短期运用免疫抑制药物对非去髓异基因骨髓移植方案诱导的混合嵌合体和移植耐受的影响

目的 探讨免疫抑制药物对CTLA4-FasL重组腺病毒和供者骨髓移植联合方案诱导的混合嵌合耐受的影响.方法 将BALB/c（H-2d）小鼠皮肤移植于C57BL/6（H-2b,B6）小鼠,同时经尾静脉给B6小鼠注射BALB/c小鼠骨髓细胞和腺病毒AdCTLA4-FasL,B6小鼠接受皮肤移植术后4周内每天注射环孢素A（CsA）或霉酚酸酯（MMF）或这两种药物同时注射;然后观察移植皮肤存活情况,通过流式细胞仪测定受体外周血Vβ11^＋的T细胞的水平和供体来源细胞的嵌合水平,通过单向混合淋巴细胞反应了解对供体抗原的耐受状态.结果 在CTLA4-FasL重组腺病毒和供者骨髓移植联合方案诱导的混合嵌合耐受模型中,短期运用免疫抑制药物均促进早期供体骨髓细胞植入,但注射CsA或CsA与MMF联用的受体小鼠在皮肤移植后140 d时混合嵌合降低到很低水平,且单用CsA或CsA与MMF联用的受体小鼠中供体皮肤平均生存时间显著低于不用免疫抑制药物或用MMF的受体小鼠（P＜0.05）;皮肤移植后21 d时单用CsA或CsA与MMF联用的受体小鼠Vβ11^＋的T细胞水平显著高于不用免疫抑制药物或用MMF的受体小鼠（P＜0.05）;皮肤移植后150 d时单用CsA或CsA与MMF联用的受体小鼠脾细胞未显示对同种抗原特异性耐受.结论 CsA或含CsA的免疫抑制方案对CTLA4-FasL重组腺病毒和供者骨髓移植联合方案诱导的混合嵌合耐受有抑制作用,其机理可能与早期外周供体特异性T细胞删除减少有关;而MMF与该嵌合耐受方案兼容.     

Recent progress in tolerance induction through mixed chimerism

Organ transplant recipients require life-long treatment with immunosuppressive drugs. Currently available immunosuppression is associated with substantial morbidity and mortality, and is ineffective in inhibiting chronic rejection and graft loss. Therefore, a permanent state of donor-specific tolerance remains a primary goal for transplantation research. The induction of mixed hematopoietic chimerism is an attractive concept in this regard. Hematopoietic chimerism modulates the immunologic repertoire by extending the mechanisms of self-tolerance to donor-specific allotolerance. Despite recent progress in developing nontoxic bone marrow transplantation protocols for rodents, translation to large animals has remained difficult. Here, we outline the concept of tolerance via mixed chimerism, and review recent progress and remaining challenges in bringing this approach to the clinical setting.     

Donor double-negative Treg promote allogeneic mixed chimerism and tolerance

Bone marrow (BM) transplantation is an efficient approach to develop donor-specific tolerance and prevent chronic rejection. Allogeneic BM transplantation is limited by donor T cell-mediated graft-versus-host disease, requirement of cytoreduction and high numbers of BM cells. In addition of these drawbacks, recent studies demonstrate that not only T cells, but also NK cells can mediate BM rejection, and long-term mixed chimerism depends on NK cell tolerance. Thus, NK cell is another potential barrier against engraftment of BM and an important target in efforts to induce transplant tolerance. We have previously identified a novel type of Treg with the phenotype TCRalphabeta+CD3+CD4-CD8- (double-negative, DN). We and others have demonstrated that DN-Treg can effectively suppress anti-donor T cell responses. In this study, we found that donor-derived DN-Treg can suppress NK cell-mediated allogeneic BM graft rejection in both parent-to-F1 and fully MHC-mismatched BM transplantation models. Perforin and FasL in DN-Treg play important roles in the suppression of NK cells. Furthermore, adoptive transfer of DN-Treg can promote a stable mixed chimerism and donor-specific tolerance without inducing graft-versus-host disease. These results demonstrate a potential approach to control innate immune responses and promote allogeneic BM engraftment.     

Preclinical and clinical studies for transplant tolerance via the mixed chimerism approach

Based upon observations in murine models, we have developed protocols to induce renal allograft tolerance by combined kidney and bone marrow transplantation (CKBMT) in non-human primates (NHP) and in humans. Induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex (MHC)-mismatched primates, with detectable chimerism typically disappearing within 30–60?days. Nevertheless, in MHC mismatched NHP, long-term immunosuppression-free renal allograft survival has been achieved reproducibly, using a non-myeloablative conditioning approach that has also been successfully extended to human kidney transplant recipients. CKBMT has also been applied to the patients with end stage renal disease with hematologic malignancies. Renal allograft tolerance and long-term remission of myeloma have been achieved by transient mixed or persistent full chimerism. This review summarizes the current status of preclinical and clinical studies for renal and non-renal allograft tolerance induction by CKBMT. Improving the consistency of tolerance induction with less morbidity, extending this approach to deceased donor transplantation and inducing tolerance of non-renal transplants, are critical next steps for bringing this strategy to a wider range of clinical applications.     

Non-myeloablative mixed chimerism approaches and tolerance, a split decision

Stable mixed chimerism has been considered the most robust tolerance strategy. However, rejection of solid donor tissues by chimeras has been observed, a state termed split tolerance. Since new non-myeloablative mixed chimerism approaches are being actively pursued, we sought to determine whether they lead to full tolerance or split tolerance and to define the mechanisms involved. Fully mismatched mixed chimeras generated by induction with various lymphocyte-depleting antibodies along with either low-dose irradiation or busulfan and temporary sirolimus, maintained stable mixed chimerism but nevertheless rejected donor skin grafts. Generation of stable mixed chimerism using antibody targeting CD40L, but not depleting antibodies to CD4 and CD8, could prevent split tolerance when skin grafts were given together with donor bone marrow. Minor antigen matching abrogated the ability of effector T cells to reject donor skin grafts. A CFSE killing assay indicated that chimeras were both directly and indirectly tolerant of donor hematopoietic cell antigens, suggesting that minor mismatches triggered a tissue-specific response. Thus, split tolerance due to tissue-restricted polymorphic antigens prevents full tolerance in a number of non-myeloablative mixed chimerism protocols and a 'tolerizing' agent is required to overcome split tolerance. A model of the requirements for split tolerance is presented.     

Induction of tolerance by mixed chimerism with nonmyeloblative host conditioning: the importance of overcoming intrathymic alloresistance.

A nonmyeloablative conditioning regimen, consisting of depleting doses of anti-CD4 and anti-CD8 monoclonal antibodies (MoAbs) given on days -6 and -1 and 3 Gy of whole body irradiation given on day 0, allows the engraftment of fully major histocompatibility complex (MHC)-mismatched allogeneic bone marrow and the induction of tolerance for the graft. If MoAbs are given on day -5 only, permanent chimerism and tolerance are not observed in most animals. The addition of thymic irradiation to the single MoAb treatment permits tolerance induction in these mice, suggesting that residual host thymocytes reject donor marrow in recipients of 1, but not 2, MoAb injections. In this study, both CD4+ and CD8+ thymocytes were found to be responsible for residual alloreactivity in mice receiving only 1 MoAb injection. Co-receptor coating and downmodulation on residual thymocytes occur to a greater extent in recipients of 2 MoAb injections than in recipients of a single MoAb injection. This downmodulation may play a role in the loss of alloreactivity. Our results suggest that a second MoAb injection inactivates mature, functional donor-alloreactive CD4+ and CD8+ host thymocytes.     

Manipulating the immune system for anti-tumor responses and transplant tolerance via mixed hematopoietic chimerism

Summary: Stem cells (SCs) with varying potentiality have the capacity to repair injured tissues. While promising animal data have been obtained, allogeneic SCs and their progeny are subject to immune-mediated rejection. Here, we review the potential of hematopoietic stem cells (HSCs) to promote immune tolerance to allogeneic and xenogeneic organs and tissues, to reverse autoimmunity, and to be used optimally to cure hematologic malignancies. We also review the mechanisms by which hematopoietic cell transplantation (HCT) can promote anti-tumor responses and establish donor-specific transplantation tolerance. We discuss the barriers to clinical translation of animal studies and describe some recent studies indicating how they can be overcome. The recent achievements of durable mixed chimerism across human leukocyte antigen barriers without graft-versus-host disease and of organ allograft tolerance through combined kidney and bone marrow transplantation suggest that the potential of this approach for use in the treatment of many human diseases may ultimately be realized.     

Deletional and regulatory mechanisms coalesce to drive transplantation tolerance through mixed chimerism

Establishing donor‐specific immunological tolerance could improve long‐term outcome by obviating the need for immunosuppressive drug therapy, which is currently required to control alloreactivity after organ transplantation. Mixed chimerism is defined as the engraftment of donor hematopoietic stem cells in the recipient, leading to viable coexistence of both donor and recipient leukocytes. In numerous experimental models, cotransplantation of donor bone marrow (BM) into preconditioned (e.g., through irradiation or cytotoxic drugs) recipients leads to transplantation tolerance through (mixed) chimerism. Mixed chimerism offers immunological advantages for clinical translation; pilot trials have established proof of concept by deliberately inducing tolerance in humans. Widespread clinical application is prevented, however, by the harsh preconditioning currently necessary for permitting BM engraftment. Recently, the immunological mechanisms inducing and maintaining tolerance in experimental mixed chimerism have been defined, revealing a more prominent role for regulation than historically assumed. The evidence from murine models suggests that both deletional and regulatory mechanisms are critical in promoting complete tolerance, encompassing also the minor histocompatibility antigens. Here, we review the current understanding of tolerance through mixed chimerism and provide an outlook on how to realize widespread clinical translation based on mechanistic insights gained from chimerism protocols, including cell therapy with polyclonal regulatory T cells.     

Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks

Organ transplantation is the treatment of choice for patients with end‐stage organ failure, but chronic immunosuppression is taking its toll in terms of morbidity and poor efficacy in preventing late graft loss. Therefore, a drug‐free state would be desirable where the recipient permanently accepts a donor organ while remaining otherwise fully immunologically competent. Mouse studies unveiled mixed chimerism as an effective approach to induce such donor‐specific tolerance deliberately and laid the foundation for a series of clinical pilot trials. Nevertheless, its widespread clinical implementation is currently prevented by cytotoxic conditioning and limited efficacy. Therefore, the use of mouse studies remains an indispensable tool for the development of novel concepts with potential for translation and for the delineation of underlying tolerance mechanisms. Recent innovations developed in mice include the use of pro‐apoptotic drugs or regulatory T cell (T_reg) transfer for promoting bone marrow engraftment in the absence of myelosuppression and new insight gained in the role of innate immunity and the interplay between deletion and regulation in maintaining tolerance in chimeras. Here, we review these and other recent advances in murine studies inducing transplantation tolerance through mixed chimerism and discuss both the advances and roadblocks of this approach.     

The requirement of intrathymic mixed chimerism and clonal deletion for a long-lasting skin allograft tolerance in cyclophosphamide-induced tolerance

Mechanisms of cyclophosphamide (CY)-induced tolerance were studied. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were primed i.v. with 1 x 10(8) viable spleen cells from H-2-identical AKR/J Sea (AKR; H-2k, Mls-1a) mice and treated with 200 mg/kg of CY 2 days later, a long-lasting skin allograft tolerance to AKR was established. When [C57BL/6 Sea (B6; H-2b, Mls-1b) x AKR]F1 (B6AKF1) cells were used as the tolerogen, however, only a moderate, but not long-lasting, skin tolerance to AKR was observed. In the C3H mice treated with AKR cells and CY, the intrathymic clonal deletion of V beta 6+ T cells, which are strongly correlated with reactivity to Mls-1a antigens, was observed in the chimeric thymus on day 35, although neither the clonal deletion of V beta 6-bearing T cells nor the mixed chimerism was observed in the thymus on day 14. In the C3H mice treated with B6AFKF1 cells followed by CY, however, neither the clonal deletion of V beta 6+ T cells nor the mixed chimerism was observed in the thymus throughout the test period. In the lymph nodes of the C3H mice treated with AKR cells and CY, only CD4+ V beta 6+ T cells, bur not CD8+V beta 6+ T cells, had selectively decreased by day 14, and they were hardly detectable on day 35. The selective decrease of CD4+V beta 6+ T cells in the lymph nodes was also observed by day 14 when B6AKF1 cells were used as the tolerogen, although CD4+V beta 6+ T cells gradually increased on day 35, at which time almost all skin grafts from AKR had already been rejected. These results strongly support the necessity of the intrathymic mixed chimerism and clonal deletion of donor-reactive T cells for a long-lasting skin allograft tolerance in CY-induced tolerance.     

Immune monitoring of transplant patients in transient mixed chimerism tolerance trials

This review focuses on mechanistic studies performed in recipients of non-myeloablative bone marrow transplant regimens developed at Massachusetts General Hospital in HLA-identical and HLA-mismatched haploidentical combinations, initially as a platform for treatment of hematologic malignancies with immunotherapy in the form of donor leukocyte infusions, and later in combination with donor kidney transplantation for the induction of allograft tolerance. In patients with permanent mixed chimerism, central deletion may be a major mechanism of long-term tolerance. In patients in whom donor chimerism is only transient, the kidney itself plays a significant role in maintaining long-term tolerance. A high throughput sequencing approach to identifying and tracking a significant portion of the alloreactive T cell receptor repertoire has demonstrated biological significance in transplant patients and has been useful in pointing to clonal deletion as a long-term tolerance mechanism in recipients of HLA-mismatched combined kidney and bone marrow transplants with only transient chimerism.     

非清髓建立混合嵌合体诱导免疫耐受实验研究新进展

迄今为止,临床上尚未能建立一种有效地诱导免疫耐受的方法,而患者长期服用免疫抑制剂会产生许多毒副作用,且免疫抑制剂对慢性排斥和供体失功无明显改善作用.非清髓建立混合嵌合体是一种非常有效的诱导免疫耐受的方法,因而许多学者致力于非清髓建立混合嵌合体的实验研究.     

Donor‐specific tolerance induction in organ transplantation via mixed splenocytes chimerism

We have shown previously that donor‐derived splenocytes can replace recipients' bone marrow and induce donor‐specific tolerance (DST). We have also shown the usefulness of the chimeric state for the induction of DST. Further analysis of mixed splenocytes chimera, especially the role of each T cells in mixed splenocytes chimera, is indispensable issue for its clinical use. A chimeric state has been shown to achieve long‐term survival in major histocompatibility complex (MHC)‐mismatched grafts. The donor‐derived splenocytes can replace recipients' bone marrow and induce DST. The long‐term survival of allogeneic skin grafts was achieved without immunosuppressants. In this study we show the role of each T cell type in a splenocyte mixed chimera. This review provides a short summary of our original work, adding some supplemental interpretations. Mixed chimerism is thus considered an attractive approach for the induction of DST without the use of immunosuppressants. In this paper, we summarize some of the findings on mixed splenocyte chimeras and review mixed chimerism in recent organ transplantation.     

Mixed hematopoietic chimerism and transplantation tolerance

Durable transplantation tolerance can be reliably achieved by inducing engraftment of hematopoietic cells in recipients initially depleted of T-lymphocytes. Engraftment of donor pluripotent hematopoietic stem cells (PPHSC) produces mixed hematopoietic chimeras in which both host and donor cells coexist and are tolerant of each other. The major mechanism of tolerance in these chimeras is central, intrathymic clonal deletion, which is induced and maintained by immigration of both host and donor marrow-derived cells to the host thymus, ensuring the ongoing central deletion of donorand host-reactive cells. In this article, approaches developed in our laboratory to induce stable mixed hematopoietic chimerism and specific central deletional allogeneic and xenogeneic tolerance without toxic or myeloablative host conditioning are reviewed.     

Gene therapy for the induction of chimerism and transplant tolerance

Technical advances in transplant surgery and the development of powerful and effective immunosuppressive drugs have contributed to the success of organ transplantation as a medical treatment for patients with end-stage diseases. Associated with this procedure, however, is a dependence on life-long immunosuppressive drugs, which are required to prevent graft rejection. These agents render the patient susceptible to infections, tumors and various side affects. The ability to achieve tolerance to organ grafts would free transplant patients from lifelong dependency on pharmacological agents with harmful side effects. Several laboratories have shown that tolerance can be achieved by the induction of mixed cell chimerism and/or by molecular chimerism achieved by gene transfer techniques prior to graft placement. Molecular chimerism, induced by transplantation of autologous bone marrow expressing either allo- or xenoantigens has the potential to induce tolerance without the development of graft vs. host disease. The application of gene transfer techniques to induce chimerism has been shown to reshape the immune repertoire by mechanisms that include clonal deletion, the induction of central tolerance or generation of regulatory T cells that would eliminate the need for immunosuppressive drugs. Optimization of this methodology for clinical use could therefore revolutionize the field of transplantation. This review summarizes the recent studies which have compared the efficacy of different vectors, conditioning regimens, and transduction conditions leading to new and improved techniques for the application of gene therapy to induce chimerism and transplant tolerance to both allografts and xenografts.     

Importance of intrathymic mixed chimerism for the maintenance of skin allograft tolerance across fully allogeneic antigens in mice

In B6 (H-2b) mice that had been given, neonatally, 1x108 B6AKF1 spleen cells intraperitoneally (i.p.), only a moderate prolongation of donor (AKR:H-2k) skin graft survival was observed. In such B6 mice, no mixed lymphocyte reaction (MLR) to AKR could be detected on day 35 (35 days after birth), but it was clearly evident on day 84. Similarly, neither Vbeta6+ (reactive to MTV-7-encoded antigens) nor Vbeta11+ (reactive to I-E+MTV-derived superantigens) T cells were detected on day 35, but both were clearly evident on day 84 in both the thymus and the lymph nodes, thus indicating the breakdown of intrathymic mixed chimerism at the antigen-presenting cell level. Furthermore, by day 84, all skin grafts from AKR had already been rejected in such B6 mice. In the periphery, however, Vbeta6+, but not Vbeta11+, T cells were clonally anergic on day 84, based on a stimulation assay with anti-T-cell receptor (TCR) monoclonal antibody (mAb), thus suggesting that tolerance to some antigens, but not to others, may be induced by the clonal anergy in fully allogeneic combinations, and that the clonal anergic state may be masked by other proliferative responses. These results therefore indicate the importance of intrathymic mixed chimerism (central tolerance) and the limitations of clonal anergy (peripheral tolerance) in maintaining tolerance across fully allogeneic antigen barriers.     

Photochemical treatment of donor lymphocytes inhibited their ability to facilitate donor engraftment or increase donor chimerism after nonmyeloablative conditioning or establishment of mixed chimerism.

Donor T-cells can provide a graft-versus-leukemia effect and help to promote donor engraftment after allogeneic BMT; however, these benefits can be outweighed by the ability of the cells to induce life-threatening GVHD. Photochemical treatment (PCT) of T-cells with S-59 psoralen and long-wavelength UV-A light can inhibit their proliferative capacity and significantly decrease their ability to induce acute GVHD after allogeneic BMT. PCT donor T-cells have been shown to facilitate donor engraftment in a myeloablative BMT model. In this study, we examined whether donor T-cells subjected to PCT ex vivo could retain the ability to facilitate engraftment or increase donor chimerism after nonmyeloablative BMT or after establishment of mixed hematopoietic chimerism. In a transplantation model in which mice were conditioned for BMT with sublethal (600 cGy) TBI, an infusion of PCT donor T-cells was unable to facilitate engraftment of donor BM. A BMT model was used in which a mixture of allogeneic and syngeneic marrow cells was infused into lethally irradiated recipients for establishment of mixed hematopoietic chimerism. The goal was to determine whether PCT donor splenocytes could increase levels of donor chimerism. Recipients of splenocytes treated with UV-A light only (no S-59 psoralen) and given at the time of BMT or in a donor lymphocyte infusion (DLI) had significantly higher levels of donor chimerism than did recipients of BM only. Although PCT donor splenocytes given at the time of BMT modestly increased donor chimerism, PCT donor splenocytes given in a DLI did not increase donor chimerism. A nonmyeloablative BMT model was employed for determining whether DLI given relatively late after BMT could increase donor chimerism. Recipient mice were conditioned for BMT with a combination of low-dose TBI (50 or 100 cGy) and anti-CD154 (anti-CD40L) monoclonal antibody for achievement of low levels of mixed chimerism. When control mixed chimeras were given a DLI 71 days after BMT, donor chimerism was significantly increased. In contrast, PCT of the donor cells eliminated the ability of the cells to increase donor chimerism after infusion. Together results from these 3 distinct BMT models indicate that PCT of donor T-cells significantly inhibited the ability of the cells to facilitate donor engraftment after nonmyeloablative BMT or to increase donor chimerism in mixed hematopoietic chimeras when the cells were administered in a DLI.