Suppressive function of the CD25⁺ line is mainly cell contact mediated in vitro. (A) Freshly isolated CD4⁺CD25⁻ T cells (25⁻) and CD25⁺ line (25⁺) (5 × 10⁵) were cultured alone or were cocultured at a 1:1 ratio for 3 days with soluble anti-CD3 and 5 × 10⁵ APCs in 24-well plates. When cocultured, the T cells either were in contact in the same well (25⁻ and (25⁺)) or were separated by a transwell membrane with the CD25⁺ line in the upper well (25⁻ and 25⁺), in which case APCs were put in both the upper and the lower wells. At day 3, [³H]-thymidine was added to the bottom wells to measure proliferation. (B) Freshly isolated CD4⁺CD25⁻ T cells (25⁻) and CD25⁺ line (25⁺) (1 × 10⁵) were cultured for 3 days alone or were cocultured at a 1:1 ratio in the presence of soluble anti-CD3 antibody and APCs (□). Anti–TGF-β–purified (⊡) or anti–IL-10–purified (▪) blocking antibodies were added to the cultures at 20 μg/mL. Results are representative of 3 independent experiments. Error bars reflect mean ± SD of triplicate cultures.