechanism for programming unique transcriptional profiles of memory CD8 T cells is not well understood, but the cell-transmissible nature of memory functions during the self-renewal process indicate that epigenetic modifications are utilized to adapt the naïve transcriptional programs into memory programs. Epigenetic modifications serve as a mechanism to instruct nucleosomal modifying factors to restrict access to specific regions of chromatin. Specifically, adaptation to epigenetic modifications during an immune response to viral infection may account for the acquired transcriptional profile of memory CD8 T cells.

We have shown here that both mouse and human antigen specific CD8 T cells that differentiate in response to acute viral infection develop a memory-specific epigenetic program at the PDCD1 conserved regions. The novel program arises through reinstating the methylation after the removal of the naïve program at the effector stage of differentiation. Interestingly, the remethylation process that occurs before T cell contraction ignores select CpG sites. In particular, CpG sites 5 and 14 in the mouse and CpG site 21 in the human remain partially demethylated. The retained demethylation of specific sites in the memory cells suggests there may be a memory-specific T cell program / mechanism for PD-1 transcriptional regulation that's different from the naïve program. The observation that terminal effector and memory precursors subsets of the effector population both recover DNA methylation indicate that the memory-specific epigenetic program arises due to signaling events that both cellular populations experience. This is consistent with prior work showing that memory CD8 T cells transition through the effector stage of differentiation (Jacob and Baltimore, 1999; Kaech et al., 2003; Sarkar et al., 2008). Furthermore, in microarray studies comparing gene expression profiles of terminal effector and memory precursor subsets, significantly fewer differences in gene expression are observed between the subsets vs. differences when compared to naïve or memory CD8 T cells (Kalia et al., 2010; Sarkar et al., 2008). Therefore any differential epigenetic programming associated with either subset may be limited, likely restricted to a master regulatory factor.

In contrast to an acute infection, where clearance of the pathogen results in a heightened functional capacity of antigen-specific CD8 T cells, the continual exposure of antigen during chronic infections results in progressive functional impairment of T cells. Indeed, we show here that the mechanism for site-specific DNA remethylation in both mouse and human is lost in CD8 T cells when antigen persists in the context of chronic infection. Interestingly, when we used (LCMV clone-13) a viral infection model system that maintains high viremia for several months but eventually resolves to an undetectable titer of infection, we observed that PD-1 expression was diminished on virus-specific CD8 T cells, yet the repressive DNA methylation marks were not reinstated. Further, the lack of remethylation is observed in two different human virus-specific CD8 T cell populations generated in response to two chronic viral infections, demonstrating that the retained demethylation of the PDCD1 locus is a result of persistent antigen. This suggests that antigen-specific CD8 T cells have a restricted timeframe for remethylation of the PDCD1 regulatory regions.

The heritable loss of chromatin repressive marks in chronic antigen environments at promoters of immunoinhibitory receptors such as PD-1 may predispose antigen-specific T cells to receive more inhibitory signals, resulting in diminished T cell function. Indeed, we observed that PD-1lo antigen-specific CD8 T cells from a chronic environment reach maximal PD-1 expression much faster than functional memory cells upon ex vivo stimulation. The striking difference in the DNA methylation program for PD-1 expression in highly-functional vs. less-functional virus-specific CD8 T cells arising from acute vs. chronic infection emphasizes two fundamental properties of CD8 T cell memory differentiation: 1) CD8 T cell memory fates are not pre-determined but rather a consequence of the environment; 2) and the epigenetic mechanism of DNA methylation in CD8 T cells is a pliable program mediated by extracellular cues. Microarray studies between functional memory and exhausted CD8 T cells have reported a large number of differentially expressed genes; thus, it will be interesting to see if other genes acquire antigen duration-dependent epigenetic modifications during CD8 T cell memory differentiation.

Differentiation of stem cells into cells with specialized functions, including lymphocytes, is coupled to development of tissue and gene-specific DNA methylation patterns (Li, 2002). Acquisition of these unique methylation patterns is catalyzed in part by a class of enzymes that perform de novo DNA methylation. Very little is known regarding the specificity determinants for these marks, but several studies have now provided a link between de novo methylation and transcription factor targeting of DNA (Giambra et al., 2008; Hervouet et al., 2009; Phillips and Corces, 2009). The locus specificity for the de novo methyltransferase Dnmt3a in CD8 T cells is likely provided by interaction with other nucleic acid binding proteins or nucleic acids. Future investigations on the role and specificity determinants of the individual isoforms of Dnmt3a in the differentiation of virus-specific CD8 T cells are of great interest.

Several recent studies have called attention to the role transcription factors play in memory CD8 T cell differentiation (Rutishauser et al., 2009; Shin et al., 2009). Indeed, the accumulating evidence suggest that certain transcription factors, often referred to as master regulators, mediate cell fate decisions by instituting a lineage specific pattern of transcriptional activation (Rothenberg, 2007). The convergence of our work highlighting the importance of epigenetic programming in T cell differentiation with work defining T cell lineage transcriptional regulatory networks puts forth the idea that transcription factors may be directing epigenetic programs. Based on the prior observation that the transcription factor NF-ATc1 interacts with the Pdcd1 promoter at CR-C (Oestreich et al., 2008), we performed a bioinformatics analysis of putative transcription factors sites upstream of the Pdcd1 transcriptional start site using PROMO. The consensus sequence for NF-AT is indeed found near mouse CpG sites -1099 and -1069 prompting speculation that NF-AT may bind within a nucleosomal footprint of the demethylated CpG sites -1069, -986 and -465 in memory CD8 T cells. A causal relationship between NF-ATc1 and epigenetic programming is yet to be determined. Further understanding of the temporal relationship between localization of transcription factors, such as NF-AT, and epigenetic modifications at lineage specific genes is needed to better define the mechanism for commitment to a memory CD8 T cell fate and potentially provide a means for reprogramming exhausted CD8 T cells to obtain a fully functional antiviral response.

The findings presented here on both mouse and human epigenetic control of PD-1 expression highlight the adaptable nature of DNA methylation in CD8 T cells undergoing infection-induced differentiation. The data presented here emphasize transcriptional regulation of PD-1 as a potential target for epigenetic reprogramming with the goal of recovering antiviral functions in exhausted antigen-specific CD8 T cells. Due to the global changes in gene expression observed in non-functional CD8 T cells future studies will need to define to a broader degree the changes that occur to the CD8 T cell epigenome during chronic infection, and the consequence of these changes on CD8 T cell adaptive immunity.

EXPERIMENTAL PROCEDURES

Generation of antigen-specific T cells

Wild type C57BL/6 mice (Jackson Laboratory) were acutely or chronically infected with lymphocytic choriomeningitis virus Armstrong (2*105 pfu i.p.) or clone 13 (2*106 pfu i.v.) respectively. Serum virus titer during an acute LCMV infection peaks at ~ 4 days post infection and is not detectable after day 8, while the virus titer in chronically LCMV infected mice stays high for several months (Matloubian et al., 1990; Wherry et al., 2003). The effector response during an acute infection ranges between 4 - 8 dpi. PD-1hi effector cells are observed at ~4 dpi and PD-1lo at 8 dpi. Memory CD8 T cells from immune mice, characterized by CD44hi and CD62Lhi, were harvested at >40 dpi. Effector and memory antigen-specific CD8 T cells were purified by Fluorescence Activated Cell Sorting (FACS) using H-2Db tetramers bound to LCMV peptide GP33-41 and GP276-286 conjugated to a fluorophore, along with a CD8-fluorophore conjugated antibody as previously described (Murali-Krishna et al., 1998).

Exhausted CD8 T cells resulting from chronic infection were obtained by depleting CD4 T cells with 500 μg of GK1.5 at -1 and 0 day post infection, then infecting mice with LCMV clone-13 (Wherry, 2007; Zajac et al., 1998). Exhaustion of CD8 T cells was determined by retention of the PD-1hi phenotype and persistent viral infection at > 40 dpi (Barber et al., 2006). All CD8 T cells were harvested from the spleen. Exhausted antigen-specific CD8 T cells were purified by FACS using H-2Db tetramers GP33-41 and GP276-286 as previously described (Murali-Krishna et al., 1998).

Transgenic P14 cells with an engineered TCR that recognize the epitope GP33-41 of LCMV were harvested from naïve mice and adoptively transferred intravenously to C57BL/6 mice (either 2000 or 1*105 antigen-specific CD8 T cells per mouse as specified in the figure legend) to generate LCMV-specific CD8 T cell chimeras (Blattman et al., 2002; Kersh, 2006; Kersh et al., 2006). Infection of chimeric mice allows for greater yield when sorting for virus-specific cells using an antibody to a congenic marker of the adoptively transferred cells, CD90.1 (Thy 1.1) (Blattman et al., 2002). Chimeric CD8 T cells were sorted using fluorescently labeled CD90.1 (Thy1.1) and CD8 antibodies as previously described (Murali-Krishna et al., 1998). Naïve antigen-specific cells obtained from transgenic P14 mice (Pircher et al., 1989) were used as an antigen-specific naïve control to compare with effector, memory and exhausted CD8 T cells.

Ex-vivo culture of antigen-specific splenocytes was performed as previously described (Wherry et al., 2003). Briefly, for cytokine analysis 1*106 splenocytes were cultured for 5 hours in a 96 well round bottom plate containing 200μl of media (RPMI, 10% FBS, L glutamine, 200 ng/ml gp33 peptide, and Golgi-plug). Cytokine staining was performed per the instruction of the Cytofix/Cytoperm kit (Becton Dickenson). Cell culture experiments lasting longer than 5 hours were performed in 96 well flat bottom plates without Golgi-plug.

Human naïve and antigen experienced CD8 T cells were obtained using blood from healthy subjects after informed consent and approval for all procedures was obtained from the Emory University Institutional review Board. Whole blood was collected in cell preparation tubes (BD) and processed for PBMCs. These were then incubated with the ACK lysis buffer (Invitrogen) to lyse RBCs, washed and stained with the relevant antibodies and / or MHC class-I tetramers and subpopulations isolated using flow cytometry based sorting. Bulk CD8 T cells were sorted by FACS based on CD3hi and CD8hi gating. This subset was further divided into CCR7hi, CCR7lo and PD-1hi populations. The naïve population was defined by CCR7hi and CD45RAhi, while the effector population was defined by CCR7lo and PD-1hi. Cytomegalovirus, Epstein Barr virus, and Yellow fever vaccine antigen-specific cells were sorted from PBMC using HLA class I tetramers. For isolation of YFV, CMV, or EBV-specific CD8 T cells, PBMCs were first incubated for 30 minutes with the YF-A2, CMV-A2, or EBV-A2-streptavidin APC class-I tetramer complexes, followed by a further 30 minute incubation with CD19-FITC, CD14-FITC, CD4-FITC, CD56-FITC and CD8-PE. Cells were washed, resuspended in sorting buffer and singlet lymphocyte gated, CD8+ Tetramer+ cells sorted. Post sort purity was ~94%.

Genomic methylation analysis

Methylation at the C-5 position of the DNA nucleotide cytosine prevents bisulfite induced deamination. Allelic frequency of cytosine deamination is assayed by PCR amplification and sequencing of the target genomic region (Trinh et al., 2001). Bisulfite modification was performed using the Zymo Research EZ DNA methylation kit. The bisulfite modified DNA was PCR amplified with locus specific primers (Supplemental Table). The PCR amplicon was cloned into the pGEM-T TA cloning vector (Promega) then transformed into XL10-Gold ultracompetent bacteria (Stratagene). Individual bacterial colonies were picked, each representing a single molecule of the amplified DNA. The cloning vector was purified and the genomic insert sequenced. It should be noted that although there is conservation between mouse and human in this genomic region, the parameters used to define a CpG island in the human genome do not result in detection of a CpG island in the mouse genome.

DNase hypersensitivity assay

Naïve antigen-specific CD8 T cells were obtained from naïve transgenic P14 mice. Wild-type (WT) C57BL/6 mice were adoptively transferred with the naïve transgenic cells and then harvested at day 4 (effector), and > than day 40 (memory) post infection with LCMV Armstrong. Due to the limited number of cells from both 3-4 dpi effector mice and >40 dpi memory mice a modified version of the DNase I hypersensitivity assay was used. Cells were purified to >60% purity by labeling cells with a Thy1.1-biotin antibody (Becton Dickenson) followed by streptavidin magnetic bead (Miltenyi) column isolation. Column purification was used in order to obtain each cell population at the same time as opposed to FACS sorting. All cell populations were simultaneously treated with the same stock of DNase I. Treatment of nuclei with the enzyme DNase I (Worthington) has previously been used to interrogate general properties of chromatin architecture; accessible vs. inaccessible. Conditions for DNase treatment were adapted from the previously described protocol (Oestreich et al.). Briefly, nuclei from 5*105 cells were extracted with the DNase buffer (10 mM HEPES pH 8.0, 50 mM KCl, 5 mM MgCl2, 3 mM CaCl2, 0.1% V/V NP40, 8% V/V glycerol, 1 mM DTT (Lu and Richardson, 2004)). Cells were brought up in DNase buffer followed by 10 strokes with a pipetman and incubated on ice for 5 minutes. Nuclei were then incubated at room temperature with 5 units of DNase I for 3 minutes. The assay was then quenched with DNase stop buffer (20 mM EGTA and 1 % W/V SDS (Lu and Richardson, 2004)). Samples were incubated with Ribonuclease A for 2 hours at 37°C and then incubated overnight at 55°C with Proteinase K. DNA was purified by phenol chloroform extraction and ethanol precipitation. DNA samples were brought up in 30 μl of 10 mM Tris. Relative hypersensitivity was determined by quantifying the amount of retained chromatin using primers specific to the genomic region of interest (Supplemental Table). Relative fold change was normalized back to samples that received no DNase I treatment.

In Vitro DNA demethylation assay

Naïve human CD8 T cells were isolated from PBMCs of healthy human donors and sorted to >95% purity. The cells were cultured with anti-CD3 anti-CD28 beads to activate cells as previously described (Parry et al., 2005). 1*106 cells were cultured in the presence and absence of the DNA demethylating compound 5-aza-2′-deoxycytidine (Sigma Aldrich) at a final concentration of 1μM. 5-aza-2′-deoxycytidine solutions were made fresh just prior to culturing the cells. Cells were analyzed for transcript and protein expression at the given time points listed in the figure.

Real-time PCR analysis of mRNA

Total RNA was isolated from antigen-specific cells. Splenocytes were harvested from mice at day 0 (naive), 4, 8, and >30 post infection with LCMV Armstrong and 40 days post infection with LCMV clone-13. Antigen-specific CD8 T cells at > 8 days post infection were purified by FACS based sorting using the MHC class I tetramer to the LCMV epitope gp33-41. Day 4 antigen-specific effector cells were obtained from acutely infected P14 chimeric mice. RNA was extracted from cells using the Qiagen RNeasy kit per the instructions of the manufacturer. Quantitative real-time PCR of PD-1 and Dnmt transcripts was performed with primers as previously described (La Salle et al., 2004; La Salle and Trasler, 2006; Lucifero et al., 2007; Oestreich et al., 2008) (Supplemental Table). Transcript expression values were normalized to the 18s ribosomal RNA. Triplicate experiments were analyzed using Prism 4. Statistically significant different transcript expression was assessed using a two-tailed unpaired Students t test.

Histone Methylation Analysis

Chromatin immunoprecipitation (ChIP) was performed as previously described (Beresford and Boss, 2001; Oestreich et al., 2008). Briefly, chromatin was isolated from 1×107 purified antigen-specific CD8 T cells. Chromatin was crosslinked with 1% formaldehyde, then precleared for 1 hour using protein A beads. Crosslinked chromatin was immunoprecipitated overnight with the antibodies described in the figure legend. Following the IP, the beads were washed and DNA was eluted. Crosslinks to the DNA were reversed by incubation at 65C overnight. DNA was then purified and assayed using real-time PCR. The values for ChIP-immunoprecipitated DNA were normalized to input DNA and plotted as fold over the normalized values of irrelevant Ab control (anti-HA). Each ChIP analysis was performed three times with chromatin from independently purified CD8 T cells. Anti-H3K9me3 and anti H3K27me3 were purchased from Millipore.

Supplementary Material

01

Highlights.

  • Longitudinal analysis of PDCD1 locus DNA methylation in antiviral CD8 T cells.

  • The PDCD1 locus becomes unmethylated during naïve to effector differentiation.

  • Memory CD8 T cells acquire an adapted DNA methylation program at the PDCD1 locus.

  • The PDCD1 locus remains unmethylated in ag-sp CD8 T cells during chronic infection.

ACKNOWLEDGMENTS

We thank R. Karaffa and S. Durham for FACS sorting at the Emory University School of Medicine Flow Cytometry Core Facility. This work was supported by the National Institutes of Health (NIH) grant 1 P01 AI080192-01 (to R.A. and J.M.B.), grant 2 R37 AI30048-17 (to R.A.), grant AHMED05GCGH0 (to R.A.), the American Cancer Society (ACS) postdoctoral fellowship PF-09-134-01-MPC (to B.A.Y.), and the Korea Research Foundation (KRF) grant funded by the Korea government (MEST) No. 2010-0004892 (to S.J.H.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Ahmed R, Gray D. Immunological memory and protective immunity: understanding their relation. Science. 1996;272:54–60. doi: 10.1126/science.272.5258.54. [DOI] [PubMed] [Google Scholar]
  2. Ahmed R, Salmi A, Butler LD, Chiller JM, Oldstone MB. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J Exp Med. 1984;160:521–540. doi: 10.1084/jem.160.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Akondy RS, Monson ND, Miller JD, Edupuganti S, Teuwen D, Wu H, Quyyumi F, Garg S, Altman JD, Del Rio C, et al. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response. J Immunol. 2009;183:7919–7930. doi: 10.4049/jimmunol.0803903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ansel KM, Djuretic I, Tanasa B, Rao A. Regulation of Th2 differentiation and Il4 locus accessibility. Annu Rev Immunol. 2006;24:607–656. doi: 10.1146/annurev.immunol.23.021704.115821. [DOI] [PubMed] [Google Scholar]
  5. Ansel KM, Lee DU, Rao A. An epigenetic view of helper T cell differentiation. Nat Immunol. 2003;4:616–623. doi: 10.1038/ni0703-616. [DOI] [PubMed] [Google Scholar]
  6. Ballestar E, Wolffe AP. Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem. 2001;268:1–6. doi: 10.1046/j.1432-1327.2001.01869.x. [DOI] [PubMed] [Google Scholar]
  7. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439:682–687. doi: 10.1038/nature04444. [DOI] [PubMed] [Google Scholar]
  8. Baylin SB. DNA methylation and gene silencing in cancer. Nat.Clin.Pract.Oncol. 2005;2(Suppl 1):S4–11. doi: 10.1038/ncponc0354. [DOI] [PubMed] [Google Scholar]
  9. Beresford GW, Boss JM. CIITA coordinates multiple histone acetylation modifications at the HLA-DRA promoter. Nat Immunol. 2001;2:652–657. doi: 10.1038/89810. [DOI] [PubMed] [Google Scholar]
  10. Bestor T, Laudano A, Mattaliano R, Ingram V. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J Mol Biol. 1988;203:971–983. doi: 10.1016/0022-2836(88)90122-2. [DOI] [PubMed] [Google Scholar]
  11. Bevan MJ, Goldrath AW. T-cell memory: You must remember this. Curr Biol. 2000;10:R338–340. doi: 10.1016/s0960-9822(00)00461-9. [DOI] [PubMed] [Google Scholar]
  12. Blackburn SD, Shin H, Freeman GJ, Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci U S A. 2008;105:15016–15021. doi: 10.1073/pnas.0801497105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Blank C, Mackensen A. Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol Immunother. 2007;56:739–745. doi: 10.1007/s00262-006-0272-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Blattman JN, Antia R, Sourdive DJ, Wang X, Kaech SM, Murali-Krishna K, Altman JD, Ahmed R. Estimating the precursor frequency of naive antigen-specific CD8 T cells. J Exp Med. 2002;195:657–664. doi: 10.1084/jem.20001021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Boettler T, Panther E, Bengsch B, Nazarova N, Spangenberg HC, Blum HE, Thimme R. Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection. J Virol. 2006;80:3532–3540. doi: 10.1128/JVI.80.7.3532-3540.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chen T, Ueda Y, Xie S, Li E. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem. 2002;277:38746–38754. doi: 10.1074/jbc.M205312200. [DOI] [PubMed] [Google Scholar]
  17. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006;443:350–354. doi: 10.1038/nature05115. [DOI] [PubMed] [Google Scholar]
  18. Doherty PC, Topham DJ, Tripp RA. Establishment and persistence of virus-specific CD4+ and CD8+ T cell memory. Immunol Rev. 1996;150:23–44. doi: 10.1111/j.1600-065x.1996.tb00694.x. [DOI] [PubMed] [Google Scholar]
  19. Duraiswamy J, Ibegbu CC, Masopust D, Miller JD, Araki K, Doho GH, Tata P, Gupta S, Zilliox MJ, Nakaya HI, et al. Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults. J Immunol. 2011;186:4200–4212. doi: 10.4049/jimmunol.1001783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Giambra V, Volpi S, Emelyanov AV, Pflugh D, Bothwell AL, Norio P, Fan Y, Ju Z, Skoultchi AI, Hardy RR, et al. Pax5 and linker histone H1 coordinate DNA methylation and histone modifications in the 3' regulatory region of the immunoglobulin heavy chain locus. Mol Cell Biol. 2008;28:6123–6133. doi: 10.1128/MCB.00233-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Golden-Mason L, Palmer B, Klarquist J, Mengshol JA, Castelblanco N, Rosen HR. Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J Virol. 2007;81:9249–9258. doi: 10.1128/JVI.00409-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol. 2005;23:515–548. doi: 10.1146/annurev.immunol.23.021704.115611. [DOI] [PubMed] [Google Scholar]
  23. Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol. 1998;18:6538–6547. doi: 10.1128/mcb.18.11.6538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hervouet E, Vallette FM, Cartron PF. Dnmt3/transcription factor interactions as crucial players in targeted DNA methylation. Epigenetics. 2009;4:487–499. doi: 10.4161/epi.4.7.9883. [DOI] [PubMed] [Google Scholar]
  25. Hirano F, Kaneko K, Tamura H, Dong H, Wang S, Ichikawa M, Rietz C, Flies DB, Lau JS, Zhu G, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res. 2005;65:1089–1096. [PubMed] [Google Scholar]
  26. Holliday R, Pugh JE. DNA modification mechanisms and gene activity during development. Science. 1975;187:226–232. [PubMed] [Google Scholar]
  27. Hutchins AS, Mullen AC, Lee HW, Sykes KJ, High FA, Hendrich BD, Bird AP, Reiner SL. Gene silencing quantitatively controls the function of a developmental trans-activator. Mol Cell. 2002;10:81–91. doi: 10.1016/s1097-2765(02)00564-6. [DOI] [PubMed] [Google Scholar]
  28. Jacob J, Baltimore D. Modelling T-cell memory by genetic marking of memory T cells in vivo. Nature. 1999;399:593–597. doi: 10.1038/21208. [DOI] [PubMed] [Google Scholar]
  29. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33(Suppl):245–254. doi: 10.1038/ng1089. [DOI] [PubMed] [Google Scholar]
  30. Johnson TB, Coghill RD. The Discovery of 5-Methyl-Cytosine in Tuberculinic Acid, The Nucleic Acid of the Tubercle Bacillus. Journal of the American Chemical Society. 1925;47 [Google Scholar]
  31. Jones PA. Epigenetics in carcinogenesis and cancer prevention. Ann.N.Y.Acad.Sci. 2003;983:213–219. doi: 10.1111/j.1749-6632.2003.tb05976.x. [DOI] [PubMed] [Google Scholar]
  32. Kaech SM, Hemby S, Kersh E, Ahmed R. Molecular and functional profiling of memory CD8 T cell differentiation. Cell. 2002;111:837–851. doi: 10.1016/s0092-8674(02)01139-x. [DOI] [PubMed] [Google Scholar]
  33. Kaech SM, Tan JT, Wherry EJ, Konieczny BT, Surh CD, Ahmed R. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat Immunol. 2003;4:1191–1198. doi: 10.1038/ni1009. [DOI] [PubMed] [Google Scholar]
  34. Kalia V, Sarkar S, Subramaniam S, Haining WN, Smith KA, Ahmed R. Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity. 2010;32:91–103. doi: 10.1016/j.immuni.2009.11.010. [DOI] [PubMed] [Google Scholar]
  35. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, et al. The UCSC Genome Browser Database. Nucleic Acids Res. 2003;31:51–54. doi: 10.1093/nar/gkg129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kaufmann DE, Walker BD. PD-1 and CTLA-4 inhibitory cosignaling pathways in HIV infection and the potential for therapeutic intervention. J Immunol. 2009;182:5891–5897. doi: 10.4049/jimmunol.0803771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kersh EN. Impaired memory CD8 T cell development in the absence of methyl-CpG-binding domain protein 2. J Immunol. 2006;177:3821–3826. doi: 10.4049/jimmunol.177.6.3821. [DOI] [PubMed] [Google Scholar]
  38. Kersh EN, Fitzpatrick DR, Murali-Krishna K, Shires J, Speck SH, Boss JM, Ahmed R. Rapid demethylation of the IFN-gamma gene occurs in memory but not naive CD8 T cells. J.Immunol. 2006;176:4083–4093. doi: 10.4049/jimmunol.176.7.4083. [DOI] [PubMed] [Google Scholar]
  39. Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31:89–97. doi: 10.1016/j.tibs.2005.12.008. [DOI] [PubMed] [Google Scholar]
  40. La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, Trasler JM. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Dev Biol. 2004;268:403–415. doi: 10.1016/j.ydbio.2003.12.031. [DOI] [PubMed] [Google Scholar]
  41. La Salle S, Trasler JM. Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse. Dev Biol. 2006;296:71–82. doi: 10.1016/j.ydbio.2006.04.436. [DOI] [PubMed] [Google Scholar]
  42. Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez-Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15:763–774. doi: 10.1016/s1074-7613(01)00227-8. [DOI] [PubMed] [Google Scholar]
  43. Lefrancois L, Masopust D. T cell immunity in lymphoid and non-lymphoid tissues. Curr Opin Immunol. 2002;14:503–508. doi: 10.1016/s0952-7915(02)00360-6. [DOI] [PubMed] [Google Scholar]
  44. Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat.Rev.Genet. 2002;3:662–673. doi: 10.1038/nrg887. [DOI] [PubMed] [Google Scholar]
  45. Lu Q, Richardson B. Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression. Biol Proced Online. 2004;6:189–203. doi: 10.1251/bpo89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lucifero D, La Salle S, Bourc'his D, Martel J, Bestor TH, Trasler JM. Coordinate regulation of DNA methyltransferase expression during oogenesis. BMC Dev Biol. 2007;7:36. doi: 10.1186/1471-213X-7-36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Matloubian M, Somasundaram T, Kolhekar SR, Selvakumar R, Ahmed R. Genetic basis of viral persistence: single amino acid change in the viral glycoprotein affects ability of lymphocytic choriomeningitis virus to persist in adult mice. J Exp Med. 1990;172:1043–1048. doi: 10.1084/jem.172.4.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. McKinstry KK, Strutt TM, Swain SL. The effector to memory transition of CD4 T cells. Immunol Res. 2008;40:114–127. doi: 10.1007/s12026-007-8004-y. [DOI] [PubMed] [Google Scholar]
  49. Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell. 1989;58:499–507. doi: 10.1016/0092-8674(89)90430-3. [DOI] [PubMed] [Google Scholar]
  50. Miller JD, van der Most RG, Akondy RS, Glidewell JT, Albott S, Masopust D, Murali-Krishna K, Mahar PL, Edupuganti S, Lalor S, et al. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity. 2008;28:710–722. doi: 10.1016/j.immuni.2008.02.020. [DOI] [PubMed] [Google Scholar]
  51. Murali-Krishna K, Altman JD, Suresh M, Sourdive DJ, Zajac AJ, Miller JD, Slansky J, Ahmed R. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity. 1998;8:177–187. doi: 10.1016/s1074-7613(00)80470-7. [DOI] [PubMed] [Google Scholar]
  52. Nishimura H, Minato N, Nakano T, Honjo T. Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses. Int Immunol. 1998;10:1563–1572. doi: 10.1093/intimm/10.10.1563. [DOI] [PubMed] [Google Scholar]
  53. Oestreich KJ, Yoon H, Ahmed R, Boss JM. NFATc1 regulates PD-1 expression upon T cell activation. J Immunol. 2008;181:4832–4839. doi: 10.4049/jimmunol.181.7.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Okazaki T, Honjo T. PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol. 2007;19:813–824. doi: 10.1093/intimm/dxm057. [DOI] [PubMed] [Google Scholar]
  55. Parish IA, Kaech SM. Diversity in CD8(+) T cell differentiation. Curr Opin Immunol. 2009;21:291–297. doi: 10.1016/j.coi.2009.05.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Parry RV, Chemnitz JM, Frauwirth KA, Lanfranco AR, Braunstein I, Kobayashi SV, Linsley PS, Thompson CB, Riley JL. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol. 2005;25:9543–9553. doi: 10.1128/MCB.25.21.9543-9553.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Phillips JE, Corces VG. CTCF: master weaver of the genome. Cell. 2009;137:1194–1211. doi: 10.1016/j.cell.2009.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Pircher H, Burki K, Lang R, Hengartner H, Zinkernagel RM. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature. 1989;342:559–561. doi: 10.1038/342559a0. [DOI] [PubMed] [Google Scholar]
  59. Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, Wehbi M, Hanson HL, Steinberg JP, Masopust D, Wherry EJ, et al. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression. J Virol. 2007;81:2545–2553. doi: 10.1128/JVI.02021-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Reiner SL. Epigenetic control in the immune response. Hum Mol Genet. 2005;14:R41–46. doi: 10.1093/hmg/ddi115. Spec No 1. [DOI] [PubMed] [Google Scholar]
  61. Riley JL. PD-1 signaling in primary T cells. Immunol Rev. 2009;229:114–125. doi: 10.1111/j.1600-065X.2009.00767.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Rothenberg EV. Cell lineage regulators in B and T cell development. Nat Immunol. 2007;8:441–444. doi: 10.1038/ni1461. [DOI] [PubMed] [Google Scholar]
  63. Rutishauser RL, Martins GA, Kalachikov S, Chandele A, Parish IA, Meffre E, Jacob J, Calame K, Kaech SM. Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties. Immunity. 2009;31:296–308. doi: 10.1016/j.immuni.2009.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Sallusto F, Lanzavecchia A. Heterogeneity of CD4+ memory T cells: functional modules for tailored immunity. Eur J Immunol. 2009;39:2076–2082. doi: 10.1002/eji.200939722. [DOI] [PubMed] [Google Scholar]
  65. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999;401:708–712. doi: 10.1038/44385. [DOI] [PubMed] [Google Scholar]
  66. Sarkar S, Kalia V, Haining WN, Konieczny BT, Subramaniam S, Ahmed R. Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J Exp Med. 2008;205:625–640. doi: 10.1084/jem.20071641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Seder RA, Ahmed R. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat Immunol. 2003;4:835–842. doi: 10.1038/ni969. [DOI] [PubMed] [Google Scholar]
  68. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol. 2007;8:239–245. doi: 10.1038/ni1443. [DOI] [PubMed] [Google Scholar]
  69. Shin H, Blackburn SD, Blattman JN, Wherry EJ. Viral antigen and extensive division maintain virus-specific CD8 T cells during chronic infection. J Exp Med. 2007;204:941–949. doi: 10.1084/jem.20061937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Shin H, Blackburn SD, Intlekofer AM, Kao C, Angelosanto JM, Reiner SL, Wherry EJ. A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Immunity. 2009;31:309–320. doi: 10.1016/j.immuni.2009.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Slifka MK, Whitton JL. Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR. Nat Immunol. 2001;2:711–717. doi: 10.1038/90650. [DOI] [PubMed] [Google Scholar]
  72. Thompson RH, Dong H, Lohse CM, Leibovich BC, Blute ML, Cheville JC, Kwon ED. PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma. Clin Cancer Res. 2007;13:1757–1761. doi: 10.1158/1078-0432.CCR-06-2599. [DOI] [PubMed] [Google Scholar]
  73. Ting AH, McGarvey KM, Baylin SB. The cancer epigenome--components and functional correlates. Genes Dev. 2006;20:3215–3231. doi: 10.1101/gad.1464906. [DOI] [PubMed] [Google Scholar]
  74. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, et al. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med. 2006;12:1198–1202. doi: 10.1038/nm1482. [DOI] [PubMed] [Google Scholar]
  75. Trinh BN, Long TI, Laird PW. DNA methylation analysis by MethyLight technology. Methods. 2001;25:456–462. doi: 10.1006/meth.2001.1268. [DOI] [PubMed] [Google Scholar]
  76. Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J Virol. 2006;80:11398–11403. doi: 10.1128/JVI.01177-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Velu V, Titanji K, Zhu B, Husain S, Pladevega A, Lai L, Vanderford TH, Chennareddi L, Silvestri G, Freeman GJ, et al. Enhancing SIV-specific immunity in vivo by PD-1 blockade. Nature. 2009;458:206–210. doi: 10.1038/nature07662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Watt F, Molloy PL. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev. 1988;2:1136–1143. doi: 10.1101/gad.2.9.1136. [DOI] [PubMed] [Google Scholar]
  79. Wherry EJ, Ahmed R. Memory CD8 T-cell differentiation during viral infection. J.Virol. 2004;78:5535–5545. doi: 10.1128/JVI.78.11.5535-5545.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol. 2003;77:4911–4927. doi: 10.1128/JVI.77.8.4911-4927.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Wherry EJ, Ha S, Kaech SM, Haining WN, Sarkar S, Kalia V, Subramaniam S, Blattman JN, Barber DL, Ahmed R. Molecular Signature of CD8 T Cell Exhaustion during Chronic Viral Infection. Immunity. 2007 doi: 10.1016/j.immuni.2007.09.006. [DOI] [PubMed] [Google Scholar]
  82. Wilson CB, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol. 2009;9:91–105. doi: 10.1038/nri2487. [DOI] [PubMed] [Google Scholar]
  83. Yamamoto R, Nishikori M, Kitawaki T, Sakai T, Hishizawa M, Tashima M, Kondo T, Ohmori K, Kurata M, Hayashi T, Uchiyama T. PD-1-PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood. 2008;111:3220–3224. doi: 10.1182/blood-2007-05-085159. [DOI] [PubMed] [Google Scholar]
  84. Youngblood B, Davis CW, Ahmed R. Making memories that last a lifetime: heritable functions of self-renewing memory CD8 T cells. Int Immunol. 2010 doi: 10.1093/intimm/dxq437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, Ahmed R. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998;188:2205–2213. doi: 10.1084/jem.188.12.2205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Zhang Z, Jin B, Zhang JY, Xu B, Wang H, Shi M, Wherry EJ, Lau GK, Wang FS. Dynamic decrease in PD-1 expression correlates with HBV-specific memory CD8 T-cell development in acute self-limited hepatitis B patients. J Hepatol. 2009;50:1163–1173. doi: 10.1016/j.jhep.2009.01.026. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS324340-supplement-01.pdf (1MB, pdf)

RESOURCES