IL-17 production by differentiated Tregs depends on IL-2 or IL-15. Sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs were stimulated with allogeneic PBMCs in the absence or presence of rIL-2, rIL-15, or both. At day 8 of the cultures, the cells were stimulated with PMA plus ionomycin in the presence of Brefeldin A and analyzed for intracellular IL-17 expression. (A) Density plots show intracellular staining of IL-17 (y-axis) and Foxp3 (x-axis) after culture of CD25^highCD27^pos Tregs using the culture condition indicated at the top. Results obtained with cells obtained from 2 different donors are shown. Numbers at the top of the plots indicate the percentage of IL-17–producing cells. Forward side scatter plot are shown in Figure S1. (B) Summary figure showing the percentages of IL-17–producing cells (y-axis) after culture of CD25^highCD27^pos Tregs using the indicated conditions (x-axis). Individual measurements and mean are indicated. Data are shown from 9 to 13 different experiments, with different donors.