Pig Cellular FLICE-like Inhibitory Protein (c-FLIP) Overexpression in Pig Xenograft Cells Induces Resistance to Human CD8+ Cytotoxic T Lymphocyte-Mediated Xenocytotoxicity

Although the use of organs from α1,3-galactosyltransferase gene knockout pigs may prolong xenograft survival, resulting in overcoming antibody-mediated hyperacute rejection, pig xenografts will be destroyed directly by cell-mediated immunity, such as NK cells, macrophages, and CD8+ cytotoxic T lymphocytes (CTLs). Therefore, conquering cell-mediated immunity, especially of human CD8+ CTLs, is of particular importance to the success of long-term xenograft survival. We have previously reported that the cytotoxicity of human CD8+ CTLs is strong against pig endothelial cells (PEC) and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as a potent inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIPS) and a long, 55-kDa form (c-FLIPL). The present study demonstrated that overexpression of c-FLIPS/L genes in PEC markedly suppressed human CD8+ CTL-mediated xenocytotoxicity; moreover, the cytoprotective effects of c-FLIPL appeared to be significantly stronger than those of c-FLIPS. Furthermore, to prove the in vivo prolongation effects of xenograft survival, we transplanted PEC transfectants with c-FLIPS/L genes under the rat kidney capsule. Prolonged survival was displayed by xenografts of FLIPS/L PEC transfectants, whereas xenografts of parental PEC were completely rejected by day 5 posttransplantation. Thus, intracellular blocking of death receptor-mediated apoptotic signals by overexpression of c-FLIPS/L in xenograft cells may prevent innate cellular attacks against xenografts opening the window of opportunity for long-term xenograft survival.