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Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

ABSTRACT: Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

SUBMITTER: Minamitani T

PROVIDER: S-EPMC5422827 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Minamitani Takeharu T Ma Yijie Y Zhou Hufeng H Kida Hiroshi H Tsai Chao-Yuan CY Obana Masanori M Okuzaki Daisuke D Fujio Yasushi Y Kumanogoh Atsushi A Zhao Bo B Kikutani Hitoshi H Kieff Elliott E Gewurz Benjamin E BE Yasui Teruhito T

Proceedings of the National Academy of Sciences of the United States of America 20170328 18

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 an ...[more]

PMID: 28351978

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Project description:Latent membrane protein 1 (LMP1) and LMP2A affect cell growth in both epithelial cells and lymphocytes. In this study, the effects on cellular gene expression were determined by microarray analysis of transgenic mice expressing LMP1, LMP2A, or both using the immunoglobulin heavy chain promoter and enhancer. Large differential changes were detected, indicating that LMP1 and LMP2A can both potently affect host gene transcription, inducing distinct transcriptional profiles. Seventy percent of the changes detected in LMP1/2A doubly transgenic lymphocytes were also modulated by LMP1 or LMP2A alone. These common and unique expression changes indicate that the combined effects of LMP1 and LMP2A may be additive, synergistic, or inhibitory. Using significant pathway analysis, the expression changes detected in LMP1, LMP2A, and LMP1/2A transgenic B lymphocytes were predicted to commonly target cancer and inflammatory pathways. Additionally, using the correlation coefficient to calculate the regulation of known c-Rel and Stat3 transcriptional targets, both were found to be enhanced in LMP1 lymphocytes and lymphomas, and a selection of Stat3 targets was further evaluated and confirmed using quantitative reverse transcription-PCR (RT-PCR). Analyses of the effects on cell growth and viability revealed that LMP2A transgenic lymphocytes had the greatest enhanced viability in vitro; however, doubly transgenic lymphocytes (LMP1/2A) did not have enhanced survival in culture and these mice were similar to negative littermates. These findings indicate that the combined expression of LMP1 and LMP2A has potentially different biological outcomes than when the two proteins are expressed individually. IMPORTANCE The Epstein-Barr virus proteins latent membrane protein 1 (LMP1) and LMP2A have potent effects on cell growth. In transgenic mice that express these proteins in B lymphocytes, the cell growth and survival properties are also affected. LMP1 transgenic mice have increased development of lymphoma, and the LMP1 lymphocytes have increased viability in culture. LMP2A transgenic lymphocytes have altered B cell development and enhanced survival. In this study, analysis of the cellular gene expression changes in transgenic LMP1 and LMP2A lymphocytes and LMP1 lymphomas revealed that both transgenes individually and in combination affected pathways important for the development of cancer and inflammation. Importantly, the combined expression of the two proteins had unique effects on cellular expression and cell viability. This is the first study to look at the combined effects of LMP1 and LMP2A on global changes in host gene expression.

Project description:Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors.

Dataset Information

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Publications

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

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