Noémi M. Nagy

Disruption of the estrogen receptor β gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor β knockout (ERβ–/–) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15–30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERβ–/– mice than in WT littermates. Some of the ERβ–/– mice also had a severe lymphoproliferative phenotype. Thus the absence of ERβ results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERβ in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERβ–/– mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERβ agonists might have clinical value in the treatment of leukemia.

Exosomes Containing Glycoprotein 350 Released by EBV-Transformed B Cells Selectively Target B Cells through CD21 and Block EBV Infection In Vitro

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV− B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350–CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.

Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization

Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter.

Epstein–Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4+ T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

Exosomes Derived from Burkitt’s Lymphoma Cell Lines Induce Proliferation, Differentiation, and Class-Switch Recombination in B Cells

Exosomes, nano-sized membrane vesicles, are released by various cells and are found in many human body fluids. They are active players in intercellular communication and have immune-suppressive, immune-regulatory, and immune-stimulatory functions. EBV is a ubiquitous human herpesvirus that is associated with various lymphoid and epithelial malignancies. EBV infection of B cells in vitro induces the release of exosomes that harbor the viral latent membrane protein 1 (LMP1). LMP1 per se mimics CD40 signaling and induces proliferation of B lymphocytes and T cell–independent class-switch recombination. Constitutive LMP1 signaling within B cells is blunted through the shedding of LMP1 via exosomes. In this study, we investigated the functional effect of exosomes derived from the DG75 Burkitt’s lymphoma cell line and its sublines (LMP1 transfected and EBV infected), with the hypothesis that they might mimic exosomes released during EBV-associated diseases. We show that exosomes released during primary EBV infection of B cells harbored LMP1, and similar levels were detected in exosomes from LMP1-transfected DG75 cells. DG75 exosomes efficiently bound to human B cells within PBMCs and were internalized by isolated B cells. In turn, this led to proliferation, induction of activation-induced cytidine deaminase, and the production of circle and germline transcripts for IgG1 in B cells. Finally, exosomes harboring LMP1 enhanced proliferation and drove B cell differentiation toward a plasmablast-like phenotype. In conclusion, our results suggest that exosomes released from EBV-infected B cells have a stimulatory capacity and interfere with the fate of human B cells.

Epstein-Barr Virus Coinfection in Children Boosts Cytomegalovirus-Induced Differentiation of Natural Killer Cells

ABSTRACT During childhood, infections with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can occur in close temporal proximity. Active, as well as latent, CMV infection is associated with enlarged subsets of differentiated natural killer (NK) and cytotoxic T cells. How EBV infection may influence CMV-driven immune differentiation is not known. We found that EBV coinfection selectively influenced the NK cell compartment of CMV-seropositive (CMV+) children. Coinfected children had significantly higher proportions of peripheral-blood NKG2C+ NK cells than CMV+ EBV− children. Ex vivo NK cell degranulation after target cell stimulation and plasma IL-15 levels were significantly higher in CMV+ children. EBV coinfection was related to the highest levels of plasma interleukin-15 (IL-15) and IL-12p70. Remarkably, in vitro EBV infection of peripheral blood mononuclear cells (PBMC) from EBV− CMV+ children increased NKG2C+ NK cell proportions. A similar tendency was seen in cocultures of PBMC with EBV+ lymphoblastoid B-cell lines (LCL) and IL-15. After K562 challenge, NKG2C+ NK cells excelled in regard to degranulation and production of gamma interferon, regardless of whether there was previous coculture with LCL. Taken together, our data suggest that dual latency with these herpesviruses during childhood could contribute to an in vivo environment supporting differentiation and maintenance of distinct NK cell populations. This viral imprint may affect subsequent immune responses through altered distributions of effector cells.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus–encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma

In line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma-derived lines and in tonsillar B cells infected with the EBNA-2-deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4(+) T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4(+) T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

Convenient preparation of 1,1-diacetates from aromatic aldehydes catalysed by zinc-montmorillonite

1,1-Diacetates were produced from a variety of aromatic aldehydes using Zn-montmorillonite as a catalyst. The procedure generally resulted in good yields (98%) of aromatic aldehydes including those carrying electron donating- or withdrawing substituents. The great advantage of the application of Zn-montmorillonite catalyst is that the reaction takes place under mild conditions. In addition, the zinc cation exchanged form of montmorillonite has a minimal environmental impact.

Concomitant increase of LMP1 and CD25 (IL‐2‐receptor α) expression induced by IL‐10 in the EBV‐positive NK lines SNK6 and KAI3

Extranodal, nasal NK/T‐cell lymphomas are regularly Epstein‐Barr virus (EBV)‐positive, with a type II latency pattern, expressing thus EBNA‐1 and LMP1. The contribution of EBV to the tumor development is not known. Similarly to normal natural killer (NK) cells, cell lines derived from malignancies with a NK phenotype require IL‐2 for in vitro proliferation. In our effort to explore the contribution of EBV, particularly the role of the LMP1 protein, to the pathogenesis of the NK lymphoma we found that its expression, studied in the NK‐lines SNK6 and KAI3, depended on the supply of IL‐2 or other cytokines. In the absence of IL‐2 other cytokines, such as IL‐10 and IFN‐γ, could maintain LMP1, but the cells did not proliferate. When grown in IL‐2, the SNK6 cells produced IL‐10 and IFN‐γ, and these cytokines mediated the expression of LMP1. IL‐10 treatment enhanced, while IFN‐γ receptor blocking antibody reduced, the expression of CD25 and CD54 in the EBV‐positive, but not in the EBV‐negative lines. IL‐10 treated cells required lower amount of IL‐2 for proliferation compared to the untreated cells. This effect was seen only with the EBV‐positive NK lines in which LMP1 and CD25 were concomitantly upregulated. By this mechanism EBV could have an important role in the development of NK lymphoma since the inflammatory component in the tumor tissue can provide these cytokines.

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2.

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein‐Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA‐1, EBERs, LMP‐1 and LMP‐2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA‐2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA‐2 and LMP‐1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21‐positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA‐1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin‐resistance‐tagged recombinant EBV strain (Akata‐Neo) we established an EBV‐positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP‐1. The EBV expression pattern could be modified in this type I subline. LMP‐1 could be induced by the histone deacetylase inhibitors TSA and n‐butyrate, by 5‐AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA‐2. Importantly, exposure to CD40 ligand and IL‐4 induced LMP‐1 without EBNA‐2 expression and lytic replication. The KMH2 EBV cells expressed LMP‐2A, but not LMP‐2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.