Extended Data Fig. 2: Migration properties and differentiation phenotype of T_β-syn cells.

a, T_β-syn and T_MBP cells follow similar migratory routes before invading the CNS. Numbers of T_β-syn and T_MBP cells in the indicated organs quantified every 24 h p.t. by flow cytometry. Data are mean + s.e.m. Representative data of three independent experiments including at least three rats per group per time point. b, T_β-syn and T_MBP cells switch from an activated state to a migratory mode. Gene expression changes between migratory T cells (isolated from blood at the onset of CNS infiltration, that is, at day 3 p.t.) and in vitro T cells (24 h after antigen challenge) as measured by RNA-seq analysis. The ratio between the expression in migratory and in vitro activated T cells for the indicated genes is displayed. Data are mean + s.e.m., n = 9–15 per group from 3 independent experiments. c, d, T_β-syn and T_MBP cells display very similar intravascular migration behaviour. Intravital TPLSM recordings were performed upon first arrival of T_β-syn or T_MBP cells in the leptomeningeal brain vessels. c, Representative migratory path of T_β-syn or T_MBP cells over a 30-min recording time. Rolling cells appear as multiple dots. Green, T cell tracks projected over time; red, blood vessels and leptomeningeal phagocytes. Representative data of at least four independent experiments (obtained from individual rats). d, Crawling velocity and track duration of T_β-syn and T_MBP cells. Data are mean ±  s.e.m. The number of analysed T cells is indicated. e, f, T_β-syn and T_MBP cells are similar in integrin and cytokine expression and chemokine responsiveness. e, Relative expression (normalized to Actb) of the indicated integrins, chemokine receptors and cytokines as measured by quantitative PCR in T_β-syn and T_MBP cells isolated from the blood at the onset of T cell CNS infiltration. Data are mean + s.d., n = 3 rats per group. f, Chemotactic response to the indicated chemokines of ex vivo-isolated T_β-syn and T_MBP cells. Data are mean + s.d., n = 2 per group. g–i, VLA-4 crucially determines T_β-syn cell invasion into the brain. g, h, T_β-syn cell motility was recorded by intravital TPLSM upon first arrival of T_β-syn cells in the leptomeningeal brain vessels before and immediately after intravenous injection of blocking anti-VLA-4 monoclonal antibody. g, Representative 30-min time-lapse recordings of T_β-syn cell migratory paths. Green, 30-min T_β-syn cell track projections; red, Texas red dextran-labelled vessels. Representative data of at least four independent experiments (obtained from individual rats). h, Percentage of crawling or rolling T_β-syn cells before and after anti-VLA-4 monoclonal antibody treatment (arrow) relative to time point 0. Data are mean ± s.e.m. of n = 3. i, Clinical disease after applications of anti-VLA-4 or isotype monoclonal antibodies (control). Arrows indicate the time point of treatment. Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. j–l, LFA-1 integrin does not contribute to T_β-syn cell invasion in the brain. Intravital TPLSM recordings were performed as in g before and after anti-LFA-1 monoclonal antibody administration. j, Representative 30-min time-lapse recordings of T_β-syn cell migratory paths. Green, T_β-syn cell tracks projected over time; red, Texas red dextran-labelled vessels. Representative data of at least four independent experiments (obtained from individual rats). k, Percentage of crawling or rolling T_β-syn cells before and after monoclonal antibody treatment quantified as in h. Data are mean ± s.e.m. of n = 3. l, Disease course in T_β-syn cell recipient rats treated either with anti-LFA-1 or isotype monoclonal antibodies (control) at the indicated time points (arrows). Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. m, Saturation of monoclonal antibody binding (in g–i and j–l experiments) was controlled by staining of blood-derived T_β-syn cells with anti-VLA-4 or anti-LFA-1 primary monoclonal antibodies followed by secondary detection antibody (red) or with secondary antibody only (black). n, o, IL-17 or IFNγ blockade partially interferes with T_β-syn cell entry into the brain. n, Disease course in T_β-syn-cell-transferred rats intrathecally treated at the indicate time points (arrows) with PBS (control), anti-IL-17 or anti-IFNγ monoclonal antibodies. Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. o, Quantification of macrophages (MΦ, CD11b⁺CD45^high), CD4⁺ and CD8⁺ T cells infiltrating the brain leptomeninges and the brain parenchyma at the peak of clinical disease. Data are mean + s.e.m. of four rats per group. Representative data of two independent experiments. n, o, Unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.