Full MHC Barriers Research Articles

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

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

The Effects of Kidney Graftectomy on the Tolerance Induced by Heart-Kidney Cotransplantation in Miniature Swine Depends on the MHC Barrier Crossed

Objective: We have previously demonstrated that isolated hearts transplanted in miniature swine across an MHC class I barrier and treated with 12 days of CyA or transplanted across a full MHC barrier and treated with FK506 rejected their allografts within 55 days. In contrast, hearts co-transplanted with kidneys and transplanted across the same MHC barriers with the identical treatment survived indefinitely. The kidney allografts were explanted from the class I mismatch recipients more than 100 days after the transplant and replaced with recipient-matched kidneys. Around one hundred days later, challenge skin grafts, including those expressing donor class I, 3rd party class II antigens, were placed which led to acute rejection of the previously stable heart grafts within 41 days. Here, we asked whether the same result would be obtained in recipients of fully mismatched heart and kidney grafts.

Costimulatory Blockade in Combination with Donor Bone Marrow Infusion Induces Immune Tolerance Across A Full MHC Barrier In A Translational Large Animal Vascularized Composite Allotransplantation Model

Costimulatory Blockade in Combination with Donor Bone Marrow Infusion Induces Immune Tolerance Across A Full MHC Barrier In A Translational Large Animal Vascularized Composite Allotransplantation Model

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

IL-17A Mediates Early Post-Transplant Lesions after Heterotopic Trachea Allotransplantation in Mice

Primary graft dysfunction (PGD) and bronchiolitis obliterans (BO) are the leading causes of morbidity and mortality after lung transplantation. Reports from clinical and rodent models suggest the implication of IL-17A in either PGD or BO. We took advantage of the heterotopic trachea transplantation model in mice to study the direct role of IL-17A in post-transplant airway lesions. Across full MHC barrier, early lesions were controlled in IL-17A-/- or anti-IL17 treated recipients. In contrast, IL-17A deficiency did not prevent subsequent obliterative airway disease (OAD). Interestingly, this early protection occurred also in syngeneic grafts and was accompanied by a decrease in cellular stress, as attested by lower HSP70 mRNA levels, suggesting the involvement of IL-17A in ischemia-reperfusion injury (IRI). Furthermore, persistence of multipotent CK14+ epithelial stem cells underlined allograft protection afforded by IL-17A deficiency or neutralisation. Recipient-derived γδ+ and CD4+ T cells were the major source of IL-17A. However, lesions still occurred in the absence of each subset, suggesting a high redundancy between the innate and adaptive IL-17A producing cells. Notably, a double depletion significantly diminished lesions. In conclusion, this work implicated IL-17A as mediator of early post-transplant airway lesions and could be considered as a potential therapeutic target in clinical transplantation.

Kidney-Induced Cardiac Allograft Tolerance across a Full MHC-Barrier in Miniature Swine

Purpose We have demonstrated previously that isolated hearts transplanted into class I disparate miniature swine treated with a 12-day course of CyA all developed cardiac allograft vaszculopathy and rejected within 55 days. In contrast, hearts co-transplanted with donor-specific kidneys survived indefinitely without CAV. Here, we asked whether tolerance can be induced in heart/kidney recipients across a full MHC barrier, which is more clinically relevant. Methods and Materials Heart/kidneys (n=5) or hearts alone (n=3) were transplanted heterotopically into the abdomen of MGH inbred miniature swine across a full MHC mismatch. Immunosuppression consisted of a 12-day course of tacrolimus. Open biopsies were taken on days 30, 60, and 100 to rule out rejection. CML and MLR assays were performed to determine responsiveness toward donor antigen. Results All control animals rejected their isolated hearts within 40 days. In contrast, all heart and kidney recipients survived for >100 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients were hyporesponsive or unresponsive toward donor antigen by CML/MLR assays and had no detectable alloantibody by flow cytometry. Conclusions Induction of long-term cardiac allograft tolerance is possible across a full MHC- mismatched barrier in large animals co-transplanted with a kidney from the same donor. Elucidating the renal element responsible for cardiac allograft tolerance could provide mechanistic information that could lead to protocols aimed at inducing tolerance in recipients of isolated heart allografts.

Immunogenicity of umbilical cord tissue–derived cells

Umbilical cord tissue provides a unique source of cells with potential for tissue repair. Umbilical cord tissue-derived cells (UTCs) are MHC class I (MHCI) dull and negative for MHC class II (MHCII), but can be activated to increase MHCI and to express MHCII with IFN-gamma stimulation. Mesenchymal stem cells with similar characteristics have been inferred to be nonimmunogenic; however, in most cases, immunogenicity was not directly assessed. Using UTC from Massachusetts General Hospital MHC-defined miniature swine, we assessed immunogenicity across a full MHC barrier. Immunogenicity was assessed by in vitro assays including mixed lymphocyte reaction (MLR) and flow cytometry to detect serum alloantibody. A single injection of MHC-mismatched unactivated UTCs did not induce a detectable immune response. When injected in an inflamed region, injected repeatedly in the same region or stimulated with IFN-gamma prior to injection, UTCs were immunogenic. As clinical cellular repair strategies may involve injection of allogeneic cells into inflamed regions of damaged tissue or repeated doses of cells to achieve the desired benefit, our results on the immunogenicity of these cells in these circumstances may have important implications for optimal success and functional improvement for this cellular treatment strategy for diseased tissues.

Rapamycin-conditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion

Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-γ and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.

Fully MHC-Disparate Mixed Hemopoietic Chimeras Show Specific Defects in the Control of Chronic Viral Infections

The establishment of mixed allogeneic chimerism can induce donor-specific transplantation tolerance across full MHC barriers. However, a theoretical disadvantage of this approach is the possibility that the state of mixed chimerism might negatively affect the recipient's immune competence to control pathogens. Previous studies using murine models have not supported this hypothesis, because they indicate that acute viral infections are cleared by chimeric animals with similar kinetics to that of unmanipulated controls. However, chronic or persistent viral infections often require a more complex and sustained response with cooperation between CD4 Th cells, CTL, and B cells for effective control. The current study indicates that profound defects become manifest in the control of chronic pathogenic infections in MHC-disparate mixed allogeneic chimeric mice. Furthermore, we show that ineffective priming of the donor-restricted CTL response leads to virus persistence, as well as severe T cell exhaustion. Our results further suggest that either T cell adoptive immunotherapy or selected MHC haplotype matching partially restore immune competence. These approaches may facilitate the translation of mixed chimerism therapeutic regimens.

Primitive hematopoietic cell populations reside in the spleen: Studies in the pig, baboon, and human

We previously observed high levels (>40%) of multilineage hematopoietic cell chimerism following spleen transplantation across full MHC barriers in immunosuppressed miniature swine. We therefore investigated the spleen as a source of hematopoietic progenitor cells (HPCs). Specific cell-surface markers were used to identify HPCs in the spleen and bone marrow (BM) of young adult (n = 15) and fetal (n = 9) miniature swine by flow cytometry. Hoechst dye-effluxing side population (SP) cells were analyzed in adult spleen, BM, and blood for their expression of c-kit. Functional HPC activity of varying repopulation potential in vitro was investigated by the ability of spleens and BM to give rise to colony-forming units (CFUs) and cobblestone area-forming cells (CAFCs) in long-term stromal cultures. Studies were also carried out on baboon and human spleens and BM. Spleen c-kit+ cells co-expressed more lymphoid markers, but equal myeloid markers, when compared with BM c-kit+ cells. BM and spleen both contained significant percentages of c-kit+ SP cells. Although the frequency of early-forming CFUs in the spleen was only 0.1 to 1.3% of that in the BM, the frequency of CAFCs developing after 8 weeks in culture was comparable to that of BM. Secondary CFUs in long-term culture-initiating cell assays confirmed the presence of long-term repopulating cells at comparable frequencies in spleen and BM. Similar findings were found with regard to baboon and human spleen cells. The adult spleen is a relatively rich source of very primitive HPCs, possibly hematopoietic stem cells (HSCs), and may be of therapeutic value.

Stable multilineage chimerism across full MHC barriers without graft-versus-host disease following in utero bone marrow transplantation in pigs

Stable engraftment of hematopoietic progenitors and multilineage chimerism following in utero bone marrow transplantation could be a promising modality for treatment of prenatally diagnosed blood dyscrasias. For treatment of these diseases, stable chimerism in the myeloid and erythroid lineages is important because it is anticipated that donor-derived cells will compensate for defects in these host lineages. In the present study, a preparation of bone marrow that includes fresh, unmanipulated marrow mixed with T-cell-depleted marrow to achieve 1.5% T-cell content, was injected into the intrahepatic portion of the umbilical vein of porcine fetuses at mid-gestation. Donor hematopoietic progenitor cell engraftment was assessed in fetal liver and recipient bone marrow postnatally by donor-specific polymerase chain reaction of colony-forming units. Chimerism was assessed in lymphoid tissues and peripheral blood by flow cytometry. Graft-versus-host disease (GVHD) was assessed by histological analysis of biopsies of skin, bone marrow, liver, and intestine. In this report, we demonstrate that stable multilineage chimerism across a full major histocompatibility complex disparity can be achieved without GVHD through in utero bone marrow transplantation.

THE ROLE OF HOST THYMIC FUNCTION IN THE INDUCTION OF TOLERANCE ACROSS FULL MHC BARRIERS BY FK506

P929 Aims: Previous studies have demonstrated the importance of an intact thymus in induction of tolerance to class I-mismatched renal allografts by Cyclosporin in miniature swine. A short course of FK506 has subsequently been shown to induce tolerance to fully mismatched renal allografts in this large animal model. In the present study, we assessed whether thymectomy likewise interferes with induction of this tolerance. Methods: Recipients of fully MHC disparate renal allografts were treated with a 12-day course of FK506 (0.15 mg/kg/day by continuous i.v. infusion), with doses adjusted to maintain trough levels of 30-50 ng/ml. Four experimental animals were thymectomized 21 days prior to transplantation, while two control animals remained euthymic. Tolerance was tested in vivo in animals with surviving grafts at day 100 by re-transplantation using another donor-matched kidney and no immunosuppression. Results: Both euthymic controls accepted their renal allografts for >100 days and accepted second grafts long-term with stable renal function. They also demonstrated donor-specific hypo-responsiveness in CML and MLR assays and showed no evidence of IgG or IgM deposition in kidney biopsies, confirming that they were tolerant. In contrast, all four thymectomized recipients developed evidence for acute and chronic rejection. The first animal rejected its graft on day 17, autopsy demonstrating a swollen and necrotic graft with patent vessels. The second animal maintained its kidney >100 days, but rejected a second donor-matched allograft acutely on day 20. The third animal was euthanized on day 99 due to chronic rejection with unstable renal function. The fourth animal accepted both its first and second renal allografts, but remnants of the native thymus were detected on autopsy, indicating incomplete thymectomy. In vitro assays of the first three (but not the fourth) animals demonstrated responsiveness to donor MHC by CML and MLR and both IgG and IgM deposition were found in the kidneys histologically. Conclusions: The host thymus is required for induction of transplant tolerance across a two-haplotype full MHC barrier by a short course of FK506, although even a small amount of thymic tissue may be sufficient to provide the required thymic function.

Complete allogeneic hematopoietic chimerism achieved by in utero hematopoietic cell transplantation and cotransplantation of LLME-treated, MHC-sensitized donor lymphocytes

Objective In utero hematopoietic cell transplantation (IUHCT) typically achieves low-level mixed hematopoietic chimerism. However, the goal of IUHCT is to achieve therapeutic levels of chimerism. We hypothesized that prenatal adoptive immunotherapy might achieve high-level donor chimerism after IUHCT. Materials and methods BALB/CE15 fetal mice were transplanted with a mixture of C57BL/6 (B6) T-cell–depleted bone marrow (TCD BM) cells and splenocytes from B6 mice presensitized to BALB/C alloantigen. The splenocytes were preincubated in L-leucyl-L-leucine methyl ester (LLME), to minimize graft vs host disease (GVHD). Recipients were followed after birth for donor cell chimerism and GVHD. Results Full donor hematopoietic chimerism following a single prenatal transplant was achieved in seven transplanted animals. Fully chimeric animals were healthy, without evidence of GVHD, and maintained their engraftment for the duration of the study (48 weeks). However, the addition of presensitized LLME-treated cells decreased survival until weaning relative to TCD BM alone, suggesting that some animals were lost to acute GVHD. Surviving chimeric animals demonstrated increased frequencies of T-regulatory cell populations in their spleen and BM, suggesting that they had successfully suppressed GVHD, allowing survival. Conclusions This study represents “proof in principle” that prenatal immunotherapeutic strategies may achieve complete hematopoietic engraftment across full MHC barriers when combined with IUHCT. However, strategies with greater hematopoietic specificity must be developed prior to consideration of clinical application.

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.

Towards a myeloablative regimen with clinical potential: I. Treosulfan conditioning and bone marrow transplantation allow induction of donor-specific tolerance for skin grafts across full MHC barriers.

To investigate whether we could create a radiation-free conditioning regimen to induce permanent mixed and multilineage chimerism and donor-specific tolerance, we treated recipient mice with anti-T-cell antibodies, varying and fractionated doses of Treosulfan and fully MHC disparate bone marrow cells. Treosulfan is mainly used in the treatment of ovarian cancer. It is a structural analog of busulfan, but it does not induce severe hepatotoxicity or veno-occlusive disease at or above the maximum tolerated dose, lacks significant nonhematological toxicity and has limited organ toxicity. We report here the successful induction of permanent mixed multilineage chimerism and donor-specific tolerance as was proven by skin transplantation and IFN-gamma ELISPOT. In conclusion, because of its lower nonhematological toxicity, compared with other myeloablative regimens (eg irradiation or busulfan admin- istration), Treosulfan could be a better candidate for conditioning to induce donor-specific allograft tolerance.

Primary and secondary immunocompetence in mixed allogeneic chimeras.

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions.

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.

AN UNEXPECTED ROLE FOR CD28 AND LACK OF A ROLE FOR CD40L SIGNALING IN PERIPHERAL DELETION OF DONOR-REACTIVE CD4+ T CELLS AND ESTABLISHMENT OF MIXED CHIMERISM THROUGH COSTIMULATORY BLOCKADE

453 Purpose: Bone marrow transplantation plus costimulatory blockade allows the establishment of long-term multilineage chimerism and tolerance across a full MHC barrier. Tolerance is associated with rapid peripheral deletion of recipient CD4+ T cells bearing donor-reactive Vβ. We evaluated the role of CD28 and CD40L in this regimen using CD28−/− and CD40L−/− mice. Methods: C57BL/6-CD28−/− and C57BL/6 wild-type (WT) mice (H-2b) received 3Gy WBI and 20×106 B10.A (H-2a) bone marrow cells on day 0, followed by single injections of anti-CD40L mAb (MR1; 0.5mg; day 0) with or without CTLA4Ig (0.5mg, day 0 or day +2). H-2b B6/129-CD40L−/− and B6/129 mice received 3Gy WBI followed by 15×106 B10.A BMC on day 0, and single injections of CTLA4Ig (0.5mg; day +2) with or without MR1 (0.5mg; day 0). Levels of donor chimerism in peripheral blood and frequency of TCR using certain Vβ were followed by FCM analysis. Tolerance was tested by grafting donor (B10.A) and third party (A.SW) tail skin. Results: In the CD28−/− experiment, five of six WT mice receiving MR1 and CTLA4Ig developed multilineage mixed chimerism, with peripheral deletion of donor-reactive CD4+ T cells at week 1 that progressed further by week 3. In contrast, CD28−/− mice failed to demonstrate measurable chimerism or deletion, regardless of CTLA4Ig administration. In the CD40L−/− experiment, two of five CD40L-deficient mice receiving only CTLA4Ig showed high levels of multilineage chimerism, similar to results in WT mice receiving both CTLA4Ig and MR1 (3 of 5 mice). Similar deletion of donor-reactive Vβ was evident among CD4+ T cells of chimeric CD40L−/− mice which received only CTLA4Ig and wild-type controls receiving MR1 and CTLA4Ig. Donor-specific skin graft tolerance (>100 days) was observed in all chimeric mice, with prompt rejection of third party skin. Conclusion: Surprisingly, signaling through CD28 on host T cells plays a role in peripheral donor-reactive T cell deletion and in permitting allogeneic marrow engraftment in mice receiving costimulatory blockade. In contrast, signaling through CD40L is not necessary in this regimen of tolerance induction. The deletion of donor-reactive CD4+ T cells observed in CD40L−/− mice which did not receive MR1 suggests that direct destruction of anti-CD40L-coated activated T cells is unlikely, and that the importance of anti-CD40L treatment in tolerance induction is due to its ability to prevent activation of APC's.

Haematopoietic stem cells can induce specific skin graft acceptance across full MHC barriers.

Previously, we and others have demonstrated in several animal models that the establishment of stable haematopoietic chimerism through allogeneic bone marrow transfusion provides an effective means for the development of specific transplantation tolerance. However, a major limitation to the clinical application of allogeneic bone marrow transfusion in immunosuppressed recipients for induction of tolerance to solid grafts, is the risk of graft-versus-host disease (GVHD). Therefore, it is important to identify the cell population needed for the induction of mixed chimerism and tolerance. Haematopoietic stem cells have the capacity of self-renewal and multilineage differentiation, and have been shown to reduce the risk of GVHD. We studied transfusion of two rich sources of stem cells, namely allogeneic fetal liver cells and a subset of purified bone marrow-derived progenitor cells (c-kit+) into anti-T cell monoclonal antibody-treated, low-dose irradiated recipient mice. Our data revealed that stable multilineage mixed chimerism and permanent donor-specific tolerance for skin, even when transplanted directly following conditioning, can be successfully achieved in this way, with no signs of GVHD.