Fig. 6.

RORC inhibition selectively blocks innate-like T cell function. a Upper panel: healthy donor CD4⁺ T cells cultured under Th17 conditions for 7 days. Data is represented as % IL-17⁺ cells as determined by intracellular flow cytometry (n = 4). (*p < 0.05, **p < 0.01 as determined by ANOVA). Lower panel: Skewed CD4⁺ Th17 cells were re-stimulated under Th17 conditions for 48 h and IL-17 levels were measured in the supernatants. Dose response of the RORγt antagonist (BIX119) for three donors is shown. b Human PBMC activated with aCD3Ab and IL-23 for 72 h. Dose response of the RORγt antagonist is shown for IL-17A and IL-22 secretion detected in culture supernatants by ELISA (n = 3). c γδ-T cells were activated with anti-CD3/CD28 beads and IL-23 cocktail for four days to produce IL-17A and IL-22 in the presence of increasing concentrations of RORγt antagonist BIX119 (n = 5). *p < 0.05 as compared to no compound (ANOVA) (d) PBMC were stimulated with αGalCer in the presence of IL-2 or IL-23 cocktail with addition of BIX119 (1 µM) or DMSO. After 14 days, cells were restimulated with PMA/CaI (with BFA) for intracellular cytokine detection. One example representative for three independent experiments is shown. The negative control (neg ctr) consisted of IL-23 skewed cells cultured in BFA alone. e quantitative overview of frequency of IL-17, IL-22 and TNFα+ iNKT cells from assays described in D (**p < 0.01 by paired t-tests). f IL-23 skewed iNKT cells were analyzed for RORγt and T-bet expression by intranuclear flow cytometry. Gating on cytokine producing iNKT cells was performed as illustrated in supplementary figure 7C (*p < 0.05, **p < 0.01 ANOVA). g FlowSOM visualization (as explained in Fig. 3F) of IL-23 skewed human iNKT cells (n = 3), cultured in the presence or absence of the BIX119 RORγt antagonist (compound). Pie chart shows markers used for automated clustering of iNKT cell types. Data throughout this figure are presented as mean ± SEM