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- Materials and Methods
Despite improvements in early posttransplant survival over the last two decades, a relentless annual attrition continues to plague recipients of previously successful heart allografts. Registry data for the International Society for Heart and Lung Transplantation (ISHLT) show that the graft half-life is only 11 years for heart recipients . Infection accounts for 33% of cardiac transplant recipient death 1-year posttransplant while after 5 years, cardiac allograft vasculopathy (CAV) (>30%) and malignancy (23%) account for most cardiac recipient deaths . These sobering statistics emphasize the limitations of chronically administered immunosuppression and make clear the need for tolerance strategies that achieve long-term graft survival and prevent chronic rejection without the use of long-term immunosuppression.
Using the preclinical, large animal model of major histocompatibility complex (MHC)-inbred miniature swine , we have previously shown that isolated, Class I-disparate hearts transplanted without immunosuppression all rejected within 8 days. The addition of a 12-day course of cyclosporine A (CsA) prolonged Class I-mismatched heart allograft survival slightly but all grafts rejected by 55 days. In contrast, hearts in recipients cotransplanted with a kidney allograft from the same Class I MHC-disparate donor and treated with the same 12-day course of CsA all developed long-term and stable tolerance of both the heart and kidney allografts [3, 4].
Although kidney-induced cardiac allograft tolerance (KICAT) was successfully achieved across a Class I MHC barrier, it was unclear whether the same effect could be achieved across a more clinically relevant full MHC barrier. Here we show a dramatic difference in outcomes between recipients of isolated, MHC-mismatched hearts, which rejected their grafts by Day 40, and recipients of cotransplanted, MHC-mismatched heart and kidneys whose cardiac allografts continued to contract strongly for over 200 days without signs of rejection or CAV on serial biopsies.
- Top of page
- Materials and Methods
We have previously shown that long-term cardiac allograft tolerance can be achieved across a Class I MHC mismatch with 12 days of CsA . Here, we established that KICAT could be consistently achieved across a full MHC incompatibility in recipients treated with 12 days of tacrolimus. Tolerance was confirmed by indefinite allograft survival, hyporesponsiveness in CML and MLR assays, absence of alloantibody production and prolongation of donor-specific skin grafts. Furthermore, the cardiac allografts maintained strong contraction as assessed by palpation and echocardiography, and exhibited no clinically relevant rejection or CAV on serial biopsies. Together, these findings strongly suggest that these heart allografts would be life-sustaining if they had been transplanted orthotopically.
It has long been recognized that the immune response to a particular organ varies with the organ transplanted and that some organ allografts, especially livers , are able to confer a survival advantage upon another organ allograft procured from the same donor and cotransplanted into the same recipient. Although less powerful, kidney allografts also seem to possess a tolerogenic effect as evidenced by the dramatic difference in survival of swine kidney and heart allografts transplanted across the same histoincompatibility into recipients treated with the same short tolerance induction regimen .
The reason for these organ-specific differences is unclear. However, our earlier studies suggest that cells or cell products intrinsic to a donor kidney, but not heart allograft, promote a thymic-dependent expansion/activation of host regulatory T cells (Tregs) in the host, which mediates tolerance of the heart graft. We found that removing the recipient thymus, a major source of natural Tregs, prevented the induction of KICAT . We also noted that a radiosensitive, lymphohematopoietic cell population intrinsic to the donor kidney but not heart appeared necessary for the development of KICAT . Using in vitro suppression assays, we found that primed PBLs from tolerant heart/kidney recipients completely suppressed lysis of Class I-mismatched target cells by naïve cells but that suppression was lost following removal of CD25+ cells from the responder population . Finally, we demonstrated that KICAT was not simply due to the additional donor antigen load presented by the cotransplanted kidney as recipients grafted with two Class I-disparate hearts showed early high grade rejection and severe CAV . Together these studies and others from this laboratory [16, 17] support a role for a regulatory mechanism in the state of tolerance induced, in part, by kidney transplantation.
The relevant clinical question then becomes what is the renal element responsible for promoting the expansion/activation of Tregs. There are two cell populations present in kidney allografts with the capacity to down-regulate alloimmune responses, (1) plasmacytoid dendritic cells (pDCs), which can promote the generation of Tregs and can induce tolerance to heart allografts in mice [18-20], and (2) renal tubular epithelial cells (RTECs), which can promote T cell unresponsiveness to self- and alloantigens in mice and humans [21-27]. The hypothesis that pDCs mediate KICAT predicts that donor pDCs transferred with the kidney allograft traffic to the host thymus where they facilitate the activation/expansion of donor-specific Tregs. It is known that abundant CD11c+ conventional DCs are present in normal mouse kidneys . There is precedent for the pDC theory as it was recently shown that hepatic stellate cells from liver allografts were able to confer unresponsiveness and long-term survival to islet allografts by inducing Tregs  and myeloid-derived suppressor cells [30, 31]. It is less likely that migrating, kidney-derived pDCs activate deletional mechanisms in the host thymus, because in our earlier studies, circulating anti-donor cytotoxic T lymphocyte precursors were shown to be present in long-term heart/kidney recipients when SLAcc skin grafting resulted in the return of anti-donor responsiveness in CML assays and rejection of donor skin graft without heart or kidney allograft injury .
The hypothesis that RTECs mediate KICAT predicts that RTECs, intrinsic to the donor kidney, down-regulate or inactivate effector T cells emigrating from the host thymus or convert them to Tregs, thus shifting the balance of the immune response away from rejection and toward tolerance. There is also precedent for this theory. Foxp3+ cells are enriched in the tubules in human  and mouse  renal allografts. Frasca et al.  have shown that IFNγ-treated human RTECs induce allospecific tolerance. They showed that overnight incubation with IFNγ-treated, antigen-pulsed RTECs induced nonresponsiveness in B7-dependent T cell clones, suggesting that MHC Class II expression on RTEC may contribute to the induction of allospecific tolerance following organ transplantation. IFNγ also induces the T cell inhibitor molecule PD-1 and IDO expressed by RTECs [34, 35]. Importantly, Amarnath et al.  have recently shown that PD-1 signaling results in the conversion of human TH1 cells into Tregs. Finally, RTECs are known to produce and activate TGFβ , which is a major inducer of Foxp3+ Treg  and tolerogenic pDCs  generation. High levels of TGFβ have been documented in spontaneously accepted DBA/2 kidneys [40, 41].
With regard to clinical applications, a tolerance induction protocol for heart allograft recipients that requires the simultaneous use of the donor kidney would only be tenable for the small number of patients requiring both organs. Instead, our goal now is to identify the renal component that is responsible for conferring tolerance to cotransplanted heart allografts, in order to use it in tolerance protocols for heart transplant recipients not requiring a kidney allograft. If the effective component should turn out to be a particular cell type, one might envision procuring such cells via a kidney biopsy at the time of organ retrieval and expanding them in vitro for use in a delayed tolerance protocol . Finally, it should be noted that a rising number of human heart allograft recipients do also require donor kidney cotransplantation for preestablished medical reasons. For this special subpopulation, KICAT may be directly applicable, using a delayed mixed chimerism strategy .
In conclusion, we achieved cardiac allograft tolerance across a full MHC mismatch via a kidney-specific mechanism in a robust and reproducible manner. We also demonstrated in this large animal model that the kidney is necessary for the induction phase but not the maintenance phase of cardiac allograft tolerance. This preclinical model will serve as the basis for further studies to isolate and apply the renal element responsible for KICAT to protocols aimed at inducing tolerance in recipients of tolerance-resistant allografts such as isolated heart, lung, composite tissue and islet allografts.