Fig. 4: Small molecule Hh inhibitors selectively block Th17 polarization in vitro.

a Schematic overview of the Hh inhibitor cyclopamine administration schedule using in panels b–d. b Naïve CD4⁺ T cells were stimulated under Th17- (top row) or iTreg- (bottom row) polarizing conditions in the presence of the indicated doses of cyclopamine (“cyclo”) or carrier control for three days. Cells were harvested for analysis by flow cytometry on day 5. Quantitation of IL-17a and FoxP3 expression for Th17 cells and iTregs respectively, viability measured by the absence of live/dead staining, and cell numbers are shown on the right. Th17 data = top row. iTreg data = bottom row. n = 3–4 independent experiments. c Naïve CD4⁺ T cells were stimulated under Th17 polarizing conditions in the presence of the indicated doses of cyclopamine or carrier control for three days. Cells were harvested for analysis by flow cytometry on day 5. Quantitation of cell-surface CCR6 is shown on the right. n = 3 independent experiments. d Naïve CD4⁺ T cells were stimulated under Th1 or Th2 polarizing conditions in the presence of 5 μM cyclopamine or carrier control for three days. Cells were harvested for analysis by flow cytometry on day 5. n = 3 independent experiments. e Naïve CD4⁺ T cells were stimulated under Th17 or iTreg polarizing conditions in the presence of the indicated dose of cyclopamine or carrier control for the final 24 h of polarization. Cells were harvested for analysis by flow cytometry on day 5. n = 3–4 independent experiments. Data are means +/− SD. p-values were calculated using one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001  .