Fig. 2. Effect of RORγt inverse agonists TF-S2 and TF-S14 on Th17 polarization of human PBMCs collected from highly sensitized transplantation candidates.
PBMCs from highly sensitized kidney transplantation candidates were treated with CD3/CD28 magnetic beads for 16 days in presence of IL6 10 ng/ml, IL1β 10 ng/ml, TGFβ 10 ng/ml, and IL23 10 ng/ml. Culture medium was changed every 3 days for the whole duration. GSK298128 (15 nM), TF-S2 (15 nM), TF-S14 (15 nM) or vehicle were added to the polarized cells on day 14 for 48 h and at the end of incubation cells were stimulated with PMA/ionomycin/monensin/prefoldin cocktail for 5 h. RORγtBV650 MFI of CD4+ cells (a), the percentages of RORγthi/ (b), IL17A+/ (c), IL21+/ (d), IL22+/ (e) and IFNγ+/CD4+ (f) were measured with flow cytometry. The ratio of Th17/Th1 was measured by dividing the percentage of IL17+/CD4+ by the percentage of IFNγ+/CD4+ cells (g). The concentration of IL17A and IL21 secreted in the culture media were both measured in supernatant using ELISA (h, i). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, two-way ANOVA and Tukey’s test, n = 6–7 per group.