Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2

Circulating lymphocytes continuously enter lymph nodes (LNs) for immune surveillance through specialised blood vessels named high endothelial venules (HEVs)(1–5), a process that increases dramatically during immune responses. How HEVs permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here, we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α)(6–8) in maintaining HEV barrier function. Mice with postnatal deletion of PDPN lost HEV integrity and exhibited spontaneous bleeding in mucosal LNs, and bleeding in the draining peripheral LN after immunisation. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells (FRCs)(7), which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2)(9,10). Mice lacking FRC PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (VE-cad), which is essential for overall vascular integrity(11,12), on HEVs. Infusion of wild-type (WT) platelets restored HEV integrity in CLEC-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate (S1P)(13,14) from platelets, which promoted expression of VE-cad on HEVs ex vivo. Furthermore, draining peripheral LNs of immunised mice lacking S1P had impaired HEV integrity similar to PDPN- and CLEC-2-deficient mice. These data demonstrate that local S1P release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.