Figure 5.

PD-1 blockade ablates tumor-specific immune suppression by T_reg. (a) C57BL/6 mice bearing s.c. B16 tumors were treated with i.t. reovirus, in combination with i.v. anti-PD-1 or isotype (control) IgG (two mice per group). Nondepleted splenocytes/LN cells (black bars), or splenocytes/LN depleted for CD8⁺ T cells (white bars) or T_reg (hatched bars) were stimulated in vitro with a. F/T lysates of B16. Forty-eight hours later supernatants were assayed for IFN-γ by enzyme-linked immunosorbent assay. Values represent levels each done in triplicate wells (mean ± standard deviation). (b) Splenocytes/LN from C57BL/6 mice treated with i.t. reovirus, in combination with i.v. anti-PD-1, isotype (control) IgG, or PBS (four mice per group) were stimulated with F/T lysates of reovirus-infected TC2 cells. Forty-eight hours later, supernatants were assayed for IFN-γ by enzyme-linked immunosorbent assay. Values represent secretion levels for three or four different mice per group, each done in triplicate wells (means ± standard deviation). (c) C57BL/6 mice bearing s.c. B16 tumors were treated with i.t. PBS or reovirus, in combination with i.v. anti-PD-1 or isotype (control) IgG. Nondepleted splenocytes/LN cells (black bars), or splenocytes/LN depleted for CD8⁺ T cells (white bars) or T_reg (hatched bars) were stimulated in vitro with F/T lysates of reovirus-infected TC2 cells, *P < 0.0001. (d) C57BL/6 mice bearing s.c. B16 tumors were treated with i.t. PBS or reovirus, with four doses of i.v. anti-PD-1 or isotype (control) IgG. On day 22 after tumor implant, tumors were harvested and stained for CD45-PerCP, CD4-FITC, CD25-Pe/Cy7 followed by intracellular staining with Foxp3-PE or with CD45-PerCP, CD8-PE. The percentages of tumor-infiltrating CD3+ T cells which are T_regs (left panel) or CD8+ T cells (right panel) are shown. Six tumors were analyzed from each treatment group with mean and standard deviation shown.