Extended Data Fig. 5: Activation of T_β-syn cells within the brain is critical for their infiltration and for secondary immune cell recruitment and clinical disease.

a, Microglia from CNS grey matter is inefficient to present antigen to T_MBP cells. Microglial cells isolated from brain parenchyma (total brain) or from cortical grey matter were cocultured for 48 h with T_MBP cells (left) or T_OVA cells (right) with or without addition of cognate antigen. Anti-MHC class II blocking monoclonal antibodies or PBS were added for each condition. IFNγ production was measured by ELISA. Representative data from three independent experiments per cell type. Data are mean + s.e.m., n = 3. b–d, Blocking antigen presentation in vivo by intrathecal injection of anti-MHC class II monoclonal antibody prevents T_β-syn cell entry into the brain and local reactivation. b, Number of T_β-syn cells in blood, brain meninges and brain parenchyma in rats treated with PBS (control) or anti-MHC-II monoclonal antibodies quantified at the onset of disease and the corresponding rate of infiltration in the brain compartments normalized to blood. Flow cytometry data. c, Quantification of the number of macrophages (CD11b⁺CD45^high cells), CD4⁺ and CD8⁺ T cells recruited to the indicated brain compartments. b, c, Representative data of two independent experiments including at least three rats per group. d, Relative expression of inflammatory cytokines (normalized to Actb) either in T_β-syn cells isolated from the indicated brain compartments (left) or in brain tissues (right) assessed by quantitative PCR. Data are mean + s.e.m. of n = 3. Representative data of two independent experiments. e–g, Effect of FK506 treatment on the infiltration of T_β-syn cells into the grey matter of the brain. e, Coronal brain sections (fluorescence overview images and magnified views of selected areas) from control (clinical score 1.5) or FK506-treated (clinical score 0) rats. Red, T_β-syn cells; green/yellow, tissue autofluorescence. f, Numbers of T _β-syn cells and recruited myeloid cells (CD11b^high) in the indicated compartments determined by flow cytometry. Data are mean + s.e.m., n = 3 per group. g, Expression of CD25 and CD134 on T_β-syn cells. a, One-way ANOVA with Dunnet’s correction for multiple comparison. b–d, f, Unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.