VALIDATION OF THE MOUSE-TO-RABBIT COMBINATION AS A MODEL FOR PIG-TO-PRIMATE XENOTRANSPLANTATION

Abstract

110 Hyperacute rejection (HAR) of porcine organs transplanted into primates is initiated by binding of preformed natural anti-porcine antibodies to the vascular endothelium of the graft and activation of the classical pathway of complement (C). Yet, in the small animal models currently employed, HAR is initiated mainly by activation of the alternative C pathway. Therefore it would be useful to develop a small animal model of more clinical relevance that reproduced the rejection process that takes place in pig-to-primate xenotransplantation. With this aim, we performed mouse-to-rabbit heterotopic cardiac transplantation to study graft survival time, histology and immunopathology of the rejected hearts. We then investigated, in vitro, the activation mechanisms that are triggered when mouse red cells are incubated with rabbit serum as a source of antibody and C. We found a mean graft survival of 37 ± 9.6 min (n=10) for mouse-to-weanling rabbit transplants and 40 ± 11.1 min (n=5) for mouse-to-adult rabbit transplants. Histology of rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage and myocyte necrosis consistent with HAR. Immunofluorescence showed moderate diffuse deposition of IgG and dense deposition of IgM and C3 on the vascular endothelium of the graft. To determine whether HAR in this combination is C-mediated, we performed 2 mouse-to-C6-deficient rabbit transplants. The first graft was rejected in 21 hr and the second recipient died with a functioning graft after 6.5 hr. Histology of the first graft showed severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate of neutrophils and some macrophages, while that of the second graft showed normal myocytes and vasculature, with minimal spotty interstitial hemorrhage. This markedly delayed rejection in C6-deficient rabbits implies that the membrane attack complex of C plays a major role in the pathogenesis of HAR in this model. To establish the role of natural antibodies and the pathway of C activation, we showed that rabbit serum 1) contains high titers of cytotoxic antibodies against mouse red cells (62 ± 11.7 units), 2) has strong classical pathway activity when tested with antibody-sensitized mouse red cells (49.6 ± 19.3 units, similar to that of human serum), and 3) has no significant alternative pathway activity against mouse red cells (in contrast to human serum which had 23.7 ± 8.5 units). Thus the cytotoxicity of rabbit serum for mouse red cells is mediated by natural antibodies and activation of the classical C pathway. In conclusion, these studies suggest that HAR in the mouse-to-rabbit combination is mediated by natural antibodies that trigger activation of the classical C pathway. Thus, the pathogenesis of HAR in this model is similar to that seen in pig-to-primate combinations. This model has potential for performing clinically relevant studies at low cost with animals that are readily accessible and easy to maintain.