Augmented priming and effector function of tumor antigen-specific T cells in PD-1^−/− mice challenged with C1498.SIY. (A) Groups of C57BL/6 or PD-1^−/− mice were challenged with 10⁶ C1498.SIY cells IV. Seven days later, spleens from tumor-bearing or control mice were harvested and the frequencies of SIY⁺CD8⁺ T cells present in each mouse were analyzed by flow cytometry after staining with SIY-K^b tetramers. Bars represent the mean frequency of SIY⁺CD8⁺/total CD8⁺ T cells (± SD) in each group (P = .05 for the comparison of the frequency of SIY⁺CD8⁺ cells present in PD-1^−/− vs C57BL/6 mice). (B) IFN-γ ELISPOT performed on spleen cells harvested from tumor-bearing or control mice as in panel A. A total of 10⁶ splenocytes from individual mice were stimulated with complete DMEM, SIY peptide (80 nM), irradiated C1498.SIY tumor cells, or PMA + ionomycin overnight; they were then analyzed for IFN-γ spot production. Bars represent the mean number of spot-forming cells (± SD) in each group (P < .001 for the comparison between IFN-γ spots produced by PD-1^−/− vs C57BL/6 spleen cells after restimulation via SIY peptide and irradiated C1498.SIY cells). (C) Survival of PD-1^−/− and C57BL/6 mice after IV injection of 10⁶ C1498.SIY cells (P = .002 for the comparison of survival between PD-1^−/− and C57BL/6 mice).