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-2K^b/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.