Extended Data Fig. 6: Antigen availability determines T cell infiltration into the brain.

a, In vitro activation of T_OVA–CAR cells. Relative expression of IFNγ in T_OVA–CAR cells challenged in vitro with biotin anti-mouse IgG at the indicated doses in the presence or absence of streptavidin. Quantitative PCR performed at the indicated time points after challenge. Stimulation of T_OVA–CAR cells at the matching time points with anti-CD3/CD28 is shown as comparison (right plot) (normalized to β-actin). b, In vivo activation prompts T_OVA–CAR and myeloid-cell entry into the brain. Plot, number of T_OVA–CAR cells in brain meninges and brain parenchyma quantified by flow cytometry 3 days p.t. Rats were injected intrathecally or i.c.v. with biotin anti-mouse IgG (anti-CAR) 6 h before analysis, to trigger T cell activation. Control, PBS-treated animals. Data are mean + s.e.m. Cumulative data of three independent experiments including two animals per group. Intravital TPLSM images of the brain cortex three days p.t. are shown. T_OVA–CAR cells show infiltration of CD11b⁺ cells labelled by intrathecal injection of SeTau647-conjugated anti-CD11b monoclonal antibodies (magenta). Recordings were performed 6 h after i.c.v. injection of PBS (control) or anti-mouse IgG1 (anti-CAR). c, d, Effector T cell entry into the brain is boosted by increased antigen availability. Plots, quantification of T_OVA cells (c) or T_MBP cells (d) in the indicated brain compartments 3 days p.t. Rats were injected i.c.v. with the cognate antigen 12 h before analysis. n = 2 per T cell specificity. Corresponding intravital TPLSM images are shown. Green, antigen-specific T cells; red, blood vessels and macrophages. e–g, Neuronal overexpression of β-synuclein strongly increases T_β-syn cell invasion into the brain. e, Fluorescence overview images and magnified views of selected areas of coronal brain sections depicting the pattern of T_β-syn cell infiltration at the peak of the T cell grey matter infiltration. The rats had been neonatally injected with AAVs expressing GFP or β-syn–GFP under the synapsin promoter. Representative data of three independent experiments. Red, T_β-syn cells; Grey (false colour), GFP- or β-synuclein-expressing neurons. f, Quantitative analyses of T_β-syn cells by intravital TPLSM in AAV-β-syn-GFP or AAV-GFP rats. Number of grey-matter-infiltrating T_β-syn cells (left) and their velocity over a 30-min period (right). Note the decreased speed of T_β-syn cells in rats overexpressing β-synuclein, indicating local T cell activation. n = 2 from 2 independent experiments. g, Neuronal overexpression of β- but not γ-synuclein increases T_β-syn cell entry into the brain. Fluorescence overview pictures and magnified cortical areas at the peak of T_β-syn cell infiltration depicting the infiltration pattern of T_β-syn cells into the brain of rats overexpressing β- or γ-synuclein in neurons. Red, T_β-syn cells; grey (false colour), neurons overexpressing β- or γ-synuclein. Representative data of two independent experiments. b–d, Unpaired two-tailed t-test. f, Mann–Whitney U-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.