IL-17 secretion by DR− Tregs is induced by IL-1β and IL-6 and inhibited by TGFβ. FACS-sorted DR− Tregs (104 cells/well) were stimulated in serum-free X-Vivo medium for 5 days with plate-bound αCD3, soluble αCD28, and IL-2 in the presence of exogenous IL-1β and IL-6. Supernatants and cells were harvested at 24-hour intervals and analyzed for IL-17 content by ELISA (A) or RNA expression of IL-17A (B) and RORC (C) by real-time polymerase chain reaction (PCR). Data are representative of 2 independent experiments. (D) IL-17 secretion measured by ELISA in day 5 supernatants and represented as percentage of max (n = 5). (E) The IL-17 levels in day 5 supernatants are shown (n = 8). (F) Percentages of FoxP3+ cells ex vivo (n = 4) or on day 5 of stimulation (n = 8). At the end of the culture, cells were pulsed for 4 hours with PMA/ionomycin and GolgiStop, and stained for intracellular IL-17 versus FoxP3 (G) or IFNγ (H). Data are representative of 4 independent experiments. (I) DR− Tregs were stimulated for 5 days in the presence of exogenous IL-1β/IL-6 or TGFβ and tested for IL-17 secretion by ELISA. (J) Although the cultures were established at identical cell numbers, the relative cell numbers at the end of culture were determined for each condition by flow cytometry and represented as relative proliferation versus untreated cells. (K) Percentages of FoxP3+ cells on day 5 of stimulation. Data are representative of 5 independent experiments. **P < .01; *P < .05.
