bluuebunny/crossref_metadata_2025_split huggingface dataset:
Memory Phenotype CD8+T Cells Are Superior to Naive CD8+T Cells in Separating Graft Anti-Tumor Activity From Gvhd After Bone Marrow Transplantation; Application to DLI.
Authors: Dutt Suparna , Baker Jeanette , Kambham Neeraja , Kohrt Holbrook E , Negrin Robert , Strober Samuel 
Published: October 2019

Abstract
Abstract 2452
Poster Board II-429
We compared the graft anti-tumor effect and GVHD activities of naturally occurring naive and memory phenotype subsets of C57BL/6 (H-2b) CD8+ T cells after bone marrow transplantation into BALB/c (H-2d) mice. The tumor used for the study was the BCL1 lymphoma, a spontaneously arising non-transfected cell line. We found that naive CD8+ T cells (CD62LhiCD44lo) express higher levels of gut homing molecules a4b7, CCR9 and CD103 compared to memory subsets comprised of both effector (CD62LloCD44hi) and central memory cells (CD62LhiCD44hi). The proliferation of the naive cells was ten-fold higher than memory phenotype cells against BALB/c stimulators and memory cells secreted significantly more IFNg in the MLR. All BALB/c hosts without tumor cells that were given donor memory CD8+T cells along with T cell depleted bone marrow cells (TCD BM) survived over 100 days of transplantation without significant weight loss compared to TCDBM controls. However, 25% of the mice given naive CD8+ T cells and TCD BM died due to GVHD and had significantly greater weight loss compared to TCD BM controls or to hosts given memory CD8+ T cells and TCD BM. BALB/c hosts given 500 BCL1 and TCD BM all died of lymphoma within 28 days of transplantation. Hosts given BCL1 cells, TCD BM and either memory or naive cells cleared the tumor by day 28 but weight loss and survival was significantly improved in the memory versus naive cell groups despite non significant differences in the GVHD scores in the liver and gut at day 100. Ex vivo imaging of mice receiving BCL1 tumor cells, TCD BM, and CD8+T cells from luciferase transgenic donors showed increased accumulation of naive phenotype CD8+T cells in the liver and the gut compared to memory phenotype cells after 6 days of transplantation. We compared the memory and naive CD8+ T cells in a model of donor lymphocyte infusion (DLI) in which BALB/c mice with progressive growth of BCL1 tumor after TCD BM transplantation were given infusion of the CD8+ T cells. Both naive and memory cells are effective in clearing the tumor, and converting the hosts from mixed to complete chimeras. Differences in GVHD are observed. In conclusion, CD8+ memory phenotype T cells are a desirable subset that mediates potent anti-tumor activity without severe GVHD, and can be used in a DLI model.

Disclosures:
No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) and Survival in a Cancer Chemotherapy Outpatient Clinic: A Retrospective Chart Review Validation of a VTE Predictive Model.
Authors: Kearney Julia C. , Rossi Suzanne , Glinert Keren , Henry David H. 
Published: October 2019

Abstract
Abstract 2503
Poster Board II-480

Because venous thromboembolism (VTE) often occurs as a complication of cancer and chemotherapy, cancer patients have a 47-fold elevation of risk of VTE compared to the general population (Khorana et al., Journal of Thrombosis and Haemostasis 2007). However, until recently there was not a model to accurately predict which cancer patients would experience a VTE during their treatment. Khorana et al. have developed such a model (Khorana et al., Blood 2008). We conducted a retrospective study to test the effectiveness of Khorana and colleagues' clinical model at predicting risk of VTE in patients with metastatic cancer. We collected data from an outpatient oncology clinic for 112 patients with solid tumors or malignant lymphoma who had undergone active chemotherapy in the last two years. The data included the five predictive variables outlined in Khorana et al.'s model: site of cancer (2 points for a very high-risk site, 1 point for a high-risk site); a platelet count of 350 × 109/L or more; hemoglobin less than 100g/L and or/use of erythropoiesis-stimulating agents; leukocyte count more than 11 × 109/L; and body mass index of 35kg/m2 or more (1 point each) (Khorana et al., Blood 2008). All data was collected on the first day of chemotherapy or the most recent labs before the start of chemotherapy. A score of 0 points represented a low risk of VTE, 1-2 points represented an intermediate risk and 3-4 points represented a high risk. There were 20 low risk patients, 63 intermediate risk and 29 high risk. We found that the clinical model accurately predicted which patients would experience a VTE (Table 1). Of the total 112 patients, 23 experienced a VTE. We determined that patients considered high risk were 8-fold more likely to experience a VTE as compared to low risk patients (see Table 1). Kuderer et al. demonstrated that this risk model also predicts overall mortality for patients receiving chemotherapy (Kuderer et al., ASH 2009). The results of our study also showed a strong correlation between overall mortality and a higher risk score. The mortality rate of high risk patients during the trial period was 69.0% compared to 30.0% of low risk patients and 42.9% of intermediate risk patients. Our retrospective review validates Khorana and colleagues' clinical model to predict VTE and survival rates in cancer patients. Our results, combined with data from previous studies, indicate that patients who are at risk for VTE also have a higher mortality rate. With an accurate predictive model, primary prophylaxis treatment for VTE could be considered for high risk patients.

Disclosures:
No relevant conflicts of interest to declare.
Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.
Authors: Han Lina , Wierenga Albertus T.J. , Rozenveld-Geugien Marjan , van de Lande Kim , Vellenga Edo , Schuringa Jan Jacob 
Published: October 2019

Abstract
Abstract 2510
Poster Board II-487
Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.
TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.
Authors: Rossi Lara , Goodell Margaret 
Published: October 2019

Abstract
Abstract 2514
Poster Board II-491
In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases.

Disclosures:
No relevant conflicts of interest to declare.
Genome Expression Profiling of the First Hematopoietic Progenitors of the Mouse Embryo.
Authors: Baron Margaret H. , Isern Joan , Fraser Stuart T. , He Zhiyong , Tuck David , Schulz Vincent , Gallagher Patrick G. 
Published: October 2019

Abstract
Abstract 2536
Poster Board II-513
The transcriptional networks that regulate lineage commitment and expansion of the earliest hematopoietic progenitors in the mammalian embryo have not been well studied, due to a lack of methods for isolating these cells. We have begun to address this problem by purifying the first hematopoietic-committed progenitors in the mouse embryo based on expression of a human ε-globin::H2B-EGFP transgene that is expressed exclusively within the primitive erythroid (EryP) lineage, as early as embryonic day (E) 7.5. EryP are the first lineage-specific cell type to form in the embryo. They arise in large numbers from yolk sac-derived progenitors at the end of gastrulation, enter the circulation as nucleated cells soon thereafter, and continue to mature in a stepwise, synchronous fashion until they enucleate. The early and lineage specific expression of the GFP reporter allowed us to isolate not only circulating EryP (E9.5-E11.5) but also a population from dispersed E7.5-8.5 embryos that is enriched in EryP progenitors. Genome expression profiling allowed us to define the transcriptome from each stage of development and revealed highly dynamic changes during the progression from progenitor to maturing erythroblast. Hierarchical clustering analysis was used to organize genes on the basis of overall similarity in expression patterns; six major cluster patterns were identified. Genes within these clusters comprised distinct functional classes. For example, particularly prominent increases in expression were detected for genes involved in ribosome biogenesis, translation, chromosome condensation, and autophagy. Genes that were downregulated included those involved in DNA replication, cell cycle, and nucleolar and organelle biogenesis. We have focused on the emergence of EryP in the yolk sac. Expression of Gata2 is high in the progenitor population at E7.5 and decreases dramatically by E8.5. In contrast, Gata1, Scl, and Eklf are all upregulated during maturation of EryP progenitors, suggesting that these transcription factors have distinct functions during primitive erythropoiesis. Consistent with expression of the GFP reporter as early as E7.5, we find that endogenous mouse embryonic globin genes are also expressed at this stage. Therefore, globin gene expression is an early feature of EryP development. Analysis of promoters of differentially expressed genes allowed us to identify candidate transcriptional regulators, some of which have not previously been implicated in erythroid development. This is the first lineage specific transcription profiling of a differentiating hematopoietic cell type in the early mouse embryo. While we have focused on the development of EryP, insights from this study should have broader relevance to the definitive erythroid lineage.

Disclosures:
No relevant conflicts of interest to declare.
AMPK Regulates Nanog Gene Expression Via p53 in Mouse Embryonic Stem Cells.
Authors: Chae Hee-Don , Broxmeyer Hal E. 
Published: October 2019

Abstract
Abstract 2537
Poster Board II-514

High glucose level is usually required for embryonic stem cell (ESC) culture but culture medium without glucose is a rather favorable condition to enhance differentiation of ESCs. However, the exact molecular relationship of how glucose metabolism affects ESC pluripotency remains unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling the metabolism of glucose and fatty acids, is activated in response to a high intracellular AMP to ATP ratio (ATP-exhausting stress). We hypothesized that AMPK activity is associated with maintenance of ESC pluripotency and tested this hypothesis utilizing aminoimidazole carboxamide ribonucleotide (AICAR) and Compound C, respectively as an activator and a selective inhibitor of AMPK. Here, we demonstrate that in the mouse ESC line, R1, in the presence of LIF, AICAR significantly reduced protein expression of Nanog and SSEA-1 (Nanog: 365+/−21 vs. 270+/−25; SSEA-1: 13195+/−707 vs. 7572+/−507, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.5 mM, 24h treatment), n=3 expts, p<0.01). In contrast, Compound C enhanced protein expression of SSEA-1 and Nanog (Nanog: 365+/−21 vs. 555+/−35; SSEA-1: 13195+/−707 vs. 15864+/−894, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.005 mM, 24h treatment), n=3 expts, p<0.02). This indicates that AMPK has a positive effect on protein expression of Nanog and SSEA-1. We extended our study to investigate whether increased Nanog protein expression level via AMPK activity resulted from enhanced stabilization of Nanog protein by measuring the half-life of nanog protein following treatment of mouse ESCs with cycloheximide, a protein synthesis inhibitor. Nanog protein stability was not affected by AMPK activity. Next, we examined whether AMPK activator or inhibitor could change Nanog and Oct-4 mRNA levels. AICAR reduced Nanog mRNA expression by 40%, but did not influence Oct-4 mRNA expression. AMPK induces cell cycle arrest or apoptosis under ATP-exhausting stresses by phosphorylating and activating the p53 tumor suppressor, which could repress Nanog gene expression. Activated AMPK induced phosphorylation of p53 at serine 15 and 392 without disturbing LIF induced STAT3 signaling in this mouse ESC line (Fig. 1). Thus, we postulate that activated AMPK might inhibit Nanog gene activity by phosphorylation of p53. Taken together, our data suggest that AMPK activity in response to metabolic ATP and AMP levels may play a primary role in controlling ESC pluripotency through p53-mediated regulation of Nanog gene expression.

Disclosures:
No relevant conflicts of interest to declare.
G0/G1 Switch Gene 2 (G0S2) Is a Positive Regulator of Quiescence in Hematopoietic Stem Cells and Naïve T Cells.
Authors: Yamada Takeshi , Park Chun Shik , Bae Leon , Lacorazza H. Daniel 
Published: October 2019

Abstract
Abstract 2538
Poster Board II-515
The G0/G1 switch gene 2 (G0S2) was identified in blood mononuclear cells activated with mitogens. G0S2 is a small basic protein located in ER/Golgi in mouse fibroblast. Even though G0S2 is upregulated in inflammatory processes (i.e. rheumatoid arthritis, psoriasis, endometriosis) and by all-trans-retinoic acid treatment in acute promyelocytic leukemia cells, its role in the proliferation of hematopoietic stem cells (HSC) and T lymphocytes is largely unknown. Similarly to HSC, a delicate balance between quiescence and homeostatic proliferation maintains the T cell pool over time. An emerging paradigm suggests that cellular quiescence is an actively regulated state. G0S2 expression in bone marrow (BM) cells is downregulated following cytoablation, suggesting a role in the maintenance of stem cell quiescence. Gain-of function experiments, using retroviral gene transfer and BM transplantation, showed that G0S2 increased number of Lin- Sca-1+ c-kit+ cells in BM by inhibiting their proliferation at steady state. In addition, ectopic G0s2 expression inhibits homeostatic expansion of CD11b+ Gr-1+ and CD3+ cells. Competitive BM transplantation showed that cells overexpressing G0S2 are outcompeted by wild type BM cells due to lower proliferative potential, based on in vivo BrdU incorporation experiments. Inhibition of T cell proliferation by ectopic G0S2 expansion is consistent with suppression of G0S2 transcription in naïve CD8+ T cells upon T-cell receptor activation via MAPK and calcium-calmodulin pathways, suggesting that G0S2 also maintains quiescence of naïve T cells. G0S2 expression is silenced in myeloid and lymphoid leukemic cell lines by gene methylation, supporting a leukemogenic role. Collectively, our study uncovered an important role of G0S2 in normal and malignant proliferation of HSC and T cells.

Disclosures:
No relevant conflicts of interest to declare.
Large Scale Generation of Functional Megakaryocytes From Human Embryonic Stem Cells (hESCs) Under Stromal-Free Conditions.
Authors: Li Feng , Lu Shi-Jiang , Feng Qiang , Lanza Robert 
Published: October 2019

Abstract
Abstract 2540
Poster Board II-517
Platelets collected from donors have very limited shelf life and are increasingly needed for transfusions. In contrast to donor dependent cord blood or bone marrow CD34+ stem cells, hESCs are a promising alternative source for continuous in vitro production of platelets under controlled conditions. Current procedures for in vitro generation of megakaryocytes/platelets from hESCs are not efficient and require undefined animal stromal cells. We have developed a novel system to generate megakaryocytes (MKs) from human ES cells under serum and stromal-free conditions. In the current system, hESCs are directed towards megakaryocytes through distinct steps including embryoid body formation and hemangioblast development (Lu et al, Nature Methods, 4:501–509, 2007). A transient bi-potential cell population expressing both CD41a and CD235a markers has been identified at the end of hemangioblast culture. These cells are capable of generating both MKs and erythroid cells as demonstrated by FACS sorting and CFU assays. TPO, SCF and IL-11 are used to further direct MK differentiation of hemangioblasts derived from human ES cells in suspension culture. Currently, up to 2.5×107 MKs (CD41a+) can be generated from 1×106 hESCs, which is approximately 10 times more efficient than recently reported methods (Takayama et al Blood, 111(11):5298–5306, 2008). Without further purification, >90% of live cells from the suspension cultures are CD41a+ and the majority of these cells are also CD42b+ (>70%). These in vitro derived MK cells have morphological characteristics of mature, polyploid MKs as shown by Giemsa staining and immunofluorescent staining of vWF in cytoplasmic granules. Importantly, proplatelet forming cells are constantly observed at the late stage of MK culture indicating that MKs generated in this system are able to undergo terminal differentiation under feeder-free conditions. Platelet-like particles are also detected in culture media by FACS. When plated on OP9 cells, these MKs generate functional platelets that are responsive to thrombin stimulation. In summary, we have established a novel system for the generation of platelet-producing MKs from human ES cells that is suitable for scale up and future preclinical and clinical studies.

Disclosures:
Li: Stem Cell & Regenerative Medicine International: Employment. Lu:Stem Cell & Regenerative Medicine International: Employment. Feng:Stem Cell & Regenerative Medicine International: Employment. Lanza:Stem Cell & Regenerative Medicine International/Advanced Cell Technology, Inc: Employment.
The Hematopoietic Stem Cell (HSC) Compartment in Tristetraprolin (TTP) Knock-out Mice Is Abnormal Phenotypically but Normal Functionally.
Authors: Kaplan Ian M , Morisot Sebastien , Heiser Diane , Cheng Wen-Chih , LaDana Christina , Civin Curt I. 
Published: October 2019

Abstract
Abstract 2541
Poster Board II-518

The RNA binding protein TTP binds to AU-rich elements (AREs) located in the 3′UTR of mRNAs and dramatically reduces their half-life through exosome-mediated RNA degradation. TTP provides a conserved mechanism for the temporal and fine-tuning regulation of gene expression. We found that the Lin−Kit+Sca+ (KSL) population, a phenotypic marker combination that enriches for HSCs, in adult (6–8 week old) C57Bl/6 marrow expressed TTP mRNA at a level 25-fold higher than whole bone marrow mononucuclear cells. Furthermore, adult TTP KO mice had twice the number of KSL cells per mouse compared to WT littermates, with no difference in bone marrow cellularity. Thus, we reasoned that TTP might represent an as yet unexplored avenue for HSC regulation. The TTP KO mouse has been described previously* and displays myeloid hyperplasia, cachexia and arthritis. Consistent with the reported phenotype there was a 2-fold increase in the number of phenotypic granulocyte-monocyte progenitors (GMPs) and normal numbers of both common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) in KO marrow. Within the KSL population of KO marrow, we observed an increase in the Flt3−CD34− fraction, which has been shown to be highly enriched for long-term HSCs (LT-HSCs). However, when we measured the frequency of functional long-term engrafting cells by limiting dilution competitive repopulation transplantation assays, there was no difference between WT and KO marrow. We also found no difference in the in vivo responses of WT and KO HSCs in chimeric transplant recipients to bi-monthly 5-FU injections for 18 weeks. We tested the hypothesis that the increase in the number of KSL cells is due to a lack of regulation of the Sca1 mRNA in KO marrow. In an RNA half-life assay in NIH3T3 cells, TTP had no effect on the half-life of a reporter message carrying the Sca1 3′UTR; whereas the TNFa 3′UTR, a known TTP target, did cause the half-life of the reporter message to decrease in the presence of TTP in our assay. Interestingly, the Lin−CD150+CD48−Kit+Sca+ labeling scheme gave a much more accurate representation of the relative frequency of HSCs in KO vs WT marrow. In studying HSC biology, the ability to prospectively identify alterations in the HSC compartment of genetically and pharmacologically manipulated marrow is of utmost importance. These studies demonstrate that in genetically manipulated marrow, the phenotypic and functional phenotypes are not always in accordance.
*Taylor GA and Blackshear PJ et al. Immunity (4) 1996

Disclosures:
No relevant conflicts of interest to declare.