bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteasome Inhibitors Decrease AAV2 Capsid-Derived Peptide Epitope Presentation On MHC Class I Following Transduction.
Authors: Finn Jonathan D , Hui Daniel J , Harre Downey , Dunn Danielle , Mingozzi Federico , Zhou Shangzhen , High Katherine A. 
Published: October 2019

Abstract
Abstract 695
Adeno-associated viral (AAV) vectors are one of the most extensively studied and highly used vector platforms for gene therapy applications. We have recently provided evidence for AAV capsid-derived antigen presentation through MHC class I on the surface of AAV-transduced cells, supporting the hypothesis that in the first clinical trial using AAV to treat Hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. Proteasome inhibitors are small molecule compounds that are able to specifically inhibit the activity of the proteasome, resulting in a buildup of ubiquitinated proteins, increased intracellular reactive oxygen species, and a general decrease in presentation of MHCI-peptide complexes. It has previously been shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here we describe using the FDA approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, while at the same time, enhancing gene expression in vivo. Using an AAV capsid specific T cell reporter line to analyze effects of proteasome inhibitor on antigen presentation, we demonstrated capsid antigen presentation at low MOI's, as well as inhibition of antigen presentation at clinically relevant levels of bortezomib. We also demonstrate that bortezomib can enhance FIX expression from an AAV2 vector in C57Bl/6 mice, however does not appear to enhance expression of AAV8. Based on the data presented here, it appears as if future studies using proteasome inhibitors in large animal models may be warranted. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease antigen presentation would be a promising solution to many of the obstacles to successful translation of AAV-mediated, liver-directed gene transfer to the clinic.

Disclosures:
No relevant conflicts of interest to declare.
Lymphoid Precursors Arise Independently at Multiple Sites During Development.
Authors: Porayette Prashanth , Yoshimoto Momoko , Glosson Nicole , Ferkowicz Michael , Shelley William C. , Kaplan Mark , Yoder Mervin C. 
Published: October 2019

Abstract
Abstract 699
Primitive hematopoietic cells arise on embryonic day 7.0 (E7.0) within the murine extra-embryonic yolk sac (YS) followed by formation of definitive hematopoietic cells on E8.25. The first cells capable of long term multi-lineage repopulation in an adult irradiated mouse arise in the embryo proper on E10.5. The temporal and spatial emergence of lymphoid precursors capable of forming mature T and B lymphocytes remains controversial. Cardiac contractions begin on E8.25, leading to the circulation of cells between various sites of hematopoiesis thus making it difficult to identify definitively the site where lymphoid precursors emerge. We have previously utilized a transgenic knock out murine mutant in which no heartbeat develops to examine hematopoietic cell emergence. NCX1 is a protein required for the initiation of cardiac contractions. The ncx1 null embryos develop normally until about E10.5, and then succumb to the lack of a systemic circulation. Single cell suspensions from within the para-aortic splanchnopleure (P-Sp) of the embryo proper and from YS at E8.5 and E9.5 were isolated and plated on the OP9-Dl1 stromal cell line that is known to support T lymphopoiesis. We observed that both YS and P-Sp independently displayed the potential to produce mature T cells including CD4 and CD8 DN, DP, and SP cells. We observed that both sites can give rise to both αaβ and γδ T cells independently at a frequency approaching 1:165 hemogenic endothelial cells plated. Cells derived from both YS and P-Sp demonstrated lymphoid specific VDJ rearrangement. Adoptive transfer of the lymphoid cells derived from these sites into NOD/SCID/IL2Rg null recipient mice revealed engraftment within multiple lymphohematopoietic organs including thymus, spleen and liver in the recipient mouse and the presence of circulating donor T cells. Stimulation of splenocytes from transplanted animals with anti-CD3 antibodies induced proliferation of these cells suggesting intact antibody responses in YS and P-Sp derived T cells. Development of mature functional T lymphocytes from precursors isolated from YS and P-Sp before the presence of a functional thymus brings forward the hypothesis for an extra-thymic multi-site origin of T cells in the early embryo. The presence of prospective lymphoid precursors more than a day earlier than the adult reconstituting hematopoietic stem cell (HSC) first observed at E10.5 during development suggests that either the HSC may be present at earlier points in development than is believed or that there might be a HSC independent emergence of lymphoid precursors in the developing embryo.

Disclosures:
No relevant conflicts of interest to declare.
Two Cycles of ABVD Followed by Involved Field Radiotherapy with 20 Gray (Gy) Is the New Standard of Care in the Treatment of Patients with Early-Stage Hodgkin Lymphoma: Final Analysis of the Randomized German Hodgkin Study Group (GHSG) HD10. Study Supported by the Deutsche Krebshilfe and in Part by the Competence Network Malignant Lymphoma.
Authors: Engert Andreas , Diehl Volker , Pluetschow Annette , Eich Hans T. , Herrmann Richard , Doerken Bernd , Kanz Lothar , Greil Richard , Markova Jana , Fuchs Michael , Borchmann Peter , Mueller-Hermelink Hans Konrad , Mueller Rolf Peter 
Published: October 2019

Abstract
Abstract 716

Background:
There has been an ongoing debate on the best treatment for patients with early favourable Hodgkin lymphoma (HL). Open questions include the choice between combined modality treatment or chemotherapy only, the number of chemotherapy cycles needed and the optimal radiation dose. The GHSG thus conducted a randomized study for patients with early-stage favourable Hodgkin lymphoma (HD10) in which these questions were addressed.

Methods:
HD10 was an international prospectively randomized multicenter trial comparing 2 and 4 cycles of ABVD as well as 20Gy or 30Gy involved field radiotherapy (IFRT) in a 2 × 2 statistical design. Between 5/1998 and 1/2003, a total of 1370 patients from 329 centers were randomized into four arms: 4 × ABVD + 30Gy; 4 × ABVD + 20Gy; 2 × ABVD + 30Gy; 2 × ABVD + 20Gy. All patients had their initial histology reviewed by a lymphoma expert panel. Documentation was complete in more than 99,1% of cases for this final analysis.

Results:
Patients were equally balanced for age, gender, stage, histology, performance status and risk factors between arms. There were significant differences in major toxicity (WHO grade III/IV) between 4 × ABVD and 2 × ABVD in the overall number of events (52% vs 33%) including leukopenia (24% vs 15%) and hair loss (28% vs 15%). In terms of radiation dose, there also was a difference in toxicity between 30Gy and 20Gy IFRT (all events: 8.7% vs 2.9%), dysphagia (3% vs 2%), mucositis (3.4% vs 0.7%). Complete remission was achieved in 97% of patients treated with 4 × ABVD, 97% with 2 × ABVD, 99% after 30Gy and 97% after 20Gy. With a median follow-up of 79–91 months, there was no significant difference between 4 × ABVD and 2 × ABVD in terms of overall survival at 5 years (OS: 4 × ABVD 97.1%; 2 × ABVD: 96.6%), freedom from treatment failure (FFTF: 93.0% vs 91.1%) and progression free survival (PFS: 93.5% vs 91.2%). For the radiotherapy question, there were also no significant differences between patients receiving 30Gy IFRT and those with 20Gy IFRT in terms of OS (97.6% vs 97.5%), FFTF (93.4% vs 92.9%) and PFS (93.7% vs 93.2%), respectively. Importantly, there was also no significant difference in terms of OS, FFTF and PFS when all four arms were compared.

Conclusion:
Two cycles of ABVD followed by 20Gy IFRT is the new GHSG standard of care for Hodgkin patients in early favourable stages.

Disclosures:
No relevant conflicts of interest to declare.
The MAPK ERK1 Is a Sensor of the Steady-State Splenic Erythropoiesis.
Authors: Guihard Soizic , Clay Denis , Cocault Laurence , Opolon Paule , Souyri Michele , Porteu Francoise , Gaudry Murielle 
Published: October 2019

Abstract
Abstract 73
In different culture models, conflicting results have been obtained with respect to the role of the ERK/MAPK pathway and the ERK kinases on erythropoiesis. There is no in vivo experimental data on the role of these kinases in adult erythropoeisis. The existence of two ERK isoforms (ERK1 and ERK2) suggests that they could play specific role, based on their expression, their activation level and/or the ratio between both of them. The ERK1−/− mice were used to study this hypothesis. Increased number of circulating erythrocytes, increased hemoglobin level and hematocrit were found in these mice. The deletion of ERK1 leads to an uncontrolled splenic erythropoiesis while the bone marrow erythropoiesis remains normal. The ERK1−/− mice display splenomegaly characterized by a marked expansion of the red pulp and an increased number in basophilic (Ery.A) and late basophilic (Ery.B) erythroblasts. This impaired erythropoiesis in ERK1−/− mice is cell autonomous as shown by bone marrow transplantation experiments. This splenic erythropoiesis is not due to an overexpression or overactivation of the ERK2 isoform in erythroblasts. It has been shown that Fas-mediated apoptosis of erythroblasts would limit the basal erythropoietic rate. In ERK1−/− mice, Ery.A expansion is associated with a decrease in cell surface expression of both Fas and FasL as compared with wild-type mice. This fall in Fas/FasL expression is correlated with a decrease in Annexin V binding on splenic Ery.A and Ery.B. In addition, cell cycle analysis revealed an increased S-phase in ERK1−/− Ery.A cells compared with wild-type Ery.A. In conclusion, these data demonstrate for the first time the in vivo involvement of the ERK/MAPK pathway in adult splenic erythropoiesis and underlies the specific role of ERK1 in this function. By regulating the cell surface expression of Fas and FasL on splenic erythroblasts, ERK1 acts as a sensor of the basal erythropoietic rate.

Disclosures:
No relevant conflicts of interest to declare.
Dysregulation of TGF-Beta Stimulated Smad Signaling Is Seen in Myelodysplasia and Points to the Potential Therapeutic Efficacy of TGF-Beta Receptor I Kinase Inhibition in Low Grade Disease.
Authors: Zhou Li , McMahon Christine , Bhagat Tushar , Alencar Christina , Yu Yiting , Fazzari M. , Sohal Davendra P. , Gundabolu Krishna , Ng Chun , Mo Yongkai , Wickrema Amittha , Kong G. , Friedman Ellen , Sokol Lubomir , Burnette Pearlie K , Steidl Ulrich , Yan L. , Yingling Jm , Lahn MM , List Alan F , Bitzer Markus , Verma Amit 
Published: October 2019

Abstract
Abstract 737
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-beta is a hematopoietic inhibitory cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS and acute leukemias. We have shown (Blood, 112(8):3434; 2008) that smad2, a component of the TGF-beta signaling pathway, is constitutively activated and upregulated in MDS progenitors. Since there is conflicting data about upregulation of TGF-beta levels in MDS, we next sought to determine the molecular basis of TGF-beta receptor-I (TBRI) overactivation and subsequent smad2 phosphorylation / activation in this disease. We observed that smad-7, a negative regulator of TBRI kinase, is markedly down regulated in a meta-analysis of gene expression studies from 89 MDS bone marrow derived CD34+ cells when compared to 61 normal controls (Adjusted P<0.001, Benjamini Hochberg). Smad-7 protein was also found to be significantly decreased in MDS BM progenitors (n=14) when compared to anemic controls (n=18) when examined immunohistochemically in a bone marrow tissue microarray (P=0.04, T test). siRNA mediated knockdown of smad-7 levels led to increased TGF mediated gene transcription over baseline, that could be effectively inhibited by a TBRI (ALK5 kinase) inhibitor. TBRI kinase inhibition effectively abrogated TGF-beta mediated activation of smad-2 in hematopoietic cells. Most importantly, treatment with the TBRI inhibitor increased erythroid and myeloid colony formation from primary MDS bone marrow specimens in a concentration-dependent fashion. Furthermore, the hematopoietic potentiating effect of the TBRI kinase inhibition was validated in a TGF-overexpressing transgenic mouse model. This mouse constitutively expresses TGF-beta through an albumin promoter, develops progressive anemia and dysplasia and thus serves as a novel model of human bone marrow failure. Treatment with the TBRI inhibitor by oral lavage at 100 mg/kg/d for two weeks led to significant increases in hematocrit when compared to placebo (Mean increase in hematocrit=48%, P value=0.01, T test). Taken together, these studies demonstrate an important role for reduction in smad-7 in contributing to ineffective hematopoiesis in MDS by activating TGF-beta signaling in hematopoietic cells. These studies also illustrate the promising therapeutic potential of TBRI inhibitors in this disease.

Disclosures:
No relevant conflicts of interest to declare.
Multiple Myeloma Cell Induction of GFI-1 in Stromal Cells Suppresses Osteoblast Differentiation in Patients with Myeloma.
Authors: D'Souza Sonia , Del Prete Davide , Esteve Flavia , Sammut Benedicte , Yu Shibing , Xiao Guozhi , Galson Deborah , Roodman G. David 
Published: October 2019

Abstract
Abstract 742
Multiple Myeloma (MM) is a plasma cell malignancy characterized by formation of lytic bone lesions in approximately 90% of the patients which do not heal even after prolonged complete remission. The basis for this selective and protracted suppression of osteoblast (OBL) differentiation from immature bone marrow stromal cells (MSC) is unknown. Although factors that inhibit OBL differentiation in MM have been identified, such as DKK-1, sFRP2, IL-3, IL-7, and TNF-a, none of these factors have been shown to be responsible for the protracted suppression of OBL differentiation in MM. Further, inhibition of Runx2 activity, a critical transcription factor required for OBL differentiation has been reported in MM, but the mechanisms responsible are still unclear. To address the basis for the protracted inhibition of OBL differentiation in MM, we have developed a murine model of MM-induced OBL suppression using a genetically modified murine myeloma cell line that expresses GFP and thymidine kinase (5TGM1-GFP-TK MM cells). Injection of these 5TGM1-GFP-TK MM cells into SCID mice resulted in persistent inhibition of OBL differentiation even when the MM cells were totally depleted by ganciclovir treatment. The MSC from these mice had selective inhibition of OBL differentiation, but not adipogenesis, and minimally differentiated to OBL even when treated with BMP2. These MSC expressed elevated levels of the SNAG family Zn-finger containing transcriptional repressor, Gfi-1, which we found can cause both acute and protracted suppression of RUNX2. In support of these results, decreased RUNX2 expression and elevated GFI-1 levels were also protracted in MSC from 7 MM patients with impaired OBL differentiation compared to normals. Further, 5TGM1 inhibition of OBL differentiation in vitro was dependent on TNF-a and IL-7, and neutralizing antibodies to TNF-a and IL-7 blocked MM-induced Runx2 suppression. In addition, TNF-a and IL-7 increased Gfi-1 in a murine OBL precursor cell line (MC4). Deletion analysis of the Runx2 P1 promoter revealed that a 943-bp region containing 27 putative Gfi-1 binding sites (AA(T/G)C core) was responsible for MM repression of Runx2 expression. Importantly, siRNA knockdown of GFI-1 expression restored RUNX2, OCN, BSP and OSX expression in both MM exposed MC4 cells and in MSC from MM patients. These results support an important role for GFI-1 in repressing RUNX2 expression in MSC exposed to MM cells, thereby inhibiting osteoblastogenesis in MM.

Disclosures:
Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.
Follicular Lymphoma Cell Niche: Identification of a Preeminent IL-4-Dependent TFH- B Cell Axis.
Authors: Tarte Karin , Celine Pangault , Ame-Thomas Patricia , Ruminy Philippe , Rossille Delphine , Caron Gersende , Bahia Maryse , De Vos John , Roussel Mikael , Monvoisin Celine , Lamy Thierry , Gaulard Philippe , Tilly Herve , Fest Thierry 
Published: October 2019

Abstract
Abstract 757
Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained on whole biopsy samples, have been associated with patient survival, independently of classical clinical features. In this study we performed, using Affymetrix U133 Plus 2.0 oligonucleotide microarrays, the gene expression profiling of purified CD19pos B-cell and CD19negCD22neg non-B cell compartments, prospectively obtained from FL and reactive lymph nodes. Unsupervised analyses of B-cell compartment in one hand (n=21) and non-B cell compartment in the other hand (n=14) allowed to differentiate FL from reactive samples. We then identified 677 nonredundant genes defining the FL synapse, i.e. the list of genes involved in the crosstalk between B cells and their microenvironment in FL. Using Ingenuity pathway analysis we pointed out 26 FL-specific functional networks among this FL synapse, including an IL-4-centered pathway. Interestingly, whereas in tonsils and reactive lymph nodes only exceptional scattered phospho-STAT6pos cells were observed, a high number of CD20posphospho-STAT6pos cells were evidenced within FL biopsies. In addition, several IL-4-target genes, including IL4I1 and HOXC4, were overexpressed in malignant B cells. Altogether, these results demonstrated that the upregulation of IL-4 within FL microenvironment was associated to a strong activation of FL B cells. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (TFH), as demonstrated through both Gene Set Enrichment Analysis (GSEA)-based transcriptomic approach and flow cytometry analysis of the CD4posCXCR5hiICOShi cell compartment. The majority of phospho-STAT6pos B cells were located at the vicinity of cells expressing the PD-1 TFH marker. Moreover, purified FL-derived TFH, unlike non-TFH T cells, expressed IL4. Finally, whereas chronically inflamed tonsils also exhibited an increase percentage of TFH cells, tonsil-derived TFH did not express IL4. Altogether, our study demonstrated that tumor-infiltrating TFH specifically express functional IL-4 in FL, creating therefore an IL-4-dependent TFH-B cell axis. This crosstalk could sustain FL pathogenesis and represent a new potential therapeutic target.

Disclosures:
No relevant conflicts of interest to declare.
Filamin A Deficiency in Platelets Reveals Functional Impairment in ITAM-Based Signaling.
Authors: Falet Hervé , Pollitt Alice Y , Weber Sarah E , Duerschmied Daniel , Wagner Denisa D , Watson Steve P , Hartwig John H 
Published: October 2019

Abstract
Abstract 769
Filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, crosslinks F-actin and controls the topology of the Von Willebrand factor (VWF) receptor and the integrin αIIbβ3 on platelets by connecting them to the underlying cytoskeleton. In mice, FlnA deficiency is embryonic lethal to hemizygous males. Heterozygous (HT) females are born, but experience premature mortality. FlnA confers survival value to platelets since HT female mice have a mild thrombocytopenia and 95% of their circulating platelets are FlnA positive. Mice specifically lacking FlnA expression in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a severe macrothrombocytopenia and increased tail bleeding time. Their platelets lack a normal cytoskeleton and have decreased GPIbα expression, which lacks cytoskeletal linkage and has an altered surface distribution by electron microscopy. Binding of VWF to FlnA-null platelets was decreased in response to botrocetin. Under arterial shear conditions (shear rate 1,500/s), fluorescent FlnA-null platelets covered ∼70% less of the collagen-coated area via collagen-bound VWF than WT platelets. Unexpectedly, immunoreceptor tyrosine-based activation motif (ITAM)-mediated signals were severely compromised in the absence of FlnA. FlnA-null platelets had decreased α-granule secretion and integrin αIIbβ3 activation in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2 (CLEC-2). Protein tyrosine phosphorylation, including that of the protein tyrosine kinase Syk and phospholipase C-γ2, was severely impaired in FlnA-null platelets stimulated through GPVI and CLEC-2. Interaction between FlnA and Syk was detected using immunoprecipitation and purified proteins. The data reveal a novel interaction between FlnA and Syk regulating ITAM-containing receptor signaling and platelet function.

Disclosures:
No relevant conflicts of interest to declare.