Tumor-associated B7-H1 inhibits T-cell immunity. (A) Purified allogeneic CD4⁺ or CD8⁺ T cells (4 × 10⁶) were stimulated with irradiated B7-H1⁺ Karpas 299 cells (5 × 10⁵) in 24-well plates. T-cell lines were harvested, washed, counted, and restimulated with newly irradiated tumor cells every 6 to 8 days, at least 3 times. The T-cell lines thus generated were harvested; viable cells counted by exclusion of Trypan Blue and either cultured alone (4 × 10⁵) or restimulated with irradiated Karpas 299 (5 × 10⁴) in triplicate in a 96-well plate and thymidine incorporation determined after 72 hours. On restimulation, either an isotype control or anti–B7-H1 (4 μg/mL) was included, as indicated. Any background thymidine incorporation by irradiated Karpas 299 cells was subtracted from the values shown. (B) CD4⁺ and CD8⁺ T-cell lines were generated, as described in panel A. An isotype control or anti–B7-H1 (4 μg/mL) was included during each stimulation cycle, as indicated. Cells were subsequently harvested and restimulated with irradiated Karpas 299 cells. (C) A CD8⁺ T-cell line (10⁶/well), generated as described in panel A, was left unstimulated or restimulated with irradiated Karpas 299 cells (2.5 × 10⁵/well) for 6 hours in a 48-well plate in the presence of anti-CD107a, as described in “Cell lines, proliferation, and cytotoxicity assays.” An isotype control or anti–B7-H1 (4 μg/mL) was included, as shown. CD107a⁺CD8⁺ cells, identified by flow cytometry, are included in the gates shown (n = 3, P = .048). (D) CFSE-labeled Karpas 299 cells were cocultured with freshly purified CD8⁺ T cells or activated effector CD8⁺ T cells (4 × 10⁵/well) at various E/T ratios (E/T ratio of 200:1 is shown). CFSE⁺ tumor cells were identified by flow cytometry after 4 days of culture. All data shown are representative of at least 3 independently performed experiments.