Prevention and restoration of CD8⁺ T-cell anergy by blockade of B7-H1/CD80 interaction. B6 mice were transferred intravenously with OT-I T cells and injected intravenously with 0.5 mg of OVA_257-264 peptide. (A) On day of peptide injection and 3 days later, the mice were treated intraperitoneally with 200 μg of 43H12 (●) or control rat IgG (○). Thirty-four days after initial peptide injection, the mice were rechallenged intravenously with 0.5 mg of OVA_257-264 peptide, and percentages of CD8/OVA-tetramer double-positive OT-I T cells in PBMCs were assessed by flow cytometry at the indicated time points. Fold expansion of OT-I T cells was calculated by dividing OT-I T-cell percentages after rechallenge by that before rechallenge in individual mice. (B) Twenty days after the initial OVA peptide injection, the mice were rechallenged with 0.5 mg of OVA_257-264 peptide and treated intraperitoneally with 200 μg of 43H12 (●) or control rat IgG (○) on day of peptide rechallenge and 3 days later. Fold expansion of OT-I T cells in PBMC was assessed as in panel A at the indicated time points. (C) Twenty days after the initial OVA peptide injection, the mice were left untreated (left panel) or rechallenged with 0.5 mg of OVA_257-264 peptide (right panel). Twenty-four hours later, CD8/OVA-tetramer double-positive OT-I T cells in the spleen were stained with anti-CD80 mAb and analyzed by flow cytometry (gray histogram). Nonstained background levels of the same cells are also shown (open histogram). All experiments were repeated at least 3 times and the representative data are shown. The numbers in the histogram indicate the percentage of positively stained cells.