The production of GalT-KO swine may represent a critical step toward the clinical reality of solid organ xenotransplantation (2
). In the present study of GalT-KO heart xenografts transplanted into baboons, HAR was prevented and graft survival improved over previous reports, even without the adsorption of anti-Gal antibodies or the continuous inhibition of complement (11
). Furthermore, one GalT-KO graft survived for almost 6 months after transplantation. We consider these results very encouraging. However, all GalT-KO heart grafts underwent graft failure, despite chronic immunosuppression. The present study characterizes the pathology associated with AHXR, ACXR and chronic rejection in GalT-KO xenografts in nonhuman primates.
The availability of GalT-KO pigs eliminated the anti-Gal antibody–Gal antigen interaction that was the major barrier to successful xenotransplantation in pig-to-nonhuman primate models (13
). This intervention did not, however, eliminate the thrombotic microangiopathy that is characteristic of AHXR (24
). Indeed, in the present study, AHXR, as characterized by thrombotic microangiopathy, developed in all the eight grafts. Non-Gal antibodies likely play an important role in the pathogenesis of AHXR and graft failure in xenogeneic transplants, as suggested by this study and several other recent reports (25
Troponin T is a clinically valuable marker of myocardial injury in heart grafts (27
), and we used it as an indicator of myocardial injury in this study. Increases in troponin T, particularly if sustained, were associated with thrombotic microangiopathy (AHXR) and progressively weaker contractions of the grafts. However, some animals demonstrated a discrepancy between troponin T levels and graft contractions. B223 showed a sudden increase and rapid recovery of troponin T around day 55, without an associated change in the beating of the graft. This troponin T leak may have been due to a localized myocardial infarction that may have been the result of an AHXR-mediated thrombus in a small myocardial artery. In contrast, B229 lost the graft in the absence of any rise of troponin T. Instead of necrosis of cardiomyocytes, which is associated with the release of troponin T, this animal might have lost its graft due to apoptotic myocardial injury that was mediated by the chronic, mild but persistent ischemic injury caused by the gradual development of AHXR.
The characterization of cellular rejection in vascularized xenograft models is still limited by the rapid development of AHXR. Initial studies in rodents concluded that graft-infiltrating cells consisted primarily of macrophages and NK cells, and, in view of the small number of T cells in the infiltrates, that these responses were T cell independent (28
). In the present study, however, the cellular response to discordant xenografts was characterized by an infiltration of CD3+
T cells, CD4+
cells and CD68+
T cells infiltrated early after transplantation. Many CD3 and TIA-1+
cytotoxic T cells were present in the infiltrate as were a small number of NK cells. These findings suggest that T-cell-dependent immunologic pathways play a significant role in xenograft rejection. Although in vitro
assays showed general unresponsiveness of recipients to pig and baboon stimulators in MLR and circulating antipig IgM and IgG were undetectable by FACS (11
), probably because of continuous immunosuppression, the T-cell infiltrates and IgG deposition seen in the grafts suggest that T cells were involved in the rejection. Several studies of human antipig cellular xenoreactivity support our findings; they have demonstrated a strong human antipig cell-mediated cytotoxic response that involves CD8+
cells, and have implicated CD4+
cells as important mediators of macrophages, T-cell and B-cell activation (30
). Therefore, as in allotransplantation, T cells may function as an initiating factor in the immune response to the xenografts and may contribute to the development of not only ACXR but also AHXR, through both direct and indirect pathways of immune recognition. In the present study, similar numbers of CD4+
T cells infiltrated the grafts, although CD8+
T cells predominate in allograft rejection, suggesting a strong role for class II-restricted T-cell-mediated reactions in ACXR. In our results, three grafts in the gradually weakened group developed interstitial mononuclear cell infiltrates, as is characteristic of ACXR. In particular, in B223, the number of cell infiltrates increased significantly as the level of MMF was decreased (see ). On the other hand, two grafts in the rapidly weakened group had cellular infiltrates comprising neutrophils and macrophages as well as CD3+ T cells, suggesting that the humoral and T-cell responses developed together. The importance of T-cell immunity in xenotransplantation may be often underestimated because humoral immunity often masks T-cell immunity and obscures the pathological findings of ACXR in xenotransplantation. As is now the case in allotransplantation, ACXR may eventually become a more important and common type of xenograft rejection.
In clinical heart transplantation, CAV is the leading obstacle to long-term graft survival (36
). It is widely established that CAV in allografts is caused by a combination of chronic immune and nonimmune injury, and that the immune mechanism involves both cellular rejection (endoarteritis or endothelialitis) in arteries and humoral rejection by circulating antidonor antibodies (36
). NK cells might also have pathogenetic significance (38
The development of chronic xenograft vasculopathy, which is considered to be a characteristic feature of chronic xenograft rejection, was previously reported in a small number of hearts transplanted from pigs transgenic for hDAF or CD46 to primates (14
). However, the pathogenic mechanism of the development of chronic xenograft vasculopathy has not been clearly determined. In the present study, chronic xenograft vasculopathy developed in the three grafts from the gradually weakened group between 78 and 179 days following transplantation. We recognized four major manifestations of chronic xenograft vasculopathy based on their histologic appearance: (1
) chronic humoral rejection-associated vasculopathy, (2
) chronic cellular rejection-associated vasculopathy, (3
) both chronic humoral and cellular rejection-associated vasculopathy and (4
) fully developed vasculopathy, which may be the end result of humoral rejection-associated and/or cellular rejection-associated vasculopathy. These histological findings suggest that prolonged antibody- and/or cell-mediated rejection can induce chronic xenograft vasculopathy. In the present study, chronic humoral rejection-associated vasculopathy was more frequently seen in the grafts than chronic cellular rejection, suggesting that chronic humoral rejection is likely the dominant mechanism for the development of chronic xenograft vasculopathy in this model.
Clinical application of xenotransplantation first requires the demonstration of efficacy in a nonhuman primate model. The ISHLT has suggested as a reasonable standard 60% graft survival of life-supporting orthotopic pig hearts in primates at 90 days (39
). The FDA Advisory Subcommittee on Xenotransplantation has a similar recommendation of a median survival of 90 days in the orthotopic position as a possible standard for clinical application (40
). In the present study, median and longest survivals of heterotopic hearts in a baboon were 78 (exclusion of euthanized animals) and 179 days, respectively. Future studies aimed at preventing acute and chronic antibody- and cell-mediated rejection in xenografts from GalT-KO donors are warranted.
In the present study, the response to GalT-KO cardiac xenografts in baboons was not controlled by our chronic immunosuppressive protocols, and acute and chronic humoral and cellular rejection developed in the grafts. We believe that improved strategies for inducing xenogeneic T-cell tolerance and preventing acute and chronic humoral and cell-mediated graft rejection may be required for long-term xenograft survival. Ongoing studies at our center are aimed at the induction of immunological tolerance to miniature swine organs in nonhuman primates by chimerism induction (41
) and donor vascularized thymus grafting (20