Importance of antigen-presenting cells in the differentiation of CD25^highCD27^pos Tregs into IL-17–producing cells. Flow cytometry and cytokine analysis of sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs and CD25^negCD27^posCD45RA^negCD4^pos (CD25^neg-CD27^pos) Teffs that were stimulated with allogeneic PBMCs or anti-CD3 plus anti-CD28 mAb–coated beads or with purified monocytes or B cells in the presence of rIL-2 plus rIL-15. (A) Density plots show intracellular staining of IL-17 (y-axis) and Foxp3 (x-axis) after 8 days of culture with PBMCs or anti-CD3 plus anti-CD28 mAb–coated beads in the presence of IL-2/IL-15 and restimulation with PMA plus ionomycin in the presence of Brefeldin A. Numbers at the top of the plots indicate the percentage of IL-17–producing cells. (B) Dot plots show intracellular staining of IL-17 (y-axis) and Foxp3 (x-axis) after 8 days of culture with PBMCs, purified monocytes, or purified B cells, and restimulation with PMA plus ionomycin in the presence of Brefeldin A. Numbers at the top of the plots indicate the percentages of IL-17–producing cells. (C) Cytokine analysis of IL-1β, IL-6, and G-CSF in culture supernatants measured at distinct time points (days; x-axis) after stimulation of sorted CD25^highCD27^pos Tregs with allogeneic PBMCs or anti-CD3 plus anti-CD28 mAb–coated beads (legends). Three experiments conducted with cells from 3 different donors are shown. Data are representative of 4 (A) and 2 (B) separate experiments conducted with cells obtained from different blood donors.