bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Regulatory T Cell Function in a Mouse Model of Beta-Thalassemia.
Authors: Bao Weili , Heck Susanne , He Wu , Yazdanbakhsh Karina
Published: October 2019

Abstract 638

The development of red blood cell (RBC) alloantibodies complicates transfusion therapy for chronically transfused patients with thalassemias. Understanding the regulation of immune responses to transfused RBCs may help in future design of therapeutic interventions to prevent RBC alloimmunization in this patient population. We have previously reported in a mouse model that key immune response regulators, CD4+CD25− regulatory T cells (Tregs) expressing Foxp3, control the rate and frequency of RBC alloimmunization in wildtype recipient mice. As an initial step to study and characterize immune regulation in thalassemias, we studied the Treg status of Hbb(th1/th1) mouse model of beta-thalassemia intermedia, known to exhibit mild anemia, reticulocytosis and splenomegaly. Compared to wildtype littermate controls (WT) (n=11), 3 month old thalassemic (THAL) mice (n=12) had significantly higher frequency of CD25+Foxp3+ Tregs in CD4+ population in peripheral blood (5.5±0.5% versus 4.1±0.3%, p=0.03) but not in the spleen (11.4%±0.9% versus 9.5±0.5%, p=0.1). Sorted splenic Tregs from THAL and WT mice were equally anergic to stimulation through the T cell receptor and Tregs from THAL mice secreted the expected low background levels of IL-2 and IFN-gamma in stimulated culture supernatants, similar to Tregs from WT mice. Surprisingly, however, the THAL stimulated Tregs, but not the WT counterparts, secreted the proinflammatory cytokine IL-17. To determine whether THAL Tregs were functionally active, we performed in vitro Treg suppressive assay. We found that thalassemic Tregs were in fact more suppressive than Tregs from their WT counterparts in their ability to suppress proliferation of CD4+CD25–T cells (at 1:4 ratio of Tregs : CD4+CD25–cells 91±3% thalassemic versus 66±3% WT , p=0.03; at 1:8 ratio, 84±4% % versus 63±7%, p=0.02 and at 1:16 ratio, 76±4% % versus 28±14%, p=0.03).

We next performed red cell transfusion studies to determine if the increased in vitro THAL Treg suppressive activity is associated with decrease in frequency of alloimmunization in THAL mice. A cohort of age- and sex-matched THAL and WT mice were transfused on a weekly basis for 4 weeks with red cells from transgenic mice expressing human glycophorin A. We found that considerably lower (2/9) alloimmunization rates in THAL compared to WT mice (8/11). Altogether, our data indicate that qualitative differences exist between Tregs from THAL mice and their littermate controls and raise the interesting possibility that THAL Tregs may represent a novel population of Tregs. Specifically, only THAL Tregs secrete the proinflammatory cytokine IL-17 normally associated with Th17 subset and yet they are functionally more suppressive. The mechanisms responsible for these differences are under further study.

Disclosures:

No relevant conflicts of interest to declare.
Modeling for Cure with Total Therapy (TT) Trials for Newly Diagnosed Multiple Myeloma (MM): Let the Math Speak.
Authors: Barlogie Bart , Shaughnessy John D , Anaissie Elias , van Rhee Frits , Alsayed Yazan , Waheed Sarah , Nair Bijay , Grazziutti Monica , Bolejack Vanessa , Crowley John
Published: October 2019

Abstract 744

Background:

Despite enormous progress over the past decade, the “cure” word has been avoided in the context of myeloma therapy. The observation of ∼90% sustained complete response (CR) with Total Therapy 3 (TT3) among the 85% with gene expression profiling (GEP)-defined low-risk disease prompted this investigation of modeling for cure in all Total Therapy (TT) trials.

Methods:

Kaplan-Meier plots of event-free survival (EFS) and complete response duration (CRD) were examined to determine whether cure fractions (CF) could be predicted from the presence of a plateau; in the case of CRD, estimating CF required the multiplication by the CR rate. Median follow-up times with TT1 (n=231), TT2 (n=668) and TT3 (n=303) were 15.7yr, 7.4yr and 4.2yr. CF estimates were examined overall and according to prognostically relevant baseline variables such as cytogenetic abnormalities (CA), available for all trials, and GEP, available to subsets of patients on TT2 and TT3. Relative survival ratios were computed by comparing patient survival with expected survival of a comparable group from the general population.

Results:

With TT1, significant CF estimates existed for EFS (6.4%) and CRD (6.0%). With TT2, significant CF estimates were noted for EFS with both control (9.9%) and thalidomide arms (23%). With TT3, CRD-derived CF for low risk patients was high at 55% (p<0.0001). Relative survival ratios revealed progressively earlier normalization of such ratios, apparent at 17yr in TT1, 10yr in TT2 and a remarkably short 5yr in TT3.

Conclusion:

Cure is a realistic goal of myeloma therapy whether or not CR was documented as evidenced through long-term follow-up in TT1. The observation of a high CF value of 55% in TT3 for low-risk myeloma with only 4.2yr of follow-up attests to the dramatically greater efficacy of this trial comprising both bortezomib and thalidomide. Therefore, cure should be an objective of contemporary trials for low-risk myeloma while intensive searches for more effective therapy are urged to improve outcomes for high-risk disease.

Disclosures:

van Rhee: Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
MCL1 Haploinsufficiency Protects Mice From MYC-Induced Acute Myeloid Leukemia.
Authors: Xiang Zhifu , Luo Hui , Payton Jacqueline E. , Wartman Lukas D , Ritchey Julie , DiPersio John F. , Ley Timothy J. , Opferman Joseph T , Tomasson Michael
Published: October 2019

Abstract 764

Anti-apoptotic Bcl2 family genes have been implicated in the pathogenesis of acute myelogenous leukemia (AML), but the functional significance and relative importance of individual proteins (i.e. BCL2, BCL-XL, MCL1) remains poorly understood. We examined the expression of BCL2, BCL-X and MCL1 in primary human hematopoietic subsets and in leukemic blasts from AML patients, and found MCL1 transcripts were consistently expressed at high levels in all samples tested (100%, n=111). Mcl1 protein was also consistently expressed at high levels in myeloid leukemic blasts in a murine Myc-induced AML model, and we used this model to test the hypothesis that Mcl1 facilitates AML development by allowing myeloid progenitor cells to evade oncogene-induced cell death. Activation of Myc for seven days in vivo substantially increased myeloid lineage cells while hematopoietic stem, progenitor and B-lineage cells were depleted. Haploinsufficiency for Mcl1 (Mcl1flox/null) abrogated the development of AML (median survival 56 days vs. not reached), and deletion of a single allele of Mcl1 from fully transformed AML cells significantly prolonged the survival of transplanted mice. In contrast, the rapid lethality of disease (median survival 25 days) was restored by co-expression of Bcl2 with Myc in Mcl1flox/null cells. Together, these data demonstrate a critical and dose-dependent role for Mcl1 in AML pathogenesis and suggest that Mcl1 may be an ideal therapeutic target in patients with de novo AML.

Disclosures:

DiPersio: Genzyme: Honoraria.
Annexin II Interactions with the Annexin II Receptor Enhance Multiple Myeloma Cell Adhesion and Growth In the Bone Marrow Microenvironment
Authors: D'Souza Sonia , Kurihara Noriyoshi , Shiozawa Yusuke , Taichman Russell , Galson Deborah , Roodman David
Published: October 2019

Abstract 130

Multiple myeloma (MM) is an incurable B-cell malignancy that develops in the bone marrow. The marrow microenvironment plays a critical role in supporting homing, lodging, and growth of MM cells by activating signaling pathways in both MM and bone marrow stromal cells (BMSC). Recently, we identified that annexin II (AXII) is involved in prostate cancer cell lodgment to the bone marrow as well as mobilization of prostate cancer cells to the peripheral blood. AXII expressed on stromal cells supports prostate cancer cell lodgment via the annexin II receptor (AXIIR) on prostate cancer cells. We hypothesized that MM cells use a similar mechanism to lodge and grow in the bone marrow. We demonstrated using bio-AXII, that MM cell lines and primary MM cells from 8 MM patients express the AXIIR protein. In addition, MM cells adhered significantly better to BMSC from wild-type mice than from AXII−/− mice. Knockdown of AXIIR by siRNA in MM.1S and ANBL.6 MM cells resulted in decreased AXII binding and decreased adherence of MM cells to KM101 stromal cells and BMSC from wild-type mice. Furthermore, the adhesion of MM.1SsiCon and MM.1SsiAXIIR cells to AXII−/− BMSC was similar indicating that AXII produced by MM cells does not act in an autocrine manner on attachment. Therefore, our studies indicate the importance of the interaction of AXII on BMSC with AXIIR on MM cells in adhesion. Our studies also revealed a role for AXIIR signaling on MM cell growth. We found that soluble AXII was released by osteoclasts into their conditioned media and stimulated the growth of MM cells via ERK1/2 and AKT phosphorylation in MM cells. AXII stimulation of MM cell growth was blocked by AXII antibody or by blocking ERK1/2 and AKT phosphorylation. In contrast, endogenous AXII produced by MM.1S cells did not stimulate MM.1S cell growth suggesting that autocrine AXII/AXIIR signaling in MM.1S cells does not contribute to MM.1S cell growth. Taken together, these results suggest that AXII/AXIIR axis in the myeloma microenvironment plays an important role in MM cell adhesion and growth through production of AXII by BMSC and osteoclasts.

Disclosures:

No relevant conflicts of interest to declare.
Discovery of a New Signaling Complex Based on Spinophilin That Regulates Platelet Activation In Vitro and In Vivo
Authors: Ma Peisong , Cierniewska Aleksandra , Signarvic Rachel , Sinnamon Andrew , Cieslak Marcin , Stalker Timothy J. , Brass Lawrence F.
Published: October 2019

Abstract 161

Platelets play an essential role in hemostasis, but excessive platelet responses to vascular injury or disease can be catastrophic. We have recently reported that members of the RGS protein family can modulate platelet responses to injury by limiting the duration of G protein-dependent signaling initiated by platelet agonists. Here we show that platelets contain a previously-unrecognized regulatory complex comprised of the 130 kDa scaffold protein, spinophilin (SPL) with at least two RGS proteins (RGS10 and RGS18) and the protein tyrosine phosphatase, SHP-1. In resting platelets, this complex is phosphorylated on spinophilin residues Y398 and Y483. Mutating both tyrosines to phenylalanine inhibits the binding of SHP-1 to spinophilin. Platelet activation by thrombin or thromboxane A2, but not ADP or collagen, stimulates a transient increase in spinophilin-associated SHP-1 tyrosine phosphatase activity and causes dephosphorylation and decay of the SPL/RGS/SHP-1 complex. Conversely, blocking SHP-1 phosphatase activity in platelets or omitting SHP-1 in transfected CHO cells inhibits dephosphorylation of spinophilin and prevents dissociation of the SPL/RGS/SHP-1 complex. While we have shown previously that inhibiting interactions between G proteins and RGS proteins produces a gain of function in platelets, knocking out spinophilin in mice inhibits platelet aggregation. This aggregation defect does not occur with all agonists, but is selective for those that are able to trigger decay and dephosphorylation of the SPL/RGS/SHP-1 complex. In addition to inhibiting platelet aggregation in vitro, the spinophilin knockout delays carotid artery occlusion in vivo following application of FeCl3 and reduces platelet accumulation following laser injury in cremaster muscle arterioles. Underlying these effects of the knockout is a decrease in Rap1 activation, an event that supports integrin activation, and attenuation of the cAMP increase otherwise caused by endothelial PGI2. Collectively, these observations show for the first time that a regulatory complex based on spinophilin helps to regulate platelet responses to injury and suggest that it does this by sequestering RGS proteins in resting platelets and releasing them after activation begins. Dissociation of the SPL/RGS complex is regulated by an agonist-induced increase in the activity of SHP-1 associated with spinophilin.

Disclosures:

No relevant conflicts of interest to declare.
Exhaled Carbon Monoxide as a Marker of Hemolysis In Transgenic Mouse Models of Sickle Cell Anemia.
Authors: Belcher John D , Nguyen Julia , Furne Julie , Vineyard Julie , Beckman Joan Denise , Marker Paul H , Hebbel Robert P , Levitt Michael D , Vercellotti Gregory M
Published: October 2019

Abstract 1642

Sickle cell disease is characterized by recurring episodes of hemolysis and painful vasoocclusion, which leads to ischemia-reperfusion injury and organ damage. Recently, the roles of heme-induced oxidative stress, nitric oxide consumption, endothelial cell activation, and inflammation in sickle cell disease have been recognized, in part because of the development of transgenic murine models of this condition. These mouse models provide insights into the pathophysiology of human sickle cell disease and the development of new therapeutic approaches. However, hemolytic rates and disease severity vary considerably between mouse models. Ferrous (Fe2+) hemoglobin (Hb) is easily oxidized in circulation to ferric (Fe3+) Hb, which readily releases free heme. Free heme is degraded by heme oxygenase which produces carbon monoxide (CO) as a byproduct. Endogenous CO production has been used as a measure of in vivo hemolysis in humans and mice, but there have been no reports of endogenous CO production in sickle mouse models. VCO was measured in sickle mouse models using a previously described rebreathing technique t (Levitt et al. J Lab Clin Med 113:241-247). Expired CO levels were highest in Hb-S knock-in Townes-SS sickle mice, almost 7-fold higher than normal control Townes-AA mice (p<0.05). Expired CO also was significantly (p<0.05) higher in HbS-BERK mice compared to normal control HbA-BERK. Injection of phenylhydrazine or hemin into normal C57BL/6 mice also raised expired CO levels 24 hours after injection (p<0.05). Expired CO levels were not significantly higher than controls in the New York, S+S-Antilles, het-BERK and Townes-AS models. These data confirm that hemolytic rates differ markedly between models. Additional data will be presented on the effects of inhaled CO on expired CO and the hemolytic rate in the Townes-SS model.

Disclosures:

Belcher: Sangart: Consultancy, Research Funding. Vercellotti:Sangart: Consultancy, Research Funding.
Novel Influenza A (2009/H1N1) Infection In Patients with Hematological Malignancies During the 2009 Pandemic Outbreak: Early Antiviral Treatment Improves Survival
Authors: Vigil Karen Joan , Adachi Javier A , Jiang Ying , Popat Uday , Mahani Mohsen Ghadimi , Granwehr Bruno P , Gaeta Susan , Malik Imrana , Champlin Richard , Raad Isaam I , Chemaly Roy F
Published: October 2019

Abstract 1731

Novel influenza A (2009/H1N1) is a new influenza virus of swine origin. The epidemiology, morbidity, and mortality are different from those of seasonal influenza viruses, with this new strain affecting younger patients. We describe the risk factors and outcome associated with 2009/H1N1 Influenza A (herein H1N1) infection in patients with hematological malignancies.

Among the first 69 cases of H1N1 infections in cancer patients occurring between April 17 to October 21, 2009, 54 (78%) had hematological malignancies (leukemia or lymphoma) and 22 (41%) had undergone hematopoietic stem cell transplantation. H1N1 strain was the only influenza A virus reported in Texas during the study period.

Most of the patients with H1N1 and hematological malignancy were old (50% were 50 years of age or older), moderately to severely immunosuppressed (67%) and obese (39%), High rates of hospitalization (48%), pneumonia (24%), and death (11%) were encountered in our patients. The majority of patients who developed pneumonia due to H1N1 were on immunosuppressive therapy at the time of diagnosis (11 (85%) of 13 cases with pneumonia vs. 20 (49%) of 41 with upper respiratory infection [URI], p=0.02). Delay in initiation of oseltamivir from onset of symptoms was the only significant predictor of progression to pneumonia (7 days in patients with pneumonia vs. 1 day in patients with URI, p=0.002) and of mortality (9 days in patients who died vs. 1 day in those who survived, p=0.003). The development of H1N1 pneumonia was significantly associated with higher mortality (p<0.001). Starting antiviral therapy within 72 hours of onset of symptoms was significantly associated with better survival (p=0.05, Figure 1) and with a trend towards less progression to pneumonia (p=0.07).

Novel influenza A (2009/H1N1) infection affected older, obese, and moderately to severely immunosuppressed patients with hematologic malignancies. Early initiation of antiviral therapy may impact outcome with less progression to pneumonia and may improve survival in this immunosuppressed patient population.

———— Patients who received antiviral therapy ≤ 72 hours after onset of symptoms

——–—- Patients who received antiviral therapy >72 hours after onset of symptoms

○ ○ ○ ○ ○ ○ Censored observations

Disclosures:

No relevant conflicts of interest to declare.
Failure of Plasmacytoid Dendritic Cells to Drive Functional Regulatory T Cell Differentiation In Human Disease
Authors: Hart Derek NJ , Ju Xinsheng , Ding Yitian , Azlan Maryam , Clark Georgina
Published: October 2019

Abstract 1746

Dendritic cells (DC) initiate and direct immune responses. Plasmacytoid DC (pDC) produce type I interferon (IFN) in response to viral and/or bacterial challenge and have been implicated in the pathogenesis of a number of chronic inflammatory disesases. pDC also induce the generation of regulatory T cells (Treg) from naïve CD4+ T cells. Treg control the balance of T cell responses and maintain immune homeostasis so the primary contribution of pDC to Treg induction in disease states is an important question. Psoriasis is a common chronic inflammatory skin disease associated with over expression of type I IFN–inducible genes in patients and this response apparently overwhelms any pDC regulatory contribution. The numbers of peripheral blood Treg cells and pDC are decreased in psoriatic patients but why pDC fail to generate a significant counterbalancing Treg response in psoriasis is unknown.

We compared purified blood pDC from normal donors with those from psoriasis donors, who were well and either untreated or only on topical therapy at the time of donation, for their ability to induce Treg. Freshly purified pDC from both normal and psoriasis donors lacked CD80 and CD83, but expressed similar amounts of HLA-DR and CD86. pDC from healthy psoriasis donors, when stimulated with TLR9 ligands, secreted high amounts of IL-6, expressed little surface inducible costimulator-ligand (ICOS-L, CD275) and exhibited low indoleamine 2, 3–dioxygenase (IDO) enzymatic activity compared to normal controls. To assess the capacity of pDC from psoriasis patients to induce the differentiation of naïve CD4+ T cells, we performed sequential co-culture experiments. In these, TLR-9 stimulated pDC from psoriasis patients failed to induce the differentiation of naïve CD4+ T cells into IL-10 secreting, functional regulatory T cells. In addition, the CD4+CD25+ T cells resulting from co-culture with psoriasis pDC were less effective in suppressing an allogeneic MLR. In contrast, the pDC from psoriasis patients, unlike those from normal donors, induced T cells capable of secreting IL-22. Further investigation as to why psoriatic pDC failed to induce Treg showed that the differentiated CD4+CD25+ (CD127-) T cells from these co-cultures expressed dramatically less Foxp3 following priming with psoriatic pDC. Exogenous kynurenine, to replace IDO restored the ability of psoriatic pDC to induce Treg.

In conclusion, our data demonstrate that aberrant pDC produce dysfunctional Treg development in psoriasis and highlights the contributions of IL-6/IDO/CD275/IL-22 to the disease pathogenesis. This insight is now driving studies in other disease states, notably graft versus host disease, where our focus is on manipulating DC for therapeutic benefit.

Disclosures:

No relevant conflicts of interest to declare.
The Effect of Vitamin D Supplementation On Calcium Excretion in Thalassemia
Authors: Balachandar Sadana , Randolph Rachel , Kleinert Dorothy A. , Sheth Sujit , Giardina Patricia , Vogiatzi Maria G
Published: October 2019

Abstract 1029

Purpose of study:

Transfusion dependent thalassemia (TM) patients are routinely supplemented with vitamin D due to their increased risk of developing osteoporosis. Recent studies from North America have found that these patients have high rates of vitamin D “deficiency” and “insufficiency,” despite supplementation. The amount of vitamin D supplementation required to raise serum 25 hydroxy-vitamin D (25-OHD) to optimal levels is not known in these patients. Recent studies have linked 25-OHD levels to hypercalciuria and nephrolithiasis in patients with TM. The purpose of this study is to determine the effect of various doses of vitamin D supplementation on vitamin D stores and calcium excretion in TM patients.

Description of project:

Prospective, single-blind, placebo-controlled study of TM patients followed in the transfusion center at Weill Cornell/New York Presbyterian Hospital. Patients with 25-OHD concentrations between 15–29 ng/mL were eligible for this 3-month study. Subjects were assigned in a block type of enrollment to the “high dose” equivalent of 2,000 IU of vitamin D per day versus placebo.

Results:

14 subjects were enrolled, with 8 assigned to the “high dose” group and 6 assigned to the placebo group. The “high dose” group consisted of 6 females, aged 15.2–45.5 years with an average baseline 25-OHD level of 22.4 ng/mL (15–26). The “placebo” group consisted of 4 females, aged 22.5–45.7 years with an average baseline 25-OHD level of 19.8 ng/mL (16–24). After the 3 month study period, hypercalciuria developed more frequently in those treated in the “high dose” group. In the placebo group, hypercalciuria was noted in 1/6 (16.7%) spot urine calcium/creatinine tests and 0/3 (0%) 24 hour urine calcium estimations. In the “high dose” group, the corresponding number of patients based on the same methods of testing were 5/8 (62.5%) and 2/5 (40%). No episodes of hypocalcemia, hypercalcemia or nephrolithiasis occurred in either group.

Conclusion:

Our findings suggest that “high dose” vitamin D supplementation results in higher rates of hypercalciuria in TM patients. Further studies are necessary to determine the optimal dose of vitamin D supplementation to minimize the risk of osteoporosis while preventing nephrolithiasis in TM patients.

Disclosures:

No relevant conflicts of interest to declare.
NK Cell Subsets Isolated From Human Thymus Differ From Peripheral Blood NK Cells in Their Cytotoxic and Cytokine Secreting Capability
Authors: Krieg Stephanie , Koehler Wolfgang , Heidkamp Gordon , Schneider Julia , Ganss Franziska , Purbojo Ariawan , Mackensen Andreas , Cesnjevar Robert , Dudziak Diana , Ullrich Evelyn
Published: October 2019

Abstract 1053

Until recently, studies on human NK cells have been mainly focused on NK cells from peripheral blood. Little is known about the functional role of human NK cell subpopulations in different lymphoid organs. We therefore aimed to characterize the phenotypical and functional profile of human NK cell subsets from different organs with a special focus on early forms of thymic NK cells in comparison to mature peripheral blood NK cells from adults and children.

Human NK cells can be classified according to the expression of CD56 and CD16 into immunoregulatory, cytokine producing CD56highCD16dim and mature cytotoxic CD56dimCD16high NK cells. It is widely accepted that NK cell development takes place in secondary lymphoid tissues leading to the maturation from CD56highCD16dim into CD56dimCD16high NK cells. Additionally, it has been reported that thymic CD34+ cells can differentiate into NK cells under treatment with IL-2 in vitro.

Here we report for the first time on a different composition of human NK cell subpopulations in the thymus compared to peripheral blood. NK cells isolated from the thymus did not only express lower levels of CD56 but also showed a different ratio of CD16high/CD16dim NK cells compared to peripheral blood NK cells. Extensive phenotypical analysis revealed significant alterations in expression patterns of killer-cell immunoglobulin-like receptors, natural cytotoxicity receptors and other maturation or differentiation markers on thymic NK cells. Functional analyses showed that the whole NK cell population from thymus as well as from peripheral blood of young children produced only low amounts of IFN-γ compared to adult peripheral blood NK cells.

Furthermore, functional assays of sorted NK cell subsets revealed important differences of CD16high and CD16dim thymic NK cells. Upon stimulation with IL-2 +/− K562 tumor cells, thymic CD16 high NK cells gain cytokine producing capacity, while CD16dim develop a tumor killing capacity comparable to naive peripheral blood NK cells.

In summary, this comparative study of NK cell subsets provides important information on the development and function of NK cells that may be of great value for the optimization of cellular immune therapy.

Disclosures:

No relevant conflicts of interest to declare.
The Study of GPIb-VWF Mediated Early-Stage Platelet Activation Triggering On a Single Cell
Authors: Ju Lining , Zhu Cheng , Cruz Miguel A. , Chen Yunfeng
Published: October 2019

Abstract 1069

Binding of GPIbα to VWF tethers platelets to disrupted vascular surface during the haemostatic process. The GPIbα –VWF interaction can also trigger outside-in signaling cascade, resulting in platelet activation, characterized by morphological transformation from discoid to a more spiky shape as well as activation of integrin α IIbβ3. Using the adhesion frequency assay with a biomembrane force probe (BFP), we studied signal initiation by repeated brief contacts of a single platelet with a glass bead coated with VWF-A1 domain and/or fibronectin III 7–10 domain (FNIII7–10) in a precisely controlled fashion (Fig. A). Contacting platelets with beads coated VWF-A1 only resulted in adhesion kinetics mediated by GPIbα –VWF interaction kinetics independent of the activation stage of the platelet. Contacting platelets with beads coated FNIII7–10 only resulted in adhesion kinetics that correlated with the activation stage of the platelet. Discoid-shaped platelets yielded low level adhesions mediated by FN interaction with inactive α IIbβ3 (Fig. B, blue). By comparison, spiky-shaped platelets produced high level adhesions mediated by FN interaction with activated α IIbβ3 (Fig. B, red)that was four times stronger than the interaction with inactive α IIbβ3. Contacting platelets with beads coated both VWF-A1 and FNIII7–10 resulted in two-stage adhesion kinetics. The first stage was mediated by GPIbα –VWF binding, which triggered a second stage consisting of an increase in adhesion after a sub-second delay. The second-stage binding coincided with morphological changes characteristic of platelet activation and matched that mediated by FN interaction with activated α IIbβ3. On the other hand, the concurrent calcium imaging showed as the platelet target was brought to the A1 bead in a repeating manner, the recorded calcium fluorescence intensity climbed up as the repeated touches continue (Fig. C). The peak temporally correlates with the morphological change. Our data indicates that binding of VWF-A1 to platelet GPIbα initiates outside-in signaling, leading to rapid irreversible platelet shape changes and calcium mobilization within a few seconds.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Kinetic Study (PKS) May Early Identify a Subset of Patients with Immune Thrombocytopenia (ITP) Probably Cured by Romiplostim. Two Years Follow-up
Authors: Iuliano Francesco , Perricelli Alessia , Iuliano Eleonora , Infusino Stefania , Pomillo Angelo , Luci Maria , Barbano Filippo
Published: October 2019

Abstract 1099

Background

(TPO)-receptor agonists (romiplostim and eltrombopag) are new therapeutic modalities in the treatment of ITP. Romiplostim treatment was associated with a number of benefits compared with the standard of care in non splenectomized ITP patients: higher platelet response rate, lower rates of treatment failure and splenectomy, fewer bleeding events, and fewer blood transfusions. Nevertheless there are not enough data on the long-term side effects and economic consequences of prolonged treatment. Furthermore, there are no criteria to identify patients potentially cured and that could stop therapy. For all these reasons we have carried out a study of platelet kinetics in all patients treated with TPO- receptor agonists and we have identified a subset who achieved a normal platelet kinetics and that is no longer relapsed two years after discontinuation of the drug

Patients and Methods

A total of 18 adult patients, female (63%),median (range) age 55 (31 – 78) years, median (range) baseline platelet count 19 (3 – 32) × 109/L.,median of 4 (1 – 7) prior ITP therapies, received romiplostim administered once weekly sc, with dose adjustments to maintain platelet counts in the target range of 50–150×109/L. The median time since ITP diagnosis was 6,8 years (range, 0.6–12.8 years) and 10% had undergone a splenectomy. Patients received romiplostim for a median of 98 weeks (range, 18–104); taking the average weekly dose of all patients, the median was 4 mcg/kg. Home administration was started by 16% of patients (3/18) but 2/3 patients discontinued home administration and resumed weekly outpatient injection. All patients achieved a platelet count ≥50×109/L.

Results

3 out of 18 experienced thrombocytosis and rebound thrombocytopenia. A PKS with (111)In oxine-labeled autologous platelets was performed in all patients failing steroid treatment, before romiplostim was started. A gamma function was used for the calculation of platelet mean life span (MLS) that was greatly reduced in 100% of patients.3 patients, who had achieved early a stable platelet count ≥150×109/L. despite the discontinuation of romiplostim maintain normal platelet count after 24 months of follow-up. Stricking a second PKS six months after starting the treatment showed in these subset, a normal platelet half-life with normal uptake on the spleen and liver.

Conclusions

In our opinion PKS, in romiplostim responding patients who discontinued treatment for the stability of response, may easily detect patients probably cured

Disclosures:

No relevant conflicts of interest to declare.
Prospective, Open-label clinical Trial of Bivalirudin in Children with Venous Thromboembolism
Authors: O'Brien Sarah H. , Yee Donald , Lira Jessica , Goldenberg Neil A , Young Guy
Published: October 2019

Abstract 1165

Background:

Bivalirudin (BIV) is a direct thrombin inhibitor with a favorable pharmacologic profile in comparison to heparin. In a previous study in children <6 months of age with venous thromboembolism (VTE), bivalirudin was demonstrated to be a safe and potentially more effective anticoagulant. The present study's aims were to assess the safety, dosing, pharmacokinetics, pharmacodynamics, and efficacy of BIV in children >6 months of age.

Methods:

This prospective, open-label study evaluated the use of BIV for initial treatment of a new VTE in children between 6 months and 18 years of age. Eligible subjects received a BIV bolus of 0.125mg/kg followed by an initial continuous infusion of 0.125mg/kg/hour which was adjusted as needed to achieve a PTT of 1.5–2.5x their baseline PTT. BIV levels by high performance liquid chromatography and PTTs were tested simultaneously 5 times in the first 24 hours, daily thereafter and following all dose adjustments. Subjects remained on BIV until they were transitioned to long-term anticoagulant treatments. Imaging was required to confirm the presence of a VTE and follow-up imaging was obtained at 2–3 days and 30 days following initiation of anticoagulation. Subjects were closely monitored for bleeding and adverse events.

Results:

18 subjects (11M:7F) ranging in age from 9 months-17 years completed the study. There were no major bleeding events and one minor bleeding event. Outcome of VTE: at 2–3 days, 2 subjects had complete resolution and 7 subjects had partial resolution while at 30 days, 7 subjects had complete resolution and 9 subjects had partial resolution. There were no symptomatic recurrent events or any asymptomatic local progressions by surveillance imaging. Seven subjects required no dose adjustments during their course of treatment ranging between 2–5 days. See figure for correlation of PTT with BIV levels [each circle represents one subject with their mean PTT and BIV level with the standard deviations (r2=0.64)].

Conclusions:

BIV offers a potentially safer, more effective and pharmacologically more predictable alternative to heparin for initial anticoagulation of children with VTE. Pharmacokinetic-pharmacodynamic correlation was excellent suggesting PTT as an acceptable monitoring strategy for BIV in this population. Further studies comparing BIV to heparin should be conducted.

Disclosures:

Off Label Use: Bivalirudin is not approved for use in children. Goldenberg:Eisai Inc: Research Funding; Pfizer Inc: Membership on an entity's Board of Directors or advisory committees; CPC Clinical Research: Consultancy.
Rescue and Primary ITI in Children and Adults with a Single FVIII/VWF Product
Authors: Aledort Louis M. , Oldenburg Johannes , Santagostino Elena , Yuste Victor Jimenez
Published: October 2019

Abstract 1174

Introduction

Inhibitors are the most serious complication of hemophilia A (HA) treatment. Current optimal intervention for inhibitor eradication is immune tolerance induction (ITI), the goal of which is for the patient to resume therapy with FVIII. Basic and clinical research have strongly supported that VWF containing FVIII concentrates may be less immunogenic and are effective in primary ITI as well as for rescue ITI for those who fail to develop tolerance with recombinant proteins.

Objective

To assess the outcome of rescue and primary ITI in children (<18 years old) and adults with a single FVIII/VWF product (Fanhdi®, Grifols, Barcelona, Spain).

Methods

Retrospective data from HA patients (FVIII< 2%) with inhibitors from 22 centers in Spain, Italy and Germany, who underwent primary or rescue ITI with FVIII/VWF concentrate were evaluated. Success, partial success and failure were defined based on the criteria of the consensus recommendations of the 2006 International ITI Workshop.

Results

The largest international multicenter study on ITI using a single product has evaluated 41 cases of primary ITI (32 children and 9 adults) and 19 cases of rescue ITI (17 children and 2 adults).

Conclusion

The response rate of 87% (CS+PS) in primary ITI exceeds that previously reported with any product class. The response rate (CS+PS) of 74% in the higher risk profile of rescue ITI is what one might expect for primary ITI. These excellent results, using a single VWF containing FVIII product, should encourage the use of this class of product for immune tolerance.

Acknowledgments:

The authors acknowledge Grifols financial support of the study. The authors also acknowledge Drs Jeffrey Spears and Roser Peiro, Grifols, for their editorial assistance in preparation of the abstract.

Disclosures:

Aledort: Grifols: Advisory Board Other, Honoraria; Kedrion: Advisory Board, Advisory Board Other; Baxter: DSMB, DSMB Other; Octapharma: DSMB, DSMB Other. Off Label Use: This FVIII is not licensed for ITI - nor is any other product. Oldenburg:d and e: Baxter, Bayer, Biotest, CSL-Behring, Grifols, Inspiration, NovoNordisk, Octapharma, Pfizer e: Biogen IDec, Swedish Orphan Biovitrum: Honoraria, Research Funding. Santagostino:Bayer, Baxter, Pfizer, CSL Behring, Novo Nordisk, Biotest, Kedrion and Grifols: Speakers Bureau; Pfizer, Baxter, Bayer, CSL Behring, Kedrion, Grifols and Novo Nordisk: Consultancy; Novo Nordisk and Pfizer: Research Funding. Jimenez Yuste:Grifols, Baxter, Pfizer, NovoNordisk: Advisory Board Other, Consultancy.
Platelet Factor 4 (PF4) Causes Cell Cycle Arrest in Megakaryocytes (Megs) by Inactivating CDC2 (CDK1) and CDK2
Authors: Xiao Liqing , Poncz Mortimer , Lambert Michele
Published: October 2019

Abstract 1238

PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts.

Disclosures:

No relevant conflicts of interest to declare.
IFNγ Licensed Human Bone Marrow Derived Mesenchymal Stromal Cells Inhibit T Cell Cytokine Production by a Mechanism Independent of Indoleamine 2 3-Dioxygenase (IDO) Activity
Authors: Chinnadurai Raghavan , Copland Ian , Patel Seema , Galipeau Jacques
Published: October 2019

Abstract 1255

Human Bone marrow derived Mesenchymal Stromal Cells (MSCs) inhibit activation-mediated T cell proliferation. This functional interaction serves nearly universally as a surrogate assay for immune suppressive activity and potency of MSCs in vitro. However the role of MSCs in inhibiting T cell function, cytokine production in particular, has not been investigated thoroughly. In the present study we have compared resting MSCs with IFNγ licensed MSCs for their ability to inhibit activated T cell function such as cytokine secretion and degranulation through intracellular cytokine staining assays. PBMCs cocultured with resting or inflammatory cytokine licensed MSCs were stimulated with the super antigen Stephylococcal Enterotoxin B (SEB) for 12–14hours in the presence of Brefeldin A. Intracellular cytokine staining was performed on the T cells to detect the Interferon gamma (IFNγ), Tumor Necrosis Alpha (TNFα) and Interleukin-2 (IL-2). Our results demonstrate that resting MSCs do not inhibit T cell cytokine secreting function. However, IFNγ and IFNγ plus TNFα licensed MSCs dose dependently inhibit IFNγ secretion by T cells. Our results also show that IFNγ and IFNγ plusTNFα licensed MSCs not only inhibit cytokine secretion but also inhibit dregranulation of activated T cells. Boolean gating analysis demonstrates that IFNγ and IFNγ plusTNFα licensed MSCs inhibit triple cytokine producing highly inflammatory IFNγ +TNFα +IL2+T cells. Phenotypic analysis of MSCs demonstrates that IFNγ licensing upregulate indoleamine 2 3-dioxygenase (IDO) and B7-H1. Blocking of IDO activity with 1-Methyl D,L Tryptophan (1MT) will abolish MSC-mediated blockade of T cell proliferation. However, 1MT does not reverse the MSC blockade of T cell cytokine secretion. Phenotypical analysis of T cells demonstrate that PD1+ cells are inhibited while PD1- cells are not inhibited suggesting the role of B7-H1 and PD-1 inhibitory pathway is involved in the inhibitory potential of IFNγ licensed MSCs. Interestingly, the inhibitory potential of IFNγ licensed MSCs has been observed only with the SEB stimulation but not with anti-CD3, CD28 stimulation. This suggests that MHC-TCR interaction is necessary for the inhibitory effect of IFNγ licensed MSCs. Our results demonstrate the superiority of IFNγ licensed MSCs in inhibiting multiple cytokine producing T cell function independent of IDO activity. Our results also provide the rationale for use of IFNγ licensed MSCs in autologous immunosuppressive cell therapy.

Disclosures:

No relevant conflicts of interest to declare.
Rb Protects B-Lineage Hematopoietic Progenitor Cells From Oxidative Stress and Exhaustion
Authors: He Zhiwen , Wang Yinan , Luo Jun , Lu Lan , Su Mack Y. , Vij Ravi , Bhattacharya Deepta , Tomasson Michael H.
Published: October 2019

Abstract 1315

Background:

Multiple myeloma (MM) is the second most common hematologic malignancy in the United States. Chromosome 13 deletions are found in approximately one half (50%) of all patient samples and is associated with worse outcomes. The retinoblastoma gene (Rb1) has been implicated as a candidate tumor suppressor at 13q14, however, unmutated Rb1 remains expressed from retained alleles in the majority of cases. We sought to explore the role of Rb1 in plasma cells by generating mice with deficiency of Rb1 targeted to late B-cell development.

Methods:

The Rb family member p107 can compensate for Rb1 function, so we crossed Rb1Flox/Flox and P107−/−mouse strains with Cγ1-Cre knock-in mice that express the Cre recombinase specifically in germinal center (GC) B-cells to generate mice with tissue-specific deletion of Rb in germinal center cells (Cg1-Rb-KO), and we aged these mice to assess disease development.

Results:

In Cg1-Rb-KO mice, we demonstrated the Rb1 locus was successfully recombined in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). Recombination was also detectable in bone marrow B-cell but not myeloid cell progenitors. Cg1-Rb-KO mice developed normally but became ill starting at 40 weeks of age. Sick mice demonstrated weight loss, hypoplastic spleens, osteopenia and significant lymphopenia. Thorough analysis of bone marrow and spleen hematopoietic progenitor cell subsets by multi-parameter flow cytometry at the time of death revealed dramatic reductions in marrow B-cell (Hardy fractions C-D, from 3.18×103to 40 cells and from 1.4×106 to 7.5×104cells respectively), suggesting an effective collapse of the B-cell lineage in mice lacking Rb. Examination of Cg1-Rb-KO mice prior to becoming sick (age 30–35 weeks) demonstrated increased numbers of B-cell progenitors in the bone marrow, consistent with Rb's known role in cell cycle regulation. Together, our results suggest Cg1-Rb-KO mice develop benign B-cell hyperplasia followed by a lethal collapse of the B-cell lineage. To help explain this B-cell exhaustion phenotype, we measured reactive oxygen species (ROS) in the B-cell of young Cg1-Rb-KO mice. We observed significantly increased ROS in Cg1-Rb-KO mice at baseline and following LPS stimulation ex vivo and in vivo compared to p107-KO control mice, suggesting that B-cells from Cg1-Rb-KO mice have elevated levels of oxidative stress.

Conclusion:

Rb1 is important for maintenance of B-cell homeostasis and protects B-lineage progenitor cells from oxidative stress and exhaustion. Rb1 deletion was not sufficient for B-cell transformation and additional mutations are likely required for MM development in the context of complete RB1 loss.

Disclosures:

Vij: Millennium: Speakers Bureau.
Tumor Suppressor Capacity of Krüppel Like Factor 4 (KLF4) in NOTCH1-Induced T-Cell Acute Lymphoblastic Leukemia
Authors: Shen Ye , Suppipat Koramit , Park Chun Shik , Yamada Takeshi , Lacorazza H. Daniel
Published: October 2019

Abstract 1325

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children, among which T-cell ALL (T-ALL) exhibits worst prognosis. Studies on genetic factors supporting T-ALL leukemogenesis will contribute to the development of alternative therapies. Previously our laboratory described that the Krüppel like factor 4 (KLF4) restricts proliferation in normal CD8+ T cells. In addition, we have found that loss of KLF4 increases self-renewal and enhances survival of normal hematopoietic stem cells, which is a feature that cancer cells acquire during transformation. There are also emerging evidences showing reduced levels of KLF4 in bone marrow of T-ALL patients compared to healthy individuals and a correlation of KLF4 expression with response of ALL patients to standard treatment. Collectively, these findings suggest a potential role of KLF4 in tumor suppression and drug resistance in hematological malignancies. To study whether KLF4 functions as a tumor suppressor in T-ALL, we used Klf4 conditional knockout mice and NOTCH1-induced T-ALL model. Bone marrow (BM) cells from control (Klf4fl/fl) and Klf4 deleted (Klf4fl/fl; Vav-iCre) mice injected with 5-FU were transduced with retrovirus carrying the NOTCH1-L1601P-DP mutant found in human T-ALL patients and then transplanted into irradiated recipient mice. Mice transplanted with transduced control BM cells showed lower incidence of leukemia (21.7%, n=23) with longer median latency (130 days). In contrast, mice transplanted with transduced Klf4-deleted BM cells showed significantly higher penetrance (76.5%, n=34) with shorter median latency (93 days, p<0.01). Moribund mice from both groups showed similar infiltration of leukemic cells in liver, kidney, testis, brain and spleen. Immunoblots of leukemic cells purified from BM of diseased mice showed increased levels of Cyclin D1 and phospho-Rb in Klf4-deleted cells, suggesting enhanced proliferation. In addition to increased proliferative activity, a global gene expression analysis revealed significant differences in metabolic pathways. Concomitantly, Klf4-deleted leukemic cells displayed increased levels of phosphorylated mTOR, an important signaling involved in the metabolic reprogramming of cancer cells. Collectively, our study shows that KLF4 has tumor suppressor function in a mouse model of NOTCH1-induced T-ALL, providing a novel molecular target to prevent expansion of pre-leukemic cells.

Disclosures:

No relevant conflicts of interest to declare.