Extended Data Fig. 7: Role of the dural lymphatics and dcLNs in EAE.

a, T cells do not preferentially accumulate in the dcLNs during EAE. Experimental set-up as in Extended Data Fig. 2. Quantification of CNS reactive T cells (T cell/g and absolute number) in the indicated peripheral compartments at the peak of T cell infiltration in the indicated rat and mouse EAE models. Flow cytometry. Mean + s.e.m. One way-ANOVA with post-hoc correction for multiple comparisons. Cumulative data of 5 (bSYN-aEAE, n = 13), 2 (MBP-aEAE, n = 6), 2 (MOG-aEAE, n = 9) and 2 (MOG-tEAE, n = 7) independent experiments, medLNs: n = 5 from 2 independent experiments. b-c, T_bSYN cells in the blood and LNs uniformly display a Th1/17 phenotype. b, Heat map of hierarchically clustered values of genes related to Th lineage. Experimental set-up as in Fig. 5d. c, Validation of the NGS data by quantitative PCR. House-keeping gene, β-actin. Cumulative data of 3 independent experiments (n = 11 animals). The number of analyzed samples is indicated. d, Effectors T cells are not activated in the dcLNs. EAEs were induced as in Extended Data Fig. 2. Flow cytometry quantification of the indicated surface activation markers in CNS-reactive T cells in bSYN-, MBP- and MOG-aEAE and percentage of Nur77⁺ T_{MOG-Nur77-GFP} cells in MOG-tEAE. Peak of the disease. Mean + s.e.m. One way-ANOVA with post-hoc correction for multiple comparisons. Cumulative data of 5 (bSYN-aEAE, n = 13), 2 (MBP-aEAE, n = 6), 2 (MOG-aEAE, n = 9) and 2 (MOG-tEAE, n = 7) independent experiments. n.s.: not significant. e-h, AAV ablation of the dural lymphatics does not affect T cell mediated CNS inflammation. e, Panel: whole-mount of the dura in Prox1-eGFP rats depicting the effective ablation of the dural lymphatics by the AAV-rVEGFR3 at the confluence of sinuses (COS) and at the skull basis 3 months after AAV treatment. Overviews images and magnified areas. Graphs: corresponding quantification of the effect of AAV-rVEGFR3 and control AAV (AAV-Sham) on the lymphatic vessel area-percentage in the indicated regions. Mean + s.e.m. Two-tailed t-test. Each dot represents an area of interest (COS: AAV-Sham n = 19; AAV-rVEGFR3 n = 19; Skull base: AAV-Sham n = 12; AAV-rVEGFR3 n = 23) from 2 animals/group. f, Lymphatic ablation prevents the drainage of Evans blue to the dcLNS. Rats were injected i.c.m. with Evans blue 3 months after AAV treatment. Representative photos depicting the lack of dye uptake in the dcLNs of AAV-rVEGFR3 lymphatic depleted rats compared to controls (AAV-Sham), 2 hours after dye injection. Arrowheads point to the dcLNs. Representative data of two independent experiments. g, T2-weighted coronal MRI sections performed 3 months after ablation of the lymphatic vessels. No enlargement of the lateral ventricles (arrowhead) suggestive of intracranial hypertension is observed. Representative data of 3-5 animals/group. h, Number of CD11b⁺ MHCII^low and CD11b⁺ MHCII^high myeloid cells at the peak of the disease in the indicated brain compartments as in Fig. 5e. Mean + s.e.m. n = 7 from 2 independent experiments. a,c,d,h: *P < 0.05, **P < 0.01, ***P < 0.001 and ****p < 0.0001. MRI, magnetic resonance imaging; MFI, median fluorescence intensity.

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