Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity

Background: The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta 2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. Methods: Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca2PO4-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in Cr-51-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. Results: The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/ NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. Conclusions: Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.