Application of xenogeneic stem cells for induction of transplantation tolerance: present state and future directions

Xenotransplantation using pig organs provides a possible solution to the severe shortage of allogeneic organ donors, one of the major limiting factors in clinical transplantation. However, because of the greater antigenic differences that exist between different species than within a species, the immune response to xenografts is much more vigorous than to allografts. Thus, tolerance induction is essential to the success of clinical xenotransplantation. Tolerance induced by mixed hematopoietic chimerism across the MHC barrier is remarkably robust, but its ability to induce tolerance across highly disparate xenogeneic barriers remains poorly studied. None of the current available regimens of host conditioning, which permit hematopoietic stem cell engraftment and chimerism induction in allogeneic or closely related (concordant) xenogeneic combinations, has been demonstrated to be effective in establishing porcine hematopoietic chimerism in a discordant xenogeneic species. Unlike bone marrow transplantation within the same species, the innate immune system and the species specificity of cytokines and adhesion molecules essential to hematopoiesis pose formidable obstacles to the establishment of donor hematopoiesis across discordant xenogeneic barriers. The genetic incompatibility between species may also impede xenograft tolerance induction by mixed chimerism. While we remain far from achieving tolerance in clinical xenotransplantation, recent studies using a transgenic mouse model have proven the principle that mixed hematopoietic chimerism may induce mouse and human T cell tolerance to porcine xenografts. This review article focuses on the barriers to porcine hematopoietic engraftment in highly disparate xenogeneic species and the possible application of mixed hematopoietic chimerism to xenograft tolerance induction.     

Reconstitution of self-tolerance after hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow or hematopoietic stem cell transplantation. GVHD is thought to be primarily due to the response of mature T cells transferred along with the bone marrow graft to foreign histocompatibility antigens expressed on host tissues. Recent studies, however, have challenged this paradigm set forth in the 1960s and have suggested that self-MHC class II antigens can be recognized in GVHD. Many questions still remain unanswered particularly in regard to the role of immune reconstitution, the ability to recognize and discriminate self and the re-establishment of self-tolerance. In fact, the failure to re-establish tolerance to self can lead to systemic autoimmunity that may exacerbate or even mimic GVHD. The present review summarizes our studies in autologous GVHD characterizing the underlying immune mechanisms and their potential impact in allogeneic hematopoietic stem cell transplantation.     

Gene therapy in the transplantation of allogeneic organs and stem cells

Most of the current hematopoietic stem cell (HSC) -directed gene therapy applications have focused on the replacement of defective or deficient genes in an autologous setting. More recently HSC gene therapy applications have also included the enhancement or improvement of HSC features. Allogeneic HSCs have been used to facilitate and improve allogeneic transplantation and to achieve tolerance to transplanted cells, tissues or organs. Different gene transfer approaches addressing a variety of immunomodulatory mediators contributing to graft tolerance or immunological ignorance may have a critical role in improving long-term graft survival. Allogeneic tissues are frequently recognized by allospecific T cells as foreign and are rapidly rejected in the absence of immunosuppression. The higher susceptibility to cancer and infectious diseases of immunosuppressed patients led to investigation of new therapies to induce graft-specific tolerance. Peripheral tolerance to allogeneic grafts can be achieved by a variety of mechanisms including clonal deletion, suppression caused by regulatory T cells and anergy induction associated with microchimerism effect. In the last decades, potential candidates to confer allograft protection were identified. In this review, we summarize ongoing strategies and developments in genetic manipulation of cells, tissues and organs for allogeneic transplantation including modulating the effector arm of the immune response.     

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.     

From stem cells to lymphocytes; biology and transplantation

Summary: We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cell This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.     

Clinical tolerance in allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) has been a curative therapeutic option for a wide range of immune hematologic malignant and non-malignant disorders including genetic diseases and inborn errors. Once in the host, allogeneic transplanted cells have not only to ensure myeloid repopulation and immunological reconstitution but also to acquire tolerance to host human leukocyte antigens via central or peripheral mechanisms. Peripheral tolerance after allogeneic HSCT depends on several regulatory mechanisms aimed at blocking alloimmune reactivity while preserving immune responses to pathogens and tumor antigens. Patients transplanted with HSCT represent an ideal model system in humans to identify and characterize the key cellular and molecular players underlying these mechanisms. The knowledge gained from these studies has allowed the development of novel therapeutic strategies aimed at inducing long-term peripheral tolerance, which can be applicable not only in allogeneic HSCT but also in autoimmune diseases and solid-organ transplantation. In the present review, we describe Type 1 regulatory T cells, initially discovered and characterized in chimeric patients transplanted with human leukocyte antigen-mismatched HSCT, and how their presence correlates to tolerance induction and maintenance. Furthermore, we summarize different cell therapy approaches with regulatory T cells, designed to facilitate tolerance induction, minimizing pharmaceutical interventions.     

Role of naturally arising regulatory T cells in hematopoietic cell transplantation.

Naturally arising CD4(+)CD25(+) regulatory T cells (Tregs) have the potential to suppress aberrant immune responses and to regulate peripheral T-cell homeostasis. In murine models of bone marrow transplantation, Tregs promote donor bone marrow engraftment and decrease the incidence and severity of graft-versus-host-disease without abrogating the beneficial graft-versus-tumor immunologic effect. These findings, in concert with observations that Tregs in mice and humans share phenotypic and functional characteristics, have led to active investigations into the use of these cells to decrease complications associated with human hematopoietic cell transplantation. Early human studies suggest that an imbalance of Tregs and effector T cells may contribute to the development of graft-versus-host-disease. However, the mechanisms of immunoregulation, in particular the allorecognition properties of Tregs, their effects on and interaction with other immune cells, and their sites of suppressive activity, are not well understood. In this review, we discuss the current knowledge of Treg biology and the potential therapeutic strategies and barriers of Treg immunotherapy in human hematopoietic cell transplantation.     

Non-myeloablative stem cell transplantation for autoimmune diseases

Treatment of life-threatening autoimmune diseases in animal models with induced or spontaneous autoimmune diseases can be accomplished by a 2-step procedure involving elimination of self-reactive lymphocytes with an immune ablative conditioning regimen followed by infusion of autologous or allogeneic stem cells, respectively. In animal models it was shown that using such a strategy, autoimmunity could be adequately controlled. It is speculated that de-novo development of the T and B cell repertoire from uncommitted progenitor cells in the presence of the autoantigens may be the best recipe for re-induction of self-tolerance, similarly to the normal ontogeny of the immune system during the induction of self tolerance in fetal stage. For both autologous and allogeneic hematopoietic stem cell transplantation, a non-myeloablative stem cell transplantation (NST) regimen may be used for safer lymphoablation rather than myeloablation. In addition, for allogeneic hematopoietic stem cell transplantation engraftment of disease resistant donor stem cells will alter the genetic predisposition towards autoimmune disease susceptibility.     

Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation.

Mesenchymal stem cells (MSCs) may be derived from adult bone marrow, fat, and several fetal tissues. In vitro, MSCs can be expanded and have the capacity to differentiate into several mesenchymal tissues, such as bone, cartilage, and fat. They escape the immune system in vitro, and this may make them candidates for cellular therapy in an allogeneic setting. They also have immunomodulatory effects, inhibit T-cell proliferation in mixed lymphocyte cultures, prolong skin allograft survival, and may decrease graft-versus-host disease (GVHD) when cotransplanted with hematopoietic stem cells. MSCs induce their immunosuppressive effect via a soluble factor. Some candidates have been suggested, and various mechanisms have also been suggested, although contradictory data exist; this may be due to differences in the cells and systems tested. A major problem has been that it has been difficult to identify and isolate MSCs after transplantation in vivo. However, MSCs seem to enhance hematopoietic engraftment in recipients of autologous and allogeneic grafts. Recently, they were found to reverse grade IV acute GVHD of the gut and liver. No tolerance was induced, however. Controlled studies are warranted. Thus, in allogeneic stem cell transplantation, MSCs may be used for hematopoiesis enhancement, as GVHD prophylaxis, and for the treatment of severe acute GVHD. They are also of potential use in the treatment of organ transplant rejection and in autoimmune inflammatory bowel disorders where immunomodulation and tissue repair are needed.     

Tolerance induction for solid organ grafts with donor-derived hematopoietic reconstitution

Tolerance of transplanted tissue has been a focus of immunologists for decades. Indeed, to some the birth of immunology and the search for tolerance of the non-self are synonymous. One of the most powerful and reproducible methods of tolerance induction to allogeneic tissue has involved infusion of donor-specific hematopoietic cells. Under certain conditions, such infusion can result in hematopoietic reconstitution that can be experimentally accomplished at a variety of different time-points in the life of an organism from the in utero period through adulthood, reconstitution at each time-point involving consideration of a different set of immunological and physiological parameters. When high levels of donor-derived hematopoietic reconstitution are achieved, tolerance induction to donor-specific antigens is reproducible and long-lasting. Unfortunately, however, clinical efforts to achieve such high levels of hematopoietic reconstitution have historically been unsuccessful or fraught with complications. Transplantation efforts have been plagued by failure of engraftment, graft-vs-host disease (GVHD), or severe immunoincompetence of the recipient. Laboratory and clinical efforts during the last decade have resulted in a variety of developments that may overcome these barriers: (1) methods have been devised in which cells that cause GVHD can be depleted from the hematopoietic graft while hematopoietic reconstitution potential is preserved, (2) methods of harvesting large numbers of cells with multilineage reconstitution potential have been devised (an accomplishment that seems to allow the immunological barrier to be overwhelmed), and (3) capitalizing on the above two principles, minimally toxic preconditioning regimens have been designed that allow allogeneic engraftment. This review will focus on some of the experimental and clinical data of the past and the experimental and clinical issues that loom ahead.     

Minor histocompatibility antigen-specific cytotoxic T lymphocytes generated with dendritic cells from DLA-identical littermates.

Donor cytotoxic T lymphocytes (CTL) specific for minor histocompatibility antigens (mHA) mediate the graft-versus-host effect whereas host mHA-specific CTL mediate graft rejection in the setting of major histocompatibility complex identical allogeneic hematopoietic stem cell transplantation. Development of a large animal model from which mHA-specific CTL can be isolated would accelerate translation in clinical studies to improve control of the graft-versus-host effect as well as prevention of graft rejection in sensitized hosts. The aims of the current study were to isolate mHA-specific CTL from dog leukocyte antigen-identical littermate nonsensitized recipients before transplantation, from stable mixed hematopoietic chimeras, and from dogs sensitized to mHA after graft rejection. Donor dendritic cells (DCs) were cultured from bone marrow-derived CD34(+) cells and were used to stimulate recipient T lymphocytes on days 1, 10, and 20 of CTL culture. We reliably generated and expanded mHA-specific CTL ex vivo from sensitized dogs that were given a donor-specific blood transfusion to boost immune recall after graft rejection after a nonmyeloablative transplantation. The mHA-specific cytotoxicity measured by (51)Cr release assay was enriched from less than 5% in the starting population of sensitized peripheral blood mononuclear cells to a median of 63% after 4 weeks in CTL culture. The expanded mHA-specific CTLs were not tissue-specific: hematopoietic cells, fibroblast, and stromal cell lines were lysed in an mHA-specific manner. Allogeneic DCs, but not peripheral blood mononuclear cells, were necessary for stimulating ex vivo expansion of mHA-specific CTL. We were unable to generate mHA-specific CTL from nonsensitized dogs before transplantation, from previously sensitized dogs but without recent recall immunization, or from stable mixed hematopoietic chimeras. We conclude that after recent in vivo sensitization, large-scale ex vivo expansion of mHA-specific CTL was feasible using allogeneic DCs.     

Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self

Summary:  Although the optimal donor for allogeneic hematopoietic stem cell transplantation (HSCT) is a human leukocyte antigen-matched sibling, 75% of patients do not have a match, and alternatives are matched unrelated volunteers, unrelated umbilical cord blood units, and full-haplotype-mismatched family members. To cure leukemia, allogeneic HSCT relies on donor T cells in the allograft, which promote engraftment, eradicate malignant cells, and reconstitute immunity. Here, we focus on the open issues of rejection, graft-versus-host disease (GVHD), and infections and the benefits of natural killer (NK) cell alloreactivity and its underlying mechanisms. Donor-versus-recipient NK cell alloreactivity derives from a mismatch between inhibitory receptors for self-major histocompatibility complex (MHC) class I molecules on donor NK clones and the MHC class I ligands on recipient cells. These NK clones sense the missing expression of the self-MHC class I allele on the allogeneic targets and mediate alloreactions. HSCT from 'NK alloreactive' donors controls acute myeloid relapse without causing GVHD. We review the translation of NK cell recognition of missing self into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia.     

Harnessing dendritic cells to improve allogeneic hematopoietic cell transplantation outcome

In clinical practice, hematopoietic cell transplantation (HCT) is now recognized as a powerful means of delivering effective cellular immunotherapy for malignant and non-malignant diseases. In patients with severe hematological malignancies, the success of allogeneic HCT is largely based on immunologic graft-versus-tumor (GVT) effects mediated by allogeneic T lymphocytes present in the graft. Unfortunately, this beneficial effect is counterbalanced by the occurrence of graft versus host reactions directed against normal host tissues resulting in graft versus host disease (GVHD), a potentially life-threatening complication that limits the success of allogeneic HCT. Therefore, while preserving beneficial GVT effects, a major objective in allogeneic HCT is the prevention of GVHD. Studies in the last decade revealed the central role of dendritic cells and macrophages in modulating graft versus host immune reactions after allogeneic HCT. In this review, we summarize recent progress and potential new therapeutic avenues using dendritic cell-based strategies to improve allogeneic HCT outcome.     

Thymic epithelium induces full tolerance to skin and heart but not to B lymphocyte grafts

Athymic nude mice reconstituted at birth with allogeneic thymic epithelia (TE) from day 10 embryos (E10), show life-long specific tolerance to skin and heart grafts, but eliminate B lymphocytes of the TE donor haplotype, nearly as well as those from a third strain. Previous immunizations with B cells do not alter the state of tolerance to skin grafts, but specifically accelerate elimination of lymphocytes. In contrast, transplantation of E15 allogeneic thymuses already seeded by hematopoietic cells resulted in chimeras tolerant to both skin and B lymphocytes. In vitro reactivities towards stimulator spleen cells of the haplotype of the thymus were observed in both E10 TE and E15 thymus chimeras. We conclude that induction of full in vivo tolerance to B cells requires hematopoietic cells, while this is not the case for induction of tolerance to skin and heart tissues; furthermore, in vitro reactivity to stimulator spleen cells of the tolerized haplotype is independent of in vivo tolerance.     

Studies of ex vivo activated and expanded CD8+ NK-T cells in humans and mice

Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. Here we describe studies of both human and murine expanded CIK cells or CD8⁺ NK-T cells. These related populations of cells are ex vivo activated and expanded T cells that express both T and NK markers. They can be generated from patients with malignancies and mediate cytotoxicity against autologous hematopoietic malignancies. Recent work in murine models show that these cells mediate cytotoxicity by using a perforin–granzyme and not through Fas ligand. In allogeneic stem cell transplantation experiments, large numbers of expanded CD8⁺ NK-T cells could be transplanted across major histocompatibility barriers without causing severe GVHD and GVT effects were retained. We conclude that expanded CD8⁺ NK-T cells are a promising form of cellular therapy in the allogeneic setting.     

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.     

MicroRNAs as Potential Diagnostic,Prognostic, and Predictive Biomarkers for Acute Graft-versus-Host Disease

Successful treatment of various hematologic diseases with allogeneic hematopoietic stem cell transplantation is often limited due to the occurrence of acute graft-versus-host disease (aGVHD). So far, there are no approved molecular biomarkers for the diagnosis and prediction of aGVHD at the clinical level due to our incomplete understanding of the molecular biology of the disease. Various studies have been conducted on animal models and humans to investigate the role of microRNAs in aGVHD pathogenesis to implicate them as biomarkers and therapeutic targets. Because of their high stability, tissue specificity, ease of measurement, low cost, and simplicity, they are excellent targets for biomarkers. In this review, we focused on microRNA expression profiling studies that were performed recently in both animal models and human cases of aGVHD to identify diagnostic and predictive biomarkers for this disease. The expression pattern of microRNAs can be specific to cells and tissues. Because aGVHD affects several organs, microRNA signatures in target tissues may help to understand the molecular pathology of the disease. Identification of organ-specific microRNAs in aGVHD can be promising to categorize patients for organ-specific therapies. Thus, microRNAs can be used as noninvasive diagnostic tests in clinic to improve prophylaxis, predict incidence and severity, and reduce morbidity.     

G-CSF、GM-CSF的临床应用现状

粒细胞集落刺激因子（Ｇｒａｎｕｌｏｃｙｔｅ ｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇ ｆａｃｔｏｒ, Ｇ－ＣＳＦ）、粒巨噬细胞集落刺激因子（ Ｇｒａｎｕｌｏｃｙｔｅ ｍａｃｒｏｐｈａｇｅ ｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇ ｆａｃｔｏｒ,ＧＭ－ＣＳＦ）分别是促粒系、粒－单系造血祖细胞增殖、分化及成熟,并增强其成熟细胞功能的特异造血调控生长因子。重组ＤＮＡ技术的发展,使得利用基因工程方法大量生产细胞因子成为可能。目前,Ｇ－ＣＳＦ、ＧＭ－ＣＳＦ基因重组产品已广泛应用于临床。在血液病的治疗,造血干细胞移植及恶…     

Treg cell therapy: How cell heterogeneity can make the difference

CD4⁺CD25^highCD127^low/−FOXP3⁺ T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy.     

Clinical impact of natural killer cell reconstitution after allogeneic hematopoietic transplantation

Although the optimal donor for allogeneic hematopoietic stem cell transplantation is a human leukocyte antigen (HLA)-matched sibling, 75% of patients do not have a match and alternatives are matched unrelated volunteers, unrelated umbilical cord blood units, and full HLA-haplotype-mismatched family members. This review will focus on the open issues of allogeneic hematopoietic transplantation and on the benefits of natural killer (NK) cell alloreactivity and its underlying mechanisms. Donor-versus-recipient NK cell alloreactivity derives from a mismatch between inhibitory receptors for self major histocompatibility complex (MHC) class I molecules on donor NK clones and the MHC class I ligands on recipient cells. These NK clones sense the missing expression of the self MHC class I allele on the allogeneic targets (“missing self”) and mediate alloreactions. Here, we review the translation of NK cell allorecognition into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia.