Clinical Stem Cell Transplantation Research Articles

Barriers and Facilitators in Continuous Medical Education Related to Allogeneic Stem Cell Transplantation: A Qualitative Study of Physicians

Introduction: This study explored qualitatively, in a sample of German hematologists working in clinical allogeneic hematopoietic stem cell transplantation (alloHSCT), perceptions of barriers and facilitators to participate in continuous medical education (CME), to provide detailed information on how to improve participation in CME activities related to alloHSCT, which may also be applicable to other areas of medicine. Methods: Based on a recruitment campaign of the German Association for Hematopoietic Stem Cell Transplantation (DAG-HSZT), 21 semi-structured telephone interviews were conducted, transcribed, and analyzed using framework analysis. Results: Three clusters of barriers were identified that explain why alloHSCT physicians may or may not participate in CME: individual constraints (e.g., better networking, young physicians being overwhelmed by the complexity of alloHSCT), structural constraints (e.g., time and financial issues, tailoring CME courses according to the targeted audience), and content-related constraints (e.g., requirement of CME sessions, provision of an overview of CME courses, more flexible offers). We discuss the ten most frequently raised issues, including the use of incentives and the need for support at the start of residency, staff shortages, and requirements for learning sessions. Conclusion: There is a need for a paradigm shift in CME related to alloHSCT toward a more individualized and needs-based approach. Close monitoring of residents’ needs and learning progress, as well as feedback systems, could help identify appropriate CME courses that should be integrated into a tiered learning system. CME should be more targeted to specific audiences (i.e., residents, fellows, and attendees) to provide training that is tailored to individual CME needs. On-demand courses can help balance work and family obligations. Finally, peer-reviewed, up-to-date information platforms should be expanded.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P<0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Low levels of CD26 on certain cellular subtypes of donor harvest is associated with better clinical outcomes post allogeneic stem cell transplantation through regulation of NF-κB pathway and pro-inflammatory cytokines

BackgroundWe had previously reported significant association of immunoectoenzyme CD26 expression on donor harvest with acute Graft-versus-Host-Disease (aGVHD) in allogeneic stem cell transplantation (ASCT) patients. The current study was aimed at analysing CD26 signaling pathway molecules and understanding their impact on immune reconstitution and clinical outcomes post-ASCT. Subjects and MethodologyThe study cohort included 26 transplant donors/patients who underwent reduced intensity (n = 21), myeloablative (n = 4) and non-myeloablative (n = 1) ASCT for hematological malignancies. Donors were matched related donors (n = 19) and haploidentical donors (n = 7). Surface expression of CD26, CD73 and ADA, and various immune cell subtypes were assessed by multicolour-flow cytometry. Soluble CD26 (sCD26) and cytokine levels were measured in plasma samples by ELISA and Multiplex Luminex assay, respectively. Immune cells from healthy individuals were stimulated with phytohemagglutinin (PHA) in the presence or absence of CD26 inhibitor. Effect of CD26 inhibition on NF-κB localization in PHA stimulated cells was analysed by immunofluorescence and confocal microscopy. Pro-inflammatory cytokines from the culture supernatants were detected with Cytometric bead array flow cytometry. Association of all measured markers with clinical outcomes was evaluated using appropriate statistical tests. ResultsCD26 surface expression on PBSC donor harvest cells showed increased risk of chronic GVHD (cGVHD, p = 0.055). Amongst the various immune cell subtypes, decreased B cells in harvest showed significant association with aGVHD (p = 0.022) whereas increased myeloid dendritic cells and CD3+T cells at Day100 in peripheral blood of transplant recipients correlated with cGVHD (p = 0.046) and aGVHD (p = 0.035), respectively. Further, high sCD26 in transplant recipients at Day100 exhibited association with reduced event-free survival (EFS) (p = 0.011). Higher CD26 expression on more & less mature NK cells, naïve & post-switched memory B cells and Treg cells in the donor harvest (p < 0.05) led to lower EFS in transplant recipients. Mechanistically, CD26 inhibitor caused dose-dependent reduction in CD26 enzyme activity and in pro-inflammatory cytokine production in post mitogen-stimulated T cell cultures. ConclusionOur study has implicated that lower CD26 expression on immune cell subtypes of the donor stem cell harvest is associated with reduced risk of GVHD and better survival. The underlying mechanism was found to be through NF-κB pathway and pro-inflammatory cytokines. Based on these observations, chemically designed or natural resources-based CD26 inhibitors can be explored further in clinical trials for improving ASCT outcomes.

P210 The impact of HLA-DPB1 disparity on clinical outcome post unrelated hematopoietic stem cell transplantation between 2016 and 2021 – A single center experience

P210 The impact of HLA-DPB1 disparity on clinical outcome post unrelated hematopoietic stem cell transplantation between 2016 and 2021 – A single center experience

Immunology of cord blood T-cells favors augmented disease response during clinical pediatric stem cell transplantation for acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) has been an important and efficacious treatment for acute leukemia in children for over 60 years. It works primarily through the graft-vs.-leukemia (GVL) effect, in which donor T-cells and other immune cells act to eliminate residual leukemia. Cord blood is an alternative source of stem cells for transplantation, with distinct biological and immunological characteristics. Retrospective clinical studies report superior relapse rates with cord blood transplantation (CBT), when compared to other stem cell sources, particularly for patients with high-risk leukemia. Xenograft models also support the superiority of cord blood T-cells in eradicating malignancy, when compared to those derived from peripheral blood. Conversely, CBT has historically been associated with an increased risk of transplant-related mortality (TRM) and morbidity, particularly from infection. Here we discuss clinical aspects of CBT, the unique immunology of cord blood T-cells, their role in the GVL effect and future methods to maximize their utility in cellular therapies for leukemia, honing and harnessing their antitumor properties whilst managing the risks of TRM.

The gut microbiome: Bridging medications and clinical outcomes post stem cell transplantation

Anti-cancer therapies are usually intertwined with prolonged use of drugs, which may lead to different clinical outcomes. Recently in Cell and Cell Host & Microbe, Nguyen etal. and Vallet etal., respectively, deconvolute the drug effects on gut microbiome dynamics underpinning clinical outcomes after allogeneic hematopoietic stem cell transplantation.

Injury-Transplantation Interval-Dependent Amelioration of Axonal Degeneration and Motor Deficit in Rats with Penetrating Traumatic Brain Injury.

Penetrating traumatic brain injury (pTBI) is increasingly survivable, but permanently disabling as adult mammalian nervous system does not regenerate. Recently, our group demonstrated transplant location-dependent neuroprotection and safety of clinical trial-grade human neural stem cell (hNSC) transplantation in a rodent model of acute pTBI. To evaluate whether longer injury-transplantation intervals marked by chronic inflammation impede engraftment, 60 male Sprague-Dawley rats were randomized to three sets. Each set was divided equally into two groups: 1) with no injury (sham) or 2) pTBI. After either 1 week (groups 1 and 2), 2 weeks (groups 3 and 4), or 4 weeks after injury (groups 5 and 6), each animal received 0.5 million hNSCs perilesionally. A seventh group of pTBI animals treated with vehicle served as the negative control. All animals were allowed to survive 12 weeks with standard chemical immunosuppression. Motor capacity was assessed pre-transplant to establish injury-induced deficit and followed by testing at 8 and 12 weeks after transplantation. Animals were euthanized, perfused, and examined for lesion size, axonal degeneration, and engraftment. Compared to vehicle, transplanted groups showed a trend for reduced lesion size and axonal injury across intervals. Remote secondary axonal injury was significantly reduced in groups 2 and 4, but not in group 6. The majority of animals showed robust engraftment independent of the injury-transplant time interval. Modest amelioration of motor deficit paralleled the axonal injury trend. In aggregate, pTBI-induced remote secondary axonal injury was resolved by early, but not delayed, hNSC transplantation.

The Promise of Next-Generation Sequencing in Improving the Diagnosis of Rare Genetic Disorders in Developing Countries

Introduction Genetic testing is frequently required for aiding and confirming the diagnosis, selecting treatment and predicting prognosis in patients with hematological disorders (1). However, large-scale genetic studies based on whole exome or genetic panel sequencing have not been a primary focus of developing countries due to lack of awareness, a dearth of genetic testing laboratories in clinical facilities, and the limited financial resources of affected families. As a result, the underlying genetic patterns in these patients often go undetected. Nevertheless, these countries constitute a rich source of clinical data with atypical phenotypes and novel mutations due to large family sizes, inter-family marriages, and potential founder effects (2). These genotype and phenotype data are worth being presented in the international data repositories for better identification of pathogenic variants in this population (3). Armed Forces Bone Marrow Transplant Centre (AFBMTC) is the country's largest hospital providing clinical hematology and stem cell transplantation services to a catchment area of over 560,000 Km2. We sought to identify underlying genetic causes of various difficult-to-diagnose rare hematological disorders with heterogenous/overlapping symptoms and challenging clinical presentation using next-generation sequencing (NGS). Materials and Methods Peripheral blood samples from 33 individuals with diverse hematological diseases were sent to Invitae Corporation (USA) or Centogene GmbH (Germany) for panel sequencing or whole exome sequencing, respectively. These samples were from patients who were difficult-to-diagnose, required risk stratification or genetic testing for selecting appropriate treatment. Results Whole exome sequencing or panel sequencing was performed in a cohort of 33 patients with various hematological disorders (figure 1). A total 115 variants were identified in these patients including 15 pathogenic variants (13%), 7 likely pathogenic variants (6%), 82 variants of uncertain significance-VUS (71%), 9 benign variants (7.8%) and 1 increased risk allele variant (0.86%). A total of 42 variants were found in musculoskeletal disorders panel, 20 in primary immunodeficiency panel, 11 each in bone marrow failure syndrome panel and dyskeratosis congenita panel, 9 in comprehensive deafness panel and 9 in hereditary hemolytic anemia panel among others. The most frequent diagnosis in our cohort was inherited bone marrow failure syndromes (27.3%), followed by hemolytic anemias (15.2%), immunodeficiency disorders (12.2%), inherited platelet function disorders (12.2%), myelofibrosis disorders (12.2%), skeletal disorders (12.2%) and miscellaneous disorders (9.1%). Patients clinical and demographic characteristics are summarized in table 1. Conclusion Use of high-throughput NGS in our clinical hematology setting has proven to be useful in identifying genetic variants for accurate diagnosis, selecting appropriate therapies, and predicting prognosis. We intend to develop a genetic facility that will provide genetic testing and identify ways to make it more accessible for people of all socioeconomic backgrounds. A second objective is establishing the prevalence of most frequent genetic disorders in the country. The high number of variants of uncertain significance (VUS) is most likely driven by a lack of sufficient population-based genetic data and proof of functional impact, though the patients identified having VUS were phenotypically disease presenting. Thus, supporting the hypothesis that these VUS may eventually be classified as pathogenic after appropriate testing on larger scales and animal models. To achieve a dependable and precise categorization of VUS, we plan to include functional genomics in our research focus to support the clinical database of this region. 1. Haferlach TJh. The time has come for next-generation sequencing in routine diagnostic workup in hematology. 2021;106(3):659. 2. MALTESE P, Poplavskaia E, Malyutkina I, Sirocco F, Bonizzato A, Capodicasa N, et al. Genetic tests for low-and middle-income countries: a literature review. 2017;16(1). 3. Gutierrez-Rodrigues F, Calado RTJH, Transfusion, Therapy C. The interpretation of rare or novel variants: Damaging vs. disease-causing. 2018;40:3-4. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P551: A PHASE 3, RANDOMIZED, OPEN-LABEL STUDY EVALUATING THE SAFETY AND EFFICACY OF MAGROLIMAB IN COMBINATION WITH AZACITIDINE IN PREVIOUSLY UNTREATED PATIENTS WITH TP53-MUTANT ACUTE MYELOID LEUKEMIA

Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancer cells, that eliminates leukemia stem cells by inducing tumor phagocytosis. Hypomethylating agents synergize with magrolimab by inducing “eat me” signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab with azacitidine (AZA) has shown encouraging activity in frontline acute myeloid leukemia (AML) unfit for intensive chemotherapy and myelodysplastic syndrome. In TP53-mutant (TP53m) AML, it demonstrated an objective response rate of 69%, complete response (CR) rate of 45%, and median overall survival (OS) of 12.9 months. The TP53 gene mutation is observed in approximately 10-15% of newly diagnosed AML and is associated with poor survival. The published first-line median OS in TP53m AML is 4-7 months, regardless of whether patients are treated with intensive chemotherapy or non-intensive approaches, such as a hypomethylating agent with venetoclax (VEN). AML patients harboring the TP53 gene mutation represent a significant unmet medical need. Aims: To evaluate the efficacy, safety, and tolerability of magrolimab+AZA vs physician’s choice of VEN+AZA or “7 + 3” chemotherapy in patients with previously untreated TP53m AML. Methods: This is a phase 3, randomized, open-label, multicenter study. Approximately 346 patients will be randomized (1:1) to magrolimab+AZA (experimental arm) or physician’s choice of VEN+AZA or 7 + 3 chemotherapy, based on patient fitness. Randomization will be stratified by appropriateness for non-intensive vs intensive therapy, geographic region (US vs non-US sites), and age (<75 vs ≥75 years). Patients are eligible if they are ≥18 years old with histologically confirmed AML with no prior antileukemic therapy, with at least one TP53 gene mutation that is not benign or likely benign (confirmed by central laboratory) or biallelic 17p deletions (based on a locally evaluated karyotype/fluorescence in situ hybridization report). During the first 28-day cycle, patients randomized to magrolimab+AZA will receive magrolimab intravenously (IV) at a priming dose of 1 mg/kg on days 1 and 4, 15 mg/kg on day 8, and 30 mg/kg on days 11, 15, and then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. For patients randomized to the VEN+AZA arm, VEN and AZA will be administered according to labeled indications. Patients treated with 7 + 3 chemotherapy will receive 1-2 induction cycle(s) with IV daunorubicin or idarubicin on days 1-3 and cytarabine 100 mg/m2 or 200 mg/m2 on days 1-7 followed by up to 4 consolidation cycles with high-dose cytarabine (3000 mg/m2). Patients receiving magrolimab+AZA or VEN+AZA will remain on treatment until disease progression, relapse, loss of clinical benefit, unacceptable toxicities, or stem cell transplant. Patients can undergo stem cell transplantation per investigator decision. The primary endpoint is OS in patients appropriate for non-intensive therapy, and the key secondary endpoint is OS in all patients. Results: Trial in progress. Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT04778397.

P910: DOES MINIMAL RESIDUAL DISEASE OF STEM CELL COLLECTION HAVE PROGNOSTIC IMPACT ON PATIENTS WITH MULTIPLE MYELOMA?

Background: Multiple myeloma (MM) is a kind of hematological malignancy involved in monoclonal plasma cells. In most clinical guidelines autologous stem cell transplantation (ASCT) is considered as the standard of care for transplant-eligible patients (TEMM) as long as patients achieving partial response (PR). However, it is unclear that under the circumstance of not achieving complete response (CR) weather it would lead to the presence of neoplastic plasma cells in the stem cell collection (SCC) and then result in negative impact on survival prognosis. Aims: Here, we evaluated the effect of the minimal residual disease (MRD) of SCC in the TEMM. Methods: We analyzed retrospectively clinical data of 90 patients with MM undergoing ASCT between January 1, 2013 to June 1, 2021 and MRD evaluation of both bone marrow (BM) and SCC were carried out at the same time. MRD was evaluated by multiparameter flow cytometry (MFC) with 10-4-10-5 sensitivity. The best response was defined as the deepest response during the follow-up. Here we defined the time from ASCT to disease progression or death as modified progression-free survival (mPFS) and the time from ASCT to death as modified overall survival (mOS). Results: A total of 90 patients met the inclusion criteria. The median age is 54 (37-69) and 62.2% were males. There were 25 (27.8%) patients presenting high-risk cytogenetic abnormalities by FISH, defined as the presence of at least one of t (4;14), t (14;16) or del (17p). Before ASCT 36.7% of patients achieved MRD negativity in BM and 76.7% in SCC. After the comparison among MRD-positivity status with different sensitivity and numbers of detectable MRD neoplastic plasma cells, we found that the percentage of patients with MRD positivity in SCC was much less than that in BM no matter the sensitivity (P <0.001). Neither mPFS (P=0.861, median mPFS, 40.83m vs. 34.17m for negativity vs. positivity) nor mOS (P=0.747, median mOS, 67.02m vs. 58.86m for negativity vs. positivity) was affected by MRD status in SCC. According to MRD status they were divided into 4 groups, namely MRD negativity in BM and SCC (Group A, 34.4%), MRD positivity in BM but negativity in SCC (Group B, 22.2%), MRD positivity in BM and SCC (Group C, 41.1%) as well as MRD negativity in BM but positivity in SCC (Group D, 2.3%). Having taken the inaccuracy of biopsy into consideration we excluded Group D. The median follow-up of the cohort was 26.8 months (15.1-105.1m). Patients among the three groups experienced similar mPFS (P=0.403, median mPFS, 41.07m vs. 34.17m vs. 40.83m, for Group A, B and C, respectively) and similar mOS (P=0.933, median mOS, 67.02m vs. 58.86m vs. 58.61m for Group A, B and C, respectively). Achievement of CR with the presence of MRD negativity was associated with prolonged mPFS and mOS compared with MRD-positive CR or ≤VGPR (P＜0.001, median mPFS, 55.88m vs. 23.03m vs. 27.10m, respectively; P=0.026, median mOS, 67.02 vs. 46.16m vs. 41.65m, respectively). Image:Summary/Conclusion: Our results demonstrated that neoplastic plasma cells in SCC have little impact on the survival prognosis in MM patients and it is sound to carry out ASCT when TEMM patients achieving PR. Also, MRD-negativity status can be considered more valuable on prognosis than CR.

Linking gastrointestinal microbiota and metabolome dynamics to clinical outcomes in paediatric haematopoietic stem cell transplantation

BackgroundHaematopoietic stem cell transplantation is a curative procedure for a variety of conditions. Despite major advances, a plethora of adverse clinical outcomes can develop post-transplantation including graft-versus-host disease and infections, which remain the major causes of morbidity and mortality. There is increasing evidence that the gastrointestinal microbiota is associated with clinical outcomes post-haematopoietic stem cell transplantation. Herein, we investigated the longitudinal dynamics of the gut microbiota and metabolome and potential associations to clinical outcomes in paediatric haematopoietic stem cell transplantation at a single centre.ResultsOn admission (baseline), the majority of patients presented with a different gut microbial composition in comparison with healthy control children with a significantly lower alpha diversity. A further, marked decrease in alpha diversity was observed immediately post-transplantation and in most microbial diversity, and composition did not return to baseline status whilst hospitalised. Longitudinal trajectories identified continuous fluctuations in microbial composition, with the dominance of a single taxon in a significant proportion of patients. Using pam clustering, three clusters were observed in the dataset. Cluster 1 was common pre-transplantation, characterised by a higher abundance of Clostridium XIVa, Bacteroides and Lachnospiraceae; cluster 2 and cluster 3 were more common post-transplantation with a higher abundance of Streptococcus and Staphylococcus in the former whilst Enterococcus, Enterobacteriaceae and Escherichia predominated in the latter. Cluster 3 was also associated with a higher risk of viraemia. Likewise, further multivariate analysis reveals Enterobacteriaceae, viraemia, use of total parenteral nutrition and various antimicrobials contributing towards cluster 3, Streptococcaceae, Staphylococcaceae, Neisseriaceae, vancomycin and metronidazole contributing towards cluster 2. Lachnospiraceae, Ruminococcaceae, Bifidobacteriaceae and not being on total parenteral nutrition contributed to cluster 1. Untargeted metabolomic analyses revealed changes that paralleled fluctuations in microbiota composition; importantly, low faecal butyrate was associated with a higher risk of viraemia.ConclusionsThese findings highlight the frequent shifts and dominations in the gut microbiota of paediatric patients undergoing haematopoietic stem cell transplantation. The study reveals associations between the faecal microbiota, metabolome and viraemia. To identify and explore the potential of microbial biomarkers that may predict the risk of complications post-HSCT, larger multi-centre studies investigating the longitudinal microbial profiling in paediatric haematopoietic stem cell transplantation are warranted.3ijjxzWkyLUsL7DWLJaC9PVideo abstract.

A Phase 3, Randomized, Open-Label Study Evaluating the Safety and Efficacy of Magrolimab in Combination with Azacitidine in Previously Untreated Patients with TP53-Mutant Acute Myeloid Leukemia

A Phase 3, Randomized, Open-Label Study Evaluating the Safety and Efficacy of Magrolimab in Combination with Azacitidine in Previously Untreated Patients with TP53-Mutant Acute Myeloid Leukemia

3131 – THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR - C IN DEVELOPMENTAL HEMATOPOIESIS

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish and replenish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) in the embryo. We use zebrafish to study endothelial-to-hematopoietic transition (EHT) because the embryos are transparent and develop externally, allowing direct observation of in vivo cellular events. My long-term goal is to identify factors that impact HSPC differentiation during EHT. One candidate, VEGFC, is expressed in the DA and specifically in long-term HSPCs of both mouse and zebrafish. VEGFC has been shown to play a role in fetal liver erythropoiesis, but its developmental role in definitive hematopoiesis has not been explored. I hypothesize that VEGFC maintains HSPC identity by negatively regulating alternative hemogenic endothelium fates. Preliminarily, we have shown in zebrafish that morpholino knock-down of vegfc results in decreased HSPC emergence and altered cell behavior during EHT. During the peak of HSPC emergence from the DA, vegfc knock-down embryos show decreased HSPC emergence, this lack of HSPCs persists into lymphoid development in the thymus. HSPCs in vegfc knock-down embryos also display altered cell behavior, they crawl similar to myeloid cells, instead of the typical and well described budding from the DA. vegfc knock-down embryos and genetic mutants also show an increase in myeloid cells in the caudal hematopoietic tissue (CHT), the zebrafish equivalent of the mammalian fetal liver. We plan to further investigate the role of VEGFC in a mammalian system using human pluripotent stem cell differentiation protocols and aorta-gonad mesonephros explants. We hope to use the results of this research to improve our ability to create HSPCs in vitro to be used in clinical stem cell transplants.

The Mayo Clinic Cellular Therapy Fellowship: Training the Next Generation of Cellular Therapy Leaders

Background & Aim The field of cellular therapy is rapidly evolving with promising clinical outcomes and multiple potential therapeutic targets. This rapid expansion, while exciting, has quickly led to a deficit of qualified directors for burgeoning human cellular therapy laboratories. Unlike conventional drug therapies, cellular based therapies require an added layer of expertise in policy, good manufacturing practices (cGMP) and regulatory elements. Academic medical centers, both nationally and internationally, are in desperate need of well-trained, experienced leaders capable of directing the labs which both oversee and produce these therapeutic agents. To meet this need, we have established the Mayo Clinic School of Graduate Medical Education (MSGME) Cellular Therapy Fellowship; an intensive one-year program for M.D. and Ph.D. candidates consisting of didactic, experiential, and project-driven training with an emphasis on laboratory management and graduated responsibility. Mayo Clinic is a multidisciplinary academic medical and research institution, which houses a lab that directly supports the bone marrow transplant service in addition to a multifaceted immune and progenitor cell program focusing on investigational cell therapies such as mesenchymal stem cells, dendritic cells, and CAR T cells. The program is robust performing more than 500 clinical stem cell transplants annually and encompassing more than 25 active investigator-initiated trials in addition to greater than 30 external manufacturing protocols. Our aim is to produce scientific and clinical leaders fluent in cGMP regulations, quality control and regulatory components, clinical laboratory management, current methodologies in cell therapy manufacturing, IND preparation and clinical trial oversight. Methods, Results & Conclusion Our curriculum mirrors this aim with rotations in the BMT and progenitor cell laboratories, our commercial processing unit, CAR-T manufacturing, quality, apheresis and infusions and leadership and management each with varying levels of graduated responsibility and on-call duties. The fellows actively participate in director level conversations and decisions, which, in combination with the aforementioned experiences and their research projects, provides a thorough and comprehensive training experience for the next generation of cell therapy leaders.

Development of a clinical hematology and stem cell transplantation program to provide state-of-the-art and cost-effective treatment to patients: a successful collaboration between a medical college in India and a leading medical university in the United States

Development of a clinical hematology and stem cell transplantation program to provide state-of-the-art and cost-effective treatment to patients: a successful collaboration between a medical college in India and a leading medical university in the United States

Progress towards improving homing and engraftment of hematopoietic stem cells for clinical transplantation.

Hematopoietic cell transplantation (HCT) is a life-saving treatment for a variety of hematological and nonhematological disorders. Successful clinical outcomes after transplantation rely on adequate hematopoietic stem cell (HSC) numbers, and the homing and subsequent short-term and long-term engraftment of these cells in the bone marrow. Enhancing the homing capability of HSCs has the potential for high impact on improving HCT and patient survival. There are a number of ways to enhance HSC engraftment. Neutralizing negative epigenetic regulation by histone deacetylase 5 (HDAC5) increases surface CXCR4 expression and promotes human HSC homing and engraftment in immune-deficient NSG (NOD.Cg-Prkdc IL2rgt/Sz) mice. Short-term treatment of cells with glucocorticoids, pharmacological stabilization of hypoxia-inducible factor (HIF)-1α, increasing membrane lipid raft aggregation, and inhibition of dipeptidyl peptidase 4 (DPP4) facilitates HSC homing and engraftment. Added to these procedures, modulating the mitochondria permeability transition pore (MPTP) to mitigate ambient air-induced extra physiological oxygen stress/shock (EPHOSS) by hypoxic harvest and processing, or using cyclosporine A during air collection increases functional HSC numbers and improves HSC engraftment. A better understanding of the regulation of human HSC homing mediated by various signaling pathways will facilitate development of more efficient means to enhance HCT efficacy.

Evolution of Peripheral Blood Stem Cell Transplantation.

Blood-derived progenitors have become the predominant source of hematopoietic stem cells for clinical transplantation. The main advantages compared to the bone marrow are as follows: harvesting blood stem cells is less painful for the donor, utilizes much less resources such as operating theater time and general anesthesia, and, above all, is associated with significantly accelerated reconstitution. The latter has ultimately improved patient safety as a consequence of significantly shortened aplastic phase and hence reduced morbidity and mortality after transplantation. Basic and translational research efforts in the 1960s to the mid-1980s have made the first blood stem cell transplantation in Heidelberg in 1985 possible. Diverse groups around the world have contributed to incremental knowledge that culminated in the first successful attempts in blood stem cell transplantation. These efforts have spawned modern research into stem cell biology and the immune modulatory effects of allogeneic transplantations.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs.We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities.Next, we employed a well-described murine model of allogeneic BMT (BL/6–>Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro.Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. [Display omitted] DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

‘We All Have a Responsibility to Each Other’: Valuing Racialised Bodies in the Neoliberal Bioeconomy

ABSTRACTIn neoliberalism, human tissue has been targeted as a source for extracting surplus value. Commercial attention on ethnic and racial minorities has resulted in products and services specifically developed for them. Here, we focus on this by exploring two empirical examples: US pharmaceutical clinical trials and UK stem cell transplantation. Both use racial taxonomies to discern biological difference and draw conclusions about the economic potential of people’s genetic constitutions. They do so by appealing to racialised minorities’ sense of responsibility towards ‘their’ communities, both buttressing the conflation of social and biological registers of human variation and demonstrating neoliberalism’s mobilisation of discourses of community. However, while the inclusion of racialised minorities is hoped to bring economic benefits, it also aims to address healthcare inequalities. Drawing on Science and Technology Studies, we argue that in our examples, economic, social and cultural values cannot be disentangled. This compels us to complement narratives of the commodification of racialised difference in neoliberal (consumer) culture, and focus on the intersections between different economic and ethical values. Ultimately we find that whilst work is being done to ameliorate racial inequities, broader socio-economic and political inequalities minority communities face go unaddressed, likely precluding the realisation of health equality.

Autograft immune effector cells and survival in autologous peripheral blood hematopoietic stem cell transplantation.

In addition to stem cells, T-cells, natural killer cells, dendritic cells, and monocytes are also collected and infused from the autograft in patients undergoing autologous peripheral blood hematopoietic stem cell transplantation. Recent reports have shown that these autograft immune effector cells can affect the clinical outcome postautologous peripheral blood hematopoietic stem cell transplantation. In this article, I will review the clinical impact on the survival of these autograft immune effector cells conferring the concept of autologous graft versus tumor effect.