A Study of Soluble HLA-G1 Protecting Porcine Endothelial Cells Against Human Natural Killer Cell-Mediated Cytotoxicity

Abstract Human natural killer (NK) cells, which can mediate direct lysis of porcine endothelial cells, play an important role in xenograft rejection. HLA-G, which is a critical molecule in maintaining maternal immune tolerance of semi-allogenic fetus, is able to protect susceptible target cells from lysis induced by NK cells. In this study, we investigated whether soluble HLA-G 1 (sHLA-G 1 ) protected porcine xenogeneic cells against human NK cell-mediated lysis. Methods The human sHLA-G 1 genomic DNA (pcDNA3-sHLA-G 1 ) was transfected into a B lymphoblastoid cell line 721.221 (LCL721.221) by nucleofector. The sHLA-G 1 expression of the transfected LCL721.221 cells was identified by RT-PCR and Dot-ELISA. The sHLA-G 1 protein was purified by affinity chromatography on anti–HLA-ImAb W6/32 coupled to cyanogen-bromide–activated Sepharose 4B from culture supernates of transfectants. Various concentrations of sHLA-G 1 protein (0, 2, 4, 6, or 8 μg/mL) were added to a NK cell-mediated xenogenic cell lysis system with either NK92 cells or fresh human peripheral blood mononuclear cells (PBMCs) cocultured with the porcine endothelial cells line. A LDH release assay was used to evaluate NK cell-mediated cytotoxicity. Results sHLA-G 1 provided significant protection of porcine endothelial cells against human NK-mediated cytotoxicity in a dose-dependent manner. The rates of NK92 cell-mediated cytotoxicity were reduced to 83.4 ± 5.7% (2 μg/mL), 56.6 ± 9.3% (4 μg/mL), 39.3 ± 10.2% (6 μg/mL), and 31.2 ± 4.9% (8 μg/mL) versus 96.9 ± 3.0% in the control group ( P n = 4) to 5.8 ± 1.6% from 23.9 ± 1.3% in the control group ( P Conclusions These results indicated that sHLA-G 1 protected xenogeneic porcine endothelial cells against attack by human NK cells, thus providing a new approach to overcome NK-mediated immunity to xenografts.