Background/Significance/Rationale: 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 which is a promising solution to the organ shortage. Despite these advancements, acute rejection remains as 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 (i) co-express HLA class I molecules in GM pig endothelial cell line null for five key xenoantigens (5GKO), and (ii) 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.HLA-G) were generated by transfection. IL-2-activated human peripheral blood mononuclear cells (PBMC) from five 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. Statistical analyses performed by one-way ANOVA and post-hoc Tukey test.
Results/Findings: We successfully co-expressed HLA-G, HLA-E, and HLA-G+E in 5GKO cells (Fig.1A). Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared to 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G (Fig.1B).
Conclusions/Discussion: 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.
Translational/Human Health Impact: As xenotransplantation continues to progress towards human clinical trials, identifying the optimal genetic modifications to produce immune compatible xenografts becomes increasingly important. This novel finding will be an important consideration in future xenotransplantation research and brings us one step closer to solving the organ shortage.