Supplementary Figure 3: Drainage of T cells and dendritic cells into the cervical lymph nodes in a CCR7-dependent manner.

a, Representative multiphoton images of endogenous T cells (Lck^tdTOMATO - red) localized within lymphatic extension (Prox1^GFP - green) along the transverse sinus. Scale bar = 20 µm. b, Representative dot plots of GFP + CD4 + T cells in the dCLNs, sCLNs and lumbar lymph nodes at different time points after injection. c, Quantification of the percentage of GFP + CD4 + T cells in the dCLNs, sCLNs, Lumbar and ILN at 2, 6 and 12h post i.c.m. injection (mean ± s.e.m.; n = 10 mice/group; F(3,78) = 20.58; two-way ANOVA with Tukey’s multiple comparisons test). d, Quantification of the number of GFP + CD4 + T cells per dCLNs, sCLNs, Lumbar and ILN at 2, 6 and 12h post i.c.m. injection (mean ± s.e.m.; n = 10 mice/group; F(3,78) = 28.35; two-way ANOVA with Tukey’s multiple comparison test). e, CD4 T cells (isolated by negative selection from wild-type spleen and lymph nodes) were activated in vitro by CD3/CD28 stimulation followed by sustained IL-2 supplementation every other day for 9 days. Cells were then labeled with CFSE and mixed with Cell Tracer Violet-labeled, freshly isolated CD4 T cells. f, Representative image of i.c.m. injected in vitro activated T cells in the dCLNs 12h post injection. Scale bar = 100 µm, 45 µm (inset). g, Quantification of the density of activated T cells/mm² of dCLNs at different time points post injection (mean ± s.e.m). h, Representative dot plots of naïve and activated T cells in the dCLNs 12h post injection. Both naïve and activated T cells are found draining into the dCLN from the subarachnoid space. The draining activated CD4 T cells bear an effector memory phenotype (CD44⁺CD62L^low/+). Representative of 3 independent mice. i, Representative images of control and PTX-treated T cells in the dCLNs of wild-type mice 12h after i.c.m. injection. Scale bar = 200 µm, 60 µm (inset). j, Quantification of the density of T cells/mm² of dCLN (mean ± s.e.m.; pooled from 2 independent experiments; t = 3.901 df = 16; two-tailed unpaired t-test). k, Gating of the CCR7-WT and CCR7-KO DCs after LPS stimulation. l, Representative histograms of expression of CCR7, CD40, CD80 and CD103 by CCR7-WT and CCR7-KO DCs after LPS stimulation. Representative of 2 independent experiment. m, Representative images of the exogenously injected DCs (TAMRA – red) located within the meningeal lymphatics (Lyve-1 – white) of the transverse sinus. Scale bar = 20 µm. Representative of 3 independent mice. n, Scheme of the experiment illustrated in panels. (O-R). Meninges of KiKGR mice were exposed for 2 min to a violet light every 12h for photo-conversion. After 2 days, mice were injected i.c.m. with Poly(I:C) and peptide. Tissues were harvested 24h after injection. o, Representative gating strategy to identify dendritic cells. p, Representative dot plots of dendritic cells in B6 control and KiKGR control mice. Representative of 3 independent mice. q, Representative dot plots of photoconverted KiKR + dendritic cells in the dCLN, sCLN and ILN 24h after Poly(I:C) injection in converted mice. Representative of 4 independent mice. r, Quantification of the percentage of KiKR + dendritic cells in the dCLNs, sCLNs and ILNs of control and converted mice 24h after Poly(I:C) injection (mean ± s.e.m.; representative of 2 independent experiments; F(3,8) = 14.96; one-way ANOVA with Tukey’s multiple comparisons test). s, Representative images of i.c.m. injected DCs (CFSE – green) in dCLNs of wild-type mice 1, 2, and 4 days after i.c.m. injection. Scale bar = 200 µm, 50 µm (insets). Representative of 5 independent mice. t, Quantification of the density of the injected DCs/mm² of dCLN and ILN at different time points after injection (mean ± s.e.m.; n = 4-5 mice/group; representative of 2 independent experiments). u, Representative image of dCLNs 24h after i.c.m. injection of CCR7-WT (CFSE – green) and CCR7-KO (TAMRA – red) DCs at a 1:1 ratio. Scale bar = 200 µm. v, Quantification of the density of CCR7-WT and CCR7-KO DCs (per mm²) in the dCLN of wild-type mice 24 and 48h post i.c.m. injection (mean ± s.e.m.; n = 4-7 mice/group; F(1,18) = 17.47; two-way ANOVA with Sidak’s multiple comparison test).