IL-1β, IL-21, and IL-23 boost differentiation of CD25^highCD27^pos Tregs into IL-17–producing cells in the obligatory presence of IL-2/IL-15; differentiation is regulated by histone deacetylases. Flow cytometry of sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs and CD25^negCD27^posCD45RA^negCD4^pos (CD25^negCD27^pos) Teffs that were stimulated with allogeneic PBMCs in the absence or presence of rIL-2, rIL-1β, rIL-6, or combinations thereof (A) or in the presence of rIL-1β or rIL-1 receptor antagonist (IL-1Ra) (B) or anti–IL-23 (D), IL-21, IL-23 (E), or the HDAC inhibitor trichostatin A (TSA) (F). Density plots show intracellular IL-17 and Foxp3 expression at day 8 of the cultures after restimulation of the cells with PMA plus ionomycin in the presence of Brefeldin A. Forward side scatter plots for panel A are shown in Figure S1. Data are representative of 4 (A) or 3 (B) separate experiments conducted with cells obtained from different blood donors. (C) Summary figure of 3 different experiments, using cells from 3 different blood donors, 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. (D) Anti–IL-23 did not hamper the differentiation of these cells into IL-17–producing cells. (E) IL-21 and IL-23 promote Treg differentiation into IL-17 cells. (F) TSA prevents Treg differentiation into IL-17 cells and promotes Foxp3 expression. Figures in panels D through F show cumulative data obtained from 3 or more experiments performed with cells obtained from different donor.