Phenotype and functional activity of CD4⁺CD25^high and CD4⁺CD25^– T-cell subpopulations sorted for coexpression of CD62L and CCR7 after in vitro expansion. (A) FOXP3 mRNA expression of human in vitro–expanded CD4⁺CD25^high and CD4⁺CD25^– T cells and sorted subpopulations thereof. CD4⁺CD25^high (CD25high) and CD4⁺CD25^– T cells (CD25neg) were expanded for 14 days, rested, and sorted into CD62L⁺CCR7⁺ (high/++ and neg/++, respectively) and CD62L⁺CCR7^– (high/+– and neg/+–, respectively) subpopulations by FACS. mRNA was extracted from expanded whole populations and sorted subpopulations (day 0), and from subpopulations after a further week of expansion (day 7) as well as after an additional resting period of 4 days (day 7, rested). Shown are combined data from 3 independent experiments with cells from 3 different donors. FOXP3 mRNA expression by CD25^– cells on day 0 was arbitrarily set to 1. (B-C) CCR7 and CD62L expression of indicated subpopulations after further expansion for 1 week. One representative example (B) and combined data (C) from 6 independent cultures with cells from 6 different donors. ***P < .001. (D-E) FOXP3 protein expression by sorted subpopulations 1 week after further expansion. Shaded histogram, high/++; thick solid line, high/+–; thin solid line, neg/++; dashed line, neg/+–; and dotted line, isotype control. One representative example (D) and combined data from 4 independent experiments (E) are shown. ***P < .001. (F-G) Cytokine production and suppressive activity of sorted subpopulations after further expansion for 1 week. Cytokine production (F) was measured by intracellular staining after 5 hours of PMA/ionomycin stimulation; 1 of 4 independent experiments is shown. Suppression of proliferation (G) of CFSE-labeled Tresp cells by the indicated T-cell subpopulations from 6 independent cultures at Treg/Tresp ratios of 1:4 (n = 6) and 1:16 (n = 3) was determined as described in “Materials and methods.” Control cultures (Tresp) contained unlabeled and CFSE-labeled Tresp cells at indicated ratios. Bars in panels A, C, E, and G represent means ± SEM.