Functional activity of in vitro–expanded CD45RA⁺ and CD45RA^– CD4⁺CD25^high T cells. (A) Cytokine production by sorted CD45RA⁺ (RA+) and CD45RA^– (RA–) subpopulations or total CD4⁺CD25^high (CD25^high) and CD4⁺CD25^– T cells (CD25^neg) after 2 weeks of in vitro expansion and restimulation with PMA/ionomycin. Representative results from 1 of 8 independent experiments. Numbers in dot plots indicate percent of cells within quadrants. (B) Suppressive activity of in vitro–expanded subpopulations compared with unseparated CD4⁺CD25^high and CD4⁺CD25^– T cells. Suppression by the indicated T-cell subpopulations at Treg/Tresp ratios of 1:4 (n = 7) and 1:16 (n = 3) was determined after polyclonal stimulation in a CFSE dilution assay. Combined data from 7 separate cultures with cells from 5 different donors. ***P < .001; *P < .05; RA⁺ (1:4) versus CD25^high (1:4) is also P < .05. Bars represent means + SEM. (C) Cytokine production by Tresp cells and different Treg-cell populations during coculture. CFSE-labeled Tresp cells and indicated autologous Treg-cell populations were stimulated with anti-CD3 on autologous antigen-presenting cells for 5 days and restimulated with PMA/ionomycin in the presence of monensin for the last 5 hours. One of 3 separate experiments with similar results is shown. Numbers in dot plots indicate percent of cells within quadrants.