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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., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., Competing Interests: The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The relative contribution of central and peripheral mechanisms to the generation and maintenance of allograft tolerance is of considerable interest. Here, we present new evidence that regulatory T cells (Foxp3(+) ) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor- and recipient-derived Foxp3(+) cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells. Foxp3(+) cell depletion did not result in the rejection of skin grafts of only MHC-disparate donors (B6.C-H2(d) /bByJ), indicating that MHC antigens were not the target in the graft. We conclude that two different mechanisms of tolerance are present in mixed chimeras. Hematopoietic chimerism, resistant to Foxp3(+) depletion, is probably due to deletional tolerance to MHC antigens, as supported by previous studies. In contrast, regulatory tolerance mechanisms involving Foxp3(+) cells are required to control reactivity against non-MHC antigens not present on hematopoietic lineages., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Making the use of daytime running lights mandatory for motor vehicles is generally documented to have had a positive impact upon traffic safety. Improving traffic safety for bicyclists is a focal point in the road traffic safety work in Denmark. In 2004 and 2005 a controlled experiment including 3845 cyclists was carried out in Odense, Denmark in order to examine, if permanent running lights mounted to bicycles would improve traffic safety for cyclists. The permanent running lights were mounted to 1845 bicycles and the accident rate was recorded through 12 months for this treatment group and 2000 other bicyclists, the latter serving as a control group without bicycle running lights. The safety effect of the running lights is analysed by comparing incidence rates - number of bicycle accidents recorded per man-month - for the treatment group and the control group. The incidence rate, including all recorded bicycle accidents with personal injury to the participating cyclist, is 19% lower for cyclists with permanent running lights mounted; indicating that the permanent bicycle running light significantly improves traffic safety for cyclists. The study shows that use of permanent bicycle running lights reduces the occurrence of multiparty accidents involving cyclists significantly. In the study the bicycle accidents were recorded trough self-reporting on the Internet. Possible shortcomings and problems related to this accident recording are discussed and analysed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

IL-2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high-dose IL-2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL-2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low-dose IL-2 (1 million IU/m(2) BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low-dose IL-2 therapy significantly expanded peripheral blood CD4(+) and CD8(+) Tregs in vivo with limited expansion of non-Treg cells. These expanded Tregs are mainly CD45RA(-) Foxp3(high) activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1(-/-) or B6.RAG1(-/-)C3(-/-) (H-2(b)) mice received B10.BR (H-2(k)) heart allografts and repeated doses of IgG2a, IgG1 or F(ab')(2) fragments of IgG2a DSA (anti-H-2(k)). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti-NK1.1 reduced significantly DSA-induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ-chain knock out) also showed decreased severity of DSA-induced CAV. Direct NK reactivity to the graft was not necessary. F(ab')(2) DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho-extracellular signal-regulated kinase (pERK), a response not elicited by F(ab')(2) DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d-negative chronic antibody-mediated rejection in humans., (© 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals., (© 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The high frequency of memory T cells present in primates is thought to represent a major barrier to tolerance induction in transplantation. Therefore, it is crucial to characterize these memory T cells and determine their functional properties. High numbers of memory T cells were detected in peripheral blood and all lymphoid tissues except lymph nodes, which were essentially the site of naïve T cells. The majority of CD8(+) memory T cells were effector memory cells located in the blood and bone marrow while most CD4(+) memory T cells were central memory cells present in the spleen. Next, memory T cells from over 100 monkeys were tested for their response to alloantigens by ELISPOT. Memory alloreactivity mediated via direct but not indirect allorecognition was detected in all animals. The frequency of allospecific memory T cells varied dramatically depending upon the nature of the responder/stimulator monkey combination tested. MHC gene matching was generally associated with a low-memory alloreactivity. Nevertheless, low anamnestic alloresponses were also found in a significant number of fully MHC-mismatched monkey combinations. These results show that selected donor/recipient combinations displaying a low memory alloresponsiveness can be found. These combinations may be more favorable for transplant tolerance induction.

Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody-mediated rejection. Complement fixation has been shown to be essential in acute antibody-mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti-H-2Kk into B6.RAG1-/- KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14-28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor-specific alloantibody (DSA, monoclonal IgG1 anti-H-2Kk) injected into B6.RAG1-/- KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti-H-2Kk) initiated endarteritis followed by CTA in B6.RAG1-/- mice genetically deficient in the third component of complement (RAG1-/-C3-/-). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivo without complement participation, in contrast to acute antibody-mediated rejection. This pathway may be relevant to C4d-negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody-mediated chronic rejection.

Viral infections including those due to cytomegalovirus have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novo formation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG(-/-)). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus but not in uninfected controls. This process was also dependent upon the presence of natural killer (NK) cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell-dependent pathway in the absence of T- and B-lymphocytes.

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.

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.

Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end-effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 x BALB/c)F(1) recipients, which then received anti-macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30-80% with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70% reduction in the development of CAV, as compared to mock-treated controls (p = 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end-effector cells in a final common pathway towards CAV independent of T-cell or B-cell alloreactivity and exert their injurious effects through mechanisms related to cytokine/growth factor production rather than phagocytosis.

Murine heterotopic cardiac allografts were used to reveal some of the fundamental interrelationships between donor-specific alloantibodies (DSA), chronic transplant arteriopathy (CTA) and capillary C4d deposition. B10.BR recipients of B10.A hearts developed transient DSA and C4d deposition that peaked on day 7 and became undetectable at day 56 while CTA developed progressively. Male cardiac grafts in female recipients showed similar degrees of CTA at day 56 but never developed DSA or C4d deposition, indicating that T cell-mediated mechanisms are sufficient to produce CTA. Passive transfer of monoclonal IgG2a anti-H-2K(k) into B6.RAG1 KO recipients of B10.BR hearts over 14-28 days led to progressive CTA. If treatment was stopped on day 14, lesions showed little progression and had no C4d deposition or detectable DSA on day 42. If treatment was stopped on day 28 when the lesions were fully developed, no regression occurred over the next 28 days, even though C4d deposition and circulating antibody became undetectable. Therefore, a minimum threshold of antibody exposure is needed to cause CTA. Once the CTA develops, C4d may become negative after DSA disappears. Thus, serial samples are needed in clinical studies to ascertain the relevance of alloantibody to the lesions of chronic graft rejection.

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.

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.

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.

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.

We previously demonstrated that cardiac allografts to fully tolerant chimeric mice developed cardiac allograft vasculopathy (CAV). Here we begin to examine which components of the immune system are responsible for the pathogenesis of CAV in such tolerant recipients. B10.A/B6 mixed chimeric mice were created by receiving injections of bone marrow cells from B10.A (H-2k) mice given to C57BL/6 (B6; H-2b) mice with some preparations. B10.A skin grafts were first placed onto B10.A/B6 mixed chimeric recipients. When the donor strain skin grafts had survived perfectly for at least 56 days, B10.A hearts were transplanted heterotopically into B10.A/B6 mixed chimeric recipients. Hearts were examined for the presence of CAV 56 days later. To determine the effector cells that contribute to the development of CAV, they were treated weekly with a combination of anti-CD4/CD8 monoclonal antibodies (mAbs) or anti-NK1.1 mAb continuing until 56 days. 14 B10.A cardiac transplants of 18 otherwise untreated B10.A/B6 chimeric recipients developed CAV; concurrent B6 isografts were unaffected (0/7). In chimeric recipients treated with anti-CD4/8 mAbs, the prevalence of CAV was greatly reduced (0/6, P < .01 compared to the untreated group). Anti-NK1.1 mAb was not effective in the prevention of CAV (4/5). These data suggest that T cells may contribute in some way to the development of CAV that occurs in those fully tolerant recipients. Host T cells that may still be responsive to non-major histocompatability complex antigens, including tissue-specific antigens presented not on skin but on heart, may also be responsible for the development of CAV in tolerant animals.

In prior experiments, we found that recipients, even though specifically tolerant of donor antigens, will develop striking cardiac allograft vasculopathy (CAV) in allogeneic mouse heart transplants. This suggested that innate immune responses, in addition to conventional adaptive immunity, may be involved in the development of CAV. We accordingly performed transplants in the parental-to-F1 combination seeking supportive evidence of NK cell activity directed against the vessels of parental donor transplants as a manifestation of "hybrid resistance." When such lesions were indeed found, we investigated their pathogenesis employing immunopathological analysis, in vitro measurements of NK cytotoxicity, and donor-specific T-cell activity in F1 recipients of parental donor hearts. We present evidence that NK cells can promote cardiac allograft vasculopathy. Since NK cell activity is not well targeted by current immunosuppressive therapy, its control may offer a valuable new possibility for improving the long-term outcome of transplanted organs.

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.

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.

Background and Aim of the Study: The study aim was to describe the long-term results and determinants of mortality after operative treatment of native and prosthetic valve endocarditis at a single institution., Methods: Between March 1985 and October 1999, 171 patients underwent surgery for native (NVE) or prosthetic valve endocarditis (PVE). NVE was present in 98 patients (57%), and PVE in 73 patients (43%). Mean follow up was 5.6+/-3.9 years (range: 0 to 15 years)., Results: Overall hospital mortality was 9.9% (n = 17). Hospital mortality was higher among patients with PVE (15.1%) than those with NVE (6.1%; p = 0.05). Overall survival at 10 years was 46+/-5%. Patients with NVE had a higher 10-year survival rate (53+/-7%) than those with PVE (37+/-7%; p = 0.02). At 10 years, overall freedom from any late complication was 47+/-6% and from residual or recurrent endocarditis was 78+/-5%. Predictors of hospital death were emergency surgery (p <0.003) and preoperative renal insufficiency (p <0.008). Predictors of late death were age >70 years (p <0.002), renal failure (p <0.03) and fungal endocarditis (p <0.04)., Conclusion: These findings demonstrate the increased perioperative, as well as postoperative, risks associated with PVE versus NVE. Cardiac and extracardiac manifestations of the disease, as well as fungal organisms, but not the activity of the endocarditis, were significant adverse determinants of late outcome.

Background: The acceptance of skin allografts has historically been among the most challenging problems in the field of transplantation, attributed, at least in part, to the existence of antigens expressed by skin but not by other tissues. Many studies have suggested the existence of skin-specific antigens in rodents, but data in large-animal models are more limited., Methods: We have recently developed protocols for attaining stable mixed hematopoietic chimerism in miniature swine, using MHC-matched donors and recipients. We have now assessed tolerance to donor-derived skin and cardiac allografts in these chimeric animals., Results: Skin-graft rejection was seen in four of six animals receiving skin grafts taken from the respective hematopoietic donors. In the other two animals, donor-derived skin grafts survived indefinitely. No cardiac-allograft rejection was observed in mixed-chimeric animals that received heart transplants from their hematopoietic donors, even in animals that had already rejected skin allografts from the same donors. In all animals assessed, in vitro hyporesponsiveness to donor hematopoietic cells persisted., Conclusion: These findings support the concept that skin expresses immunogenic alloantigens that either are not expressed or are not immunogenic in cardiac or hematopoietic tissue.

The most common cause of chronic allograft loss is an incompletely understood clinicopathological entity called chronic rejection (CR). Recent reports suggest an improvement in long-term renal allograft survival, although it is not clear from these data whether a true reduction of biopsy-proven CR has occurred. Although newer immunosuppressive medications have greatly reduced the incidence of acute rejection (AR) in the early post-transplantation period, the ideal therapy for both AR and CR would be to achieve a state of tolerance. By definition, such a state should allow for indefinite allograft survival, with no histopathological evidence of CR, despite immunocompetence in the host (i.e. without the need for chronic immunosuppression). Although several experimental studies are able to achieve tolerance, with clear improvement in allograft survival, detailed studies on graft function and morphology are often not included. This review will discuss possible ways that tolerance induction could lead to a CR-free state. General mechanisms of CR and transplantation tolerance induction are discussed as well as the difficulties in translating small animals studies into large animals and humans.

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.

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the alpha(1) domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/-1.7 vs. 54.7 +/-3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

The mechanisms by which CTLA4Ig exerts its powerful immunomodulatory effects are not clear. We show here that CTLA4Ig can induce linked regulation of allogeneic porcine T cell responses in vitro. Naive miniature swine SLA(dd) T cells were rendered hyporesponsive to specific allogeneic Ag after coculturing with MHC-mismatched SLA(cc) stimulators in the presence of CTLA4Ig. These Ag-specific hyporesponsive T cells were subsequently able to actively inhibit the allogeneic responses of naive syngeneic T cells in an MHC-linked fashion, as the responses of naive SLA(dd) responders against specific SLA(cc) and (SLA(ac))F(1) stimulators were inhibited, but allogeneic responses against a 1:1 mixture of SLA(aa) (I(a), II(a)) and SLA(cc) (I(c), II(c)) were maintained. This inhibition could be generated against either class I or class II Ags, was radiosensitive, and required cell-cell contact. Furthermore, the mechanism of inhibition was not dependent upon a deletional, apoptotic pathway, but it was reversed by anti-IL-10 mAb. These data suggest that CTLA4Ig-induced inhibition of naive allogeneic T cell responses can be mediated through the generation of regulatory T cells via an IL-10-dependent mechanism.

Objective: To evaluate the influence of inflammatory activity and glucocorticoid (GC) treatment on serum parathyroid hormone (s-PTH) and bone metabolism in patients with rheumatoid arthritis (RA). Furthermore, in patients with active RA, to examine the PTH secretion and Ca2+ set point before and after treatment with GC., Methods: A range of biochemical markers of bone metabolism and calcium homeostasis were measured in 95 patients with definite RA stratified into groups according to disease activity and GC treatment. In a subgroup of 12 patients with active disease, initiating slow-acting-anti-rheumatic-drugs (SAARDs) +/- GC, the PTH secretion and calcium set point were evaluated by use of the Cica clamp technique before and after 1 month of treatment., Results: S-osteocalcin, s-total alkaline phosphatase (s-TAP) and s-carboxyterminal cross-linked telopeptide of type I collagen (s-ICTP) were elevated in all groups. The levels of urine pyridinoline (Pyr) and s-albumin-corrected calcium (s-AlbCorrCa2+) were elevated in patients with active disease and patients treated with GC. S-PTH and s-phosphate were within normal ranges. S-TAP, s-ICTP, Pyr and s-AlbCorrCa2+ correlated positively with indices of disease activity. In the subgroups undergoing the Cica clamp technique, no difference in PTH responsiveness of B-Ca2+ was unveiled., Conclusion: Neither active disease nor GC therapy appears to induce secondary hyperparathyroidism, nor is there an alteration in PTH responsiveness of B-Ca2+ in patients with RA. The increased levels of markers of type I collagen metabolism (s-ICTP, Pyr) and s-AlbCorrCa2+ in patients with active disease and patients treated with GC may be a result of increased degradation in synovium, cartilage and bone due to the inflammatory process.

Infection of an ascending aortic prosthesis is a grave complication associated with a high mortality. In most cases, extraanatomic bypass and removal of the infected vascular graft are not possible. Furthermore, the standard approach to this problem, which includes excision and replacement or debridement and repair of infected thoracic aortic grafts, carries a high early mortality. We report the successful treatment of this life-threatening complication using a conservative strategy in which the aortic prosthesis was salvaged by in situ disinfection followed by coverage with tissue flaps.

Background: Tolerance to cardiac allografts has not been achieved in large animals using methods that are readily applicable to human recipients. We investigated the effects of mixed hematopoietic chimerism on cardiac allograft survival and chronic rejection in miniature swine, Methods: Recipients were T-cell depleted using a porcine CD3 immunotoxin, and each received either of two nonmyeloablative preparative regimens previously demonstrated to permit the establishment of stable mixed hematopoietic chimerism across MHC-matched, minor antigen-mismatched histocompatibility barriers. Five to 12 months after the chimerism was induced, hearts from the original cell donors were heterotopically transplanted into the stable mixed chimeras., Results: Cardiac allografts transplanted into untreated recipients across similar minor antigen barriers were rejected within 44 days (within 21, 28, 35, 39, 44 days among individual study subjects). In contrast, hearts transplanted into the mixed chimeras were all accepted long term ( > 153, > 225, > 286, > 362 days) without immunosuppressive drugs and developed minimal vasculopathy., Conclusions: Mixed hematopoietic chimerism, established in miniature swine using clinically relevant, non-myeloablative conditioning regimens, permits long-term cardiac allograft survival without chronic immunosuppressive therapy, significant vasculopathy, or graft-versus-host disease.

Objective: Cotransplantation of a donor kidney along with a heart allograft can induce tolerance to both organs and prevent cardiac allograft vasculopathy in miniature swine. To determine whether the tolerogenic effect of donor kidney cotransplantation was due to an effect specific to the kidney graft or to an increase in donor antigen load, we compared heart-kidney recipients with recipients receiving two class I disparate hearts or with recipients receiving donor peripheral mononuclear cells at the time of isolated heart transplantation., Methods: Recipients of major histocompatibility complex class I disparate allografts received 12 days of cyclosporine (INN: cyclosporin; 10-13 mg/kg administered intravenously on days 0-11). Group 1 animals received a heart alone (n = 5). Group 2 animals received heart and kidney allografts (n = 4). Group 3 animals received two major histocompatibility complex-matched heart allografts (n = 4). Two double-heart recipients were thymectomized 21 days before transplantation. Group 4 animals received a heart allograft and an infusion of high-dose donor peripheral blood leukocytes (2.5 x 10(9) cells/kg, n = 2)., Results: Vasculopathy developed in group 1 recipients and the allografts were rejected within 55 days. Group 2 recipients accepted their heart and kidney allografts indefinitely without vasculopathy. Euthymic recipients from group 3 accepted their hearts long-term (>190 and >197 days), but vascular lesions developed. In thymectomized recipients from group 3, the hearts were rejected in 63 and 96 days with severe vasculopathy. Group 4 recipients demonstrated transient macrochimerism but their hearts were rejected within 47 and 63 days., Conclusions: The beneficial effects of donor kidney cotransplantation on cardiac allograft survival and prevention of cardiac allograft vasculopathy are likely to involve both an increase in donor antigen load and an effect specific to the kidney allograft.

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.