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.

Integrating APOL1 Gene Variants Into Renal Transplantation: Considerations Arising From the American Society of Transplantation Expert Conference.

Thirteen percent of individuals of African ancestry express two variant copies of the gene encoding apolipoprotein 1 (APOL1) that has been associated with an increased risk of end-stage renal disease (ESRD) in the general population. Limited studies suggest that the survival of transplanted kidneys from donors expressing two APOL1 risk alleles is inferior to that of kidneys from donors with zero or one risk allele. In living kidney donation, two case reports describe donors expressing two APOL1 risk alleles who developed ESRD. Given the potential impact of APOL1 variants on the utility and safety of kidney transplantation and living kidney donation, the American Society of Transplantation convened a meeting with the goals of summarizing the current state of knowledge with respect to transplantation and APOL1, identifying knowledge gaps and studies to address these gaps, and considering approaches to integrating APOL1 into clinical practice. The authors recognize that current data are not sufficient to support traditional evidence-based guidelines but also recognize that it may require several years to generate the necessary data. Thus, approaches as to how APOL1 might currently be integrated into the clinical decision-making process were considered. This report summarizes the group's deliberations.

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.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.

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.

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.

Induction of cardiac allograft tolerance across a full MHC barrier in miniature swine by donor kidney cotransplantation.

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Donor brain death inhibits tolerance induction in miniature swine recipients of fully MHC-disparate pulmonary allografts.

We have previously shown that a short course of high-dose tacrolimus induces long-term tolerance to fully mismatched lung allografts procured from healthy MHC-inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12-day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL-1, TNF-α and IL-10 were seen after brain death. Upregulation of IL-1 and IL-6 gene expression was also observed.

Comparison of lung and kidney allografts in induction of tolerance by a mixed-chimerism approach in cynomolgus monkeys.

BACKGROUND: We have previously reported the successful induction of renal allograft tolerance in non-human primates using a nonmyeloablative conditioning regimen to produce a mixed-chimeric state in the recipient. In the present study, we applied this same technique to lung allotransplantation in cynomolgus monkeys. METHODS: Nine pairs of fully major histocompatibility complex (MHC)-mismatched cynomolgus monkeys were used. The conditioning regimen consisted of total body irradiation, thymic irradiation, and antithymocyte globulin. The recipients underwent lung and bone marrow transplantation, followed by anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine. The regimen included anti-CD8 mAb in the last 5 recipients and alpha 1-antitripsin in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same regimen. RESULTS: Transient chimerism developed in all lung recipients, as was previously seen in the kidney recipients. Nonetheless, the lung recipients rejected their allografts significant earlier than the kidney recipients (P < .01). CONCLUSIONS: Despite the successful induction of mixed chimerism in recipients of fully MHC-mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. Strategies to overcome this problem include organ-specific modifications of the transplant regimen.

The indirect alloresponse impairs the induction but not maintenance of tolerance to MHC class I-disparate allografts.

We studied the effects of indirect allorecognition on the induction and maintenance phases of tolerance in miniature swine cotransplanted with heart and kidney allografts. MHC class I-mismatched heart and kidney grafts were cotransplanted in recipients receiving CyA for 12 days. Recipients were unimmunized or immunized with a set of donor-derived or control third-party MHC class I peptides either 21 days prior to transplantation or over 100 days after transplantation. T-cell proliferation, delayed type hypersensitivity reaction (DTH) and antibody production were assessed. All animals injected with donor MHC class I peptides developed potent indirect alloresponses specific to the immunizing peptides. While untreated recipients developed stable tolerance, all animals preimmunized with donor allopeptides rejected kidney-heart transplants acutely. In contrast, when peptide immunization was delayed until over 100 days after kidney-heart transplantation, no effects were observed on graft function or in vitro measures of alloimmunity. Donor peptide immunization prevented tolerance when administered to recipients pre transplantation but did not abrogate tolerance when administered to long-term survivors post transplantation. This suggests that the presence of T cells activated via indirect allorecognition represent a barrier to the induction but not the maintenance of tolerance.

Effects of tolerance induction on the actions of interferon-gamma on porcine cardiac allografts.

It is well known that interferon-gamma (IFN-gamma) not only plays a critical role in antigen-dependent but also in antigen-independent tissue injury; however, it is not clear how tolerance induction affects the actions of IFN-gamma in the transplant setting. To address this question, we compared the effects of IFN-gamma on porcine recipients of near-syngeneic, rejecting, and tolerant heart transplants. IFN-gamma was infused continuously into the left anterior descending artery of hearts transplanted into 3 groups of major histocompatibility complex (MHC) inbred miniature swine, each treated with a 12-day course of cyclosporine A (CyA). Group 1 recipients received a MHC class I disparate heart, group 2 recipients received a near-syngeneic heart, and group 3 recipients were cotransplanted with a MHC class I disparate heart and kidney, which uniformly induces tolerance to both grafts. An additional group of animals was not transplanted but received intracoronary IFN-gamma infusion into their native hearts. IFN-gamma perfusion not only accelerated the acute rejection of MHC class I disparate hearts (mean survival time = 19 +/- 7.21 vs 38 +/- 8.19 days, P = .025), but caused near-syngeneic heart transplants, which otherwise survive indefinitely, to reject within 35 days (n = 3). In contrast, IFN-gamma perfusion had no demonstrable effects on interstitial rejection, the development of vascular lesions, or graft survival in tolerant heart plus kidney allograft recipients (n = 4) or in autologous hearts (n = 2). These results suggest that tolerance induction mitigates the damaging effects of IFN-gamma itself and that the beneficial effects of tolerance induction on acute and chronic rejection may extend to antigen-independent factors like ischemia/reperfusion injury.

Thymectomy does not abrogate long-term acceptance of MHC class I-disparate lung allografts in miniature Swine.

UNLABELLED: We have previously reported that tolerance to class I disparate lung allografts in miniature swine could be induced using an intensive 12-day course of tacrolimus and that pretransplant sensitization with immunogenic MHC class I allopeptides failed to block the induction of tolerance. We also have previously reported the importance of the presence of the thymus in the induction of tolerance to isolated heart, kidney, and combined heart-kidney transplants. In this study, we examined the impact of thymectomy on tolerance induction in lung transplantation. METHODS: Orthotopic left lung transplantation was performed using MHC class I-disparate donors. The recipients received a 12-day course of high-dose tacrolimus (n = 6). Total thymectomies were performed in three of the swine 21 days prior to transplantation. Lung grafts were monitored by chest radiography and serial open lung biopsy. RESULTS: All euthymic recipients maintained their grafts for over 1 year. None of the thymectomized recipients has experienced graft loss in the 6 to 10 months following transplantation. Although isolated lesions of obliterative bronchiolitis were occasionally seen in one thymectomized animal on biopsy, donor-specific unresponsiveness has been observed on assays of cell-mediated lymphocytotoxicity in all recipients. Moreover, co-culture assays have shown that recipient lymphocytes can strongly inhibit the normally robust response of naïve recipient-matched lymphocytes to donor antigen. This inhibition was not seen when using stimulators primed with third-party antigens against appropriate targets. CONCLUSIONS: These data suggest that thymus-independent peripheral regulatory mechanisms may be sufficient to induce and maintain long-term acceptance of the lung allografts.

The role of indirect recognition of MHC class I and II allopeptides in a fully mismatched miniature swine model of lung transplantation.

UNLABELLED: Considerable evidence suggests that indirect recognition of MHC allopeptides plays an important role in solid-organ rejection. Here, we examine whether immunization with class I or class II allopeptides accelerates rejection in a fully MHC-mismatched lung transplant model in miniature swine. METHODS: Recipients were immunized with either donor-derived class I or class II peptides. Sensitization to the peptides was confirmed by DTH testing and in vitro proliferation assays. Nonimmunized control (n = 6), class I peptide-immunized (n = 3), and class II peptide-immunized (n = 3) swine were transplanted with fully mismatched lungs using only a 12-day course of tacrolimus. RESULTS: One control animal rejected its graft on postoperative day 103, while the others maintained their grafts for over 1 year. In the class I peptide-immunized group, two recipients rejected their grafts (days 14 and 52). The third animal has not rejected the graft (day 120, experiment is ongoing). In contrast, in the class II-peptide immunized group, only one animal rejected its graft on day 52, while the others maintained their grafts over 1 year. Both anti-donor IgM and IgG antibodies were detectable in all acute rejectors, although no alloantibody was detectable in long-term acceptors. Regardless of the fate of the graft, all animals have maintained their proliferative responses to the peptides. However, only acceptors maintained donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity and mixed lymphocyte reaction assays. CONCLUSIONS: Pretransplant sensitization of lung allograft recipients to donor allopeptides accelerates graft rejection. This appears particularly true for class I-derived allopeptides, suggesting that class II molecules may be less antigenic when presented indirectly.

An MHC class II disparity raises the threshold for tolerance induction in pulmonary allografts in miniature swine.

OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35-50 ng/mL) as their only immunosuppression. RESULTS: All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis. CONCLUSIONS: We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.

Long-term acceptance of porcine pulmonary allografts without chronic rejection.

OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. Using a strategy to induce tolerance across strong allogeneic barriers, we have employed a brief, intensive course of immunosuppression to determine whether the induction of donor-specific hyporesponsiveness would prevent allograft rejection in a preclinical model of lung transplantation using MHC-inbred miniature swine. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate donors. The recipients received a 12-day postoperative course of cyclosporine (n = 6) or a 12-day postoperative course of high-dose tacrolimus (n = 3) as their only immunosuppression. Control animals received no immunosuppression (n = 3). RESULTS: Cyclosporine-treated recipients exhibited graft survival ranging from 67 to >605 days. All six animals developed acute cellular rejection between postoperative days (PODs) 27 and 108. Two animals lost their grafts on PODs 67 and 69, before developing obliterative bronchiolitis (OB). The other four recipients developed OB between PODs 119 and 238. In contrast, all tacrolimus-treated recipients maintained their grafts long term, without developing chronic rejection (>339, >308, and >231). These recipients also exhibited donor-specific hyporesponsiveness in assays of cell-mediated lymphocytotoxity. All untreated control animals lost their grafts to acute rejection by POD 11. CONCLUSIONS: This study demonstrates the ability of a brief course of high-dose tacrolimus to induce long-term graft acceptance with donor-specific hyporesponsiveness in a class I-disparate preclinical lung transplant model.

Hyperacute rejection is attenuated in GalT knockout swine lungs perfused ex vivo with human blood.

BACKGROUND: Hyperacute rejection (HAR) is one of the principal obstacles to successful xenotransplantation. Homozygous alpha-1,3-galactosyltransferase knockout (GalT-KO) miniature swine now offer the prospect of overcoming this barrier to xenotransplantation. In this study, the short-term function of GalT-KO swine lungs was evaluated in a well-established ex vivo model of swine-to-human lung xenotransplantation. METHODS: Lungs from homozygous GalT-KO swine (n = 3) and control lungs from pigs of the background strain used to create the GalT-KO pig line (n = 2) were perfused ex vivo with freshly collected heparinized human blood. Graft function was assessed by various physiologic measurements, serial histologic and immunohistochemical evaluation, and assays of complement and platelet activation. RESULTS: Xenoperfused control swine lungs exhibited HAR with graft survival times <5 minutes. In contrast, GalT-KO swine lungs retained their function for approximately 2 hours, on average. GalT-KO swine lungs showed decreased complement and platelet activation compared with controls. Nonetheless, activation of complement and coagulation cascades was not completely eliminated in the GalT-KO swine lungs. CONCLUSIONS: The survival of xenoperfused GalT-KO swine lungs was significantly prolonged, as compared with control lungs expressing Gal. This appears to have been due largely to substantially reduced complement activation. Nonetheless, the xenoperfused GalT-KO lungs still showed some evidence of complement fixation and intravascular coagulopathy by the time of graft demise.

Understanding xenotransplantation risks from nonhuman primate retroviruses.

Significant progress in making animal-to-human transplantation a viable adjunct to human organ donation will require a greater understanding of the intricacies of immunologic rejection. Recent success in generating cloned knockout piglets increases the possibility that xenotransplantation may find its way into the clinics. Nonhuman primates' organs have been used for human transplants in the past and there is reason to believe that if ethical considerations and inherent problems with supply were overcome, their close genetic proximity to humans would lessen complications of rejection. Unfortunately, nonhuman primates harbor several pathogens known to be infectious in humans and the potential of other viral infections has precluded further use of monkeys in this setting. Baboons are generally considered the nonhuman primate species of choice yet this species carries several retroviruses considered a threat to humans in transplantation. Both known and potentially undiscovered retroviruses pose an important risk that is the focus of this review.

Morphometric analysis of miniature swine hearts as potential human xenografts.

Miniature swine are considered to be potential donors for clinical cardiac transplantation. However, it is unclear how an appropriately sized porcine donor will be selected for a particular human recipient. To address this issue, we performed a morphometric study of the swine heart using transthoracic echocardiography (n = 26) to determine the diameters of the aortic annulus and root, pulmonary artery annulus, and mitral valve annulus. We also obtained direct ex vivo measurements of swine heart weight and linear dimensions (n = 71). Relationships between a swine's height, weight, length, chest circumference and these internal and external cardiac dimensions are described. The strongest correlations were found between a pig's body length and its aortic annulus and root diameters (r-values = 0.97). These relationships are accurately described by univariate linear regression models. By cross-relating our morphometric measurements of aortic annulus diameter in the miniature swine with normative human data, we were able to develop a nomogram, relating swine length and human height, which predicts which miniature swine would donate the best size-matched heart for a particular human recipient.