Figure 3: Role of the NF-κB pathways in GITR-mediated suppression of iTregs.

(a) Immunoblot analysis of the induction of the canonical (p50, RelA) and non-canonical (p52, RelB) NF-κB pathways in the cytosol and nucleus in WT B6 naive T cells activated for 1 to 3 days under iTreg-polarizing conditions with DTA-1 or Ctrl IgG. Data are representative of three independent experiments. (b,c) Naive CD4⁺ T cells from WT B6, p50^−/− and p52^−/− mice were activated under iTreg conditions, with or without GITR ligation for 3 days, and induction of Foxp3⁺ T cells and IL-9+ T cells was analysed by FACS and shown. Numbers in the quadrants indicate the percentage of cells. (c) Graphs depict Mean±s.d. of five experiments with triplicate cultures. (d,e) ChIP analysis of H3Ac and H4Ac modifications at Foxp3 (d) and Il9 (e) promoter and CNS regions from naive WT and p50^−/− CD4⁺ T cells, either left untreated (Unstim) or activated under iTreg-polarizing conditions for 2 days in the presence of DTA-1 or Ctrl IgG. Data represent mean values±s.d. (n=3). P values were determined by Student’s t-test (*P<0.05).