Differentiated IL-17–producing CD25^highCD27^pos Tregs are characterized by the expression of CCR6. Sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs and CD25^negCD27^pos CD45RA^negCD4^pos (CD25^negCD27^pos) Teffs that were stimulated with allogeneic PBMCs and rIL-2 plus rIL-15 for 8 days. (A) Cell surface analysis of the indicated receptors (top) at day 8 of culture in the CD27^neg and CD27^pos cell gates (indicated at the left) after stimulation of CD25^highCD27^pos Tregs (top 2 rows) and CD25^negCD27^pos Teffs (bottom 2 rows). Histograms show forward scatter (FSC; y-axis) and fluorescence intensity (x-axis); numbers indicated percentage of positive cells. (B) Dot plots show combined CCR6 and CCR4 expression of the cells as described in panel A. (C) Sorted CD25^highCD27^pos Tregs were cultured for 8 days with allogeneic PBMCs in the presence of the indicated recombinant cytokines (left). After restimulation with PMA plus ionomycin in the presence of Brefeldin A intracellular IL-17 expression in CCR4- (left) and CCR6- (right) expressing cells was analyzed in the CD27^neg cell gate. (D) Sorted CD25^highCD27^pos Tregs were cultured with allogeneic PBMCs in the presence of rIL-2, rIL-15, and rIL-1β. At day 8 of the culture the cells were stained with anti–CD27-FITC plus anti–CCR6-PE mAb, and subsequently the CD27^negCCR6^pos (CCR6^pos) and CD27^negCCR6^neg (CCR6^neg) cell populations were sorted by high-purity flow cytometric cell sorting. IL-17 production was measured as described in panel C. Density plots show IL-17 production (x-axis) and CD3 expression (y-axis). Explanatory figures are provided in Figure S3. (E) Real-time quantitative RT-PCR of the mRNA expression of RORγt and IL-17 in sorted CD27^negCCR6^pos (CCR6^pos) and CD27^negCCR6^neg (CCR6^neg) cell populations. The RT-PCR data shown were normalized to human HPRT1 levels, and expression in sorted CD27^negCCR6^neg cells obtained after differentiation of CD25^negCD27^pos Teffs was set as 1.0. Data in panels A through E are representative of 3 separate experiments performed with cells obtained form different blood donors. (F) Freshly peripheral blood sorted CD25^highCD27^posCD45RA^negCCR6^pos Tregs and CD25^highCD27^posCD45RA^negCCR6^neg Tregs were stimulated and analyzed as described in panel C. One of 3 similar experiments is shown. (Explanatory figures are provided in Figure S3.)