Antigens in the posterior eye elicit immune responses in the brain.

a, Schematic of the schedule of procedures for the experiments described below. b, Wild-type C57BL/6J mice were immunized using heat-inactivated HSV-2 injection through i.p., i.c., AC and IVT administration. Survival was monitored after i.c. challenge with a lethal dose of HSV-2 30 days later (naive, n = 18; i.p., n = 12; i.c., n = 6; AC, n = 6; IVT, n = 18). c, dCLNs of mice were ligated using a cauterizer. Seven days later, mice were injected through the IVT route with heat-inactivated HSV-2. Their survival was monitored after i.c. challenge with a lethal dose of HSV-2 30 days later (naive, n = 5; IVT immunized, n = 5; LN ligation, n = 6). d, Schematic of the parabiosis mouse model and treatment plans. e, Mice were injected through the IVT route with heat-inactivated HSV-2. Four weeks later, the immunized mice were joined to naive mice. The immunized mice or naive mice were challenged through the i.c. route with a lethal dose of HSV-2 after 3 weeks, and their survival was monitored (naive, n = 4; IVT, n = 4; IVT–naive (IVT challenge), n = 6; IVT–naive (naive challenge), n = 2; naive–naive (naive challenge), n = 2). f, Anti-HSV-specific antibody was measured by enzyme-linked immunosorbent assay after different routes of HSV-2 immunization (i.p., n = 12; i.c., n = 6; AC, n = 10; IVT, n = 10). Data are shown as mean ± s.e.m. g, Wild-type C57BL/6J mice were injected with heat-inactivated HSV-1 through i.p., i.c., AC or IVT administration. Their survival was monitored after i.c. challenge with a lethal dose of HSV-1 30 days later (naive, n = 15; i.p., n = 6; i.c., n = 6; AC, n = 6; IVT, n = 18). h, As in a, but S. pneumoniae strain TIGR4 was used (naive, n = 8; i.p., n = 5; i.c., n = 5; AC, n = 8; IVT, n = 8). i, Mice were inoculated through the i.c. route with 50,000 GL261 luciferase-expressing (GL261–Luc) brain tumour cells, treated with irradiated GL261–Luc cells through s.c., i.c., AC or IVT administration (day 7) along with anti-PD1 (RMP1-14) antibodies (days 7, 9 and 11) and monitored for survival (naive, n = 6; s.c., n = 6; i.c., n = 6; AC, n = 6; IVT, n = 12). Data are representative of two independent experiments. The graphics in a,d were created with BioRender.com.

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Eyes have a compartmentalized lymphatic drainage system.

a, Schematic of AC and IVT injection of dye. b, C57BL/6J mice were injected with dye through the AC or IVT route. The percentage of dye retention in the eye was analysed from 6 h to day 5 post injection (AC, n = 6; IVT, n = 6). c, Dye was injected into the eye through the AC or IVT route. sCLNs and dCLNs were collected and measured using a fluorescence plate reader 1 h after injection. RFU, relative fluorescence unit (AC, n = 8; IVT, n = 8). Data are shown as mean ± s.e.m. ***P = 0.0002, AC sCLN; *P = 0.0167, IVT sCLN; ***P = 0.0009, IVT dCLN. d, Dye was injected into the left eye through the AC or IVT route, and eyes, sCLNs and dCLNs were collected for IVIS epifluorescence imaging. Representative background-subtracted heat maps of dye in the eye, sCLNs and dCLNs 1 h after injection are shown. e, Schematic of the anatomical locations of the sCLNs and dCLNs. f,g, sCLNs, dCLNs or both CLNs were surgically ligated. Two days later, dye was injected into the eye through the AC (f) or IVT (g) route. The percentage of dye retention in the eye was measured 12 h later. Data are shown as mean ± s.e.m. in f and g (sham AC, n = 3; n = 4 in all other conditions). ****P < 0.0001; ***P = 0.0002, IVT sCLN; ***P = 0.0003, IVT dCLN; NS, not significant. P values were calculated using a one-way analysis of variance (ANOVA) with multiple comparisons testing (Dunnett). The graphics in a,e were created with BioRender.com.

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Optic nerve sheath lymphatics drain the posterior eye.

a,b, Immunostaining of sections of optic nerve from zebrafish with lymphatics labelled for Mrc1a (white arrows; a, top left) and iDISCO immunolabelling of meningeal lymphatic vessels of rabbit (a, top right), pig (a, bottom left), non-human primate (a, bottom right) and human (b) optic nerve and chiasma, with lymphatics showing colocalization of LYVE1 and VEGFR3 (white arrows). DAPI, 4′,6-diamidino-2-phenylindole. c, Whole-mount wild-type mouse optic nerve sheaths stained for LYVE1, CD31, PROX1 and VEGFR3. The images at the bottom show a higher-magnification view of the area highlighted in the merged image at the top right. Scale bars, 50 μm (a, top left; c, bottom merged), 500 μm (a, top right; c, top merged), 1,000 μm (a, bottom left and right) and 3,000 μm (b). d, Schematic of ICM injection and the eye and optic nerve dissection for e. e, Dye was ICM injected, and fluorescence signal intensity was measured 1 h later in the eye and optic nerve (control, n = 11; CSF injection, n = 11). ****P < 0.0001. f, Schematic of injection methods for g. g, VEGFC was injected through the AC, IVT or ICM administration route. Two days later, dye was IVT injected into the eye, and the percentage of dye retention was measured 12 h after dye injection (control, n = 12; AC, n = 19; IVT, n = 20; CSF (2 μg), n = 20; CSF (6 μg), n = 10) ****P < 0.0001; ***P < 0.0002. Data are shown as mean ± s.e.m. P values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett) or two-tailed unpaired Student’s t-test. The graphics in d,f were created with BioRender.com.

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Lymphatic inhibition enables repeat rAAV administration.

a,b, Mice were injected with rAAV-RFP through the IVT or AC route. Their dCLNs, sCLNs and retinas were collected 10 days later, and rAAV-specific immune responses were quantified using an ELISpot assay. For c–g, C57BL/6J mice were IVT injected with rAAV, and, 1 month later, were rechallenged with rAAV-RFP. The efficiency of rAAV-RFP transduction was analysed by imaging 1 month later. c, Schematic of experimental plans. d, In vivo fluorescence fundus imaging to visualize vessels (green) and RFP transduction (red) in different LN ligation conditions. Scale bar, 500 μm. e, Quantification of RFP intensity from d (primary, n = 6; secondary, n = 5; sCLN ligation, n = 5; dCLN ligation, n = 6). **P = 0.0095, primary versus secondary; **P = 0.0044, secondary versus dCLN ligation. f, In vivo fluorescence fundus imaging to visualize vessels (green) and RFP transduction (red) with addition of VEGFC or sVEGFR3. Scale bar, 500 μm. g, Quantification of RFP intensity from f (primary, n = 9; secondary, n = 10; VEGFC, n = 9; sVEGFR3, n = 8). *P = 0.0167, primary versus secondary; *P = 0.0334, secondary versus VEGFC; **P = 0.0096, secondary versus sVEGFR3. P values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett). Data are shown as mean ± s.e.m in b, e and g. The graphics in c were created with BioRender.com.

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