bluuebunny/crossref_metadata_2025_split huggingface dataset:
ALL Progression Induces PD1 on T Cells and Blockade of PD1 Enhances Adoptive Immunotherapy
Authors: Burk Chad R , Fix William , Qin Haiying , Fry Terry J 
Published: October 2019

Abstract
Abstract 2587

Background:
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and, despite tremendous success in therapy over the past 3 decades, remains a primary cause of cancer-related mortality in children. Enthusiasm for the use cellular immunotherapy for ALL has been tempered by the poor response to donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation. However, ALL blasts are susceptible to T cell and NK cell mediated lysis in vitro suggesting that poor response to in vivo immune interventions may be due to events occurring during the priming of the immune response. Using a murine model of precursor B cell ALL we examined the impact of leukemia progression on T cells in vivo. Methods: We developed a transplantable syngeneic model of pediatric ALL derived from transgeneic mice expressing human E2aPBX1, a recurring translocation present in 5% of pediatric leukemia (Bijl et al, Genes and Development, 2005). This murine line displays a precursor B cell phenotype and results in 100% lethality following injection of 100,000 cells (Qin et al, ASH, 2010). Using congenic (CD45.1) B6 recipients, we tracked the early progression of ALL in vivo and examined the T cells in the leukemia-containing compartments by flow cytometry and PCR. Results: Using congenic markers, ALL cells can be detected in bone marrow as early as 3 days following intravenous injection of 1,000,000 cells with a sensitivity of 0.01%. Spleen and lymph node involvement was seen later (10 days) followed by the detection of circulating blasts by 2 weeks. E2aPBX1 cells express variable levels of costimulatory molecules in vitro with no change in expression during in vivo progression. Notably, PDL1 and PDL2 are expressed both in vitro and in vivo at higher levels than on non-malignant precursor B cells in leukemia-bearing mice. Remarkably, although PD1+ T cells are not seen in the bone marrow of non-leukemia-bearing mice, PD1 expression on bone marrow T cells was markedly increased during progression such that 60–80% of all bone marrow CD4 and CD8 T cells were positive by 2 weeks following leukemia injection (figure). In addition to expression of PD1, these T cells also co-expressed Tim3, a phenotype associated with T cell exhaustion. Blockade of PD1 or PDL1 starting 3 days following leukemia injection had no impact on leukemia progression. However, combining PD1 blockade with the adoptive transfer of T cells from leukemia-primed donors resulted in improved survival compared to primed T cells alone (p=0.0004). Conclusions: Early progression of ALL results in the induction of PD1 and Tim3 on T cells in vivo. Combination of PD1 blockade plus adoptive T cell therapy results in therapeutic benefit suggesting that this axis may be an attractive target in ALL.

Disclosures:
No relevant conflicts of interest to declare.
CXCR3 Is Required for the Efficient Recruitment of Bone Marrow-Resident Memory T Cells to Inflamed Tissues,
Authors: van der Voort Robbert , Brandao Claudia , Volman Thomas J. , Verweij Viviènne , van Gisbergen Klaas , Lu Bao , Gerard Craig , Nolte Martijn A. , Dolstra Harry 
Published: October 2019

Abstract
Abstract 3242
Although the importance of the bone marrow (BM) in hematopoiesis is well known, its function in adaptive immune responses has only recently been acknowledged. Currently it is known that the BM contains fully functional CD4+ and CD8+ T cells that can engage in both primary and secondary immune responses. Interestingly, most of these T cells belong to the memory T cell lineage, identifying the BM as one of the largest memory T cell reservoirs in the body. Since not much is known about the trafficking of BM T cells, we compared the homing phenotype and function of T cell subsets in the BM, blood, spleen and peripheral lymph nodes (pLN). In addition, we determined the expression of chemokine mRNA and protein levels in the BM and other lymphoid organs. We confirmed that at least 80% of the CD4+ and 60% of the CD8+ BM T cells have a memory phenotype, and that most CD4+ T cells belong to the effector memory lineage, while the CD8+ population predominantly consists of central memory T cells. Most BM T cells expressed the chemokine receptor CXCR3, the adhesion molecules P-selectin glycoprotein ligand 1 and VLA-4, and increased levels of CD44 and LFA-1, as compared to T cells from the spleen. In addition, L-selectin was absent from most CD4+ BM T cells, but present on virtually all CD8+ T cells. Notably, the percentage of CXCR3+ T cells within the effector memory and central memory subsets from BM was higher than within the same subsets from pLN. Furthermore, BM contained significant mRNA levels of the CXCR3 ligands CXCL9, CXCL10 and CXCL11. An in vivo migration assay using a mixture of fluorescent-labeled T cells from CXCR3-deficient mice and control mice indicated however that during homeostasis CXCR3 does not play a major role in BM entry or retention. These data suggest that CXCR3 expressed by memory T cells is rather involved in BM exit, than in BM entry. Indeed, we observed that, as compared to control mice, CXCR3−/− mice contained significantly more CD4+ and CD8+ T cells in their BM. Additional in vitro assays demonstrated that CD4+ and CD8+ BM T cells migrated vigorously in response to CXCL9 and CXCL10, generally released in high concentrations during inflammation. Finally, we demonstrate that CXCR3−/− effector/effector memory T cells, but not wild type T cells, accumulate in the BM of mice infected with lymphocytic choriomeningitis virus. Altogether, these data demonstrate that the BM is a major reservoir of memory T cells that employ CXCR3 to quickly respond to chemotactic signals from inflamed tissues.

Disclosures:
No relevant conflicts of interest to declare.
Analysis of Platelet Receptor Expression in ITP,
Authors: Qiao Jianlin , Tran Huy , Mu Fi-Tjen , Andrews Robert K , Gardiner Elizabeth E 
Published: October 2019

Abstract
Abstract 3289
In this study we assess platelet receptor expression and shedding in patients with immune thrombocytopenia (ITP) before and during treatment. The aim is to evaluate the potential value of quantitative measurement of platelet receptors for diagnosis and/or monitoring treatment in thrombocytopenia due to immune or other causes. The platelet-specific collagen receptor, glycoprotein (GP)VI, is associated with the Fc receptor γ-chain (FcRγ). GPVI/FcRγ is coassociated on platelet surface with the GPIb-IX-V complex; GPIbα of GPIb-IX-V binds von Willebrand factor and other ligands. Our previous studies showed engagement of platelet FcγRIIa by antiplatelet antibodies induced ectodomain shedding of GPVI, generating soluble ectodomain (sGPVI) in plasma. However, apart from one individual with an anti-GPVI antibody, whether anti-platelet antibodies associated with ITP affect GPVI/GPIb expression/shedding has not been addressed. In this study we used flow cytometry and a sGPVI ELISA to assess 1) whether patients with ITP had dysregulated expression/shedding of GPVI or GPIbα, and 2) whether platelet receptor expression changes prior to recovery of platelet count in individuals undergoing treatment for ITP. In 9 ITP patients (mean age=48.6, range 29–79; 6 female) with platelet count 61±9 × 109/L (range, 33–122 × 109/L), GPVI surface expression (GeoMean±SE, 137±17) was lower than healthy controls (274±26; n=17; platelet count 247±13), and sGPVI in patient plasma was significantly higher (39±4 ng/mL) compared to 17 healthy donors (19±3 ng/mL) (P=0.0006). In longitudinal samples analysed at weekly intervals during 2-month treatment with steroids, decreased GPVI surface expression and increased sGPVI in plasma remained essentially unchanged as the platelet count normalized, consistent with persistent anti-platelet antibody. However, while levels of intact platelet GPIbα were significantly reduced in ITP compared to healthy donors (P=0.0053), they approached healthy levels within 1 week of treatment, preceding improvement in platelet count or other measures. GPIbα expression/cleavage has been previously implicated in platelet clearance in experimental models, and our analysis suggests the proteolytic status of human GPIbα may be a novel early marker for evaluating response to treatment in ITP.

Disclosures:
No relevant conflicts of interest to declare.
Activated Protein C and Septic Shock: A Propensity-Matched Cohort Study,
Authors: Rimmer Emily K. , Kumar Anand , Doucette Steve , Marshall John , Dial Sandra , Gurka David , Dellinger Phillip , Sharma Sat , Penner Charles , Kramer Andreas , Wood Kenneth , Ronald John , Zarychanski Ryan 
Published: October 2019

Abstract
Abstract 3307

Background:
Septic shock is a highly inflammatory and pro-coagulant state associated with high mortality. In a single randomized controlled clinical trial, recombinant human activated protein C (rhAPC, drotrecogin alfa) reduced mortality in patients with severe sepsis at high risk of death. Further clinical trials in patients with a lower severity of illness and in children have failed to reproduce these results, and the effectiveness of rhAPC remains the subject of ongoing debate. Insufficient data exists regarding the use of rhAPC outside of a clinical trial.

Objective:
To evaluate the effectiveness of rhAPC on mortality in a cohort of patients with septic shock.

Methods:
We conducted a retrospective, propensity-matched, multi-center cohort study in 28 academic and community intensive care units in 3 countries. Propensity scores were based more 33 variables that were either known or suspected to be associated with rhAPC use. Our primary outcomes were mortality over 30 days and 30-day mortality stratified by APACHE II quartile. Secondary outcomes were hospital and ICU mortality and length of stay, ventilator free and vasopressor free days, and the use of acute dialysis.

Results:
Using a propensity matched Cox proportional hazard model we observed a 5.9% absolute reduction in 30-day mortality associated with activated protein C use [115/342 (33.6%) vs. 135/342 (39.5%), HR 0.83, 95% CI 0.65–1.07, p=0.15]; however this reduction failed to reach statistical significance. Similarly, we observed non-significant reductions in mortality among the 3 lowest APACHE II quartiles (APACHE II <20: 12.5% vs. 18.2%, HR 0.66, 95%CI 0.29 – 1.49, p=0.32; APACHE II 21–25: 20% vs 33.3%, HR 0.55, 95% CI 0.30–1.02, p=0.06; and APACHE II 26–30: 36.7% vs. 48.7%, HR 0.73, 95% CI 0.45 – 1.19, p=0.21). Hospital mortality (40.6% vs. 46.8%, HR 0.83, 95%CI 0.66–1.04, p=0.10) and ICU mortality (31.0 vs. 35.1%, HR 0.87, 95%CI 0.67–1.13, p=0.28) were not significantly reduced among patients who received rhAPC compared to matched controls. The use of rhAPC was not associated with significant differences in ICU or hospital length of stay, ventilator free days, vasopressor free days or the use of acute dialysis.

Conclusion:
In this retrospective, multi-center propensity matched cohort study, the use of rhAPC in patients with septic shock was not associated with statistically significant reductions in 30-day mortality. However, the absolute reduction in 30-day mortality was in similar to that observed in previously published clinical trials. Prospective clinical trials evaluating the efficacy of rhAPC are currently ongoing and will serve to define the role of rhAPC in patients with septic shock.

Disclosures:
Kumar: Pfizer: Research Funding; Astellas: Research Funding; GSK: Research Funding; Roche: Research Funding.
Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,
Authors: Zhang Wei , Dang Suying , Wisniewski Thomas 
Published: October 2019

Abstract
Abstract 3350
HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Mir-130a-Mediated Downregulation of SMAD4 Contributes to Reduced Sensitivity to TGFβ Stimulation in Promyelocytic Cells,
Authors: Hager Mattias , Pedersen Corinna Cavan , Larsen Maria Torp , Andersen Mette Klarskov , Hother Christoffer , Grønbæk Kirsten , Jarmer Hanne , Borregaard Niels , Cowland Jack B 
Published: October 2019

Abstract
Abstract 3383
MicroRNAs (miRNA) are noncoding RNA molecules that regulate the synthesis of proteins and, if dysregulated, can result in development of various form of cancers. We have found that miR-130a is highly expressed in immature proliferating granulocytic precursors. In more mature granulocytes the miR-130a expression is significant lower. In acute myeloid leukemia the granulocyte precursors have lost the ability to undergo terminal maturation, leading to accumulation of non-functional, immature granulocytes (myeloblasts). We hypothesize that a sustained high expression of miR-130a during granulopoiesis may sustain continuous cell proliferation. TGF-β is a strong inhibitor of cell proliferation and lack of TGF-β expression is associated with various form of cancer. Smad4 is an essential compound in the TGF-β signaling pathway. Using microRNA target-prediction software, we identified Smad4 as a putative target for miR-130a. This was confirmed experimentally by demonstrating that transient overexpression of miR-130a results in reduction in the amount of Smad4 protein. Luciferase reporter constructs with the 3`-UTR of Smad4 also respond to miR-130a – an effect that is abolished by point mutations in the miRNA–binding site. In agreement, we observed that stable overexpression of miR-130a in a granulocytic cell line reduces the level of Smad4 protein, and render the cells less sensitive to TGF-β-induced growth inhibition. This was also confirmed with cell cycles analysis. Furthermore, the effect was diminished when transfecting the same clones with SMAD4 lacking the 3-‘UTR. In line with our hypothesis, the most immature granulocyte precursors demonstrate the highest expression of miR-130a is highest, and the lowest expression of Smad4 protein. As the granulocyte precursors mature, the expression of miR-130a decreases dramatically whereas the level of Smad4 protein expression increases demonstrating inverse correlation between miR-130a and Smad4 protein. The level of Smad4 mRNA is comparable at all stages of granulopoiesis. High miR-130a levels and low or no expression of Smad4 was found in primary cells from patients with acute myeloid leukemia and in a cell line (Kasumi-1) with the t(8;21)(q22;q22) chromosomal translocation. The level of Smad4 increased in Kasumi-1 cells when the endogenous level of miR-130a was inhibited by anti-miR-130a LNA. Our data indicate that miR-130a is involved in cell cycle regulation of normal and malignant granulocytic cells through engagement of Smad4 in the TGF-β-pathway. Grant acknowledgment: Lundbeck foundation, Carlsberg foundation, Swedish Research Council.

Disclosures:
No relevant conflicts of interest to declare.
Two Distinct Transactivation Domains of GATA1 Contribute Megakaryopoiesis,
Authors: Kaneko Hiroshi , Kobayashi Eri , Shimizu Ritsuko , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 3384
Transcription factor GATA1 orchestrates erythroid/megakaryocytic cell differentiation by coordinately regulating the expression of multiple genes encoding proteins responsible for the proliferation, differentiation and survival of progenitors. Mice lacking GATA1 expression die in utero due to insufficient primitive erythropoiesis. GATA1 retains three functional domains; N-terminus (NT) transactivation domain (TAD) and N-terminus and C-terminus zinc finger domains that are both critical for DNA-binding and interaction with co-factors. We previously showed that each domain of GATA1 confers distinguishable properties to GATA1 during primitive and definitive hematopoiesis in vivo. Especially, GATA1 mutant lacking NT domain (GATA1s) is sufficient to rescue the GATA1-deficient mice from embryonic lethality, but the rescued mice suffered from anemia and thrombocytopenia. Showing very good agreement with this finding, inherited mutation yielding GATA1s was found in a family with dyserythropoiesis and dysmegakaryopoiesis. Whereas previous biochemical studies argued that NT domain was a unique TAD in GATA1, these findings suggest that alternative TAD domain is vital for the GATA1 activity in vivo. In this study, we focus on the C-terminus (CT) region of GATA1. The transactivation activity of GATA1 lacking the CT region (G1-DCT) was significantly reduced to approximately 30% of the wild-type GATA1, which was similar to the GATA1s or NT deletion level. Practically no transactivation activity was found in the GATA1 lacking both NT and CT domains. We also examined transactivation potential of the NT and CT fused with GAL4-DBD. CT-GAL4-DBD fusion protein showed Luciferase reporter activity over 20-fold higher than the GAL4-DBD. Furthermore, retrovirally transduced G1-DCT failed to control the hyperproliferation of megakaryocyte progenitors lacking endogenous GATA1, similar to the salient feature reported for GATA1s. These results thus indicate that the coordinated function of CT- and NT-TADs of GATA1 is required in the megakaryopoiesis. GATA1s mutation was found in majority cases of Down syndrome patients with transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Since CT domain is rich in Ser, Thr and Pro residues, while the NT domain is an acidic transactivation domain rich in Glu, we surmise that GATA1 regulates target gene expression by utilizing two distinct transactivation domains. The imbalance in gene expression in megakaryocytic progenitors, which is caused by GATA1s loosing the function of NT-TAD, may be the key aspect for the understanding of the onset of TMD and AMKL.

Disclosures:
No relevant conflicts of interest to declare.
The Tibetan PHD2 Polymorphism Asp4Glu Is Associated with Hypersensitivity of Erythroid Progenitors to EPO and Upregulation of HIF-1 Regulated Genes Hexokinase (HK1) and Glucose Transporter 1 (SLC2A/GLUT1),
Authors: Lorenzo Felipe R , Swierczek Sabina , Huff Chad Daniel , Prchal Josef T. 
Published: October 2019

Abstract
Abstract 3388
The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. As a high-altitude population, many Tibetans have developed a remarkable ability to protect against several hypoxic complications, including polycythemia and other harmful responses exhibited by non-adapted populations upon exposure to severe hypoxia. We have identified 10 genes involved in high-altitude adaptation in Tibetans, including a principal negative regulator of HIF-1a and HIF-2 a peptides, i.e. PHD2 (EGLN1), as well as HIF2A (EPAS1) (Simonson, Science 2010). At this meeting last year (Lorenzo, Abstract# 2602 ASH 2010), we reported a novel PHD2 Asp4Glu mutation that we found in 57 of 94 Tibetan, 2 of 88 Asian and 0 of 38 Caucasian chromosomes. In most Tibetan samples, this variant is associated with a previously reported, unvalidated PHD2 polymorphism, Cys127Ser (found in 70 of 94 Tibetan, 27 of 88 Asian and 4 of 38 Caucasian chromosomes).
To study the functional consequences of this PHD2 Asp4Glu mutation, we recruited five Tibetan volunteers living in Utah, four of whom were homozygous and 1 heterozygous for PHD2 Asp4Glu and Cys127Ser. We unexpectedly found that homozygotes for the exon 1 PHD2 mutation had markedly hypersensitive erythroid BFU-E (Fig.1) compared to the range of normal controls we have standardized over several decades. Interestingly, erythroid progenitors from individuals with Chuvash polycythemia or a HIF-2a gain-of-function mutation also have hypersensitive BFU-E. To determine if the Tibetan erythroid hypersensitivity data may be explained by increased HIF activity, we have quantified HIF target gene expression in subject granulocytes and found a significant increase in hexokinase (HK1) and glucose transporter (GLUT1/SLC2A) mRNA levels.
These data report the first molecular defect with functional consequences that is associated with the complex Tibetan adaptation to extreme hypoxia.

Disclosures:
No relevant conflicts of interest to declare.
The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,
Authors: He Zhixu , Shu Liping , Yang Xiaoyan , Deng Min , Liu Tingxi 
Published: October 2019

Abstract
Abstract 3413
Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown.
Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish.

Disclosures:
No relevant conflicts of interest to declare.