Vascularized bone marrow transplantation model in rats as an alternative to conventional cellular bone marrow transplantation: preliminary results

The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMT) versus conventional bone marrow transplantation (BMT). In one group, a VBMT model consisted of donor Brown Norway rat hind limb heterotopic transplanted on recipient Lewis rats. An intravenous infusion of donor bone marrow cells in suspension equivalent to that grafted in the vascularized femur limb was administered intravenously to recipient rats in the second group. Cellular microchimerism was investigated in recipients of VBMT versus BMT. Donor-derived cells could be detected in VBMT recipients at 30 and 60 days but not in recipients of intravenous suspension of BMC. VBMT provides a theoretical alternative to conventional cellular bone marrow transplantation by addressing crucial clinical problems such as failure of engraftment or graft-versus-host disease.     

Review of vascularized bone marrow transplantation: current status and future clinical applications

In this review, we examine the applicability of the vascularized bone marrow transplant (VBMT) as an alternative to conventional bone marrow transplantation (BMT). As a new surgical approach, the VBMT is unique by transplantation of the stromal environment that eliminates the need for an engraftment period, provides critical signaling and modulatory functions, and may potentiate tolerance induction. Thus far, VBMT studies have demonstrated an absence of graft-versus-host disease (GVHD) and robust engraftment into nonmanipulated as well as irradiated recipients with evidence of immunological tolerance. Further investigation is needed to determine the applicability of VBMT as an alternative to BMT.     

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.     

Induction of kidney transplantation tolerance across major histocompatibility complex barriers by bone marrow transplantation in miniature swine

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.     

A novel rat full‐thickness hemi‐abdominal wall/hindlimb osteomyocutaneous combined flap: influence of allograft mass and vascularized bone marrow content on vascularized composite allograft survival

Vascularized bone marrow transplantation (VBMT) appears to promote tolerance for vascularized composite allotransplantation (VCA). However, it is unclear whether VBMT is critical for tolerance induction and, if so, whether there is a finite amount of VCA that VBMT can support. We investigated this with a novel VCA combined flap model incorporating full‐thickness hemiabdominal wall and hindlimb osteomyocutaneous (HAW/HLOMC) flaps. Effects of allograft mass (AM) and VBMT on VCA outcome were studied by comparing HAW/HLOMC VCAs with fully MHC‐mismatched BN donors and Lewis recipients. Control groups did not receive treatments following transplantation. Treatment groups received a short course of cyclosporine A (CsA), antilymphocyte serum, and three doses of adipocyte‐derived stem cells (POD 1, 8, and 15). The results showed that all flaps in control allogeneic groups rejected soon after VCAs. Treatment significantly prolonged allograft survival. Three of eight recipients in HLOMC treatment group had allografts survive long‐term and developed donor‐specific tolerance. Significantly higher peripheral chimerism was observed in HLOMC than other groups. It is concluded that the relative amount of AM to VBMT is a critical factor influencing long‐term allograft survival. Accordingly, VBMT content compared with VCA mass may be an important consideration for VCA in humans.     

骨髓移植诱导临床心脏移植后供者特异性免疫耐受方案的探讨

目的 探讨骨髓移植诱导临床心脏移植后供者特异性免疫耐受的可行性.方法 采取供心的同时采用改良"灌流法"获取供者的骨髓350 ml,经过滤及离心处理后,加入细胞冷冻保护液共80ml,分装于低温冻存袋,经程序降温,置于-80℃冰箱中保存.在常规原位心脏移植术后40 d,取冻存骨髓快速复温,穿刺受者双侧髂后上嵴,立即行骨髓腔内骨髓细胞输注(IBM-BMT),共输注单核细胞1.2×107/kg,CD34+细胞2.38×105/kg.骨髓输注前3 d行预处理,包括应用氟达拉滨、抗胸腺细胞球蛋白及全身淋巴结照射.骨髓移植后静脉应用他克莫司(Tac),维持血Tac浓度谷值在10～20μg/L;3周后改为口服Tac+吗替麦考酚酯(MMF);6周后改为环孢素A及MMF.分别于心脏移植后2、4、8和12周采集受者外周血,分别于术后4、8和12周采集受者的骨髓,应用短串联重复序列-聚合酶链反应法检测供者嵌合体.心脏移植后每周行心肌内心电图检查,每月行心肌活检1次.术后3个月,取受者及第三者外周血单核细胞,行混合淋巴细胞反应(MLR).结果心脏移植后1、2及3个月时受者的外周血及髂骨内骨髓细胞中供者来源的细胞比例分别为26.3%、19.1%、4.8%和46.3%、24.4%、7.6%.IBM-BMT后心肌内心电图监测显示心肌阻抗及R波波幅无明显变化.术后3个月行心内膜心肌活检,未见排斥反应征象.术后3个月时行超声心动图检查,提示心脏舒张、收缩功能良好.MLR提示受者对供者特异性刺激呈现低反应性,而对第三者仍保持良好的免疫活性(P＜0.01).结论 采取分期骨髓移植免疫耐受诱导方案可安全、有效地建立嵌合体,成功诱导心脏移植后供者特异性免疫耐受,但远期效果有待进一步研究.

Abstract：

Objective To investigate a new strategy of bone marrow transplantation (BMT) for donor-specific tolerance induction after heart transplantation. Methods Donor bone marrow cells (BMCs)were harvested simultaneously with donor cardiac graft using modified perfusion method (PM) ,then stored in a -80 ℃ refrigerator after filtration and centrifugation. Whole BMCs (IBM-BMT) (monocytes 1.2 ×107/kg,CD34+ cells 2.38× 105/kg) in host iliac bones were injected into the bone marrow cavity 40 days after heart transplantation. Preconditoning regimens that consisted of fludarabine, antithymoctye globin and total lymphoid irradiation were performed 3 days before BMT. Tacrolimus (Tac) was administrated intravenously after BMT or orally in conjunction with mycophenolate mofetil (MMF) 3 weeks later.Cyclosporine and MMF were orally administrated 6 weeks later. Donor chimerism was detected using short tandem repeats-polymerase chain reaction in monocytes from peripheral blood at the 2nd,4th, 8th or 12th week after BMT or BMCs at the 4th, 8th or 12th week after BMT. Intramyocardium electrocardiography examination or endomyocardial biopsy was performed weekly or monthly respectively. Mixed lymphocyte reactions (MLR) were performed 3 months after BMT. Results Donor chimerism in monocytes in peripheral blood or BMCs in iliac bones measured at the 1 st,2nd and 3rd month after BMT was 26.3%, 19.1%,4.8% ,and 46.3%, 24.4%, 7.6%, respectively. After 3-month follow-up, there was no rejection confirmed by endomyocardial biopsy or intramyocardium electrocardiography. Echocardiography revealed that the diastolic and systolic function of the cardiac graft was maintained well 3 months after BMT. MLR revealed donor-specific hyporesponsiveness while immunocompetence was preserved to third-party antigens. Conclusion These findings indicate that the two-stage BMT strategy is a safe and feasible method for the induction of donor-specific tolerance via stable mixed chimerism and needs to be further confirmed after a long-term observation.     

Mechanisms of alloimmune tolerance associated with mixed chimerism induced by vascularized bone marrow transplants

Rat limb allograft recipients represent surgically induced, immediately vascularized bone marrow transplant (VBMT) chimeras. The majority of these chimeras undergo tolerance while a minority develop graft versus host disease (GVHD). T-cell chimerism and associated mechanisms of cellular immune nonresponsiveness were investigated in tolerant VBMT chimeras. A strong correlation (p < 0.01) was observed between the clinical onset of GVHD and levels of donor T-cell chimerism approximating or greater than 50%. However, stable mixed chimerism was associated with tolerance. In conclusion, three major sequential mechanisms of immune nonresponsiveness were elucidated in tolerant VBMT chimeras over time and included development of nonspecific suppressor cells (which potentially represent natural suppressor cells), maturation of antigen-specific suppressor cell circuits, and eventually putative clonal inactivation.     

Induction of donor-specific tolerance to rat cardiac allografts by intrathymic inoculation of bone marrow.

BACKGROUND. Induction of donor-specific tolerance to tissue or organ allografts can readily be achieved by administration of allogeneic bone marrow to neonatal rodents; however, in adult recipients induction of transplantation tolerance by this strategy generally requires intensive cytoablative conditioning. Described here is a novel method of promoting transplantation tolerance that involves inoculation of donor bone marrow into the thymus of transiently immunosuppressed adult recipients. METHODS. Prospective Wistar-Furth recipients were inoculated with allogeneic Lewis bone marrow cells (BMCs) either intrathymically or intravenously in conjunction with a single dose of antilymphocyte serum 2 to 3 weeks before receiving donor-strain cardiac allografts. Recipients were monitored for graft survival and examined for presence of hematopoietic chimerism. RESULTS. Intrathymic but not intravenous inoculation of donor BMCs led to permanent survival of donor-strain cardiac allografts, whereas third-party Dark agouti cardiac allografts were rejected promptly. Persistence of donor chimerism was demonstrated in the thymus of Wistar-Furth recipients of intrathymic Lewis BMCs for as long as 3 weeks after BMC inoculation. CONCLUSIONS. Intrathymic inoculation of BMCs concurrently with a single dose of antilymphocyte serum induces donor-specific unresponsiveness to rat cardiac allografts. The unresponsiveness may be the result of deletion or functional inactivation of alloreactive clones maturing in a thymus bearing donor alloantigen. Intrathymic inoculation of BMCs deserves further evaluation as a possible clinical strategy for the induction of transplantation tolerance.     

In utero bone marrow transplantation induces kidney allograft tolerance across a full major histocompatibility complex barrier in Swine

BACKGROUND: In utero hematopoietic stem-cell transplantation has been shown to induce donor-specific tolerance in small-animal models. However, tolerance has been difficult to achieve in large-animal studies. METHODS: Outbred swine underwent in utero transplantation of fully major histocompatibility complex (MHC)-mismatched CD3-depleted bone marrow mixed with fresh bone marrow to achieve a final CD3 content of 1.5%. Transplantation was performed at 50 to 55 days' gestation and two animals survived long term and demonstrated multilineage peripheral blood hematopoietic chimerism. These two long-term survivors were analyzed for in vitro evidence of donor-specific tolerance by mixed leukocyte reaction (MLR), cell-mediated lysis (CML), and antibody testing and in vivo by kidney transplantation. RESULTS: Both animals demonstrated in vitro donor-specific unresponsiveness by MLR and CML and did not demonstrate anti-donor antibody production. Donor matched kidney transplants were performed without immunosuppression and functioned for more than 100 days, with no evidence for rejection. CONCLUSIONS: The authors demonstrate conclusively that in utero transplantation of fully MHC-mismatched bone marrow in swine can lead to engraftment and stable multilineage hematopoietic chimerism and tolerance to postnatal donor MHC-matched kidney transplantation without the need for immunosuppression.     

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.     

Targeted bone marrow radioablation with 153Samarium-lexidronam promotes allogeneic hematopoietic chimerism and donor-specific immunologic hyporesponsiveness

BACKGROUND: Transplantation tolerance, defined as acceptance of a graft by an otherwise fully immunocompetent host, has been an elusive goal. Although robust tolerance has been achieved by the induction of stable hematopoietic chimerism after bone marrow transplantation, lethal or sublethal radiation conditioning used to induce long-term chimerism precludes its clinical use. We studied whether targeted delivery of radiation to bone marrow could allow for bone marrow cell (BMC) engraftment, chimerism, and donor-specific tolerance in the absence of the side effects associated with external irradiation. METHODS: We administered a radioactive bone-seeking compound (Samarium-Lexidronam, Quadramet, Berlex Laboratories, Wayne, NJ) together with transient T-cell costimulatory blockade to recipient mice. Allogeneic BMCs were given 7 or 14 days after preconditioning. Costimulatory blockade was obtained by the use of an anti-CD154 antibody for 4 weeks. Chimerism was assessed by flow cytometry. Mice then received donor-specific and third-party skin grafts. Graft survival was analyzed with mechanisms of donor-specific hyporesponsiveness. RESULTS: High levels of stable chimerism across an allogeneic barrier were achieved in mice by a single administration of Samarium-Lexidronam, transient T-cell costimulatory blockade, and BMC transplantation. A large percentage of chimeric animals retained donor-derived skin grafts for more than 120 days without requiring additional immunosuppression, suggesting that harsh cytotoxic preconditioning is not necessary to achieve stable chimerism and donor specific hyporesponsiveness. Analysis of the T-cell repertoire in chimeras indicates T-cell deletional mechanisms. CONCLUSIONS: These data broaden the potential use of BMC transplantation for tolerance induction and argue for its potential in treating autoimmune diseases.     

Permanent acceptance of both cardiac and skin allografts using a mild conditioning regimen for the induction of stable mixed chimerism in mice

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.     

Simultaneous vascularized bone marrow transplantation to promote acceptance of hind limb allografts and its effects on central and peripheral chimerism

Administration of donor bone marrow (BM) cells can improve the outcome of transplantation. The ability of donor vascularized bone marrow transplantation (VBM) to provide an ongoing source of donor cells and improve survival in a rigorous rat model of hind limb transplantation (HLTX) was investigated. HLTX were performed between Brown Norway (BN) donors and Lewis recipients in three groups: HLTX; HLTX plus intravenous donor BM cells and HLTX plus simultaneous VBM transplantation. Animals received 12 weeks triple immunosuppression. Survival was compared at 4 months and donor chimerism was evaluated. Simultaneous VBM transplantation led to slight but nonsignificant prolongation of survival (P=0.056). Donor cells in the VBM were eventually replaced by recipient and there was no long-term increase in chimerism. Few donor cells were observed in thymus. Simultaneous VBM transplantation showed a trend for improved survival of HLTX however the VBM failed to provide a sustained increase in chimerism.     

Impact of Bone Marrow Transplantation on Type I Diabetes

Type I diabetes is a systemic autoimmune disease. Evidence is accumulating that autoimmune diseases such as type I diabetes are linked to the bone marrow hematopoietic stem cell (HSC) itself rather than its derivatives. HSC chimerism achieved through bone marrow transplantation (BMT) may affect type I diabetes in two ways: first, to induce tolerance to pancreas and islet cell transplants; and second, to reverse the autoimmune process prior to the development of terminal complications. Transplantation of bone marrow from normal donors into patients with hematologic malignancy and coexistent type I diabetes has reversed the systemic diabetic autoimmune process. Donor HSCs can also be utilized for the induction of donor-specific tolerance to islet cell transplants. Islet or whole pancreas transplantation is the most physiologic approach to treating type I diabetes. Currently, this is limited by the requirement for high-dose chronic nonspecific immunosuppression to prevent rejection. Despite these agents, chronic rejection remains the primary cause for late graft loss. Donor-specific tolerance eliminates the requirement for immunosuppression and prevents the development of chronic rejection. Bone marrow transplantation does have limitations. In particular these limitations include the morbidity associated with lethal conditioning, graft-versus-host disease, and failure of engraftment. Currently the morbidity and mortality associated with lethal conditioning could not be justified for tolerance induction or interruption of the autoimmune state in type I diabetes. The goal of current research is to identify those factors in both recipient and donor that optimize engraftment to reverse the risk/benefit ratio associated with BMT. This article reviews the state of the art for HSC chimerism affecting diabetes.     

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.     

Induction of stable chimerism and elimination of graft-versus-host disease by depletion of T lymphocytes from bone marrow using immunomagnetic beads

The goal of transplantation is the induction of immunologic tolerance. At present, nonspecific immunosuppression is used to prevent graft rejection and, commonly, graft-versus-host disease (GVHD). Nevertheless, nonspecific immunosuppressive therapy is frequently complicated by infection, malignant tumors, and drug toxicity. In order to examine whether hematopoietic chimerism can be used to induce specific allograft tolerance, we have reconstituted lethally irradiated Lewis rats with ACI bone marrow that has been depleted of T cells with use of immunomagnetic beads. This technique consists of binding OX-19, a mouse anti-rat pan-T lymphocyte monoclonal antibody, to magnetic polymer beads. Mixing of bone marrow or splenocytes with the bead/OX-19 complexes, followed by magnetic separation, results in significant depletion of T cells with minimal nonspecific cell loss. Immunomagnetic T-cell depletion of bone marrow, followed by reconstitution of a lethally irradiated host, allows for the development of stable, mixed hematopoietic chimerae without evidence of GVHD. These hosts are immunocompetent by clinical criteria. Recipients of untreated donor bone marrow that did or did not receive nonspecific immunosuppression demonstrated varying degrees of GVHD and reduced survival. The ability to rapidly and simply deplete T lymphocytes from bone marrow and produce stable, immunocompetent hematopoietic chimerae without GVHD may be an important method for tolerance induction to vascularized allografts.     

Tolerance through bone marrow transplantation with costimulation blockade

The routine induction of tolerance in organ transplant recipients remains an unattained goal. The creation of a state of mixed chimerism through allogeneic bone marrow transplantation leads to robust donor-specific tolerance in several experimental models and this approach has several features making it attractive for clinical development. One of its major drawbacks, however, has been the toxicity of the required host conditioning. The use of costimulation blocking reagents (anti-CD 154 monoclonal antibodies and the fusion protein CTLA4Ig) has led to much less toxic models of mixed chimerism in which global T cell depletion of the host is no longer necessary and which has even allowed the elimination of all cytoreductive treatment when combined with the injection of very high doses of bone marrow cells. In this overview we will briefly discuss general features of tolerance induction through bone marrow transplantation, will then describe recent models using costimulation blockade to induce mixed chimerism and will review the mechanisms of tolerance found with these regimens. Finally we will attempt to identify issues related to the clinical introduction of bone marrow transplantation with costimulation blockade which remain unresolved.     

Donor bone marrow transplantation: chimerism and tolerance

Infusion of donor bone marrow (DBM)-derived cells continue to be tested in clinical protocols intended to induce specific immunologic tolerance. Central clonal deletion of donor-specific alloreactive cells associated with mixed chimerism reliably produced long-term graft tolerance. In this setting, depletion of recipient T cells by antilymphocyte antibodies and subsequent repopulation by donor hematopoietic cells after donor bone marrow infusion (DBMI) are prerequisites for tolerance induction. Major advances have been made in animal models and in pilot clinical trials and the key questions with the future perspectives are presented in this article.     

Tolerance induction through megadose bone marrow transplantation with two-signal blockade

BACKGROUND: Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. MATERIALS AND METHODS: Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. RESULTS: High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. CONCLUSIONS: The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.