Fig. 2. TGF-β1 produced by Th17 cells is required for the stability of Th17 cells.

a Naive CD4⁺ T cells from Tgfb1^fl/flIl17a^CreR26^YFP or WT mice were differentiated into Th17 cells for 3 days and analyzed for the expression of cytokines by flow cytometry. Representative FACS plots and quantification of IFN-γ- and/or IL-17-expressing cells. b Experimental scheme. Tgfb1^fl/flIl17a^CreR26^YFP and WT mice were immunized with MOG_35-55 peptide in CFA. Eight to nine days after immunization, lymphocytes from the dLNs were stimulated with MOG_35-55 peptide in the presence of IL-23 and an anti-IFN-γ antibody. After 5 days, the expression of cytokines was determined by flow cytometry. c Representative contour plots and quantification of IFN-γ- and/or IL-17-expressing cells (IL-17⁺IFN-γ⁻: IL-17 SP, IL-17⁻IFN-γ⁺: IFN-γ SP) in CD4⁺CD44^hi cells from the indicated mice. d Representative FACS plots and quantification of IFN-γ- and/or IL-17-expressing cells in CD4⁺YFP⁺ cells. e Schematic representation of the T cell adoptive cotransfer experimental autoimmune encephalomyelitis (EAE) model. Tgfb1^fl/flIl17a^CreR26^YFP (CD45.2⁺) and WT (CD45.1⁺CD45.2⁺) mice were immunized with MOG_35-55 peptide in CFA. After 8–9 days, lymphocytes from the dLNs were stimulated with MOG_35-55 peptide in the presence of IL-23 and an anti-IFN-γ antibody. After 5 days of stimulation, CD4⁺YFP⁺ T cells were sorted on a FACSAria^TM III, mixed at a 1:1 ratio and adoptively transferred into Tcrb^−/− mice. Recipient mice were immunized with MOG_35-55/CFA and intraperitoneally injected with pertussis toxin (PTX) (n = 6). f Representative contour plots and quantification of IFN-γ- and/or IL-17-expressing cells in CD4⁺YFP⁺ cells from the dLNs after restimulation with PMA and ionomycin (n = 6). Data are representative of three independent experiments. Quantification plots show the mean ± SD (a, c, and d); *p < 0.05 and **p < 0.01. A two-tailed Student’s t-test (c and d) and the Wilcoxon signed-rank test (f) were performed.