Phenotype and function of CD4⁺CD25^high T_reg cells from human peripheral blood. (A) Expression of CD4 and CD25 on human PBMCs and sort gates (i) used to isolate CD4⁺CD25^- (ii) and CD4⁺CD25^high T cells (iii). Numbers indicate the precentage of cells in each quadrant. (B) Intracellular expression of CTLA-4 and surface expression of CD62L and CCR7 on sorted CD4⁺CD25^- T cells (solid line histogram in i; also ii) and CD4⁺CD25^high T_reg cells (gray histogram in i; also iii). Dotted line indicates isotype control. (C) CD4⁺ T cells (T_resp) were cultured either for 5 days with irradiated allogeneic stimulator cells (i) or for 4 days with irradiated autologous PBMCs and soluble anti-CD3 (ii) and variable numbers of CD4⁺CD25^high or CD4⁺CD25^- T cells to obtain the indicated ratios. ^***P < .001 (Student t test). ^*P < .05. Panels show 1 of at least 3 independent experiments using cells from different donors. Error bars represent standard deviation from the triplicate wells.