Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA‐E double transgenic pig forelimbs with human blood

Abstract

Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Transgenic Expression of Human CD46 on Porcine Endothelium

“…In contrast, a significantly reduced C5b-9 deposition in transgenic tissue was shown, confirming our previous data on reduction of soluble C5b-9 by transgenic expression of hCD46/HLA-E during xenoperfusion. 9 These data corroborate that the expression of hCD46 indeed inhibits the terminal pathway of complement activation by blocking the central complement proteins C3b and C4b. The pig limbs, which we used for perfusion with human blood, also expressed HLA-E to inhibit NK cell activation.…”

“…Wildtype (n = 8) and GalTKO-heterozygous, hCD46/HLA-E double transgenic (n = 10) forelimbs of large white pigs (30-40 kg) of both sexes were used to perform ex vivo xenoperfusion with heparinized whole human blood as described previously. 9,40 Briefly, 500 mL of whole blood were withdrawn from individual human donors into standard transfusion bags containing 10,000 IU of heparin (Liquemin). The amputated porcine forelimbs were connected to extracorporeal circuits and xenogeneic perfusions were performed for 12 hours.…”

“…We have already shown that the transgenic expression of hCD46 provided protection against complement-mediated responses. 9,40 Our hypothesis for the current study was that the expression of CD46 would also attenuate activation of the coagulation system and prevent the induction of an anti-fibrinolytic state of the endothelium during pig-to-human xenoperfusion.…”

Porcine Extrahepatic Vascular Endothelial Asialoglycoprotein Receptor 1 Mediates Xenogeneic Platelet Phagocytosis In Vitro and in Human-to-Pig Ex Vivo Xenoperfusion

“…Forelimbs of nontransgenic, large white pigs of both sexes, weighing 30 to 40 kg, were used to perform ex vivo perfusion with 500 mL of whole, heparinized (Liquemin, 10,000 IU/ 500 mL) anticoagulated human blood (xenoperfusion) or autologous blood (autologous perfusion), for up to 12 hours as described by Bongoni et al 22 Biopsies and blood samples were collected at regular time intervals and blood cell counts performed using an automated hematology analyzer (Sysmex KX21N; Sysmex Suisse AG, Horgen, Switzerland) to measure changes in platelet counts.…”

“…In contrast, little is known on platelet phagocytosis mediated by other endothelial sites. Here, we report that ex vivo xenoperfusion of amputated porcine forelimbs with human blood using extracorporeal perfusion 22 leads to an immediate loss of human platelets from circulation. We therefore examined ASGR1 expression on porcine vascular endothelial cells (EC) including aortic EC (PAEC), femoral arterial (PFAEC), and PLSEC.…”