Effect of Transgenic hCD46 Expression During Ex Vivo Xenoperfusion of hCD46/HLA-E Double Transgenic Pig Limbs with Human Blood

Abstract

Background: To overcome rejection of pig-to-human xenografts, a new founder line with overexpression of human CD46 and HLA-E was analyzed in ex vivo perfusion experiments with respect to complement activation and cytokine responses. Methods: Six forelimbs (2 wild type and 4 hCD46/HLA-E transgenic pigs) were ex vivo perfused with whole, heparin-anticoagulated human blood. Blood samples were analyzed for production of porcine and/or human (p/h) cytokines and complement activation. Total complement activity was assessed by CH50 assay. Tissue samples were stained for deposition of human IgM, MBL, C3b/c, C4b/c, C6 and co-localization of MBL with IgM and C4b/c to determine the role of the lectin pathway of complement activation in xenotransplantation. Results: No hyperacute rejection was observed in this model and extremity perfusions lasted for up to 18 h, irrespective of the presence of the transgenes. Serum levels of p/h cytokines IL1b, IL-6, IL-8, IL-10, as well as sC5b-9 and sC4d were significantly lower in blood perfused through transgenic as compared to wild type limbs. CH50 results revealed early (1h) decrease of complement activity in all limb perfusions. Deposition of hIgM, MBL, C3b/c, C4b/c, and C6 were observed in tissue of wild type limbs, but no C6 deposition was found in transgenic limbs. Co-localization of MBL with IgM and C4b/c was also observed. Conclusion: The terminal pathway of complement activation was down-regulated by transgenic expression of CD46 and was associated with decreased cytokine production. In addition, deposition of MBL and it's co-localization with IgM and C4b/c suggested a role of antibodydependent activation of the lectin pathway of complement activation in xenotransplantation. Overall, this ex vivo perfusion system represents a powerful tool to study the basic mechanisms occurring in the setting of vascularized xenotransplantation and to evaluate the protection from early rejection mechanisms in tissues originating from transgenic pigs.