Extended Data Fig. 4: T_β-syn cells are reactivated in the brain.

a–e, T_β-syn cells isolated from their target organ display an activated phenotype. a, Expression of selected sets of genes (grouped in functional classes) determined by RNA-seq analysis of T_β-syn cells isolated from blood, brain meninges and brain parenchyma at the onset of T_β-syn cell CNS infiltration. RPKM, reads per kilobase per million mapped reads. Data are mean + s.e.m., n = 9–15 per group from 3 independent experiments. Genes for which the expression in T_β-syn cells from brain was significantly different from the expression in T_β-syn cells from blood are highlighted by a red overlay colour. Unpaired two-tailed t-test. b, IFNγ and IL-17 expression (intracellular staining) in T_β-syn cells isolated from blood and brain at the peak of T cell infiltration. Representative data of three independent experiments. c, Surface membrane activation marker (CD25 and CD134) expression in T_β-syn cells isolated from the indicated compartments at the onset of T_β-syn cell CNS infiltration determined by flow cytometry. d, Expression of Ifng and Il17a mRNA in T_β-syn cells isolated from brain meninges, brain parenchyma, cortical grey matter and corpus callosum (white matter) measured by quantitative PCR (normalized to Actb). Data are mean + s.e.m. Representative data from two independent experiments. Unpaired two-tailed t-test. *P < 0.05, **P < 0.01. e, Top 50 differentially expressed genes in T_β-syn cells isolated from blood, brain meninges and parenchyma at day 3 p.t. determined by RNA-seq. Heat map shows z-transformed relative expression values. Each column represents a gene, each row represents a biological replicate. Genes known to be regulated by TCR-driven activation are indicated. f, T_β-syn cells, in contrast to T_MBP cells, are strongly activated in the brain. Left, Ifng and Il17a relative expression (normalized to Actb) in T_β-syn or T_MBP cells isolated from blood or from the indicated CNS compartments at the indicated time points p.t. determined by quantitative PCR. Right, expression of the cytokines in the corresponding total CNS tissues. Data are mean + s.d. of three independent experiments. g, T_β-syn cells do not proliferate in the brain. Histogram plots, Ki-67 expression of in vitro-cultured T_β-syn cells (two and six days after antigenic stimulation) or of T_β-syn cells isolated ex vivo from blood and brain at the peak of T cell infiltration. Grey, isotype control. Dot plots, corresponding quantification of BrdU incorporation. Representative data of three independent experiments. h, Kinetics of NFAT translocations in activated T_{β-syn–NFAT} cells in vitro. Plot, quantification of T cells with nuclear or cytoplasmic NFAT reporter over the period of 36 h after stimulation. Numbers of counted T_{β-syn–NFAT} cells are indicated. Representative fluorescent images showing T_{β-syn–NFAT} cells with nuclear (closed arrowheads) or cytoplasmic (open arrowheads) NFAT reporter are shown. i–k, Detection of NFAT translocations in situ. i, Histologic analysis of brain tissue at the onset of disease. Top, fluorescent overview images of a coronal brain section with magnified views. bottom, selected region acquired at higher magnification. Green, T_{β-syn–NFAT} cells; red, MHC class II⁺ cells; blue, DAPI (not depicted in the top panel). Filled and open arrowheads indicate T cells with nuclear and cytoplasmic localization of the NFAT–YFP reporter, respectively. Dashed line indicates the position of an area presented in Fig. 3a. j, Confocal images showing representative T_{β-syn–NFAT} cells with nuclear NFAT reporter in the parenchyma and meninges of the cortex. Individual z planes. k, In vivo visualization of de novo NFAT reporter translocation. Top, intravital TPLSM recording of NFAT translocation from the cytoplasm (open arrowhead) to the nucleus (closed arrowhead). Bottom, single z planes (shown as single colours and merged channels), maximum intensity projections (MIP) and 3D-rendered images depict the same T_{β-syn–NFAT-CherryH2B} cell before (0 min) and after (30 min) NFAT translocation. Histogram profile shows YFP and mCherry fluorescence intensities along the line indicated on the adjacent image.