Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning

BACKGROUND: We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. METHODS: Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day -2. Cyclosporine was administered for 30 to 60 days beginning on day -1. A total of 1 to 2 x 10(10) /kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. RESULTS: Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. CONCLUSIONS: These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.     

Delayed non-myeloablative irradiation to induce long-term allograft acceptance in a large animal lung transplantation model

We previously induced long-term allograft acceptance in an allogeneic lung transplantation (LTx) model in miniature swine using perioperative non-myeloablative irradiation (IRR) combined with infusion of donor specific alloantigen. In order to improve clinical applicability, we delayed induction with irradiation in this study. Left sided single LTx was performed in minipigs. Group 1 received non-myeloablative irradiation (7Gy thymus and 1.5Gy whole body IRR) before LTx and a perioperative donor specific splenocyte infusion (SpTx). Group 2 received perioperative SpTx but delayed IRR three days after LTx. Group 3 was exposed to delayed IRR without SpTx. Whereas 4 out of 7 animals from the non-delayed group never rejected their grafts and were electively sacrificed on postoperative day (POD) +500, all animals from group 2 rejected their grafts before POD 108. In group 3, 3 out of 8 animals developed long-term allograft acceptance. In all groups, donor leukocyte chimerism peaked up to 20% in peripheral blood one hour after reperfusion of the lung. Group 1 maintained prolonged chimerism beyond POD 7, whereas chimerism levels in groups 2 and 3 decreased continuously thereafter. Delayed irradiation has the potential to improve long-term graft survival, yet not as efficient as a perioperative conditioning protocol.     

Augmentation of Transient Donor Cell Chimerism and Alloantigen‐Specific Regulation of Lung Transplants in Miniature Swine

Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major histocompatibility complex–mismatched single lung transplantation was performed in 28 minipigs followed by a 28‐day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low‐dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4⁺CD25^high+ T cell frequencies were detected in peripheral blood associated with decreased interferon‐γ production of leukocytes. Secondary third‐party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third‐party splenocytes or donor splenocyte protein extracts. While animals treated with third‐party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third‐party, splenocyte infusions may develop long‐term donor‐specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.     

CTLA4-Ig-based conditioning regimen to induce tolerance to cardiac allografts

BACKGROUND: Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. METHODS: Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. RESULTS: Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. CONCLUSIONS: The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.     

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

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

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

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

The Indirect Alloresponse Impairs the Induction but Not Maintenance of Tolerance to MHC Class I-Disparate Allografts

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

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine

BACKGROUND: Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. METHODS: Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. RESULTS: All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. CONCLUSIONS: This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.     

Mixed chimerism achieved by a nonlethal conditioning regimen induces donor-specific tolerance to lung allografts

BACKGROUND: Graft rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem in lung transplantation (Tx). Mixed hematopoietic chimerism has been shown to produce long-lasting donor-specific transplant tolerance without immunosuppressive drugs in animal models; however, most conditioning regimens required to achieve mixed chimerism are too toxic for clinical use. The aim of this study was to develop a nonlethal conditioning regimen to induce tolerance to lung allografts. METHODS: Four to 6-wk old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as organ donors and recipients, respectively. The recipient conditioning regimen included: 10 mg/animal antilymphocyte globulin (on day-5), 1 mg/kg/d tacrolimus (days 1 to 10), total body irradiation (500 cGy; day 0), and donor bone marrow (DBM) Tx (100 x 10(6) T-cell depleted cells on day 0 following irradiation). Six weeks after DBM Tx, chimeric animals received orthotopic left lung Tx. Graft survival was monitored by chest X-ray and histology. RESULTS: Long-term DBM engraftment was observed: hematopoietic chimerism in the peripheral blood was 12.4 +/- 3.4%, 36.7 +/- 14.1%, and 31.9 +/- 14.1% at 30 d, 6 mo, and 16 mo following DBM Tx, respectively. There was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>400 d) donor-specific lungs (RT1.A(a); n = 8), yet rapidly rejected (<8 d) third party hearts (RT1.A(l); n = 5). Graft (lung) tolerant (>150 d) chimeric recipients accepted secondary donor-specific heart grafts (>150 d; n = 4) but rejected third party heart grafts (<7 d; n = 3). Graft tolerant recipients demonstrated reduced (P < 0.05) in vitro donor-specific lymphoproliferative response and cytotoxicity, and no evidence of acute or chronic graft rejection. CONCLUSION: Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung 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.     

Transplantation tolerance through mixed chimerism: From allo to xeno

To date, the only successful means of achieving allogeneic transplantation tolerance in the clinic has involved induction of mixed lymphohematopoietic chimerism. Such chimerism was first achieved in mice and subsequently in large animals, including miniature swine, monkeys and most recently humans. The mechanism of tolerance has differed between models, involving both deletional and regulatory mechanisms, in varying proportions, depending on the model. Considerable progress has also been made toward induction of tolerance across the xenogeneic pig‐to‐primate barrier, although complete success has not yet been achieved. The two approaches toward xenograft tolerance currently being investigated both involve establishment of a mixture of host and donor cells in the thymus, in one case through administration of donor bone marrow to the recipient and in the other through vascularized donor thymus transplantation to a thymectomized recipient. Hopefully, a combination of these approaches may provide an effective means for achieving full tolerance and thereby bringing xenograft organ transplantation to the clinic.     

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA^cc of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA^cc swine donor was T‐cell depleted and injected intravenously into the fetuses between days 50–55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell‐mediated lympholysis (CML) assays to assess their response to donor. Donor‐matched VCAs from SLA^cc donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21 days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor‐specific tolerance of VCAs across a full MHC barrier in this animal model.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

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

Tolerance 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.     

The Induction of Tolerance of Renal Allografts by Adoptive Transfer in Miniature Swine

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I‐mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In this study, we attempted to induce tolerance of class I‐mismatched kidneys by adoptive transfer of cells and/or kidneys from long‐term tolerant animals. Fifteen SLA^dd miniature swine received 1.5 Gy whole body irradiation and class I‐mismatched (SLA^gg) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from long‐term tolerant (LTT) SLA^dd recipients of SLA^gg grafts. In addition, three SLA^dd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLA^dd recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.     

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 small bowel allograft function induced by short‐term FK 506 application is associated with split tolerance

Abstract Functional long‐term allograft survival after experimental small bowel transplantation (SBT) is limited by chronic rejection. Initial application of high‐dose FK 506 has been shown to induce stable long‐term graft function. In order to examine whether this long‐term function is associated with donor‐specific tolerance, we analyzed the functional status of recipient T cells in vivo and in vitro. One‐step orthotopic SBT was performed in the allogeneic Brown Norway (BN)‐to‐Lewis rat strain combination. FK 506 was given daily at a dose of 2 mg/kg from days 0‐5 in the rejection model and from days 0‐9 in the long‐term functional model. Mean survival time in the rejection model was 98 ± 2.8 days. Histological examination of these small bowel allografts disclosed signs of chronic rejection. In contrast, all animals of the long‐term functional model survived long term (> 250 days) without clinical signs of chronic rejection. The latter model, furthermore, produced evidence of donor‐specific tolerance. Whereas heterotopic Dark Agouti (DA) hearts were rejected regularly within 7 days, BN hearts survived indefinitely (> 70 days). In vitro, mixed leukocyte reactivity of CD4 + T cells was similarly strong against donor (BN) antigens as against third‐party (DA) antigens. The split tolerance revealed by our in vivo and in vitro results enabled acceptance of both the small bowel allograft without signs of chronic rejection and of donor‐specific heart allografts.     

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.     

Persistent chimerism despite antidonor MHC in vitro responses in miniature swine following allogeneic hematopoietic cell transplantation

BACKGROUND: T-cell chimerism predominates in miniature swine receiving hematopoietic-cell transplantation without myelosuppressive conditioning. Several chimeric recipients have become hyporesponsive to donor-major histocompatibility complex (MHC) in vitro and accepted donor-matched renal transplants without immunosuppression. However, some retained antidonor in vitro responses and subsequently rejected donor renal allografts despite the persistence of peripheral blood chimerism. In this study, we characterize the donor cells in both "tolerant" and "nontolerant" chimeric miniature swine. METHODS: Peripheral blood chimerism was determined by flow cytometry. In vitro antidonor responsiveness was determined by mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Donor cells were separated from chimeras by immunomagnetic bead separation and used as stimulators or targets in CML assays. Phenotypic analysis of donor cells in chimeras was performed using flow cytometry. RESULTS: Peripheral blood chimerism stabilized beyond 100 days and was made up almost entirely of T cells. PBMC from nontolerant chimeras could be stimulated in vitro to kill donor cells isolated from the mixed chimera itself. In contrast, PBMC from tolerant chimeras hyporesponsive to donor-type cells could not be stimulated in vitro to kill their own sorted donor cells. CONCLUSIONS: The in vivo persistence of donor T cells in mixed chimeric animals with in vitro antidonor responsiveness is not caused by an inability of these cells to be killed but rather by the poor stimulating capacity of these donor T cells. The nature of donor T cells that persist in the face of in vitro antidonor responses, has important implications for the induction of transplant tolerance by way of the generation of mixed chimerism.     

Donor Brain Death Inhibits Tolerance Induction in Miniature Swine Recipients of Fully MHC‐Disparate Pulmonary Allografts

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