OX40 inhibits T reg–suppressive activity. Naive CD4⁺CD25^- (5 × 10⁴) and CD4⁺CD25⁺ T cells (named as CD25^- and CD25⁺, respectively) were purified from BALB/c mice and cultured at the indicated ratio together or alone. Cells were cultured in the presence of 5 μg/mL of anti-OX40 (A), anti-GITR (B), left untreated (NT), or treated with the respective isotype controls rat IgG1 (A) and rat IgG2a (B) mAbs. In a dose-response assay, CD25^- T cells (5 × 10⁴) were cultured (C) or not (D) with CD25⁺ T cells (2.5 × 10⁴) at different concentrations of anti-CD3 Ab as indicated with or without (NT) anti-OX40 or anti-GITR Ab (a-OX40 or a-GITR, respectively). In another dose-response assay, CD25^- (5 × 10⁴) T cells were cultured with CD25⁺ T cells at the indicated ratio with or without (NT) anti-OX40 (E) or anti-GITR (F) mAbs at variable concentration, as indicated. All samples were stimulated with 1 μg/mL of anti-CD3 and irradiated accessory cells (5 × 10⁴); proliferation was measured after 72 hours. ³[H]TdR pulse was during the last 10 hours. One representative experiment out of 3 performed in triplicate is shown as mean ± standard deviation (SD). The significance of the data was evaluated by Student t test (^***P < .005).