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

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.

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 8 postinfection. The phenotype and function of P14 cells were analyzed at days 1, 8, 30, and 45 posttransfer. (b) The ratios of acute and chronic P14 cells among total P14 cells in blood are shown on the left. The numbers of acute and chronic P14 cells in blood were examined at days 1, 8, 30, and 45 days posttransfer and are shown on the right. (c) Expression of PD-1 on acute and chronic P14 cells at days 1, 8, and 30 posttransfer is shown on the left. A summary graph is shown on the right. (d) The ratios of acute and chronic P14 cells in spleen and liver were examined at day 45 posttransfer. A summary graph is shown on the right. (e) Expression levels of PD-1 were analyzed in spleen and liver at day 45 posttransfer. Data are representative of results from two or three independent experiments with three to five mice per group. Error bars show SEM.

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 expression of Tim-3 and 2B4 (a) and CD127 and CD62L (b) on acute and chronic P14 cells were examined at day 1, 8, and 30 posttransfer. Data are representative of those from two or three independent experiments with three to five mice per group. Error bars show SEM.

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 these P14 chimeric mice were stimulated with gp33 peptide for 6 h at days 1, 8, and 30 posttransfer. The percentages of IFN-γ⁺ P14 cells able to produce TNF-α or IL-2 were examined. Data are representative of those from three independent experiments with five mice per group. Error bars show SEM.

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 at day 8 postinfection. After 30 days of transfer, acute and chronic P14 cells were subjected to fluorescence-activated cell sorting to analyze DNA methylation. (b) Bisulfite sequencing of PD-1 promoter was performed to analyze DNA methylation on genomic DNA from sorted donor P14 cells (Arm day 8 and CL13 day 8) and sorted acute and chronic P14 cells (day 30 posttransfer). Each line represents an individual clone. The filled circles indicate methylated cytosine, and the open circles indicate unmethylated cytosine. Summary graphs of methylations are shown at the bottom. Data are representative of those from five independent experiments with three to five mice per group. Error bars show SEM.

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 mutant day 8) mice. Levels of PD-1 expression of P14 cells were analyzed at days 1, 8, 15, and 30 posttransfer. (b and c) Expression kinetics of PD-1 on acute and chronic P14 cells in blood of WT-infected (b) or V35A mutant-infected (c) mice are shown. (d and e) Methylation of the PD-1 promoter on acute and chronic P14 cells was examined in WT-infected (d) or V35A mutant-infected (e) mice at day 30 posttransfer. Data are representative of those from three independent experiments with three mice per group. Error bars show SEM.

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) recipients at day 8 postinfection. (b) After 6 h of stimulation with gp33 peptide, PD-1 expression was analyzed on acute and chronic P14 cells in spleen at day 30 posttransfer. Data are representative of those from two independent experiments with five mice per group. Error bars show SEM.

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 by LCMV Arm infection. (b) Flow plots on the left show the ratios of acute and chronic P14 cells in blood at days 0, 8, 15, and 40 post-secondary challenge. Numbers of acute and chronic P14 cells in blood are shown on the right. (c) Levels of PD-1 expression on acute and chronic P14 cells in blood were analyzed at days 8, 15, and 40 post-secondary challenge and are shown on the left. A summary graph of PD-1 expression level is shown on the right. (d) The numbers of acute and chronic P14 cells in spleen and liver were examined at day 40 post-secondary challenge. (e) PD-1 expressions on acute and chronic P14 cells were analyzed at day 40 post rechallenge. Data are representative of those from three independent experiments with five mice per group. Error bars show SEM.

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, and 2B4 were examined on P14 cells in blood at days 8, 15, and 40 post-secondary infection. (c) On day 40 postinfection, granzyme B expression was analyzed on acute and chronic P14 cells in spleen (left). After peptide stimulation for 6 h, cytokine production was examined on acute and chronic P14 cells in spleen (right). Data are representative of those from two independent experiments with five mice per group. Error bars show SEM.

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 day 8 postinfection. Then an equal number of acute and chronic P14 cells were sorted on day 30 posttransfer and then transferred into naive B6 mice, followed by LCMV Arm infection. Anti-PD-L1 antibody or control Ig was administered to mice at days 0, 3, 6, and 9 after rechallenge. (b) Numbers of acute (right) and chronic (left) P14 cells in blood of control Ig- or anti-PD-L1-treated mice during secondary challenge. (c) Thirty-five days after infection, granzyme B expression was analyzed on acute and chronic P14 cells in spleens of control Ig- and anti-PD-L1-treated mice (left). After peptide stimulation for 6 h, cytokine production on acute and chronic P14 cells was examined in spleens of control Ig- and anti-PD-L1-treated mice (right). Data are representative of those from two independent experiments with five mice per group. Error bars show SEM.