Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo
Fig 3
Cpd 1 specifically impairs human Th17 cell polarization and Th17-signature cytokine production.
(A) Total CD4+ T-cells were incubated with the RORγt inhibitor and were activated under Th17 cell favoring conditions for 72 hrs followed by quantification of IL-17A production. Data are representative of five experiments with triplicate readings. (B) Th17 cells were treated with PMA/ionomycin and Brefeldin A for 4 hrs and frequencies of IL-17A producing cells in gated CD4+ T-cells were determined by intracellular staining. Data represent duplicate measurements of two independent experiments. (C and D) Supernatants from polarized Th17 cells were taken to determine IL-17F and IL-22 cytokine concentrations. Naïve (E) and memory (F) human CD4+ T-cells were purified from PBMCs and were cultured under Th17 skewing conditions for 7 days in the presence of cpd 1. IL-17A production was quantified by ELISA. Representative examples of concentration-response curves from two experiments with duplicate readings are shown. (G) Human CD4+ T-cells were stimulated with anti-CD3 plus anti-CD28 antibodies only (Th0) or incubated with IL-12 and anti-IL-4 antibody (Th1) or with IL-4 and anti-IFN-γ antibody (Th2). After 48 hrs, Th subset signature cytokines including IL-2, IFN-γ and IL-13 were analyzed by ELISAs. Representative examples from two experiments with triplicate readings are shown.