Multiple genetically modified GTKO/hCD46/HLA-E/h2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood

BackgroundIn pig-to-human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA-E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA-E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. MethodsCardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA-E/h2-microglobulin) and wild-type (wt) controls (n=6 each) were reperfused and tested in an 8hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165-minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. ResultsDeposition of IgG (1.21x10(7) vs 8.8 +/- 2.9x10(6); P<.01), IgM (4.4 +/- 3.7x10(6) vs 1.7 +/- 1.2x10(6); P<.01), and the complement activation product C4b/c (3.5 +/- 1.3x10(6) vs 2.3x10(6)+/- 9.4x10(5); P>.01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94 +/- 0.77% to 0.21 +/- 0.25% (P=.04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480minutes of perfusion was lower than in wt organs (2.5 +/- 3.7x10(4)/mm(3) vs 1.3 +/- 1.4x10(5)/mm(3); P=.0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2hours of ex vivo perfusion. ConclusionGTKO, hCD46, and HLA-E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human-anti-pig rejection reactions and improve myocardial function after xenotransplantation.