bluuebunny/crossref_metadata_2025_split huggingface dataset:
Platelet Birth.
Authors: Cramer-Bordé Elisabeth , Morison Ian M. , Capron Claude , Dunois-Lardé Claire , Baruch Dominique 
Published: October 2019

Abstract
How and where human platelets get born are two questions still harboring great mystery. In vivo and in vitro approaches have contributed to a better understanding of the phenomenon. The recent study of a family with congenital autosomal-dominant thrombocytopenia showed ectopic platelet release in the bone marrow of the affected individuals. Further, molecular studies identified a new mutation in the gene encoding cytochrome c, which yields a cytochrome c with enhanced apoptotic activity. It is known that platelet birth occurs when megakaryocytes (MKs) undergo compartmentalized apoptotic activity. In this family case, enhanced apoptosis in MKs led to the premature release of platelets in the bone marrow, preventing their natural birth into the circulation after MK migration and causing thrombocytopenia. A similar finding, i.e., platelet birth in the marrow, was made in Wiskott Aldrich syndrome, providing a pathophysiological explanation for the thrombocytopenia, related to impaired migration capacities of MKs upon SDF chemotaxis. Indeed, direct platelet delivery in the human bone marrow space is not currently observed. However, maturing MKs are located close to the marrow sinusoids whose barrier they are able to cross, and entire MKs have been seen in the circulating blood where they become exposed to circulatory shear forces. Since the rate of platelet shedding in culture systems is remarkably low, we hypothesised that a missing element for efficient platelet production in static culture systems would be shear stress. Thus, we exposed human MKs, cultured in vitro until full maturation, to a substrate of matrix protein and high shear rates equivalent to those encountered in capillaries and small arteries. Cells were observed by real-time videomicroscopy, immunofluorescence, and electron microscopy. Shear forces specifically induced a sequence of morphological changes, converting mature MKs into proplatelets, and platelets, which consistently detached from the mother cells into the flow,, at a rate 20 times higher than static culture conditions. Using specific antibody inhibition, we showed the major involvement of GPIb in platelet formation, since its blockade inhibited MK adhesion and subsequent platelet formation. In addition, aIIbb3 was essential for firm MK anchorage, which was required for subsequent platelet formation. These experiments show that MK exposure to high shear rates promotes platelet production via GPIb (and secondarily aIIbb3) interactions with vascular matrix proteins. In conclusion, the above studies converge to present evidence that platelet birth takes place in circulating blood, being regulated by the migratory and apoptotic capacities of MKs and dependent on GPIb and hemodynamic forces.
Notch Signaling Is Required for Mast Cell Development in the Zebrafish.
Authors: Da'as Sahar , Klein Lauren C. , Ferrando Adolfo A. , Berman Jason N. 
Published: October 2019

Abstract
Abstract 3588
Poster Board III-525
The molecular pathways regulating mast cell (MC) development in vertebrates remain to be elucidated. The Notch signaling pathway is highly conserved in all metazoans and has been implicated in regulating hematopoietic stem cell induction and lineage cell fate decisions. Notch receptors and their ligands are expressed in a number of hematopoietic cells, including MCs. We were the first to identify zebrafish MC equivalents (Dobson et al., Blood 2008) and examine vertebrate MC transcriptional regulation in vivo. These studies demonstrated the significance of carboxypeptidase A 5 (cpa5) as a zebrafish MC-specific marker. Co-localization studies reveal zebrafish notch3 (a homologue of human NOTCH3) is expressed in a proportion of cpa5 positive cells in 7 day old embryos. Moreover, the zebrafish Notch signaling mutant, mind bomb, displays profound loss of cpa-5 expression, as do wild type zebrafish embryos treated with Compound E (Cpd E), a gamma-secretase inhibitor that blocks Notch signaling. We previously identified pu.1 and gata2 as essential transcription factors for early MC development. Interestingly, we observed a dose-dependent response, with reduced cpa5 and gata2 but preserved pu.1 expression at 50 μM Cpd E, compared with profound decreased expression of all these factors, as well as gata1 and mpo at 75 μM Cpd E. These data suggest a particular role for Notch signaling in regulating MC development, as well as a potentially broader role in regulating the myeloid and erythroid lineages. These studies are currently being validated through reciprocal experiments overexpressing notch mRNA in wild type embryos and rescue experiments overexpressing the notch intracellular domain and the above-mentioned transcription factors in Notch deficient embryos (mind bomb and Cpd E treated). We have also developed a transgenic zebrafish line expressing the human c-KIT D816V mutation found in systemic mastocytosis, which exhibits increased mast cells at the expense of erythroid cells, features in keeping with the human condition. These transgenic fish provide an opportunity to examine if Notch pathway inhibition alone, or in combination with other therapies, such as those targeting the c-KIT kinase, have a therapeutic impact in this condition. Parallel approaches in a human mastocytosis cell line are also being undertaken. These studies promise key insight into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model for identifying novel therapeutic strategies in MC diseases.

Disclosures:
Ferrando: Merck, Pfizer: Research Funding.
Regulation of Hematopoietic Stem Cell Quiescence by Ku70.
Authors: Qing Yulan , Wang Zhengqi , Liu Min , Bunting Kevin D. , Gerson Stanton L. 
Published: October 2019

Abstract
Abstract 3613
Poster Board III-549
Genomic integrity is essential for organism development and longevity, and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is essential for DNA double-strand break repair in all cells and for VDJ processing in B and T cells. NHEJ is also critical for hematopoietic stem cell (HSC) maintenance and function, but the mechanisms by which the NHEJ pathway regulates HSC function are not known. Ku70 is a key component of NHEJ; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Previously we have shown that HSC from Ku70-deficient mice are defective in repopulation, self-renewal and competitive repopulation. Also, mice defective in other NHEJ components, Ku80 or ligase 4, display impaired HSC function and reduced HSC numbers during aging. Here we used Ku70-deficient mice to investigate the role of the NHEJ pathway in HSC function.
Ku70-deficient mice show comparable HSC (Sca1+,c-Kit+,Lin-) frequency to Wild-type (WT) mice, and HSCs from Ku70-deficient mice display similar apoptosis rates as HSCs from WT mice. SKL cells from both Ku70-deficient and WT mice proliferate at similar rates in vitro in the presence of IL3, SCF, Flt3-L and Tpo. However, a greater percentage of HSCs from Ku70-deficient mice are actively cycling, and fewer are quiescent than those obtained from WT mice. Further, recipients of Ku70-deficient BM are more sensitive to 5-FU treatment than those receiving WT BM. In addition, WT HSCs efficiently replace endogenous Ku70-deficient HSCs when WT BM cells are transplanted into Ku70-deficient mice in the absence of any conditioning.
Together, our data indicate that loss of quiescence in Ku70-deficient HSCs correlates with dramatic defects observed in repopulation, self-renewal and competitive repopulation. These results suggest that the NHEJ pathway, either through the processes of DNA repair and genome maintenance, or through a stress response signaling pathway, is critical for maintaining HSCs in a quiescent state, avoiding age-related physiological stress, and allowing functional stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.
Authors: Guo Feng , Zhou Peng , Ma Liang 
Published: October 2019

Abstract
Abstract 3665
Poster Board III-601

Introduction
Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear.

Methods
Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector.

Results
In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells.

Conclusions
Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells.

Disclosures:
No relevant conflicts of interest to declare.
Rapid Reconstitution of Human Antibody Secreting Cells After Haploidentical Allogeneic Stem Cell Transplantation.
Authors: Steurer Martina , Malenke Elke , Federmann Birgit , Kanz Lothar , Bethge Wolfgang Andreas , Wirths Stefan 
Published: October 2019

Abstract
Abstract 3682
Poster Board III-618
Innate immunity including granulocytes, monocytes, and NK cells is reported to recover rapidly after allogeneic stem cell transplantation within weeks. In contrast, adaptive immunity, including T- and B-cells, has delayed recovery over months. In murine models innate type marginal zone and B1 B cells, established at fetal age and providing natural antibodies, are distinguished from adaptive B2 or follicular B cells. A crucial maturation and survival factor for adaptive murine B cells was shown to be TNF-family member BAFF (B cell-activating factor), while development of innate B1 B cells is BAFF independent. Kinetics in reconstitution of innate and adaptive immunity after ablation in adults may give insight into hierarchy and attribution to innate and adaptive immunity of defined lymphocyte populations. Reconstitution of lymphopoiesis after CD3 and CD19 depleted haploidentical stem cell transplantation was analyzed in 10 patients, which received monoclonal anti-CD3 antibody OKT3 as immunosuppressant only. This model may enable detailed in vivo evaluation of de novo B cell formation. Weekly samples before and after reduced-intensity conditioning were analyzed by flow cytometry for absolute numbers of T-cell, NK-, and B-cell subsets. Their origin of host or donor hematopoiesis was differentiated by HLA-FACS. Antibody secreting cells (ASC) were enumerated by ELISPOT. Plasma cytokine concentrations were determined by bead based arrays and ELISA. Complete reconstitution of allogeneic NK cells was found at day +21 after transplantation. CD4+ and CD8+ T-cells and their subsets had delayed reconstitution with less then 100 cells/μl at 3 months after transplantation. CD19+ B-lymphocytes of naïve and memory phenotype (>0,5% of all lymphocytes) were detected not before day +60. In contrast, complete reconstitution of antibody-secreting cells after a nadir (<0,05/μl) was observed at day +14. Absolute numbers of ASC were comparable to those of healthy controls (d+14: 72 ASC/μl vs. control: 12 ASC/μl). ASC secreting the isotypes IgM and IgA were more prevalent than IgG compared to controls (time increase: IgM 20; IgA 10; IgG 2,9). These ASC appear CD19low/neg, CD38+, and intracellular Ig+ in flow cytometry and carried donor HLA-haplotype. Reconstitution of ASC occurred without detectable circulating T-cells and before increase of BAFF concentrations were observed. In summary, the rapid and complete reconstitution of peripheral blood ASC after allogeneic transplantation, far proceeding detection of naïve and memory type B-cells, is a novel observation. Incidence before T-cell reconstitution and increase in BAFF concentrations indicates a T-cell and BAFF independent mechanism allocating these early ASC to innate immunity, potentially maintaining natural antibody levels.

Disclosures:
No relevant conflicts of interest to declare.
Stimulation of B-CLL Cells with Interleukin 21 and Toll Like Receptor Agonists Induces a CTL-Like Transcriptional Profile.
Authors: Hagn Magdalena , Ebel Verena , Sontheimer Kai , Beyer Thamara , Blackwell Sue E. , Simmet Thomas , Weiner George J. , Jahrsdoerfer Bernd 
Published: October 2019

Abstract
Abstract 3685
Poster Board III-621
Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Using various techniques including FACS analysis, Western immunoblotting and RT-PCR we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of transcription factors, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, Eomesodermin (EOMES) and nuclear factor of activated T cells (NFAT), a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by simultaneous upregulation of phosphorylated STAT3 and complete abrogation of GrB expression by the pan-JAK inhibitor pyridone 6. Stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86), and GrB uptake (CD222), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. Similar findings resulted when the toll-like receptor (TLR)7 agonist imiquimod was used instead of the TLR9 agonist CpG ODN, suggesting that comparable differentiation programs are initiated by TLR7 and TLR9. In summary, B-CLL cells can express transcription factors involved in cytotoxic differentiation of CTL as well as GrB in response to IL-21 and TLR stimulation. The B-CLL cell differentiation program described in this study could explain our recent findings of GrB-dependent apoptosis in B-CLL cells but not in benign B cells. Moreover, our data provide novel insights into the aberrant signaling state of B-CLL cells.

Disclosures:
No relevant conflicts of interest to declare.
Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress.
Authors: Asai Takashi , Liu Yan , Giandomenico Silvana Di , Deblasio Anthony , Menendez Silvia , Antipin Jack , Reva Boris , Wevrick Rachel , Nimer Stephen D. 
Published: October 2019

Abstract
Abstract 379
Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model.

Disclosures:
No relevant conflicts of interest to declare.
Bcl-Xl Mitigates p53 Activity in AML1-ETO-Expressing Human Hematopoietic Cells.
Authors: Chou Fu-Sheng , Wunderlich Mark , Mulloy James C. 
Published: October 2019

Abstract
Abstract 387
A subset of acute myeloid leukemia (AML) is caused by chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. We have recently shown that AE represses DNA damage repair genes, resulting in increased DNA damage and upregulation of p53. These findings suggest that AE contributes to leukemogenesis by promoting additional mutations. However, it remains undefined how AE cells survive in the context of increased p53. In the current study, we showed that Bcl-xL is upregulated upon AE expression. Bcl-xL knockdown resulted in growth disadvantage and increased apoptosis in AE cells, but not in normal CD34+ cells. On the other hand, Bcl-xL overexpression in AE cells leads to an expansion of colony-forming cells and an increase in the frequency of long-term culture-initiating cells (LTC-IC). Interestingly, similar results were obtained when a Bcl-xL mutant (Bcl-xLG138A) that is defective in BH3 binding is overexpressed in AE cells, suggesting that the additional cytoprotection is not contributed by pro-apoptotic protein binding. Instead, published evidence on the p53 sequestering function of Bcl-xL prompted us to speculate that AE expression may lead to the establishment of a new balance between pro-apoptotic signals conveyed by activated p53 and the pro-survival characteristic of Bcl-xL. Indeed, using confocal microscopy, we observed colocalization of Bcl-xL and p53 in CD34+ AE cells. In addition, we also observed an increase in p53 target gene expression in AE cells following Bcl-xL knockdown, implicating interplay between Bcl-xL levels and p53 activity. Strikingly, examination of the two critical apoptosis-inducing p53 downstream targets, Bim and Puma, revealed that, whereas Bim is upregluated in AE cells, Puma is downregulated upon AE expression. AE ablation in an established cell line induces Puma expression. A chromatin immunoprecipitation assay revealed that AE binds to the first intron of the Puma gene. These data suggest that AE directly represses Puma gene expression, thereby preventing Puma-induced dissociation of p53 from Bcl-xL. Taken together, our in vitro findings demonstrated that the interplay among p53, Bcl-xL and Puma in response to DNA damage in normal cellular physiology is disrupted in the presence of AE to ensure cell survival. To recapitulate the critical role of Bcl-xL in AE expressing cells in an in vivo xenotransplantation assay, we transduced scrambled and Bcl-xL shRNA into AE/N-RASG12D cells, followed by transplantation into the bone marrow cavity of immunodeficient mice. We found that Bcl-xL knockdown is associated with a significant reduction in the long-term engraftment capability of AE/N-RASG12D cells. In conclusion, our data indicate that AE expressing human hematopoietic cells depend on Bcl-xL for their survival and growth.

Disclosures:
No relevant conflicts of interest to declare.