Transplantation tolerance and mixed chimerism: at the frontier of clinical application

Although the persistence of donor-type hematopoietic cells in low numbers (microchimerism) is well established in some transplant recipients, its relevance for graft acceptance is still a matter of debate. On the other hand, clonal deletion of donor-specific alloreactive cells associated with mixed chimerism (macrochimerism) has reliably produced long-term graft tolerance in pre-clinical models. So far, the cytoablative conditioning regimens required to achieve mixed chimerism have hampered the clinical development of such protocols. Here, we discuss recent observations suggesting that the deliberate induction of hematopoietic cell chimerism might become a feasible strategy to achieve transplantation tolerance in clinics.     

Chimerism induction in vascularized bone marrow transplants augmented with bone marrow cells

Composite tissue allografts (CTAs) are currently accepted in the clinic; however, long-term immunosuppression is still needed for allograft survival. The presence of donor-specific chimerism may induce tolerance. Thirty-six vascularized bone marrow transplantation (VBMT) allotransplantation were performed across MHC barrier under short-term protocol of 7-day alphabeta-TCRmAb and Cyclosporin A therapy to determine the efficacy of VBMT alone and VBMT augmented with donor bone marrow transplantation (BMT) in chimerism induction. Flow cytometry analysis revealed that VBMT supported with donor BMT directly into the bone resulted in chimerism augmentation and maintenance compared to VBMT. In vivo and in vitro tolerance testing showed prolonged survival of donor skin graft up to 35 days and moderate reactivity in MLR assay that suggests only tolerance induction. Transplantation of vascularized bone without chronic immunosuppression provides a substantial source of bone marrow cells, leading to the development of stable donor-specific chimerism.     

New composite tissue allograft model of vascularized bone marrow transplant: the iliac osteomyocutaneous flap

Vascularized bone marrow transplant (VBMT) induces donor-specific chimerism in experimental models across the major histocompatibility barrier. An experimental model for immunotolerance studies should sustain a high antigenicity with low morbidity. Accordingly, we introduced an iliac bone osteomusculocutaneous (IBOMC) transplant model in rat. It consists of a large skin component and an abundant bone marrow cells (BMC) population. We tested this model with isograft transplantations between Lewis rats (RT1^l) and with allograft transplantation between Lewis-Brown Norway (LBN RT1^l + n) donors and Lewis (RT1^l) recipients under low dose of cyclosporine A monotherapy. Immunologic responses were tested for donor cell engraftment and chimerism induction. All isografts survived indefinitely and allografts were viable at 200 days post-transplant under low dose of cyclosporine A. Microangiography of the graft revealed preservation of skin, muscle, and bone components. Histologic examination confirmed viability of all allograft components without signs of rejection. Long-term engraftment of donor-origin (RT1ⁿ) BMC was confirmed by donor-specific chimerism (1.2%) in peripheral blood and bone marrow (1.65%) compartments and by engraftment into lymphoid organs of recipients. The IBOMC transplant proved to be a reliable composite tissue allotransplantation (CTA) model. Moreover, because of its robust bone marrow component and large skin component, it is applicable to studies on immunologic responses in CTA.     

Lack of Role for CsA-Sensitive or Fas Pathways in the Tolerization of CD4 T Cells Via BMT and Anti-CD40L

Anti-CD40L mAb plus bone marrow transplantation (BMT) and recipient CD8 T-cell depletion permits long-term mixed hematopoietic chimerism and systemic donor-specific tolerance to be achieved across full MHC barriers. Initial tolerance is characterized by peripheral deletion of donor-reactive CD4 cells. In regimens using costimulatory blockade without BMT to achieve allograft survival, cyclosporine inhibited graft survival, suggesting that the combination may not be clinically applicable. We assessed the role of cyclosporine-sensitive mechanisms and the mechanisms of T-cell apoptosis involved in the induction of early peripheral CD4+ T-cell tolerance by BMT with anti-CD40L. Neither a short course of cyclosporine (14 days) nor the absence of FAS-mediated activation-induced cell death (AICD) blocked the induction or maintenance of donor-specific tolerance. IL-2 production was not associated with tolerance induction, consistent with the lack of a role for Fas-mediated AICD. Mice in which passive T-cell death was impaired because of constitutive expression of a Bcl-xL transgene did not develop tolerance with this protocol. These data confirm that deletion of donor-reactive T cells is critical for the induction of mixed chimerism and tolerance. However, the mechanisms involved may differ from those involved in costimulatory blockade regimens that do not include BMT.     

Conventional Immunosuppression is Compatible with Costimulation Blockade-Based, Mixed Chimerism Tolerance Induction

T-cell costimulatory blockade has emerged as an effective strategy to prevent allograft rejection in experimental models. We and others have reported that the beneficial effects of costimulation blockade can be negated when combined with certain immunosuppressants. The current study evaluates the compatibility of various immunosuppressive agents in a costimulation blockade-based, mixed chimerism tolerance protocol. The addition of conventional agents, including calcineurin inhibitors, did not interfere with tolerance induction. All mice developed multilineage macrochimerism and accepted donor allografts. Analysis of specific T-cell receptor utilization demonstrated selective deletion of donor-reactive T cells. Challenge with donor and third-party allografts confirmed donor-specific tolerance. Clinical introduction of costimulation blockade-based strategies will likely incorporate currently approved immunosuppressive agents. While it has been reported that certain conventional agents are detrimental to costimulation blockade-based strategies, our results suggest that these agents could safely be combined in clinical trials when used as part of a nonmyelosuppressive, mixed chimerism-based tolerance strategy.     

Amnion‐Derived Multipotent Progenitor Cells Support Allograft Tolerance Induction

Donor‐specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism‐based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co‐infusion of limited numbers of donor unfractionated BMCs with human amnion‐derived multipotent progenitor cells (AMPs) 7 days post–allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co‐infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor‐reactive T cells, expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_regs) and long‐term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro‐tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.     

Lack of donor hyporesponsiveness and donor chimerism after clinical transplantation of the hand

BACKGROUND: Composite tissue allografts offer great potential in reconstructive surgery. However, the risks of immunosuppression and graft-versus-host disease (GVHD) after transplantation of vascularized bone in these grafts are significant. Transplantation of vascularized bone also may confer donor hematopoietic chimerism and, potentially, tolerance. We have followed two hand transplant recipients for more than 1 year to determine the level of chimerism and possible donor-specific tolerance, in addition to possible GVHD. METHODS: We performed kinetic studies on peripheral blood of two subjects after hand transplantation that included portions of the radius and ulna. We evaluated donor-specific reactivity, chimerism, and antibody production. RESULTS: Donor-specific tolerance did not develop clinically or in mixed lymphocyte reaction. The first subject recovered an excellent in vitro response to phytohemagglutinin, donor and third-party alloantigen, and by month 4 and at month 12 also recovered the ability to respond to Epstein-Barr virus. The second subject also demonstrated good in vitro proliferative responses, which were attenuated by immunosuppression. No phenotypic changes in mature hematopoietic lineages were detected by four-color flow cytometry other than those expected in response to immunosuppression. Donor chimerism was not detectable using four-color flow cytometry. Microchimerism (approximately 1:75,000 cells) was observed at the level of detection in some of the early posttransplantation specimens and was undetectable thereafter. CONCLUSIONS: In this particular transplantation and immunosuppressive regimen, the composite tissue allograft with vascularized bone marrow did not provide the immunologic benefit of tolerance induction nor cause GVHD.     

Induction of tolerance in a rat model of laryngeal transplantation

BACKGROUND: The major limitation preventing expansion of laryngeal transplantation as a therapeutic modality is the necessity of lifelong immunosuppression. In this report, we describe an immunomodulatory strategy for tolerance induction in laryngeal allotransplantation that permits escape from chronic immunosuppression. MATERIALS AND METHODS: Larynges were transplanted from Lewis-Brown-Norway (RT1l/n, F1) donors to Lewis (RT1l) recipients. Recipients received 7 days of treatment with tacrolimus and mouse anti-rat alphabeta T-cell-receptor (TCR) monoclonal antibodies. Histology, mixed lymphocyte reaction (MLR), skin grafting, and flow cytometry assessed functional tolerance, efficacy of immunodepletion, and donor-specific chimerism. RESULTS: All 10 recipients survived until sacrifice at 100 days. Histology suggested functional allograft tolerance. Skin grafting, MLR, and flow cytometry revealed that tolerance is neither donor-specific nor related to systemic immunocompromise. CONCLUSIONS: In this rat laryngeal-transplantation model, functional tolerance was induced under combined tacrolimus and alphabeta TCR protocol. Mechanisms responsible for this tolerance induction require future elucidation.     

Prolonged survival of experimental extremity allografts: A new protocol with total body irradiation,granulocyte‐colony stimulation factor,and FK506

The induction of a high‐level of chimerism (macrochimerism) may be the most reliable strategy for achieving donor‐specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using total‐body irradiation (TBI) and granulocyte‐colony stimulation factor (G‐CSF) to induce high‐level chimerism following rat whole‐limb allotransplantation. In this study, we investigated whether the timing of TBI influenced the period of graft survival. In total, 50 whole‐limb allotransplants from LacZ transgenic rats to LEW rats were performed. TBI was performed at days 0 and 14, and G‐CSF was given for 4 days after TBI. FK506 was given for 28 days after transplant. Nontreated limb allografts were rejected after 4.2 days. The survival time was prolonged to 64 days in the FK506 monotherapy group. In the group receiving TBI at day 14, limb allograft survival was significantly prolonged to 81 days. In the group receiving TBI at day 0, 26% of recipients died but in the surviving recipients the grafts survived for longer than 1 year without lethal graft‐versus‐host disease (GVHD). Polymerase chain reaction (PCR) analysis revealed a high level of intrabone marrow chimerism in the recipient, thus demonstrating successful induction of macrochimerism. A new protocol of pretransplant TBI followed by treatment with G‐CSF and FK506 was found to induce a high level of chimerism and to significantly prolong the survival of limb allografts in recipients without lethal GVHD. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:457–461, 2010     

Chimerism and bone marrow based therapies in transplantation

Composite tissue allotransplantation holds a great potential for providing increased knowledge of anatomy and microsurgical experience for life-enhancing reconstructions. Many transplant cases around the world have made this a clinical reality at the present time. Composite tissue allotransplants contain multiple tissue types, including bone, muscle, vessels, nerves, skin, and immune cells and bear a huge antigenic load. Although immunosuppressive drugs are applied successfully to prevent allograft rejection, their side effects pose a barrier to worldwide use. Bone marrow therapy in many tolerance induction protocols, therefore, provides a guide to reaching the target of permanent immunotolerance. Multiple studies suggest that bone marrow is immunomodulatory and may facilitate allograft acceptance. In this review, bone marrow based therapy protocols of clinical and experimental models are presented in two major categories: solid organ and composite tissue transplantation.     

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.     

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.     

Composite tissue allotransplantation in chimeric hosts part II. A clinically relevant protocol to induce tolerance in a rat model

BACKGROUND: We and others have shown that mixed allogeneic chimerism induces donor-specific tolerance to composite tissue allografts across major histocompatibility complex barriers without the need for immunosuppression. However, a delay period between bone marrow transplantation and limb allotransplantation is required, making such protocols impractical for clinical application. This study eliminates this delay period in a rat hind limb allotransplantation model by performing mixed allogeneic chimerism induction and transplantation "simultaneously." METHODS: Group 1 included controls in which na?ve Wistar Furth (WF) hosts received ACI hind limbs. Group 2 included (ACI-->WF) chimeras that received limbs from third-party donors (Fisher), and group 3 included chimeras that received irradiated (1,050 cGy) ACI limbs. In group 4, WF hosts conditioned with 950 cGy received irradiated (1,050 cGy) ACI limbs followed by infusion of 100 x 10(6) ACI T-cell-depleted bone marrow cells and immunotherapy (tacrolimus and mycophenolate mofetil) for 28 days. Group 5 animals received the same treatment as group 4 animals without immunotherapy. RESULTS: The rats in groups 1 and 2 rejected their limbs within 10 days. Only one rat in group 4 survived to the end of the study. Groups 3 and 5 demonstrated long-term limb survival without rejection or graft-versus-host disease. High levels of donor chimerism (>80%) were achieved and maintained throughout the study. Mixed lymphocyte reaction assays in both groups revealed donor-specific hyporesponsiveness with vigorous third-party reactivity. CONCLUSIONS: This study demonstrated that infusion of donor bone marrow cells into conditioned hosts immediately after limb transplantation results in stable mixed chimerism, robust tolerance, and reliable limb allograft survival.     

A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts

Composite tissue allotransplantation (CTA) is the new frontier in transplantation. More than 25 hand allograft transplants have been performed worldwide, and the feasibility has been well established. The classical experimental model of CTA involves rat orthotopic hindlimb transplantation, a time-consuming procedure associated with high mortality and morbidity. We describe a rat heterotopic osteomyocutaneous flap that serves as a nonfunctional CTA, allowing the study of tolerance induction to a highly antigenic vascularized allograft of bone, muscle, and skin while minimizing the morbidity and mortality of full hind limb transplantation. In the present studies, we explored whether establishing chimerism by nonmyeloablative conditioning would induce tolerance to CTA. When compared with the classic hind limb transplantation model, these results demonstrate that our heterotopic hind limb flap is less morbid and as an effective experimental model for the study of CTA tolerance.     

The role of cyclophosphamide and granulocyte colony-stimulation factor in achieving high-level chimerism in allotransplanted limbs.

The establishment of a high-level of chimerism may be the most stable strategy for donor-specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Seventy-three whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP was injected at day 2, and G-CSF was given from day 0 to 3. Nontreated limb allografts were rejected after 4.2 days. In FK506-treated group for 28 days, the survival time was prolonged to 64 days. In the group treated with CYP/G-CSF, limb allografts were rejected after 5.4 days and 5 of 15 recipients showed acute lethal graft-versus-host disease (GVHD). Polymerase chain reaction (PCR) study showed a high level of chimerism even within 1 week after transplantation. Fourteen of 30 recipients given CYP/G-CSF/FK506 died within 2 weeks. The limb survival was significantly prolonged, however, with three grafts surviving more than 300 days. Seven recipients (24%) showed chronic GVHD. A high-level of chimerism was maintained when limb allografts were not rejected by recipients. Limb allografting could function as a vascularized carrier for bone marrow transplantation, provide a continuous source of donor cells and contribute to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolonged the survival of limb allografts but frequently caused chronic GVHD in the recipients.     

Earlier Low-Dose TBI or DST Overcomes CD8+ T-Cell-Mediated Alloresistance to Allogeneic Marrow in Recipients of Anti-CD40L

Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day -1 or day -2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.     

Tolerance induced by bone marrow chimerism prevents transplant vascular sclerosis in a rat model of small bowel transplant chronic rejection

BACKGROUND: The major impediment to success in solid organ transplantation is chronic rejection (CR). The characteristic lesion of CR is transplant vascular sclerosis (TVS). Although the mechanism of TVS is thought to have an immunologic basis, in humans immunosuppression does not prevent or reverse it. One possible therapy to prevent TVS is induction of donor-specific tolerance. Bone marrow chimerism has been successful in inducing tolerance in acute and chronic rejection heart and kidney transplant models. The highly immunogenic small bowel (SB) allograft provides a rigorous test of the efficacy of this tolerance regimen. We examined whether induction of tolerance by bone marrow chimerism could prevent TVS in a model of Fisher 344 (F344) to Lewis (LEW) rat SB transplantation. METHODS: Bone marrow chimeras (BMC) were created by transplantation of T-cell-depleted F344 bone marrow into irradiated LEW rats. Chimerism was assessed by flow cytometric method. F344 SB, heterotopically transplanted into the chimeras, was clinically and histologically assessed for CR. F344 SB grafts, transplanted into cyclosporine-A-treated LEW recipients, served as control grafts for CR. RESULTS: Cyclosporine-A-treated LEW rats chronically rejected F344 SB grafts. By contrast, the BMC group demonstrated tolerance and had long-term SB graft survival (>120 days) without TVS. The BMC demonstrated immunocompetence by prompt rejection of third party ACI (RT1av1) SB allografts. CONCLUSIONS: Bone marrow chimerism prevents chronic graft failure secondary to TVS in a model of chronic SB rejection. TVS fails to develop when tolerance is established, suggesting that the mechanisms involved in TVS are, in part, immunologically mediated.     

Induction of tolerance to composite tissue allograft in a rat model

The goal of this study was to establish a rat model that can be used to determine the variables in influencing induction of tolerance to composite tissue allografts. An anti T-cell depleting agent (R73) and 15-deoxyspergualin were given in different doses and schedule to four groups of Lewis rats receiving a limb transplant from Brown-Norway donors. Graft survival prolongation was maximal combining a single dose of R73 and a 20-day administration of 15-deoxyspergualin. Long-term survivors accepted a skin graft from Brown-Norway donors at 80 days, but rejected grafts from an unrelated donor. Skin grafting did not influence survival of the transplanted limb. Mixed allogeneic chimerism was not detectable in peripheral blood by flow cytometry, but immunohistochemistry identified donor-derived cells in the thymus of tolerant recipients at 100 days. These results suggest a state of donor-specific, dynamic tolerance, with potential for future application in human composite tissue allotransplantation.     

Role of thymus in operational tolerance induction in limb allograft transplant model

BACKGROUND: In this study, we evaluated the role of host thymus in tolerance induction in composite tissue allografts (CTA) across major histocompatibility complex (MHC) barrier during a 7-day alphabeta- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. MATERIALS AND METHODS: A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1(1)) received vascularized hind-limb allografts from hybrid Lewis x Brown-Norway (F1), (LBN, RT1(1+n)) donors. Mixed lymphocyte reaction (MLR) and skin grafting assessed donor-specific tolerance in vitro and in vivo, respectively. Flow cytometry determined the efficacy of immunosuppressive protocols and the presence of donor-specific chimerism. Immunocytochemistry revealed the presence of donor-specific cells in the lymphoid organs of recipients. RESULTS: Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the alphabeta-TCR/CsA protocol, the MST of allografts was 51 days. For euthymic rats, the MST of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with alphabeta-TCR/CsA resulted in indefinite allografts survival (MST=370 days). MLR and skin grafting confirmed donor-specific tolerance in euthymic recipients. Flow cytometry showed stable chimerism in the euthymic rats and transient chimerism in thymectomized limb recipients. Immunoperoxidase staining revealed the persistence of donor-derived cells in the lymphoid tissues of euthymic recipients. CONCLUSION: We found that the presence of thymus was imperative for the induction of donor-specific tolerance in rat hind-limb composite tissue allografts using a alphabeta-TCR/CsA protocol.     

Development and maintenance of donor-specific chimerism in semi-allogenic and fully major histocompatibility complex mismatched facial allograft transplants

BACKGROUND: The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. MATERIAL AND METHODS: Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. RESULTS: Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. CONCLUSION: Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.