Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy by Fumihiko Tsushima, Sheng Yao, Tahiro Shin, Andrew Flies, Sarah Flies, Haiying Xu, Koji Tamada, Drew M. Pardoll, and Lieping Chen Blood Volume 110(1):180-185 July 1, 2007 ©2007 by American Society of Hematology

Phenotype and kinetics of PD-1 expression. Phenotype and kinetics of PD-1 expression. (A) B6 mice that had received OT-1 T cells were given 0.5 mg OVA peptide intravenously. At the indicated time point, PBMCs were prepared, and surface expression of PD-1 on OT-1 cells was determined by triple staining with APC-labeled anti-CD8 mAb, PE-labeled H-2Kb/OVA tetramer (tetramer), and FITC-anti-PD-1 mAb. Numbers represent percentage of tetramer+ cells within the CD8+ subset (top row) or percentage of PD-1+ cells within the tetramer/CD8 double-positive subset from the same experiment (bottom). The data are from 1 of 3 representative experiments. (B) Blood, lymph nodes (LNs), or spleen cells at 60 hours with (+OVA; bottom) or without (−OVA; top row) OVA peptide injection were stained with PE-labeled tetramer and APC-labeled CD8 mAb. Numbers represent percentage of tetramer+ cells within the CD8 subset. The data are from 1 representative experiment of at least 3. (C) LN cells were collected from B6 mice at 48 hours with (+OVA; bottom row) or without (−OVA; top row) OVA peptide treatment. Cells were stained with tetramer/CD8 (OT-1 cells; left panels) together with FITC-anti-PD-1, anti-CD25, or anti-CD69 mAb, respectively. Numbers in the left panels represent percentage of tetramer+ cells within the CD8+ subset. Numbers in the right panels represent percentage of PD-1, CD25, or CD69+ cells within the tetramer+/CD8+ (OT-1) cells. Fumihiko Tsushima et al. Blood 2007;110:180-185 ©2007 by American Society of Hematology

Genetic ablation of B7-H1 or PD-1 prevents T-cell anergy. 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]). Fumihiko Tsushima et al. Blood 2007;110:180-185 ©2007 by American Society of Hematology

B7-H1 or PD-1 blockade by mAb prevents OT-1 T-cell anergy. 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. Fumihiko Tsushima et al. Blood 2007;110:180-185 ©2007 by American Society of Hematology

Interaction between B7-H1 and PD-1 in lymphoid organs is sufficient to induce OT-1 T-cell anergy. Interaction between B7-H1 and PD-1 in lymphoid organs is sufficient to induce OT-1 T-cell anergy. B6 mice were given OT-1 cells prior to intravenous administration of 0.5 mg OVA peptide. On 0, 24, or 48 hours after OVA peptide injection, mice were given 100 μg intravenously of control hamster IgG, B7-H1 mAb (clone 10B5), or PD-1 mAb (clone G4). Second doses were administered 3 days after the first dose. Blood was taken from mice at the time points indicated, and the percentage of OT-1 cells observed in each mouse was analyzed by flow cytometry as described previously. The data shown are the means ± SD of 3 mice in each group and are representative of at least 3 independently performed experiments. Fumihiko Tsushima et al. Blood 2007;110:180-185 ©2007 by American Society of Hematology

Blockade of B7-H1 or PD-1 reverses established T-cell anergy. Blockade of B7-H1 or PD-1 reverses established T-cell anergy. B6 mice were given OT-1 cells prior to intravenous administration of 0.5 mg OVA peptide. On day 10 after OVA peptide injection, mice were given 100 μg intravenously of control hamster IgG, B7-H1 mAb (clone 10B5), or PD-1 mAb (clone G4) together with (A) or without (B) intravenous administration of 0.5 mg OVA peptide. Blood was taken from mice at the time points indicated, and the percentage of OT-1 cells observed in each mouse was analyzed by flow cytometry as described previously. The data shown are the means ± SD of 3 mice in each group and are representative of at least 3 independently performed experiments. Fumihiko Tsushima et al. Blood 2007;110:180-185 ©2007 by American Society of Hematology