No statistical methods were used to predetermine sample size.

Animals

Male and female C57Bl/6, NOD.Cd11c-YFP and Prox1tdT mice were purchased from Jackson Laboratories and were housed in temperature and humidity controlled rooms, maintained on a 12 h/12 h light/dark cycle (lights on at 7:00). All strains were kept in identical housing conditions. All procedures complied with regulations of the Institutional Animal Care and Use Committee at University of Virginia. Only adult animals (eight to ten weeks) were used in this study. Sample size was chosen in accordance with similar, previously published experiments. Animals from different cages in the same experimental group were selected to assure randomization. For all experiments, investigators were blinded from the time of euthanasia to the end of the analysis.

Human samples

Autopsy specimens of human dura including the superior sagittal sinus were obtained from the Departments of Pathology and Neurosurgery at the University of Virginia. All samples are from consenting patients that gave no restriction to the use of their body for research and teaching (through the Virginia Anatomical Board, Richmond, VA). All obtained samples were fixed and stored in a 10% formalin solution for prolonged time periods.

Meninges immunohistochemistry

Mice were euthanized with an intraperitoneal (i.p.) injection of Euthasol and perfused with 0.1 M of PBS for 5 min. Skin was removed from the head and the muscle was stripped from the bone. After removal of the mandibles and the skull rostral to maxillae, the top of the skull was removed with surgical scissors. Whole-mount meninges were fixed while still attached to the skull cap in PBS with 2% paraformaldehyde (PFA) for 24 h at 4 °C or in 1:1 ethanol:acetone solution for 20 min at −20 °C depending on the antibody. The dura/arachnoid was then dissected from the skullcap. For analysis of the pia mater, brains extracted from the skull were flash frozen, and 40 µm-thick transverse sections were sliced using a cryostat (Leica). The choroid plexus was dissected out of the ventricles of non-fixed brain and fixed with 2% PFA in PBS for 24 h. For coronal sectioning of whole-mount meninges, 100 µl of Matrigel (Corning) was injected into the dural sinuses before dissection. The meninges (dura mater/arachnoid) were dissected from the skullcap, flash frozen, and 10-µm thick sections were cut using a cryostat, and the slices were placed on gelatin-coated slides.

Whole mounts and sections were incubated with PBS containing 2% of normal serum (either goat or chicken), 1% BSA, 0.1% Triton-X-100 and 0.05% of Tween 20 for 1 h at room temperature, followed by incubation with appropriate dilutions of primary antibodies: anti-CD31 (eBioscience, clone 390, 1:100); anti-CD3e (eBioscience, clone 17A2, 1:500), anti-MHC II (eBioscience, clone M5/114.15.2, 1:500), anti-Lyve-1 (eBioscience, clone ALY7, 1:200), anti-Prox1 (Angiobio, 11-002, 1:500), anti-podoplanin (eBioscience, clone 8.1.1, 1:100), anti-VEGFR3 (R&D Systems, AF743, 1:100), anti-α-SMA (Sigma-Aldrich, clone 1A4, 1:500), anti-VE-Cadherin (eBioscience, clone BV13 1:100), anti-Claudin-5 (Molecular Probes, 352588, 1:200), anti-CCL21 (R&D Systems, AF457, 1:100), anti integrin-α9 (R&D Systems, AF3827, 1:100), anti-B220 (eBioscience, clone RA3-6B2, 1:100), anti CD11c (eBioscience, clone N418, 1:100) overnight at 4 °C in PBS containing 1% BSA and 0.5% Triton-X-100. Whole mounts and sections were then washed 3 times for 5 min at room temperature in PBS followed by incubation with Alexa-fluor 488/594/647 chicken/goat anti rabbit/goat IgG antibodies (Invitrogen, 1:1,000) or PE/Cy3 conjugated streptavidin (eBioscience, 1:1,000) for 1 h at room temperature in PBS with 1% BSA and 0.5% Triton-X-100. After 5 min in 1:10,000 DAPI reagent, whole mount and section were washed with PBS and mounted with Aqua-Mount (Lerner) under coverslips. For the pre-absorption experiments, the anti-VEGFR3 and anti-podoplanin antibodies were incubated respectively with recombinant mouse VEGFR3 (743-R3-100, R&D Systems) or recombinant mouse podoplanin (3244-PL-050, R&D Systems) at the concentration ratio of 1:10 overnight at 4 °C in PBS containing 1% BSA and 0.5% Triton-X-100 before staining.

For human samples, formalin-fixed superior sagittal sinuses were dissected in 2-mm thick sections and fixed overnight in PBS with 4% PFA. Tissues were then embedded in OCT and 10-µm thick sections were sliced onto gelatin-coated slides on a cryostat (Leica). Antigen retrieval was performed by incubation of the slides for 20 min in sodium citrate buffer pH 6.0 at 80 °C. After washing with PBS, endogenous biotins were blocked with a 30 min incubation in PBS with 3% H 2 O 2 , then the slides were blocked in PBS containing 2% of normal goat serum, 1% BSA, 0.1% Triton-X-100 and 0.05% Tween 20 for 1 h at room temperature. Slides were then incubated overnight at 4 °C with anti-Lyve-1 (ab36993, Abcam, 1:200), anti-podoplanin (HPA007534, Sigma-Aldrich, 1:200) or anti-CD68 (HPA048982, Sigma-Aldrich, 1:1,000) diluted in PBS with 1% BSA and 0.5% Triton-X-100. Sections were then washed 3 times for 5 min at room temperature in PBS followed by incubation with biotin-conjugated goat anti-rabbit antibody (Jackson Immunoresearch, 1:1,000) for 1 h at room temperature, then for 30 min at room temperature with ABC kit solutions (Vectastain, Vector Labs). Slides were then incubated with the peroxidase substrate DAB (Sigma-Aldrich) for several minutes, counterstained with haematoxylin, dehydrated, and mounted in Cytoseal 60 (Thermo Scientific) under coverslips. Nine human samples were labelled and analysed; the lymphatic structures were identified in two of them.

Image analysis

Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) using the LAS AF Software. For the images of the complete whole mount, images were acquired with a ×10 objective with 0.25 NA. Other confocal images were acquired using a ×20 objective with 0.70 NA or a ×40 oil immersion objective with 1.30 NA. All images were acquired with at a 512 × 512 pixel resolution and with a z-step of 4 µm. Quantitative assessments were performed using FIJI software (NIH). Percentage of luminal T cells was determined by counting the number of T cells with luminal localization in the sinuses area. T cell density was established by dividing the number of T lymphocytes by the area of meninges. Prox1-positive cell density was defined by dividing the number of Prox1+ nuclei by the area of lymphatic vessels. Statistical analyses were performed using GraphPad Prism software. Specific statistical tests are presented in the text for each experiment. Outlier samples were eliminated using the Grubbs’ test with a significance level of 0.01 (only for the rh-VEGF-c experiment). No estimate of variation between groups was performed.

Electron microscopy

Meninges were harvested as previously described and fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer with 0.15% potassium ferrocyanide. After rinsing in buffer, the tissue was dehydrated through a series of graded ethanol to propylene oxide, infiltrated and embedded in epoxy resin and polymerized at 70 °C overnight. Semi-thin sections (0.5 μm) were stained with toluidine blue for light microscope examination. Ultrathin sections (80 nm) were cut (Integrated microscopy centre, University of Memphis) and imaged using the Tecnai TF20 TEM with an AMT XR41 camera (Extended Data Fig. 5i; Integrated microscopy centre, University of Memphis) or using the Tecnai F20 TEM with an UltraScan CCD camera (Fig. 2h; Advanced Microscopy core, University of Virginia).

Multiphoton microscopy

Mice were anaesthetized by ketamine/xylazine injection i.p. and injected i.c.v. (into the cisterna magna) with 5 µl of QDot655 (Invitrogen) or 5 µl of Alexa488-conjugated anti-Lyve-1 antibody (ALY7, eBioscience). Mice were co-injected i.v. with 25 µl of 10% fluorescein sodium salt (Sigma-Aldrich) or 5 µl of QDot655 (Invitrogen). Meningeal lymphatic vessels were imaged through a thinned skull preparation. The core temperature of the mice was monitored and maintained at 37 °C. Imaging was performed with a Leica TCS SP8 multiphoton microscopy system (Leica Microsystems) equipped with a Chameleon Ultra II tunable Ti:Sapphire laser (Coherent). GFP and QDot655 were excited with an excitation wavelength of 880 nm. Images were obtained using a ×25 water immersion objective with 0.95 NA and external HyD non-descanned detectors (Leica Microsystems). Four-dimensional imaging data were collected by obtaining images from the x, y, and z-planes over time. The resulting images were analysed with Imaris software (Bitplane).

Labelling of the vascular compartment

To assess the abluminal localization of the sinusal T cells, mice were injected i.v. with 10 µg of FITC-conjugated anti-CD45 antibody (eBioscience, clone 30-F11) or control isotype 1 h before euthanasia. To assess that the meningeal lymphatic vessels are not part of the cardiovasculature, mice were injected with 100 µl of DyLight 488 Lycopersicon Esculentum Lectin (Vector Laboratories) 5 min before euthanasia.

In vivo VEGFR3 activation

Mice were injected i.c.v. with 4 µg of rh-VEGF-c (Cys156Ser, R&D Systems) or with PBS. Meninges were harvested 7 and 14 days after the injection.

Evans blue injection and detection

Mice were anaesthetized by ketamine/xylazine injection i.p., and then 5 µl of 10% Evans blue (Sigma-Aldrich) was injected i.c.v. into the cisterna magna or intranasally. Thirty minutes after injection, mice were euthanized with CO 2 and the central nervous system draining lymph nodes were dissected for assessment of Evans blue content. The dura mater was also harvested to analyse Evans Blue localization using confocal microscopy. The intensity of the Evans blue was measured using the plot profile function of FIJI.

Flow cytometric analysis of meninges

Mice were perfused with 0.1 M PBS for 5 min. Heads were removed and skulls were quickly stripped. Mandibles were removed, as well as all skull material rostral to maxillae. Surgical scissors were used to remove the top of the skull, cutting clockwise, beginning and ending inferior to the right post-tympanic hook. Meninges (dura mater, arachnoid and pia mater) were carefully removed from the interior aspect of the skulls and surfaces of the brain with Dumont #5 forceps (Fine Science Tools). Meninges were gently pressed through 70 µm nylon mesh cell strainers with sterile plastic plunger (BD Biosciences) to yield a single-cell suspension. For lymphatic endothelial cells isolation, meninges (along with diaphragm and ear skin) were digested for 1 h in 0.41 U ml−1 of Liberase TM (Roche) and 60 U ml−1 of DNase 1. Cells were then centrifuged at 280g at 4 °C for 10 min, the supernatant was removed and cells were resuspended in ice-cold FACS buffer (pH 7,4; 0.1 M PBS; 1 mM EDTA; 1% BSA). Cells were stained for extracellular marker with antibodies to CD45-PacificBlue (BD Bioscience), CD45-phycoerythrin (PE)-Cy7 or eFluor 450 (eBioscience), TCRβ-Alexa780 (eBioscience), CD4-Alexa488 (eBioscience), CD8-peridinin chlorophyll (PerCP)-Cy5.5 (eBioscience), CD44-allophycocyanin (APC) (eBioscience), CD62L-PE (eBioscience), CD71-APC (eBioscience), podoplanin-PE (eBioscience), CD31-Alexa647 (eBioscience), B220-PE (eBioscience), CD19-BB515 (BD Bioscience). Except for the lymphatic endothelial cells identification experiment, all cells were fixed in 1% PFA in 0.1 M pH 7.4 PBS. Fluorescence data were collected with a CyAn ADP High-Performance Flow Cytometer (Dako) or a Gallios (Beckman Coulter) then analysed using FlowJo software (Treestar). To obtain accurate cells counts, single cells were gated using the height, area and the pulse width of the forward and side scatter, then cells were selected for being live cells using the LIVE/DEAD Fixable Dead Cell Stain Kit per the manufacturer’s instructions (Invitrogen). The cells were then gated for the appropriate markers for cell type (Extended Data Figs 3, 9). Experiments were performed on meninges from n = 3 mice per group. Data processing was done with Excel and statistical analysis was performed using GraphPad Prism.

Deep cervical lymph node resection, ligation and sham surgery

Eight-week old mice were anaesthetized with ketamine/xylazine, shaved at the neck and cleaned with iodine and 70% ethanol, and an ophthalmic solution was put on the eyes to prevent drying. An incision was made midline 5 mm superior to the clavicle. The sternocleidomastoid muscle (SCM) was retracted, and the deep cervical lymph node was removed with forceps. For the ligation experiment, the collecting lymphatic vessels anterior to the deep cervical lymph nodes were ligated using a nylon suture (9-0 Ethilon black 6”VAS100-4). Sham-operated mice received the incision and had the SCM retracted, but were not ligated or the lymph nodes were not removed. Mice were then sutured and allowed to recover on a heating pad until responsive. Post-surgery, mice were given analgesic in the drinking water: 50 mg l−1 for 3 days post-surgery and 0.16 mg for the next 2 weeks.