Characterization of the 4C8 antigen involved in transendothelial migrationof CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers

In extravasation of T cells, little is known about the mechanisms of transe ndothelial migration subsequent to the T cells' tight adhesion to endotheli um. To investigate these mechanisms, we developed a monoclonal antibody (mA b), termed anti-4C8, that blocks transmigration but not adhesion in a cultu re system in which high CD26-expressing (CD26(hi)) T cells preferentially m igrate through human umbilical vein endothelial cell (HUVEC) monolayers cul tured on collagen gels. Anti-4C8 reacted with all CD3(+) T cells and monocy tes but not neutrophils or HUVECs. The structure defined by this antibody w as an 80-kD molecule. The mAb at 1 mu g/ml inhibited 80-90% of migration of CD3(+) T cells through unstimulated and interferon gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of and-4C8. Anti-4C8 exhibited agonistic effects on restin g T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels , the antibody promoted chemokinetic migration of the cells in a dose-depen dent manner from 0.1 to 10 mu g/ml. The predominant population of T cells t hat migrated into collagen gels with impregnated anti-4C8 were CD26(hi). Se cond, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the s ubstrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin conten t preferentially in CD26(hi) T cells when added to T cells at a high dose o f 10 mu g/ml. Finally, both anti-4C8-induced chemokinetic migration and tra nsendothelial migration were inhibited by pretreatment of T cells with pert ussis toxin. These findings suggest that stimulation via the 4C8 antigen in creases cell motility of CD26(hi) cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be inv olved in preferential transmigration of CD26(hi) cells adherent to HUVECs.