Figure 1.

Retinoic acid receptor-related orphan nuclear receptor γt (RORγt) inverse agonist inhibits mouse interleukin-17 (IL-17) expression and T helper type 17 (Th17) cell differentiation in vitro. (a) Mouse splenocytes were stimulated with anti-CD3/anti-CD28 monoclonal antibody (mAb) plus the indicated cytokine and antibody for 3 days. The IL-17 titres in the supernatants were then determined by Meso Scale Discovery (MSD). (b) Mouse splenocytes were stimulated with anti-CD3/anti-CD28 mAb plus IL-6 and transforming growth factor-β (TGFβ) in the presence of 1 µm of indicated RORγt inverse agonists for 3 days. The IL-17 titres in the supernatants were then determined by MSD. (c) Different doses of TMP778 were added into the culture as described in Fig.2(b) for 3 days. The IL-17 titres in the supernatants were then determined and the IC₅₀ was calculated. (d) Cell proliferation from (c) was determined using CellTiter-Glo. (e) Naive mouse CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 plus IL-6/TGF-β in the presence of small molecule compound for 4 days. Cells were then re-stimulated with PMA/ionomycin for 3 hr before intracellular staining for IL-17 and interferon-γ (IFN-γ). (f) Mouse splenocytes were stimulated under ThN (anti-CD3/28 mAb), Th17 (anti-CD3/28 mAb, IL-6, TGFβ), Th2 (anti-CD3/28 mAb, IL-4, anti-IL12 mAb), or Th1 (anti-CD3/28 mAb, IL-12, anti-IL4) skewing conditions with or without 1 μmTMP778 for 3 days. The Th1/Th2/Th17 cytokines were then determined by Meso Scale Discovery. Data shown are the log₂-fold change between TMP778 and DMSO control. (g) The percentage of IL-17-positive T cells after treatment with TMP778 or digoxin as in (e) was used to calculate their IC₅₀. Data are representative of two to five separate experiments.