Genetic ablation of B7-H1 or PD-1 prevents T-cell anergy. (A-B) OT-1 T cells in RAG-1KO background (WT) were transferred into WT B6 (WT → WT), B7-H1KO (WT → B7-H1KO) mice (A) or into B7-DCKO (WT → B7-DCKO) mice. (B) In addition, OT-1/RAG-1KO T cells in PD-1KO background was also transferred into WT B6 mice (PD-1KO→WT) (A). After transfer, the mice were treated with 0.5 mg OVA peptide at the same day. From day 3, PBMCs were prepared from tail veins of individual mice, and OT-1 T cells were enumerated by tetramer and CD8 mAb in flow cytometry. At day 20, the mice were rechallenged with 0.5 mg of OVA peptide intravenously, and OT-1 T cells were similarly enumerated. (C) At day 3 after primary peptide injection, sera of individual mice were taken and assayed by sandwich ELISA for IFN-γ using specific mAb. Data from 3 mice were shown, which is a representative of 2 experiments. (D) LN cells were collected from B6 or B7-H1KO mice, which had been transferred with OT-1 T cells, at 48 hours after OVA peptide injection, and were stained with OVA tetramer/CD8 mAb and anti-CD25 or anti-CD69 mAb. Numbers represent percentage of CD25⁺ or CD69⁺ cells within OT-1 T cells (OVA tetramer⁺/CD8 mAb⁺). Error bars represent average of data from 3 mice (SD [standard deviation]).