Fig 2: PD-1 Blockade at the Peak of Viremia Enhances CD8 T Cell Activation with No Immediate Impact on Viral Control(A) Survival LCMV-c13-infected B6 mice following anti-PD-L1 treatment. n = 25 (control), n = 6 (days 0–8), and n = 30 (days 8–22) pooled from 3 experiments.(B) Survival of LCMV-c13-infected B6 mice after adoptive transfer of unexhausted LCMV-specific CD8 T cells and treatment with anti-PD-L1 or isotype control. Data are from 3 experiments.(C) Expression of CD4, CD8, CD44, and LCMV-gp276-specific T cell receptor (TCR) in peripheral blood mononuclear cells (PBMCs) from control and anti-PD-L1-treated mice on 14 dpi with LCMV-c13 infection. Representative plots are shown with mean ± SD from 3 experiments.(D and E) Expression of CD4, CD8, CD44, PD-1, LAG-3, and LCMV-gp33-specific TCR in PBMCs on 22 dpi. Representative plots (D) and mean ± SD from 2 experiments (E) are shown.(F) Plasma viral RNA load in LCMV-c13-infected mice on 22 dpi. Data pooled from 5 experiments are shown with median. The statistical difference was tested by the Mann-Whitney U test.See also Figure S1.

Fig 3: Type I Interferon Receptor Blockade Rescues Immunopathology Caused by Loss of PD-1 Signaling in LCMV-Infected Mice(A) Survival of LCMV-c13-infected B6 mice treated with anti-IFNAR 1 day prior to infection, anti-PD-L1 (days 0, 2, 4, 6, and 8), or both. Data pooled from 3 experiments with n > 6 mice per group are shown.(B and C) Frequencies of total CD8 T cells, LCMV-gp33-specific CD8 T cells, and PD-1+ cells in indicated PBMC populations on 8 dpi in LCMV-c13-infected B6 mice with indicated treatment. Representative plots (B) with mean ± SD from 2 experiments (C) are shown.(D) Plasma LCMV viral RNA load in LCMV-c13-infected B6 mice with indicated treatment from 2 experiments shown with median.(E) Macroscopic images of spleen and splenocyte counts of LCMV-c-13-infected B6 mice with indicated treatment as in (A) on 22 dpi. Data from two experiments are shown.See also Figures S3 and S4.

Fig 4: Characterization of Tet2/3-deficient Treg cells. a Representative flow cytometry analyses of the percentage of Treg cells (% of Foxp3+ cells in gated CD4+ T cells) in spleen and pLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13–16 weeks-old). b Quantification of the percentages (left panel) and cell numbers (right panel) of Treg cells in spleen, pLN and mLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13-16 weeks old, n = 9). c–f Flow cytometry analysis of Tet2/3-deficient Treg cells from spleen and pLN for the expression of CD25 and Nrp1 (c); Helios, ICOS, CD103, GITR, CTLA4, PD1 and CD127 (d); CD62L versus CD44 (e); and CD69 (f). Shown are WT CD4+ T cells (shaded gray); WT Tregs (black line); DKO Tregs (red line). Statistical analysis was performed using two-tailed unpaired student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars show mean ± s.d. from at least three independent experiments

Fig 5: PD-1 Blockade at the Peak of Viral Load Results in Reduced Spleen Sizes and Pan-lymphopenia(A) Gross images of spleens from control and anti-PD-L1-treated mice on 22 dpi.(B) Splenocyte counts of control and anti-PD-L1-treated mice on 22 dpi. n > 8 mice per group, shown by mean and SD.(C) H&E staining of spleen sections from control and day 8–22 anti-PD-L1-treated mice on 22 dpi. Scale bars, 200 µm. Representative images of 6 mice per group are shown.(D) Expression of CD44 and LCMV-gp33-specific TCR of total splenocytes and expression of TCF-1 and TIM-3 by LCMV-gp33-specific CD8 T cells in control and anti-PD-L1-treated mice on 22 dpi.(E) Statistical analysis of frequencies and absolute numbers of the indicated cell populations pooled from 2 experiments. n = 4–6 mice per group.(F) Expression of IFN-? and TNF-a by splenocytes from LCMV-c13-infected control and anti-PD-L1-treated B6 mice following stimulation with the gp276–284 peptide. Representative data from 2 experiments are shown with mean ± SD; n = 4.(G) Expression of Ki-67 by gp33-specific CD8 T cells.(H) Frequencies of CD45.2 PD-1+ donor CD8 T cells in PBMCs of CD45.1-recipient mice that received PD-1+ CD8 T cells from LCMV-c13-infected anti-PD-L1-treated B6 mice and subsequently challenged with LCMV-c13. Data are pooled from two experiments.(I) Expression of Fas and GL7 expression in B220+ splenocytes from control and anti-PD-L1-treated mice. Data from 2 experiments are shown with mean and SD.(J) Anti-LCMV IgG2c antibody titers on 22 dpi.See also Figure S2.