Fig. 1: Suppression of IL-17A production in CD8⁺ T cells by DMF is ROS-dependent.

a Flow cytometry of IL-17A in CD8⁺CD45RA⁻ or CD4⁺CD45RA⁻ cells from blood of the same MS patients before and after DMF therapy fulfilling (reponders, n = 18), or not (non-responders, n = 18) NEDA-3 criteria after treatment (Supplementary Tables 1-3, Supplementary Fig. 1a, b). The observer was blinded to experimental groups. b Frequency of CD8⁺CD45RA⁻IL-17A⁺ cells before DMF therapy in responders and non-responders. c-j Naive CD62L⁺CD44⁻CD8⁺ or CD62L⁺CD44⁻CD4⁺ T cells from WT mice were primed with anti-CD3/CD28 antibodies and TGF-β + IL-6 + IL-2 (murine Tc17) or TGF-β + IL-6 + IL-23 + IL-2 (murine Th17), in the presence of DMSO (Ctrl), 20 µM DMF or 20 µM DMF + 50 µM GSH (DMF + GSH) or 20 µM DMF + 1 mM NAC or ± 250 µM 2-DG or ± 10 nM Rotenone or ± 15 nM Oligomycin. c, g, i, j Flow cytometry of IL-17A in Tc17 c, i, j or Th17 cells g differentiated for 72 h with indicated treatment. d Ratio of reduced to oxidized glutathione (GSH/GSSG) contents in Tc17 cells differentiated for 2 h. e, f Flow cytometry of ROS levels in Tc17 or Th17 f cells differentiated for 2 h determined by CM-H₂DCFDA staining (fold of geometric mean fluorescence intensity (MFI), normalized to the corresponding control, which was arbitrarily set to 1. h Extracellular acidification rates (ECAR) and glycolytic capacity of Tc17 cells ± DMF after addition of 10 mM Glucose, 2.5 µM Oligomycin and 100 mM 2-DG. Bars show mean ± s.d. from seven e, or five c, d, i, or four to three f, g, j combined experiments, or one representative with 9 replicates of four experiments h; individual values are plotted. P-values in a from the same patient by two-tailed, paired t-test, while for the change in percentages CD8⁺CD45RA⁻IL-17⁺ or CD4⁺CD45RA⁻IL-17⁺ after DMF treatment by two-tailed, unpaired t-test (value between responders and non-responders), in b P value and in g, h ***p < 0.05 or non-significant (ns), by two-tailed, unpaired t-test, f *p < 0.05 by two-tailed, unpaired t-test with Welch’s correction, c, i, j ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) multiple comparison test, in d, e *p < 0.05, **p < 0.01, ***p < 0.001 by one-way Welch’s ANOVA with Games–Howell multiple comparison test.