Figure 5. Generation of IL-17-producing cells from inducible regulatory T cells.

(A-B) FACS sorted CD4⁺Foxp3-GFP^-CD44^low T cells from Foxp3-GFP mice were stimulated under iTreg polarizing conditions (TGF-β, IL-2, anti-IFN-γ and anti-IL-4) for 5 days. Foxp3-GFP⁺ cells were FACS-sorted and stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of the indicated cytokines. (A) 4 days later, IL-17 and Foxp3-GFP expression was determined by flow cytometry, and (B) mRNA expression of indicated genes was analyzed by real-time RT-PCR. Numbers in FACS quadrants represent the percentages. The data shown in B was normalized to expression of a reference gene Actb. The lowest expression for each gene was referred as 1. Graph shows means ± s.d. The data represent at least three independent experiments with consistent results. (C) Naive T cells from Il17f^rft/Foxp3^gfp mice were stimulated with plate-bound anti-CD3, anti-CD28, TGF-β, IL-2 in the presence or absence of all-trans retinoic acid (RA). 3 days later, Foxp3-GFP⁺IL-17F-RFP^- cells were sorted and cultured with plate-bound anti-CD3 and anti-CD28 in the presence of TGF-β, IL-1, IL-6 and IL-23. 4 days later, IL-17, IL-17F-RFP and Foxp3-GFP expression was determined by flow cytometry. Numbers in FACS quadrants represent the percentages. The data represent at least two independent experiments with consistent results.