PSL2 is an adhesion receptor.

(A) Activated T cells expressing PSGL1 and/or PSL2. P-selectin binding profiles of activated T cells generated using donor/recipient combinations specified expressing PSGL1 and PSL2, PSL2 alone, or neither. Staining with P-selectin was performed on untreated cells or on cells after EDTA pre-washing revealing the independent contributions of PSGL1 (EDTA-resistant) and PSL2 (EDTA-strippable). Independent triplicate staining profiles are shown. Fig 4A is representative of five independent analyses. (B) Activated platelets expressing P-selectin can bind activated T cells via PSGL1 and/or PSL2. Platelets from P-selectin+/+ and P-selectin-/- mice were isolated, labeled with anti-CD41-APC, activated with thrombin, paraformaldehyde fixed, and washed. Cells shown in (A) were then assessed for capacity to bind CD41-APC labeled platelets by flow cytometry. Free platelets were excluded from analysis by forward/side light scatter characteristics. Platelets from P-selectin-/- mice were assayed in parallel to resolve P-selectin dependent platelet adhesion to responding T cells. Expected P-selectin ligand displayed in parentheses. Mean and standard deviation of percentage CD41-APC+ (platelet bound) cells indicated for P-selectin+/+ platelets and P-selectin-/- platelets. P-values for differences in P-selectin+/+ platelet binding determined by Student’s T test, with confidence levels reaching 2, 3 or 4 SD as indicated by **,***,**** respectively. Fig 4B is representative five independent analyses. (C) PSL2 supports adhesive interaction with immobilized P-selectin. Tracking dye-labeled donor T cells responding in OT1→ PSGLnull, OT1→ C2GnT1null, OT1 C2GnT1null→ PSGLnull, and OT1 C2GnT1null → C2GnT1null adoptive transfers yielded activated cells expressing PSGL1 and PSL2, PSGL1, PSL2, or neither ligand respectively. These cells were compared for adhesion to P-selectin immobilized on V-bottom wells under shear generated by centrifugation. Wells were coated with blocking agent alone (no P-selectin) or with PSel-hIgG chimera (immobilized P-selectin). Cells were also centrifuged in wells pre-coated with P-selectin but in the presence of EDTA to prevent selectin mediated binding (immobilized P-selectin + EDTA). Non-adherent cells pelleting to the nadir were harvested directly and cells adhering to the tapered well sides were subsequently harvested using EDTA elution (Adherent). Donor derived activated T cells in non-adherent vs adherent fractions were quantified by flow cytometry-based counting and each normalized to input cell numbers. Triplicate counts of pooled quadruplicate samples were used to generate mean and standard deviation values of cell yields as shown. The significance of differences in cell yields were determined by applying two-tailed Student’s T-tests with equal variance. P-values reaching 1–5 sigma levels of significance were indicated by *, **, ***, ****, ***** respectively. The results shown are representative of four independent analyses.