Treatment with anti-PD-1/GITR mAb induced a tumor antigen-specific CTL response in treated mice. A, Mice (5 mice/group) were transplanted i.p. with 5 × 106 ID8 cells on day 1 and treated with 250 μg of control, anti-PD-1, anti-GITR and anti-PD-1/GITR mAb on day 8, 11 and 15. Seven days after last mAb injection, splenocytes from treated mice were cultured in the presence or absence of H-2Db-restricted mesothelin or control GP33-41 epitope peptide for 3 days and IFN-γ production in the supernatants were determined by ELISA. B, Pooled splenocytes (5 × 106) from control or combined anti-PD-1/GITR mAb were incubated with 5 × 105 UV-irradiated ID8 cells for 4 days prior to subject to analysis of antigen-specific CTL activity by CytoTox 96 Non-radioactive cytotoxicity assay using EL4 cells pulsed with H-2Db-restricted mesothelin as target cells. C, As a specific control, pooled splenocytes were tested cytotoxicity against EL4 target cells pulsed with control H-2Db-restricted GP33-41 peptide.