Human CD4⁺CD25^hi T cells suppress proliferation and IFNγ production of CD4⁺CD25^-T cells. CD4⁺CD25^- responders (5 × 10⁴/well) and CD4⁺CD25^hi Tregs (5 × 10⁴/well) were cultured with plate-bound anti-CD3 (1 μg/well) either alone or at a 1:1 ratio. (A) After 72 hours, ³H-thymidine incorporation was determined. Results are the mean ± SEM of 40 separate experiments using individual donors. (B) The percent inhibition of proliferation of these 40 experiments. Data are shown as means ± SEM. (C) Culture supernatants were diluted and analyzed to determine the amount of interferon γ. Data represent the mean ± SE of triplicate cultures and are expressed as nanogram per milliliter. (D) The suppressive activity of human CD4⁺CD25^hi Tregs is independent of TGFβ1, IL-10, or GITR. CD4⁺CD25^- T cells (5 × 10⁴/well) or CD4⁺CD25^hi Tregs (5 × 10⁴/well) either alone or mixed together at a ratio of 1:1 were stimulated with plate-bound anti-CD3 (1 μg/well). Neutralizing antibodies against TGFβ1 (2 μg/well), IL-10 (2 μg/well), or anti-GITR (0.2 μg/well) were added as indicated. ³H-thymidine incorporation was determined after 72 hours and the data shown are the mean ± SEM of 3 independent experiments.