Figure 3. T-cell-derived SIGIRR regulates Th17 cell development in vivo.

Wild-type and Sigirr^−/− naïve T cells were intravenousely transferred into Rag1^−/− mice and EAE was induced by MOG_35–55 immunization. (A) Mean clinical scores were calculated each day for Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells. n=5 *, p<0.05; (ANOVA). (B) Hematoxylin and eosin staining of spinal cord of Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells 15 days after immunization with MOG_35–55. (C) Real-time PCR analysis of relative expression of IFN-γ, IL-17, TNF-α and IL-6 in spinal cords of MOG_35–55 immunized Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells (n=3, 10 days after disease onset). (D–E) Draining lymph node cells from SIGIRR-TG-KO (TG-KO) and littermate Sigirr^−/− mice were collected 10 days after immunization with MOG_35–55 emulsified in complete Freund's adjuvant and were re-stimulated with MOG_35–55 in vitro for 4 days, followed by ELISA (D) and ELISPOT analysis (E) (See also Figure S1), Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of three independent experiments. (F–H) Naïve T cells (CD4⁺CD44^low) from wild-type and Sigirr^−/− mice were co-cultured with wild-type splenic antigen-presenting cells (APCs) in the presence of anti-CD3 and polarized them under Th1 (IL-12 and anti-IL-4), or Th17 (TGF-β, IL-6, anti-IFN-γ and anti-IL-4) conditions, followed by intracellular cytokine staining for IL-17 and IFN-γ (F) and ELISA for IL-17 (H). (G) Wild-type and Sigirr^−/− mice Th1 cells were treated with 5ng/ml IL-18 for 48 h, followed by ELISA for IFN-γ. (I) Real-time PCR analysis of relative expression of IL-17, IL-22, IL-23R, ROR-γt and IL-10 in wild-type and Sigirr^−/− Th17 cells as compared to naïve T cells. Data are representative of at least three (A–C) independent experiments. Error bars (A–C), s.d. *, p<0.05; **.p<0.01 (two tailed t-test).