Figure 6. Gprc5a deficiency decreases pT_reg-cell differentiation and promotes autoimmune inflammation.

(a,b) Splenocytes were prepared from unimmunized control mice (Ctr) or EAE mice 10 days after immunization (n=3–4). qPCR analysis of Gprc5a expression in total splenocytes or in sorted CD4⁺ T cells. (c,d) Western blot analysis of Gprc5a in splenocytes and CNS infiltrating cells derived from healthy controls (Ctr) or EAE mice. (e) Naive T cells were sorted from Gprc5a^+/+ and Gprc5a^−/− mice. Flow cytometry of polarized iT_reg cells in the presence of different concentrations of TGF-β1. (f) Flow cytometric analysis of T_reg cells in thymus from 6-week-old Gprc5a^+/+ and Gprc5a^−/− mice (gated on CD4⁺ T cells). (g,h) Flow cytometric analysis of T_reg cells in the inflamed spleen and lymph nodes Gprc5a^+/+ and Gprc5a^−/− mice 14 days after the induction of EAE (gated on CD4⁺ T cells). Numbers adjacent to outlined areas indicate per cent cells in each. (i,j) Clinical scores and weight loss (mean±s.e.m.) of Gprc5a^+/+ or Gprc5a^−/− mice after the induction of EAE were assessed every day (n=7). *P<0.05, **P<0.01, ***P<0.001, NS, not significant, two-tailed Student's t-test for a,b,f,g and h; one-way analysis of variance for i,j. Data are representative of at least two independent experiments (mean±s.e.m.).