Synergistic Effects on the Inhibition of Human CD8+ Cytotoxic T Lymphocytes-Mediated Killing Against Xenograft Cells by Coexpression of Membrane-Bound Human FasL and Decoy Fas Antigen

Abstract: The principal barrier to the use of pigs as donors to humans is hyperacute rejection mediated by the interaction of α-gal abundantly expressed on pig cells and the natural anti-Gal antibody, abundantly produced in humans. This antibody-mediated hyperacute rejection may be overcome by an α1, 3 galactosyltransferase gene-knockout pig. However, xenograft cells could be rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenografted cells. We previously demonstrated that the Fas/FasL pathway is a major contributor to CD8+ CTL function. Furthermore, we sought to prevent this cytotoxicity by overexpression of membrane-bound FasL carrying the deletion at the metalloproteinase cleavage site or by decoy Fas antigen that does not contain the death domain in its cytoplasmic region. To investigate the effects of coexpression of these molecules, we cotransfected both genes into swine endothelial cells (SEC). The double-overexpression effectively prevented CD8+ CTL-mediated killing. Although cotransfectants and single transfectants of either membrane-bound FasL or decoy Fas gene showed similar inhibition of cytotoxicity, the expression levels of decoy Fas in SEC cotransfectants were much lower than those of decoy Fas single transfectants. These data suggest that beneficial effects for prevention of CTL-mediated xenocytotoxicity may be produced by the double expression of these molecules. The overexpression of both molecules on xenografted cells may decrease the innate cellular response to xenografts creating a window of opportunity to facilitate xenograft survival. [Copyright &y& Elsevier]