Posttransplant lymphoproliferative disorder after liver transplantation in miniature swine.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a well-known, life-threatening complication of immunosuppressive therapy, occurring in both adult and pediatric transplant recipients. METHODS: To study the effect of major histocompatibility complex on tolerance induction to primarily vascularized liver allograft, a semi-identical miniature swine model was developed to mimic the clinical situation of parent-into-infant liver transplantation. Long-term acceptance of semi-identical liver allograft was obtained by a transient course of FK506. In a subgroup of six animals, three developed a lethal PTLD. These animals were studied by histology and immunohistochemistry and the anti-donor cellular immune response was assessed. In addition, the possible viral origin of the proliferative process was evoked. RESULTS: Histology and immunohistochemistry revealed an abnormal B-cell proliferation in many organs of swine suffering from PTLD. Evidence of human Epstein-Barr virus, cytomegalovirus, and adenovirus was not evidenced, but a porcine virus responsible for a respiratory and reproductive syndrome (PRRS) was identified in the lymphoid tissue of these animals. In mixed lymphocyte reaction, a significant antiself immune response confirmed an infection by a virus. CONCLUSIONS: This is the first report suggesting that PRRS virus might provoke PTLD in immunosuppressed miniature swine after orthotopic liver transplantation. Whether PTLD could be induced by injection of the PRRS virus in immunosuppressed animals, a pig model of PTLD might be developed and would represent an interesting preclinical model for testing anti-PTLD therapies.     

Immunological unresponsiveness in chimeric miniature swine following MHC-mismatched spleen transplantation

BACKGROUND: In rodents, spleen allotransplantation (SpTx) induces tolerance. We investigated the induction of chimerism and donor-specific unresponsiveness following pig SpTx. METHODS: Thirteen pigs underwent splenectomy (day 0); all received a blood transfusion. In 11/13 pigs, SpTx was performed across a MHC class I (n=1) or full (n=10) barrier; two control pigs received no SpTx. All pigs were monitored for chimerism, and anti-donor immune responses, including suppressor assays. Four pigs (two asplenic controls and two with SpTx) underwent delayed donor-matched kidney transplantation without immunosuppression. RESULTS: Six of the 11 spleen grafts were lost from rejection (n=5) or splenic vein thrombosis (n=1), and five remained viable. All 11 SpTx recipients developed multilineage chimerism, but chimerism was rapidly lost if the graft failed. Two control pigs showed <6% blood chimerism for 4 and 11 days only. Pigs with functioning spleen grafts had multilineage chimerism in blood, thymus and bone marrow for at least 2-6 months, without graft-versus-host disease. These pigs developed in vitro donor-specific hyporesponsiveness and suppression. In 2 pigs tolerant to the spleen graft, donor MHC-matched kidney grafts survived for >4 and >7 months in the absence of exogenous immunosuppression; in two asplenic pigs, kidney grafts were rejected on days 4 and 15. CONCLUSIONS: Successful SpTx can result in hematopoietic cell engraftment and in vitro donor-specific unresponsiveness, enabling prolonged survival of subsequent donor-matched kidney grafts without immunosuppression.     

Simultaneous en-bloc allotransplantation of pancreas and kidney in the animal model. Comparison of separate organ and en-bloc pancreas/kidney transplantation in swine]

The high technical complication rate of pancreas transplantation requires large animal models to improve clinical transplant survival rates. The pig is a very suitable animal due to its anatomy, physiology and immunology which are similar to humans. In this study a model of en-bloc simultaneous pancreas and kidney transplantation was established which--in contrast to separate transplantation of both organs--decreases preservation time, operation time, and clamp time. Furthermore, the rates of intra- and postoperative complications were reduced compared with separate transplantation. The donor aorta (encompassing celiac axis, superior mesenteric artery, and left renal artery) is anastomosed en-bloc to the recipients aorta in a an oblique-to-side fashion. The portal vein is anastomosed end-to-side to the left common iliac vein. The exocrine pancreatic secretions are drained via duodenocystostomy to allow for monitoring of urinary amylase for rejection. The en-bloc technique is an alternative for pediatric donor organs since the risk of vascular complications is lower compared with separate implantation of the donor vessels. Based on our results in a large animal model the en-bloc technique could be used in adult uremic diabetic patients who receive a combined pancreas-kidney transplant from a pediatric cadaver donor.     

Tacrolimus versus cyclosporine induction therapy in pulmonary transplantation in miniature swine.

OBJECTIVE: Tacrolimus has been shown to provide superior immunosuppression in various solid organ transplant settings. The purpose of our study was to compare the survival of porcine lung allografts after induction with either cyclosporine A (CsA) or tacrolimus. METHODS: Single lung transplantation from MHC mismatched donors was performed in 10 minipigs. Immunosuppression included 1.5 mg/kg per day methylprednisolone and 1.0 mg/kg per day azathioprine. CsA (n=5) was adjusted to trough levels of 300-500 ng/ml, tacrolimus (n=5) was adjusted to 16-26 ng/ml. All immunosuppressive drugs were discontinued on postoperative day (POD) 28. Allograft survival was monitored by sequential chest radiographs, bronchoscopy and transbronchial biopsy histology. Peripheral blood leukocytes were scanned for donor chimerism and CD3, CD4, CD8 and CD25 expression. RESULTS: The animals survived a 4-week course of immunosuppression without radiological or histological signs of rejection on POD 28. Median allograft survival in CsA-treated animals was 55+/-15 days and all animals rejected their grafts within 42 days after withdrawal of immunosuppression. In tacrolimus-treated animals, median survival was 152+/-65 days with the longest survivor being electively sacrificed on POD 390 (P=0.0064). The degree of donor leukocyte chimerism and the frequency of CD4+CD25+ T-cells were higher in the tacrolimus group, however, these differences were not statistically significant. CONCLUSION: The results of our study show that primary immunosuppression with tacrolimus is superior to cyclosporine after pulmonary allotransplantation in a large animal model.     

Long-term acceptance of fully allogeneic cardiac grafts by cotransplantation of vascularized thymus in miniature swine

BACKGROUND: We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. METHODS: Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. RESULTS: All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. CONCLUSION: This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.     

Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I-mismatched renal allografts

BACKGROUND: Previous studies have demonstrated that long-term tolerance of class I mismatched renal allografts in miniature swine is induced by a short course of cyclosporine (CyA), and that a total thymectomy 21 days before transplantation abrogates the induction of stable tolerance. We have now examined the effects of surgical manipulation of the thymus, with or without a reduction in the thymic volume, on the induction of tolerance. MATERIALS AND METHODS: Miniature swine receiving a transplant of a class I-mismatched renal allograft and 12 days of CyA underwent either (1) a partial thymectomy 21 days before kidney transplantation (day -21), (2) serial thymic biopsies (to evaluate the effect of surgical trauma and reduction in volume of the thymus) or serial incisions of the thymus thymus (to evaluate the effect of surgical trauma without changes in thymic volume), (3) a sham thymectomy on day -21, or serial sham thymic surgery on the same POD as the thymic biopsies and incisions (control animals). RESULTS: Control animals had a stable plasma creatinine, had donor-specific unresponsiveness in cell-mediated lympholysis (CML) assays, had absence of rejection in kidney biopsy specimens, and did not develop anti-donor class I immunoglobulin (Ig)G alloantibodies. Animals undergoing a partial thymectomy on day -21 or serial thymic biopsies showed severe renal dysfunction, histological evidence of rejection in kidney biopsy specimens and anti-donor reactivity in CML assays; all but one animal developed anti-donor class I IgG alloantibodies. Serial incisions of the thymus induced an increase in plasma creatinine and histological rejection in 1 of 3 animals and anti-donor cytotoxic T cells in vitro in all 3 animals. CONCLUSIONS: A partial thymectomy or serial thymic biopsies markedly interfere with the induction of tolerance to renal allografts. Serial thymic incisions also interfere with the induction of tolerance, but to a lesser degree. These studies may have implications for tolerance-inducing protocols that involve thymic manipulation.     

Posttransplant lymphoproliferative disease after allogeneic transplantation of the spleen in miniature swine

Spleen transplantation (SpTx) was performed in miniature swine across full major histocompatibility complex barriers to study the tolerogenic effect of the spleen. This study describes the development of posttransplant lymphoproliferative disease (PTLD) after allogeneic SpTx. Recipient pigs underwent whole body irradiation (100 cGy), thymic irradiation (700 cGy), and native splenectomy (day 0), and received a 45-day course of intravenous cyclosporine (trough level 400-800 ng/ml). After SpTx, two of seven pigs developed PTLD (1 donor-type, 1 host-type). These two pigs had greater T cell depletion and higher trough levels of cyclosporine. Early changes that occurred prior to the development of clinical features of PTLD were increased porcine lymphotropic herpesvirus-1 viral loads in blood and tissues, and increased numbers of leukocytes, B cells, and total serum IgM. PTLD can occur after allogeneic SpTx in swine. This model may be useful in studies of the pathogenesis of PTLD.     

Skin-specific alloantigens in miniature swine

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.     

Role of the thymus and kidney graft in the maintenance of tolerance to heart grafts in miniature swine

BACKGROUND: The authors have examined the mechanism whereby co-transplantation of a kidney and heart from the same donor induces and maintains tolerance to both organs in miniature swine. METHODS: Transplants were performed across a major histocompatibility complex class I mismatch, and recipients received cyclosporine for 12 days. Group 1 animals received heart transplants alone (n=5), and all other groups received both heart and kidney allografts. Group 2 animals received no further intervention (n=2). Group 3 animals underwent transplant nephrectomy 8 days after heart and kidney co-transplantation (n=2). Group 4 animals underwent transplant nephrectomy 100 days after co-transplantation (n=2). Skin grafts were placed on group 4 animals, on one group 3 animal, and on two animals from group 2. Group 5 animals underwent thymectomy 100 days after co-transplantation (n=4). RESULTS: Group 1 animals developed cardiac allograft vasculopathy (CAV) and rejection. Group 2 animals never developed CAV and demonstrated in vitro donor-specific unresponsiveness. Group 3 animals suffered CAV and rejection. Group 4 animals developed CAV without concomitant donor-specific cell-mediated lympholysis reactivity, interstitial rejection, or cessation of graft function. Skin grafts on group 3 and group 4 animals led to fulminant rejection of heart and skin grafts, in contrast to grafts on group 2 animals that had no in vivo effect. Group 5 animals developed CAV but no significant increase in interstitial infiltrates. CONCLUSIONS: Both the kidney and thymus were necessary for maintenance of tolerance to heart allografts.     

Bone marrow transplantation in miniature swine. I. Development of the model

Procedures for successful autologous and MHC-matched allogeneic bone marrow transplantation in partially inbred, MHC-defined miniature swine have been established. All marrow recipients were conditioned with single-dose total-body irradiation at the upper level of tolerance, and supported with antibiotics and irradiated blood products during aplasia. Surgical harvest of autologous and allogeneic marrow yielded sufficient numbers of cells to successfully reconstitute recipients. Radiation control animals that received no marrow failed to show recovery of marrow function. Pigs transplanted with autologous marrow at doses greater than 10(8) cells/kg routinely engrafted and recovered normal marrow function. The major clinical complications were acute and chronic infections and hemorrhage. T cell-depleted autologous marrow also engrafted, and there was no observed increase in clinical complications. In bone marrow transplantation across non-MHC allogeneic differences, engraftment and survival were similar to that observed for autologous transplants. The T cell depletion of marrow in such MHC-matched allogeneic recipients was associated in one animal, however, with early reconstitution by cells of autologous origin.     

Role of the thymus in transplantation tolerance in miniature swine: V. Deficiency of the graft-to-thymus pathway of tolerance induction in recipients of cardiac transplants

BACKGROUND: We have previously shown that both thymic immigrants (graft to thymus pathway) and thymic emigrants (thymus to graft pathway) are involved in tolerance to renal allografts in miniature swine treated with a short course of calcineurin inhibitors. This study investigates the role of these pathways in cardiac transplant survival in recipients treated with a short course of tacrolimus. METHODS: Eleven animals received two-haplotype fully MHC-mismatched cardiac grafts with a 12-day course of tacrolimus. Recipients were thymectomized on day -21 (n=5) or day 0 (n=3), or were left euthymic (n=3). Two of the day -21 thymectomized animals received a day 0 host-MHC matched thymocyte infusion. RESULTS: Euthymic recipients of cardiac grafts treated with an immunosuppressive regimen identical to that previously shown to induce tolerance in euthymic recipients of renal allografts all rejected their grafts. Although no animal became tolerant, animals that were euthymic or thymectomized on day 0, as well as recipients of day 0 host-type thymocyte infusions following thymectomy on day -21, developed donor-specific hyporesponsiveness and maintained their cardiac grafts for markedly prolonged periods. In contrast, all animals thymectomized on day -21 that did not receive thymocyte infusions developed strong antidonor CTL responses and rejected their grafts by day 35. CONCLUSIONS: The graft-to-thymus pathway that plays an important role in tolerance induction to renal allografts appears to be relatively deficient in recipients of cardiac grafts. Strategies to increase donor antigen migration to the host thymus might therefore assist in tolerance induction to cardiac allografts.     

Induction of kidney transplantation tolerance across major histocompatibility complex barriers by bone marrow transplantation in miniature swine

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.     

Vascularized islet-cell transplantation in miniature swine. I. Preparation of vascularized islet kidneys

BACKGROUND: Whereas clinical pancreatic transplantation has been highly successful in correcting the hyperglycemia of insulin-dependent diabetes mellitus (type 1), the results of islet transplantation have been disappointing. This discrepancy may be because of, at least in part, nonspecific loss of islets during the time required for revascularization. To test this hypothesis, we have designed composite kidney grafts containing vascularized autologous islets that can be used to compare the engraftment potential of vascularized versus nonvascularized islet tissue. METHODS: (1) Islet-cell isolation: miniature swine underwent either partial pancreatectomy to isolate autologous islets or total pancreatectomy to isolate minor antigen-mismatched islets. Islets were purified from excised pancreatic tissue by enzymatic digestion and discontinuous density gradient purification. Isolated islets were cultured for 3 days before transplant. (2) Creation of vascularized islet kidneys (IK): autologous islets alone (n=6), minor-mismatched islets alone (n=3), and minor-mismatched islets plus simultaneous autologous thymic tissue (n=3) were transplanted beneath the renal capsule of juvenile miniature swine. Minor antigen-mismatched islets were also transplanted into both the vascularized thymic graft of a thymokidney (to produce a thymo-islet kidney [TIK]) and the contralateral native kidney (n=3) and both the host thymus and beneath the renal capsule (n=2). All recipients receiving minor-mismatched islets were treated with a 12-day intravenous (IV) course of either cyclosporine A (CsA) at 10 mg/kg per day or FK506 at 0.15 mg/kg per day. (3) Assessment of Function: to evaluate the function of the transplanted islets, three animals bearing TIK and IK underwent total pancreatectomy 3 months following islet transplantation. RESULTS: (1) Islet-cell yields: an average of 254,960+/-51,879 (4,452+/-932 islet equivalents [IEQ]/gram of pancreas) and 374,410+/-9,548 (4,183+/-721 IEQ/gram of pancreas) viable islets were obtained by partial pancreatectomy and complete pancreatectomy, respectively. (2) Creation of IK: autologous islets engrafted indefinitely, whereas recipients of minor-mismatched islets alone rejected the islets within 2 months. However, when minor-mismatched islets were implanted into both the thymokidney and the contralateral kidney of animals bearing a thymokidney, the islets engrafted indefinitely in both sites (>3 months). Simultaneous implantation of islets into the host thymus and under the renal capsule also led to permanent engraftment of minor-mismatched islets. (3) Function of vascularized islets: three animals with both a TIK and an IK in place for 3 months underwent total pancreatectomy. All three animals maintained normoglycemia thereafter. In two of these animals, the IKs were removed 2 months after the pancreatectomy, and in both cases normoglycemia was maintained thereafter by the TIK. CONCLUSIONS: The implantation of islets beneath the autologous renal capsule permitted the establishment of a vascular supply and thereby supported normal islet-cell growth and function. The presence of thymic tissue beneath the autologous renal capsule facilitated the engraftment of minor-mismatched islets, and such grafts achieved results similar to autologous islet transplants. Therefore, the ability to create vascularized islet grafts may provide a strategy for successful islet transplantation across allogeneic and potentially across xenogeneic barriers.     

Transplantation in miniature swine. VIII. Recombination within the major histocompatibility complex of miniature swine

Offspring of heterozygous parents derived from three herds of miniature swine, each of which is homozygous at the major histocompatibility complex (MHC), were screened for recombination within the MHC. The swine were typed serologically at weaning and later typed by mixed lymphocyte reaction (MLR). Two intra-MHC recombinants were discovered, both of which involved the exchange of D region specificities without apparent dissociation of ABC region specificities, confirming the localization of the SLA-D region outside of the SLA-ABC regions. The first recombinant was the offspring of an SLAc/d (cd) by dd mating and typed serologically as cd but typed by MLR as dd. The second recombinant was the offspring of a cd by cd mating. It typed serologically as cc but stimulated cc in one-way MLR and retained its reactivity to dd, thus suggesting a possible recombination within the D region. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 3H-leucine-labeled lymphocyte surface antigens demonstrated that corresponding Ia antigens were also exchanged during these recombinant events supporting the hypothesis that genes coding for Ia antigens are identical or closely linked to D region genes encoding the MLR specificities.