Fig 2: SR12418 Potently Suppresses the Development and Severity of EAE(A) Clinical EAE scores (left) from mice subjected to MOG-induced EAE and treated with vehicle (10/10/80 formulation of DMSO/Tween80/H2O) or SR12418 (i.p., 50mg/kg, b.i.d.) for the duration of the experiment. Middle and right graphs demonstrate the percent incidence of disease and percent change in body weight overtime between groups, respectively (n = 8–10/group).(B) Frequencies and cell counts of CD3+CD4+ cells in the draining LNs and CNS of mice at peak of disease. V, vehicle; SR, SR12418-treated mice (n = 8, V; n = 7, SR).(C) Graphs depicting the frequencies and cell counts of CD4+GM−CSF+ cells in LN and CNS of mice at peak of disease.(D) FACS analysis, frequencies, and cell counts of RORγt+ cells in LNs and CNS of mice at peak of disease.(E) FACS analysis, frequencies, and cell counts of IL-17A+IFNγ−, IL-17A+IFNγ+, and IL-17A−IFNγ+ cells in the LN and CNS of mice at peak of disease.Cells were gated on live, CD45+CD3+CD4+ cells. Data are mean ± SEM and representative of three separate, independent experiments with similar results. Two-way ANOVA (clinical score) and Student’s t tests were performed for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: SR12418 Is Effective When Used in Intervention Studies of Colitis and Relapsing-Remitting EAE(A) Schematic of the adoptive T cell transfer colitis model treatment design.(B-E) FACS plots and graphs demonstrating frequencies of (B) α4β7+, (C) RORγt+, (D) IL-17A+IFNγ−, IL-17A+IFNγ+, and IL-17A−IFNγ+, and (E) CD25+Foxp3+ T cells in the colons of recipient mice. Cells were gated on live, CD45+CD3+CD4+ cells. V, vehicle; SR, SR12418. Data are mean ± SEM (n = 10/group; PBS, n = 2).(F) Clinical EAE scores from mice subjected to PLP-induced EAE and treated with vehicle or SR12418 (i.p., 50 mg/kg, b.i.d.) starting on day 18 and continued for the duration of the experiment (n = 17/group).(G-K) Graphs demonstrating decreased frequencies and cell numbers of (G) effector CD4 and CD8 T cells and (H) CCR6+ T cells in the CNS of mice treated with SR12418. FACS plots and graphs demonstrating frequencies and/or cell numbers of (I) RORγt+, (J) RORγt+ GM-CSF+, and (K) IL-17A+IFNγ−, IL-17A+IFNγ+, and IL-17A−IFNγ+ T cells in the CNS of SR12418-treated mice relative to vehicle controls.Cells were gated on live, CD45+CD3+CD4+CD44+cells (n = 7, vehicle; n = 6, SR12418). Data represent mean ± SEM. Two-way ANOVA (clinical score) and Student’s t tests were performed for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: RORγtK256R/K256R mice display impaired Th17-mediated EAE induction(A) Mean clinical scores at indicated time points for EAE in Rag1−/− mice adoptively transferred with 3 × 106 naive CD4+ T cells from WT or RORγtK256R/K256R mice (n = 11–12, three independent experiments), followed by immunization with MOG35–55.(B) Incidence of EAE in mice shown in A on day 30 post immunization.(C) The number of CD4+ T cells infiltrated into the CNS of indicated mice shown in (A).(D and E) Representative flow cytometric analysis (left panels) and percentage (right panels) of IFN-γ+/CD4+, GM-CSF+/CD4+, or IL-17A+/CD4+ cells recovered from CNS (D) or spleen (E) of mice as shown in (A).(F) Representative flow cytometric analysis of RORγt (left panels) and percentage of RORγt+ cells among CD4+ T cells recovered from CNS of mice shown in (A).(G) Representative flow cytometric analysis of IL-17A/RORγt (left panels) and percentage of IL-17A+ cells among RORγt+ cells in CD4+ T cells recovered from CNS of mice shown in (A). Data are presented as mean ± standard error mean (SEM). Statistical significance is indicated as ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see Figure S1.