Antigen specificity of the in vitro expanded Treg cells. (A) Vβ TCR-usage of the alloantigen-stimulated Treg cells. Fresh CD4⁺CD25⁺ T cells (⊡) and CD25⁺ line (▪) were stained with 15 different FITC-labeled anti–TCR Vβ mAbs and analyzed by flow cytometry. (B) CD25⁻ line (1 × 10⁵; 25⁻) were cultured for 3 days alone, with 1:1 CD25⁻ line (25⁻/25⁻), with decreasing ratios (25⁻/25⁺; ratios, 1:1, 1:0.5, 1:0.25) of CD25⁺ line, in the presence of syngeneic irradiated DCs pulsed with K^b peptide. (C) Freshly isolated CD4⁺CD25⁻ responder (25⁻) or CD25⁺ line cells (1 × 10⁵) were cultured alone or were cocultured 1:1 for 4 days in the presence of soluble anti-CD3 antibody and APCs (□), irradiated mature syngeneic CBA DCs (H2^k) (⊡), CBK DCs (H2^k+K^b) (▪), or B10.A DCs (H2^k+D^d) (). Results are representative of 3 independent experiments. Error bars reflect mean ± SD of triplicate cultures.