Adenovirus-Mediated Gene Expression of the Human c-FLIP_L Gene Protects Pig Islets Against Human CD8⁺ Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP_S/L genes, which are potent inhibitors of death receptor-mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP_L seem to be significantly stronger than those of c-FLIP_S. Accordingly, in the present study, expression of c-FLIP_L was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% ± 15.9% and 64.0% ± 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP_L gene were markedly protected from lysis (30.5% ± 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP_L with adenovirus. Pig islets expressing c-FLIP_L are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.