bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface HLA-DR Expression in Monocyte Subpopulations During Adverse Events After Hematopoietic Stem Cell Transplantation
Authors: Döring Michaela , Haufe Susanne , Erbacher Annika , Müller Ingo , Handgretinger Rupert , Hofbeck Michael , Kerst Gunter
Published: October 2019

Abstract 2161

Background:

Human leukocyte antigen DR (HLA-DR) surface expression in CD14++ monocytes reflects the activation state of these cells. Decreased HLA-DR expression levels have been described as an indicator of immunosuppression. According to CD14 and CD16 expression levels, monocyte subpopulations have been classified into “classical” CD14++/CD16- (M1), “non-classical” CD14+/CD16++ (M3), “intermediate” CD14++/CD16+ (M2) and CD14-/CD16++ (M4) monocytes (Figure 1). This study analyzes HLA-DR expression in these monocyte subpopulations after hematopoietic stem cell transplantation (HSCT) and in particular during transplant-related adverse events.

Patient and Methods:

30 pediatric patients (< 15 years) (n = 23) and young adults (> 15 years) (n = 7) (7 autologous, 12 allogeneic and 11 allogeneic haploidentical transplanted) with hemato-oncological malignancies and immunodeficiency disorders were consecutively included after informed written consent. Median age was 9.5 years (range 0.5–38 years). Flowcytometric assessment of HLA-DR expression was started the day before conditioning therapy and continued up to 379 days after HSCT. Normal values of HLA-DR expression in monocyte subpopulations were established from a control group of healthy children and young adults (n = 20). Surface marker expression of CD14, CD16, CD56, and HLA-DR was determined by four-color flowcytometry.

Results:

Median data acquisiton period was 228 days (range of 43–379 days). By analysis of covariance (ANCOVA), HLA-DR expression in healthy controls varied in dependency of age. In young adults (> 15 years), HLA-DR expression was significantly (P < 0.05) lower in M2, M3 and M4 but not in M1 monocyte subpopulations (P = 0.084) compared to children and adolescents (< 15 years). After HSCT, a significant decrease of HLA-DR expression in M1 and M4 monocytes was observed during sepsis or bacteremia (P < 0.01). The onset of graft-versus-host-disease (GvHD)°III-°IV was associated with a significant increase of HLA-DR expression in M1 monocytes (P < 0.05). HLA-DR expression in monocyte subpopulations was not altered before or during veno-occlusive disease (VOD), a relapse of underlying disease or before death.

Conclusion:

The distinct HLA-DR expression pattern in monocyte subpopulations during sepsis and GvHD may facilitate identification of patients at risk after HSCT. Moreover, HLA-DR expression in monocyte subpopulations may be used to monitor treatment response in these entities.

CD14++/CD16- (M1), CD14++/CD16+ (M2), CD14+/CD16++ (M3) and CD14-/CD16++(M4)

Disclosures:

No relevant conflicts of interest to declare.
Cellular and Biochemical Properties of Cell-Free DNA: A Prognostic Marker In Severe Sepsis Patients
Authors: Gould Travis J , Dwivedi Dhruva , Pepler Laura , Swystun Laura , Lysov Zakhar , Liaw Patricia
Published: October 2019

Abstract 2169

Background:

Sepsis is the leading cause of morbidity and mortality in patients in the non-coronary ICU. However, the heterogeneity of patients with severe sepsis makes the identification of high-risk patients (ie. those who are sickest and thus would require more aggressive and expensive therapies) an ongoing challenge. Previously, we have shown that high levels of cell-free DNA (cfDNA) appear to be a powerful predictor of death in severe sepsis patients. Using Receiver Operating Characteristic curves, we have demonstrated that the area under the curve for cfDNA is 0.96 (95% CI=0.93–1.00). The purpose of this study is to characterize the source and biochemical properties of cfDNA in sepsis.

Methods:

Toll-like receptor (TLR) cells (which are stimulated by microbial-specific, unmethylated DNA) were used to determine whether the cfDNA found in septic patients is host- or microbe-derived. Whole blood, monocytes, and neutrophils were isolated from healthy volunteers and stimulated with either lipoteichoic acid and peptidoglycan (from Gram-positive bacteria) or lipopolysaccharide (LPS) from Gram-negative bacteria. Monocytes and neutrophils were also incubated with LPS-induced apoptosis inhibitors, cilostamide and cilostazol, prior to stimulation with LPS. DNAse enzymatic activity was quantified using Org 590 DNAse activity ELISA (Orgentec Diagnostica, GmbH), while DNAse protein was quantified by Western blot analyses.

Results:

Exposure of TLR9-reporter cells to cfDNA from sepsis patients revealed that the cfDNA is host-derived. Stimulation of whole blood, monocytes, neutrophils, and endothelial cells to microbial cell wall components revealed that monocytes and neutrophils are the major sources of cfDNA in sepsis. Of the total amount of nuclear DNA present within monocytes and neutrophils, nearly all was released into the supernatant upon stimulation. Inhibition of LPS-induced apoptosis with cilostamide and cilostazol did not completely prevent the release of cfDNA from monocytes and neutrophils. We also found that cfDNA levels correlated with levels of histones in septic patients and that the cfDNA was degraded to nucleosomal units. Finally, we observed an inverse correlation between levels of DNAse activity/DNAse protein and levels of cfDNA.

Conclusions:

These studies are the first to characterize the source and biochemical properties of cfDNA in sepsis. A portion of cfDNA release from monocytes and neutrophils appears to occur via an apoptosis-independent mechanism. Plasma levels of DNAse enzyme were reduced in septic patients compared to healthy volunteers.

Disclosures:

No relevant conflicts of interest to declare.
G6PC3 Deficiency Associated with Congenital Neutropenia and Enterocolitis
Authors: Diestelhorst Jana , Kotlarz Daniel , Appaswamy Giridharan , Beier Rita , Krawitz Peter M , Robinson Peter N , Hecht Jochen , Puchalka Jacek , Schäffer Alejandro A. , Pfeifer Dietmar , Baumann Ulrich , Pfister Eva-Doreen , Brandes Gudrun , Palla Gabriella , Jacobs Roland , Kreipe Hans H. , Mundlos Stefan , Klein Christoph
Published: October 2019

Abstract 2170

Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency causes congenital neutropenia in conjunction with various cardiac or urogenital developmental aberrations.

We here describe a consanguineous pedigree of maroccan descent. Four patients presented with early-onset inflammatory bowel diseases associated with severe congenital neutropenia. Two children died in early childhood, two patients (19-years old female, 17-years old male) are alive with signs of recurrent infections (mouth ulcera, otitis, upper and lower respiratory tract infections) and refractory Crohn's like enterocolitis. Mutations in the Glucose-6-phosphate translocase (SLC37A4) gene were excluded.

In an attempt to discover the underlying molecular pathophysiology, we performed SNP-based homozygosity mapping followed by in depth next-generation exome sequencing. We identified two genes with potentially deleterious homozygous sequence variations: P2RY11 (exon 13; c.C2160G, p.Y720X), encoding a protein with antiapoptotic function, and G6PC3 (exon 2; c.C323T, p.P108L). The G6PC3 mutation segregates with the disease phenotype, whereas the P2RY11 variant was also found in a healthy sibling.

Functional experiments in patient's neutrophil granulocytes showed an accelerated oxidative burst capacity (OxyBurst Assay, Phagoburst Assay). Furthermore, G6PC3-deficient individuals showed a rapid dissipation of the mitochondrial membrane potential upon treatment with valinomycin, whereas the inner mitochondrial membrane potential in neutrophils from healthy individuals was maintained. We observed increased apoptosis in G6PC3-deficient neutrophils upon exposure to staurosporine. Interestingly, increased apoptosis was also seen in granulocytes from the healthy sibling with the P2RY11 sequence variant, suggesting that P2RY11 deficiency may aggravate the phenotype of G6PC3 deficiency.

In summary, our study indicates that the clinical spectrum of G6PC3 deficiency may be more diverse than previously appreciated and that P2RY11 may have a modifying effect on G6PC3-deficient neutrophil granulocytes.

J.D. and D. K. contributed equally to this work and should be considered aequo loco.

Disclosures:

Schäffer: National Institues of Health: Employment; Intramural Research Program of the National Institues of Health, NLM: Research Funding.
The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood
Authors: Nagamura-Inoue Tokiko , Kobayashi Seiichiro , Ogami Kazuo , Yamamoto Yuki , Izawa Kiyoko , Tojo Arinobu
Published: October 2019

Abstract 2180

Background:

Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting.

Disclosures:

No relevant conflicts of interest to declare.
MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo
Authors: Zou Tao , Satake Atsushi , Maltzman Jonathan , Kambayashi Taku
Published: October 2019

Abstract 2181

Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s.

Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency.

We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway.

In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Fibronectin Isoform Containing the Alternatively-Spliced Extra Domain A in Circulation Accelerates the Development of Atherosclerosis in Mice
Authors: Gandhi Chintan , Khan Mohammad Moshahid , Chauhan Anil K
Published: October 2019

Abstract 2205

Background and Objective:

The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several pathological conditions including atherosclerosis. It remains unclear in humans whether these elevated levels of EDA+-FN are actively contributing to disease pathogenesis, or rather simply serving as a marker associated with vascular stress and/or injury. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses We hypothesize that presence of EDA+-FN in plasma promotes inflammation and accelerates atherosclerotic plaque formation.

Model and Method:

We generated EDA+/+/ApoE−/− mice, which contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+-FN, and EDA−/−/ApoE−/− mice, which contain an EDA-null allele of the EDA exon and express only FN lacking EDA. ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain.

Results:

We report that atherosclerotic plaque (% of total aorta) formation in the aorta of EDA+/+/ApoE−/− mice was increased by two-fold compared to control ApoE−/− mice (P<0.0001). Deletion of the alternatively spliced EDA domain in the ApoE−/− mice (EDA−/−/ApoE−/−) significantly reduced atherosclerotic plaque formation in the aorta (P<0.05) compared to ApoE−/− mice. Total cholesterol and triglycerides levels were similar in ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− mice. Similarly, atherosclerotic plaque formation was significantly increased in the aortic sinus of EDA+/+/ApoE−/− mice, intermediate in control ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice (P<0.05). Additionally, we found that macrophage content, as analyzed by immunohistochemistry, was significantly elevated in the aortic root lesions of EDA+/+/ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice compared to ApoE−/− mice (P<0.05). Moreover, EDA+-FN did not affect the sex-dependent regulation of atherosclerosis in ApoE−/− mice. Future experiments using EDA+/+/ApoE−/−/TLR4−/− are under progress to determine whether EDA+-FN exacerbate atherosclerosis via upregulating TLR4 signaling.

Conclusions:

Our findings reveal that EDA+-FN is pro-inflammatory and promotes atherosclerotic lesion formation and that monitoring plasma EDA+-FN might have prognostic value in patients at high risk for atherosclerosis.

Disclosures:

No relevant conflicts of interest to declare.
The Von Willebrand Factor D'D3 Domains Modulate the Interacton of Platelet Receptor Glycoprotein Ib with the VWF-A1 Domain Under Conditions of Shear Stress
Authors: Nowak Agata Anna , Shah Amar , Laffan Michael , McKinnon Thomas A J
Published: October 2019

Abstract 2212

The VWF A1 domain contains the binding site for the platelet receptor glycoprotein (GP)Ib receptor, and this interaction forms the initial tethering event to capture platelets at sites of injury under high shear stress. The VWF-A1:GPIbα interaction has a high association and dissociation rate and as such platelets translocate along the VWF surface until stable attachment can occur. It has been previously shown that under static conditions the VWF D'D3 domains shield the A1 from interaction with GPIbα, however it has not been established if this occurs under conditions of shear stress. To this end we constructed VWF fragments spanning the D'-A3 domains and the A1A2A3 domains. These were expressed and purified and the ability of these constructs to interact with platelets was analysed in an in vitro flow assays system. Purified and fluorescently labelled platelets resuspended in plasma free blood were perfused over immobilised VWF at a range of shear rates (400–2000s−1), and real time movies of the interactions captured. Interestingly, platelets translocated slower across VWF-A1A2A3, with lower rolling velocities compared to VWF-D'A3 at all shear rates tested. Significantly the dissociation rate constant (koff) of the transient tethers formed between GPIbα and VWF was similar for both constructs, indicating that that the observed difference in platelet rolling velocity is not due to an alteration in bond lifetime. Frame-by-frame analysis of platelet translocation demonstrated that platelets paused more often on VWF lacking the D'D3 domains and therefore translocated a shorter distance in a set time when compared to VWF D'A3 resulting in decreased rolling velocities. We hypothesise that the accessibility of the GPIb binding site in the A1 domain is increased in the absence of the D'D3 domains, resulting in more interactions. Together these data demonstrate the D'D3 domains are able to shield the A1 domain under shear stress conditions.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation of C10orf55 Expression in Megakaryocytic Cell Lineage From Quebec Platelet Disorder Individuals
Authors: Bunimov Natalia , Blavignac Jessica , Tasneem Subia , Waye John S , Paterson Andrew D , Rivard Georges E , Hayward Catherine PM
Published: October 2019

Abstract 2274

Quebec platelet disorder (QPD) is a unique and serious bleeding disorder, associated with a gain-of-function defect in fibrinolysis that leads to a delayed onset bleeding after surgery or trauma in affected individuals. It has an autosomal dominant pattern of inheritance and affected individuals typically demonstrate an extensive personal and family history of bleeding. The disorder is caused by a tandem duplication mutation of a 78-kb genomic region located on chromosome 10 that contains PLAU (the gene that encodes urokinase plasminogen activator, uPA) in addition to the gene C10orf55, a gene of unknown function that encodes a transcript with partial complementary regions to PLAU mRNA. While QPD has minimal effects on uPA in urine, plasma and many other cell types, it elevates uPA levels >100 fold in QPD platelets compared to controls, due to overexpression of PLAU during megakaryopoiesis. As C10orf55 expression is postulated to have negative regulatory effects on uPA expression, we investigated if there is evidence of C10orf55 dysregulation in QPD. TaqMan assays were utilized for quantitative real-time RT-PCR analysis of C10orf55 transcripts in QPD and control CD34+ peripheral hematopoietic progenitors, platelets, lymphoblasts and saliva buccal cells (n=4–8 control and patient subjects for each cell type). QPD platelets contained elevated levels of C10orf55 mRNA that were >130 fold higher than in control platelets (p<0.001). Whereas in the other QPD cell types that were examined, the levels of C10orf55 mRNA were within the range expected for one additional copy of this gene, based on transcript levels measured in the control cells. These data demonstrate that the tandem duplication mutation of QPD leads to megakaryocyte specific increases in C10orf55 expression that parallel the pathological changes in PLAU expression during megakaryopoiesis. Although C10orf55 has been postulated to downregulate PLAU expression, due to complementarity of its transcripts, this seems unlikely given our findings. Our study indicates that the pathogenesis of QPD involves megakaryocyte-differentiation specific dysregulation of both genes contained in the 78-kb tandem duplicated locus on chromosome 10.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of the Novel Function of Cyclin A1 to Influence the Stem Cell Niche and Microenvironment Signaling in Mouse Model
Authors: Miftakhova Regina R , Hedblom Andreas , Bredberg Anders , Wolgemuth Debra J , Persson Jenny L
Published: October 2019

Abstract 2338

The molecules and cellular mechanisms that regulate pool size of hematopoietic stem cells and its association with stem cell niches to protect HSC from cell cycle-dependent injury are unclear. The cell cycle regulatory factor, cyclin A1 is overexpressed in patients with hematopoietic malignancies. Further, targeted overexpression of cyclin A1 in myeloid progenitor cells initiated acute myeloid leukemia in transgenic mice.

In the present study, we investigated the role of cyclin A1 in controlling the HSC pool and its functional association with key molecules that regulate stem cell niches under steady-state conditions or following the cytokine stimulation or radiation exposure in vivo and in vitro.

We reported that cyclin A1 null bone marrow displayed a significant increase in the frequency of stem cells (P<0,01) and increased expression of P27kip and increased phosphorylation of Akt at ser-473 site in HSCs and hematopoietic progenitors.

We further showed that increased frequency and number of cyclin A1 null HSCs was associated with the increased expression BMP receptor type IA that is known as a key molecule controlling the HSC niche. In addition, cyclin A1 null HSCs exhibited increased ability to migrate as determined by in vitro migration assay, and bone marrow transplantation assay, and this correlated with the increased expression of MMP9, that is known for controlling the osteoblast cell expansion, and the accumulated nuclear localization of angiogenic and vascularization factor VEGFR2 in cyclin A1 null bone marrow cells. We also observed that IRSp53 that is a regulator for extracellular matrix signaling, was present in the nuclear compartments of cyclin A1 null bone marrow progenitor cells, but was absent in that of the wild-type controls. Further, flow cytometry and immunoblot analyses showed that cyclin A1 null HSCs and progenitor cells exhibited relatively resistant to TNF stimulation and the radiation exposure, and this was associated with the great increase in the expression of phosporylated of ser-473 Akt. Our findings suggest that the microenvironment may be altered in bone marrows from cyclin A1 null mice. Thus cyclin A1 may have important function in the decision of maintaining the HSC pools and protecting the HSCs and progenitors from exposure to the external agents by regulating the interaction between the HSCs/progenitor cells and bone marrow environment.

Disclosures:

No relevant conflicts of interest to declare.
Enhanced Cytokine Responsiveness Counteracts Age-Induced Decline in Hematopoietic Stem Cell Function
Authors: Norddahl Gudmundur L , Wahlestedt Martin , Gisler Santiago , Sigvardsson Mikael , Bryder David
Published: October 2019

Abstract 2342

In recent years, it has become increasingly clear that physiologic aging has several profound cell intrinsic effects on hematopoietic stem cell (HSC) aging. This includes an altered output of mature progeny and an expansion of the HSC pool, although the latter is accompanied with several functional shortcomings. Because of the hierarchical structure of hematopoiesis, improving HSC function should present a promising avenue to restore aberrant hematopoietic function.

The signal adaptor protein Lnk, a relay of cytokine signaling, has been shown to negatively regulate hematopoiesis at several cellular stages. As a consequence of Lnk deficiency, mice display increased B lymphopoiesis and increased HSC numbers. The increase in HSC numbers has been suggested to arise by an increased ability of HSCs to self-renew. However, it has remained unclear how the enhanced sensitivity to cytokine signaling would affect hematopoiesis upon physiological age. In the present study, we investigated the effects of physiological aging on hematopoiesis in the Lnk−/− mice. Aged wild-type mice showed a number of age-related alterations in the hematopoietic system including; increased HSC numbers, decreased lymphopoiesis and decreased reconstitution potential upon competitive transplantation. Most of these parameters were dramatically altered as a consequence of Lnk deficiency. When competitively transplanted, aged Lnk−/− HSCs displayed a dramatically increased overall reconstitution potential compared to WT mice. Increases in reconstitution potential were observed in both primary and serially reconstituted recipient animals. Upon physiologic age, the lineage distribution of transplanted aged wild-type HSCs is skewed towards the myeloid lineage. By contrast, transplanted aged Lnk−/− HSCs gave rise to a hematopoietic system with a balanced lineage distribution resembling that generated by young wild-type HSCs. The enhanced HSC potential of aged Lnk−/− mice was uncoupled from effects on telomere length maintenance but displayed a distinct molecular profile in whole genome expression analysis. In summary, we demonstrate that deficiency of the signal adaptor molecule Lnk appears to functionally protect HSCs from many of the consequences of physiologic age, which at a broader level perhaps suggest a potential of cytokines to counteract age-related HSC decline.

Disclosures:

No relevant conflicts of interest to declare.
Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1
Authors: Montel-Hagen Amelie , Handel Ben Van , Ferrari Roberto , Sasidharan Rajkumar , Org Tonis , Boogerd Kees , Nakano Haruko , Zhou Jian , Li Xinmin , Pellegrini Matteo , Orkin Stuart H. , Nakano Austin , Kurdistani Siavash , Evans Sylvia , Mikkola Hanna
Published: October 2019

Abstract 2362

The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of Role for flt3L in Human Early B and T/NK Lymphopoiesis in a Novel Stromal Cell Culture System
Authors: Nakamori Yoshiki , Ohishi Kohshi , Liu Bing , Masuya Masahiro , Hamada Hirofumi , Katayama Naoyuki
Published: October 2019

Abstract 2385

The regulatory mechanism of human early lymphopoiesis remains less defined. A major limitation of conventional in vitro assay is that B and T lymphopoiesis cannot be assessed simultaneously. We exploded a novel culture assay and found that the hTERT-transduced telomerized human stromal cells support the generation of CD19+CD34lo/-CD10+cyCD79a+CD20+/−VpreB− pro B- and CD7+CD34+CD45RA+CD56−cyCD3− early T-cell precursors from human hematopoietic progenitors without cytokines, which was enhanced by flt3L (2010, ASH). We further characterized the generated lymphoid precursors, and verified that lineage-specific transcription factors for B (Pax-5 and EBF) and T/NK-cell precursors (GATA-3, HEB, Id2) were expressed by the CD19+ and CD7+CD56− cells, respectively. The CD7+CD56− cells showed the differentiation potential to T- and NK-lineage cells, by replating with OP9-Delta1 in the presence of flt3L+IL-7, and with the telomerized-stromal cells in the presence of flt3L+IL-7+IL-15, respectively. In serum-free culture condition, B-cell differentiation was minimally supported by the stromal cells, while low number of CD7+ cells was observed. Nonetheless, a significant number of CD7+, CD19-cyCD79a+, and CD19+ cells developed in the presence of flt3L, suggesting an important role for flt3L in the generation of early T/NK- and, particularly, B-lineage cell precursors on the stromal cells. The cellular interaction between Notch and Notch ligand Delta-1 or -4 in the presence of appropriate cytokines is considered to be crucial in T-lineage cell development in mice. We therefore examined whether Notch pathway is involved in the T/NK-cell development under this culture condition. While the gene expression of Delta-1 or -4 was detected in the telomerized stromal cells, the protein expression of these ligands was not detected with Western-blotting analysis. After co-culture of hematopoietic progenitor cells with the telomerized stromal cells, the gene expression of Notch target gene, HES1, was not increased. These data suggest that Notch signaling is not involved in the generation of early T/NK cell precursors on the stromal cells. This novel in vitro culture system suggests that the development of early B- and T/NK- cell precursors from hematopoietic precursors take places on the stromal cells in a Notch-independent manner and that flt3L plays a principal role in the stimulation of the early B and T/NK lymphopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Flt3 Is Required for MLL-ENL-Induced Leukemogenesis In Vivo, but Not for LSC Function in Vitro
Authors: Kamezaki Kenjiro , Snoeck Hans-Willem
Published: October 2019

Abstract 2450

Flt3 is a type III tyrosine kinase receptor expressed on hematopoietic multipotential progenitors and more downstream progenitor cells in the mouse bone marrow (BM).Flt3 is one of the most frequently mutated genes in acute myeloblastic leukemia (AML) patients, leading to constitutive activation. Several Flt3 kinase inhibitors are in clinical development in AML with Flt3 mutations, although disappointing results suggest that Flt3-inhibitors as monotherapy are unlikely to become reality. Wild type (wt) Flt3, which differs in its signaling characteristics and downstream targets from mutated Flt3, is highly expressed in particular in AML and ALL (acute lymphoblastic leukemia) with MLL (Mixed Lineage Leukemia) rearrangements. These observations suggest a role for wt Flt3 in pathogenesis as well, although little experimental or clinical evidence has been published yet supporting this notion. To our knowledge, there is only one report suggesting that wt Flt3 is required for the development of leukemia in a murine model of c-cbl mutation-induced leukemogenesis (Rathinam C, et al. Cancer Cell 2010). For this reason, we focused on the role of wt Flt3 in leukemogenesis and using leukemia mouse models established by retroviral transduction of MLL-ENL, MLL-AF9 and AML1-ETO9a. Mice transplanted with MLL-ENL-transduced Flt3−/− BM cells showed a higher survival rate (wt 61.3%, n=34 vs Flt3−/− 96.8%, n=31, median follow-up period; 255 days, p=0.038). No difference in survival was observed in the MLL-AF9 and AML1-ETO9a models however. Surprisingly, MLL-ENL-transduced Flt3−/− BM cells showed no significant difference in serial replating capacity of BM cells in methylcellulose colony forming assays in vitro compared to wt BM cells, and putative lin-kit+ leukemic stem cells (LSCs) did not express Flt3. In vivo, however, putative MLL-ENL LSCs, but not MLL-AF9 LSCs expressed Flt3. Furthermore, in non- and pre-leukemic MLL-ENL-transduced mice BM cells the frequency of lin-kit+ putative LSCs among MLL-ENL positive populations was decreased in Flt3−/− group (wt 1.468% vs Flt3−/− 0.466%, p=0.031). Flt3+ lin-kit+ populations were increased in MLL-ENL transduced leukemic wt mice compared to non-leukemic mice (leukemic mice 26.7% vs non-leukemic mice 9.9%, p=0.028). These results suggest that Flt3 signaling is required for MLL-ENL-induced leukemogenesis and maintenance of LSCs in vivo, but not in vitro. Importantly, the data suggest that the in vitro replatable leukemia stem cell (LSC) paradigm might not reflect LSC function or phenotype in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Clofarabine Combinations in Adults with Refractory/Relapsed Acute Lymphoblastic Leukemia (ALL): A GRAALL Report
Authors: Pigneux Arnaud , Sauvezie Mathieu , Vey Norbert , Raffoux Emmanuel , Marcais Ambroise , Huguet Françoise , Rousselot Philippe , Marolleau Jean Pierre , Leprêtre Stéphane , Turlure Pascal , Chevallier Patrice , Hunault Mathilde , Bonmati Caroline , Cluzeau Thomas , Leguay Thibaut , Ifrah Norbert , Dombret Herve
Published: October 2019

Abstract 2586

Even when intensively treated within pediatric-inspired protocols, about 25 to 30% of adults with ALL eventually relapse. With standard 4-drug or Hyper-CVAD salvages, post-relapse outcome remains very poor. Clofarabine is one of the new agents approved in US and EU to treat relapsing B-cell precursor (BCP) and T-cell ALL in children and young adults (up to 21 at initial diagnosis). In these patients, clofarabine is associated with a salvage rate around 30% when used as single agent. Combining clofarabine with conventional drugs in an intensive schedule may provide a chance to improve results in this difficult to treat population.

Methods:

Fifty-five patients were treated between March 2008 and February 2011. Thirty-seven patients received the VANDEVOL chemotherapy combining dexamethasone 10 mg/m2/12h day 1–5, mitoxantrone 8 mg/m2/d day 3–4, etoposide 150 mg/m2/d day 3–5, Peg-asparaginase 2.500 UI/m2 day 7, and Clofarabine 30 mg/m2/day day 1–5 and eighteen patients received the ENDEVOL chemotherapy combining cyclophosphamide 300 mg/m2/d day 1–3 and clofarabine 30 mg/m2/d day 1–5. Median age was 34 (19–67) and 53 (18–78) years in the VANDEVOL and ENDEVOL cohort, respectively. The proportion of first relapsing patients was 68% in the VANDEVOL cohort and 39% in the ENDEVOL cohort. Fifty patients had BCP-ALL (including 8 Ph+ ALL) and 5 patient had T-ALL.

Results:

Complete remission was achieved in 15/37 (41%) VANDEVOL patients and in 9/18 (50%) ENDEVOL patients. Early death rate was 5/37 (14%) in patients treated with VANDEVOL and 1/18 (6%) in patients treated by ENDEVOL. Grade 3–4 infectious, neurological, GI, and liver toxicities were observed in 22, 5, 5, and 11 VANDEVOL patients and in 10, 0, 0, and 2 ENDEVOL patients, respectively. Thirteen patients in the VANDEVOL group and three in the ENDEVOL group received subsequent allogeneic stem cell transplantation (overall transplant rate, 29%). After a median follow-up of 6 months, the median OS was 6.5 months.

Conclusion:

Combination of clofarabine with standard chemotherapy seems to be a promising and relatively safe approach to treat adult patients with refractory/relapsing ALL. This approach may be considered as a bridge to transplant in patients eligible for subsequent allogeneic stem cell transplantation. Updated data will be presented. The GRAALL will soon initiate a large multicenter prospective Phase II study using the VANDEVOL regimen.

Disclosures:

No relevant conflicts of interest to declare.
Mitotic Spindle Assembly Inhibition Through Aurora A (MLN8237) Plus Vincristine Is Synthetic Lethal and Synergizes with Rituximab As a Curative Therapy in Aggressive B-Cell Non-Hodgkin Lymphoma
Authors: Mahadevan Daruka , Cooke Laurence , Stejskal Amy , Manziello Ann , Morales Carla , Persky Daniel O. , Fisher Richard I , Miller Thomas P. , Qi Wenqing
Published: October 2019

Abstract 2721

Most aggressive B-cell non-Hodgkin lymphomas (B-NHL) are not curable with current chemo-immunotherapy combinations. Synthetic lethal interactions with novel agents may yield effective combination therapies with minimal toxicity for aggressive B-NHL. Auroras (A and B) are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. Aberrant over-expression of Auroras leads to genetic instability, polyploidy, tumor initiation and progression. Over-expression of Auroras in aggressive B-NHL promotes resistance to microtubule targeted agents (MTA, taxanes and vinca alkaloids). Si-RNA knockdown or pharmacologic inhibition of Aurora with MLN8237 [M], an ATP-site small molecule inhibitor, leads to enhanced sensitivity of B-NHL cells to MTAs. We hypothesized that promotion of microtubule de-polymerization with vincristine [V] would be more effective in synergizing with M than a microtubule polymerizing agent docetaxel [D]. We demonstrated that M plus V is synergistic while M plus D is additive in B-NHL cell culture models. Further, the addition of rituximab [R] enhanced apoptosis of B-NHL cells treated with MV or MD therapy. Mouse xenograft models of mantle cell lymphoma show modest single agent activity for M, R, D and V with tumor growth inhibition (TGI) of ∼10–15% (p=0.01). Of the doublets, MV caused tumor regression while MD and MR caused TGI (∼55–60%) and (∼20–25% (p=0.001) respectively. Although MV caused tumor regression, mice relapsed after 2 weeks of stopping therapy. In contrast, MVR had no relapses 120 days after stopping therapy, while MDR led to TGI of ∼85% (p=0.001). Kaplan-Meier analysis of overall survival showed that the mice treated with MV and MVR had a statistically significant improvement in overall survival when compared with the control (p<0.0001) or MR (p=0.0043). Gene expression profiling (human HG-U133A) and confirmatory Western blotting of harvested tumors at the end of treatment (3 weeks) confirmed reactivation of cell cycle regulatory genes including PCNA, Aurora B, cyclin B1 and cyclin D1, in MV versus MVR. Moreover, MVR therapy continues to inhibit Aurora B by repressing genes regulating mitotic sister chromatid segregation by repressed gene expression analyzed by Gene Ontology. Thus, addition of R to MV represents a novel therapeutic strategy that warrants clinical trial evaluation in aggressive B-NHL [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1].

Disclosures:

No relevant conflicts of interest to declare.
Longitudinal Changes in Iron Overload Parameters and Iron Chelation Therapy in Young Patients with Thalassemia Major,
Authors: Kattamis Antonis , Stokidis Konstantinos , Delaporta Polyxeni , Dimitra Kyriakopoulou , Petropoulou Theoni , Boiu Sorina , Ladis Vassilis
Published: October 2019

Abstract 3202

Background:

New modalities for the assessment of iron overload and the availability of new oral chelators have led to important changes in the iron load status and its treatment for patients with thalassemia major (TM). The goals of this retrospective analysis were to evaluate the changes that occurred in regards to the degree of iron overload, as well as to the therapeutic regimen of iron chelation over the last decade in young patients with TM.

Methods:

All patients with TM followed in our unit, who were <18 years at certain time points, were included in this study. Group A included all patients who were younger than 18 years old on 1/1/2001, while group B, C, D, E and F on 1/1/2003, 1/1/2005, 1/1/2007, 1/1/2009, and 1/1/2011, respectively. Liver iron concentration (LIC) and cardiac siderosis (T2*) were evaluated by MRI. Cardiac iron concentration (CIC) was calculated based on the recently prescribed formula CIC= 45 × (T2*Heart)−1.22.The closest MRI, which was <12 months from the time point, was recorded for each patient at each group. The therapeutic regimen for iron chelation, being deferoxamine (DFO), deferiprone (DFP), combination therapy of DFO and DFP (DFO+DFP) and deferasirox (DFX), used at the different time points were also recorded.

Results:

The results of the analysis are shown in the following table:

Ferritin levels did not change significantly over the last decade (p>0.05). There was a trend for decreasing values of LIC (Independent Samples Kruskal-Wallis test, p=0.075) with the mean LIC of group E and F being significantly lower than group C (Mann-Whitney test, p<0.05). Similarly, there was a trend for improvement in the indexes of cardiac iron load. Of note is, that cardiac overload was not documented in this group of patients. None of the patients has significant (T2*<10 msec), and only 3 patients had moderate cardiac siderosis (T2*>10 <20 msec).

Conclusions:

A steady decrease in the number of young patient with thalassemia has been observed, reflecting the efficacy of the thalassemia prevention program. As expected, the utilization of MRI to evaluate iron overload has increased significantly especially in the second part of the last decade, but it remains limited mainly to older children and teenagers. Oral chelation has become the preferable mode of treatment of hemosiderosis in young patients with TM. While DFX is, currently, the most used iron chelator, the use of DFO is becoming limited as an additive therapy to DFP. Despite presumed better compliance with oral chelation therapy, the iron overload indexes have not improved dramatically. This may reflect the short period of using the oral chelators or/and the need for further treatment intensification.

Disclosures:

Kattamis: Novartis Oncology: Honoraria, Research Funding, Speakers Bureau; Apopharma: Honoraria.
Regulation of Plasmacytoid Dendritic Cells by Commonly Used Anti-Viral Vaccines,
Authors: Jahrsdörfer Bernd , Nussbaum Benedikt , Mandel Birgit , Busch Daniel , Panitz Verena , Vollmer Angelika , Beyer Thamara , Debatin Klaus-Michael , Fabricius Dorit
Published: October 2019

Abstract 3221

Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. Recently, we demonstrated that apart from their pro-inflammatory effects, which are mainly mediated by secretion of interferon-alpha (IFN-a), pDC can also exhibit potent anti-inflammatory functions borne by active secretion of granzyme B (GrB), a serine protease classically known from CTL, NK cells and regulatory T cells. Here, we hypothesized, that commonly used anti-viral vaccines may affect pDC on several levels including their immunophenotype as well as their capacity to secrete either IFN-a or GrB. Using various methods including FACS, ELISpot, ELISA, spinning-disk confocal microscopy and RT-PCR, we could demonstrate that various anti-viral vaccines including vaccines against TBEV, yellow fever, polio, measles, rotavirus, varicella and hepatitis B were able to affect pDC by modulating expression of a series of surface molecules involved in cell adhesion, antigen-presentation and co-stimulation. In addition, major differences between the vaccines were found in terms of their effects on secretion of IFN-a and GrB. Interestingly, while only one vaccine, FSME Immun (TBEV) induced substantial IFN-a responses in pDC, all others tested did not. In contrast, virtually all vaccines tested induced more or less strong suppression of GrB secretion by maturing pDC. Of note, pDC that secreted high amounts of GrB induced by far lower allogeneic T cell proliferation as compared to pDC that secreted little or no GrB. Moreover, suppression of pDC-derived GrB by a substrate-specific GrB inhibitor resulted in significant enhancement of T cell proliferation in co-cultures of GrB-secreting pDC with allogeneic T cells. Our data demonstrate 1) that anti-viral vaccines may have distinct effects on both the immunophenotype and the secretory potential of pDC, and 2) that GrB is an important novel variable affecting the capacity of pDC to either trigger or dampen adaptive T cell responses. Our results may have implications for further study of the role pDC play in the regulation of adaptive immune responses, and for the potential application of this knowledge in the development of novel adjuvants admixed to anti-viral vaccines.

Disclosures:

No relevant conflicts of interest to declare.