B7-H1 or PD-1 blockade by mAb prevents OT-1 T-cell anergy. (A-B) B6 mice were given OT-1 cells prior to intravenous administration of 0.5 mg OVA peptide. On the day of peptide administration and again 3 days later, mice were given 100 μg control hamster IgG (A-B), anti–B7-H1 (clone 10B5) (A), anti–B7-DC (clone YL-1) (A) or anti-PD-1 (clone G4) (B). Blood was taken from mice at the time points indicated, and the percentage of OT-1 cells present in each mouse was analyzed by flow cytometry analysis as described in the Figure 2 legend. The data shown are the means ± SD of 2 mice in each group and are representative of at least 3 independently performed experiments. (C) At 20 days following initial injection of OVA peptide, spleens were harvested and stimulated with OVA peptide (10 ng/mL) in 96–round-well plates. OT-1 cells were gated by anti-CD8 mAb and OVA tetramer, and OVA-specific IL-2 and IFN-γ production were determined 24 hours later, respectively, by mAb intracellular staining. Numbers represent percentage of OT-1 cells which are positive for indicated intracellular cytokines. The data are from 1 of 3 experiments. It compared with unstained control (data not shown). Horizontal bars represent gating of positive cells versus negative cells in flow cytometry analysis.