bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunosuppressive FVIII-Specific Human Cartregs in Hemophilia a
Authors: Yoon Jeong Heon , Schmidt Anja , Kim Yong Chan , Koenigs Christoph , Scott David William 
Published: October 2019

Abstract
Hemophilia A is an X-linked disorder, in which mutations in the coagulation Factor VIII (FVIII) gene lead to a loss of FVIII function and serious bleeding episodes. These episodes can be treated with recombinant FVIII protein replacement. Unfortunately, ~25% of hemophilia A patients produce inhibitory anti-FVIII antibodies because of lack of tolerance. Thus, it is necessary to develop effective tolerogenic therapies to prevent, as well as reverse, inhibitor formation. Previously, we generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T cell clone. The resulting engineered T cells bind MHC tetramers, proliferate in response to a specific FVIII epitope, and suppress effector responses to FVIII.
In this study, we engineered a FVIII-specific chimeric antigen receptor (ANS8CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Following initial experiments in naïve CD4 T cells, this CAR was introduced into human Tregs. Western blot and specific staining with FVIII verified CAR expression. Transduced ANS8CAR Tregs proliferated in response to FVIII and were able to suppress the proliferation of FVIII-specific T effector cells in vitro. Additionally, the proliferation of T effector cells with different FVIII domain specificity was suppressed as well when ANS8CAR-transduced Tregs were activated with FVIII. Thus, engineered cells are able to promote bystander suppression. Cytokine expression of ANS8CAR-transduced Tregs was comparable to expression of untransduced and TCR-transduced Tregs indicating that the regulatory phenotype of Tregs was not negatively influenced by ANS8CAR expression.
In conclusion, CAR-transduced Tregs seem to be a promising alternative to TCR-transduced Tregs for a future tolerogenic treatment of hemophilia A patients with inhibitory FVIII-specific antibodies.
Supported by NIH grants HL061883 and HL126727 (DWS), the Society of Thrombosis and Hemostasis Research, and the Günter Landbeck Excellence Award (AS).

Disclosures
Kim: Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment
Authors: ten Hacken Elisa , Oellerich Thomas , Gounari Maria , Hoellenriegel Julia , Pan Kuan-Ting , O'Brien Susan , Wierda William , Ferrajoli Alessandra , Estrov Zeev , Keating Michael J. , Urlaub Henning , Ghia Paolo , Burger Jan A. 
Published: October 2019

Abstract
Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined.
Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells.
Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands.
Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases.
Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment.

Disclosures
Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.
BAR-CD8 T-Cell Mediated Targeted Killing of Inhibitor Producing FVIII-Specific B Cells
Authors: Parvathaneni Kalpana , Zhang Aihong , Kim Yong Chan , Scott David William 
Published: October 2019

Abstract
Anti-FVIII antibody (inhibitor) formation is a major complication in the treatment of Hemophilia A. Our lab has focused on developing novel approaches to modulate inhibitor formation by regulation of FVIII-specific T effectors that recognize FVIII and "help" B cells to produce antibodies to FVIII. As an alternative approach, in this presentation, we directly target anti-FVIII B cells. Therefore, we have developed a novel chimeric antigen-like receptor called B-cell antibody recognizing receptor (BAR) to target and directly kill the FVIII-specific B cells and antiobody-producing cells . Our BAR comprises of FVIII-domain C2 attached to T cell signaling CD28 and CD3ζ elements. We reasoned that CD8 T cells transduced to express the FVIII C2 should trigger cytotoxicity upon recognition by and interaction with specific B cells. We expressed the C2-BAR or Ova-BAR (control) in human T cells and demonstrated expression by flow cytometry. These BAR CD8 T cells also proliferated upon addition of anti-C2 or anti-Ova, respectively post 72 hrs. For proof of principle that these BAR-expressing CD8 T cells can kill specific B cells, the following basic experiment was designed. Mouse CD8 T cells transduced to express C2-BAR were co-cultured with FVIII-specific hybridomas (expressing surface IgG anti-C2) at a various ratios. After 5 hours, we measured antibody secretion by ELIspot assay. Our results demonstrated suppression of antibody secretion by C2 BAR expressing CD8 T cells, but not by control Ova-expressing BAR CD8s. These results provide evidence that BAR CD8 T cells expressing FVIII domains can be used to target specific B cells to suppress inhibitor formation, and may also provide a platform to treat other undesirable antibody responses. (Supported in part by a Pfizer ASPIRE award and NIH grant HL061883)

Disclosures
Zhang: Henry Jacksopn Foundation: Other: patent filed. Kim:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
UCHL1 Is a Biomarker of Aggressive Multiple Myeloma Required for Disease Progression
Authors: Hussain Sajjad , Bedekovics Tibor , Chesi Marta , Bergsagel Leif , Galardy Paul J. 
Published: October 2019

Abstract
The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. In an unbiased screen of de-ubiquitinase activity, we previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma. Using a transgenic mouse model, we also found that aberrant expression of UCH-L1 is sufficient to drive the development of spontaneous B-cell lymphomas and plasmacytomas, demonstrating its oncogenic activity. Whether and how UCH-L1 affects the clinical behavior of myeloma, however, is not known. Here we show that UCH-L1 is a potent negative prognostic factor that is essential for the dissemination and progression of myeloma. We found high levels of UCHL1 to predict early progression in newly diagnosed patients; a finding reversed in studies including bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease.

Disclosures
Galardy: Mission Therapeutics: Research Funding.
Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals That the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance
Authors: Driscoll James J. , Malek Ehsan 
Published: October 2019

Abstract
The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance.

Disclosures
No relevant conflicts of interest to declare.
Prenatal Allospecific Tolerance Is Characterized By Early Treg Expansion and a Surprisingly Slow Pace for Teff Clonal Deletion
Authors: Strong Beverly SI , Lee Amanda E , Newkold Tess J , Turner Lucas E , Shaaban Aimen F 
Published: October 2019

Abstract
Prenatal transplantation capitalizes on the unique fetal environment, allowing for life-long engraftment of allogeneic stem cells without the need for harsh conditioning regimens. A prerequisite for stable engraftment of allogeneic cells likely requires the negative selection of donor-specific host effector T cells (Teff) and the support of donor-specific regulatory T cells (Tregs). However, little is known about the interplay between these cell types during development. The purpose of this study was to characterize the dynamic relationship between donor-specific Teff and Tregs as they emerge during development.
Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature T cells from B6 mice expressing TCRv-beta-5, 11, and 12 are negatively selected by mtv-8 superantigen complexed with I-E class MHC II on Balb/c cells. As alpha/beta TCR rearrangement does not occur until E16, this established transplantation model allows for alloantigen to be present from the earliest stages of thymic selection. Kinetic analysis of donor-specific T cell populations was performed in peripheral blood paired with in depth analysis in thymus and spleen in control and chimeric mice.
Negative selection of donor-specific Teff cells occurs at an unexpectedly slow pace in Balb/c to B6 prenatal chimeras. Donor-specific CD4 and CD8 Teff are significantly decreased in frequency at 4 weeks of age but do not reach maximal deletion until 12 weeks of age (TCRv-beta-5 data shown in Figure A). Further analysis demonstrated that this slow elimination of donor-specific Teff was paired with an early increase in the frequency of donor-specific Tregs at 4 weeks of age (TCRv-beta-5 data shown in Figure B.) This increase in donor-specific Tregs likely occurred as a result of peripheral expansion as there was no change in the frequency of donor-specific Tregs in the thymus (Figure B) and no change in the frequency of these cells that expressed the markers of thymically derived natural Tregs neuropilin-1 or helios (data not shown). In agreement with this hypothesis, donor-specific splenic Tregs incorporated BrdU at a higher rate than other Tregs in young mice indicating a potential expansion of donor-specific Tregs in the periphery (TCRv-beta-5 data shown in Figure C.)
Collectively, these data demonstrate that prenatal transplantation is characterized by: 1) a surprisingly slow reduction of donor-specific Teff subsets; 2) an early expansion of donor-specific Tregs. Further studies will explore the role of donor-specific Tregs in controlling early immunity to prenatally encountered antigens.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
RIG-I-Induced Type I IFNs Promote Regeneration of the Intestinal Stem Cell Compartment during Acute Tissue Damage
Authors: Fischer Julius C , Bscheider Michael , Eisenkolb Gabriel , Wintges Alexander , Lindemans Caroline A , Heidegger Simon , Monette Sebastien , Calafiore Marco , Porosnicu Rodriguez Kori A. , Lieberman Sophie , Liu Chen , Peschel Christian , Docampo Melissa , Velardi Enrico , Jenq Robert R. , Hanash Alan M , Dudakov Jarrod A , Haas Tobias , van den Brink Marcel , Poeck Hendrik 
Published: October 2019

Abstract
Introduction: Although the role of Type I IFNs in initiating host defense against pathogens is well established, recent work highlights the regenerative function of this cytokine family. Yet, despite its involvement in tissue repair, the cellular targets and mechanisms of action by which Type I IFNs act during tissue regeneration are poorly understood.
Results: Here, we describe a hitherto unrecognized regenerative function for the RIG-I/MAVS/IFN-I pathway through direct effects on epithelial regeneration. Mice deficient in the RIG-I adaptor MAVS were more sensitive to intestinal barrier damage after total body irradiation (TBI) and, like RIG-I deficient mice, developed worse graft-versus-host disease (GVHD) in a preclinical model for allogeneic hematopoietic stem cell transplantation (allo-HSCT). This phenotype was not associated with changes in the intestinal microbiota, but with a defect in epithelial regeneration. Moreover, in contrast to previous reports in steady-state conditions and after viral challenge, we found that interferon-α/β receptor (IFNAR) signaling in non-hematopoietic epithelial cells was crucial for tissue regeneration after acute damage. Importantly, we could demonstrate that this pathway could be therapeutically targeted with RIG-I agonists, which promoted barrier integrity and prevented GVHD. Mechanistically, Type I IFNs (either RIG-I-induced or recombinant) could promote intestinal stem cell (ISC) growth in crypt organoid cultures, suggesting that stimulation of the ISC compartment led to epithelial regeneration.
Conclusion: Our findings suggest that activation of RIG-I and IFN-I can promote regeneration of intestinal epithelial cells and thus offers an innovative therapeutic strategy for the management of acute intestinal injury.

Disclosures
van den Brink: Merck: Honoraria; Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee.
Outcomes of Grades II-IV Acute Graft-Versus-Host Disease Post-Allogeneic Hematopoietic Stem Cell Transplantation: How Much Progress Was Achieved?
Authors: Khoury Hanna Jean , Wang Tao , Arora Mukta , Hemmer Michael , Spellman Stephen R. , Cutler Corey S , Couriel Daniel R. , Alousi Amin M. , Joseph Joseph A. 
Published: October 2019

Abstract
Despite recent reduction in mortality following hematopoietic cell transplant (HCT), graft versus host disease (GVHD) remains a major causes of failure. It is unclear if advances in antiviral, antifungal and antibacterial prophylaxis/treatment, and immunosuppression, have led to better survivals following the onset of acute GVHD (aGVHD). We therefore analyzed within the CIBMTR database outcomes over time of allogeneic HCT recipients who developed grade II-IV aGVHD. The objectives were to determine the change in overall survival (OS), disease-free survival (DFS), treatment-related mortality (TRM) and to identify risk factors.
Methods: Between 1999 and 2012, 7295 patients with AML (57%), ALL (29%) or MDS (14%) received sibling (35%) or unrelated donor (65%) blood (65%) or marrow (35%) HCT (1216 for 1999-2001, 2329 for 2002-2005, 3750 for 2006-2010), and 3312 (45%) developed grades II-IV aGVHD. Median follow-up was 141 (3-174), 96 (3-144) and 49 (3-95) months for 1999-2001, 2002-2005, and 2006-2010 periods respectively. Multivariate models were built using the Cox's proportional hazards model. Clinical variables were tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption were adjusted through stratification. Stepwise model building procedure was used in developing models for each outcome with a threshold of 0.05 for both entry and stay in the model. Interactions between the main variable, (i.e. year of transplant) and the adjusted covariates were tested.
Results: Univariate analyses showed a decrease in the incidence of grades III-IV aGVHD: 56 % (52-60), 47 %(44-50), and 38% (36-40) for 1999-2001, 2002-2005, and 2006-2010, respectively (p= <0.001). Multivariate analyses showed that age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), disease status at transplant and KPS influenced OS; while age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), CMV serostatus matching, disease status at transplant, unrelated donor age and KPS affected OS and TRM. After adjusting for these factors, recipients of tacrolimus-based GVHD prophylaxis who developed grade II aGVHD had better OS after 2006 (HR 0.78, p=0.02), while a trend for lower TRM (HR 0.79, p=0.056) and better OS (HR 0.83, p=0.08) were observed for those with grades III-IV aGVHD.
Conclusions: Incidence of grades III-IV aGVHD has overall decreased and OS and TRM have improved over time for recipients of tacrolimus-based GVHD prophylaxis who developed grades II-IV aGVHD.

Disclosures
Alousi: Therakos, Inc: Research Funding.
The Long Non-Coding RNA NEAT1 Modulates Imatinib-Induced Apoptosis in CML Cells
Authors: Zeng Chengwu , Liu Sichu , Yang Lijian , Chen Shaohua , Li Yangqiu 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a clonal disease characterizedby the presence of a constitutively active tyrosine kinase BCR-ABL oncoprotein. Although BCR-ABL is crucially important for pathogenesis and responsiveness to treatment, it is thought that some additional factors might involve in regulation in this process. For example, aberrant expression of Long noncoding RNAs (lncRNAs) have recently been identified to be involved in various diseases including cancer, suggesting that lncRNAs may play a role in BCR-ABL-mediated CML. In order to characterize the effect of lncRNAs in CML, we firstly performed lncRNA expression profiling microarray in newly diagnosed CML patients and healthy individuals¡¯ samples. We found that nuclear enriched abundant transcript1 (NEAT1), an lncRNA essential for the formation of nuclear body paraspeckles, is significantly repressed in in CML patients samplescompared with those of healthy donors (figure 1A). We found that NEAT1 expression was repressed by BCR-ABL, and the expression of NEAT1 could be restored after treatment with imatinib and BCR-ABL mediated pathway inhibitor or knockdown BCR-ABL in K562 cells (figure 1B). Moreover, knockdown of NEAT1 combining with imatinib treatment, could promote apoptosis of K562 cells (figure 1C). Importantly, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for NEAT1-mediated apoptosis of K562 cells. Furthermore, NEAT1 was shown to be regulated by c-Myc in K562 cells (figure 1D). Taken together, these results are the first to assign a biological function to the nuclear long noncoding RNANEAT1 inapoptosis CML cells and may lead to a fuller understanding of the molecular events leading to CML.
Figure 1. Figure 1.

Disclosures
Zeng: National Science Foundation of China (No. 81400102) and China Postdoctoral Science FoundationiNo. 2015M570751): Research Funding. Li:National Science Foundation of China (No. U1301226): Research Funding.