bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing Natural Killer Cell-Mediated Lysis of Lymphoma Cells By Combining Therapeutic Antibodies with CD20-Specific Immunoligands Engaging NKG2D or NKp30
Authors: Peipp Matthias , Kellner Christian , Günther Andreas , Humpe Andreas , Repp Roland , Klausz Katja , Derer Stefanie , Valerius Thomas , van de Winkel Jan G.J. , Parren Paul W.H.I. , Gramatzki Martin
Published: October 2019

Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a major effector function of many therapeutic antibodies. Thus, enhancing ADCC is a promising approach to further improve antibody therapy. Here, the CD20-specific immunoligands ULBP2:7D8 and B7-H6:7D8, which engage the stimulatory NK cell receptors natural killer group 2 member D (NKG2D) and NKp30, respectively, were compared for their abilities to boost ADCC in an attempt to design an effective antibody combination strategy.

The immunoligands are designed as single chain molecules, with a single chain fragment variable (scFv) of the CD20 antibody 7D8 fused to UL16-binding protein (ULBP) 2 or B7 homologue 6 (B7-H6), which are ligands of the activating NK cell receptors NKG2D and NKp30, respectively. By binding to lymphoma cells the immunoligands designated as ULBP2:7D8 and B7-H6:7D8 mimicked an induced self phenotype and thereby triggered NK cells to kill lymphoma and leukemia cells. Both immunoligands augmented ADCC by NK cells synergistically when combined with the lymphoma-directed antibodies rituximab or daratumumab recognizing CD20 and CD38, respectively. Antibody combinations with ULBP2:7D8 resulted in higher cytotoxicity (up to 10-fold lower EC50-values) in comparison to combinations with B7-H6:7D8, which in individual experiments failed to boost ADCC. Thus, NK cells were triggered more efficiently when NKG2D rather than NKp30 was co-ligated together with FcγRIIIA. Although a combination of ULBP2:7D8 and B7-H6:7D8 produced synergistic effects, no significant improvements were obtained by combining the three agents rituximab, B7-H6:7D8 and ULBP2:7D8. Enhancement of ADCC by the immunoligands was also achieved when NK cells from lymphoma or leukemia patients were analyzed as effector cells. ULBP2:7D8 in particular increased lysis not only of allogeneic but also of autologous tumor cells.

In summary, co-targeting of NKG2D was more effective in promoting NK cell-mediated ADCC than co-ligation of NKp30 and may represent a promising approach to further enhance the efficacy of therapeutic antibodies. Based on these results we propose a ‘dual-dual-targeting’ concept by co-targeting of two surface antigens on tumor cells and concomitant engagement of two different activating NK cell receptors.

Disclosures

van de Winkel: Genmab BV: Employment, Patents & Royalties. Parren:Genmab: Employment, Equity Ownership.
Whole-Genome Epigenomic Analysis in Multiple Myeloma Reveals DNA Hypermethylation of B-Cell Specific Enhancers
Authors: Agirre Xabier , Castellano Giancarlo , Pascual Marien , Health Simon , Kulis Marta , Segura Victor , Bergmann Anke , Esteve Anna , Merkel Angelika , Raineri Emanuele , Agueda Lidia , Blanc Julie , Richardson David , Clarke Laura , Russiñol Nuria , Queirós Ana C. , Beekman Renée , Rodriguez-Madoz Juan R. , San José-Enériz Edurne , Fang Fang , Gutierrez Norma Carmen , García-Verdugo José M , Robson Michael I. , Schirmer Eric C. , Guruceaga Elisabeth , Martens Joost H.A. , Gut Marta , Calasanz María José , Flicek Paul , Siebert Reiner , Campo Elias , San Miguel Jesus F. , Melnick Ari M , Stunnenberg Hendrik G. , Gut Ivo G. , Prosper Felipe , Martín-Subero Jose Ignacio
Published: October 2019

Analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed regional DNA hypermethylation embedded in extensive global hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM as compared to normal plasma cells were located outside CpG islands and were unexpectedly associated with intronic enhancer regions active in normal B cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with downregulation of its host genes. ChIP-seq and DNAseI-seq further revealed that DNA hypermethylation in these regions was related to enhancer decommissioning. Hypermethylated enhancer regions overlap with binding sites of B-cell specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation suggesting that MM cells reacquire epigenetic features of undifferentiated cells upon loss of expression of B-cell specific TFs. Overall, we have identified DNA hypermethylation of developmentally-regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

Disclosures

No relevant conflicts of interest to declare.
Epigenetic Signature of Leukemia Stem Cells Defines Subgroups Associated with Clinical Outcome and Cell of Origin in AML
Authors: Jung Namyoung , Dai Bo , Gentles Andrew J. , Murakami Peter , Majeti Ravindra , Feinberg Andrew P.
Published: October 2019

Acute myeloid leukemia (AML) is a hematologic malignancy initiated by leukemia-initiating or leukemia stem cells (LSC) which can differentiate into clonally related leukemic blast cells. This leukemia stem cell model proposes that functional properties of LSC and their blast progeny must be derived by epigenetic differences. Here, we examined genome wide DNA methylation of LSC-enriched populations and blast cells from 15 AML patients, along with 6 well-defined hematopoietic stem and progenitor cell (HSPC) populations from 5 normal controls using Illumina Infinium Human Methylation 450 BeadChip array. Strikingly, LSC-enriched populations exhibited global hypomethylation compared to non-engrafting blast cells, demonstrating that epigenetic change could drive the functional difference of LSC and their blast progeny. We defined an LSC epigenetic signature by integrating DNA methylation and gene expression analysis. The signature independently predicted overall survival of patients in both DNA methylation and gene expression data sets. Finally, we identified that LSC-enriched populations formed two major clusters when compared to normal HSPC: a granulocyte-macrophage progenitor (GMP)-like and a lymphoid-primed multipotential progenitor (L-MPP)-like subgroup that may reflect the cell of origin for these cases. These subgroups showed strong association with cytogenetic abnormalities and molecular mutations associated with the cell of origin. These results provide the first evidence for epigenetic variation between LSC and their blast progeny that are prognostic, and for epigenetically defined cell of origin of AML LSC.

Disclosures

No relevant conflicts of interest to declare.
Absolute Lymphocyte Counts at the End of Induction Is a Prognostic Indicator in Childhood Acute Lymphoblastic Leukemia
Authors: Hasegawa Daiichiro , Hirase Satoshi , Takahashi Hironobu , Saito Atsuro , Kozaki Aiko , Ishida Toshiaki , Yanai Tomoko , Kawasaki Keiichiro , Yamamoto Noriyuki , Kubokawa Ikuko , Mori Takeshi , Hayakawa Akira , Nishimura Noriyuki , Iijima Kazumoto , Kosaka Yoshiyuki
Published: October 2019

BACKGROUND: Recently, several studies have demonstrated that absolute lymphocyte counts (ALC) after induction therapy predicted treatment outcome. To address this issue, we here assessed the impact of the ALC at the end of induction therapy on outcomes in childhood ALL.

METHODS: We reviewed 141 cases of pediatric ALL with 1-21 years of age treated on the Japan Association Childhood Leukemia Study group ALL-02 series of treatment trials between 2002 and 2013. Patients with Philadelphia chromosome-positive ALL were excluded. Variables retrospectively analyzed included ALC at several time points during remission induction, age at diagnosis, gender, initial white blood cell count (WBC), cytogenetics, immunological phenotype, stratified risk, treatment response for bone marrow (the percentage of blasts at day 15), and outcome. Events in the analysis of event-free survival (EFS) included induction failure, death, relapse and secondary malignant neoplasm. The comparison of categorical variables between groups was performed by chi-square test. The probability of EFS and overall survival (OS) were analyzed with the use of the Kaplan–Meier method and a stratified log-rank test. A multivariate analysis of survival was performed with the use of a Cox proportional-hazard model to evaluate the treatment effect with adjustment for stratification factors.

RESULTS: The subjects included 121 of B-precursor ALL, 10 of T cell ALL, and 4 of acute mixed lineage leukemia/ acute unclassified leukemia. We found high WBC count at diagnosis (>100K/microL) and slow early responder for bone marrow at day 15 to be an unfavorable prognostic indicator, and also the ALC at the end of induction (day29) to be a statistically significant predictor of improved OS and EFS in our cohort. Patients with ALC ≥ 800/microL had a superior 5-year overall survival (100 ± 1.7% vs 88.1 ± 4.3 %, p=0.0001) and EFS (98.3 ± 1.7% vs 81.8 ± 5.0 %, p=0.0001). Multivariate analysis demonstrated that ALC at day29 was an independent, clinically significant predictor of improved EFS and OS after controlling WBC at diagnosis, gender, age at diagnosis, and cytogenetics. Multiple regression analysis adjusting for initial WBC count, peripheral blast counts at day8, and cytogenetics, also revealed an independent relationship (p=0.005) between treatment response (the percentage of blasts at day 15) and ALC at day29.

CONCLUSIONS: ALC is a simple, statistically significant prognostic factor in childhood ALL that may refine current risk stratification algorithms.

Disclosures

No relevant conflicts of interest to declare.
Embryologic Origin of Functional Granulopoiesis
Authors: McGrath Kathleen E , Palis James , Fegan Katherine H. , Catherman Seana C
Published: October 2019

The first progenitors with granulocyte potential arise in the murine yolk sac beginning at embryonic day 8.5 (E8.5), 48 hours before hematopoietic stem cells (HSCs) are formed. These granulocyte progenitors are part of a wave of definitive erythro-myeloid progenitors (EMPs) that display a unique immunophenotype. By E10.5, we observe EMPs in the bloodstream and enriched in the liver, the site of fetal hematopoiesis, consistent with previous reports of GM-CFC presence in the early embryo. HSCs begin to colonize the fetal liver between E11.5 and E12.5, where they subsequently expand and differentiate. Thus, the presence of maturing blood cells at this time-point likely represent the output of EMP that colonize the fetal liver before HSCs. In vitro culture of purified EMPs results in the complete myeloid repertoire found in the adult, including neutrophils, basophils, eosinophils and mast cells. To see which of these potentials is actually realized in the embryo, we examined myelopoiesis in the liver at E11.5- E12.5. There was no morphological evidence of eosinophils, basophils or mast cells in the early fetal liver, and there were no lineage specific transcripts for these cells types (EPX, FCepsilonR) detected by qPCR before E15.5. However, rare cells with neutrophil morphology were found in the fetal liver and in the bloodstream at E12.5. We utilized flow cytometry to enumerate granulocytes (GF1+, Mac1+) in both liver and the bloodstream during early embryogenesis. In order to rule out contamination from maternal cells, we analyzed embryos generated from GFP+ male mice mated with wild-type females. Per embryo equivalent, we consistently found a small number of granulocytes already present in both fetal liver and circulation at E11.5. The number of granulocytes increases to over one hundred at E12.5 and thousands by E13.5. We used imaging flow cytometry to examine the maturational state of the granulocytes in the fetal liver. Consistent with their recent differentiation, fetal liver granulocytes were predominately at the most immature stages as compared to bone marrow samples. Interestingly, at E11.5 and E12.5, a large proportion the circulating granulocytes were maternal derived (GFP-). The presence of these maternal granulocytes could not be accounted for by contamination of maternal blood given the levels of maternal RBCs in the samples. These data indicate that both maternal- and embryonic EMP-derived neutrophils co-circulate in the early embryo. Furthermore, when fetal blood was stimulated with bacteria-like BioParticles, fetal derived (GFP+) and maternal granulocytes (GFP-) each responded with oxidative bursts. Taken together, these data indicate that the early mammalian embryo utilizes both yolk sac-derived transient definitive progenitors and maternal granulocytes to provide host defense before HSC-derived hematopoiesis is established.

Disclosures

No relevant conflicts of interest to declare.
Quantitative Assessment of Translocation Transcript Ratio Predicts for Relapse Free Survival Among Patients with Core Binding Factor Acute Myeloid Leukemia
Authors: Hoang Thuyvan , Qiao Wei , Cortes Jorge E. , Patel Keyur , Ravandi Farhad , Kadia Tapan M. , Brandt Mark , Luthra Raja , Kantarjian Hagop M. , Borthakur Gautam
Published: October 2019

Presence of unique translocation events allows us to monitor minimal residual disease by quantitative polymerase chain reaction (qPCR) in patients with core binding factor acute myeloid leukemia (CBF-AML) that includes Inversion(16), t(16;16) and t (8;21) cytogenetic abnormalities. Fludarabine based regimens; Fludarabine, cytarabine, G-CSF and gemtuzumab ozogamicin (FLAG-GO) and FLAG, idarubicin (FLAG-Ida) have been two consecutive regimens used as front-line therapy for all new patients with CBF-AML presenting to MD Anderson Cancer Center since 2007. MRD has been monitored by baseline and periodic qPCR studies from bone marrow samples during induction/consolidation and follow up. Based on recent literature we investigated whether time to achievement of lowest qPCR value or the lowest qPCR value are important to predict for relapse free survival (RFS) in a multi-variate analysis.

Between 2007 and early 2014, 89 patients (pts) have achieved remission with frontline induction regimens; FLAG-GO=41 (46%) and FLAG-Ida=48 pts (54%); 44 patients with Inv (16) and 45 pts with t(8;21). Median presenting WBC count is 12.5x 106/L (range 1.9-97.2) and median qPCR ratio with ABL1 as control at presentation is >100. Median lowest qPCR following induction/consolidation is 0 (range 0-15.9) and is the same for inv (16) and t(8;21) (p=0.14). The median lowest qPCR value by regimen is 0 for FLAG-GO and the same for FLAG-Ida is 0.01 (p=0.003). RFS with a median follow up of more than 3 years is 80% and is not different among regimens (p=0.5). Because of infrequency of events, the analysis was mostly done using both cytogenetic groups and both regimens together.

Median time to lowest qPCR was 7 months for both cytogenetic subgroups as well as for both FLAG-GO and FLAG-Ida regimens (range 1-28 months). In a univariate analysis that included age, log WBC and platelet counts, cytogenetics, regimen, time to lowest qPCR value and the log lowest qPCR value as variables; log lowest qPCR is the only variable significantly predicting for RFS (p<0.01) while time to lowest qPCR is not (p=0.7).

Conclusion: RFS remains high among patients treated with FLAG-GO or FLAG-Ida and lowest qPCR value rather than the time to achieve lowest qPCR.

Disclosures

No relevant conflicts of interest to declare.
Erythroid Cells Adapt to L-Leucine Scarcity By Reducing Hemoglobin Production Via the mTORC1/4E-BP Pathway
Authors: Chung Jacky , Bauer Daniel E. , Ghamari Alireza , Nizzi Christopher P. , Deck Kathryn M. , Kingsley Paul D. , Yien Yvette Y. , Huston Nicholas C. , Chen Caiyong , Wittig Johannes G. , Palis James , Orkin Stuart H. , Lodish Harvey F. , Eisenstein Richard S. , Cantor Alan B. , Paw Barry H.
Published: October 2019

In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we find that maturing erythroid cells increase L-leucine uptake via transcriptional up-regulation of the L-leucine transporter, LAT3. Inadequate L-leucine uptake by L-leucine starvation or LAT3 inhibition triggers a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells via the mTORC1/4E-BP pathway. CRISPR-mediated deletion of 4E-BPs in murine erythroid cells renders them resistant to mTORC1 and LAT3 inhibition, markedly restoring hemoglobinization. Our complementary results demonstrate that globins are direct translational mTORC1 targets during normal development. This pathway is distinct from the previously reported translational regulatory mechanisms mediated by the heme-regulated inhibitor (HRI) kinase or by severe amino acid deprivation via the general control nonderepressible 2 (GCN2) kinase. We propose that, in red cells, mTORC1 serves as a homeostatic sensor coupling hemoglobin production to sufficient L-leucine uptake.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Mcl-1 Inhibitors in Preclinical Models of Multiple Myeloma
Authors: Belmonte Matthew A , Adam Ammar , Borrelli Deanna , Bhavsar Deepa , Bebernitz Christina , Hird Alexander , Secrist Paul
Published: October 2019

Mcl-1, an anti-apoptotic Bcl-2 family member, is a known resistance factor to many cancer therapies, and a historically difficult target to drug. Recently however, we have made significant progress optimizing the potency and characterizing the mechanism of action of a novel class of selective Mcl-1 inhibitors. Here, we characterize the activity of one of our lead compounds and confirm on-target mechanistic activity in vitro and in vivo.

Using an Mcl-1 binding assay we demonstrate very potent activity (IC50 < 3nM), while maintaining selectivity over other anti-apoptotic Bcl-2 family proteins, Bcl-2 (IC50 > 33 µM) and Bcl-xL (IC50 > 33 µM). In an Mcl-1 dependent multiple myeloma cell line, MOLP-8, we demonstrate rapid Mcl-1/Bak complex disruption with 10 nM inhibitor by 15 min followed by caspase 9-dependent apoptosis by 1 hr (EC50= 50 nM), consistent with an Mcl-1 dependent mechanism of action. Importantly, by depleting Bak from cells using siRNA, we also confirm that the observed apoptosis is through a Bak-dependent mechanism. Moreover, our Mcl-1 inhibitor exhibits broad submicromolar activity across a panel of multiple myeloma cell lines.

In the absence of a validated selective Mcl-1 reference compound to benchmark in vivo activity, we engineered MOLP-8 cells to express the pro-apoptotic protein, Noxa, using a Tet-on expression system and grew these as subcutaneous tumors on the flanks of SCID mice. Within 24 hours of switching the mice to a diet containing doxycycline, we observe significant upregulation of Noxa, leading to an increase in cleaved caspase 3 (CC3) and cleaved PARP in tumor lysates, resulting in rapid tumor regression. In the same MOLP-8 xenograft model, we demonstrate that our Mcl-1 inhibitor induces rapid dissociation of Bak from Mcl-1 in tumors leading to accumulation of CC3 and cleaved PARP by 1 hr. Complete tumor regression was observed after a single 60 mg/kg or 100 mg/kg dose, while partial regression was observed after a single 30 mg/kg dose, and tumor growth inhibition was observed after a single 10 mg/kg dose. All doses were well tolerated with no significant body weight loss. Together, these data reinforce the potential utility of selective Mcl-1 inhibitors in hematological malignancies.

Disclosures

Belmonte: Astrazeneca: Employment, Equity Ownership. Adam:Astrazenenca: Employment, Equity Ownership. Borrelli:AstraZeneca: Employment, Equity Ownership. Bhavsar:AstraZeneca: Employment, Equity Ownership. Bebernitz:AstraZeneca: Employment, Equity Ownership. Hird:AstraZeneca: Employment, Equity Ownership. Secrist:AstraZeneca: Employment, Equity Ownership.
Relationship Between Metabolic Tumor Volume and Rituximab Pharmacokinetic Parameters in Non-Hodgkin’s Lymphoma
Authors: Monjanel Helene , Ternant David , Venel Yann , Prunier-Aesch Caroline , Tournamille Jean-François , Arbion Flavie , Colombat Philippe , Paintaud Gilles , Gyan Emmanuel
Published: October 2019

The pharmacokinetics (PK) of the monoclonal antibody rituximab in B-cell lymphomas presents a high interindividual variability. In the pivotal study, inverse correlation between rituximab concentration and tumor burden estimated by the measurement of tumor bulk at baseline has been observed (Berinstein and al., Ann. Oncol. 1998). Using a pharmacokinetic/pharmacodynamic (PK/PD) model, it has been demonstrated in a murine model of lymphoma-expressing CD20 that tumor burden influences rituximab concentration (Dayde and al., Blood 2009). The aim of the present study was to evaluate the relationship between rituximab pharmacokinetic (PK) parameters and metabolic tumor volume (MTV) in patients with a previously untreated CD20-positive non-Hodgkin's lymphoma.

Twenty-five patients (12 follicular and 13 diffuse large B-cell lymphoma) received a combination of rituximab (dose of 375 mg/m2) with 6 to 8 cycles of CHOP. All patients provided written informed consent for the study. Rituximab serum levels were measured using a validated enzyme-linked immunosorbent assay (ELISA). Tumor volume delineations were performed on 18F-FDG PET-scan at baseline (Planet Onco, Dosisoft, France). Rituximab PK was assessed using population compartmental modelling. A relationship between MTV and survival was described.

Estimations of typical (interindividual standard deviation) systemic clearance (CLc) and volume of distribution (Vc) were 0.00972 L.h-1 and 3.12 L, respectively. Median MTV was 600 mL (range, 7 - 6217 mL). The central volume of distribution and the systemic clearance increased with MTV (p<0.05) (Table). A high MTV (>600 mL) was significantly associated with decreased event-free survival (p<0.05).

This pilot study is the first to describe a correlation between initial imaging-calculated metabolic tumor volume as a continuous variable and rituximab PK parameters in patients treated with R-chemotherapy for B-cell lymphomas. An increase in MTV leaded to a decrease in exposure to rituximab. Further prospective investigations on rituximab PK and dose adjustments in relationship with tumor burden are needed. Increased rituximab dose should be considered in patients with high tumor burden.

Table. Estimated pharmacokinetic parameters Parameter (units) Estimate Precision (%) p Vc (L) 3,12 10 b Vc (MTV) (L) 0,00012 36 0,0059 Cl (L.h-1) 0,00972 14 b Cl (MTV) (L.h-1) 0,000146 46 0,03

Disclosures

Paintaud: LFB and Pierre-Fabre Laboratoire: Consultancy; Roche Pharma, Chugai, Pfizer, Novartis, Janssen: Research Funding. Gyan:Roche: Research Funding.
Sensory Neuropeptides Prime the Splenic Marginal Zone for Optimal Humoral Immunity
Authors: Tsui Hubert , Ghazarian Magar , Winer Shawn , Adissu Hibret , Dosch Michael , Reis Marciano D. , Winer Daniel , Keating Armand
Published: October 2019

Effective spleen-derived humoral responses are essential for protecting hosts from blood-borne pathogens. Increased susceptibility to infection is therefore a risk post-splenectomy but is also associated with smoking, chemotherapy and aging. In fact, decreased vaccine efficacy in the elderly may be due to splenic hypofunction. While investigating the role of sensory nerves in hematopoiesis, we discovered a lymphocytosis phenotype involving the splenic marginal zone (MZ). Dividing red and white pulp in the spleen, the MZ is a specialized micro-anatomic structure facilitating innate and adaptive crosstalk to incoming antigen. Sensory nerves were found to synapse at this site and in mice deficient in the major sensory neurotransmitter substance P (Tac-1-/-), MZs were expanded histologically and confirmed by flow cytometry as an increase involving the IgMhiIgDloCD21hiCD23lo MZ B cell population. Tac-1-/- mice also exhibited splenomegaly with overall Ki-67 proliferation indices of 40% vs. 5% in controls. In the peripheral blood, Tac-1-/- mice had persistent polyclonal pan-B cell lymphocytosis with an absolute increase of 200% relative to wildtype. Aberrant B cell homeostasis had functional consequences as demonstrated by significantly decreased total basal plasma IgG (P<0.001) but not IgM, suggesting a transitional defect from the low-affinity T-independent (TI) polyspecific response to T-dependent (TD) mature germinal center derived antibodies. Mechanistically, MZ B cell migration to follicles is required for optimal TD responses where they shuttle antigen-laden IgM immune complexes. Since MZ B cell activation can occur independently of the B cell receptor, relying on Toll-like receptors or other pattern recognition receptor ligation – it is reasonable to include neuropeptide release as an additional stimulus coordinating appropriate MZ B cell mobilization. A working hypothesis therefore is inflammatory stimuli conveyed by the sensory nervous system, which may or may not involve perceptible pain sensation, is rapidly conveyed to the splenic MZ via release of substance P. This may provide an integrated communication network facilitating optimal antibody formation, more efficient than relying on diffusion of conventional danger signals or chemokines alone. TI and TD antigen challenge, cell tracing and electrode stimulation experiments are underway to confirm this theory. Splenic sensory nerve manipulation or pharmacological modulation of the substance P axis may have therapeutic applications for boosting humoral immunity in a variety of immune compromised states.

Disclosures

No relevant conflicts of interest to declare.
BCL6 Orchestrates Tfh Differentiation Via Multiple Distinct Mechanisms
Authors: Hatzi Katerina , Nance J. Philip , Kroenke Mark , Haddad Elias K. , Melnick Ari M. , Crotty Shane
Published: October 2019

Follicular Helper CD4 T Cells (Tfh) cells are required for T cell help to B cells and are essential for germinal centers and the development of most high affinity antibody responses that are a hallmark of protective immunity. BCL6 is the defining transcription factor of Tfh cells. However, the functions of Bcl6 in Tfh have largely remained unclear. Intriguingly, Bcl6 is essential in both Tfh cells and germinal center B cells, two cells with very different functions that exist in the same place at the same time and interact extensively. How Bcl6 accomplishes different tasks in these two different cell types has remained unknown. We defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. Bcl6 binds over 3,000 genes in GC Tfh cells and predominantly occupies promoters, but also enhancers. In these cells BCL6 primarily acts as a repressor and BCL6 binding was associated with control of Tfh cell migration, TCR signaling, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.

Disclosures

Melnick: Genentech: Speakers Bureau; Janssen: Research Funding; Calgene: Consultancy; Bioreference: Scientific Advisory Board, Scientific Advisory Board Other.
Mitochondrial Uncoupling Protein 2 Is Present in Human Platelets and Regulates Platelet Activation
Authors: Shiva Sruti , Novelli Enrico M , Bullock Grant C , Kenny Elizabeth , Hill Gabrielle , Corey Catherine G
Published: October 2019

It has long been recognized that platelets contain functional mitochondria and accumulating data suggest that several aspects of mitochondrial function, including reactive oxygen species production, modulate platelet thrombotic function. We have recently shown that platelet mitochondria isolated from patients with Sickle Cell Disease generate significantly greater concentrations of reactive oxygen species than healthy African American subjects and that this mitochondrial oxidant generation contributes to higher levels of basal platelet activation in these patients (Blood. 2014 May 1;123(18):2864-72). Based on these data, we have now investigated the factors that regulate platelet mitochondrial superoxide generation in healthy and Sickle Cell Disease platelets. Here we demonstrate that human platelets express mitochondrial uncoupling protein-2 (UCP2), a protein that is known to decrease the efficiency of oxidative phosphorylation and oxidant generation in other cell types but has previously not been identified in platelets. In this study we show that UCP2 protein is expressed in healthy human platelets and is fully functional as it facilitates proton leak across the inner mitochondrial membrane, leading to decreased mitochondrial membrane potential. Further, we demonstrate that the expression of this protein attenuates platelet mitochondrial superoxide generation, as treatment of platelets with Genipin (2-10µM), a pharmacological inhibitor of UCPs, concentration-dependently increases mitochondrial membrane potential and reactive oxygen species production. Further, an approximately 70% inhibition of UCP activity results in platelet activation demonstrated by increased membrane p-selectin expression (65±7% versus 6±3% in untreated controls) and augmented glycoprotein IIb/IIIa activation (57±9% versus 9±4% in untreated controls). The use of the mitochondrial-targeted antioxidant mitoTEMPO (10µM) decreases genipin-induced superoxide generation and significantly attenuates platelet activation. Notably, preliminary data presented here also suggest that UCP expression is decreased in platelets isolated from Sickle Cell Disease patients (3-fold) compared to healthy African American subjects. Additionally, ongoing studies are investigating platelet function in mice deficient in UCP2. Taken together, these data demonstrate a novel mechanism of regulation of platelet thrombotic function whose physiological relevance is apparent in the context of Sickle Cell Disease. More broadly this study advances the understanding of the role of the mitochondrion in platelet biology and thrombotic disease.

Disclosures

No relevant conflicts of interest to declare.
Integrin αIIbβ3-Mediated Outside-in Signaling: Brake or Amplifier of Platelet Activation?
Authors: Dai Baiyun , Wu Peng , Xue Feng , Yang Renchi , Yu Ziqiang , Dai Kesheng , Ruan Changgeng , Zhu Jieqing , Newman Peter J. , Gao Cunji
Published: October 2019

αIIbβ3 is the most prominent integrin in platelets, and binding to its ligands, in addition to supporting platelet aggregation, also results in the transmission of so-called αIIbβ3-mediated outside-in signals into the cell interior. While it is well accepted that integrin-mediated outside-in signaling functions as an amplifier of platelet activation, accumulating evidence suggests that outside-in signaling can, under certain conditions, function as an inhibitor of platelet activation. In this regard, previous studies have shown that ligand binding and platelet aggregation activate the inositol phosphatase SHIP-1, a negative regulator of the PI3K/Akt signaling pathway, to shift activated integrins back to their resting state, leading to dissociation of platelet aggregates. Because the PI3K/Akt signaling pathway is also involved in platelet granule secretion, we examined whether ligand binding to αIIbβ3 might transmit inhibitory signals that suppress platelet granule secretion. Interestingly, we found that antagonists of integrin αIIbβ3 promote both platelet dense- and α-granule secretion stimulated by low dose agonists. In support of this finding, both mouse and human platelets lacking expression of αIIbβ3 exhibited increased granule secretion compared to their wild-type counterparts. Conversely, Mn++-induced fibrinogen binding to αIIbβ3 inhibited low-dose agonist-induced platelet granule secretion. Biochemical analysis revealed that blocking ligand binding to, or absence of, αIIbβ3, enhanced agonist-induced Akt phosphorylation, while at the same time prevented the activation of the inhibitory enzyme, SHIP-1. To further investigate the role of SHIP-1 in inhibitory signaling, we examined the effect on platelet secretion of 3AC, a specific inhibitor of SHIP-1. We found that 3AC not only restores ADP-induced platelet granule secretion, but also increases CRP- or TRAP-induced platelet granule secretion. Taken together, these data demonstrate that integrin αIIbβ3-mediated outside-in signaling act as a brake to restrict unnecessary platelet activation that occurs in the presence of low-dose agonist stimulation.

Disclosures

No relevant conflicts of interest to declare.
Novel Mouse Hemostasis Model for Real-Time Determination of Bleeding Time and Hemostatic Plug Composition
Authors: Bergmeier Wolfgang , Piatt Raymond , Petrich Brian G. , Monroe Dougald , Mackman Nigel , Getz Todd M
Published: October 2019

Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiological conditions. However, measuring hemostasis in mice is quite variable and current methods do not quantify platelet adhesion or fibrin formation at the site of injury.

Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation, and time to hemostatic plug formation in real-time. Repeated vessel injuries of ~50-100µm in diameter were induced using laser ablation technology in the saphenous vein of mice.

Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein (GP)Ibα (IL4R/GPIb-tg) or talin-1 (talin1f/f PF4-Cre), important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice defective in the extrinsic (tissue factor (TF)low) or the intrinsic (FIX-/-) coagulation pathways, even though platelet adhesion was significantly reduced. Interestingly, fibrin accumulation was markedly increased in lesions of talin1f/f PF4-Cre, IL4R/GPIb-tg and FIX-/- mice, while it was decreased in TFlow mice. These findings suggest that prolonged plasma exposure to TF leads to increased thrombin and fibrin generation in the surrounding tissue. A partial reduction of platelet adhesiveness using clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TFlow mice.

Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. This new model has several defining characteristics, including the use of intravital fluorescence microscopy to monitor the hemostatic process, the novel use of laser ablation technology to generate vascular lesions with a defined diameter, the ability to repeatedly disrupt the hemostatic process at the same site of injury, and the possibility to perform multiple injuries along the exposed saphenous vein. Based on its sensitivity towards platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool to study the efficacy and safety of antiplatelet agents.

Disclosures

No relevant conflicts of interest to declare.
A Lesson from the Use of Optional Inferior Vena Cava Filters of Type Optease in Trauma Patients- a Single Type of Filter in a Single Medical Center
Authors: Salomon Ophira , Lavan Orly , Rimon Uri , Simon Dani , Khaitovich Boris , Segal Batia , Grossman Ehud , Kleinbaum Yeroham , Steinberg David M
Published: October 2019

Venous thromboembolism (VTE) is a frequent complication of patients who experienced major trauma and is accountable for almost 11% of deaths in those who survive at least 24 hours. Prevention of VTE is usually feasible through thromboprophylaxis and/or mechanical devices, but some trauma patients have injuries that make thromboprophylaxis impossible. Using inferior vena cava filter (IVC) is optional but it is not devoid of complications. Therefore the ASH in 2013 choosing Wisely® recommended avoiding routine usage of IVC filter. In this work our goal was to assess complications related to a single type of filter used prophylactically in trauma patients in a tertiary center and to search for risk factors associated with failure to remove the filter. The study included 136 patients aged 16 through 60 who experienced a major trauma and underwent prophylactic IVC filter insertion of type Optease at Sheba Medical Center during the years 2007-2013. The mean age of the patients was 36.7 with 79.6% men and 20.4% women. The predominant cause of injury was motor vehicle accidents (52%) and the mean Glasgow coma scale of patients when first seen by a traumatologist was 7.5. The median time from trauma to filter insertion was 2 days. Anticoagulation in prophylactic dose was started in 92.1% patients (117/127, no information for 9) once filter was inserted. The filter was removed in 68 (50%) of patients. In 13 patients the attempted filter removal was not successful for mechanical reasons. Nine patients developed IVC thrombosis and one of them died because of development of bilateral phelgmasia cerulea dolens leading to ischemic foot. Another 8 patients developed lower extremities DVT and therefore the filter was left in place. Among patients with a successful removal, the median dwelling time was 30 days, with a minimum of 8 days and a maximum of 207 days. As seen in the table, no relationship was found between age, sex, BMI, or GCS to successful filter removal.

Table 1. Successful Unsuccessful Mean age 35.3 (sd = 12.8) 38.1 (sd = 12.7) p = 0.20 Sex 81% Male 80% Male p = 0.93 BMI 25.3(sd = 4.7)kg/m2 26.9(sd = 7.3) kg/m2 p = 0.17 GCS 7.4(sd = 6.1) 7.8(sd = 5.8) p = 0.70

Of concern are the persistent complications related to the filter such as IVC thrombosis even with the prophylactic use of anticoagulants. Further, the long-term effects of radiation needed for insertion and extraction of the filter are worrisome, especially when it involves young patients. Thus the routine use of IVC filter in trauma patients may not be desirable. Careful assessment of the risks and the benefits is needed especially for young patients.

Disclosures

No relevant conflicts of interest to declare.
STAT5-Regulated miRNA193b Controls Hematopoietic Stem and Progenitor Cell Expansion By Fine Tuning Cytokine Signaling
Authors: Haetscher Nadine , Feuermann Yonatan D , Wingert Susanne , Rehage Maike , Weiser Christian , Thalheimer Frederic B. , Bohnenberger Hanibal , Schroeder Timm , Serve Hubert , Oellerich Thomas , Hennighausen Lothar , Rieger Michael A.
Published: October 2019

Hematopoietic stem cells (HSCs) regenerate the blood system life-long. Their number must be strictly controlled to avoid either exhaustion or excessive expansion leading to leukemia. Not only proteins but also regulatory RNAs are necessary to maintain HSCs. Hematopoiesis requires the right composition of microRNAs (miRNA), small, non-coding RNAs with the ability to simultaneously target multiple mRNAs for the orchestration of complex gene expression networks.

We identified miR193b to be induced in long-term repopulating (LT)-HSCs by the self-renewal promoting cytokine thrombopoietin via STAT5 signaling. To understand the physiological function of miR193b expression in HSCs, we generated a miR193b knock-out mouse model. These mice showed a selective expansion of the pool of functional LT-HSCs over time that were fully capable of multilineage blood reconstitution. MiR193b-deficient LT-HSCs were less quiescent than wildtype LT-HSCs in homeostasis, and were therefore more susceptible for repeated 5-Fluorouracil-induced stress. Further, they showed a higher expansion in vitro, not by changing the cell cycle length or death rate, but by slowing differentiation. Conversely, ectopic miR193b expression in LT-HSCs restricted their expansion and they were unable to repopulate the blood of lethally irradiated recipients. Long-term videomicroscopy-based tracking of LT-HSCs and their progeny revealed an increased cell death and slowed cell cycle upon miR193b expression. MiR193b-deficient hematopoietic stem and progenitor cells (HSPCs) showed a higher basal level of cytokine receptor signaling (pSTAT5 and pAKT), and an extended signal after cytokine stimulation, determined by phosphoflow cytometry at single cell resolution and in vitro PamGene kinase activity assays. Differential next generation RNA-sequencing of HPSCs with and without miR193b confirmed the regulation of signaling-related gene clusters and revealed potential mRNA targets. We identified the receptor tyrosine kinase ckit as a direct target of the miR193b, suggesting that miR193 modulates cytokine receptor expression and thereby signaling in HSCs.

Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR193b upon self-renewal promoting thrombopoietin – Mpl - STAT5 signaling, that in turn restricts cytokine signaling by targeting cytokine receptor expression. Thereby, we shed light into the essential function of miR193b as a modulator of HSC self-renewal.

Disclosures

No relevant conflicts of interest to declare.
Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cell Function and Promotes G-CSF Mediated HSC Mobilization
Authors: Herman Angela , Romine Molly , Monlish Darlene , Schuettpelz Laura G.
Published: October 2019

Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function.

To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs.

Disclosures

No relevant conflicts of interest to declare.
Molecular Regulation of Adult Hematopoiesis By GATA-2
Authors: Li Haiyan , Jin Jin , Sun shao-Cong , Watowich Stephanie S.
Published: October 2019

GATA-2 is a zinc finger-containing transcriptional regulator that plays important roles in embryonic and adult hematopoiesis. Mutations in human GATA2 are associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as immunodeficiency disorders that present with a profound loss of monocytes, dendritic cells and other myeloid lineage populations. Recent work reveals crucial roles for GATA-2 in definitive hematopoietic stem/progenitor cell activity, vascular integrity and lymphatic development. However, the molecular mechanisms by which GATA-2 controls adult hematopoiesis via hematopoietic-cell autonomous functions are largely unknown. To address this question, we generated a tamoxifen-inducible Gata2-deficient mouse strain by breeding Gata2flox/flox mice with Cre-ER transgenic animals. Following tamoxifen treatment, Cre-ER Gata2flox/flox mice show a rapid and profound loss of circulating neutrophils, monocytes and lymphocytes, concomitant with development of anemia. These results are consistent with the requirement for GATA-2 in hematopoietic stem/progenitor cells, and may also reflect GATA-2 function in endothelial cells within the vascular niche. To explore hematopoietic-specific GATA-2 activity, we generated bone marrow chimeric mice with hematopoietic-restricted Gata2-deficiency by transplanting Cre-ER Gata2flox/flox hematopoietic cells into wild type recipients. Cre-ER Gata2flox/flox bone marrow chimeras show rapid development of cytopenias upon tamoxifen exposure, suggesting a cell autonomous role for GATA-2 in maintaining adult hematopoiesis. Strikingly, hematopoietic progenitor cells rapidly lose c-Kit expression upon inducible Gata2 deletion. Chromatin immunoprecipitation and reporter assays suggest GATA-2 cooperates with C/EBPa in regulating kit transcription. Our study suggests conditional deletion of Gata2 restricted to the hematopoietic compartment provides a model for bone marrow failure associated with MDS and mutant GATA2 human immunodeficiencies that may enable further insight into the molecular network by which GATA-2 mediates definitive hematopoiesis.

Supported by grants from NIH (AI098099) and the MD Anderson Center for Cancer Epigenetics.(SSW) and the MD Anderson Center for Cancer and Inflammation (HSL).

Disclosures

No relevant conflicts of interest to declare.