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 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.