Ameliorated EAE in IL-17RA −/− mice. Wild type mice and IL-17RA −/− mice that had been backcrossed onto C57BL/6 background were immunized with MOG_35–55 peptide to elicit EAE. Clinical signs and scores were recorded as described in Materials and Methods. Data are the average clinical scores from mice that have shown clinical signs of EAE. Data are representative of five independent experiments. *. P<0.05 determined by Mann-Whitney statistics.

MOG-specific Th1 responses were reduced in IL-17RA −/− mice. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. At ten days post-immunization, lymphocytes were isolated from draining lymph nodes, spleens, and CNS and stimulated with MOG_35–55 peptide as described in Materials and Methods. Frequencies of CD4⁺ T cells that made IL-17 (A, C, E) or IFNγ (B, D, F) among total lymphocytes were determined by intracellular cytokine staining assay. The number of CD4⁺ T cells infiltrating CNS (G) was calculated based on flow cytometry and cell count. Six IL-17RA −/− and six wild type and IL-17RA +/− mice were used. Student T test was performed.

Reduced CNS stroma infiltration by leukocytes in IL-17RA −/− mice. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. At 15 days post-immunization, immuno-histochemistry was done as described in Materials and Methods to determine the degree of leukocyte infiltration using anti-CD11b antibody.

Blockade of TGFβ recovered normal Th1 responses against the MOG peptide in IL-17RA −/− mice. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. Anti-TGFβ antibodies were administered at day 5 and day 9. At ten days post-immunization, lymphocytes were isolated from draining lymph nodes and stimulated with MOG_35–55 peptide as described in Materials and Methods. Frequencies of CD4⁺ T cells that made IL-17 (A) (C) or IFN-γ (B) (D) among total lymphocytes were determined by intracellular cytokine staining assay. More than seven mice were used in each experimental group. Student T test was performed.

Blockade of TGFβ exacerbated EAE in IL-17RA −/− mice. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. Anti-TGFβ antibodies were administered at day 5 and day 9. Clinical signs and scores were recorded as described in Materials and Methods. Data were the average clinical scores from mice that have shown clinical signs of EAE. Each point represents the mean of 5–8 animals and vertical lines are s.e.m. Where no error bar appears, the s.e.m = 0. ^* p < 0.05 compared to the IL-17RA−/− group. Statistical analysis was performed using one-way analysis of variance test (ANOVA) followed by Tukey’s post hoc t-test.

Severe CNS inflammation in IL-17RA −/− mice treated with anti-TGFβ antibodies. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. Anti-TGFβ antibodies were administered at day 5 and day 9. At 24 days post-immunization, immuno-histochemistry was done as described in Materials and Methods to determine the degree of leukocyte infiltration using anti-CD11b antibody. (A) IL-17RA +/− injected with control Ig, peak clinical score 4. (B) IL-17RA +/− injected with anti-TGFβ, peak clinical score 4. (C) IL-17RA −/− injected with control Ig, peak clinical score 1. (D) IL-17RA −/− injected with anti-TGFβ, peak clinical score 2. Wild type mice (WT), IL-17RA +/− (Het), and IL-17RA −/− littermates were immunized with MOG_35–55 peptide to elicit EAE. Anti-TGFβ antibodies were administered at day 5 and day 9. Mice developed EAE were used around day 20 after the induction of EAE. The number of CD4⁺ T cells infiltrating CNS was calculated based on flow cytometry and cell count (E). CNS infiltrating cells were analyzed for Gr-1 and CD11b positive cells (F). CNS infiltrating lymphocytes were stimulated for 16 hours with MOG_35–55 peptide, and then cells making IFNγ or IL-17 were analyzed by flow cytometry (G). The result was the average of data obtained from 4 independent experiments.

Effects of TGFβ on IFNγ production and CCR5 expression. CD4⁺ T cells were cultured under Th1 conditions in the absence or presence of TGFβ (3 ng/ml) for 4 days and then restimulated for 4 hours with plate-bound anti-mouse CD3 (10 μg/ml or 2 μg/ml) or alternatively with PMA (5 μg/m) and ionomycin (1 μg/ml). A) Intracellular staining using anti-IFNγ mAb. The lower panel shows specific inhibition of IFNγ production with TGFβ. B) qPCR assessment of CCR5 expression from Control (■) and TGFβ (□) cultures. RQ, relative quantity calculated by the Step one real time PCR software.