Fig. 10: The VE-cadherin GFP reporter mouse allows for visualization of immune cell trafficking across the leptomeninges in vivo during health and neuroinflammation.

Representative images of spinal cord window 2P-IVM imaging of a VE-cadherin-GFP knock-in reporter mouse suffering from a CD8⁺ T-cell-mediated neuroinflammatory disease. On day 7 after viral infection, the mice were injected intravenously with a fluorescently labeled anti-endoglin antibody (white) to highlight the blood vessel lumen (BV). The dura mater is visible in blue due to the second-harmonic generation of the collagen type 1 fibers in the dura. OT-I CD8⁺ T cells are depicted in red. A X–Y MIP of the pia mater. White arrowhead shows an OT I CD8⁺ T cell (red) that has crossed the pia mater, and white arrow highlights one above the pia mater. See also Supplementary Movie 9. Data are representative of ten independent experiments. B, C YZ MIP of the meningeal layers of the spinal cord. The arachnoid mater (A.M.) and pia mater are visible in green due to VE-cadherin-GFP expression. OT I CD8⁺ T cells (red) are visible inside the blood vessel (BV), in the SAS, above and below the pia mater. White asterisk indicates a CD8⁺ T cell in the arachnoid mater. C Zoom-in of the magenta box in B showing the fluorescence signals (top panel) and the segmented surfaces (bottom panel) of the dura mater, arachnoid mater, pia mater, and OT I CD8⁺ T cells. Surfaces were rendered with Imaris 9.8 software. White arrow points to an OT I CD8⁺ T cell above the pia mater, white arrowhead points to an OT I CD8⁺ T cell below the pia mater and asterisks point to OT I CD8⁺ T cells interacting with arachnoid mater. White asterisks indicate CD8⁺ T cells in the arachnoid mater. Data are representative of seven mice imaged in seven different experiments. D, E XY and YZ MIP images of OT I CD8⁺ T cells (red) localized right above (D) or below (E) pia mater (green) in a VE-cadherin-GFP knock-in mouse. See also Supplementary Movie 10. Data are representative of three independent experiments. F Quantification of the number of OT I CD8⁺ T cells observed in different CNS compartments during immune surveillance and neuroinflammation. Data are pooled from three control mice (“immunosurveillance”) and 2 under neuroinflammation and shown as mean values. Source data are provided as a Source Data file. G Brains and spinal cords from ODC-OVA; VE-Cadherin-GFP knock-in mice were harvested on day 7 after induction of autoimmune neuroinflammation. GFAP immunofluorescence staining was performed on 20 μm cryosections. Images show the surface segmentations of the signals of VE-cadherin-GFP at the pia mater (green), glia limitans (purple), and OT I CD8⁺ T cells (red) rendered with Imaris 9.8 software. Original fluorescence images are shown on the right panel. White arrows point to OT-I CD8⁺ T-cell above the pia mater, white arrowhead points to an OT I CD8⁺ T cell below the pia mater. Images are representative of three independent experiments. H Violin plots of the crawling speed (μm/min), displacement, and directionality of OT I CD8⁺ T cells (a total of 30 cells per condition were analyzed in immunosurveillance from a total of 3 VE-cadherin-GFP knock-in mice and 30 cells per condition were analyzed during neuroinflammation from a total of two ODC-OVA; VE-cadherin-GFP knock-in mice). Cell tracking was performed with Imaris 9.8 software. Data were analyzed using two-sided unpaired parametric T test. Source data are provided as a Source Data file.