The sorted CD25^highCD27^posCD45RA^negCD4^posTreg population produces IL-17 upon stimulation. (A) Cytokine measurements of sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs and CD25^negCD27^posCD45RA^negCD4^pos (CD25^negCD27^pos) Teffs measured in culture supernatants at 20 hours after stimulation with PMA and ionomycin. Data shown were normalized to cytokine production by CD25^negCD27^pos cells that was set as 1.0. Fold reduction or increase in cytokine production was calculated from 6 independent experiments performed with cells from different donors. Mean and SD from different experiments are shown. * indicates statistically significant differences (P < .05) according to the Wilcoxon matched pairs test. (B) IL-17 concentration measured at distinct time points (days; x-axis) after stimulation with allogeneic PBMCs in the presence of rIL-2 and rIL-15. Data show 3 experiments of 6 (A), or 8 (B), using cells from 3 different donors. (C) A single representative experiment showing flow cytometric analysis of IL-17 production in the sorted Treg and Teff populations that were cultured for 8 days with allogeneic PBMCs and rIL-2 plus rIL-15, and then stimulated for 4 hours with PMA plus ionomycin, in the presence of Brefeldin A. Numbers in the plots indicate the percentages of IL-17–producing CD3⁺ cells as assessed by intracellular cytokine staining. (D) Data summary of intracellular IL-17 staining in sorted Treg and Teff populations of 9 different donors at day 8 of allogeneic stimulation, such as that described in panel C.