Regulatory capacity is contained within the CD25^highCD27⁻CD62L⁻CD45RO⁺ T-cell subset that expresses FoxP3, CTLA-4, and GITR. (A) CD4⁺CD25⁻ T cells were stimulated with allogeneic stimulator cells. CD25 expression was analyzed in the CD4⁺ T-cell fraction of FK778-treated cells (top plot) and untreated control cells (bottom plot). (B) The subsets formed upon FK778 treatment, separated using flow cytometric cell sorting, and were restimulated (2 × 10⁴) with allogeneic stimulator cells (5 × 10⁴). Proliferation during the secondary culture, in the absence of FK778, was examined in the absence (left panel) or presence (right panel) of 12.5 U/mL IL-2. At the peak of proliferation, *P < .002 for differences between untreated and FK778-treated CD25^high cells. (C) The suppressive capacity of the sorted subsets, after stimulation in the presence (top graph) or absence (bottom graph) of FK778, was investigated in a coculture assay. At day 6, proliferation of naive CD4⁺CD25⁻ responder T cells (5 × 10⁴) stimulated with allogeneic stimulator cells (5 × 10⁴) was studied in the absence or presence of increasing numbers of the CD25⁻ (•), CD25^intermediate (), or CD25^high (○) cells (*P < .05; **P < .01 for differences between untreated and FK778-treated cells). (D) Flow cytometric FoxP3 analysis (x-axis) plotted against CD4 expression (y-axis) of the sorted CD25^high and CD25^intermediate subsets from the FK778-treated (left panels) and the untreated control groups (right panels). (E) Flow cytometry was performed directly after cell sorting of the CD4⁺CD25^high T cells at day 6. One representative experiment of 2 is presented. For panels B and C, error bars represent SD counts.