New strategies for bone marrow transplantation

Adoptive immunotherapy of hematologic malignancies and metastatic solid tumors by donor lymphocyte infusion following induction of host-versus-graft transplantation tolerance against can best be achieved following nonmyeloablative stem-cell transplantation (NST). Induction of mixed chimerism may represent the best approach for induction of transplantation tolerance to donor alloantigens. Thus NST may become the optimal approach for the treatment of nonmalignant diseases, where replacement of host with donor hematopoietic cells is indicated: for correction of genetic or stem cell deficiency diseases; as a platform for immunotherapy of autoimmune or infectious diseases; or for induction of tolerance to organ allografts.     

Transplantation tolerance: from theory to clinic

Tolerance induction and alloreactivity can be applied to the clinic for the transplantation of solid organs and in the treatment of human cancers respectively. Hematopoietic chimerism, the stable coexistence of host and donor blood cells, guarantees that a solid organ from the same donor will be tolerated without a requirement for maintenance immunosuppression, and it also serves as a platform for the adoptive immunotherapy of hematologic malignancies using donor lymphocyte infusions. This review focuses on clinically relevant methods for inducing hematopoietic chimerism and transplantation tolerance, with a special emphasis on reduced intensity transplantation conditioning and high dose, post-transplantation cyclophosphamide to prevent graft rejection and graft-versus-host disease (GVHD). Reduced intensity transplantation regimens permit a transient cooperation between donor and host immune systems to eradicate malignancy without producing GVHD. Their favorable toxicity profile also enables the application of allogeneic stem cell transplantation to treat non-malignant disorders of hematopoiesis and to induce tolerance for solid organ transplantation.     

Essential role for B cells in transplantation tolerance

T lymphocytes are the primary targets of immunotherapy in clinical transplantation. However, B lymphocytes are detrimental to graft survival by virtue of their capacity to present antigen to T cells via the indirect pathway of allorecognition and the generation of donor specific alloantibody. Furthermore, the long-term survival of organ allografts remains challenged by chronic rejection, a process in which activated B cells have been found to play a significant role. Therefore, the achievement of transplantation tolerance will likely require induction of both T and B cell tolerance to alloantigens. Moreover, human and animal investigations have shown that subsets of B cells, Transitional and Regulatory, are inherently tolerogenic. Developing therapeutic strategies that exploit these populations may be key to achieving transplantation tolerance. In this review we describe the current evidence for the essential role of B cells in transplant tolerance and discuss emerging B cell directed strategies to achieve allograft tolerance.     

Transplantation tolerance with monoclonal antibodies

Monoclonal antibodies (Mabs) are powerful tools for manipulating the immune system as they are capable of triggering, amplifying or suppressing immune responses. The resultant effect of monoclonal antibody (MAb) manipulation is related not only to the antigenic specificity of the antibody used, but also to its structural properties and mechanism of action. There has been much interest in using MAbs to control or regulate immune responses in vivo and they are now commonly used successfully in clinical transplantation for the treatment of graft rejection. The induction of transplantation tolerance in adult recipients is an important goal of transplantation research. The development of MAbs for immunotherapy has allowed new approaches for the induction of tolerance to vascularised organ grafts to be explored.     

Monoclonal antibody therapy for the induction of transplantation tolerance

There are three ways in which monoclonal antibodies could be used to facilitate the induction of tolerance to foreign tissues after organ transplantation. First, depleting monoclonal antibodies could be directed against the T cells responsible, thereby reducing their number and acting to non-specifically immunosuppress the patient. This is generally not sufficient to allow tolerance induction in the T cells which repopulate the periphery. Second, depleting monoclonal antibodies could be used to remove donor passenger leukocytes and antigen-presenting cells from the donor organ, which may both reduce immunogenicity and increase the chance of tolerance induction. Third, non-depleting, but functionally blocking, monoclonal antibodies to T cell molecules such as CD4 and CD8 can allow the specific induction of transplantation tolerance in mouse models, an approach which might be applicable to man, not only for organ transplantation, but also in the treatment of autoimmune diseases. These three approaches are, in time, likely to complement each other in clinical practice. Monoclonal antibodies can be tailored to each approach by choosing appropriate specificities and isotypes, and further refinements can be made where necessary by making monovalent or humanised antibodies. The application of each of these approaches to clinical therapy is described.     

Relative Immunogenicity and Tolerogenicity of Individual Regions or Segments of the MHC

Mixed lymphocyte culture (MIX) reactivity, induction of cytotoxicity in vitro, survival of skin allografts, and induction of neonatal transplantation tolerance were compared in mice of the strain combinations differing in the entire H-2 complex or in individual segments of it. The results showed that antigenic products of the K end of the H-2 complex were more immunogenic and also more resistant to tolerance induction than the products of the D end of H-2. I-region products that elicited transplantation reactions of variable strength, depending on the detection system, were relatively easier to overcome in tolerance induction and could, at least in some combinations, contribute to tolerance induction across the barrier represented by the products of the K region of the H-2 complex.     

Intrathymic Tolerance Induction: Determination of Tolerance to Class II Major Histocompatibility Complex Antigens in Maturing T Lymphocytes by a Bone Marrow-Derived Non-Lymphoid Thymus Cell

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.     

Lymphocyte apoptosis: Mechanism of specific immunotherapy of atopic diseases

Effect of specific immunotherapy on the expression of activation markers, apoptosis marker CD95, and peripheral blood lymphocyte morphology were studied in patients with atopic bronchial asthma. Specific therapy activated T and B cells and triggered lymphocyte apoptosis. Apoptosis may play an important role in induction of allergen tolerance during specific therapy. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 4, pp. 434–436, April, 1998     

Markers of tolerance development to food allergens

IgE‐mediated reactions to food allergens are the most common cause of anaphylaxis in childhood. Although allergies to cow's milk, egg, or soy proteins, in contrast to peanut and tree nut allergens, resolve within the first 6 years of life in up to 60% due to natural tolerance development, this process is not well understood. At present, there is no cure or treatment for food allergy that would result in an induction of tolerance to the symptom‐eliciting food. Avoidance, providing an emergency plan and education, is the standard of treatment. Oral immunotherapeutic approaches have been proven reasonable efficacy; however, they are associated with high rates of side‐effects and low numbers of patients achieving tolerance. Nevertheless, mechanisms that take place during oral immunotherapy may help to understand tolerance development. On the basis of these therapeutic interventions, events like loss of basophil activation and induction of regulatory lymphocyte subsets and of blocking antibodies have been described. Their functional importance at a clinical level, however, remains to be investigated in detail. Consequently, there is eminent need to understand the process of tolerance development to food allergens and define biomarkers to develop and monitor new treatment strategies for food allergy.     

Induction of tolerance to alloantigen

The study of transplantation tolerance has been a major area of immunological research since the pioneering work by Medawar and colleagues. It has been classically defined as the absence of an immune response to a specific antigen in the setting of an otherwise normal immune system. The induction of tolerance to alloantigen in the transplantation setting would not only allow better graft survival but would also obviate the need for immunosuppressive therapy. The induction of tolerance is considered by many to be the "holy grail" of organ transplantation but, despite many years of experimental investigation, reliable induction of allograft tolerance has not been achieved in humans. There exists four fundamental mechanisms of T cell tolerance that may be operational in the transplantation setting: deletion, functional anergy, ignorance and regulation/suppression. Several strategies to induce tolerance in transplantation have been employed, but most of these tolerogenic strategies have been investigated in rodents and it remains to be determined whether they can be transferred into large outbred animal models or clinical transplantation.     

Mechanisms of induction of tolerance to organ allografts

The long-term acceptance of organ allografts can be induced in numerous rodent and some preclinical outbred models. Induction methods can use donor alloantigen in various forms, including spontaneous acceptance of grafts such as livers, to deviate the immune response so a subsequent graft will be accepted. Establishment of lymphohemopoietic chimerism is not essential. Short-term immunosuppressive treatments that prevent acute rejection can also induce tolerance. These include nonspecific immunosuppressive drugs and immunotherapy that blocks cell surface molecular interactions or cytokine function. There is variation in the effect of these protocols on different strain combinations that may be due to innate differences in the cell subpopulations and cytokines activated in the hosts. Th1 cytokines, although important in the mediation of rejection, are also required for induction of tolerance. Th2 cytokines may facilitate tolerance induction but are not essential. The tolerant state takes weeks to fully mature after exposure to alloantigen. Tolerance is associated with a loss or change in dendritic cells and the development of suppressor cells, which in all cases include CD4+ T cells. In the near future precise understanding of the function of these two cell types may allow diagnosis and induction of tolerance in clinical transplantation.     

Chimerism‐based tolerance in organ transplantation: preclinical and clinical studies

Induction of allograft tolerance has been considered the ultimate goal in organ transplantation. Although numerous protocols to induce allograft tolerance have been reported in mice, a chimerism‐based approach through donor haematopoietic stem cell transplantation has been the only approach to date that induced allograft tolerance reproducibly following kidney transplantation in man. Renal allograft tolerance has been achieved by induction of either transient mixed chimerism or persistent full donor chimerism. Although the risk of rejection may be low in tolerance achieved via durable full donor chimerism, the development of graft‐versus‐host disease (GVHD) has limited the wider clinical application of this approach. In contrast, tolerance induced by transient mixed chimerism has not been associated with GVHD, but the risk of allograft rejection is more difficult to predict after the disappearance of haematopoietic chimerism. Current efforts are directed towards the development of more clinically feasible and reliable approaches to induce more durable mixed chimerism in order to widen the clinical applicability of these treatment regimens.     

Tr1 cell immunotherapy promotes transplant tolerance via de novo Tr1 cell induction in mice and is safe and effective during acute viral infection

Tr1 cell therapy is considered an emerging approach to improve transplant tolerance and enhance allogeneic graft survival. However, it remains unclear how Tr1 cells promote transplant tolerance and whether they will be safe and stable in the face of an acute viral infection. By employing a mouse model of pancreatic islet transplantation, we report that Tr1 cell therapy promoted transplant tolerance via de novo induction of Tr1 cells in the recipients. Acute viral infection with lymphocytic choriomeningitis virus (LCMV) had no impact on Tr1 cell number and function, neither on the Tr1 cells infused nor on the ones induced, and that was reflected in the robust maintenance of the graft. Moreover, Tr1 cell immunotherapy had no detrimental effect on CD8 and CD4 anti‐LCMV effector T‐cell responses and viral control. Together, these data suggest that Tr1 cells did not convert to effector cells during acute infection with LCMV, maintained transplant tolerance and did not inhibit antiviral immunity.     

Transplantation of hematopoietic stem cells for induction of unresponsiveness to organ allografts

Although it has been recognized since the early days of Owen and Medawar that engraftment of donor stem cells, induced in utero spontaneously or intentionally neonatally, results in life-long unresponsiveness to donor alloantigens. However, successful induction of transplantation tolerance in adult life still represents an unsolved problem. Engraftment of donor stem cells using conventional modalities involves intensive myeloablative or lymphoablative immunosuppression, which is associated with toxicity and mortality and such methods are not suitable for organ allograft recipients. In this chapter, we present an innovative approach for induction of donor-specific unresponsiveness to bone marrow and organ allografts without myeloablative conditioning. Our methods is based on cyclophosphamide-induced, alloantigen-primed lymphocyte depletion. Cyclophosphamide is administered 1 day following infusion of donor hematopoietic cells, thus eliminating predominantly host T lymphocytes reacting against donor cell challenge, and resulting in relative unresponsiveness to donor alloantigens. Subsequently, life-long tolerance to fully mismatched donor skin allografts can be accomplished by a second infusion of stem cells from the same donor, with donor T cells displacing residual alloreactive host cells that may have escaped deletion. Taken together, we believe that induction of true permanent and specific tolerance to organ allografts using donor hematopoietic cells could become a clinical reality in the foreseeable future.     

Permanent protection of PLG scaffold transplanted allogeneic islet grafts in diabetic mice treated with ECDI-fixed donor splenocyte infusions

Allogeneic islet cell transplantation is a promising treatment for human type 1 diabetes. Currently, human islets are transplanted via intra-portal infusions. While successful, it leads to significant early islet attrition from instant blood-mediated inflammatory reaction. An extra-hepatic site was established by transplanting islet-loaded microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad in syngeneic islet transplant models. This study examined this technology in allogeneic islet transplantation and determined whether transplant tolerance could be effectively induced to protect PLG scaffold transplanted allogeneic islets. The efficacy of an established tolerance induction strategy using donor splenocytes treated with ethylcarbodiimide(ECDI) was tested. ECDI-fixed donor splenocytes were infused 7 days before and 1 day after islet transplantation. Immediate normoglycemia was restored, and treated mice maintained indefinite normoglycemia whereas untreated mice rejected islet grafts within 20 days of transplantation. Interestingly, efficacy of tolerance induction was superior in PLG scaffold compared with intra-portal transplanted islets. Protection of PLG scaffold islet allografts was associated with several mechanisms of immune regulation. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that does not impair tolerance induction. This approach of combining tolerance induction with scaffold islet transplantation has potential therapeutic implications for human islet transplantation.     

Antigen-specific regulatory T-cell subsets in transplantation tolerance regulatory T-cell subset quality reduces the need for quantity

Regulatory T cells (Treg) are critical controllers of the immune response. Disturbed Treg function results in autoimmunity, whereas in transplantation Treg are crucial in graft survival and transplant tolerance. Hence therapeutic modalities that influence Treg numbers or function hold great clinical opportunity. Ahead of us are clinical trails studying in vivo Treg induction protocols and immunotherapy with ex vivo expanded Treg. Here we discuss the preferential use and/or induction of antigen-specific Treg subsets with high suppressive power and migratory capacity as a potential therapeutic tool to prevent solid organ transplantation rejection. Accordingly, ex vivoselection procedures to induce and isolate highly suppressive antigen-specific Treg (subsets) are needed. This subject, as well as the Treg-facilitating potential of immunosuppressive agents, is discussed.     

RNAi DC在移植免疫耐受诱导研究中的应用进展

致耐受树突状细胞(Tol DC)可以诱导T细胞免疫耐受,近年来在移植免疫耐受诱导及自身免疫病治疗等方面展示出独特的应用价值。利用近年来发展的RNA干扰(RNAi)技术可以特异性沉默DC某些基因表达构建出RNAi DC,是诱导致耐受性DC产生的又一新方法。     

A critical role for Stat3 signaling in immune tolerance

Antigen-presenting cells (APCs) can induce T cell activation as well as T cell tolerance. The molecular mechanisms by which APCs regulate this critical decision of the immune system are not well understood. Here we show that Stat3 signaling plays a critical role in the induction of antigen-specific T cell tolerance. Targeted disruption of Stat3 signaling in APCs resulted in priming of antigen-specific CD4(+) T cells in response to an otherwise tolerogenic stimulus in vivo. Furthermore, APCs devoid of Stat3 effectively break antigen-specific T cell anergy in vitro. Conversely, increased Stat3 activity in APCs led to impaired antigen-specific T cell responses. Stat3 signaling provides, therefore, a novel molecular target for manipulation of immune activation/tolerance, a central decision with profound implications in autoimmunity, transplantation, and cancer immunotherapy.     

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.     

In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display “empty” HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8⁺ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.