Human NK cells can lyse porcine endothelial cells independent of their expression of Galalpha(1,3)-Gal and killing is enhanced by activation of either effector or target cells.

:  Background:  Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galα(1,3)-Gal (αGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of αGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory. Methods:  We investigated the recognition of αGal by human NK cells using endo-β-galactosidase C, an enzyme that cleaves αGal, and endothelial cells (EC) from α1,3-galactosyltransferase null pigs that do not synthesize αGal. Endo-β-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. Results:  Removal of αGal from porcine EC using endo-β-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from αGal−/− pigs were marginally, and not significantly, less susceptible to lysis by naive human NK cells compared with αGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P > 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-α, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on αGal−/− cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-α stimulation, that could explain the differential susceptibility to lysis. Strikingly, all αGal−/− and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2. Conclusions:  These findings demonstrate that human NK cells can kill porcine targets in the absence of αGal, and donor variability plays a major role in whether αGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of αGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of αGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.