CD69^posand CD69^negTregs in SPL and CD69^negTregs in PB have similar suppressive capacity, but different proliferative and cytokine production potentials. CD4^pos T cells were isolated from SPL and PB samples of healthy human donors, and sorted into CD69^neg and CD69^pos Tregs (SPL) or only CD69^neg Tregs (PB). (A) Suppressive capacity of Treg subsets was determined in coculture assays using flow cytometry. CFSE-labeled allogeneic CD4^pos Tresp isolated from PB were activated in vitro with anti-CD3/CD28 microbeads (bead:cell ratio, 1:5). Treg subsets were titrated into these cultures at indicated Tresp:Treg subset ratios and percentage of suppression is shown (N = 3). Data were compared using 2-way ANOVA and no significant differences were found between suppression capacities of Treg subsets. (B) Proliferative capacity of Treg subsets without or with anti-CD3/CD28 microbeads stimulation in the absence or presence of exogenously added IL-2. Proliferation was determined by measuring [³H]thymidine incorporation at day 4 (mean for each donor plus SD, N = 3). Data were compared using 1-way ANOVA. Significant differences are indicated: *P < .05; **P < .01; ***P < .001. (C) Treg subsets were stimulated for 24 hours with PMA and ionomycin, and culture supernatants were analyzed for the concentration of indicated cytokines by Luminex (N = 3). Data were compared using 1-way ANOVA. Significant differences are indicated: *P < .05; **P < .01; ***P < .001. (D) PB and SPL-derived CD4^pos T cells were stimulated for 24 hours with PMA and ionomycin in the presence of Brefeldin A and analyzed for expression of FoxP3 and production of IL-2 by flow cytometry (N = 3 for each tissue, representative examples are shown).