EBV-transformed Cell Lines Research Articles

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

Biallelic PI4KA mutations Disrupt B Cell Mitochondrial Metabolism and Cause Hypogammaglobulinemia

Introduction Biallelic mutations in the PI4KA gene cause PI4KA-related disorder, a novel condition with neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus), gastrointestinal (GI; inflammatory bowel disease [IBD] and multiple intestinal atresia [MIA]) and immunological manifestations. PI4KA encodes phosphatidylinositol (PI) 4-kinase alpha, which catalyzes the first step of phosphoinositide metabolism, phosphorylating PI to PI4P (phosphatidylinositol 4-phosphate). Phosphoinositides collectively have fundamental signalling roles in the plasma membrane and other organelles. We collected comprehensive clinical and laboratory data on 13 patients and performed a multiomic strategy, combining transcriptome, proteome, lipidome and metabolome analyses of three patients' EBV-transformed cell lines to unravel the burden of B cell deficiency in PI4KA-related disorder and define the role of PI4KA in B cells. Results Recurrent or severe infections (8/13) and GI (either gastroesophageal reflux disease, MIA or IBD; 6/13) manifestations were frequent. Thyroid disease (either hypo- or hyperthyroidism; 2/13), juvenile idiopathic arthritis (JIA; 1/13) and non-Hodgkin lymphoma (1/13) were reported. At last follow up 11 patients were alive. Ten had either hypogammaglobulinemia, decreased absolute numbers of B cells or decreased B cell subsets. B cell subsets showed increased transitional (4/7), decreased naive (2/7) and decreased switched memory (4/7) B cells. B cell proliferation to T independent mitogens, specifically IgM + CpG or pokeweed, was impaired in 2/2. T-cell (either CD3+, CD4+ or CD8+) lymphopenia was detected in 4/13. T-cell proliferation was maintained in 5/5. NK cells were decreased in 6/13. Ig replacement therapy (4/13) and immunosuppressive treatment (due to JIA [1/13] or IBD [2/13]) were given. Lipidome analysis showed that different glycerophospholipids profile (i.e., phosphatidylcholine [PC], phosphatidylethanolamines [PE] and phosphatidylserine [PS]) was altered. Metabolome analysis and pathway enrichment analysis confirmed impaired phosphatidylethanolamine biosynthesis and additionally showed altered PIP metabolism and mitochondrial dysfunction (i.e., beta-oxidation of short and long fatty acids). Although the total number of mitochondria was not affected, the mitochondrial activity was significantly reduced. Integration analyses of proteomics, metabolomic and lipidomic data reveals biochemical mechanisms underlying biallelic PI4KA mutation. In particular, dysregulation of metabolic enzymes and pathways involved in glutathione metabolism and tricarboxylic acid cycle was found (Fig. 1). These metabolic impairments ultimately impacted on oxidative phosphorylation, thus disrupting mitochondrial integrity. Gene set enrichment analysis of differentially expressed genes suggested a skewing towards naive B cell gene sets. Pathways enrichment analysis of transcriptome and proteome showed enrichment in B cell receptor (BCR) pathway and complex, PI3K/mTOR pathway and phospholipids metabolism. PI4P and its metabolite PI(4,5)P2 are determinants of PM identity, specifically controlling cytoskeletal dynamics. Deranged basal actin organization was reverted after anti-IgM stimulation. Hyperactivation of PI3K targets (pAKT473, p4EBP1 and pS6) was observed. Dysregulation of RABGAP1, RAB8B and LAMP3 mRNA prompted us to investigate autophagy. Indeed, LC3 was increased either in the presence or absence of chloroquine, showing that PI4KA mutations enhanced autophagosome formation and inhibited its degradation. Conclusion Overall, by altering the production of several key lipids, biallelic PI4KA mutations disrupt B cell metabolism causing mitochondrial dysfunction, mTOR hyperactivation, increased autophagy and deranged cytoskeleton organization. PI4KA-related disorder is a novel syndromic inborn error of immunity causing B cell deficiency and hypogammaglobulinemia. Figure 1. Integration of proteomics, metabolomics and lipidomics reveals biochemical mechanisms underlying biallelic PI4KA mutations.

Variable Clinical Presentation in Individuals with Truncating Variants in DDX41

Background and AimsDefects in DDX41 have previously been associated with increased risk of myeloid neoplasms, lymphoid neoplasms, cytopenia, and red blood cell macrocytosis. DDX41-related phenotypes have a variable age of onset typically in the 60 s. A previous study analyzed a cohort of 1,385 individuals with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), identifying 33 individuals with a pathogenic variant in DDX41, 9 of whom had a familial history of MDS/AML. In our cohort of 6,103 participants with diverse clinical manifestations, we identified 7 individuals with truncating variants in DDX41. We sought to characterize age-related penetrance and the phenotypic spectrum of individuals with DDX41 truncating variants. MethodsWe performed exome or genome sequencing on DNA extracted from blood, saliva, fibroblasts, or EBV-transformed cell lines in individuals referred to the Centralized Sequencing Program at the NIH for immune-related, neurological, psychiatric, and other clinical indications. ResultsWe identified 7 subjects with truncating variants in DDX41, including 3 different frameshift variants in 5 subjects and 2 different nonsense variants in the other 2 subjects. The average age of subjects was 37 years old, ranging from 5 to 76 years old at time of enrollment. There were 3 males and 4 females. Family history of DDX41-related phenotypes was not reported in the families; furthermore, there were no familial cases, thought segregation data was incomplete. Clinical features were highly variable, ranging from a healthy volunteer to an individual with JAK2 p. Val617Phe-associated eosinophilic myeloid neoplasm. Previously associated DDX41-related phenotypes were not observed in the 7 subjects even include those above 60. ConclusionsHere, we report 7 subjects with truncating variants in DDX41. These individuals have variable immune phenotypes and further studies are necessary to delineate whether they are attributed to DDX41 truncating variants. Detection of DDX41-defects in younger individuals prior to symptom onset poses genetic counseling challenges given the long latency period and minimal evidence-based management guidelines at present. Nonetheless, current expert-based clinical recommendations for management and monitoring may provide clinical benefit upon further study. Additional studies of clinical and sub-clinical features in expanded cohorts are warranted to further delineate DDX41-related disease risk.

Lymphocyte activation gene-3-associated protein networks are associated with HDL-cholesterol and mortality in the Trans-omics for Precision Medicine program

Deficiency of the immune checkpoint lymphocyte activation gene-3 (LAG3) protein is significantly associated with both elevated HDL-cholesterol (HDL-C) and myocardial infarction risk. We determined the association of genetic variants within ±500 kb of LAG3 with plasma LAG3 and defined LAG3-associated plasma proteins with HDL-C and clinical outcomes. Whole genome sequencing and plasma proteomics were obtained from the Multi-Ethnic Study of Atherosclerosis (MESA) and the Framingham Heart Study (FHS) cohorts as part of the Trans-Omics for Precision Medicine program. In situ Hi-C chromatin capture was performed in EBV-transformed cell lines isolated from four MESA participants. Genetic association analyses were performed in MESA using multivariate regression models, with validation in FHS. A LAG3-associated protein network was tested for association with HDL-C, coronary heart disease, and all-cause mortality. We identify an association between the LAG3 rs3782735 variant and plasma LAG3 protein. Proteomics analysis reveals 183 proteins significantly associated with LAG3 with four proteins associated with HDL-C. Four proteins discovered for association with all-cause mortality in FHS shows nominal associations in MESA. Chromatin capture analysis reveals significant cis interactions between LAG3 and C1S, LRIG3, TNFRSF1A, and trans interactions between LAG3 and B2M. A LAG3-associated protein network has significant associations with HDL-C and mortality.

P62-mediated Selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress.

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.

Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation.

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

Functional characterization of DcR3 in EBV transformed cell lines from IBD patients of different allelic background and role in disease pathogenesis

Functional characterization of DcR3 in EBV transformed cell lines from IBD patients of different allelic background and role in disease pathogenesis

SU32 - PATIENT-SPECIFIC AD IN VITRO MODELS FOR THE ANALYSIS OF LATE-ONSET ALZHEIMER'S DISEASE

Background Alzheimer´s Disease (AD) is the most common form of dementia. Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly understood. AD is genetically complex and shows heritability of up to 80%. Glia cells including astrocytes and microglia are affected in AD patients contributing to the tau pathology and the accumulation of neurotoxic amyloid as well. In recent years, Genome-Wide Association Studies (GWAS) allowed the identification of DNA variations associated with an elevated risk for AD. Together with other groups, we identified a number of AD susceptibility genes including CD33, SORL1, ABCA7 and TREM2, which highly recommends that the immune system plays a major role in onset, progression and treatment of AD. Reprogramming of EBV-transformed patient specific cell lines for the generation of induced pluripotent stem cells (iPS) enables the patient-specific differentiation into neurons, astrocytes, and microglia. Patient-specific cells can be used as a model to functionally characterize disease associated variants. Methods Blood was collected from AD patients carrying risk variants in AD susceptibility genes for the generation of iPS cells. SNP variants in those genes were genotyped based on their potential function according to literature and genomic localization (exonic non-synonymous, binding domain, or promoter affecting SNPs). Reprogrammed iPS cells carried the genetic background of a certain AD patient. Pluripotency was characterized by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ layers. Crucial pluripotency markers are OCT4, SOX2, NANOG, KLF4, MYC, and LIN28. AD iPS cells were differentiated into astrocytes and microglia and the differentiation status was characterized by the expression of crucial glia cell markers. Further, AD susceptibility genes recently published in GWAS were analysed. Results The protein expression was successfully induced shown by immunofluorescence and western-blot analysis. Cells were also screened for the most efficient induction of the three germ layers and the induction of neural cell fates including glia cell fates. The established AD-specific iPS cell lines from AD patients represent a powerful tool for the analysis of molecular and cellular disease mechanisms. We established 4-step protocol for the generation of AD-specific microglia enabling the focused analysis of AD-associated risk variants. The protocol was verified by morphology, FACS analysis, immunofluorescence and RNA expression of crucial microglia marker, including hematopoetic lineage markers and the induction of well-described microglia markers including IBA1. The disease-specific analysis of the genetic background of AD patients represents a completely new approach for the understanding of AD genetics and AD-associated risk variants. Discussion Together, combining molecular genetics of AD for the investigation of risk variants and iPS cell technology for the generation of patient- and disease-specific stem cells provides a promising approach to characterize known disease mechanisms, to deepen the understanding of known disease mechanisms, and to discover unknown disease aspects.

Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection.

Human cell lines have been used in a variety of research fields as an in vitro model. These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.

T cell mediated immunity induced by the live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S in humans

BackgroundShigellosis persists as a public health problem worldwide causing ~ 165,000 deaths every year, of which ~ 55,000 are in children less than 5 years of age. No vaccine against shigellosis is currently licensed. The live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S (S. flexneri 2a; ΔguaBA, Δset, Δsen) demonstrated to be safe and immunogenic in phase 1 and 2 clinical trials. Earlier reports focused on humoral immunity. However, Shigella is an intracellular pathogen and therefore, T cell mediated immunity (T-CMI) is also expected to play an important role. T-CMI responses after CVD 1208S immunization are the focus of the current study.MethodsConsenting volunteers were immunized orally (3 doses, 108 CFU/dose, 28 days apart) with CVD 1208S. T-CMI to IpaB was assessed using autologous EBV-transformed B-Lymphocytic cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines (IFN-γ, IL-2, IL-17A and TNF-α) and/or expression of the degranulation marker CD107a in 14 volunteers (11 vaccine and 3 placebo recipients).ResultsFollowing the first immunization, T-CMI was detected in CD8 and CD4 T cells obtained from CVD 1208S recipients. Among CD8 T cells, the T effector memory (TEM) and central memory (TCM) subsets were the main cytokine/CD107a producers/expressors. Multifunctional (MF) cells were also detected in CD8 TEM cells. Cells with 2 and 3 functions were the most abundant. Interestingly, TNF-α appeared to be dominant in CD8 TEM MF cells. In CD4 T cells, TEM responses predominated. Following subsequent immunizations, no booster effect was detected. However, production of cytokines/expression of CD107a was detected in individuals who had previously not responded. After three doses, production of at least one cytokine/CD107a was detected in 8 vaccinees (73%) in CD8 TEM cells and in 10 vaccinees (90%) in CD4 TEM cells.ConclusionsCVD 1208S induces diverse T-CMI responses, which likely complement the humoral responses in protection from disease.Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT01531530)

Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties

BackgroundMany plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants. Hypothesis/PurposeHere we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine. Study DesignThis work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA). ResultsOf the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine. ConclusionThe results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8+ and CD4+ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8+ and CD4+ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4+ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4+ T cells was significantly correlated with that of CD8+ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4+ T cell responses showed more significant changes than CD8+ T cell responses. CD8+ and CD4+ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4+ T cells secreted significantly higher cytokine levels than did CD8+ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.

BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.

Human leukocyte telomere length is associated with DNA methylation levels in multiple subtelomeric and imprinted loci.

In humans, leukocyte telomere length (LTL) is positively correlated with lifespan, and shorter LTL is associated with increased risk of age-related disease. In this study we tested for association between telomere length and methylated cytosine levels. Measurements of mean telomere length and DNA methylation at >450,000 CpG sites were obtained for both blood (N = 24) and EBV-transformed cell-line (N = 36) DNA samples from men aged 44–45 years. We identified 65 gene promoters enriched for CpG sites at which methylation levels are associated with leukocyte telomere length, and 36 gene promoters enriched for CpG sites at which methylation levels are associated with telomere length in DNA from EBV-transformed cell-lines. We observed significant enrichment of positively associated methylated CpG sites in subtelomeric loci (within 4 Mb of the telomere) (P < 0.01), and also at loci in imprinted regions (P < 0.001). Our results pave the way for further investigations to help elucidate the relationships between telomere length, DNA methylation and gene expression in health and disease.

Expansion of CD16 positive and negative human NK cells in response to tumor stimulation

NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.

Visualization and probability-based scoring of structural variants within repetitive sequences

Motivation: Repetitive sequences account for approximately half of the human genome. Accurately ascertaining sequences in these regions with next generation sequencers is challenging, and requires a different set of analytical techniques than for reads originating from unique sequences. Complicating the matter are repetitive regions subject to programmed rearrangements, as is the case with the antigen-binding domains in the Immunoglobulin (Ig) and T-cell receptor (TCR) loci.Results: We developed a probability-based score and visualization method to aid in distinguishing true structural variants from alignment artifacts. We demonstrate the usefulness of this method in its ability to separate real structural variants from false positives generated with existing upstream analysis tools. We validated our approach using both target-capture and whole-genome experiments. Capture sequencing reads were generated from primary lymphoid tumors, cancer cell lines and an EBV-transformed lymphoblast cell line over the Ig and TCR loci. Whole-genome sequencing reads were from a lymphoblastoid cell-line.Availability: We implement our method as an R package available at https://github.com/Eitan177/targetSeqView. Code to reproduce the figures and results are also available.Contact: ehalper2@jhmi.eduSupplementary information: Supplementary data are available at Bioinformatics online.

CMV-Specific CD8+ T-CELL Function Is NOT Impaired In CLL

IntroductionT-cell exhaustion is a state of acquired T-cell dysfunction that arises during chronic infections, but also in the presence of cancer. Chronic lymphocytic leukemia (CLL)-derived CD8+ T cells were recently found to exhibit features of exhaustion as shown by increased expression of inhibitory receptors (including PD-1), impaired proliferative and cytotoxic capacity and disturbed immunological synapse (IS) formation (Riches et al, Blood '13). In contrast, we have reported a decreased expression of PD-1 on effector-type CD45RA+/-CD27- CD8+ T cells in CLL patients (Tonino et al., Leuk&Lymph ‘12). As this population is enriched for CMV-specific T cells, this implies that PD-1 expression might also be decreased on CMV-specific T cells. Importantly, CMV viral loads are generally undetectable in untreated CLL patients. Taken together these data suggest that the phenotypical and functional changes found in the total CD8+ T-cell population in CLL may not be applicable to CMV-specific CD8+ T cells. In this study, we have thoroughly analyzed phenotype and function of CMV-specific CD8+T cells in CLL. Methods and ResultsIn agreement with Riches et al. we found an increased expression of the exhaustion markers PD-1, CD160 and CD244 (assessed by flow cytometry) on CLL-derived total CD8+ T cells (n=38) as compared to CD8+ T cells from age-matched healthy controls (HC; n=13). In sharp contrast, we found a significantly lower expression of these markers on CMV-tetramer+CD8+ T cells in CLL patients (n=18) as compared to HC (n=7). Next we analyzed the functionality of CMV-specific CD8+ T cells derived from CLL patients and HC. First, the sensitivity of the functional assays used was verified by treating CD8+ T cells with cyclosporine, which mimics intrinsic T-cell defects in vitro. Cytokine production was measured upon stimulation with PMA/Ionomycin. We found no differences between CLL patients and age-matched HC as to IFNγ, TNFα and IL-2 production by CMV-tetramer+CD8+ cells. Interestingly, the fraction of CMV-tetramer+CD8+ T cells producing cytokines after stimulation with CMV-pp65-peptide loaded EBV-transformed B-lymphoblastoid cell lines (EBV-LCLs) derived from normal B lymphocytes also did no differ between CLL patients and HC. Next, the cytotoxic potential of these cells was examined. CMV-specific CD8+ T cells from CLL patients were as effective as those from HC in killing CMV-peptide-loaded EBV-LCLs at varying effector:target ratios. Finally, CMV-specific IS formation was assessed by imaging flow cytometry analysis. This technique combines quantitative and qualitative data and has advantages over the current gold standard in the study of IS, i.e. confocal laser-scanning microscopy, since it is able to measure large cell populations in an automated fashion. On average 0.6x105 total cells per condition were acquired containing approximately 550 doublets. Again, percentages of CD8+T cells forming specific proper IS with CMV-peptide loaded EBV-LCLs were comparable in CLL and HC T cells (around 45%). ConclusionIn contrast to evidence for a global disturbed T-cell functionality in CLL, characterized by T cells exhibiting phenotypic and functional signs of exhaustion, we now show that CMV-specific CD8+ T cells derived from CLL patients have a lower expression of exhaustion markers and are functionally intact. These data shed new light on the complexity of interactions between T-lymphocytes and CLL cells and indicate that changes found in the total CD8+ T-cell population in CLL cannot be extrapolated to CMV-specific CD8+ T cells. Disclosures:No relevant conflicts of interest to declare.

A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR– B cells

Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre–B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors.

Clinical and cellular features in patients with primary autosomal recessive microcephaly and a novel CDK5RAP2 mutation

BackgroundPrimary autosomal recessive microcephaly (MCPH) is a rare neurodevelopmental disorder that results in severe microcephaly at birth with pronounced reduction in brain volume, particularly of the neocortex, simplified cortical gyration and intellectual disability. Homozygous mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 are the cause of MCPH3. Despite considerable interest in MCPH as a model disorder for brain development, the underlying pathomechanism has not been definitively established and only four pedigrees with three CDK5RAP2 mutations have been reported. Specifically for MCPH3, no detailed radiological or histological descriptions exist.Methods/ResultsWe sought to characterize the clinical and radiological features and pathological cellular processes that contribute to the human MCPH3 phenotype. Haplotype analysis using microsatellite markers around the MCPH1-7 and PNKP loci in an Italian family with two sons with primary microcephaly, revealed possible linkage to the MCPH3 locus. Sequencing of the coding exons and exon/intron splice junctions of the CDK5RAP2 gene identified homozygosity for the novel nonsense mutation, c.4441C > T (p.Arg1481*), in both affected sons. cMRI showed microcephaly, simplified gyral pattern and hypogenesis of the corpus callosum. The cellular phenotype was assessed in EBV-transformed lymphocyte cell lines established from the two affected sons and compared with healthy male controls. CDK5RAP2 protein levels were below detection level in immortalized lymphocytes from the patients. Moreover, mitotic spindle defects and disrupted γ-tubulin localization to the centrosome were apparent.ConclusionThese results suggest that spindle defects and a disruption of centrosome integrity play an important role in the development of microcephaly in MCPH3.

EBV-Specific Micro-RNA Via Exosome: A Key Inter-Cellular machinery between EBV+ Tumor and Tumor-Surrounding Immune Cells?

Abstract 50The Epstein-Barr virus (EBV) is one of the most major human pathogen that establish long-term latent, or chronic infections, which is associated with a heterogeneous group of lymphoma, including Burkitt’s lymphoma, Hodgkin’s lymphoma (HL), NK-T lymphomas and lymphoproliferative disease. These malignancies are subdivided into in terms of EBV latent infection pattern, with typical three types of latency: type I to type III. HL is characterized by a minority of neoplastic Hodgkin and Reed-Sternberg (HRS), which are embedded in non-neoplastic bystanders, mostly B and T cells, but also macrophages. Without these bystander cells, these HL cells are incapable of being engrafted in immunodeficient mice. In this context, the non-tumor immune cells are tumor-supportive “inflammatory niche”. Because of the complexity of interplay between tumor and tumor surrounding immune cells, the detailed mechanism and how tumor cells escape from the attack of host immune cells remains an open question. Small RNAs including miRNAs are well known intra-cellular regulatory elements of gene expression. Recently, it was reported that they are conjugated in exosomes and transferred to cells and are involved in tumor metastasis by educating tumor surrounding niche. Moreover, it was also reported that EBV-infected lymphocytes produce exosomes that contain viral encoded, EBV specific miRNAs (BART-miRNA) and that these could be transferred in host cells and decrease the levels of known cellular targets. Accordingly, we hypothesized that EBV+ tumor derived exosomal BART-miRNA might redirect tumor surrounding immune cells from tumor reactive into tumor- -supportive “inflammatory niche”, which ultimately leads to tumor progression. To this aim, first, we evaluated tumor derived viral encoded BART-miRNA in EBV+HL clinical specimens by using BART-miRNA specific probe in situ hybridization. As expected, these EBV specific BART-miRNA could be detected in HRS as well as in tumor surrounding inflammatory niche, especially macrophage. This result indicated that tumor derived EBV specific BART-miRNA could transfer to the non-tumor cells in the tumor inflammatory niche, supporting the in vivo relevance of secretary EBV specific miRNA. Next, we evaluated the properties of exosomes produced by EBV+ cells (EBV-Ex). To this aim, EBV-Ex was harvested either from the media of the type III or type I EBV-transformed lymphoid cell line. Then, by using transwell co-culture system, we tested the delivery and the effect of EBV-Ex on human peripheral blood mononuclear cells (PBMC) derived monocyte/macrophage (Mo/Mf). As a result, we detected uptake of fluorochrome dye-labeled EBV-Exo in Mo/Mf. We also confirmed exosomal BART miRNA transfer in Mo/Mf. Surprisingly, exosome from Type III latency (Type III-Ex) was relatively enriched in BART miRNA, and were potent on Mo/Mf in inducing surface CD69 expression (Fig.A). This is in contrast to that of exosome from Type I latency (Type I-Ex), in which BART miRNA were relatively vacant and were weak in inducing surface CD69 expression (Fig.A). Panels of cytokine analysis by Q-PCR revealed that type III-Ex treated Mo/Mf displayed an anti-inflammatory/immunosuppressive cytokine rich signature, especially IL-10, compared to type I-Ex treated Mo/Mf, suggesting the possibility that type III-Ex might polarize macrophage into immunosuppressive M2-like phenotype. Intriguingly, type III-Ex from BART miRNA deletion mutant derivative cell lines totally lack the type III -Ex signature. Moreover, ectopic expression of a part of BART in Type I cells changed the EBV-Ex signature from type III to type I (Fig.B), suggesting the importance of specific BART lesion in functional EBV-Ex production in terms of Mo/Mf polarization. Taken these together, secretary tumor derived miRNAs in EBV associated malignancy, specifically in EBV+HL, might play a certain role in tumor inflammation niche. EBV might utilize the exosomal machinery to secrete key viral-encoded miRNAs, through which a small number of neoplastic EBV+ cells could modulate the tumor microenvironment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.