In vitro and in vivo prevention of human CD8+ CTL-mediated xenocytotoxicity by pig c-FLIP expression in porcine endothelial cells

Abstract

Overcoming cell-mediated immunity, especially of human CD8+ CTLs, is important for the success of xenotransplantation. Our group has previously reported that the cytotoxicity of human CD8+ CTLs against pig endothelial cells (PEC) is highly detrimental and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as an 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 cytoprotective effects of c-FLIPS/L in xenograft cells remain controversial. This study demonstrates that the overexpression of c-FLIPS/L genes markedly suppress human CD8 + CTL-mediated xenocytotoxicity and, in addition, the cytoprotective effects of c-FLIPL appear to be significantly stronger than those of c-FLIPS. Furthermore, to prove the prolonged effects of xenograft survival, PEC transfectants with c-FLIPS/L genes were transplanted under rat kidney capsules. Prolonged survival was elicited from FLIP S/L transfectants, whereas parental PEC was completely rejected through day 5, posttransplant. Thus, intracellular remodeling with the overexpression of c-FLIPS/L in xenograft cells may avoid innate cellular attacks against xenografts and facilitate long-term xenograft survival. © 2008 The Authors.