Clinical Hematopoietic Stem Cell Transplantation Research Articles

Allogeneic Hematopoietic Stem Cell Transplantation in Immunodeficiency—Centromeric Instability—Facial Dysmorphism (ICF) Syndrome: an EBMT/ESID Inborn Errors Working Party Study

Immunodeficiency–Centromeric instability–Facial dysmorphism (ICF) syndrome is an inborn error of immunity characterized by progressive immune dysfunction and multi-organ disease usually treated with antimicrobial prophylaxis and immunoglobulin substitution. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment, but data on outcome are scarce. We provide a detailed description of disease characteristics and HSCT outcome in an international cohort of ICF syndrome patients. Eighteen patients (including all four genotypes) were enrolled. Main HSCT indications were infections (83%), enteropathy/failure to thrive (56%), immune dysregulation (22%) and myelodysplasia/haematological malignancy (17%). Two patients underwent pre-emptive HSCT after early diagnosis. Patients were transplanted between 2003–2021, at median age 4.3 years (range 0.5–19), after myeloablative or reduced-intensity conditioning, from matched sibling or matched family donors, matched unrelated or mismatched donors in 39%, 50% and 12% of cases respectively. Overall survival was 83% (all deaths occurred within the first 5 months post-HSCT; mean follow-up 54 months (range 1–185)). Acute GvHD occurred in 35% of patients, severe (grade III) in two (12%), while none developed chronic GvHD. At latest follow-up (median 2.2 years (range 0.1–14)), complete donor chimerism was achieved in 15/17 surviving patients. All survivors demonstrated normalized T and B cell numbers. Immunoglobulin substitution independence was achieved in all but two patients. All survivors recovered from pre-transplant infections, enteropathy/failure to thrive and immune dysregulation. All three patients transplanted at young age (≤ 3 years), after early diagnosis, survived. The favourable clinical and immunological HSCT outcome in this cohort of patients supports the timely use of this curative treatment in ICF syndrome.

Tgfb1 deficiency impairs the self-renewal capacity of murine hematopoietic stem/progenitor cells in vivo

Transforming growth factor β1 (TGFB1) refers to a pleiotropic cytokine exerting contrasting roles in hematopoietic stem cells (HSCs) functions in vitro and in vivo. However, the understanding of hematopoiesis in vivo, when TGFB1 is constantly deactivated, is still unclear, mainly due to significant embryonic lethality and the emergence of a fatal inflammatory condition, which makes doing these investigations challenging. Our study aims to find the specific role of TGFB1 in regulating hematopoiesis in vivo. We engineered mice strains (Vav1 or Mx1 promoter-driven TGFB1 knockout) with conditional knockout of TGFB1 to study its role in hematopoiesis in vivo. In fetal and adult hematopoiesis, TGFB1 KO mice displayed deficiency and decreased self-renewal capacity of HSCs with myeloid-biased differentiation. The results were different from the regulating role of TGFB1 in vitro. Additionally, our results showed that TGFB1 deficiency from fetal hematopoiesis stage caused more severe defect of HSCs than in the adult stage. Mechanistically, our findings identified TGFB1-SOX9-FOS/JUNB/TWIST1 signal axis as an essential regulating pathway in HSCs homeostasis. Our study may provide a scientific basis for clinical HSC transplantation and expansion.

Altered Serum Alpha1-Antitrypsin Protease Inhibition before and after Clinical Hematopoietic Stem Cell Transplantation: Association with Risk for Non-Relapse Mortality.

α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients.

Ex vivo hematopoietic stem cell expansion technologies: recent progress, applications, and open questions

Hematopoietic stem cells (HSCs) are a rare but potent cell type that support life-long hematopoiesis and stably regenerate the entire blood and immune system following transplantation. HSC transplantation represents a mainstay treatment for various diseases of the blood and immune systems. The ex vivo expansion and manipulation of HSCs therefore represents an important approach to ask biological questions in experimental hematology and to help improve clinical HSC transplantation therapies. However, it has remained challenging to expand transplantable HSCs ex vivo. This review summarizes recent progress in ex vivo HSC expansion technologies and their applications to biological and clinical problems and discusses current questions in the field.

Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs invitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Identification and Separation of Distinct Subpopulations of Hematopoietic Stem Cells and Multipotent Progenitors By Deep Learning

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are crucial for maintaining lifelong hematopoiesis. Several decades of successful clinical HSC transplantation have demonstrated the therapeutic importance of HSCs and MPPs. For basic research, HSCs and MPPs are routinely analyzed and separated by fluorescence-activated cell sorting (FACS) based on cell surface marker staining, a procedure that requires incubation with various antibodies and expensive laser-equipped flow cytometers. Both antibody staining and laser impair cell viability and stem cell activity. To develop a new staining-free, laser-free method for predicting and separating subpopulations of HSCs and MPPs, we used a deep learning approach that can extract minutiae from large-scale datasets of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) to distinguish subpopulations of hematopoietic precursor solely based on their morphology. Remarkably, our deep learning model can achieve predictable identification of subsets of HSCs with at least 85% accuracy; this demonstrates for the first time that HSC subpopulations can be differentiated solely based on their morphology exhibited in light microscopic images through deep learning. We anticipate that our robust and accurate deep-learning-based platform for hematopoietic precursors will provide a basis for the future development of a next-generation cell sorting system.

Top 100 Cited Articles on Clinical Hematopoietic Stem Cell Transplantation: A Bibliometric Analysis.

We conducted a bibliometric analysis to identify scholarly impact and factors associated with the top 100 cited articles on clinical hematopoietic stem cell transplantation (HSCT). In January 2021, a title-specific search was conducted. Non-HSCT and pre-clinical (in-vitro and animal) studies were excluded. A total of 39,406 records were identified and a list of the top 100 articles was made. Articles included in our study were characterized by the citations received, publication year, topic, study design, authors, h-index, and institutions. Linear regression analyses were performed. The 100 most cited articles were published over 52 years from 1968 to 2020, with a maximum number of articles (n = 40) published in the 1990s decade. Top-100 articles were cited 62,002 times with a median citation count of 465 (range 336–2240). The top-cited articles originated from 12 countries. United States contributed 69 articles. The University of Washington Fred Hutchinson Cancer Center (n = 15) was the leading institution. Blood (n = 32) and New England Journal of Medicine (n = 31) made the greatest contribution, and 52 manuscripts were clinical trials. The first author's H-index significantly correlated with citation count while journal impact factor, years since publication, first author's gender, and the number of authors did not have a significant association with the number of citations. In a multivariate regression model, the first author's h-index (regression coefficient 5.46, 95% confidence interval 2.99 to 7.93, p < 0.001) independently correlated with the citation count. Our study highlights the most influential articles on clinical HSCT and provides valuable insight for future research needs of the specialty.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Top 100 Cited Articles on Hematopoietic Stem Cell Transplantation: A Bibliometric Analysis

Background: Research performance is evaluated by bibliometric analysis of citations, and this can help to identify the most impactful articles in the field of hematopoietic stem cell transplantation (HSCT). We conducted a bibliometric analysis to identify scholarly impact and factors associated with the top 100 cited articles on HSCT.Methods: Thomas Reuters Web of Science core collection (WoS) was accessed in January 2021 and a title-specific search was conducted using the keywords “hematopoietic stem-cell transplantation ”, “transplantation, hematopoietic stem cell,” “stem cell transplantation, hematopoietic,” “bone-marrow transplantation,” “transplantation, bone marrow,” “grafting, bone marrow,” “transplantation bone marrow cell,” “bone marrow cell transplantation,” “stem cell transplant,” “allogeneic hematopoietic stem cell transplant,” “autologous hematopoietic stem cell transplant”. Non-Human and non-HSCT studies were excluded. A total of 39,406 records were identified and a list of the top 100 articles was made. Articles included in our study were characterized by the number of citations, year of publication, topic, study design, authors, h-index, and institutions. Data were analyzed using SPSS in April 2021. Bivariate analyses, using chi-square and t-test, and linear regression analyses were performed.Results: The 100 most cited articles in the field were published over 52 years from 1968 to 2020, with a maximum number of articles (n=40) published in the 1990s decade. Top-100 articles were cited 62,002 times with a median citation count of 465 (range 336-2240). The top-cited articles originated from 12 countries and the United States (US) contributed 69 articles. The University of Washington Fred Hutchinson Cancer Center (n=15) was the leading institution. Blood (n=32) and New England Journal of Medicine (n=31) made the greatest contribution, and 41 manuscripts were clinical trials. In a multivariate regression model, the first author's h-index (regression coefficient 5.46, 95% confidence interval 2.99-7.93, p<0.001) correlated with the citation count while gender, journal impact factor, years since publication, and the number of authors did not have a significant association.Conclusion: Our study highlights the most influential articles on clinical HSCT, centralization of research to North American and European institutions, and provides valuable insight for future research needs of the specialty. [Display omitted] DisclosuresAbhyankar: Incyte/Therakos: Consultancy, Research Funding, Speakers Bureau. McGuirk: Novartis: Research Funding; Pluristem Therapeutics: Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Astelllas Pharma: Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; Allovir: Consultancy, Honoraria, Research Funding; Gamida Cell: Research Funding; EcoR1 Capital: Consultancy; Bellicum Pharmaceuticals: Research Funding; Novartis: Research Funding; Fresenius Biotech: Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau.

Microtubule Polymerization Inhibition Enhances Human Hematopoietic Stem Cell Homing and Engraftment

Hematopoietic stem cells (HSCs) serve as the origin of the hematopoietic system, with the ability to differentiate into all blood cell lineages and self-renewal to sustain the hematopoiesis throughout life. Hematopoietic stem cell transplantation (HSCT) currently represents the most effective therapeutic strategies to treat hematological and non-hematological diseases. However, limited numbers of HSCs or poor homing capabilities into the bone marrow are still major hurdles for successful HSCT. Moreover, graft failure and delayed reconstitution due to inefficient engraftment, remains an important complication because of the high morbidity and mortality. Although ex vivo expansion of HSCs has been well studied for decades, which displays huge potentials for clinical application, exploration of novel targets to improve HSC homing and engraftment will provide new insights to enhance HSCT efficacy.To explore the chemical compounds enhancing the capabilities of homing and engraftment, we used CXCR4 (CD184) as the readout bio-marker, which is considered as the most essential chemokine receptor of HSPCs interacting with CXCL12 (SDF1) secreted in BM niche to support HSPCs homing, migration, proliferation and survival. We first performed chemical screening of 139 small molecules that can increase CD184 expression on cord blood (CB) CD34 + hematopoietic stem and progenitor cells (HSPCs). We concluded that treatment of CB CD34 + HSPCs for 16 hours with Lexibulin (Lex) or Vinblastine Sulfate (VS), both of which were microtubule polymerization (MP) inhibitors, could significantly promote the CD184 expression.Next, we optimized the MP inhibitors treatment conditions including dosage, treatment duration and culture time prior to treatment. The results proved that treatment with Lex or VS for 16 hours at 1μM was the optimal conditions to significantly enhance the CD184 expression of CD34 + HPSCs and LT-HSCs (CD34 +CD90 +CD45RA -), while maintaining robust cell survival, when compared with the DMSO control group. Moreover, we found that only when HSPCs were under culture within two days prior to small molecules treatment, CD184 expression was significantly increased by MP inhibitors while maintaining high viability, compared with DMSO control group.In order to assess the in vivo repopulating potential of the CB CD34 + HSPCs post treatment with MP inhibitors, we transplanted CB-HSPCs 16 hours post-treatment with Lex and VS respectively into irradiated nonobese diabetic (NOD)/Prkdc scid/IL-2Rγ null (NPG) mice. All transplanted mice of MP inhibitors-treated groups presented efficient engraftment, in multiple immune organs at 4-16 weeks post-transplantation, suggesting greater engraftment potential than the mock group, as measured by human CD45 of total CD45. Furthermore, hematopoietic reconstitution analysis indicated that the MP inhibitors -treated cells maintained different lineage distribution in peripheral blood (PB), bone marrow (BM) and spleen. Moreover, the equivalent phenotypes of pre- and post-treatment reveal the better reconstitution by MP inhibitors was independent of HSC-enrichment, Thus, short-term MP inhibitors treatment of CB CD34 + HSPCs enhances their homing and long-term engraftment.In conclusion, we demonstrated that short-term microtubule polymerization inhibition on human CB CD34 + HSPCs could not only enhance CD184 cell surface expression but also the capabilities of in vivo human HSCs homing and reconstitution via screening chemical compounds to increase CD184 expression and the following function evaluation study. Vinblastine Sulfate and Lexbulin were applied or registered as anti-cancer drugs for clinical use. Our study also indicates that MP inhibitors pretreatment of cells possesses significant translational implications, designating MP inhibitors as promising drug candidates to facilitate clinical HSCT. [Display omitted] DisclosuresFang: EdiGene, Inc.: Current Employment.

The Determinative Role of Hematopoietic Stem and Progenitor Cell Graft Composition on the Engraftment Kinetics after HSCT

The importance of hematopoietic stem and progenitor cell (HSPC) dose in the outcome of hematopoietic stem cell transplant (HSCT) has been demonstrated by analyses of the threshold dose of CD34+ cells required to achieve donor engraftment, usually defined as endpoints of donor-derived neutrophil (PMN) or platelet (PLT) absolute numbers, and RBC transfusion independence. We recently described the heterogeneous HSPC composition of prospectively obtained umbilical cord blood samples and lack of correlation between CD34+ cell dose and the frequency of hematopoietic stem cells (HSC)(Mantri S et al, Blood Adv 2020).We describe here a pilot analysis of the relationship between HSPC graft composition and engraftment after HSCT. The study population is comprised of 17 children (3.4-22 years, median 13 years) treated with αβT/CD19B - cell depleted mobilized peripheral blood stem cell (PBSC) HSCT (αβhaplo-HSCT). Eight patients had acute lymphoblastic leukemia, seven had acute myeloid leukemia, and two myelodysplastic syndrome. All patients received serotherapy with Thymoglobulin and Rituximab, and a myeloablative conditioning consisting of either TBI 1200 cGy in combination with Fludarabine/Thiotepa (15 patients), Melphalan/Thiotepa (1 patient), or TBI (400 cGy) with Fludarabine/Thiotepa (1 patient receiving a second HSCT). Only 2/17 patients received 1-2 doses of G-CSF before Day+30.The CD34+ Lin- cells in the PBSC products were analyzed for HSPC composition, including HSC (CD38- CD90+ CD45RA-), Multipotent Progenitors (MPP, CD38- CD90- CD45RA-), Common Myeloid Progenitors (CMP, CD38+ CD123+ CD45RA-), Granulocyte-Monocyte Progenitors (GMP, CD38+ CD123+ CD45RA+), Megakaryocyte-Erythroid Progenitors (MEP, CD38+ CD123- CD45RA-) and Common Lymphoid Progenitors (CLP, CD38+ CD127+). Similar HSPC analyses were performed on marrow obtained at Days +30, +60, and +90 post-HSCT. The CD34+ cell dose in the grafts ranged from 8.5-40 x 10e6/kg recipient body weight (mean 15.6). The median time to Absolute Neutrophil Count (ANC) > 500 or 1000/mm 3 were Days +12 and +14, respectively, and for PLT > 50K or 100K/mm 3 Days +14 and +15, respectively. The time to either PMN or PLT engraftment did not correlate with the HSC dose. In contrast, the GMP dose was predictive of PMN engraftment: 7/8 patients receiving the highest GMP dose achieved ANC > 500 at 0-3 days before the median day of engraftment, while PMN engraftment was delayed by 1-5 days beyond the median in 6 of the 9 receiving the lowest GMP dose (χ 2 = 8.14, p=0.004)(Fig A). Of the patients with the highest MEP dose, 7/8 achieved PLT >50K/mm 3 0-4 days before the median, while 5/9 receiving the lowest dose engrafted 1-6 days beyond the median (χ 2 4.9, p=0.026) (Fig B). In the first 100 days post-HSCT, naïve CD4+ T-cells were all CD31+ CD45RA+ Recent Thymic Emigrants (RTE), indicating that they were newly produced T cells and not adoptively transferred from the αβhaplo-HSCT. The HSC dose did not correlate with the number of naïve CD4+ T cells until Day+180 (Fig C). Like PMN and PLT recovery, early T lymphoid recovery after HSCT was mainly derived from infused CLP, and likely switched to HSC-derived lymphopoiesis by Day +180. Consistent with this concept, HSC dose in the PBSC products was unrelated to the numbers of committed progenitors, e.g., CMP, MEP, CLP, in early (Day +30) post-transplant marrow samples.These data are consistent with clonal analyses of patients receiving lentiviral gene therapy and murine experiments demonstrating prolonged steady-state contribution of committed progenitors to peripheral blood cell maintenance (Biasco L et al, Cell Stem Cell 2016; Sun J et al, Nature 2014). While post-HSCT PMN or PLT numbers are frequently equated with “HSC engraftment” and naïve T-cell numbers with HSC-derived immune reconstitution, these early events reflect blood cell production by committed progenitors, which are variably present in the grafts. Although CD34+ cell dose is currently used to predict post-transplant engraftment and to qualify stem cell products for release, more accurate clinical predictions may be determined by the HSPC grafts' composition. Further, engineering of the progenitor composition of clinical HSCT products, e.g., co-transplantation of additional committed progenitors like GMP or CLP, could be strategically used to control post-transplant lymphohematopoietic recovery. [Display omitted] DisclosuresParkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

Inducible Sbds deletion impairs bone marrow niche capacity to engraft donor bone marrow after transplantation

AbstractBone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited BM failure syndromes (iBMFSs). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes BM dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC after HSCT, a hypothesis that could explain morbidity noted after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have also identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and are accentuated by or develop specifically after myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets, including mesenchymal lineage cells, macrophages, and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.

Low Soluble Programmed Cell Death Protein 1 Levels After Allogeneic Stem Cell Transplantation Predict Moderate or Severe Chronic GvHD and Inferior Overall Survival.

Programmed cell death protein-1 (PD-1) is an inhibitory co-receptor required for regulating immune responsiveness and maintaining immune homeostasis. As PD-1 can be released as bioactive soluble molecule, we investigated the clinical significance of soluble PD-1 (sPD-1) after allogeneic hematopoietic stem cell transplantation (HSCT) regarding graft-versus-host disease (GvHD), relapse, and overall survival (OS) in a mono-centric cohort of 82 patients. Compared to pre-HSCT and to healthy controls, post-HSCT sPD-1 plasma levels were significantly increased during an observation time of three months. Univariate analysis revealed that low sPD-1 plasma levels at month one, two or three post HSCT were associated with acute GvHD grade III-IV, the onset of moderate/severe chronic GvHD (cGvHD) and inferior OS, DFS, and TRM, respectively. No relationship was detected to relapse rates. sPD-1 plasma levels were significantly increased in ATG-treated patients compared to ATG-untreated patients. Multivariate analysis revealed that a low sPD-1 plasma levels status at one or two month(s) after HSCT is an independent indicator for inferior OS, DFS, or TRM. A low sPD-1 plasma levels status at month three post HSCT is predictive for the onset of moderate/severe cGvHD. Thus, our study pinpoints the soluble inhibitory co-receptor PD-1 as a promising candidate molecule for the prediction of clinical HSCT outcome.

Murine bone marrow mesenchymal stromal cells have reduced hematopoietic maintenance ability in sickle cell disease

AbstractSickle cell disease (SCD) is characterized by hemolytic anemia, which can trigger oxidative stress, inflammation, and tissue injury that contribute to disease complications. Bone marrow mesenchymal stromal cells (MSCs) tightly regulate hematopoietic stem cell (HSC) homeostasis in health and disease, but their functionality in SCD remains unclear. We identified for the first time that murine SCD MSCs have altered gene signatures, reduced stem cell properties, and increased oxidative stress, due in part to hemolysis. Murine SCD MSCs had lower HSC maintenance ability in vitro and in vivo, as manifested by increased HSC mobilization and decreased HSC engraftment after transplant. Activation of Toll-like receptor-4 through p65 in MSCs further contributed to MSC dysfunction. Transfusions led to an improved MSC and HSC oxidative state in SCD mice. Improving the regulation between MSCs and HSCs has vital implications for enhancing clinical HSC transplantation and gene therapy outcomes and for identification of new molecular targets for alleviating SCD complications.

G-CSF Signaling Counteracts TGF-β—PAI-1-Dependent Retention of Hematopoietic Stem Cells in the Niche

Hematopoietic stem cells (HSCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but are usually stayed in the niche. Recently, we have reported that niche cell-producing TGF-β induces intracellular PAI-1 expression in HSCs, which inhibits the proteolytic activity of proprotein convertase Furin-dependent maturation of MT1-MMP, a protease involved in the mobilization of HSPCs, thereby keeping HSCs retaining in the BM niche. Pharmacological inhibition or genetic disruption of the TGF-β−PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSCs from the TGF-β-expressing niche cells, demonstrated a crucial role for TGF-β−PAI-1 signal in the retention of HSCs in the BM. On the other hand, HSCs frequently egress from the BM to circulation and replenish blood supply in order to maintain the homeostasis of blood system. At this moment, however, how environmental stimuli, such as a blood G-CSF concentration rise, counteract the TGF-β-PAI-1 signaling and releases the motility restriction of HSCs remains unknown.To identify the molecular mechanism that govern the dynamic motion of HSCs in the niche, we chose to investigate the cellular activities of HSCs in response to G-CSF. Here, we provide evidence that G-CSF inhibits Smad-dependent signaling by regulating cAMP-PKA-dependent signaling pathway in HSCs. G-CSF-induced activation of this pathway inhibits Smad-dependent expression of PAI-1 through an increase in phosphorylation levels of CREB protein. Mechanistically, increased pCREB competitively inhibited the Smad from binding to the p300/CBP, which ultimately resulting in a transcriptional suppression of Smad-p300/CBP transcription regulatory complex-dependent PAI-1 gene expression. In line with this, administration of G-CSF to mice strongly downregulated intracellular PAI-1 expression in HSCs, which resulted in enhanced Furin-dependent MT1-MMP maturation. The enhanced expression of MT1-MMP results in MT1-MMP-mediated CD44 cleavage as well as CXCR4 and VLA-4 activation, which in turn stimulates detachment of HSPCs from the niche and active trafficking into the blood stream. Expectedly, pharmacological inhibition of intracellular PAI-1 activity synergized with G-CSF-induced HSC mobilization.Our study reveals a novel role for G-CSF signaling in controlling TGF-β−PAI-1-dependent retention of HSCs to recruit them into the circulation, which potentially improve clinical HSC mobilization and transplantation protocols. DisclosuresAndo:CHUGAI PHARMACEUTICAL: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; NOVARTIS: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; Eisai: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; MSD: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; ALEXION: Other: Donation to institute; Takeda: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; NIHON PHARMACEUTICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute.

Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor Ameliorates Acute Graft-versus-Host Disease by Inhibiting Effector T Cell Differentiation while Sparing Regulatory T Cells.

Graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). IL-2-inducible T cell kinase (ITK), a TEC cytoplasmic tyrosine kinase, has an essential role in T cell development and receptor signaling. The ITK/Bruton tyrosine kinase inhibitor ibrutinib has been shown to improve chronic GVHD symptoms; however, the effect of ITK selective inhibition on acute GVHD remains unclear. In this study, we evaluated the pharmacological effects of an ITK selective inhibitor (ITKsi) on acute GVHD using murine bone marrow transplantation models. First, we found that CD4+ T cell differentiation toward Th1, Th2, or Th17 was inhibited following ITKsi treatment in a dose-dependent manner while maintaining regulatory T cells in the presence of alloantigens both invitro and invivo. ITKsi preferentially inhibited inflammatory cytokine production and invivo proliferation of alloreactive T cells. We then demonstrated that short-term exposure of donor graft cells to ITKsi significantly delayed the onset of GVHD-associated mortality without compromising the donor cell engraftment and the graft-versus-tumor effect, indicating the potential of ITK selective inhibition in the setting of clinical allogeneic HSCT. These findings suggest that ITK is a potential therapeutic target against GVHD, and the pharmacological ITK inhibitor may serve as a novel strategy for immune regulation after HSCT.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after (“post-transplant,” PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular “immune privilege.” PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

IntroductionQuiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because Thomsen–Friedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomarkers.MethodsWe evaluated the co-expression of both antigens on CD34+ peripheral blood mononuclear cells (PBMCs) from both healthy volunteers and CML patients, using flow cytometry. Then, we used site-directed mutagenesis to induce knob-in-hole mutations in the human IgG heavy chain and the human lambda light chain to generate the bi-specific antibody (Bis-Ab) TF/RAP that binds both antigens simultaneously. We measured complement-directed cytotoxicity (CDC) in CML samples with the Bis-Ab by flow cytometry.ResultsIn contrast to healthy volunteers, CML samples displayed a highly significant co-expression of CD176 and IL1RAP. When either a double-positive cell line or CML samples were treated with increasing doses of Bis-Ab, increased binding and CDC was observed indicating co-operative binding of the Bis-Ab as compared to monoclonal antibodies.DiscussionThese results show that the bi-specific antibody is capable of targeting IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells in CDC assay. We hereby offer a novel strategy for the depletion of CML stem cells from the bulk population in clinical hematopoietic stem cell transplantation.

Broad Virus Detection and Variant Discovery in Fecal Samples of Hematopoietic Transplant Recipients Using Targeted Sequence Capture Metagenomics.

Pediatric allogeneic hematopoietic stem cell transplantation (HSCT) patients often suffer from gastro-intestinal (GI) disease caused by viruses, Graft-versus-Host Disease (GVHD) or a combination of the two. Currently, the GI eukaryotic virome of HSCT recipients remains relatively understudied, which complicates the understanding of its role in GVHD pathogenicity. As decisions regarding immunosuppressive therapy in the treatment of virus infection or GVHD, respectively, can be completely contradicting, it is crucial to better understand the prevalence and relevance of viruses in the GI tract in the HSCT setting. A real time PCR panel for a set of specific viruses widely used to diagnose the most common causes of GI viral gastroenteritis is possibly insufficient to grasp the full extent of viruses present. Therefore, we applied the targeted sequence capture method ViroCap to residual fecal samples of 11 pediatric allogeneic HSCT recipients with GI symptoms and a suspicion of GVHD, to enrich for nucleic acids of viruses that are known to infect vertebrate hosts. After enrichment, NGS was applied to broadly detect viral sequences. Using ViroCap, we were able to detect viruses such as norovirus and adenovirus (ADV), that had been previously detected using clinical diagnostic PCR on the same sample. In addition, multiple, some of which clinically relevant viruses were detected, including ADV, human rhinovirus (HRV) and BK polyomavirus (BKV). Interestingly, in samples in which specific PCR testing for regular viral GI pathogens did not result in a diagnosis, the ViroCap pipeline led to the detection of viral sequences of human herpesvirus (HHV)-7, BKV, HRV, KI polyomavirus and astrovirus. The latter was an only recently described variant and showed extensive sequence mismatches with the applied real time PCR primers and would therefore not have been detected if tested. Our results indicate that target enrichment of viral nucleic acids through ViroCap leads to sensitive and broad possibly clinically relevant virus detection, including the detection of newer variants in clinical HSCT recipient samples. As such, ViroCap could be a useful detection tool clinically, but also in studying the associations between viral presence and GVHD.

Optimized culture medium for enhanced ex vivo expansion of human hematopoietic stem cells

Background & Aim Development of ex vivo culture systems to expand human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research goal that would enhance clinical hematopoietic stem cell (HSCs) transplantation and gene therapies. To address the problem that HSCs generally die or differentiate rapidly in current ex vivo culture media, we developed a xeno-free, serum-free medium – CTS™ StemPro™ HSC Expansion Medium. Methods, Results & Conclusion In a Design of Experiments approach, media constituents were systematically varied and iterations were evaluated with the goal to maximize ex vivo expansion of HSCP immunophenotypes. Culture of normal primary human CD34+ cells immunopurified from cord blood, mobilized peripheral blood and bone marrow in HSC Expansion Medium supplemented with FLT3L, SCF, TPO, IL3, and IL6 (FKT36), resulted in higher numbers of immunophenotype-defined HSPCs, as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood (mPB) for 7 days in FKT36-containing HSC Expansion Medium resulted in ∼100-fold increased numbers of CD34+CD45+Lin- cells and ∼2000-fold increased numbers of CD34+Lin- CD90+CD45RA- cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. CD34+ cells expanded in this medium maintained differentiation capacity in in-vitro colony forming assays, forming erythroid and non-erythroid cell colonies. In addition, using a directed differentiation method we showed that CD34+ cells were able to differentiate into erythroid lineage which generated red blood cells at > 80% efficiency as indicated by co-expression of CD71 and CD235a markers. Further, we demonstrated CTS™ StemPro™ HSC Expansion Medium enabled transduction of CD34+ cells with lentiviral vector generated using CTS™ LV-MAX System, and can be gene edited with CRISPR-Cas9 Gene editing tools that are suitable for cell and gene therapies CD34+ cells expanded with CTS™ StemPro™ HSC Expansion Medium exhibited long-term engraftment potential and multilineage chimerism at 6 months post-transplant. We demonstrated that ex vivo-cultured CD34+ HSPCs generate transplanted cell dose-dependent engraftment in NRG mouse bone marrows and spleens at 26 weeks post-transplant. Thus, CTS™ StemPro™ HSC Expansion Medium will be an impactful tool to enhance expansion of HSPCs that are suited for translational purposes such as transplantation, and gene therapies.