The in vitro–cultured CD4⁺CD25⁺ T-cell line retains suppressive properties. (A-F) CD4⁺CD25^– T cells were stimulated by DCs pulsed with the A2 (138-170) peptide in the presence of equal numbers (A-C, E-F) or different numbers (D) of CD4⁺CD25⁺ T cells. After 20 hours or 48 hours (F) of culture, supernatants were harvested and their IL-2 contents were measured by CTLL-2 cell proliferation. (A) Heterozygous autologous HLA-DR 7, DR10 DCs were used as APCs. For panels B and D-F, HLA-DR7 homozygous DCs were used as APCs, and for panel C, HLA-DR10, DR16 DCs were used as APCs. (G) CD4⁺CD25^– T cells were stimulated by CD3/CD28 Dynal beads in the presence of different numbers of the CD4⁺CD25⁺ T cells for 20 hours. IL-2 secretion in the culture was measured by CTLL-2 cell proliferation. Each bar shows the mean cpm ± SD. Results are representative of at least 2 independent experiments.