Expression of Human Ecto-5′-Nucleotidase in Pig Endothelium Increases Adenosine Production and Protects from NK Cell-Mediated Lysis


*Corresponding author: Ryszard T. Smolenski,


Ecto-5′-nucleotidase (E5′N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5′N on pig endothelial cells (EC) attenuates human NK cell-mediated cytotoxicity. A pig EC line was stably transfected with human E5′N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5′N activity in pig EC lysates increased from 0.68 ± 0.07 to 1013 ± 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 ± 0.05 to >300 nmol/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. 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 the addition of adenosine, demonstrating the key role of adenosine produced by E5′N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5′N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xenotransplantation.