Co-expression of HLA-E and HLA-G in Porcine Endothelial Cells Attenuates Human Natural Killer Cell-Mediated Cytotoxicity

Abstract

INTRODUCTION: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically-modified (GM) pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute rejection remains a barrier to clinical XTx. Natural killer cells (NK) play a crucial role in transplantation. In the context of XTx, suppressing human NK activity by expressing HLA class I molecules in pig organs may protect xenografts from immune attack. Therefore, we aimed to co-express HLA class I molecules in GM pig endothelial cell line null for 5 key xenoantigens (5GKO) and to evaluate human NK response to 5GKO+HLA class I+ cells. METHODS: DNA sequences encoding HLA-E and HLA-G were designed, synthesized, and cloned into expression vectors. Three GM porcine cell lines (5GKO.HLA-E, 5GKO.HLA-G, 5GKO.HLA-E+G) were generated by transfection. IL-2–activated human peripheral blood mononuclear cells (PBMCs) from 5 donors were co-cultured with GM porcine cell lines to initiate NK cell–mediated immune responses. NK activation (CD107a expression) was evaluated by flow cytometry. Three experimental replicates were averaged and analyzed by one-way ANOVA and a post hoc Tukey test. RESULTS: We successfully co-expressed HLA-G, HLA-E, and HLA-E+G in 5GKO cells. Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared with 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G. CONCLUSION: Co-expression of HLA-E and HLA-G in porcine cells significantly reduces NK cell–mediated cytotoxicity and shows promise for incorporation into GM pigs for pig-to-human xenotransplantation.