Induction of HLA-A2 (138-170) allopeptide-specific CD4⁺CD25^– and CD4⁺CD25⁺ T-cell lines. (A) CD25 expression on purified CD4⁺, CD4⁺CD25^–, and CD4⁺CD25⁺ cells by flow cytometric analysis. (B) CD4⁺CD25^– and CD4⁺CD25⁺ T-cell lines were stimulated by autologous DCs in the absence or presence of the A2 (138-170) allopeptide. After 48 hours of culture, [³H]thymidine was added and the cells were harvested 16 hours later. The concentration of rIL-2 was 5 U/mL. Each bar shows the mean cpm ± standard deviation (SD). (C) The CD4⁺CD25^– and CD4⁺CD25⁺ T-cell lines were stimulated by autologous DCs pulsed with the A2 peptide in the presence of exogenous IL-2 (10 U/mL; 10 ng/ml IL-7 was added in the culture of CD4⁺CD25⁺ T cells) for one week and then cell numbers were counted. (D-E) CD25 expression on gated CD4⁺ cells was analyzed after CD4⁺CD25^– (gray open curve) and CD4⁺CD25⁺ (filled curve) T cells were stimulated by the A2 (138-170) peptide–pulsed autologous DCs in the presence of IL-2 for one week (D) and then reactivated for 16 hours by CD3/CD28 Dynalbeads (E). (F) To measure intracellular cytokines IFN-γ and IL-10, the CD4⁺CD25^– and CD4⁺CD25⁺ T cells were stimulated by ionomycin and PMA for 6 hours; Brefeldin A was added for the last 5 hours. Cells were then stained and analyzed by flow cytometry. The numbers of cytokine-producing cells are shown in percentages. Results are representative of at least 2 independent experiments.