Expanded CD4⁺CD25⁺ T cells become more potent suppressors than fresh Treg cells while remaining anergic in vitro. Freshly isolated CD4⁺CD25⁻ responder T cells (1 × 10⁵; CD25⁻) were cultured alone, with 1:1 freshly isolated CD4⁺CD25⁻ (25⁻/25⁻), with decreasing ratios (25⁻/25⁺; ratios 1:1, 1:0.5, 1:0.25) of freshly isolated CD4⁺CD25⁺ T cells (⊡), or with CD25⁺ line cells (▪) in the presence of soluble anti-CD3 and 1 × 10⁵ APCs. (A) T-cell proliferation was measured after 3 days of culture. (B-C) Supernatants were collected at days 1 and 2 of cocultures for the determination of IL-2, as measured by CTLL-2 proliferation (B) and IFN-γ by ELISA (C). (D) Freshly isolated CD4⁺CD25⁻ T cells and CD25⁺ line (25⁺) (1 × 10⁵) (25⁻) were cultured alone or were cocultured at a 1:1 ratio in the presence of soluble anti-CD3 antibody and APCs (□). To reverse suppression, 10 U/mL IL-2 (⊡) or 20 μg/mL purified anti-CD28 antibody (▪) was added to the cultures, and T-cell proliferation was measured at 72 hours. Results are representative of 4 independent experiments. Error bars reflect mean ± SD of triplicate cultures.