302 INHIBITION OF HUMAN NATURAL KILLER (NK) CELL-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS K5 PROTEIN

Abstract

Human natural killer (NK) cell-mediated response plays an important role in xenograft rejection. In the case of pig-to-human xenotransplantation, it has been suggested that NK cells are involved in delayed-type rejection, which is characterized by pig endothelial cell activation, direct lysis, and secretion of proinflammatory cytokines. Natural killer cell activation can be a direct barrier to the potential use of pig organs for human xenograft transplantation. Therefore, it is important to suppress NK cell activity on pig-to-human xenografts. Expression of Kaposi's sarcoma-associated herpesvirus (KSHV)-K5 molecules inhibits the cytotoxic activity of NK-activating receptor (B7-2, ICAM-1). As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. In this study, we produced cell lines expressing K5 to control NK-mediated cytotoxicity in minipig cells. We transfected the K5 gene into minipig fetal fibroblasts and established 2 transgenic clonal cell lines. Presence of the K5 gene was confirmed by PCR, and expression of the gene was identified by real-time PCR and flow cytometry. In an NK cytotoxicity assay, the rate of NK-92MI-mediated cytotoxicity was significantly reduced, to 48.4 ± 5.9% compared with the control (75.6 ± 5.8%; P < 0.05, n = 8, paired t-testing). In conclusion, these results indicate that the expression of K5 molecules on porcine cells can efficiently control NK-mediated cytotoxicity. This strategy can be used in transgenic pig production in which porcine organs would be protected from NK-mediated rejection.