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J Virol. 2016 October 1; 90(19): 8934–8946.
Published online 2016 September 12. Prepublished online 2016 July 27. doi:  10.1128/JVI.00798-16
PMCID: PMC5021389

Demethylation of the PD-1 Promoter Is Imprinted during the Effector Phase of CD8 T Cell Exhaustion

M. S. Diamond, Editor
Washington University School of Medicine

ABSTRACT

PD-1 is an inhibitory receptor that has a major role in T cell dysfunction during chronic infections and cancer. While demethylation of the PD-1 promoter DNA is observed in exhausted T cells isolated from chronically infected individuals, little is known about when this stable demethylation of PD-1 promoter DNA is programmed during the course of a chronic infection. To assess if PD-1 promoter DNA demethylation is impacted by prolonged stimulation during effector phase of chronic infection, we adoptively transferred virus-specific day 8 effector CD8 T cells from mice infected with lymphocytic choriomeningitis virus (LCMV) clone 13 into recipient mice that had cleared an acute infection. We observed that LCMV-specific CD8 T cells from chronically infected mice maintained their surface expression of PD-1 even after transfer into acute immune mice until day 45 posttransfer. Interestingly, the PD-1 transcriptional regulatory region continued to remain unmethylated in these donor CD8 T cells generated from a chronic infection. The observed maintenance of PD-1 surface expression and the demethylated PD-1 promoter were not a result of residual antigen in the recipient mice, because similar results were seen when chronic infection-induced effector cells were transferred into mice infected with a variant strain of LCMV (LCMV V35A) bearing a mutation in the cognate major histocompatibility complex class I (MHC-I) epitope that is recognized by the donor CD8 T cells. Importantly, the maintenance of PD-1 promoter demethylation in memory CD8 T cells was coupled with impaired clonal expansion and higher PD-1 re-expression upon secondary challenge. These data show that the imprinting of the epigenetic program of the inhibitory receptor PD-1 occurs during the effector phase of chronic viral infection.

IMPORTANCE Since PD-1 is a major inhibitory receptor regulating T cell dysfunction during chronic viral infection and cancers, a better understanding of the mechanisms that regulate PD-1 expression is important. In this work, we demonstrate that the PD-1 epigenetic program in antigen-specific CD8 T cells is fixed during the priming phase of chronic infection.

INTRODUCTION

CD8 T cells play a critical role in controlling acute viral infections and, upon control, can establish antigen-specific memory that provides the host with long-lived immunity to the previously experienced pathogen. While chronic pathogens also generate robust CD8 T cell responses, prolonged exposure to high levels of antigen results in the decline of CD8 T cell effector functions. The progressive decline of effector function in antigen-specific CD8 T cells is coupled with reduced proliferative potential and repression in the ability to express the cytokines interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) upon antigenic restimulation (1, 2). This progressive decline in T cell effector function, known as T cell exhaustion, poses a major challenge for controlling chronic diseases, including HIV, hepatitis C virus (HCV), and HBV infections (3) and cancer (4,6). Thus, there is considerable interest in developing therapeutic strategies to reverse T cell exhaustion for enhancing antipathogen and antitumor CD8 T cell immunity.

Recent investigation into the molecular mechanisms regulating CD8 T cell exhaustion has revealed that expression of inhibitory receptors is causal in the maintenance of the exhausted state (7). Specifically, programmed cell death 1 (PD-1) is the major characterized inhibitory receptor and has been shown to inhibit T cell proliferation as well as reduce the effector function of antigen-specific CD8 T cells. The impact of PD-1 signaling on T cell exhaustion and the resulting implications for cancer immunotherapy came from studies in which PD-1 signals were blocked in mice chronically infected with lymphocytic choriomeningitis virus (LCMV) (8). Treatment of chronically infected mice with a PD-1 blocking antibody resulted in the expansion of antigen-specific CD8 T cells that had increased effector functions. These findings were immediately extended to several chronic human viral infections, such as HIV, HCV, and many different cancers, including melanoma and lung, bladder, kidney, and head and neck cancers (9,18). The rapid translation of this fundamental discovery into a therapeutic strategy for treating chronic infections and cancer further highlights the untapped potential for PD-1-targeted immunotherapy.

Given the central role that PD-1 plays in current cancer immunotherapy strategies, a better understanding of the transcriptional regulatory mechanisms that govern PD-1 expression is required. Many labs have shown that upregulated expression of PD-1 on virus-specific CD8 T cells is maintained during chronic infection, while PD-1 is rapidly downregulated on antigen-specific CD8 T cells after viral clearance during an acute infection (8, 19). Accordingly, it is generally accepted that initial upregulation, as well as continued expression of PD-1, is predominantly driven by T cell receptor (TCR) signaling. The molecular mechanisms regulating PD-1 expression were recently explored by examining the roles of transcription factors, including NFAT, FoxO1, T-bet, and Blimp-1, in controlling PD-1 transcription (20,24). Both NFAT and FoxO1 serve as transcriptional activators of the PD-1 promoter by direct binding (22, 24). Blimp-1 also directly binds to the PD-1 locus but represses PD-1 expression in the early phase of acute infection, while T-bet represses PD-1 during chronic infection (20, 21, 23). Along with transcription factors, epigenetic mechanisms play a major role in controlling gene expression, mediating stable maintenance of transcriptional programs. We have recently reported that the PD-1 transcriptional regulatory region underwent dynamic change in epigenetic programming during the development of functional memory and exhausted CD8 T cells. Specifically, we observed that functional memory CD8 T cells reacquired a methylated PD-1 promoter while the PD-1 promoter remained unmethylated in exhausted CD8 T cells even when viral loads were reduced to undetectable levels (25, 26). These reports suggest that long-term antigen exposure enforces the demethylation of the PD-1 promoter in fully exhausted CD8 T cells. However, it remains unclear when stabilization of PD-1 promoter methylation programming sets in during chronic infection. To address the stabilization of effector-phase DNA methylation changes, we used the mouse model of LCMV infection to track the methylation state of the PD-1 promoter in antigen-specific effector CD8 T cells adoptively transferred from chronically infected mice into acutely infected animals. Here we report that effector CD8 T cells generated from a chronic viral infection maintain the demethylated state of the PD-1 locus even when they are transferred and rested in immune recipient mice. Additionally, maintenance of the demethylated locus on virus-specific CD8 T cells is associated with heightened PD-1 re-expression and decreased clonal expansion during a secondary response.

MATERIALS AND METHODS

Mice and infection.

Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory. Lymphocytic choriomeningitis virus (LCMV) gp33-41 specific TCR transgenic mice (P14) were used. Mice were infected with LCMV Arm (2 × 105 PFU intraperitoneally [i.p.]), clone 13 (CL13) (2 × 104 or 2 × 106 PFU intravenously [i.v.]), or CL13 V35A (2 × 104 i.v.). All mice were used in accordance with National Institutes of Health and the Emory University Institutional Animal Care and Use Committee guidelines.

Adoptive transfer.

A total of 5 × 103 P14 (Thy1.1/1.1 or Ly5.1/5.1) cells were adoptively transferred into congenically distinct B6 mice (Thy1.2/1.2 or Ly5.2/5.2). P14 chimeric mice were then infected with LCMV Arm (2 × 105 PFU i.p.) or CL13 (2 × 106 PFU i.v.). CD8 T cells were purified from spleen using a CD8 T cell purification kit (Miltenyie Biotech) at day 8 postinfection. A total of 2 × 106 (Thy1.1/1.1 or Ly5.1/5.1) P14 cells from LCMV Arm-infected P14 chimeric mice and 2 × 106 (Thy1.1/1.2 or Ly5.1/5.2) P14 cells from LCMV CL13-infected P14 chimeric mice were cotransferred into infection-matched (day 8 LCMV Arm infection) host mice. To examine the recall response, sorted 5 × 103 memory P14 cells derived from acute P14 (Ly5.1/5.1) cells and chronic P14 cells (Ly5.1/5.2) were cotransferred into naive B6 mice, followed by LCMV Arm (2 × 105 PFU i.p.) infection.

Flow cytometry and staining.

Lymphocytes were collected from peripheral blood mononuclear cells (PBMCs), spleen, and liver, as previously described (27). LIVE/DEAD Near-IR (Invitrogen) was used to remove the dead cells. For intracellular cytokine staining, splenocytes were incubated with gp33-41 peptide (0.2 μg/ml) for 6 h at 37°C in the presence of brefeldin and monensin. Cells were stained with anti-IFN-γ, anti-TNF-α, and anti-IL-2 antibodies (Abs) (BD) using Cytofix/Cytoperm (BD) according to the manufacturer's recommendations. Cells were analyzed on a Canto II flow cytometer (BD Immunocytometry Systems). Data were analyzed with FlowJo v.9.2 (TreeStar).

Genomic DNA methylation analysis.

Genomic DNA was purified from sorted P14 cells at day 30 posttransfer. Bisulfite-induced deamination of cytosine was performed using the Zymo Research EZ DNA methylation kit. This bisulfite-modified DNA was amplified by PCR with locus-specific primers as previously described (25).

RESULTS

LCMV-specific effector CD8 T cells from chronic infection maintain surface PD-1 expression after transfer into acute immune mice.

We have previously reported that demethylation of both the mouse and human PD-1 promoters is coupled to PD-1 expression during effector differentiation of virus-specific CD8 T cells (25). Furthermore, the demethylated state of the PD-1 promoter persists in CD8 T cells that have prolonged exposure to antigen during chronic infections. Surprisingly, we found that the demethylated state continued even when virus levels were reduced and PD-1 expression was downregulated (25). In this study, we sought to further understand the direct relationship between duration of antigen exposure and maintenance of the demethylated PD-1 promoter in virus-specific CD8 T cells. To investigate the relationship between stability in DNA methylation programming and antigen persistence, we utilized the lymphocytic choriomeningitis virus (LCMV) mouse model of acute and chronic viral infections. This model allows for the examination of effector and memory differentiation of antigen-specific CD8 T cells that develop in response to either acute or chronic antigen presentation. Furthermore, adoptive-transfer experiments using P14 TCR transgenic CD8 T cells that recognize a dominant LCMV epitope (gp33-41) facilitates tracking of phenotype, function, and epigenetic programs longitudinally in mice that had cleared an acute LCMV infection. To track the plasticity of the DNA methylation program during LCMV infection, we generated chimeric mice by adoptively transferring congenically distinct naive P14 cells into WT animals. The chimeric animals were then infected with the acute or chronic strain of LCMV. At 8 days postinfection, equal numbers of “acute” and “chronic” effector P14 cells were cotransferred into infection-matched mice that were infected with the acute strain of LCMV. Thus, the existing effector pool of CD8 T cells in the recipient mice were capable of clearing any residual virus that was potentially transferred with the effector P14 cells from LCMV CL13-infected mice (Fig. 1a).

FIG 1
LCMV-specific day 8 P14 cells from chronically infected mice show sustained expression of PD-1 after transfer into acute immune mice. (a) P14 cells from either LCMV Arm or CL13 infection were cotransferred into infection-matched (Arm day 8) mice at day ...

To first assess the ability of donor chronic P14 cells to persist after transfer, we longitudinally measured the number of acute and chronic P14 cells in the blood of recipient mice until 45 days after adoptive transfer. Chronic P14 cells underwent a much greater level of contraction than did acute P14 cells between days 1 and 8 posttransfer, but following the contraction phase of the immune response, the ratio of chronic P14 cells among total P14 cells was maintained at a steady frequency (~20% of total P14) until day 45 posttransfer (Fig. 1b). We next examined changes in PD-1 expression on acute and chronic P14 cells in the blood of acute immune mice. Since the acute strain of LCMV is controlled to undetectable levels by day 8 postinfection, donor P14 cells from the acutely infected mice had already low levels of the PD-1 expression. In contrast, donor P14 cells isolated from the chronically infected mice had high levels of PD-1 expression (Fig. 1c, day 0). Upon transfer into the infection-matched recipient mice, the PD-1 expression on the acute P14 cells was further downregulated. Likewise, the PD-1 expression on chronic P14 cells also decreased in mice that had cleared the acute LCMV infection, although the level of PD-1 expression on chronic P14 cells at the memory phase of the immune response was significantly higher than on acute P14 cells (P = 0.0009 at day 8 and P = 0.0018 at day 30) (Fig. 1c). We also assessed the quantity and level of PD-1 expression on acute and chronic P14 cells in the lymphoid and nonlymphoid tissues of the recipient mice. At day 45 posttransfer, we found that the ratio of acute and chronic P14 cells among total P14 cells in the spleen and liver was similar to the ratio that we observed in the blood (Fig. 1d). Importantly, chronic P14 cells continued to retain a higher constitutive level of PD-1 expression than did acute P14 cells in both lymphoid and nonlymphoid tissues at day 45 posttransfer (Fig. 1e). Hence, while the chronic P14 cells acquired some memory-associated characteristics, such as antigen-independent long-term survival, they also retained an expression program for higher constitutive expression of the PD-1 inhibitory receptor.

After observing the changes in PD-1 expression, we proceeded to more broadly examine the phenotypic changes of chronic P14 cells following rest in the acute immune mice. Interestingly, the downregulated expression of other inhibitory receptors on chronic P14 cells, including Tim3 and 2B4, within the first week upon transfer into the immune environment occurred much faster than the downregulation of PD-1. Furthermore, the level of these inhibitory receptors on chronic P14 cells decreased to similar levels of expression on the acute P14 cells at day 30 posttransfer (Fig. 2a). We next examined the re-expression of several memory-associated cell surface molecules. After viral clearance, antigen-specific CD8 T cells are known to re-express CD127 and l-selectin (CD62L), which are critical for antigen-independent survival and lymphoid homing, respectively. Surviving chronic and acute P14 cells both upregulated CD127, but the chronic P14 cells were impaired in their ability to re-express l-selectin (CD62L) compared to the acute P14 cells (Fig. 2b).

FIG 2
Acute and chronic P14 cells share similar phenotypic properties after transfer into acute immune mice. P14 cells from either LCMV Arm- or CL13-infected mice were transferred into infection-matched (LCMV Arm) recipients at day 8 postinfection. Levels of ...

To determine if the sustained expression of PD-1 in the chronic P14 cells after transfer into acute LCMV mice was associated with decreased functionality, we next assessed the ability of the P14 cells to recall effector cytokine expression. At day 1 posttransfer, the pool of chronic P14 cells was impaired in the ability to express TNF-α and IL-2 compared to acute P14 cells. The impairment in TNF-α and IL-2 expression among the pool of chronic versus acute P14 cells progressively diminished over time, although the chronic P14 cells continued to retain a slight defect in TNF-α expression even after 30 days posttransfer (Fig. 3). While it is well established that exhausted virus-specific CD8 T cells retain elevated levels of PD-1 expression and have an impaired effector function, our data demonstrate that some aspects of the early exhaustion program instilled at the onset of a chronic infection can be stably maintained even after transfer into acute immune mice.

FIG 3
Function of chronic P14 cells was progressively recovered after transfer into acute immune mice. P14 cells from either LCMV Arm- or CL13-infected mice were transferred into infection-matched (LCMV Arm) recipients at day 8 postinfection. Splenocytes of ...

PD-1 promoter demethylation is imprinted in antigen-specific CD8 T cells at the effector phase of chronic infection.

We have previously reported that the fully methylated PD-1 promoter of naive CD8 T cells is demethylated during effector differentiation, but it only becomes remethylated in functional memory CD8 T cells that emerge after viral clearance (25). Therefore, we sought to determine if the higher constitutive PD-1 expression of the chronic P14 cells in acute immune mice was coupled to a defect in PD-1 promoter remethylation. To test if PD-1 promoter demethylation was stabilized at the effector phase of T cell exhaustion, we cotransferred day 8 acute and chronic P14 cells into acute infection-matched mice, allowed the cells to rest in the immune environment for 30 days, and then assessed the PD-1 promoter methylation (Fig. 4a). Bisulfite sequencing analysis of the genomic DNA from sorted P14 cells revealed significant remethylation of CpG sites in the PD-1 promoter of acute P14 cells at day 30 posttransfer compared to that in day 8 donor P14 cells (~50% of methylation at day 30 versus ~30% of methylation in day 8 donor cells). However, the PD-1 promoter in chronic P14 cells at day 30 posttransfer remained unmethylated, similar to the day 8 donor P14 cells (Fig. 4b). These data suggest that chronic P14 cells acquire a stable defect in PD-1 promoter DNA remethylation during the effector phase of chronic infection.

FIG 4
PD-1 promoter DNA demethylation is preserved in chronic P14 cells after transfer into mice that had cleared an acute LCMV infection. (a) Equal numbers of P14 cells from LCMV Arm- or CL13-infected mice were transferred into LCMV Arm infection-matched mice ...

Maintenance of the demethylated PD-1 promoter on LCMV-specific effector CD8 T cells from chronically infected mice is antigen independent.

To further probe the possibility that minimal TCR signaling from residual viral antigen in recipient mice is responsible for retaining the unmethylated PD-1 promoter on chronic P14 cells, we used the LCMV V35A variant virus, which has a mutated gp33 epitope and is incapable of activating P14 cells (28). Using this model, we cotransferred acute and chronic P14 cells into recipient mice that were acutely infected with LCMV WT or LCMV V35A mutant virus at day 8 postinfection (Fig. 5a). We found that similar to the experiments whose results are shown in Fig. 1c, PD-1 expression on chronic P14 cells underwent a significant reduction after ~1 week in both LCMV WT- and V35A mutant-infected recipient mice, but it then was maintained at a higher constitutive level of expression than on acute P14 cells after 30 days of rest (Fig. 5b and andc).c). Importantly, methylation profiling of P14 cells revealed that the chronic P14 cells still retained a demethylated PD-1 promoter in both WT- and V35A mutant virus-infected recipient mice at day 30 posttransfer, whereas acute P14 cells had remethylated the PD-1 regulatory region (Fig. 5d and ande).e). These data demonstrate that the mechanism for maintenance of PD-1 promoter demethylation in antigen-specific CD8 T cells that is initially generated in response to a chronic infection does not rely on continuous antigen exposure.

FIG 5
Sustained demethylation of PD-1 promoter on LCMV-specific day 8 P14 cells from chronically infected mice is antigen independent. (a) Equal numbers of P14 cells from Arm- and CL13-infected mice were cotransferred into infection-matched (LCMV WT or V35A ...

Sustained DNA demethylation of the PD-1 promoter is coupled to impaired T cell expansion during a recall response.

Preservation of the demethylated PD-1 promoter along with higher constitutive PD-1 expression suggests that the chronic P14 cells are poised for rapid upregulation of PD-1. To test this, we first stimulated acute and chronic P14 cells with cognate peptide (gp33) ex vivo on day 30 posttransfer (Fig. 6a). Indeed, chronic P14 cells expressed a much higher level of PD-1 than did the acute P14 cells upon restimulation, although the levels of induction of PD-1 by gp33 stimulation were comparable between acute and chronic P14 cells (Fig. 6b).

FIG 6
Chronic P14 cells (day 8 effector) rested in acute immune mice for 30 days retain a heightened ability to re-express PD-1. (a) Equal numbers of P14 cells from either LCMV Arm- or CL13-infected mice were cotransferred into infection-matched (LCMV Arm) ...

We next examined the relationship between the poised PD-1 expression and the magnitude of the secondary response. To assess the ability of the chronic P14 cells to mount an in vivo recall response, acute and chronic P14 memory cells were sorted at day 30 posttransfer and cotransferred into naive mice. The chimeric mice were then infected with LCMV Arm, and the quantity and quality of the secondary effector and memory cells were measured (Fig. 7a). At day 8 post-secondary infection, we found that the number of chronic P14 cells was significantly lower than the number of acute P14 cells in the blood (Fig. 7b). While the number of cells available for adoptive transfer precluded us from assessing proliferation kinetics, it is likely that the defect in expansion of chronic P14 cells resulted from impairment in both survival and proliferation capacity of the cells. Longitudinal analysis of the P14 cells revealed that the chronic P14 cells persisted at a lower quantity as they developed into secondary memory cells. We further observed that the chronic P14 cells had a higher level of PD-1 expression than did acute P14 cells during the secondary effector response. Specifically, ~50% of acute P14 cells expressed PD-1, while most of the chronic P14 cells (80 to 90%) expressed PD-1, at day 8 post-secondary infection. Additionally, the level of PD-1 expression was sustained on chronic P14 cells until day 40 post-secondary infection (20 to 30%) (Fig. 7c). Interestingly, both acute and chronic P14 cells had much slower kinetics for downregulation of PD-1 expression during the contraction after the recall response than after the primary response. We further observed that the absolute numbers of chronic P14 cells were also much lower than those of acute P14 cells in the spleen and liver at day 40 post-secondary infection (Fig. 7d). Also, the levels of PD-1 expression on chronic P14 cells in blood, spleen, and liver were all significantly higher than on acute P14 cells in these compartments (Fig. 7e). Given that the ratio of chronic P14 cells among total P14 cells was closely maintained from day 8 to day 40 postinfection (21.3% at day 8 versus 27.1% at day 40), it is likely that the impaired secondary memory formation of chronic P14 cells was due to a defect in expansion during the recall response.

FIG 7
Chronic P14 cells (day 8 effector) rested in acute immune mice for 30 days still show defect in recall response in vivo. (a) Equal numbers of acute and chronic P14 cells were sorted at day 30 posttransfer and cotransferred into naive B6 mice, followed ...

Upon observing a higher level of PD-1 expression on chronic P14 cells than on acute P14 cells, we next examined if expression of other inhibitory receptors was also differentially regulated during secondary response. As before, we transferred an equal number of acute and chronic P14 memory cells into naive mice and then acutely infected the mice with LCMV Arm (Fig. 8a). Again, PD-1 expression on chronic P14 cells was significantly elevated compared to that on acute P14 cells, but surprisingly, the levels of Tim-3 and 2B4 expression on the both chronic and acute P14 cells were comparable at all time points between 8 and 40 days post-secondary infection. We further examined whether higher PD-1 expression was coupled to changes in functionality of chronic P14 cells. We found that at day 40 postinfection, levels of cytokine production among chronic and acute P14 cells were comparable upon restimulation. Interestingly, though, chronic P14 cells had a lower level of granzyme B expression (Fig. 8c). We next proceeded to determine if the higher PD-1 re-expression on chronic P14 cells was responsible for the expansion defect during the recall response. To test this, anti-PD-L1 antibody or control Ig was administered at days 0, 3, 6, and 9 post-secondary infection (Fig. 9a). Indeed, inhibition of the PD-1 pathway led to a significant increase in the number of chronic P14 cells at day 8, and this recovery in the number P14 cells was also maintained until day 35 postinfection, while the number of acute P14 cells was not greatly affected by PD-L1 blockade (Fig. 9b). However, PD-L1 blockade did not result in significant differences in granzyme B expression and cytokine production from either acute or chronic P14 cells at day 40 after secondary challenge (Fig. 9c). Collectively, these data indicate that stable demethylation of the PD-1 promoter in chronic P14 memory cells facilitates heightened re-expression of PD-1 during a recall response and is broadly coupled to a reduced ability of the cell to undergo expansion during a secondary immune response.

FIG 8
Phenotype and function of acute and chronic P14 cells during recall response. (a) Acute and chronic P14 cells were sorted at day 30 posttransfer and cotransferred into naive B6 mice, followed by LCMV Arm infection. (b) surface expressions of PD-1, Tim-3, ...
FIG 9
PD-L1 blockade improves the clonal expansion of chronic P14 cells transferred into acute immune mice during secondary challenge. (a) P14 cells from either LCMV Arm- or CL13-infected mice were transferred into infection-matched (LCMV Arm) recipients at ...

DISCUSSION

Continuous antigen exposure during chronic infection results in sustained expression of the inhibitory receptor PD-1 and a progressive decline in virus-specific CD8 T cell effector function. Our previous study revealed that PD-1 expression on exhausted CD8 T cells was coupled to DNA demethylation of the PD-1 transcriptional regulatory region (25). Interestingly, exhausted CD8 T cells retained the unmethylated PD-1 loci even when the viral load was reduced to undetectable levels at the late phase of mouse and human chronic infection (25, 26). This study generated several important questions: was the retained demethylated state simply due to the persistence of a low level of antigen exposure? If not, when does the demethylation “on” program for PD-1 expression become stably reinforced? In the current study, we found that when antigen-specific CD8 T cells were transferred from day 8 chronic infection into infection-matched immune mice, while the PD-1 surface expression was downregulated, CD8 T cells still maintained an unmethylated PD-1 promoter. One caveat of this experimental setting is that although acute and chronic P14 cells were transferred into the same recipient, their microenvironment inside tissue might be different. For example, chronic P14 cells might preferentially localize to a relatively antigen-rich area compared to acute P14 cells, which could cause the continuous surface expression of PD-1 and stable demethylation of the PD-1 promoter. However, we demonstrated that maintenance of PD-1 promoter demethylation does not require a persistent TCR signal, as it still occurred in mice infected with the variant LCMV V35A mutant virus. Thus, these data support a model whereby demethylation of the PD-1 transcriptional regulatory region is imprinted during effector differentiation in response to a chronic infection.

Consistent with the data presented here, it has been previously reported that HIV-specific CD8 T cells from patients who had controlled viral loads by antiviral therapy or naturally, as in the case of HIV elite controllers, also maintained an unmethylated PD-1 promoter even after the viral load was reduced for several years (26). Given that similar results have been obtained with various human and mouse model systems of chronic viral infection, it is tempting to speculate that this may be a general mechanism observed during development of T cell exhaustion and as such may also be observed during the development of cancer. Indeed, continuous exposure to cancer antigen and the inflammatory environments results in CD8 T cell exhaustion, but it remains to be determined if tumor-specific CD8 T cells maintain a demethylated PD-1 promoter after therapy-induced clearance of the cancer. Moreover, future efforts are needed to broadly assess if PD-1 pathway blockade results in long-lived changes in gene expression programs in tumor-specific CD8 T cell following therapy.

Both acute and chronic LCMV infections initially result in demethylation of the PD-1 promoter in virus-specific effector CD8 T cells, yet remethylation of the PD-1 promoter occurs only in CD8 T cells with acute exposure to the virus antigen and inflammatory environment. Indeed, T-cell priming prior to day 8 of a chronic infection likely contributes to the differential regulation of PD-1 promoter methylation. Although challenging, future assessment of early priming events will require adoptive transfer of day 5 effector CD8 T cells from acute and chronic infections into infection-matched mice. Such studies will provide additional insight into the roles viral persistence and tropism (29) play in establishing long-lasting epigenetic programs in memory CD8 T cells. In addition to the potential role of viral tropism (differences in antigen-presenting cells), poor CD4 help and higher level of inflammatory cytokines such as type I interferon (30,33) likely also contribute to the initial imprinting of the DNA methylation state during the early stage of chronic infection. Therefore, the combination of longer exposure to antigen and these various signals may culminate in the differential expression or changes in specificity of the DNA methyltransferases involved in epigenetic modification.

In a previous study using the same model system as ours, it was reported that antigen-specific effector CD8 T cells isolated from chronically LCMV-infected mice on day 5 or 8 could escape from exhaustion if they were removed from continuous antigen exposure (34, 35). Angelosanto et al. showed that these effector CD8 T cells arising from chronic infection were maintained over time, downregulated PD-1, and re-expressed CD127 in infection controlled recipients, which was consistent with our data (35). The authors concluded that partially exhausted virus-specific CD8 T cells retained the potential to differentiate into memory CD8 T cells. While we have several overlapping observations, our phenotypic and epigenetic analyses revealed that the virus-specific effector CD8 T cells derived from chronic infection are restricted in their ability to develop memory T cell properties. First, chronic P14 cells undergo a severe contraction within a week of adoptive transfer into acute immune mice, which results in a reduced quantity of memory T cells. Second, re-expression of the homing molecule l-selectin (CD62L) on chronic P14 cells was limited compared to that on acute P14 cells during memory differentiation. Third, and most important, the demethylated transcriptional regulatory region of PD-1 on memory CD8 T cells arising from chronic infection-derived effector T cells was coupled to accelerated PD-1 expression upon restimulation. Prior analyses of exhausted T cells transferred and rested in naive mice for 56 days also revealed higher PD-1 expression than in memory CD8 T cells upon secondary acute infection until day 30 (36). Our results presented here provide a possible mechanism of the stable poisoning of PD-1 expression in exhausted CD8 T cells. This poised PD-1-mediated inhibitory signal appeared to impair the production of cytokines following ex vivo restimulation and ultimately reduced the clonal expansion of chronic P14 cells during a recall response. Lastly, the expansion defect was restored by PD-1 signal blockade, confirming that PD-1 was responsible for this defective recall response. Prior studies investigating PD-1 blockade during a recall response of memory CD8 T cells generated from vaccinia virus with and without CD4 help and expressing higher PD-1 than CD4-helped memory CD8 cells also noted that the expansion defect of helpless memory CD8 T cells could be restored by PD-1 blockade during the recall response (37). Although the mechanism was linked to a higher level of PD-1 expression in the helpless memory cells, it is unclear if it is also due to the epigenetic regulation of PD-1 promoter. Therefore, although effector CD8 T cells derived from chronic infection retain the ability to re-express several surface markers associated with memory T cell differentiation, effector T cells from a chronic environment may be epigenetically limited in regaining the capacity to differentiate into fully functional memory CD8 T cells.

In summary, we determined that the priming phase of chronic infection is critical for instilling a stable PD-1 DNA methylation program in antigen-specific CD8 T cells, and these data provide a mechanism to explain why memory CD8 T cells derived from a chronic infection may retain a functional impairment. These results shed new insight into the molecular mechanisms that govern the generation of long-lived functional and nonfunctional memory T cells.

ACKNOWLEDGMENTS

We thank B. T. Konieczny and H. Wu for technical assistance and R. Karaffa and S. Durham for fluorescence-activated cell sorting (FACS) at the Emory University School of Medicine Flow Cytometry Core Facility.

E. Ahn received a postdoctoral fellowship (E00024) through the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education.

REFERENCES

1. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, Ahmed R 1998. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 188:2205–2213. doi:.10.1084/jem.188.12.2205 [PMC free article] [PubMed] [Cross Ref]
2. Wherry EJ. 2011. T cell exhaustion. Nat Immunol 12:492–499. [PubMed]
3. Virgin HW, Wherry EJ, Ahmed R 2009. Redefining chronic viral infection. Cell 138:30–50. doi:.10.1016/j.cell.2009.06.036 [PubMed] [Cross Ref]
4. Jiang Y, Li Y, Zhu B 2015. T-cell exhaustion in the tumor microenvironment. Cell Death Dis 6:e1792. doi:.10.1038/cddis.2015.162 [PMC free article] [PubMed] [Cross Ref]
5. Kim PS, Ahmed R 2010. Features of responding T cells in cancer and chronic infection. Curr Opin Immunol 22:223–230. doi:.10.1016/j.coi.2010.02.005 [PMC free article] [PubMed] [Cross Ref]
6. Zou W. 2005. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 5:263–274. doi:.10.1038/nrc1586 [PubMed] [Cross Ref]
7. Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ 2009. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 10:29–37. doi:.10.1038/ni.1679 [PMC free article] [PubMed] [Cross Ref]
8. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682–687. doi:.10.1038/nature04444 [PubMed] [Cross Ref]
9. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ, Klenerman P, Ahmed R, Freeman GJ, Walker BD 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443:350–354. doi:.10.1038/nature05115 [PubMed] [Cross Ref]
10. Gardiner D, Lalezari J, Lawitz E, DiMicco M, Ghalib R, Reddy KR, Chang KM, Sulkowski M, Marro SO, Anderson J, He B, Kansra V, McPhee F, Wind-Rotolo M, Grasela D, Selby M, Korman AJ, Lowy I 2013. A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1), in patients with chronic hepatitis C virus infection. PLoS One 8:e63818. doi:.10.1371/journal.pone.0063818 [PMC free article] [PubMed] [Cross Ref]
11. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, Carcereny E, Ahn MJ, Felip E, Lee JS, Hellmann MD, Hamid O, Goldman JW, Soria JC, Dolled-Filhart M, Rutledge RZ, Zhang J, Lunceford JK, Rangwala R, Lubiniecki GM, Roach C, Emancipator K, Gandhi L, KEYNOTE-001 Investigators 2015. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372:2018–2028. doi:.10.1056/NEJMoa1501824 [PubMed] [Cross Ref]
12. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, Routy JP, Haddad EK, Sekaly RP 2006. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12:1198–1202. doi:.10.1038/nm1482 [PubMed] [Cross Ref]
13. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, Pitot HC, Hamid O, Bhatia S, Martins R, Eaton K, Chen S, Salay TM, Alaparthy S, Grosso JF, Korman AJ, Parker SM, Agrawal S, Goldberg SM, Pardoll DM, Gupta A, Wigginton JM 2012. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366:2455–2465. doi:.10.1056/NEJMoa1200694 [PMC free article] [PubMed] [Cross Ref]
14. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, Dronca R, Gangadhar TC, Patnaik A, Zarour H, Joshua AM, Gergich K, Elassaiss-Schaap J, Algazi A, Mateus C, Boasberg P, Tumeh PC, Chmielowski B, Ebbinghaus SW, Li XN, Kang SP, Ribas A 2013. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369:134–144. doi:.10.1056/NEJMoa1305133 [PMC free article] [PubMed] [Cross Ref]
15. Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, Sosman JA, McDermott DF, Powderly JD, Gettinger SN, Kohrt HE, Horn L, Lawrence DP, Rost S, Leabman M, Xiao Y, Mokatrin A, Koeppen H, Hegde PS, Mellman I, Chen DS, Hodi FS 2014. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515:563–567. doi:.10.1038/nature14011 [PMC free article] [PubMed] [Cross Ref]
16. Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, Bellmunt J, Burris HA, Petrylak DP, Teng SL, Shen X, Boyd Z, Hegde PS, Chen DS, Vogelzang NJ 2014. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515:558–562. doi:.10.1038/nature13904 [PubMed] [Cross Ref]
17. Schmidt LH, Kummel A, Gorlich D, Mohr M, Brockling S, Mikesch JH, Grunewald I, Marra A, Schultheis AM, Wardelmann E, Muller-Tidow C, Spieker T, Schliemann C, Berdel WE, Wiewrodt R, Hartmann W 2015. PD-1 and PD-L1 expression in NSCLC indicate a favorable prognosis in defined subgroups. PLoS One 10:e0136023. doi:.10.1371/journal.pone.0136023 [PMC free article] [PubMed] [Cross Ref]
18. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS 2014. Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol 32:1020–1030. doi:.10.1200/JCO.2013.53.0105 [PMC free article] [PubMed] [Cross Ref]
19. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, Subramaniam S, Blattman JN, Barber DL, Ahmed R 2007. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27:670–684. doi:.10.1016/j.immuni.2007.09.006 [PubMed] [Cross Ref]
20. Kao C, Oestreich KJ, Paley MA, Crawford A, Angelosanto JM, Ali MA, Intlekofer AM, Boss JM, Reiner SL, Weinmann AS, Wherry EJ 2011. Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection. Nat Immunol 12:663–671. doi:.10.1038/ni.2046 [PMC free article] [PubMed] [Cross Ref]
21. Lu P, Youngblood BA, Austin JW, Mohammed AU, Butler R, Ahmed R, Boss JM 2014. Blimp-1 represses CD8 T cell expression of PD-1 using a feed-forward transcriptional circuit during acute viral infection. J Exp Med 211:515–527. doi:.10.1084/jem.20130208 [PMC free article] [PubMed] [Cross Ref]
22. Oestreich KJ, Yoon H, Ahmed R, Boss JM 2008. NFATc1 regulates PD-1 expression upon T cell activation. J Immunol 181:4832–4839. doi:.10.4049/jimmunol.181.7.4832 [PMC free article] [PubMed] [Cross Ref]
23. Shin H, Blackburn SD, Intlekofer AM, Kao C, Angelosanto JM, Reiner SL, Wherry EJ 2009. A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Immunity 31:309–320. doi:.10.1016/j.immuni.2009.06.019 [PMC free article] [PubMed] [Cross Ref]
24. Staron MM, Gray SM, Marshall HD, Parish IA, Chen JH, Perry CJ, Cui G, Li MO, Kaech SM 2014. The transcription factor FoxO1 sustains expression of the inhibitory receptor PD-1 and survival of antiviral CD8(+) T cells during chronic infection. Immunity 41:802–814. doi:.10.1016/j.immuni.2014.10.013 [PMC free article] [PubMed] [Cross Ref]
25. Youngblood B, Oestreich KJ, Ha SJ, Duraiswamy J, Akondy RS, West EE, Wei Z, Lu P, Austin JW, Riley JL, Boss JM, Ahmed R 2011. Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells. Immunity 35:400–412. doi:.10.1016/j.immuni.2011.06.015 [PMC free article] [PubMed] [Cross Ref]
26. Youngblood B, Noto A, Porichis F, Akondy RS, Ndhlovu ZM, Austin JW, Bordi R, Procopio FA, Miura T, Allen TM, Sidney J, Sette A, Walker BD, Ahmed R, Boss JM, Sekaly RP, Kaufmann DE 2013. Cutting edge: prolonged exposure to HIV reinforces a poised epigenetic program for PD-1 expression in virus-specific CD8 T cells. J Immunol 191:540–544. doi:.10.4049/jimmunol.1203161 [PMC free article] [PubMed] [Cross Ref]
27. Masopust D, Vezys V, Marzo AL, Lefrancois L 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413–2417. doi:.10.1126/science.1058867 [PubMed] [Cross Ref]
28. Puglielli MT, Zajac AJ, van der Most RG, Dzuris JL, Sette A, Altman JD, Ahmed R 2001. In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb. J Virol 75:5099–5107. doi:.10.1128/JVI.75.11.5099-5107.2001 [PMC free article] [PubMed] [Cross Ref]
29. Matloubian M, Kolhekar SR, Somasundaram T, Ahmed R 1993. Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus. J Virol 67:7340–7349. [PMC free article] [PubMed]
30. Borrow P, Evans CF, Oldstone MB 1995. Virus-induced immunosuppression: immune system-mediated destruction of virus-infected dendritic cells results in generalized immune suppression. J Virol 69:1059–1070. [PMC free article] [PubMed]
31. Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de La Torre JC, Oldstone MB 2000. Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J Exp Med 192:1249–1260. doi:.10.1084/jem.192.9.1249 [PMC free article] [PubMed] [Cross Ref]
32. Mueller SN, Matloubian M, Clemens DM, Sharpe AH, Freeman GJ, Gangappa S, Larsen CP, Ahmed R 2007. Viral targeting of fibroblastic reticular cells contributes to immunosuppression and persistence during chronic infection. Proc Natl Acad Sci U S A 104:15430–15435. doi:.10.1073/pnas.0702579104 [PubMed] [Cross Ref]
33. Teijaro JR, Ng C, Lee AM, Sullivan BM, Sheehan KC, Welch M, Schreiber RD, de la Torre JC, Oldstone MB 2013. Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340:207–211. doi:.10.1126/science.1235214 [PMC free article] [PubMed] [Cross Ref]
34. Brooks DG, McGavern DB, Oldstone MB 2006. Reprogramming of antiviral T cells prevents inactivation and restores T cell activity during persistent viral infection. J Clin Invest 116:1675–1685. doi:.10.1172/JCI26856 [PubMed] [Cross Ref]
35. Angelosanto JM, Blackburn SD, Crawford A, Wherry EJ 2012. Progressive loss of memory T cell potential and commitment to exhaustion during chronic viral infection. J Virol 86:8161–8170. doi:.10.1128/JVI.00889-12 [PMC free article] [PubMed] [Cross Ref]
36. Utzschneider DT, Legat A, Fuertes Marraco SA, Carrie L, Luescher I, Speiser DE, Zehn D 2013. T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion. Nat Immunol 14:603–610. doi:.10.1038/ni.2606 [PubMed] [Cross Ref]
37. Fuse S, Tsai CY, Molloy MJ, Allie SR, Zhang W, Yagita H, Usherwood EJ 2009. Recall responses by helpless memory CD8+ T cells are restricted by the up-regulation of PD-1. J Immunol 182:4244–4254. doi:.10.4049/jimmunol.0802041 [PMC free article] [PubMed] [Cross Ref]

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