CD25^highCD27^pos Treg differentiation into CD27^neg IL-17–producing cells and pass through into a Foxp3/IL-17 double-positive stage. Flow cytometry of sorted CD4^posCD25^highCD27^posCD45RA^neg (CD25^highCD27^pos) Tregs and CD4^posCD25^negCD27^posCD45RA^neg (CD25^negCD27^pos) Teffs that were stimulated with allogeneic PBMCs in the presence of both rIL-2 and rIL-15 and analyzed for cell division, IL-17 production, and Foxp3 and CD27 expression. To analyze IL-17 production, cells were restimulated with PMA plus ionomycin in the presence of Brefeldin A. (A) Density plots at the top show intracellular expression of IL-17 (y-axis) and Foxp3 (x-axis) at days 5, 7, and 8 after culture of CD25^highCD27^pos Tregs. Numbers at the top of the plots indicate the percentages of IL-17–producing cells. The lower density plots show CFSE content and costaining of Foxp3 of CFSE-labeled CD25^highCD27^pos Tregs at day 6 of the culture. (B) Summary plot showing percentage of IL-17– and Foxp3–expressing cells, such as analyzed under panel A, at day 8 of the cultures (data from 12 individual experiments, using 12 different blood donors). (C,D) Sorted CD25^highCD27^pos Tregs and CD25^negCD27^pos Teffs were CFSE labeled and subsequently stimulated with allogeneic PBMCs and rIL-2 plus rIL-15. Plots show CFSE content (x-axis) and expression of CD27 (C) or IL-17 (D, y-axis) at day 8 of the culture. (E) Density plots show CD27 (x-axis) expression and intracellular IL-17 expression (y-axis). (F) Cytokine measurements of differentiated CD27^pos and CD27^neg cells obtained after differentiation of sorted peripheral CD25^highCD27^pos Tregs and CD25^negCD27^pos Teffs. CD25^highCD27^pos Tregs and CD25^negCD27^pos Teffs were sorted from peripheral blood, labeled with CFSE, and cultured in the presence of allogeneic stimulator PBMCs and rIL-2 plus rIL-15 for 10 days. At day 10, the resultant CD27^pos and CD27^neg cells in the divided CFSE-low cell population were isolated by cell sorting using flow cytometry. The sorted cell populations (20 × 10³) were stimulated for 20 hours with PMA plus ionomycin; thereafter IL-17 concentrations were analyzed in the culture supernatants. Means plus or minus SD are shown for 3 independent experiment performed with cells obtained from different donors. Data are representative of 2 (A) or 3 or more (C-E) separate experiments conducted with cells obtained from different blood donors.