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

Abstract

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 naïve 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.

Multiple Receptors Trigger Human NK Cell-Mediated Cytotoxicity against Porcine Chondrocytes

“…This finding was consistent with previous observations made with pig endothelial cells treated with hTNF-a (22). Adhesion certainly contributed to this effect, as it paralleled well the level of cytotoxicity observed under the multiple conditions tested.…”

“…Furthermore, human NK cells can adhere and lyse porcine endothelial cells both directly and by Ab-dependent cell-mediated cytotoxicity (ADCC) in the presence of human serum (20). The aGal Ag is involved in both of these mechanisms because it is a main target of xenogeneic natural Abs (XNA) (20) and can contribute to the direct triggering of natural cytotoxicity against pig cells (21,22). However, the aGal Ag does not seem to be key in the direct activation of human NK cells (20,22) compared with other porcine ligands recognized by human NK cells.…”

“…The aGal Ag is involved in both of these mechanisms because it is a main target of xenogeneic natural Abs (XNA) (20) and can contribute to the direct triggering of natural cytotoxicity against pig cells (21,22). However, the aGal Ag does not seem to be key in the direct activation of human NK cells (20,22) compared with other porcine ligands recognized by human NK cells. In particular, it has been recently described that cytotoxicity against porcine endothelial cells is triggered by the NK receptors NKG2D and NKp44, whereas other activating receptors such as NKp30 and NKp46 have not been implicated in this setting (16).…”

Immune responses to α1,3 galactosyltransferase knockout pigs

“…Some studies demonstrated that direct recognition of Gal by human NK cells was able to induce cytotoxicity [47][48][49]. In contrast, other studies found no role of Gal in direct NK cytotoxicity [12,13,[50][51][52][53], whereas NK cytotoxicity via ADCC is largely dependent on the presence of Gal [13]. The only available data on human anti-pig T cell response against GalT-KO was presented as an abstract and showed a somewhat weaker response than against wildtype pig PBMC, suggesting that the GalT-KO pig may provide advantages with regard to both humoral and cellular immunity [54].…”

Immunological Challenges and Therapies in Xenotransplantation

“…Ligands on porcine cells, one of which has recently been identified (Lilienfeld et al 2006), can interact efficiently with the activating receptors NKp44 or NKG2D, resulting in direct human NK cell cytotoxicity (Forte et al 2005). Interaction between the porcine costimulatory molecule CD86 and a variant form of CD28 on hNK cells may also be involved in direct NK cytotoxicity (Costa et al 2002), whereas the expression of aGal residues on pig endothelium does not appear to have a major role, because the use of cells lacking aGal expression did not prevent direct NK-mediated cell lysis (Baumann et al 2004;Horvath-Arcidiacono et al 2006). Second, it has also been shown that, on activation by porcine endothelial cells, NK cells produce INF-g, ultimately potentiating T-cell activity (Xu et al 2002).…”