Production of Minipig's Cells Harboring HLA-G1, hDAF, and CD59 Genes.

Abstract

Hyperacute and cellular rejection in xenotransplantation is caused by activation of complement and human natural killer cells (NK cells) on endothelium. The combination of human leucocyte antigen-G1 (HLA-G1), decay accelerating factor (DAF) and CD59 could be an effective strategy to overcome host complement and NK cell-mediated rejection in xenotransplantation. In this study, we established the HLA-G1, hDAF and CD59 combination cell lines by transfection of hDAF and CD59 gene into ear cells obtained from the transgenic cloned minipig harboring HLA-G1 gene. Presence and expression of HLA-G1, hDAF and CD59 gene were confirmed by PCR, FACS and Immunocytochemistry, respectively. Especially, the cytotoxic inhibition was assessed by NK cytotoxicity assay and LDH assay. These result showed that expression of these genes directly inhibited the complement cascade reaction and NK cytotoxicity against clonal cell lines. In our experiment, the rate of the LDH cytotoxicity was significantly reduced to 40.8 ± 12.1% comparing to the control (84.6 ± 11.2%, p < .05). The rate of NK-92MI cytotoxicity was significantly reduced to 45.5 ± 11.0% comparing to the control (85.3 ± 10.5%, p < .01). In conclusion, these results indicate that the expression of HLA-G1, hDAF and CD59 genes on porcine cells can effectively inhibit complement and NK cell mediated cytotoxicity.