Histopathology of livers and native organs of recipients following genetically-engineered pig-to-baboon liver xenotransplantation

Abstract

In project 1, orthotopic liver transplantation was carried out in baboons using wild-type (WT,n=1) or genetically-engineered (GE) pigs (alpha1,3-galactosyltransferase gene-knockout, GTKO), n=1; GTKO pigs transgenic for human CD46, n=7). Biopsies of GE livers were obtained pre-transplantation, 2h after reperfusion and at necropsy (4 7 days after transplantation). The histopathology of livers and major native organs and lymph nodes were studied by direct light microscopy, immunohistology, and electron microscopy to determine morphological changes in the liver and native organs. In project 2, 12 baboons received carotid artery patch transplants from GE pigs (either GTKO/CD46/CIITA-DN; Group 1,n=8 or GTKO/CD46; Group 2,n=4). One baboon in Group 1 received no immunosuppressive therapy. Group 1A(n=2) received abatacept-based regimen. Groups 1B(n=3) received belatacept-based regimen and Group 1C(n=2) received a regimen based on costimulation blockade of both the CD28/B7 and CD40/CD154 pathways.

Results: In project 1, after GE pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4 7 days due to repeated spontaneous bleeding as a result of profound thrombocytopenia which necessitated euthanasia. At 2h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.

In project 2, when GTKO/CD46/CIITA-DN grafts were transplanted (Group 1), but no immunosuppression was administered, an elicited anti-pig antibody response, a proliferative T cell response on MLR, and intense cell infiltration of the graft were documented. When CD28/B7 pathway blockade was administered (Group 1A), no proliferative response was seen in T cells on MLR, and the elicited antibody response was greatly attenuated. It was only when both pathways were blocked that no elicited antibody response was documented (Groups 1C and 2). In these cases, cell infiltration of the graft was minimal or absent. In recipients of CIITA-DN grafts with both CD28/B7 and CD40/CD154 costimulatory pathway blockades (Group 1C), there was no proliferative response on T cells post-transplantation, elicited antibody response was the lowest. Only blockade of both pathways (Group 1C) prevented T cell proliferative and elicited antibody responses and cell infiltration of the graft. In order to determine whether the CIITA-DN mutation was playing a role, this regimen was administered to four baboons receiving grafts from GTKO/CD46 pigs (Group 2). No proliferative or elicited antibody responses were documented and there was no significant cell infiltration of the graft.

Conclusions: In project 1, after GTKO and GTKO/CD46 pig liver Tx in baboons, the rapid development of a profound thrombocytopenia was by far the major problem seen. The histopathologic features described can largely be explained on this basis. If platelet activation and aggregation/phagocytosis can be prevented, possibly by further genetic modification of the pigs or by novel therapeutic agents, bridging by a pig liver to allotransplantation may become a feasible clinical option. In project 2, blockade of both the CD40/CD154 and CD28/B7 pathways is required to prevent the baboon adaptive response to a pig artery patch graft. If the graft is taken from a CIITA-DN pig, then the adaptive response is reduced. The presence of a CIITA-DN graft reduced the baboon T cell-dependent anti-nonGal IgG response.