Background.Natural killer (NK) cells are major component of the cellular response in xenotransplantation. NK cell activation and NK cell-mediated cytotoxicity can be a direct barrier to the potential use of xenogeneic organs in human transplantation.Methods.To determine if HLA-G would protect porcine xenogeneic cells from human NK cell lysis, human full-length HLA-G genomic DNA was transfected into porcine aortic endothelial cell (PAECs) by the lipofection method. Surface expression of HLA-G in transfected PAECs was confirmed by immunofluorescence staining with anti-HLA class I framework antibody, PA2.6. Fresh human peripheral blood lymphocytes were used as NK effector cells with HLA-G-transfected PAECs as targets in a51Cr release assay. The inhibition of human polyclonal NK cells by HLA-G expression on PAECs was confirmed by antibody blocking using purified F(ab′)2 portion of anti-human HLA class I antibody PA2.6.Results.Expression of HLA-G on PAECs conferred a significant protection against NK-mediated lysis (range: 52-100% inhibition) when peripheral blood lymphocytes from seven healthy donors, bearing either homozygous HLA-Cw3 or -Cw4 used as NK effector cells. Such protection was inhibited by purified F(ab′)2 portion of anti-HLA class I antibody, indicating that the protection of PAECs was directly mediated by HLA-G expression.Conclusions.Expression of HLA-G on PAECs protected xenogeneic PAECs against human polyclonal NK cell-mediated lysis. These results indicate that the expression of HLA-G alone in the absence of other nonclassical HLA class I molecules is sufficient to inhibit human NK cell lysis. These findings suggest methods utilizing HLA-G to overcome NK cell-mediated cytotoxicity against porcine endothelial cells, considered the first cell type effected during xenograft cellular rejection.