Clinical experience with mixed chimerism to induce transplantation tolerance

Lymphohematopoietic chimerism was first shown to be associated with donor‐specific allograft tolerance more than 60 years ago. However, early clinical experience with bone marrow transplantation soon revealed that conventional, myeloablative approaches were far too toxic and the risk of graft‐versus‐host disease too great to justify using this technology for the purpose of organ allograft tolerance induction in the absence of malignant disease. In this review, we discuss a step‐wise approach that has been applied by several centers to establish less toxic approaches to using hematopoietic cell transplantation (HCT) for tolerance induction. These steps include (i) feasibility and efficacy data for tolerance induction in large animal models; (ii) safety data in clinical trials for patients with hematologic malignancies; and (iii) pilot trials of combined HCT and kidney transplantation for tolerance induction. Thus far, only one published trial conducted at the Massachusetts General Hospital in Boston has achieved long‐term acceptance of human leukocyte antigen‐mismatched kidney allografts without chronic immunosuppressive therapy. Alternative protocols have been successful in large animals, but long‐term organ allograft tolerance has not been reported in patients. Thus, proof‐of‐principle that nonmyeloablative induction of mixed chimerism can be used intentionally to induce organ allograft tolerance has now been achieved. Directions for further research to make this approach applicable for a broader patient population are discussed.     

Allogeneic hematopoietic stem-cell transplantation, mixed chimerism, and tolerance in living related donor renal allograft recipients

OBJECTIVE: We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts. PATIENTS AND METHODS: Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad x 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW x 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4+/CD 8+ T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 x 10(8) cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy. RESULTS: Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 +/- 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients. CONCLUSION: We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.     

Mixed chimerism to induce tolerance for solid organ transplantation

Chimerism, or the coexistence of tissue elements from more than one genetically different strain or species in an organism, is the only experimental state that results in the induction of donor-specific transplantation tolerance. Transplantation of a mixture of T-cell-depleted syngeneic (host-type) plus T-cell-depleted allogeneic (donor) bone marrow into a normal adult recipient mouse (A + B----A) results in mixed allogeneic chimerism. Recipient mice exhibit donor-specific transplantation tolerance, yet have full immunocompetence to recognize and respond to third-party transplantation antigens. After complete hematolymphopoietic repopulation at 28 days, animals accept a donor-specific skin graft but reject major histocompatibility complex (MHC) locus-disparate third-party grafts. We now report that permanent graft acceptance can also be achieved when the graft is placed at the time of bone marrow transplantation. Histologically, grafts were viable and had only minimal inflammatory changes. This model may have potential future clinical application for the induction of donor-specific transplantation tolerance.     

Targeted T-cell depletion or CD154 blockade generates mixed hemopoietic chimerism and donor-specific tolerance in mice treated with sirolimus and donor bone marrow

BACKGROUND: The administration of donor specific bone marrow (DSBM) to mice conditioned with antilymphocyte serum (ALS) and sirolimus can result in stable multilineage mixed chimerism and long-term graft survival. This study seeks to determine if either the targeted depletion of CD4 and/or CD8 pos T cells or costimulation blockade can substitute for ALS and preserve the efficacy of this regimen. METHODS: C57BL/6 recipients of BALB/c skin allografts were treated with DSBM (150 x 10(6) cells), sirolimus (24 mg/kg intraperitonealy), and either ALS or various monoclonal antibodies (alphaCD4, alphaCD8, alphaCD154 alone or in combination). Recipient peripheral blood mononuclear cell (PBMC) depletion, donor chimerism, and deletion of donor reactive T cells were assessed using flow cytometry. The specificity of immunologic nonreactivity and the presence of immunoregulatory activity were assessed through a mixed lymphocyte reaction assay. RESULTS: The administration of ALS, sirolimus, and DSBM resulted in sustained recipient PBMC depletion, transient chimerism, and prolonged graft survival. The substitution of an equivalent degree and duration of targeted depletion of either CD4 or CD8 pos T cells alone for ALS failed to produce chimerism or prolonged graft survival. In contrast, depletion of both CD4 and CD8 pos T cells resulted in durable multilineage chimerism, indefinite allograft acceptance (>350 days), and donor-specific tolerance to secondary skin grafts. Substitution of alphaCD154 monoclonal antibody for ALS also resulted in a state of mixed chimerism and donor specific tolerance. This tolerant state appears to be maintained at least partially through clonal deletion and suppression. CONCLUSION: Either combined CD4 and CD8 T-cell depletion or alphaCD154 blockade can effectively substitute for ALS in producing chimerism and tolerance in this model.     

Mixed chimerism and transplantation tolerance

Establishment of mixed hematopoietic chimerism carries with it the induction of transplantation tolerance to any other tissue or organ from the same donor. This strategy has been studied extensively for induction of tolerance in mice. During the past decade, we have extended the same strategy, with modifications, to cynomolgus monkeys and most recently to renal transplant patients. In this report we review the evolution of these studies from preclinical applications to our current clinical experience with two therapeutic protocols sponsored by the Immune Tolerance Network. The first of these studies is for patients with myeloma and end-stage renal disease with an HLA-matched sibling donor; the second for patients with end-stage renal disease and HLA-mismatched donors. Although it is still early in the course of these studies, the results to date are very encouraging.     

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.     

供者NK细胞及其嵌合状态在异基因造血干细胞移植中的研究进展

目前,异基因造血干细胞移植(allo-HSCT)已广泛应用于造血系统疾病的治疗,但移植术后也存在一系列并发症。NK细胞的运用为改善allo-HSCT受者预后带来希望,供者来源NK细胞通过其细胞膜上的杀伤细胞免疫球蛋白样受体与其配体错配发挥同种异体反应,该过程具有保留移植物抗白血病和减少移植物抗宿主病双重效应。NK细胞是allo-HSCT后受者体内最早重建的免疫细胞群,因此移植后供、受者NK细胞嵌合状态评估对预测疾病预后及指导干预治疗具有重要意义。基于NK细胞嵌合状态的供者NK细胞输注免疫干预疗法可改善疾病预后,在血液系统疾病治疗中表现出良好的应用前景。本文就近年来供者NK细胞及其嵌合状态在allo-HSCT中的研究进展作一综述。     

Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning

BACKGROUND: We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. METHODS: Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day -2. Cyclosporine was administered for 30 to 60 days beginning on day -1. A total of 1 to 2 x 10(10) /kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. RESULTS: Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. CONCLUSIONS: These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.     

AdCTLA-4Ig combined with donor splenocytes,bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti–ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.Abbreviations BMC bone marrow cells - ICOS inducible co-stimulator - pfu plaque-forming units - SPC spleen cell     

Efficacy of donor splenocytes mixed with bone marrow cells for induction of tolerance in sublethally irradiated mice

BACKGROUND: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice. METHODS: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3 x 10(6) BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation. RESULTS: Recipients receiving 3 X 106 BMCs mixed with 10 x 10(6) T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients. CONCLUSIONS: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.     

Transplantation tolerance through mixed chimerism: From allo to xeno

To date, the only successful means of achieving allogeneic transplantation tolerance in the clinic has involved induction of mixed lymphohematopoietic chimerism. Such chimerism was first achieved in mice and subsequently in large animals, including miniature swine, monkeys and most recently humans. The mechanism of tolerance has differed between models, involving both deletional and regulatory mechanisms, in varying proportions, depending on the model. Considerable progress has also been made toward induction of tolerance across the xenogeneic pig‐to‐primate barrier, although complete success has not yet been achieved. The two approaches toward xenograft tolerance currently being investigated both involve establishment of a mixture of host and donor cells in the thymus, in one case through administration of donor bone marrow to the recipient and in the other through vascularized donor thymus transplantation to a thymectomized recipient. Hopefully, a combination of these approaches may provide an effective means for achieving full tolerance and thereby bringing xenograft organ transplantation to the clinic.     

Use of DNA restriction fragment length polymorphisms to document marrow engraftment and mixed hematopoietic chimerism following bone marrow transplantation

We have studied the feasibility of using DNA restriction fragment-length polymorphisms (RFLP) to study marrow engraftment in 27 patients after allogeneic bone marrow transplantation, and have compared these results with those obtained using red blood cell antigens, cytogenetics, and immunoglobulin allotypes. Using highly polymorphic DNA probes, we have documented stable chronic mixed hematopoietic chimerism, have identified transient mixed chimeras, have excluded mixed chimerism with high probability in retrospective studies even when a pretransplant DNA sample was not available, have documented marrow engraftment in the early posttransplant period, and have studied the origin of leukemic cells in patients with recurrent disease. We have evaluated the advantages and disadvantages of several genetic markers and have developed tentative statements concerning the prognosis of patients with mixed chimerism. We conclude that DNA RFLP are powerful and practical genetic markers in bone marrow transplantation studies and that further studies of mixed hematopoietic chimerism are warranted.     

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.     

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.     

Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning

BACKGROUND: Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. METHODS: B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. RESULTS: IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. CONCLUSIONS: Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.