Functional Evaluation of hCD46 and HLA-E Transgenes in an Ex Vivo Pig-to-Human Xenoperfusion Model.

Abstract

Background: Human (h)CD46/HLA-E double transgenic pigs were tested for prevention of complement- and NK cell-mediated xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, ASGR1 expression on porcine aortic endothelial cells (PAEC) as well as PAEC/ASGR1 mediated xenogeneic platelet phagocytosis was examined in vitro. Methods: hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for complement deposition, NK infiltration and endothelial activation. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. Results: No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL-1b, and IL-8 as well as human C3a, C5a and soluble C5b-9 were significantly lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, C6 and fibrin deposition as well as E-selectin, VCAM-1, tissue factor and PAI-1 expression were significantly higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell (NKp46) tissue infiltration. A rapid decrease of platelets was observed in all xenoperfusions. By blocking ASGR1 with anti-ASGR1 antibody and ASGR1 ligands, human platelet phagocytosis by PAEC was significantly reduced in vitro and suggesting that ASGR1 molecule is involved in xenogenic human platelet phagocytosis. Conclusions: Transgenic hCD46/HLA-E expression clearly reduced complement- and NK mediated humoral xenoresponses. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human.