Model of P-selectin ligand expression and activity on primary activated CD8+ T cells consistent with prior literature and data.

PSL2 structure: PSL2 association with activated T cells requires calcium and is quantitatively dependent on L-selectin. PSL2 is likely an L-selectin ligand produced by lymph node HEV and bearing 6-O-sulfo sLex, the L-selectin-binding structure shared by all known L-selectin ligands. 6-O-sulfo sLex is also likely present on the exposed, unengaged, face of PSL2 since Meca79 staining correlated with PSL2 expression on both recipient and responding-donor CD8+ T cells (Figs 3F and 4D); Meca79 recognizes 6-O-sulfo sLex on core 1 extensions. P-selectin recognition of the canonical P-selectin ligand PSGL1 requires both sLex and proximal tyrosine sulfation; whether PSL2 bound to L-selectin on T cells also presents sLex/Tyr-SO4 for P-selectin binding or whether the externally exposed 6-O-sulfo sLex detected by Meca79 can substitute as P-selectin ligand is unknown. In either case PSL2 must present (multiple?) selectin ligands on two faces, available for concurrent binding of L-selectin and P-selectin, and most consistent with PSL2 being a mucin-type glycoprotein. It is possible that PSL2 can only serve as a P-selectin ligand after binding/stabilization by L-selectin. PSL2 loading onto activated T cells. PSL2 loading onto CD8+ T cells occurs most clearly after their activation in lymph nodes consistent with available literature that activation is accompanied by enhanced functional affinity of L-selectin for its ligands, perhaps through L-selectin dimerization. L-selectin on activated cells effectively acquires PSL2 by extraction from either resident lymphocytes (Fig 5) or from the fluid phase after its presumed secretion by HEV. Contribution of PSL2 to P-selectin driven signaling and adhesion. Both L-selectin and PSGL1 are thought to co-reside at tips of microvilli. Once bound to L-selectin, sLex glycans on PSL2 may support L-selectin relocation to lipid raft signaling platforms and in proximity to PSGL1. Upon engagement of P-selectin on activated platelets or activated endothelia, the two proximate P-selectin ligands may provide avidity enhancements along with distinct signaling inputs through PSGL1 and L-selectin supporting integrin activation and chemokine responses respectively.