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Vascular endothelial cells (EC) have been shown to play a critical role in the recruitment of leukocytes into inflammatory sites. In addition, allograft EC express MHC class II molecules in vitro and in viva in acute and chronic rejection. Therefore, it is likely that allograft EC can provide signal 1 to CD4+ T cells as they transmigrate into the allograft. However, It is not known whether EC provide the second costimulatory signal via CD28:B7 interactions that is necessary for full CD4+ T cell activation or whether it is provided "in trans" within the graft. We utilized an in vitro transmigration model in which EC were grown to confluence on 8_m pore Transwell membranes to assess the induction of costimulatory molecules on leukocytes following interactions with EC. PBL (5x106), or purified human monocytes (2x106 ) were added to the upper chamber of transwells and were allowed to transmigrate across unactivated, or TNF_- activated EC. Transmigrated cells were harvested at 6 hrs and were analyzed by semiquantitative RT-PCR for the expression of B7-2. We found that transmigrated PBL or monocytcs had a greater than 2 fold induction of B7-2 mRNA. To confirm that EC induction of leukocyte B7- 2 involves contact-dependent signals, we generated membrane fractions of TNF_-activated EC by hypotonic lysis of cells and centrifugation. Addition of activated EC membranes from 3x10to the sixth EC to monocytcs or CD4+ T cells (2x10to the sixth) for 6 hrs resulted in the induction of B7-2 mRNA and B7-2 protein (by FACS analysis). We next sought to determine whether the B7-2 on infiltrating leukocytes may be functional ("in trans') following partial activation of CD4+ T cells by allogeneic-EC. EC were treated with IFN-y (1000_/ml) for 48hrs to Induce MHC class II expression and were cocultured with unactivated naive CD4+ T cells and B7-2.transfected CHO cells. B7-1 or mock transfected cells were used as a control. EC were irradiated and CHO cells were treated with paraformaldehyde to inhibit proliferation. B7-2. and B7-1.transfected CHO cells but not mock transfected cells enhanced allogeneic-EC mediated CD4+ T cell proliferation and IL2 production in a dose dependent manner that is inhibited by CTLA-Ig. Thus, EC may provide signal 1 to CD4+ T cell and in the absence of B7-2 costimulation, they may promote effective costimulation indirectly via the induction of B7-2 on neighboring transmigrating leukocytes. We conclude that allogeneic EC arc unique in that they 1) provide signal 1 to transmigrating CD4+ T cells, and 2) modify the phenotype of graft infiltrating leukocytes to provide signal 2 "in trans". Thus, EC may promote persistence of local intragraft CD4+ T cell activation responses.