bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allogeneic Virus-Specific T Cells with HLA Alloreactivity Do Not Produce Graft-Versus-Host Disease In Human Subjects
Authors: Melenhorst J. Joseph , Leen Ann M. , Bollard Catherine M. , Quigley Máire F , Price David A , Rooney Cliona M , Brenner Malcolm K. , Barrett A. John , Heslop Helen E. 
Published: October 2019

Abstract
Abstract 1252
We and others have recently established that T cell reactivity with non-self HLA (HLA alloreactivity) arises not only from naive T cells but also from the antigen-experienced T cell pool, including Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific T cells. Virus-specific T cells could therefore mediate graft-versus-host disease (GvHD) if infused into partially HLA mismatched recipients. We reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplant donor-derived virus-specific T cell lines in 153 recipients who received donor-derived EBV-specific CTLs (N=114), bivirus CTLs specific for adenovirus and EBV (n=14), and trivirus CTLs specific for CMV, adenovirus and EBV (n=25). Seventy three donor-recipient pairs were partially HLA-mismatched, with the degree of HLA mismatching varying from one allele to a full haplotype. De novo GvHD did not develop after infusion of cytotoxic T lymphocytes (CTL), and the incidence of GvHD reactivation was 6.5% and not significantly different between recipients of HLA matched or mismatched CTL. Thus, virus-specific CTL did not mediate GvHD, even in recipients of partially matched CTL. Next we analyzed the HLA alloreactivity of four donor-infused bivirus-specific T cell lines, using activated T cells, that are known to lack CMV and EBV antigen expression, as antigen presenting cells (TAPC). We used a panel of 44 TAPC covering the most frequent HLA class I and II alleles. The CTL lines were labeled with CFSE and stimulated with TAPC for 6 hours, after which production of TNFα and IFNγ/IL-2 by CD4+ and CD8+ T cells in the CFSE-positive fraction was analyzed by flow cytometry. All CTLs responded to a number of TAPC, with some APC being recognized strongly. The majority elicited only weak or no response from the CTLs. We then assessed whether the CTLs recognized TAPC expressing the recipient's HLA alleles. We found moderate reactivity of the CTL with 1–5 TAPC expressing recipient HLA alleles. Taken together, our data indicate that reactivity of virus-specific CTLs with hematopoietic APC does not correlate with the risk of developing GvHD, and that virus-specific CTL can safely be infused into HLA class I and/or II mismatched recipients.

Disclosures:
No relevant conflicts of interest to declare.
Immune Phenotyping and Naive T Cells as a Predictor of Response to Therapy In Chronic Lymphocytic Leukemia (CLL)
Authors: Zent Clive S. , Abraham Roshini , Lin Yi , Gustafson Michael , Wu Wenting , Dietz Allan 
Published: October 2019

Abstract
Abstract 1362
The role of anti-tumor immunity is not well understood in CLL but does have the potential to be manipulated to improve treatment responses. We studied the predictive values of 43 immune phenotypes in patients enrolled in a clinical trial of early treatment of high risk CLL. The study enrolled 33 patients with CLL diagnosed using standard NCI-WG96 criteria who had previously untreated early - intermediate stage disease and did not meet NCI-WG96 guidelines for conventional treatment. High risk disease was defined as at least one of the following: 17p13 deletion; 11q22 deletion or unmutated IGHV together with either expression of ZAP-70 (>20%) or CD38 (>30%). Patients were treated with GM-CSF 250 mcg sc 3 × week × 5 weeks, alemtuzumab sc with dose escalation over 3 days then 30 mg/d 3 × week × 4 weeks, and rituximab 375 mg/m2 weekly × 4 weeks for a total duration of therapy of 5 weeks. We measured immune phenotypes (representing Tregs, naïve, effector memory, central memory T cells, B cells, NK cells, monocytes, among others) at the time of enrollment, through treatment and at 1 year after enrollment in 28 patients. We identified pre-treatment immune phenotypes that correlated with disease burden (as measured by the number of circulating B cells). Increased disease burden was correlated with increased absolute numbers of CD3+, CD8+, CD4+ T cells, NK (CD56+CD16+), Dr+CD8+ T cells, and Tregs (CD4+CD25+CD127+); p<0.001. The disease burden was inversely related to the number of circulating monocytes (p<0.01) and unrelated to the circulating number of naïve T cells. We repeated the pre-treatment phenotype correlation studies using the CD19+ cell counts 12 months after treatment for 13 patients who have completed the final analysis. Interestingly, the number of naïve CD4 and CD8 cells (CD4+RA+62L+CD27+ and CD8+RA+62L+CD27+) of the patient prior to treatment correlated with CD19+ cells 12 months after treatment (p=0.024 and p=0.002 respectively; n=13). Finally, we correlated pre-treatment immune phenotypes with time to progression. Again, an elevated T cell phenotype (CD4+RA+62L+CD27- as a percent of CD4+ cells) correlated to increased time to progression (p=0.04; n=28). It is noteworthy that the naïve T cell phenotype did not correlate with tumor burden prior to therapy. Thus, while pre-treatment lymphocyte expansion is commensurate with tumor burden in CLL, the frequency and numbers of naïve T cells prior to treatment has some predictive value to response to treatment. These data suggest that CLL induces the expansion of lymphocytes, suppresses monocytes with an induction in naïve T cells acting as an indicator of higher B cell counts at 12 months in patients with durable remissions.

Disclosures:
Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.
Dependency of CLL Cell Survival on Direct Physical Interaction with Bone Marrow Stromal Cells In Vitro Is Largely Outweighed by Secretion of Soluble Factors with Time In Culture
Authors: Gehrke Iris , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl-Anton 
Published: October 2019

Abstract
Abstract 1365
The major pathophysiology feature of chronic lymphocytic leukemia (CLL) cells is their extended life span due to a pronounced resistance towards apoptotic stimuli in vivo. Despite this, CLL cells die within a few days when isolated from their natural microenvironment and are placed under cell culture conditions. That is why the bone marrow microenvironment has been ascribed an essential role in maintenance of the apoptotic resistance of the CLL cell. Thereby both, the physical interaction between bone marrow stromal cells and CLL cells and the secretion of soluble factors have been described to be essentially involved. We analysed the survival capacity of CLL cells in monoculture and in coculture with the bone marrow-derived stromal cell line HS5 with and without physical separation using transwells for up to 7 days by flow cytometric determination of Annexin-V/PI status. As expected, in vitro CLL cell survival was significantly reduced when physical contact between CLL cells and bone marrow stromal cells was prevented. Interestingly, this was only the case for short term cultivation for up to three days. With time under culture conditions CLL cell survival became less dependent on direct physical contact with the HS5 feederlayer, suggesting the secretion of soluble factors to compensate for the loss of pro-survival signals obtained from direct cell-cell interactions over time. This was further supported by the fact of reduced survival support for CLL cells when HS5 proliferation, hence production and secretion of soluble factors, was prevented by mitomycin treatment or formaldehyde fixation. The use of an expanded human Cytokine Antibody Array (Affymetrix), which analyses the presence of the most common 36 cytokine proteins, might offer information about the composition of soluble factors present in the supernatant which are essential for CLL cell survival in vitro. In conclusion, while direct cell-cell contact between CLL cells and bone marrow stromal cells provides an immediate protection against in vitro apoptosis of CLL cells, the secretion of soluble factors, most likely by both, CLL and bone marrow stormal cells, leads to the creation of an in vitro environment which can to a certain extent compensate for the loss of prosurvival signals obtained by direct physical interactions.

Disclosures:
No relevant conflicts of interest to declare.
Investigation of the Morphology of Recombinant Von Willebrand Factor by Two High-Resolution Microscopic Techniques
Authors: Rottensteiner Hanspeter , Seyfried Birgit K , Friedbacher Gernot , Allmaier Guenter , Ilk Nicola , Sleytr Uwe B , Ehrlich Hartmut J. , Turecek Peter L 
Published: October 2019

Abstract
Abstract 1409
Von Willebrand factor (VWF) is the largest known glycoprotein circulating in human blood plasma and is composed of a series of multimers with molecular weights ranging from 600 to 20,000 kDa or even more. In this study, we investigated the morphology of recombinant VWF (rVWF) and compared it to that of plasma-derived VWF (pdVWF) by tapping mode atomic force microscopy and transmission electron microscopy.
Tapping mode atomic force microscopy showed that both rVWF and pdVWF contain globular and stretched domains. Mean chain lengths of the filaments and diameters of the core globular domains were determined and analyzed on a statistical basis. About 70% of the rVWF and pdVWF molecules were 100–300 nm long. The portion of very long molecules (>300 nm) was only slightly greater in rVWF than in pdVWF (20% versus 18%). The diameters of the globular core structures were in the range of 12 to 30 nm for both types of VWF. Inspection of a purified rVWF dimer revealed a similar range for the globular domain (14-32 nm). Upon exposure to high shear stress, a dramatic conformational change was observed for rVWF, just as has been reported for pdVWF.
Investigation of negatively stained preparations of rVWF by transmission electron microscopy showed a highly organized structure. The heterogeneity in size observed was as expected for a polymeric VWF that consists of a mixture of multimers of varying sizes. The micrographs also revealed the presence of coiled and elongated structures. Analysis of pdVWF led to similar results, which were also in agreement with data from the literature.
The results confirmed that the morphology of rVWF is similar to that observed for VWF purified from human normal plasma.

Disclosures:
Rottensteiner: Baxter Innovations GmbH: Employment. Seyfried:Baxter Innovations GmbH: Employment. Friedbacher:Vienna University of Technology: Consultancy. Allmaier:Vienna University of Technology: Consultancy. Ilk:University of Natural Resources and Applied Life Sciences: Consultancy. Sleytr:University of Natural Resources and Applied Life Sciences: Consultancy. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment.
Recombinant Dimeric IgA Antibodies as Tumor-Specific Agents.
Authors: Dechant Michael , Lohse Stefan , Derer Stefanie , Beyer Thomas , Klausz Katja , Peipp Matthias , Leusen Jeanette H. W. , van de Winkel Jan G. J. , Valerius Thomas 
Published: October 2019

Abstract
Abstract 1488
Dimeric IgA antibodies contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA, exemplarily directed against the epidermal growth factor receptor (EGF-R). Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGF-R specific monomeric IgA with a vector coding for the His-tagged human J-chain. For purification of J-chain-containing dimeric IgA two different affinity (anti-human-kappa and anti-His-tag) and one size exclusion chromatography were combined, resulting in a homogenous preparation of highly pure IgA dimers, as determined by gel electrophoresis under denaturing and native conditions using silver stain, Coomassie blue or Western blots for protein detection, respectively. Functional studies demonstrated dimeric IgA (dIgA) to be at least as effective as monomeric IgA (mIgA) in triggering ADCC of A431 tumor cells by isolated monocytes (EC50 0.35 and 0.30 μg/ml for mIgA and dIgA, 0.23 μg/ml for the corresponding IgG1; maximal killing 51.2 vs 43.3 vs 56.3 %), by isolated PMN (EC50 0.96, 1.20 and 0.8 μg/ml for mIgA, dIgA and IgG1; maximal killing 52.6 vs 50.6 vs 19.9 %) and in human whole blood assays (EC50 0.32 and 0.32 μg/ml for mIgA and dIgA; maximal killing 7.9 vs 12.5 %; IgG1: maximal killing 0.7 %, EC50 not determinable). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells by dimeric IgA was achieved at significantly lower concentrations than by monomeric IgA (EC50 7.1 vs 18.1 μg/ml, respectively). In addition, growth of EGF-R expressing DiFi colon carcinoma cells was inhibited at significantly lower concentrations by dimeric than by monomeric IgA (EC50 1.6 vs 3.7 μg/ml). Both IgA isoforms and their IgG1 variant were similarly effective in triggering apoptosis of DiFi cells, as analyzed by PARP cleavage. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric immunoglobulin receptor (pIgR) through an epithelial cell monolayer, as analyzed by an experimental transcytosis assay with polarized MDCK cells stably transfected with human pIgR. Together, these studies demonstrate that recombinant dimeric IgA antibodies recruit a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies, trigger important biological functions and constitute an interesting antibody format for future tumor therapies.

Disclosures:
van de Winkel: Genmab: Employment, Membership on an entity's Board of Directors or advisory committees. Valerius:Genmab: Consultancy, Research Funding.
Extended Molecular and Clinical Phenotype of Human G6PC3 Deficiency.
Authors: Boztug Kaan , Rosenberg Philip S. , Böhm Marie , Moulton Thomas , Curtin Julie , Rezaei Nima , Corns John , Innis Jeffrey , Avci Zekai , Tran Hung Chi , Pellier Isabelle , Gatti Simona , Fruge Rachel , Parvaneh Nima , Darbyshire Phil , Buchanan George R. , Alter Blanche P , Boxer Larry , Donadieu Jean , Welte Karl , Klein Christoph 
Published: October 2019

Abstract
Abstract 1495
Severe congenital neutropenia (SCN) is a heterogenous group of disorders characterized by an increased susceptibility to bacterial infections. Recently, our discovery of G6PC3 deficiency in 12 patients with SCN and various developmental features highlighted the role of glucose metabolism in the viability of neutrophil granulocytes. To further delineate the molecular and clinical phenotype of this complex syndrome, we analyzed a second cohort of 23 SCN patients referred to us for molecular analysis of G6PC3 mutations. All patients had at least one syndromic feature in addition to congenital neutropenia such as such as congenital heart defects, urogenital malformations or increased venous marking. Among these 23 patients, we identified 14 patients with biallelic mutations in G6PC3. 10 patients had novel mutations in G6PC3. A comprehensive review of the clinical characteristics of these patients underlined the phenotypic variability of G6PC3 deficiency. In addition to known manifestations including cardiac (14/14) and urogenital malformations such as cryptorchidism (3/14), novel features such as facial dysmorphy (11/14) or malformation of the outer genitalia (4/14) were found. No obvious genotype-phenotype correlations could be established. All patients except one had a good response to treatment with G-CSF characterized by increased peripheral neutrophil counts and decreased frequency and severity of infections.
To assess the risk of leukemogenesis, we performed a meta-analysis comparing the 14 G6PC3-deficient SCN patients combined with the 12 patients in the original publication with a cohort of 374 patients SCN patients bearing mutations in ELANE or HAX1, in which 61 developed MDS or AML. The rate of MDS/AML was found to be significantly lower in G6PC3-deficient patients (p=0.02). Our analysis suggests that the risk of transition to MDS/AML may be lower in G6PC3-deficient SCN compared with other genetically defined SCN subgroups.

Disclosures:
No relevant conflicts of interest to declare.
Patterns of HLA-DR Surface Expression on CD14+ Monocytes During Adverse Events After Hematopoietic Stem Cell Transplantation.
Authors: Döring Michaela , Müller Ingo , Erbacher Annika , Handgretinger Rupert , Hofbeck Michael , Kerst Gunter 
Published: October 2019

Abstract
Abstract 1499
The human leukocyte antigen DR (HLA-DR) surface expression on CD14+ monocytes reflects the degree of activation of these cells. In line with the central role of monocytes and macrophages in the immune system, a decreased HLA DR expression on CD14+ monocytes has been shown to be a hallmark of an altered immune status during the systemic inflammatory response syndrome (SIRS). We therefore hypothesized that HLA-DR expression might likewise be altered after hematopoietic stem cell transplantation (HSCT).
HLA-DR surface expression of CD14+ monocytes was assessed by flow cytometry in 30 pediatric patients (below 15 years, n=23) and young adults (15 years and above, n=7) up to one year after HSCT (7 autologous and 23 allogeneic stem cell transplants). Normal values were derived from a control group of healthy children (n=18) and young adults (n=22).
During the conditioning period, a significant increased HLA-DR expression in pediatric patients and young adults was observed. This increase could not be attributed to the administration of anti-thymocyte globulin (ATG). After HSCT, HLA-DR expression was not altered in general. However, HLA-DR expression decreased significantly up to 5 days before (p<0.05) and during (p<0.01) bacterial infections or sepsis. In contrast, HLA-DR expression levels were increased 7–10 days before and at the time of diagnosis of viral infections. HLA-DR expression was also elevated during acute graft-versus-host disease (GVHD). At the time of hepatic veno-occlusive disease (VOD), HLA-DR expression on CD14+ monocytes was reduced. Neither the administration of granulocyte colony-stimulating factor (G-CSF) nor the occurrence of a relapse was associated with a change of HLA-DR expression.
In conclusion, adverse events after HSCT were associated with altered HLA-DR expression levels. Therefore, HLA-DR expression on CD14+ monocytes appears as a promising parameter which might allow identifying patients at risk after HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Increased Prevalence of Neoplasms In Patients Diagnosed with Common Variable Immunodeficiency.
Authors: Grossman William J , Bonnet Patrick O , Xiong Yan 
Published: October 2019

Abstract
Abstract 1531

Introduction:
Common variable immunodeficiency (CVID) is a heterogenous disorder characterized by hypogammaglobulinemia, poor specific antibody responses, and increased numbers of infections. In addition, patients with CVID have been reported to carry an increased risk of developing other co-morbidities, including autoimmunity and certain types of neoplasms. Data establishing the overall risk and prevalence of these co-morbidities in patients with CVID has been limited due to the small sizes of patient cohorts examined to-date, and the lack of comprehensive primary immunodeficiency registries. The objective of this study was to document the prevalence rates of lymphoma select neoplasms in a large cohort of patients with CVID.

Methods:
The MedStat MarketScan database, which contains approximately 48 million patients, was evaluated for the number of IVIG-treated CVID patients using the 279.06 ICD-9 code for CVID over a 6-year period (2002-2008). Ten-fold the number of randomly selected patients, based on age, gender and length of enrollment, were used for matched control comparisons to our CVID cohort. Each cohort was evaluated for the rate of select neoplasms using ICD-9 codes for malignant lymphatic and hematopoietic tissue neoplasms (ICD-9 codes 200 to 208).

Results:
814 CVID patients with at least 18 months of data were identified in the database, with 61% being female and 39% being male. The average age for females and males was 45 and 37 years, respectively (p<.0001). The CVID cohort carried a significantly higher prevalence of having a diagnosis code for a malignant neoplasm of the lymphatic and hematopoietic tissue than the control cohort [10.1% vs. 0.6%, respectively (p<.0001)]. Most of these neoplasm diagnoses occurred after the diagnosis of their CVID; however, 26.8% of neoplasm diagnoses occurred before the diagnosis of CVID. Additionally, the rates for each of the select neoplasms examined showed a significantly higher rate in the CVID cohort than in the control cohort.

Conclusions:
This study showed that CIVD patients have a higher prevalence of being co-diagnosed with certain neoplasms than matched controls. These data also suggest that CVID patients should not only be evaluated longitudinally for the development of neoplasms, but that certain cancer patients may also need to be considered and evaluated for underlying immune deficiencies such as CVID.

Disclosures:
Grossman: Baxter BioScience: Employment. Bonnet:Baxter BioScience: Employment. Xiong:BioScience: Employment.