Figure 3: cpLECs capture and present CNS-derived antigen.

(a – c): EAE was induced in CNP-OVA transgenic mice and analyzed on day 15 post-immunization at score 3.0, in which OVA-GFP^{+ fl/fl} expression is driven by the oligodendrocyte specific CNPase-Cre. Immunohistochemistry of layers V/VI of the cortex confirm OVA-GFP expression by CNPase⁺ oligodendrocytes within the CNS when immunolabeled with an anti-GFP antibody. Scale bar = 100 μm

(d – h): Immunolabeling of cpLVs confirm MHC II expression and OVA-GFP⁺ signal by a subset of Podoplanin⁺ cpLECs. Perpendicular yellow lines highlight two orthogonal views (E_i) and (E_ii) along x and y axis indicating colocalization. White dotted line denotes profile intensity of ROI analyzed in (m).

(i – l): 3-D reconstruction of (e – h). Yellow arrowheads indicate MHC II⁺ OVA-GFP⁺ cpLECs. Scale bar = 50 μm

(m): Representative plot profile intensity of cells as indicated by the white line shown in (e) showing 5 representative cells, consisting of MHC II⁺ OVA-GFP⁺ cpLECs (i and ii), MHC II⁺ non-cpLECs (iii and v), and MHC II⁺ OVA-GFP⁺ non-cpLEC (iv).

(n – p): Control immunolabeling of Podoplanin⁺ cpLECs with MHCII and without an anti-GFP primary antibody, confirming that there is no unspecific labeling of GFP with the secondary antibody. Scale bar = 100 μm

(q): The median fluorescence intensity histogram of MHCII of cpLECs.

(r – v): Immunolabeling of cpLVs with Podoplanin and dendritic cells near the cribriform plate with CD11c along with MHC II and OVA-GFP. Yellow arrowheads indicate MHC II⁺ OVA-GFP⁺ CD11c⁺ dendritic cells in contact with Podoplanin⁺ cpLECs. Scale bar = 100 μm (r), 50 μm (s – v)

(w – y_ii): Immunolabeling of cpLVs with Lyve-1 and CD4⁺ T cells near the cribriform plate with MHC II and OVA-GFP. Yellow arrowheads indicate CD4⁺ T cells in contact with MHC II⁺ OVA-GFP⁺ Lyve-1⁺ cpLECs. Scale bar = 100 μm (w), 50 μm (x_i – y_ii)