Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.     

Dystrophin isoforms DP71 and DP427 have distinct roles in myogenic cells

The efficacy of MHC class I-derived peptides to induce tolerance was tested in a cardiac transplantation model. Two 25-mer peptides from the polymorphic region of the DA class I molecule (RT1.Aa) were synthesized by F-moc chemistry and injected intrathymically or intraperitoneally into LEW (RT1.1) responder animals. Intrathymic treatment of the recipient animals with peptide 1 (residues 56-80) accompanied by intraperitoneal treatment with peptide 4 (residues 96-120) led to indefinite survival of allogeneic DA cardiac allografts (n = 7; > 100 days). The tolerogenicity of both peptides differed according to the site of inoculation, as donor-specific tolerance was only observed after administration of peptide 1 into the thymus and injection of peptide 2 into the abdominal cavity of LEW recipients, but not vice versa. Donor-specific tolerance was confirmed in vivo by grafting of full-thickness skin and in vitro by appropriate proliferation and cytotoxicity assays using donor and third-party rats. Donor-specific tolerance was associated with up-regulation of interleukin-4, transforming growth factor beta, and interleukin-10 gene expression within cardiac allografts, thus suggesting intrathymic clonal deletion and external suppression with expansion of T-helper 2-type lymphocytes as the underlying mechanisms of tolerance induction.     

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.     

Clinicopathologic features of retinoblastoma after primary chemoreduction

BACKGROUND: Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS: In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS: In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION: This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.     

Permanent and specific transplantation tolerance induced by a nonmyeloablative treatment to a wide variety of allogeneic tissues: I. Induction of tolerance by a short course of total lymphoid irradiation and selective elimination of the donor-specific host lymphocytes

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.     

Induction of immunologic tolerance for transplantation

In the second half of the 20th century, the transplantation of replacement organs and tissues to cure disease has become a clinical reality. Success has been achieved as a direct result of progress in understanding the cellular and molecular biology of the immune system. This understanding has led to the development of immunosuppressive pharmaceuticals that are part of nearly every transplantation procedure. All such drugs are toxic to some degree, however, and their chronic use, mandatory in transplantation, predisposes the patient to the development of infection and cancer. In addition, many of them may have deleterious long-term effects on the function of grafts. New immunosuppressive agents are constantly under development, but organ transplantation remains a therapy that requires patients to choose between the risks of their primary illness and its treatment on the one hand, and the risks of life-long systemic immunosuppression on the other. Alternatives to immunosuppression include modulation of donor grafts to reduce immunogenicity, removal of passenger leukocytes, transplantation into immunologically privileged sites like the testis or thymus, encapsulation of tissue, and the induction of a state of immunologic tolerance. It is the last of these alternatives that has, perhaps, the most promise and most generic applicability as a future therapy. Recent reports documenting long-term graft survival in the absence of immunosuppression suggest that tolerance-based therapies may soon become a clinical reality. Of particular interest to our laboratory are transplantation strategies that focus on the induction of donor-specific T-cell unresponsiveness. The basic biology, protocols, experimental outcomes, and clinical implications of tolerance-based transplantation are the focus of this review.     

Immunosuppression by inhibition of cellular adhesion mediated by leukocyte function-associated antigen-1/intercellular adhesion molecule-1 in murine cardiac transplantation

BACKGROUND: Donor alloantigen-specific tolerance to vascularized allografts can be induced by several treatments, but the immunological mechanism(s) of these effects remain unclear. One hypothesis is that allograft unresponsiveness is correlated with a shift in the pattern of expression of the T helper 1 versus T helper 2 T-cell cytokines. We report here an extensive analysis of murine cardiac allografts, during normal first set rejection and in mice treated with anti-adhesion molecule monoclonal antibodies (mAbs), a regimen that results in prolonged unresponsiveness. METHODS: A combination of immunohistochemical staining with a panel of mAbs, and in situ hybridization with a panel of digoxigenin-labeled riboprobes, was performed on frozen-tissue sections of cardiac allografts. RESULTS: In several strain combinations, injection of anti-leukocyte function-associated antigen-1 and anti-intercellular adhesion molecule-1, from day 0 to day 6 after transplantation, results in significant long-term survival. Examination of tolerated cardiac allografts by in situ hybridization and immunohistochemical staining shows an altered cytokine expression pattern, although the frequency of CD3 and CD4 cells is not dramatically reduced. These allografts show a decreased frequency of interferon-gamma and interleukin (IL)-2-expressing cells and a slightly increased frequency of cells expressing IL-4 and IL-10, compared with unmodified acute rejection. A direct role of these changes in T-cell cytokine expression is demonstrated by reversal of tolerance induction and rejection of the allograft by in vivo injection of either anti-IL-10 or anti-IL-4 mAb. CONCLUSIONS: Although there are significant differences in the frequency of different cellular subsets and patterns of cytokine gene expression, these differences are quantitatively subtle, suggesting a delicately balanced immune response that can develop a pattern of specific unresponsiveness, with relatively minor alterations in the specific T-cell response.     

Induction of donor-specific ACAID can prolong orthotopic corneal allograft survival in "high-risk" eyes

PURPOSE: To examine the effect of donor-specific anterior chamber-associated immune deviation (ACAID) induction on the survival of orthotopic corneal allografts in neovascularized graft beds. METHODS: To induce donor-specific ACAID in recipients, peritoneal exudate cells (PEC) from C57BL/6 mice were incubated overnight with transforming growth factor (TGF)-beta. Cultured PEC were injected intravenously (i.v.) into BALB/c mice, and, 1 week later, these animals received orthotopic corneal allografts from C57BL/6 donors into neovascularized graft beds. Control mice received i.v. injection of syngeneic (BALB/c) PEC, cultured overnight with TGF-beta, and then received orthotopic corneal allografts from C57BL/6 donors. RESULTS: All corneal allografts (15 out of 15) were rejected within 2 weeks after grafting in the neovascularized graft beds of control animals. However, only 6 out of 16 (37.5%) of corneal allografts were rejected in recipients in which donor-specific ACAID had been induced by injection of allogeneic PEC cultured with TGF-beta. CONCLUSION: Previous studies revealed that rejection of orthotopic corneal allografts in neovascularized graft beds in mice correlated with acquisition of donor-specific delayed hypersensitivity (DH). The results of this study suggest that induction of donor-specific ACAID, which selectively impairs DH responses to donor antigens, effectively prolongs corneal allograft survival in "high-risk" eyes.     

Tolerance to experimental cardiac allografts produced by neonatal intrathymic injection of donor cells

Intrathymic inoculation of allogeneic cells after systemic administration of antilymphocyte serum in adult experimental animals has produced donor-specific tolerance to cardiac allografts. We investigated whether thymic injection of allogeneic cells without antilymphocyte serum in neonatal Lewis rats (day 1 of life) with immature immune systems also produced tolerance to cardiac grafts. Intrathymic or intraperitoneal injection of 5 x 10(7) Lewis (control) or Lewis-Brown Norway (allogeneic) spleen cells in Lewis neonates was followed by heterotopic cardiac transplantation using Lewis, Lewis-Brown Norway, or Wistar Furth (third-party allograft) hearts at 6 to 8 weeks of age. Graft survival was prolonged with both intraperitoneal and intrathymic allogeneic cells. Recipients of cells by the intrathymic route had longer graft survival, and 2 of 5 animals achieved permanent graft acceptance (longer than 100 days). As expected, Lewis isografts survived indefinitely, whereas third-party Wistar Furth allografts were rejected in the usual time frame. Intrathymic introduction of allogeneic cells in a neonatal recipient with an immature immune system can produce donor-specific tolerance to a subsequent graft without the need for a systemic immunosuppression regimen, even transiently.     

Long-lasting unresponsiveness to polyclonal T cell-binding immunoglobulins

We have shown that mice after a single injection of anti-T cell antibody followed by multiple injections of a second xeno-, allo- or syngeneic anti-T cell antibody differing from the former in species origin developed specific, long-lasting tolerance to the second antibody. To characterize the mechanism of this anti-antibody unresponsiveness and the modalities accompanying the preinjection step, we injected mice with anti-pan T, anti-CD4 or anti-CD8 monoclonal antibodies, followed by multiple injections of polyclonal rabbit anti-mouse thymocyte globulin (RbATG). Our observations indicate that: (i) Depletion of CD4+ cells is the most important factor for tolerance induction to subsequently injected RbATG. Non-depleting mAb were less effective, and antibody-induced CD4 modulation or blockade were inconsequential. (ii) The prevention of anti-antibody responses involves specific B cell tolerance, as shown by suppression of anti-RbATG but not anti-bovine serum albumin (BSA) antibodies after challenge of tolerant mice with RbATG-BSA conjugates. (iii) Suppression of anti-antibody responses involves T cell unresponsiveness rather marginally, as demonstrated by in vitro spleen cell restimulation with RbATG and in vivo antibody response to RbATG-fluorescein isothiocyanate hapten conjugate. (iv) Analysis of isotypes of anti-RbATG antibodies does not suggest alterations in Th1/Th2 tuning as being responsible for this kind of tolerance. (v) Over 150-day survival of fully allogeneic skin grafts (CBA-to-C57BL/6) was observed in mice preinjected with anti pan-T or anti-CD4 mAb followed by RbATG. Mice treated with anti-CD4 mAb alone rejected allografts within 21 days and induced anti-antibodies. Taken together, our experiments suggest B cell tolerance as main mechanism in this type of acquired long-term humoral unresponsiveness to foreign, polyclonal, T cell-binding immunoglobulins.     

Intimal thickening develops without humoral immunity in a mouse aortic allograft model of chronic vascular rejection

BACKGROUND: The major threat to the long-term survival of cardiac allograft recipients is the development of diffuse intimal thickening in the allograft coronary arteries through mechanisms that are poorly understood. Although antidonor antibodies have been associated with the development of this condition, a causal relationship has not been established. METHODS AND RESULTS: To determine whether humoral immune responses are necessary for the development of graft vascular disease, we performed abdominal aortic allografts from normal donor mice into different immunodeficient recipient mice: those lacking all donor-specific immune responses (severe combined immunodeficient [SCID] mice and recombination activating gene-1 [RAG-1]-deficient mice) and those lacking humoral immune responses alone owing to a targeted deletion of the joining region (JH) gene segments for the immunoglobulin heavy chain. At 6 to 9 weeks after transplantation, aortic allografts in normal immunocompetent recipients showed concentric intimal thickening extending the full length of the graft (percent luminal reduction, [%LR], 31.2 +/- 9.1 [mean +/- SD] and 38.5 +/- 3.6 in different donor-recipient strain combinations). In contrast, syngeneic (histocompatible) aortic grafts showed a normal-appearing vessel wall (%LR, 1.6 +/- 0.7). In both SCID and RAG-1-deficient recipients, aortic allografts showed a virtual absence of neointimal formation (%LR, 3.7 +/- 2.1 and 3.8 +/- 1.6 in SCID and RAG-1-deficient recipients, respectively), indicating a critical etiological role for alloimmune responses in this model. Importantly, allografts in JH-deficient mice showed marked intimal thickening (%LR, 35.7 +/- 7.9), with an appearance histologically indistinguishable from that of normal immunocompetent recipients. CONCLUSIONS: Neointimal formation in graft vascular disease is critically dependent on alloimmune responses of the host. Humoral effector mechanisms, however, may not be required.     

Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.     

Characterization of SCML1, a new gene in Xp22, with homology to developmental polycomb genes

BACKGROUND: Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS: 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS: Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS: Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.     

The presentation of self and allogeneic MHC peptides to T lymphocytes

The presentation of donor-derived MHC peptides by recipient APCs to T cells is an essential component of the rejection of allografts (indirect allorecognition). Initial alloreactive T cell response is confined to a few well processed and presented dominant determinants on donor MHC. However, during long-term graft rejection, T cell response spreads to formerly poorly presented cryptic allogeneic MHC peptides. This phenomenon is likely to play an important role in the amplification and the perpetuation of the rejection process. Additionally, we present evidence that T cell repertoire selection to allogeneic MHC peptides is acquired via recognition of self-MHC peptides presented in the thymus during ontogeny. Supporting this view, we have shown that indirect alloresponses can lead to self-T cell tolerance breakdown to cross-reactive determinants on self-MHC molecules or alternatively that sensitization of recipients to self-MHC peptides can lead to accelerated graft rejection. It is therefore essential to determine the factors which govern the processing and presentation of self and allogeneic MHC molecules and to elucidate the mechanisms regulating subsequent T cell responses in order to design antigen-specific based immune therapies in transplantation.     

Suppressor cells and their precursors in A/J mice, tolerant to heterologous gamma globulin

The role of suppressor cells and of their precursors was examined in A/J mice, immunized or tolerized-immunized with rabbit gamma globulin. Antibody response and tolerance were assessed by antigen elimination, followed by an indirect plaque-forming assay. Reconstitution experiments were performed to estimate loss of cooperative capacity in thymus and spleen cells. Infectious tolerance was examined by reconstitution with mixtures of spleen or thymus cells of normal and tolerant donors. Infectious tolerance could not be detected after neonatally induced tolerance. It could be detected when tolerance was induced 11-16 days after birth. Under these circumstances, loss of cooperative capacity and increased capacity for infectious tolerance occurred rapidly over the first 2 days and reached completion by the 10th-20th day after administration of tolerogen. Thymectomy, after tolerance induction, resulted in relative recovery of responsiveness of spleen cells and loss of capacity for infectious tolerance. Pretreatment with cyclophosphamide resulted in a less profound state of unresponsiveness and in the disappearance of the capacity for infectious tolerance. Simultaneous treatment with tolerogen and colchicine also resulted in a less profound state of tolerance. This effect of colchicine was more profound when a low dose of tolerogen was used or when animals were thymectomized before administration of tolerogen and colchicine.     

The induction of transplantation tolerance by intrathymic (i.t.) delivery of alloantigen: a critical relationship between i.t. deletion, thymic export of new T cells and the timing of transplantation

Intrathymic (i.t.) injection of donor alloantigens has proved to be an effective strategy for the induction of tolerance. However, the mechanisms by which tolerance is induced and maintained after transplantation remain unclear. In this report we show that tolerance to donor cardiac allografts can be induced across a MHC class I difference by i.t. injection of donor splenocytes and transient T cell depletion. Furthermore, using H-2K(b)-specific TCR transgenic mice (BM3), we demonstrate that prolonged deletion of donor-reactive thymocytes was essential to induce tolerance by i.t. injection and this was dependent upon donor cells persisting in the thymus. Examination of the kinetics of thymic export following i.t. injection revealed that prolonged deletion of thymocytes was required to delay export of new T cells to the periphery until the time of transplantation. Importantly, after transplantation donor cell persistence in the thymus and i.t. deletion were no longer necessary to maintain tolerance. The graft itself or cells from the graft was responsible for maintaining tolerance at this stage. These findings reveal that multiple mechanisms are responsible for the induction and maintenance phases of tolerance to alloantigens in vivo after i.t. delivery, and that a complex inter-relationship between donor cell persistence in the thymus, i.t. deletion, thymic export of T cells and the timing of transplantation is involved.     

A nonlethal conditioning approach to achieve engraftment of xenogeneic rat bone marrow in mice and to induce donor-specific tolerance

BACKGROUND: The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance. METHODS: C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting. RESULTS: One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80 x 10(6) Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks. CONCLUSION: The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.