Abstract 1823: Attenuation of Platelet and NK-Cell Mediated Xenograft Rejection by Expressing Human Ecto-5′-Nucleotidase in Pig Endothelial Cells
Xenotransplantation is one possible solution to circumvent the shortage of human hearts for allotransplantation. Pigs are considered as optimal candidates and major obstacle - hyperacute rejection has been partially resolved. However, acute vascular rejection mediated by immune and haemostatic mechanisms involving antibodies, natural killer (NK) cells and monocytes remains unresolved. Ecto-5′-nucleotidase (E5′N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides that triggers thrombosis and immune response into adenosine that oppose these efects. We evaluated whether expression of human E5′N on pig endothelial cells (EC) attenuates inhibit platelet aggregation/adhesion, human NK cell mediated as well as antibody/complement mediated cytotoxicity. A pig EC line was stably transfected with human E5′N and adhesion and cytotoxicity towards pig EC was measured by flow cytometry and intracellular enzyme release. E5′N activity in pig EC lysates increased from 0.68±0.07 to 1013±293nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37±0.05 to >300nmol/min/mg protein in non-transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Platelet aggregation was reduced up to 80% by adding supernatants of cells transfected with E5′N to human platelet reach plasma. Adhesion of fluorescently labelled human platelets to E5′N transfected pig endothelial cells was reduced to 39±9% of the adhesion observed in non-transfected cells. Both effects on platelet function were abolished by the E5′N inhibitor. Cytotoxicity of human NK cells was reduced from 10.7±0.4% and 11.1±1.1% with non-transfected pig EC to 5.2±0.2% and 5.0±0.2% in transfected cells with 50 μM and 250 μM AMP respectively. Reduction of cytotoxicity in E5′N-transfected EC was abolished by the E5′N inhibitor and was mimicked in non-transfected EC by adenosine. However, antibody mediated cytotoxicity was not attenuated by E5′N expression. We suggest that over-expression of E5′N in EC of transgenic pigs is a possible strategy to ameliorate platelet and NK-cell mediated rejection after xenotransplantation.