Advances in the treatment of acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologic malignant and non‐malignant hematologic diseases and other diseases. However, acute graft‐versus‐host disease (GVHD) is a life‐threatening complication of allogeneic transplantation. Acute GVHD may occur in 30% of transplant recipients, which is a syndrome of erythematous skin eruption, cholestatic liver disease and intestinal dysfunction, resulting from the activation of donor T lymphocytes by host antigen‐presenting cells, resulting in an immune‐mediated inflammatory response. Recent scientific advances in the understanding of the pathogenesis involved in the development of acute GVHD and clinical investigation have provided more effective therapeutic strategies for acute GVHD. This review focuses on major scientific and clinical advances in the treatment of acute GVHD.     

Umbilical cord-derived mesenchymal stem cells promote myeloid-derived suppressor cell enrichment by secreting CXCL1 to prevent graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsHuman mesenchymal stem cells (MSCs) from various tissues have emerged as attractive candidates for the prevention and treatment of graft-versus-host disease (GVHD). However, the molecular machinery that defines and channels the behavior of these cells remains poorly understood.MethodsIn this study, the authors compared the efficacy of four tissue-derived MSC types in controlling GVHD in a murine model and investigated their immunomodulatory effects.ResultsHuman umbilical cord-derived mesenchymal stem cells (hUCMSCs) effectively decreased the incidence and severity of GVHD, which was mediated by the enrichment of myeloid-derived suppressor cells in GVHD target tissues. RNA sequencing results showed that hUCMSCs highly expressed CXCL1.ConclusionsThese results suggest a novel prophylactic application of hUCMSCs for controlling GVHD after allogeneic hematopoietic stem cell transplantation.     

CD4^＋CD25^＋Foxp3^＋调节性T细胞的体外扩增及其在移植物抗宿主病中的应用

同种异基因造血干细胞移植是急、慢性白血病及其他恶性血液病重要的治疗方法,但急慢性移植物抗宿主病（graft—versus-host disease,GVHD）作为异基因造血干细胞移植的主要并发症严重影响移植患者的存活率,阻碍移植的临床推广。很多研究发现,高表达Foxp3的CD4^＋CD25^＋调节性T细胞（regulatory T cells,Treg）不仅能控制急慢性GVHD的发生,而且不影响移植物抗白血病效应（graft-versusleukemia,GVL）,在急慢性GVHD发生发展及治疗方面有重要的作用。但Treg细胞在体内的数量很少,不能满足临床应用需求。目前应用外源的IL-2联合TCR、CD28信号通路共同刺激以及运用树突状细胞（dendritic cell,DC）刺激均能达到体外有效扩增Treg细胞的目的。这些扩增的Treg细胞在控制造血干细胞移植过程中急慢性GVHD的发生及防治自身免疫性疾病和移植排斥等方面具有明显作用,在疾病控制和临床应用中具有广阔前景。     

生长因子诱导人脐带/胎盘间充质干细胞定向分化

近年来,间充质干细胞(mesenchymal stem cell,MSC)已成为干细胞领域的研究热点,其不仅支持造血系统,还可在特定的培养条件下向多种组织细胞分化。人脐带和胎盘来源的MSC取材容易,较骨髓间充质干细胞有更广泛的应用前景。本文就含有特定生长因子的培养基诱导人脐带MSC和人胎盘MSC定向分化的研究进展作一简要的综述。     

Harnessing and honing mesenchymal stem/stromal cells for the amelioration of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency. Despite its increased use, the mortality rate for patients undergoing this procedure remains high, mainly due to the perceived risk of exacerbating graft-versus-host disease (GVHD). However, even with immunosuppressive agents, some patients still develop GVHD. Advanced mesenchymal stem/stromal cell (MSC) strategies have been proposed to achieve better therapeutic outcomes, given their immunosuppressive potential. However, the efficacy and trial designs have varied among the studies, and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs. This review aims to provide real insights into this clinical entity, emphasizing diagnostic, and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues. The indications and timing for the clinical application of MSCs are still subject to debate.     

Bone marrow mesenchymal stem cells suppress lymphocyte proliferation in vitro but fail to prevent graft-versus-host disease in mice

Several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect in vitro, and thus may have a therapeutic potential for T cell-dependent pathologies. We aimed to establish whether MSCs could be used to control graft-vs-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation. From C57BL/6 and BALB/c mouse bone marrow cells, we purified and expanded MSCs characterized by the lack of expression of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. These MSCs suppressed alloantigen-induced T cell proliferation in vitro in a dose-dependent manner, independently of their MHC haplotype. However, when MSCs were added to a bone marrow transplant at a MSCs:T cells ratio that provided a strong inhibition of the allogeneic responses in vitro, they yielded no clinical benefit on the incidence or severity of GVHD. The absence of clinical effect was not due to MSC rejection because they still could be detected in grafted animals, but rather to an absence of suppressive effect on donor T cell division in vivo. Thus, in these murine models, experimental data do not support a significant immunosuppressive effect of MSCs in vivo for the treatment of GVHD.     

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)-expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver-injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co-cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions.     

Reduced Intensity Conditioning,Combined Transplantation of Haploidentical Hematopoietic Stem Cells and Mesenchymal Stem Cells in Patients with Severe Aplastic Anemia

We examined if transplantation of combined haploidentical hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) affected graft failure and graft-versus-host disease (GVHD) in patients with severe aplastic anemia (SAA). Patients with SAA-I (N = 17) received haploidentical HSCT plus MSC infusion. Stem cell grafts used a combination of granulocyte colony-stimulating factor (G-CSF)-primed bone marrow and G-CSF-mobilized peripheral blood stem cells of haploidentical donors and the culture-expanded third-party donor-derived umbilical cord MSCs (UC-MSCs), respectively. Reduced intensity conditioning consisted of fludarabine (30 mg/m²·d)+cyclosphamide (500 mg/m²·d)+anti-human thymocyte IgG. Transplant recipients also received cyclosporin A, mycophenolatemofetil, and CD25 monoclonal antibody. A total of 16 patients achieved hematopoietic reconstitution. The median mononuclear cell and CD34 count was 9.3×10⁸/kg and 4.5×10⁶/kg. Median time to ANC was >0.5×10⁹/L and PLT count >20×10⁹/L were 12 and 14 days, respectively. Grade III-IV acute GVHD was seen in 23.5% of the cases, while moderate and severe chronic GVHD were seen in 14.2% of the cases. The 3-month and 6-month survival rates for all patients were 88.2% and 76.5%, respectively; mean survival time was 56.5 months. Combined transplantation of haploidentical HSCs and MSCs on SAA without an HLA-identical sibling donor was safe, effectively reduced the incidence of severe GVHD, and improved patient survival.     

The Additive Effect of Mesenchymal Stem Cells and Bone Morphogenetic Protein 2 on γ-Irradiated Bone Marrow in Mice

Irradiation from γ-rays can cause severe damage to bone marrow and hematopoietic tissues. Presently, the most effective method available to treat severe hematopoietic injury is a bone marrow transplant (BMT). Allogeneic BMT is a difficult technique to perform due to the differences in human leukocyte antigen proteins between the donor and recipient, with acute graft-versus-host disease being a major complication of the technique. This limits the widespread applicability of allogeneic BMT. To develop a novel treatment for acute hematopoietic damage, we transplanted bone marrow derived mesenchymal stem cells (MSCs) into recipient mice and treated them with recombinant human bone morphogenetic protein 2 (rhBMP2) to investigate whether MSCs and rhBMP2 could additively promote the restoration of hematopoietic function. MSCs are vital components of the hematopoietic microenvironment that supports hematopoiesis, and bone morphogenic protein is a key factor in hematopoiesis. The 30-day survival rate as well as the numbers of nucleated cells, bone marrow colony-forming unit-granulocyte macrophages, spleen colony-forming units and peripheral blood cells were enumerated. The results showed that, after γ-irradiation and transplantation, MSCs and rhBMP2 additively promoted and improved hematopoietic restoration and function in vivo and in vitro. This additive effect of MSCs and rhBMP2 may one day provide a novel means of treating acute hematopoietic damage.     

Hematopoietic stem cell transplantation with umbilical cord multipotent stromal cell infusion for the treatment of aplastic anemia—a single-center experience

Background aimsThe purpose of this study was to observe the outcome of co-transfusion of umbilical cord multipotent stromal cells (UC-MSC) and allogeneic hematopoietic stem cells in the treatment of heavily-transfused patients with severe aplastic anemia.MethodsOf the 22 patients, eight cases received haploidentical hematopoietic stem cells from granulocyte colony-stimulating factor–primed bone marrow and peripheral blood grafts; the other patients received granulocyte colony-stimulating factor–mobilized peripheral blood grafts from human leukocyte antigen–matched related (six cases) and unrelated donors (eight cases). MSCs were intravenously infused at a mean dose of 1.2 × 10⁶/ kg (range, 0.27–2.5 × 10⁶/kg). Fludarabine-based conditioning was conducted, and graft-versus-host disease prophylaxis containing cyclosporine A, methotrexate and mycophenolate mofetil with or without addition of anti-CD25 monoclonal antibody was performed. Hematopoietic engraftment, the occurrence of graft-versus-host disease (GVHD) and infections and overall survival were documented.ResultsAll patients had rapid engraftment; mean time for neutrophil and platelet recovery was 13.95 d and 20.27 d, respectively. No acute toxicity associated with UC-MSC transfusion was observed. Acute GVHD developed in seven cases (grade I–II), and none had development of chronic GVHD. Cytomegalovirus reactivation was observed in 11 cases. One patient died of pulmonary complication 6 months after transplantation. Twenty-one patients are currently alive, at a median follow-up of 15 months; they are transfusion-independent and reached full donor chimerism at the time of reporting.ConclusionsUC-MSC infusion might be an alternative option to promote hematopoietic engraftment and reduce the occurrence of GHVD in hematopoietic stem cell transplantation in the treatment of heavily transfused patients with severe aplastic anemia.     

Allogeneic transplantation across the HLA barriers

In high-risk acute leukemia patients, a 10-fold increase in the dose of extensively T-cell-depleted hematopoietic stem cells ensures sustained full-donor engraftment of one-haplotype-mismatched transplants without graft-vs.-host disease. Since our first successful pilot study, which exploited the principle of a megadose stem cell transplant, our efforts have concentrated on developing new conditioning regimens, optimizing graft processing and improving the post-transplant immunologic recovery. The results so far achieved in more than 100 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype-mismatched family donor, a T-cell-depleted mismatched transplant can be offered with curative intent, thus extending allogeneic transplantation procedures to virtually all candidates.     

Immunophenotypic characteristics of multipotent mesenchymal stromal cells that affect the efficacy of their use in the prevention of acute graft vs host disease

BACKGROUNDMultipotent mesenchymal stromal cells (MSCs) are widely used in the clinic due to their unique properties, namely, their ability to differentiate in all mesenchymal directions and their immunomodulatory activity. Healthy donor MSCs were used to prevent the development of acute graft vs host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). The administration of MSCs to patients was not always effective. The MSCs obtained from different donors have individual characteristics. The differences between MSC samples may affect their clinical efficacy.AIMTo study the differences between effective and ineffective MSCs.METHODSMSCs derived from the bone marrow of a hematopoietic stem cells donor were injected intravenously into allo-BMT recipients for GVHD prophylaxis at the moment of blood cell reconstitution. Aliquots of 52 MSC samples that were administered to patients were examined, and the same cells were cultured in the presence of peripheral blood mononuclear cells (PBMCs) from a third-party donor or treated with the pro-inflammatory cytokines IL-1β, IFN and TNF. Flow cytometry revealed the immunophenotype of the nontreated MSCs, the MSCs cocultured with PBMCs for 4 d and the MSCs exposed to cytokines. The proportions of CD25-, CD146-, CD69-, HLA-DR- and PD-1-positive CD4+ and CD8+ cells and the distribution of various effector and memory cell subpopulations in the PBMCs cocultured with the MSCs were also determined.RESULTSDifferences in the immunophenotypes of effective and ineffective MSCs were observed. In the effective samples, the mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD105, and CD146 was significantly higher. After MSCs were treated with IFN or cocultured with PBMCs, the HLA-ABC, HLA-DR, CD90 and CD54 MFI showed a stronger increase in the effective MSCs, which indicated an increase in the immunomodulatory activity of these cells. When PBMCs were cocultured with effective MSCs, the proportions of CD4+ and CD8+central memory cells significantly decreased, and the proportion of CD8+CD146+ lymphocytes increased more than in the subpopulations of lymphocytes cocultured with MSC samples that were ineffective in the prevention of GVHD; in addition, the proportion of CD8+effector memory lymphocytes decreased in the PBMCs cocultured with the effective MSC samples but increased in the PBMCs cocultured with the ineffective MSC samples. The proportion of CD4+CD146+ lymphocytes increased only when cocultured with the inefficient samples.CONCLUSIONFor the first time, differences were observed between MSC samples that were effective for GVHD prophylaxis and those that were ineffective. Thus, it was shown that the immunomodulatory activity of MSCs depends on the individual characteristics of the MSC population.     

Hematopoietic stem cell transplantation from alternative donors for high-risk acute leukemia: the haploidentical option

Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted full-haplotype mismatched transplants for acute leukemia. Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immuno-myeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal graft-vs-host disease (GvHD) without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in more than 200 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.     

Allogeneic peripheral blood stem cell transplantation

Granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) are now widely used instead of bone marrow for autologous transplantation due to earlier hematopoietic recovery after transplant. The low toxicity of G-CSF has prompted phase I and II studies to evaluate PBSC for allogeneic transplantation; these studies have demonstrated that engraftment of neutrophils, red blood cells and platelets is faster with peripheral blood cells compared to marrow. In randomized studies comparing mobilized PBSC and marrow for allogeneic transplantation, most trials have confirmed significantly earlier engraftment with PBSC and similar risks of acute graft-vs.-host disease (GVHD). In some trials, an increase of 10-15% in grade II-IV GVHD has been noted with PBSC. All studies showed a trend towards more chronic GVHD with PBSC. Some randomized studies have shown improved survival and disease-free survival with the use of PBSC due to lowered transplant-related mortality and fewer relapses in recipients of PBSC as a result of improved immune reconstitution and a graft-vs.-leukemia (GVL) effect. This survival benefit is most apparent in patients with more advanced hematologic malignancies, but further studies are needed to define the relative benefits of PBSC for patients with less advanced disease. The GVL effect of PBSC is currently being exploited with the use of non-ablative allografts.     

Virotherapy Using Myxoma Virus Prevents Lethal Graft-versus-Host Disease following Xeno-Transplantation with Primary Human Hematopoietic Stem Cells

Graft-versus-host disease (GVHD) is a potentially lethal clinical complication arising from the transfer of alloreactive T lymphocytes into immunocompromised recipients. Despite conventional methods of T cell depletion, GVHD remains a major challenge in allogeneic hematopoietic cell transplant. Here, we demonstrate a novel method of preventing GVHD by ex vivo treatment of primary human hematopoietic cell sources with myxoma virus, a rabbit specific poxvirus currently under development for oncolytic virotherapy. This pretreatment dramatically increases post-transplant survival of immunocompromised mice injected with primary human bone marrow or peripheral blood cells and prevents the expansion of human CD3(+) lymphocytes in major recipient organs. Similar viral treatment also prevents human-human mixed alloreactive T lymphocyte reactions in vitro. Our data suggest that ex vivo virotherapy with myxoma virus can be a simple and effective method for preventing GVHD following infusion of hematopoietic products containing alloreactive T lymphocytes such as: allogeneic hematopoietic stem and progenitor cells, donor leukocyte infusions and blood transfusions.     

Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologi-cal malignancies and congenital deficiencies. In recent years, non-myeloablative and reduced-intensity condi-tioning regimens have significantly expanded t…     

Tolerance might be stably induced by both mesenchymal stem cell and hepatocyte growth factor in heart transplantation

Heart transplantation still remains the best choice of treatment for many kinds of end-stage heart diseases, but the side-effects of immunosuppressive agents and cardiac allograft vasculopathy (CAV) remain the main two obstacles in improving the long outcome of cardiac allografts. Mesenchymal stem cells (MSCs) are nonhematopoietic pluripotent cells that retain the ability to undergo differentiation into cells of different lineages. What's more, MSCs have been demonstrated to exert many profound inhibitory effects on many lymphocytic subpopulations such as T cells, regulatory T cells, and dendritic cells in vitro. In vivo studies also suggested that MSCs could be used to attenuate immune-mediated disorders such as transplant rejection and autoimmune diseases including rheumatoid arthritis, multiple sclerosis and GVHD. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in protecting infarcted myocardial by its antiapoptotic, antifibrotic and angiogenic effects. And also it was suggested that the administration of HGF could effectively suppress acute and chronic cardiac allograft rejection. We based our hypothesis on that HGF-modified MSCs would engraft stably in recipient and interact with important immune cells such as T cells, B cells and dendritic cells, then stable immune tolerance be induced possibly by both immunomodulation of MSCs and cardioprotective and immunomodulative effects of HGF. Further work is necessary to highlight the specific underlying mechanisms.     

International Society for Cell & Gene Therapy Stem Cell Engineering Committee: Cellular therapies for the treatment of graft-versus-host-disease after hematopoietic stem cell transplant

Background aimsAllogeneic hematopoietic stem cell transplant is a curative approach for many malignant and non-malignant hematologic conditions. Despite advances in its prevention and treatment, the morbidity and mortality related to graft-versus-host disease (GVHD) remains. The mechanisms by which currently used pharmacologic agents impair the activation and proliferation of potentially alloreactive T cells reveal pathways essential for the detrimental activities of these cell populations. Importantly, these same pathways can be important in mediating the graft-versus-leukemia effect in recipients transplanted for malignant disease. This knowledge informs potential roles for cellular therapies such as mesenchymal stromal cells and regulatory T cells in preventing or treating GVHD. This article reviews the current state of adoptive cellular therapies focused on GVHD treatment.MethodsWe conducted a search for scientific literature in PubMed® and ongoing clinical trials in clinicaltrial.gov with the keywords “Graft-versus-Host Disease (GVHD),” “Cellular Therapies,” “Regulatory T cells (Tregs),” “Mesenchymal Stromal (Stem) Cells (MSCs),” “Natural Killer (NK) Cells,” “Myeloid-derived suppressor cells (MDSCs),” and “Regulatory B-Cells (B-regs).” All the published and available clinical studies were included.ResultsAlthough most of the existing clinical data focus on cellular therapies for GVHD prevention, there are observational and interventional clinical studies that explore the potential for cellular therapies to be safe modalities for GVHD treatment while maintaining the graft-versus-leukemia effect in the context of malignant diseases. However, there are multiple challenges that limit the broader use of these approaches in the clinical scenario.ConclusionsThere are many ongoing clinical trials to date with the promise to expand our actual knowledge on the role of cellular therapies for GVHD treatment in an attempt to improve GVHD-related outcomes in the near future.     

Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion

Adult bone marrow mesenchymal stromal cells (MSCs) have cross-functional, intrinsic potency that is of therapeutic interest. Their ability to regenerate bone, fat, and cartilage, modulate the immune system, and nurture the growth and function of other bone marrow hematopoietic stem/progenitor cells have all been evaluated by transplant applications of MSCs. These applications require the isolation and expansion scaled cell production. To investigate biophysical properties of MSCs that can be feasibly utilized as predictors of bioactivity during biomanufacturing, we used a low-density seeding model to drive MSCs into proliferative stress and exhibit the hallmark characteristics of in vitro aging. A low-density seeding method was used to generate MSCs from passages 1–7 to simulate serial expansion of these cells to maximize yield from a single donor. MSCs were subjected to three bioactivity assays in parallel to ascertain whether patterns in MSC age, size, and shape were associated with the outcomes of the potency assays. MSC age was found to be a predictor of adipogenesis, while cell and nuclear shape was strongly associated to hematopoietic-supportive potency. Together, these data evaluate morphological changes associated with cell potency and highlight new strategies for purification or alternatives to assessing MSC quality.