Resistance of Gas6^−/− endothelial cells to TNF-α activation prevented donor T-lymphocyte transmigration. (A) Expression of E-selectin by activated endothelial cells (ECs). Primary ECs isolated from WT and Gas6^−/− mice were stimulated by murine TNF-α (100 ng/mL) in 96-well plates. E-selectin expresion was determined at the cell surface by enzyme-linked immunosorbent assay. Data are mean ± SEM; n = 10. ***P < .001 (analysis of variance). (B) Transmigration experiments were performed in transwells with gelatin-coated membrane inserts covered with ECs isolated from WT or Gas6^−/− mice. ECs were then stimulated or not for 4 hours with murine TNF-α (100 ng/mL). WT splenic mononuclear cells were placed in the upper transwell chamber in the presence of 15 ng/mL TNF-α. The number of cells in the upper and lower transwell chambers was assessed by a cell counter. Data are percentage of transmigrated cells (mean ± SEM, n = 3). *P < .05 (analysis of variance).