New immunosuppressive drugs in heart transplantation

Only a few randomized clinical trials have been performed so far in heart transplant recipients, mainly because of the relatively small number of heart transplants performed worldwide each year. The main focus of the few controlled trials that have been completed has been the prevention and treatment of heart allograft rejection. In the area of pharmacologic immunosuppression, both biological agents and drugs have been the subject of investigation. Among the biological agents, chimeric monoclonal antibodies directed against the interleukin (IL)-2 receptor, which have been found to be safe and effective in renal transplant recipients, are now undergoing the test of controlled trials in heart transplant recipients. Immunosuppressive drugs that have been studied in controlled trials include calcineurin inhibitors (such as the microemulsion formulation of cyclosporine and tacrolimus) and inhibitors of purine synthesis, such as mycophenolate mofetil. Non-pharmacologic prophylactic immunosuppression with photopheresis has also been tested in a prospective, multicenter, randomized trial. New immunosuppressive regimens, such as mycophenolate mofetil combined with a monoclonal antibody against the IL-2 receptor, are being tested with the aim to reduce or eliminate calcineurin inhibitors or corticosteroids. Although clinical approaches to the induction of tolerance have undergone preliminary clinical evaluation, the ability to induce tolerance to an allograft in humans remains an elusive goal.     

Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells

Composite-tissue (e.g., hand allograft) allotransplantation is currently limited by the need for immunosuppression to prevent graft rejection. Inducing a state of tolerance in the recipient could potentially eliminate the need for immunosuppression but requires reprogramming of the immunological repertoire of the recipient. Skin is the most antigenic tissue in the body and is consistently refractory to tolerance induction regimens using bone marrow transplantation alone. It was hypothesized that tolerance to skin allografts could be induced in rats by injecting epidermal cells with bone marrow cells during the first 24 hours of life of the recipients. Brown Norway rats (RT1n) served as donors for the epidermal cells, bone marrow cells, and skin grafts. Epidermal cells were injected intraperitoneally and bone marrow cells were injected intravenously into Lewis (RT1l) newborn recipient rats. In control groups, recipients received saline solution with no cells (group I, n = 12), bone marrow cells only (group II, n = 15), or epidermal cells only (group III, n = 15). In the experimental group (group IV, n = 18), recipients received epidermal and bone marrow cells simultaneously. Skin grafts were transplanted from Brown Norway (RT1n) rats to the Lewis (RT1l) rats 8 weeks after cell injections. Skin grafts survived an average of 8.5 days in group I (10 grafts), 9.2 days in group II (12 grafts), and 12 days in group III (14 grafts). Grafts survived 15.5 days (8 to 26 days) in group IV (15 grafts). The difference was statistically significant (p < 0.05). Hair growth was observed in some accepted grafts in group IV but never in the control groups. This is the first report of prolonged survival of skin allografts in a rat model after epidermal and bone marrow cell injections. Survival prolongation was achieved across a major immunological barrier, without irradiation, myeloablation, or immunosuppression. It is concluded that the presentation of skin-specific antigens generated a temporary state of tolerance to the skin in the recipients that could have delayed the rejection of skin allografts.     

Cyclosporin A: a powerful immunosuppressant.

Cyclosporin A (CyA) is a powerful immunosuppressive agent whose lack of myelotoxicity makes it unique among nonsteroidal drugs currently given for immunosuppression. It has been used with initial success in recipients of kidney, liver, bone marrow and pancreas transplants, and it may also have clinical application in the treatment of autoimmune disorders. In regard to its use in transplant recipients, there are many remaining questions about its mechanism of action, the optimum dose, whether it should be used alone or with other immunosuppressants, whether it can suppress chronic rejection and what its long-term side effects may be. These questions can only be answered by further careful laboratory investigation and controlled clinical trials. Until then, CyA should only be administered in centres experienced in its use.     

Advances in haplo-identical stem cell transplantation in adults with high-risk hematological malignancies

Allogeneic bone marrow transplant is a life-saving procedure for adults and children that have high-risk or relapsed hematological malignancies. Incremental advances in the procedure, as well as expanded sources of donor hematopoietic cell grafts have significantly improved overall rates of success. Yet, the outcomes for patients for whom suitable donors cannot be found remain a significant limitation. These patients may benefit from a hematopoietic cell transplant wherein a relative donor is fully haplotype mismatched. Previously this procedure was limited by graft rejection, lethal graft-versus-host disease, and increased treatmentrelated toxicity. Recent approaches in haplo-identical transplantation have demonstrated significantly improved outcomes. Based on years of incremental preclinical research into this unique form of bone marrow transplant, a range of approaches have now been studied in patients in relatively large phase Ⅱ trials that will be summarized in this review.     

Clinical use of immunoglobulin titers of anti-cytomegalovirus antibodies

The frequency and severity of CMV infections in kidney, bone marrow, heart transplant recipients, in polytransfused patients, in immunodeficiencies and in newborns with congenital or peri-natal infection prompted clinical trials of immunoglobulins (Ig) with anti-CMV activity. Among the various methods for titrating Ig of plasmatic or placental origin, polyvalent or hyperimmune, ELISA would be the most sensitive assay and the neutralizing reaction might be the better reflection of a protective effect. In kidney transplantation, two non-randomized clinical trials have suggested a curative effect in association to other treatments, especially when given early. In bone marrow transplantation, three randomized studies have shown a prophylactic effect on incidence of symptomatic infections, interstitial pneumonias due to CMV, but only in seronegative patients not treated with leucocyte transfusions. No effect was observed on the time of infection, the survival of patients, the incidence of surinfections or graft versus host disease. The prophylactic effect of anti-CMV Ig in kidney and heart transplantation as well as in newborns has still to be documented in randomized trials. The possible clinical benefit for the patients and the mode of preparation of effective anti-CMV antibodies need further investigations.     

The Role of Bone Marrow Cells in the Phenotypic Changes Associated with Diabetic Nephropathy

The aim of our study was to investigate the role of bone marrow cells in the phenotypic changes that occur in diabetic nephropathy. Bone marrow cells were obtained from either streptozotocin-induced diabetic or untreated control C3H/He mice and transplanted into control C3H/He mice. Eight weeks after bone marrow cell transplantation, renal morphologic changes and clinical parameters of diabetic nephropathy, including the urine albumin/creatinine ratio and glucose tolerance, were measured in vivo. Expression levels of the genes encoding α1 type IV collagen and transforming growth factor-β1 in the kidney were assayed. Our results demonstrated that glucose tolerance was normal in the recipients of bone marrow transplants from both diabetic and control donors. However, compared with recipients of the control bone marrow transplant, the urinary albumin/creatinine ratio, glomerular size, and the mesangial/glomerular area ratio increased 3.3-fold (p < 0.01), 1.23-fold (p < 0.01), and 2.13-fold (p < 0.001), respectively, in the recipients of the diabetic bone marrow transplant. Expression levels of the genes encoding glomerular α1 type IV collagen and transforming growth factor-β1 were also significantly increased (p < 0.01) in the recipients of the diabetic bone marrow transplant. Our data suggest that bone marrow cells from the STZ-induced diabetic mice can confer a diabetic phenotype to recipient control mice without the presence of hyperglycemia.     

Pre-Transplant Donor-Specific T-Cell Alloreactivity Is Strongly Associated with Early Acute Cellular Rejection in Kidney Transplant Recipients Not Receiving T-Cell Depleting Induction Therapy

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.     

Treatment with immunotoxin

T-cell depletion prior to or beginning at the time of transplantation has been shown to be a valuable adjunct to the induction of immunological unresponsiveness. Both total lymphoid irradiation and anti-lymphocyte globulin have been used for this purpose in experimental models of transplantation as well as in human organ transplant recipients. However, these methods of T-cell depletion are limited in their ability to deplete T cells selectively due to non-specific targeting and limited efficacy. A new anti-CD3 immunotoxin has been developed with a far more potent ability to deplete T cells selectively as measured by flow cytometry analysis of peripheral blood T lymphocytes as well as lymph node lymphocytes. This immunotoxin is well tolerated by rhesus monkeys when administered in vivo. When administered as a single immunosuppressive agent pretransplant, it substantially promotes allograft survival, inducing tolerance in at least one-third of recipients as measured by subsequent acceptance of donor skin grafts and rejection of third-party skin grafts. When administered on the day of transplant in combination with steroid pretreatment and a brief course of deoxyspergualin or mycophenolate mofetil (4 to 14 days), long-term unresponsiveness is also produced and in a more reliable manner than using immunotoxin alone. A new immunotoxin directed at the human CD3epsilon has been developed with excellent potency in T-cell killing and lacking the Fc portion of the CD3 antibody. This construct may be useful for T-cell depletion in humans and has a potential application in tolerance induction in human organ transplantation. Lessons learned from anti-CD3 immunotoxin in the non-human primate model to date include (i) profound (2-3 log) depletion of T-cells can be accomplished safely without inducing lymphoma or infection, (ii) such depletion is a useful adjunct for tolerance induction to allogeneic organ transplants, and (iii) tolerance to both allogeneic renal transplants and xenogeneic islet transplants has been accomplished using such strategies to date in non-human primates and in pigs. Immunotoxin may be useful for the induction of chimerism using strategies that include donor bone marrow infusion. Successful strategies for tolerance induction have also been developed using immunotoxin without the adjunct of donor bone marrow or stem cell infusion. Clinical application of immunotoxin will use a newly engineered construct with the potential for causing cytokine release, less susceptibility to neutralization by anti-diphtheria antibody and not dependent on chemical conjugation of an antibody and toxin. The usefulness of immunotoxin is directly related to its tremendous potency for depleting T cells. Based on results in nonhuman primates, it is anticipated that it will become a useful agent in tolerance induction in humans.     

Viral abrogation of stem cell transplantation tolerance causes graft rejection and host death by different mechanisms

Tolerance-based stem cell transplantation using sublethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. We have shown that mouse bone marrow recipients treated with sublethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. We now report that infection with lymphocytic choriomeningitis virus at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Infected allograft recipients also failed to clear the virus and died. Postmortem study revealed hypoplastic bone marrow and spleens. The cause of death was virus-induced IFN-alphabeta. The rejection of allogeneic bone marrow was mediated by a radioresistant CD8(+)TCR-alphabeta(+)NK1.1(-) T cell population. We conclude that a noncytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sublethally irradiated mice treated with costimulation blockade. Clinical application of stem cell transplantation protocols based on costimulation blockade and tolerance induction may require patient isolation to facilitate the procedure and to protect recipients.     

Relationship between chimerism and tolerance in a kidney transplantation model

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.     

Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in nonhuman primates

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.     

Nonhuman primate models for islet transplantation in type 1 diabetes research

Insulin-dependent diabetes mellitus is an autoimmune disease that causes a progressive destruction of the pancreatic beta cells. As a result, the patient requires exogenous insulin to maintain normal blood glucose levels. Both the pancreas and the islets of Langerhans have been transplanted successfully in humans and in animal models, resulting in full normalization of glucose homeostasis. However, insulin independence, transient or persistent, was documented in only a small fraction of cases until recently. The chronic immunosuppression required to avoid immunological rejection appears to be toxic to the islets and adds the risk of lymphoproliferative disease reported earlier. For islet transplantation to become the method of choice, it is essential first to identify islet-friendly immunosuppressive regimens and/or to develop methods that induce donor-specific tolerance and improve islet isolation and transplantation protocols. Indeed, researchers have already successfully allografted islets in the presence of nonsteroidal immunosuppression in a process known as the Edmonton protocol. An alternative method, gene therapy, could replace these other methods and better meet the insulin requirement of an individual without requiring pancreatic or islet transplantation. This alternative, however, requires animal models to develop and test clinical protocols and to demonstrate the feasibility of preclinical trials. Nonhuman primates are ideally suited to achieve these goals. The efforts toward developing a nonhuman primate diabetic model with demonstrable insulin dependence are discussed and include pancreatic and islet transplant trials to reverse the diabetic state and achieve insulin independence. Also described are the various protocols that have been tested in primates to circumvent immunosuppression by using tolerance induction strategies in lieu of immunosuppression, thus exploring the field of donor-specific tolerance that extends beyond islet transplantation.     

The abrogation of allosensitization following the induction of mixed allogeneic chimerism

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.     

Immunosuppressive drug-free operational immune tolerance in human kidney transplant recipients: Part I. Blood gene expression statistical analysis

Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous "operational tolerance" can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.     

Induction of transplantation tolerance in non-human primate preclinical models

Short-term outcomes following organ transplantation have improved considerably since the availability of cyclosporine ushered in the modern era of immunosuppression. In spite of this, many of the current limitations to progress in the field are directly related to the existing practice of relatively non-specific immunosuppression. These include increased risks of opportunistic infection and cancer, and toxicity associated with long-term immunosuppressive drug exposure. In addition, long-term graft loss continues to result in part from a failure to adequately control the anti-donor immune response. The development of a safe and reliable means of inducing tolerance would ameliorate these issues and improve the lives of transplant recipients, yet given the improving clinical standard of care, the translation of new therapies has become appropriately more cautious and dependent on increasingly predictive preclinical models. While convenient and easy to use, rodent tolerance models have not to date been reliably capable of predicting a therapy's potential efficacy in humans. Non-human primates possess an immune system that more closely approximates that found in humans, and have served as a more rigorous preclinical testing ground for novel therapies. Prior to clinical adaptation therefore, tolerance regimens should be vetted in non-human primates to ensure that there is sufficient potential for efficacy to justify the risk of its application.     

ES-cell derived hematopoietic cells induce transplantation tolerance

Background

Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells.

Methodology/Principal Findings

Here, we derived CD45⁺ HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts.

Conclusions

Our data show, for the first time, the efficacy of ES-derived CD45⁺ HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.     

Virus-induced abrogation of transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody

Treatment with a 2-week course of anti-CD154 antibody and a single transfusion of donor leukocytes (a donor-specific transfusion or DST) permits skin allografts to survive for >100 days in thymectomized mice. As clinical trials of this methodology in humans are contemplated, concern has been expressed that viral infection of graft recipients may disrupt tolerance to the allograft. We report that acute infection with lymphocytic choriomeningitis virus (LCMV) induced allograft rejection in mice treated with DST and anti-CD154 antibody if inoculated shortly after transplantation. Isografts resisted LCMV-induced rejection, and the interferon-inducing agent polyinosinic:polycytidylic acid did not induce allograft rejection, suggesting that the effect of LCMV is not simply a consequence of nonspecific inflammation. Administration of anti-CD8 antibody to engrafted mice delayed LCMV-induced allograft rejection. Pichinde virus also induced acute allograft rejection, but murine cytomegalovirus and vaccinia virus (VV) did not. Injection of LCMV approximately 50 days after tolerance induction and transplantation had minimal effect on subsequent allograft survival. Treatment with DST and anti-CD154 antibody did not interfere with clearance of LCMV, but a normally nonlethal high dose of VV during tolerance induction and transplantation killed graft recipients. We conclude that DST and anti-CD154 antibody induce a tolerant state that can be broken shortly after transplantation by certain viral infections. Clinical application of transplantation tolerance protocols may require patient isolation to facilitate the procedure and to protect recipients.     

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants.

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.