OX40 specifically triggers T reg's. (A) CD4⁺ lymphocytes from normal BALB/c mice or Wistar rats were stained with biotin anti–mouse OX40, biotin anti–rat OX40 (thin lines) or the relative biotin isotype control (filled histograms) and FITC-streptavidin. (B) CD4⁺CD25^- (5 × 10⁴) or CD25⁺ (2.5 × 10⁴) T cells from BALB/c mice were cultured with irradiated rat accessory cells (ACs) in the presence or absence of anti-OX40 mAb (5 μg/mL; left panel). Rat CD4⁺ T cells (5 × 10⁴) and mouse CD4⁺CD25⁺ (2.5 × 10⁴) T cells were cultured with irradiated rat ACs in the presence or absence of anti-OX40 mAb as indicated (right panel). (C) CD25^- T cells (5 × 10⁴) cultured with CD25⁺ T cells at the indicated ratio were stimulated with ACs in the presence or absence (NT) of anti-OX40 antibody (5 μg/mL; ▪); where indicated, CD25⁺ T cells were preincubated for 2 hours with 30 μg/mL of anti-OX40 mAb (dark gray bars) or their respective isotype control rat IgG1 or rat IgG2a (light gray bars), washed 2 times with PBS and added to CD25^- T cells. (D) Cells were prepared and treated as in panel C, except that anti-GITR mAb (5 μg/mL; ▪) was added to coculture or preincubated (30 μg/mL; dark gray bars) or with purified T reg's. All samples were stimulated with 1 μg/mL of anti-CD3 in the presence of ACs. Proliferation was measured after 72 hours and pulsed with ³[H]TdR for the last 10 hours. Data (mean ± SD) are from 1 of 3 independent experiments with similar results. The significance of the data was evaluated by Student t test (^**P < .01).