Supplementary Figure 3: Rhbdd3 promotes the generation of Treg and inhibits the differentiation of Th1 and Th17 by inhibiting IL-6 production from DC.

(a-c) CFSE-labeled OT-II CD4⁺ T cells were co-cultured with TLR-activated BMDCs (a) or splenic DCs (b, c) which were pretreated with OVA_323-339 peptide at a ratio of 10:1. After 4 days of culture, proliferation rate of T cells were determined by CFSE dilution (a, b) and secretion of IFN-γ and IL-17 in co-culture supernatants were measured by CBA kit (c). (d) Percentages of CD4⁺CD25⁺ and CD4⁺Foxp3⁺ Treg in the spleens of Rhbdd3^+/+, Rhbdd3^–/–, Rhbdd3^–/–Il6^–/– or Il6^–/– mice were measured by FACS (n=3 mice per group). (e) Production of IFN-γ and IL-17 by CD4⁺ T cells from Rhbdd3^+/+, Rhbdd3^–/–, Rhbdd3^–/–Il6^–/– or Il6^–/– mice stimulated with α-CD3/CD28 were determined by CBA test. (f, g) CD11c-DTR mice depleted of DC were administrated with Rhbdd3^+/+ or Rhbdd3^–/– BMDCs. After 3 days, TNBS-induced colitis was performed in recipient mice. Weight loss were monitored daily (f, n=3 mice per group) and histology of colon sections were examined by H&E staining at day3 after TNBS induction (g). *P<0.05, **P<0.01 (two tailed Student's t-test). Data are from three independent experiments (mean±s.d. of three mice (d, f) or of technical triplicates (a, c, e) or are representative of three independent experiments with similar results (b, g).