1. Volume 35, Issue 3, Pages 400-412 (September 2011)
Chronic Virus Infection Enforces Demethylation of the Locus that Encodes PD-1 in Antigen-Specific CD8+ T Cells 
Ben Youngblood, Kenneth J. Oestreich, Sang-Jun Ha, Jaikumar Duraiswamy, Rama S. Akondy, Erin E. West, Zhengyu Wei, Peiyuan Lu, James W. Austin, James L. Riley, Jeremy M. Boss, Rafi Ahmed 
Immunity 
Volume 35, Issue 3, Pages (September 2011)
DOI: /j.immuni
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
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 ( (Karolchik et al., 2003) and a CpG island in the human genome as defined by CpG Plot from the European Bioinformatics Institute.
(B) Genomic DNA from EL4 (PD-1hi) and A20 (PD-1lo) was digested with either HpaII (H, methylation sensitive) or MspI (M, nonsensitive) along with HindIII (HIII, nonsensitive). A Southern blot of the methylation-sensitive restriction endonuclease challenge showing an undigested Hind III fragment from A20 cells (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 indicate methylated cytosine; open circles indicate nonmethylated 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 with qRT-PCR. The values obtained were normalized to 18S rRNA and expressed as fold over the amount of transcript from day 0 cells. Error bars were generated with the SEM from triplicate experiments.
Immunity  , DOI: ( /j.immuni )
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3. Figure 2
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. Red line indicates LCMV-specific CD8+ T cells; filled gray histogram indicates naive CD8+ T cells.
(B) Real-time PCR analysis of PD-1 transcript from purified naive, day 4 effector, day 8 effector, and memory antigen-specific CD8+ T cells. Error bars were generated with the SEM from triplicate experiments.
(C) Bisulfite sequencing analysis and graphical summary of Pdcd1 CR-C and CR-B from FACS-purified naive, 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 postinfection with 2 × 105 LCMV Armstrong. Filled circles indicate methylated cytosine. Each line represents a sequenced clone. Open circles indicate nonmethylated cytosine. CpG sites 1–14 correspond to sites −1280, −1158, −1099, −1069, −986, −778, −672, −667, −636, −612, −535, −496, −491, and −465.
Error bars were generated with the SEM from triplicate experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 3
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. Red line indicates LCMV-specific CD8+ T cells; filled gray histogram indicates naive CD8+ T cells.
(B) Real-time PCR analysis of PD-1 transcript from purified naive 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) PD-1 expression was measured on the LCMV-specific CD8+ T cells harvested at day 8 postinfection from the acutely (2 × 105 and 2 × 106 pfu of LCMV Armstrong) and chronically (2 × 106 pfu of LCMV clone-13) infected mice.
(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) Bisulfite sequencing analysis and graphical summary of CR-C and CR-B in fully exhausted antigen-specific cells at 40 days postinfection from CD4-depleted chronically infected mice.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
CR-C Chromatin Is Hypersensitive to Digestion by DNase I in PD-1hi Virus-Specific CD8+ T Cells
(A) Experimental setup for generating antigen-specific transgenic cells at 4, 9, and >40 dpi with LCMV clone-13 or Armstrong. Viremia is detectable only in mice infected with clone-13 at 9 dpi versus mice infected with Armstrong at 9 dpi (data not shown).
(B) Histogram panels show PD-1 expression on naive (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 naive, day 4 effector, day 9 effector, and memory CD8+ T cells is plotted. p values were determined with the Student's t test. Not shown, the p value for the difference in DNase I hypersensitivity of CR-C between naive and day 4 effector cells is <0.005.
(D) Chromatin immunoprecipitation analysis for histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) at Pdcd1 CR-C was performed on chromatin extracted from the purified naive, D8 effector Armstrong, and D8 effector clone-13 CD8+ T cells. The bar graphs were generated from an average of three independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 5
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 after LCMV clone-13 infection reveals that PD-1 expression decreases subsequent to the clearance of virus. Serum virus titers (PFU/ml) for days 8, 30, and 135 postinfection were 6 × 104, 6 × 103, and undetectable, respectively. Day 135 p.i., antigen-specific cells were sorted for methylation analysis.
(B) Methylation analysis of DNA from antigen-specific naive CD8+ T cells and virus-specific CD8+ T cells isolated from clone-13-infected mice at >135 dpi.
(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) Congenically marked antigen-specific PD-1lo cells from either immune mice or chronically infected mice were cultured for 0, 6, and 12 hr in media containing the gp33 peptide. FACS analysis of PD-1 re-expression in virus-specific CD8+ T cells from the long-term chronic environment (blue line) and from the acute environment (red line) (gated on Thy1.1+ cells).
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 6
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 via real-time RT-PCR. Analysis was performed on cDNA generated from total RNA from naive, 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 with two different sets of primers that distinguish between the two isoforms. Transcript expression values were normalized to 18S ribosomal RNA. p values were determined with the Student's t test from triplicate experiments.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
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 naive and virus-specific CD8+ T cells. Bisulfite sequencing and graphical summary of CR-C of the PDCD1 promoter in human naive (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 CpG sites 18–26 of the PDCD1 promoter in CMV- and EBV-specific CD8+ T cells (cytomegalovirus and Epstein-Barr virus). 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 naive 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.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions