T_reg-cell characteristics in expanded CD4⁺CD25^high T cells. Expanded CD4⁺CD25^high T cells retain phenotypical and functional characteristics of T_reg cells. (A) Surface expression of CD25, CD62L, and CCR7, and intracellular expression of CTLA-4 of CD4⁺CD25^high T cells (gray histograms and right dot plots) and CD4⁺CD25^- T cells (solid line histograms and left dot plots) expanded for 2 weeks (i-ii) or 3.5 weeks (iii-iv) on CD32⁺ L cells. Dotted line in i and iii indicates isotype control. Numbers indicate the percentage of cells within each quadrant. (B) CD4⁺ T_resp cells were stimulated with allogeneic PBMCs (i), or with anti-CD3 Ab and autologous PBMCs (ii) for 5 or 4 days, respectively. CD4⁺CD25^high or CD25^- T cells, expanded on CD32⁺ L cells for 23 days and then rested for 2 days, were added at variable numbers to obtain the indicated ratios. ^***P < .001 (Student t test). ^*P < .05. One of at least 4 independent experiments using cells from different donors. Error bars represent standard deviations for triplicate wells.