The effects of brain death and ischemia on tolerance induction are organ-specific.

We have previously shown that 12 days of high-dose calcineurin inhibition induced tolerance in MHC inbred miniature swine receiving MHC-mismatched lung, kidney, or co-transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19-45 days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12 days of calcineurin inhibition received organs from donors rendered brain dead for 4 hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4 hours, 8 hours, or 4 hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long-term survival (>100 days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ-specific, which has implications for the application of tolerance to clinical transplantation.

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts.

The emergence of skin-containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin-resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II-mismatched chimeras were tolerant of VCAs. MHC class I-mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient-type CD8+ lymphocytes. Systemic donor-specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Prolonged Survival Following Pig-to-Primate Liver Xenotransplantation Utilizing Exogenous Coagulation Factors and Costimulation Blockade.

Since the first attempt of pig-to-primate liver xenotransplantation (LXT) in 1968, survival has been limited. We evaluated a model utilizing α-1,3-galactosyltransferase knockout donors, continuous posttransplant infusion of human prothrombin concentrate complex, and immunosuppression including anti-thymocyte globulin, FK-506, methylprednisone, and costimulation blockade (belatacept, n = 3 or anti-CD40 mAb, n = 1) to extend survival. Baboon 1 remained well until postoperative day (POD) 25, when euthanasia was required because of cholestasis and plantar ulcers. Baboon 2 was euthanized following a seizure on POD 5, despite normal liver function tests (LFTs) and no apparent pathology. Baboon 3 demonstrated initial stable liver function but was euthanized on POD 8 because of worsening LFTs. Pathology revealed C4d positivity, extensive hemorrhagic necrosis, and a focal cytomegalovirus inclusion. Baboon 4 was clinically well with stable LFTs until POD29, when euthanasia was again necessitated by plantar ulcerations and rising LFTs. Final pathology was C4d negative and without evidence of rejection, inflammation, or thrombotic microangiopathy. Thus, nearly 1-mo rejection-free survival has been achieved following LXT in two of four consecutive recipients, demonstrating that the porcine liver can support life in primates for several weeks and has encouraging potential for clinical application as a bridge to allotransplantation for patients with acute-on-chronic or fulminant hepatic failure.

Tolerance of Vascularized Islet-Kidney Transplants in Rhesus Monkeys.

We previously reported that transplantation (Tx) of prevascularized donor islets as composite islet-kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cell (HSC) Tx in an attempt to induce tolerance in nonhuman primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IK Tx. HSC Tx was performed after mobilization and leukapheresis of the donors and conditioning of the recipients with total body irradiation, T cell depletion, and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IK Tx was performed either 20-22 (n = 3) or 208 (n = 1) days after HSC Tx. All animals accepted IKs without rejection. All recipients required >20 U/day insulin before IK Tx to maintain <200 mg/dL, whereas after IK Tx, three animals required minimal doses of insulin (1-3 U/day) and one animal was insulin free. These results constitute a proof-of-principle that this IK tolerance strategy may provide a cure for both end-stage renal disease and diabetes without the need for immunosuppression.

Role of Intrinsic (Graft) Versus Extrinsic (Host) Factors in the Growth of Transplanted Organs Following Allogeneic and Xenogeneic Transplantation.

In our studies of life-supporting α-1,3-galactocyltransferase knockout (GalT-KO) pig-to-baboon kidneys, we found that some recipients developed increased serum creatinine with growth of the grafts, without histological or immunological evidence of rejection. We hypothesized that the rapid growth of orthotopic pig grafts in smaller baboon recipients may have led to deterioration of organ function. To test this hypothesis for both kidneys and lungs, we assessed whether the growth of outbred (Yorkshire) organ transplants in miniature swine was regulated by intrinsic (graft) or extrinsic (host environment) factors. Yorkshire kidneys exhibited persistent growth in miniature swine, reaching 3.7 times their initial volume over 3 mo versus 1.2 times for miniature swine kidneys over the same time period. Similar rapid early growth of lung allografts was observed and, in this case, led to organ dysfunction. For xenograft kidneys, a review of our results suggests that there is a threshold for kidney graft volume of 25 cm3 /kg of recipient body weight at which cortical ischemia is induced in transplanted GalT-KO kidneys in baboons. These results suggest that intrinsic factors are responsible, at least in part, for growth of donor organs and that this property should be taken into consideration for growth-curve-mismatched transplants, especially for life-supporting organs transplanted into a limited recipient space.

Persistence of Indirect but Not Direct T Cell Xenoresponses in Baboon Recipients of Pig Cell and Organ Transplants.

We investigated the contributions of direct and indirect T cell antigen recognition pathways to the immune response to porcine antigens in naïve baboons and baboon recipients of pig xenografts. In naïve baboons, in vitro culture of peripheral blood T cells with intact pig cells (direct xenorecognition pathway) or pig cell sonicates and baboon antigen-presenting cells (indirect xenorecognition pathway) induced the activation and expansion of xenoreactive T cells producing proinflammatory cytokines, interleukin-2 and interferon-γ. Primary indirect xenoresponses were mediated by preexisting memory T cells, whose presence is not typically observed in primary alloresponses. Next, baboons were conditioned with a nonmyeloablative regimen before short-term immunosuppression and transplantation of xenogeneic peripheral blood progenitor cells and a kidney, heart, or pancreatic islets from a miniature swine. All transplants were rejected acutely within 30 days after their placement. Posttransplantation, we observed an inhibition of the direct xenoresponse but a significant expansion of indirectly activated proinflammatory T cells. These results suggest that additional treatment to suppress indirect T cell immunity in primates may be required to achieve tolerance of pig xenografts through hematopoietic chimerism.

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine.

A 12-day course of high-dose tacrolimus induces tolerance of major histocompatibility complex-mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart-lung cotransplantation using the same induction protocol. Hearts (n = 3), heart-kidneys (n = 3), lungs (n = 6), and hearts-lungs (n = 3) were transplanted into fully major histocompatibility complex-mismatched recipients treated with high-dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart-kidney recipients and five of six lung recipients demonstrated long-term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor-specific unresponsiveness by cell-mediated lympholysis/mixed-lymphocyte reaction. In contrast, heart-lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.

The Effects of Exogenous Administration of Human Coagulation Factors Following Pig-to-Baboon Liver Xenotransplantation.

We sought to determine the effects of exogenous administration of human coagulation factors following pig-to-baboon liver xenotransplantation (LXT) using GalT-KO swine donors. After LXT, baboons received no coagulation factors (historical control, n = 1), bolus administration of a human prothrombin concentrate complex (hPCC; 2.5 mL/kg, n = 2), continuous infusion of hPCC (1.0 mL/h, n = 1) or continuous infusion of human recombinant factor VIIa (1 µg/kg per hour, n = 3). The historical control recipient demonstrated persistent thrombocytopenia despite platelet administration after transplant, along with widespread thrombotic microangiopathy (TMA). In contrast, platelet levels were maintained in bolus hPCC recipients; however, these animals quickly developed large-vessel thrombosis and TMA, leading to graft failure with shortened survival. Recipients of continuous coagulation factor administration experienced either stabilization or an increase in their circulating platelets with escalating doses. Furthermore, transfusion requirements were decreased, and hepatic TMA was noticeably absent in recipients of continuous coagulation factor infusions compared with the historical control and bolus hPCC recipients. This effect was most profound with a continuous, escalating dose of factor VIIa. Further studies are warranted because this regimen may allow for prolonged survival following LXT.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead Box Protein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism.

While the induction of transient mixed chimerism has tolerized MHC-mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed-tolerance-induction protocol resulting in three out of four monkeys achieving long-term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post-mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC-mismatched lungs in primates.

Long-term lung transplantation in nonhuman primates.

Despite advances in surgical technique and clinical care, lung transplantation still remains a short-term solution for the treatment of end-stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long-term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre-existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple-drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse-derived anti-thymocyte globulin and a monoclonal antibody to the IL-6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.

High incidence of xenogenic bone marrow engraftment in pig-to-baboon intra-bone bone marrow transplantation.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Pilot Study Evaluating Regulatory T Cell-Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model.

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, anti-interleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor-specific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

Evidence for a gene controlling the induction of transplantation tolerance.

Class I mismatched kidney transplantation in Massachusetts General Hospital MHC-defined miniature swine has been studied extensively as a model for induction of systemic allograft tolerance. In a large series of juvenile swine, long-term graft acceptance has been observed consistently following a 12-day course of cyclosporine. It was therefore surprising when three of five recipients in one of our studies rejected their grafts. Examination of the origins of the rejecting animals revealed that they were derived from a subline of the SLA(dd) miniature swine herd that was intentionally being inbred toward full homozygosity and had been inbred for eight generations prior to these experiments. A blinded study of additional class I mismatched renal transplants into animals from this subline confirmed the genetic basis of this rejection. We present here preliminary evidence suggesting that a likely explanation for this phenomenon is that the rejectors in this subline are homozygous for a recessive mutant allele of a gene normally involved in the induction of tolerance. Subsequent studies will be directed toward identification and characterization of the gene(s) involved, since existence of a similar genetic locus in humans might have implications for assessing an individual's likelihood of graft rejection versus tolerance induction prior to organ transplantation.

Abrogation of renal allograft tolerance in MGH miniature swine: the role of intra-graft and peripheral factors in long-term tolerance.

We have previously demonstrated that long-term tolerance (LTT) of an MHC class-I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class-I mismatched primary kidneys were subjected to a treatment consisting of donor-specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor-MHC-matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4(+) Foxp3(+) Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class-I mismatched allotransplants.