Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine

BACKGROUND: Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. METHODS: Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day coprse of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. RESULTS: Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6-9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. CONCLUSIONS: Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.     

Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft

BACKGROUND: An important component of a composite tissue limb allograft (CTA) is the vascularized bone marrow and bone marrow stroma, which when transplanted could create immediate marrow space and engraftment. We have previously demonstrated that tolerance to musculoskeletal allografts can be achieved with a 12-day course of cyclosporine without the presence of long-term peripheral donor cell chimerism. The objective of this study was to determine the fate of the donor bone marrow after transplantation of a limb allograft in a miniature swine model. METHODS: CTAs from donor swine were heterotopically transplanted into six MHC-matched, minor-antigen-mismatched recipients, and a 12-day course of cyclosporine was given. Previous animals transplanted without cyclosporine rejected their grafts in less than 42 days. A non-MHC-linked marker, pig allelic antigen (PAA), was used to distinguish host and donor cells. Three PAA- animals received PAA+ CTAs, and three PAA+ animals received PAA- CTAs. Bone marrow was harvested from the donor limb grafts and the recipient and analyzed by flow cytometry and histology. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine and evaluated for the presence of donor cells by flow cytometry. RESULTS: All animals receiving cyclosporine demonstrated permanent tolerance to their allografts. Donor bone marrow cells were present in all grafts at the time of transplantation and during the immediate postoperative period. By 48 weeks, donor cells were no longer detectable within the marrow space of the allograft. In long-term animals host bone marrow cells replaced donor cells in the graft marrow space. No evidence of donor cell engraftment was found in recipient animals. CONCLUSION: This study demonstrates that in long-term tolerant recipients of musculoskeletal allografts there is no evidence of persistent donor bone marrow cells in the hematopoietic tissues of the graft or the host. Rather, the recipient's bone marrow cells and lymphocytes repopulate the donor marrow space of the graft.     

Long-term acceptance of renal allografts following prenatal inoculation with adult bone marrow

BACKGROUND: The aim was to investigate if intravascular in utero injection of adult bone marrow into swine fetuses could lead to macrochimerism and tolerance to the donor. METHODS: Outbred Yorkshire sows and boars screening negative for MHC allele SLA of MGH miniature swine were bred. A laparotomy was performed on the sows at 50 days gestation to expose the uterus. Bone marrow harvested from SLA miniature swine was T-cell depleted and injected intravascularly into seventeen fetuses. Flow cytometry was performed to detect donor cells (chimerism) in the peripheral blood after birth. Mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays were used to assess the response to donor MHC. Previously frozen skin grafts from the bone marrow donor were placed on the offspring from the first litter. Donor-matched renal transplant from SLA donors were performed on chimeric swine, with and without a short 12-day course of cyclosporine, and one nonchimeric littermate. RESULTS: Nine inoculated offspring demonstrated donor cell chimerism in the peripheral blood and lymphohematopoietic tissues. All animals with detectable chimerism within the first three weeks were consistently nonreactive to donor MHC in vitro. Animals challenged with donor skin grafts displayed prolonged graft survival without producing antidonor antibodies. All chimeric animals accepted donor-matched kidney allografts, even one without cyclosporine. The kidney in the nonchimeric littermate rejected by day 21. CONCLUSIONS: Transplantation of allogeneic adult bone marrow into immunocompetent fetal recipients resulted in chimerism. In utero inoculation led to operational tolerance to the donor's major histocompatibility antigens and long-term acceptance to organ allografts.     

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.     

The failure of skin grafting to break tolerance to class I-disparate renal allografts in miniature swine despite inducing marked antidonor cellular immunity.

Long-term specific tolerance to one haplotype class I plus minor antigen disparate renal allografts develops without exogenous immunosuppression in approximately 35% of miniature swine (n = 128). Previous studies have suggested that this phenomenon is related to limited class I-specific helper T cell activity as evidenced by the failure of antibody class switching in vivo and the ability of exogenous interleukin 2 to elicit antidonor responses in vitro. To determine whether tolerance could be broken by inducing antidonor reactivity with donor antigen and a source of T cell help, multiple skin grafts bearing donor class I plus third-party class II antigens were placed on tolerant animals. Skin grafts were placed at least 3 months after the kidney transplant, at which time all recipients had normal renal function as measured by blood urea nitrogen and serum creatinine. First-set rejection of skin grafts by SLAad and SLAdd hosts occurred in 11.8 +/- 1.1 days (mean +/- SEM, n = 6) and in 9.3 +/- 0.9 days (n = 4), respectively. Coincident with skin rejection, most animals developed a transient rise in BUN to 62 +/- 11 mg/dl (n = 10) and a similar rise in Cr to 4.9 +/- 1.2 mg/dl (n = 10), with normal levels returning in all animals within two weeks. Subsequent skin grafts with the same disparity did not undergo second-set rejection and did not induce BUN or Cr elevations. Prior to skin grafting, animals showed no antidonor activity in mixed lymphocyte reaction or cell-mediated lymphocytotoxicity assays. After two skin grafts, all animals developed donor-specific CML and secondary MLR responses, and additional skin grafts amplified this cellular immunity. Development of marked antidonor immunity without a break in tolerance suggested that either graft adaptation or local suppression might be involved in maintaining tolerance to class I MHC antigens. In preliminary studies, an immunized SLAad animal and an immunized SLAdd animal were retransplanted with kidneys MHC matched to their first allografts. In both cases, the second graft was accepted permanently without immunosuppression, suggesting that graft adaptation is not necessary for the maintenance of tolerance to renal allografts in miniature swine.     

Transplantation in miniature swine. VI. Factors influencing survival of renal allografts.

Renal allografts were performed between and among animals from three herds of miniature swine that were selectively inbred to homozygosity at the major histocompatibility complex, MSLA. The results suggest several genetic factors which influence the survival of renal allografts in these animals. As expected, the major histocompatibility complex (MHC) was of dominant importance, and all MSLA-mismatched grafts were rejected promptly (12 +/- 3.7 days). Some MSLA-matched grafts were also rejected (30 +/- 15.0 days), indicating that non-MSLA loci also determine antigens which can lead to kidney rejection. Other MSLA-matched grafts were accepted indefinitely. At least one immune response gene that determined ability to reject kidneys across non-MSLA differences seemed to be segregating in our swine population. Animals that had accepted MSLA-matched renal grafts for extended periods demonstrated markedly prolonged survival of subsequent donor skin grafts compared to skin graft survival across the same non-MSLA difference in normal animals. This finding suggests that failure to reject kidneys across non-MSLA differences indicates systemic tolerance, and that there may be a relationship between the induction of such tolerance and the proposed immune response gene controlling rejection.     

Induction of Tolerance to an Allogeneic Skin Flap Transplant in a Preclinical Large Animal Model

Clinical composite tissue allotransplantation can adequately reconstruct defects that are not possible by other means. However, immunosuppressant toxicity limits the use of these techniques. Clinical attempts to reduce the amount of immunosuppression required by induction of an immunologically permissive state have so far been unsuccessful. The aim of this study was to induce tolerance in a preclinical large animal model. Donor hematopoietic stem cell (HSC) engraftment was induced by T-cell depletion, irradiation, and a short course of cyclosporine administered to the recipient, along with a hematopoietic cell infusion from a single haplotype major histocompatibility complex (MHC) mismatched donor. Skin was then allotransplanted from the donor. Both primarily vascularized skin flaps and secondarily vascularized conventional skin grafts were allotransplanted to investigate if the mode of transplantation affected outcome. Control animals received the skin allotransplants without conditioning. Tolerance was defined as no evidence of rejection at 90 days following transplantation. Conventional skin grafts only achieved prolonged survival (<65 days) in HSC-engrafted animals (P < .01). In contrast, there was indefinite skin flap survival with the achievement of tolerance in HSC-engrafted animals; this was confirmed on histology with donor-specific unresponsiveness on MLR and CML. Furthermore, a conventional skin donor graft subsequently applied to an animal tolerant to a skin flap was not rejected and did not trigger skin flap rejection. To our knowledge, this is the first time skin tolerance has been achieved across a MHC barrier in a large animal model. This is a significant step toward the goal of clinical skin tolerance induction.     

Induction of tolerance to heart transplants by simultaneous cotransplantation of donor kidneys may depend on a radiation-sensitive renal-cell population

BACKGROUND: To determine the mechanism by which cotransplantation of a donor kidney and heart allograft induces tolerance to both organs in miniature swine, we examined the renal elements responsible for tolerance induction. METHODS: Recipients received 12 days of cyclosporine, and transplants were performed across a major histocompatibility complex (MHC) class I mismatch. Group 1 animals received heart transplants (n=5); group 2 animals received heart and kidney allografts with no other manipulation (n=4); group 3 animals received heart transplants and donor-specific renal parenchymal cells (n=4); group 4 animals received heart and kidney allografts from lethally irradiated donors (n=7); group 5 animals received irradiated hearts and nonirradiated kidneys (n=2); group 6 animals received nonirradiated hearts and peripheral blood leukocytes from swine MHC matched to recipients and becoming tolerant to donor antigen (n=2); group 7 animals received nonirradiated hearts and donor-specific peripheral blood monocyte cells (PBMC) (n=2). RESULTS: Animals in group 1 developed vasculopathy and fulminant rejection by day 55. Animals in group 2 never developed vascular lesions. Parenchymal kidney cell infusion (group 3) did not prolong cardiac survival. Animals in group 4 developed arteriopathy by postoperative day (POD) 28. Group 5 recipients accepted allografts without vascular lesions. Adoptive transfer of leukocytes from tolerant swine (group 6) prolonged cardiac graft survival as much as 123 days, whereas donor PBMC infusion (group 7) did not affect cardiac survival or development of arteriopathy. CONCLUSIONS: Radiosensitive elements in kidney allograft may be responsible for tolerance induction and prevention of chronic vascular lesions in recipients of simultaneous heart and kidney allografts.     

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.     

Experimental vascularized bone allografting

Presented here is a compendium of studies investigating the fate of vascularized bone allografts. The first set of experiments employ the posterior rib graft in two canine models. The rib-to-mandible model was used to evaluate the rejection phenomena of vascularized bone allografts in an outbred dog model. This ascertained the time course of rejection and histological characteristics of the grafts. Immunosuppression of the graft recipients was attempted with azathioprine and cyclosporine. The results demonstrated that azathioprine was not an effective immunosuppressant, whereas cyclosporine resulted in survival of cortical osteons. The use of the vascularized rib allograft, with and without azathioprine, to bridge the defect in the dog femur was met with failure. Further studies employed a genetically defined rat model to determine the effect of different histocompatibilities on the survival of vascularized knee allografts. Grafts were transplanted from Lewis rats to syngeneic Lewis rats as isografts and to Fischer-344 rats (F-344) and Brown-Norway rats (BN) as allografts. Grafts across a major histocompatibility barrier to BN were rejected by 7 days, whereas grafts across a weak histocompatibility barrier to F-344 were rejected more slowly. The use of cyclosporine in this model abrogated the rejection response when administered to both groups continuously. However, a short course of cyclosporine was effective in preventing rejection in the F-344 animals. Efforts to induce tolerance by blood transfusions, from the donor strain or from a third-party donor, were not effective in preventing rejection.     

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.     

Long-term tolerance to kidney allografts in a preclinical canine model

Durable immune tolerance supporting vascularized allotransplantation offers the possibility of extending graft survival and avoiding harmful complications of chronic immunosuppression. Immune tolerance to renal allografts was induced in a preclinical canine model through engraftment of donor hematopoietic cells using a combination of low-dose total body irradiation and a short course of immunosuppression. Subsequently, donor renal allografts were transplanted accompanied by bilateral native nephrectomies. With 5-year follow up, we found normal renal function in all recipients and no histological evidence of acute or chronic rejection. This tolerance does not extend universally to donor skin grafts, however, with two of four animals rejecting delayed donor skin grafts. Hematopoietic chimerism produces durable and robust immune tolerance to kidney allografts, although incomplete tolerance to donor skin grafting.     

Spontaneous acceptance or rejection of orthotopic liver transplants in outbred and partially inbred miniature swine.

BACKGROUND: Results of clinical liver transplantation have shown that rejection and loss of human liver allografts occurs despite immunosuppression. Because genetic disparity and liver immunogenicity remain a matter of controversy, we reexamined the fate of outbred liver allografts without immunosuppression and used partially inbred miniature swine, in which the genetics of major histocompatibility complex (MHC) antigens have been characterized and can be controlled. METHODS: Orthotopic liver transplantation was performed between pairs of outbred domestic farm pigs and between pairs of inbred miniature swine with genetically defined major histocompatibility (SLA) loci. A passive splenic and vena caval to jugular vein shunt with systemic heparinization prevented hypotension during the anhepatic phase. Immunological responses were monitored by mixed lymphocyte culture (MLC), CML, skin graft rejection, liver biopsies, and serial serum chemistries. RESULTS: Median survival of technically successful liver allografts between pairs of outbred pigs (n=20) was 38 days and between partially inbred swine matched at the SLA locus (n=17) was 79 days. MLC responsiveness did not correlate with the development of rejection. Five of 20 (25%) outbred pigs and 6 of 17 (35%) MHC matched inbred miniature swine survived more than 100 days. In the long-term survivors, donor, but not third party, MHC matched skin graft survival times were prolonged. In contrast, all SLA-mismatched inbred recipients (n=26) died rapidly from massive liver rejection, with a median survival time of 9 days. In these rejecting animals, the marked MLC responsiveness to donor lymphocytes evident pretransplant diminished rapidly after transplantation, but an undiminished PHA responsiveness and a blunted third party MLC response persisted. CONCLUSION: The length of survival and the degree and incidence of rejection were similar in outbred pigs and in SLA-matched inbred miniature pigs, indicating that the outbred animals were, therefore, probably closely related and shared relevant genes. However, survival was significantly shortened and liver allograft rejection was accelerated in SLA-mismatched inbred swine. These results indicate that major histocompatibility differences play an important role in the rejection of liver allografts, as is true for other vascularized grafts in the unimmunosuppressed recipient. The development of liver allograft rejection across non-MHC differences is variable and, when present, appears to be a chronic process.     

Heart and en-bloc thymus transplantation in miniature swine

BACKGROUND: Donor-specific tolerance to organ allografts might be induced by cotransplantation of a sufficient amount of vascularized donor thymus. To facilitate donor thymus-induced cardiac allograft tolerance, we have developed a novel technique for heart and en-bloc thymus transplantation in swine. METHODS: Donor heart and en-bloc thymus grafts were prepared by a technique that preserves the entire arterial supply and venous drainage of the right thymic lobe. En-bloc grafts (n = 4) were transplanted heterotopically into the abdomens of major histocompatibility complex-matched miniature swine. Recipients received 12 days of cyclosporine intravenously. Grafts were monitored by palpation, electrocardiographic monitoring, and periodic open biopsy. Engraftment of the donor thymus was demonstrated by measuring the proportion of recipient-type thymocytes in the donor thymus with flow cytometry. RESULTS: All of the heart and en-bloc thymus grafts had normal cardiac contractility and immediate perfusion of the thymus. All en-bloc grafts were accepted for more than 200 days without significant acute cellular rejection or cardiac allograft vasculopathy. Thymic tissue of en-bloc grafts displayed normal architecture and supported thymopoiesis of recipient-type cells. CONCLUSION: We have validated a new technique of donor thymus transplantation that could have utility in human heart transplantation.     

Mechanism of tolerance following class I--disparate renal allografts in miniature swine. Cellular responses of tolerant animals

Previous studies utilizing a recombinant MHC haplo-type in our partially inbred miniature swine herd have demonstrated that some recipients matched only for SLA class II show long-term acceptance of renal allografts without exogenous immunosuppression. Such animals have been shown to develop systemic tolerance as evidenced by prolonged rejection times of subsequent donor-specific, but not third-party, skin grafts. In the present studies in vitro cellular responses of long-term tolerant animals and of 7 animals studied sequentially are presented. Long-term tolerant animals demonstrated responses consistent with the absence of the class I reactive helper populations normally present in naive controls. Animals studied sequentially segregated into two groups based on cellular reactivity and survival. All animals showed complete loss of antidonor class I cell-mediated lymphocytolytic (CML) reactivity by postoperative day 10. However, animals surviving less than 20 days maintained CML reactivity to donor class I plus third-party class II in the posttransplant period, while animals surviving greater than 40 days lost such reactivity. Addition of exogenous interleukin 2 could not reverse this loss. These studies suggest that tolerance induction to a renal allograft across a class I only difference involves effects on both helper and killer class I reactive cell populations.     

Role of persistence of antigen and indirect recognition in the maintenance of tolerance to renal allografts

BACKGROUND: We have previously shown that a 12-day treatment with cyclosporine A (CyA) facilitates induction of tolerance to class-I disparate kidneys, as demonstrated by acceptance of second, donor-matched kidneys without immunosuppression. In the present study, we have examined 1) the duration of tolerance in the absence of donor antigen and 2) the pathway of antigen recognition determining maintenance or loss of tolerance. METHODS: Seventeen miniature swine received class-I mismatched kidneys with 12 days of CyA, and received second donor-matched kidneys without immunosuppression at 0, 1, 3, or 4 months after nephrectomy of the primary graft. Five were sensitized 6 weeks after nephrectomy of the primary graft, three with donor-matched skin grafts, and two with donor class-I peptides to eliminate direct pathway involvement. In addition, two long-term tolerant animals received class-I peptides. RESULTS: Rejection of second grafts required at least a 3 month absence of donor antigen. Although donor-matched skin grafts in animals tolerant to kidneys induced antidonor cytotoxic T lymphocyte responses, second renal transplants revealed no evidence of sensitization. In contrast, immunization of recipients with donor class-I peptides after nephrectomy of the primary graft led to loss of tolerance at both T-cell and B-cell levels, as evidenced by rejection of the second graft in 5 days and development of antidonor immunoglobulin G. Peptide immunization of long-term tolerant in recipients bearing long-term renal grafts did not break tolerance. CONCLUSIONS: These data indicate that the renal allograft is required for the indefinite maintenance of tolerance, that indirect antigen presentation is capable of breaking tolerance, and that in tolerant animals, direct antigen presentation may suppress rejection, allowing tolerance to persist.     

Detection of regulatory cells as an assay for allograft tolerance in miniature swine.

BACKGROUND: There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized. METHODS: Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen. RESULTS: Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells. CONCLUSIONS: This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.     

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

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

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.     

The effect of thymectomy on tolerance induction and cardiac allograft vasculopathy in a miniature swine heart/kidney transplantation model.

BACKGROUND: We have previously demonstrated that MHC class I disparate hearts transplanted into miniature swine treated with a short course of cyclosporine developed florid cardiac allograft vasculopathy (CAV) and were rejected within 55 days. However, when a donor-specific kidney is cotransplanted with the heart allograft, recipients become tolerant to donor antigen and accept both allografts long-term without the development of CAV. In the present study, we have investigated the role of the host thymus in the induction of tolerance and prevention of CAV in heart/kidney recipients. METHODS: Total thymectomies were performed in six animals (postoperative day [POD]-21), which on day 0 received either an isolated MHC class I disparate heart allograft (n=3) or combined class I disparate heart and kidney allografts (n=3), followed in both cases by a 12-day course of cyclosporine (POD 0-11). Graft survival and the development of CAV in these thymectomized recipients were compared to the same parameters in non-thymectomized, cyclosporine-treated recipients bearing either class I disparate heart allografts (n=5) or heart and kidney allografts (n=4). RESULTS: In the group of animals bearing isolated class I disparate heart allografts, the thymectomized recipients rejected their allografts earlier (POD 8, 22, 27) than the non-thymectomized recipients (POD 33,35,45,47,55). The donor hearts in both the thymectomized and non-thymectomized animals developed florid CAV. In the group of animals bearing combined class I disparate heart and kidney allografts, the nonthymectomized recipients accepted both donor organs long term with no evidence of CAV. In contrast, none of the thymectomized heart/kidney recipients survived >100 days, and they all developed the intimal proliferation of CAV. CONCLUSION: Thymic-dependent mechanisms are necessary for the induction of acquired tolerance and prevention of CAV in porcine heart/kidney recipients.