Improved survival and augmented immune responses in PD-1^−/− mice challenged with C1498.GFP. (A) Survival rates of C57BL/6 (●) or PD-1^−/− mice (○) challenged with 10⁶ C1498.GFP cells IV (P = .002 for the comparison of survival between PD-1^−/− and C57BL/6 mice). (B) Peripheral blood was sampled from a PD-1^−/− mouse and a C57BL/6 mouse 13 days after IV injection of C1498.GFP and was analyzed by flow cytometry for GFP⁺ cells. Gating was initially performed on the total WBC population on the FSC versus SSC gate. GFP⁺ cells present in the WBC population were gated in a SSC versus GFP plot. The numbers above the GFP⁺ cell gate represent the percentage of GFP⁺ cells divided by the percentage of WBCs in the FSC versus SSC plot and multiplied by 100. (C) Cohorts of 5 PD-1^−/− and C57BL/6 mice received 10⁶ C1498.GFP cells IV. On days 10, 13, 15, and 19 after tumor challenge, peripheral blood was sampled from each mouse. Red blood cells were removed via exposure to ACK lysis buffer, and samples were analyzed by flow cytometry. The percentage of leukemia cells was calculated as the GFP⁺ cells/% total WBCs. Closed circles and open circles represent the mean percentage of GFP⁺ cells at each time point from C57BL/6 and PD-1^−/− mice, respectively. (D) Spleens were harvested from C57BL/6 or PD-1^−/− mice 12 days after IV injection with C1498.GFP cells. A total of 10⁶ splenocytes from individual mice were restimulated overnight with either complete DMEM, 5 × 10⁴ irradiated C1498.GFP cells (10 000 rad) or PMA + ionomycin in an IFN-γ ELISPOT assay. Bars represent the mean number of spot-forming cells (± SD) in each group (P < .001 for the comparison between PD-1^−/− and C57BL/6 mice). Similar results were obtained in 2 independent experiments.