Epstein-Barr virus LMP1 and LMP2A transgenic B lymphocytes and lymphoma cells
ABSTRACT: Gene expression profile of splenic B cells (CD19+) from transgenic mice expressing the Epstein-Barr virus (EBV) latent membrane proteins (LMP) 1 and/or LMP2A. Freshly harvested primary B cells were profiled. B lymphocytes from transgenic LMP1, LMP2A, LMP1/2A mice and negative littermates were profiled from 6 month old adult mice; lymphoma cells were passaged in SCID mice and profiled for three LMP1 positive lymphomas and one negative lymphoma. 12 total samples. 4 transgenic B lymphocyte samples pooled from multiple biological replicates were hybridized to duplicate microarrays: LMP1 (pooled from 2 replicates), LMP2A (pooled from 3 replicates); LMP1/2A (pooled from 5 replicates), negative littermates (pooled from 4 replicates). 3 biological replicates of LMP1 lymphomas expressing high, medium and low levels of LMP1 and; 1 negative lymphoma was hybridized to 1 microarray chip. The reference sample consisted of 4 biological replicates of splenic B cells (CD19+) pooled from 4-7 month old non-transgenic Balb/c mice. The same reference was used for all hybridizations.
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.
Project description: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.
Project description:A mechanism used by Epstein-Barr virus (EBV) for in vitro transformation of B cells into lymphoblastoid cell lines (LCLs) is activation of the NF-kappaB pathway, which is largely mediated by the EBV latent membrane protein 1 (LMP1). LMP1 is coexpressed with LMP2A in many EBV-associated lymphoid malignancies. Since inhibition of NF-kappaB leads to apoptosis of EBV-infected LCLs and lymphoma cell lines, we sought to determine whether LMP1 alone, or in combination with other viral proteins, is responsible for initiating NF-kappaB activation in these cells, thereby playing a role in cell survival. We found that suppression of LMP1 by RNA interference results in inhibition of basal NF-kappaB and induction of apoptosis. Unexpectedly, knockdown of LMP2A also resulted in comparable decrease of NF-kappaB activity and apoptosis. We report that LMP2A protein controls the expression of TRAF2 mRNA, which in turn is necessary for signaling by LMP1. Our data contrast with previous studies showing that transfected LMP1 can signal in the absence of LMP2A or TRAF2, and demonstrate that both LMP2A and TRAF2 are required for survival in naturally infected lymphoma cells and LCLs. These results also support LMP1, LMP2A, and TRAF2 as potential therapeutic targets in a subset of EBV-associated lymphoid malignancies.
Project description:Epstein-Barr virus (EBV) is associated with several B and epithelial cell cancers. EBV-encoded latent membrane protein 2A (LMP2A) contributes to cellular transformation by mimicking B cell receptor signaling. LMP2A/MYC double transgenic mice develop splenomegaly and B cell lymphoma much faster than MYC transgenic mice do. In this study, we explored the potential therapeutic efficacy of a novel spleen tyrosine kinase (SYK) and FLT3 inhibitor TAK-659 for development of a treatment option for EBV-associated malignancies. In our transgenic model, TAK-659 treatment totally abrogated splenomegaly and tumor development in LMP2A/MYC mice in both pretumor and tumor cell transfer experiments. TAK-659 treatment killed tumor cells, but not host cells within the spleen and tumors. Furthermore, TAK-659 treatment abrogated metastasis of tumor cells into bone marrow. Our data also show that TAK-659 inhibits SYK phosphorylation and induces apoptosis in LMP2A/MYC tumor cells at low nanomolar concentrations. Therefore, TAK-659 may provide an effective therapeutic option for treatment of LMP2A-positive EBV-associated malignancies and should be explored further in clinical trials.IMPORTANCE The novel SYK and FLT3 inhibitor TAK-659 prevents the enlargement of spleen and tumor development in a mouse model of EBV-associated lymphoma by counteracting the activation of cellular kinase SYK through the viral LMP2A gene by inducing cell death in tumor cells but not in nontumor cells. These findings indicate that TAK-659 may be a very effective nontoxic therapeutic molecule especially for EBV-positive hematologic malignancies.
Project description:Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.
Project description:Epstein-Barr virus (EBV) is a major cause of immunosuppression-related lymphomas. EB-driven lymphoproliferative disease complicates up to 20% of transplants, and EBV is a major cause of human immunodeficieciency virus associated lymphomas. Despite successful antiretroviral therapy, the incidence of EBV-associated Hodgkin lymphoma continues to increase in HIV+ individuals. To gain insights into EBV membrane oncoprotein effects on B-cell growth, survival and pathogenesis in vivo, we generated transgenic mouse models, in which knock-in mice transgenically express control GFP or EBV latent membrane proteins (LMP) 1 and 2A under the control of the AICDA promoter. Upon T and NK-cell depletion by antibody cocktail, LMP1 and 2A co-expression drove explosive growth of plasmablastic lymphoma-like cells, which proliferated in the spleen, caused severe end-organ damage and death. RNAseq profiling identified genome-wide LMP1 and 2A effects on B-cell gene expression, including dramatic effects on chemokine and cytokine production. While cells exhibited plasmablast features, LMP1 and 2A co-expression also induced mixed hematopoietic lineage markers, a well described but incompletely understood feature of Hodgkin lymphoma. Collectively, our results identify synergistic effects of EBV membrane oncoprotein expression, and highlight their role in lymphoproliferative diseases of immunocompromised hosts. Overall design: B220 positive B splenocytes were isolated from GFP and LMP1/2a transgenic mice, which are T/NK cell depleted by antibody cocktails, and subjected to RNA-seq analysis to compare the transcriptomic changes in LMP1/2a transgenic mice compared to GFP transgenic mice. Two mice from each transgenic group were used.
Project description:In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.
Project description:Epstein-Barr virus (EBV) establishes lifelong infection in B lymphocytes of most human hosts and is associated with several B lymphomas. During latent infection, EBV encodes latent membrane protein 2A (LMP2A) to promote the survival of B cells by mimicking host B-cell receptor signaling. By studying the roles of LMP2A during lymphoma development in vivo, we found that LMP2A mediates rapid MYC-driven lymphoma onset by allowing B cells to bypass MYC-induced apoptosis mediated by the p53 pathway in our transgenic mouse model. However, the mechanisms used by LMP2A to facilitate transformation remain elusive. In this study, we demonstrate a key role of LMP2A in promoting hyperproliferation of B cells by enhancing MYC expression and MYC-dependent degradation of the p27kip1 tumor suppressor. Loss of the adaptor protein cyclin-dependent kinase regulatory subunit 1 (Cks1), a cofactor of the SCFSkp2 ubiquitin ligase complex and a downstream target of MYC, increases p27kip1 expression during a premalignant stage. In mice that express LMP2A, Cks1 deficiency reduces spleen weights, restores B-cell follicle formation, impedes cell cycle progression of pretumor B cells, and eventually prolongs MYC-driven tumor onset. This study demonstrates that LMP2A uses the role of MYC in the cell cycle, particularly in the p27kip1 degradation process, to accelerate lymphomagenesis in vivo. Thus, our results reveal a novel mechanism of EBV in diverting the functions of MYC in malignant transformation and provide a rationale for targeting EBV's roles in cell cycle modulation.
Project description:Elevated expression of MYC is a shared property of many human cancers. Epstein-Barr virus (EBV) has been associated with lymphoid malignancies, yet collaborative roles between MYC and EBV in lymphomagenesis are unclear. EBV latent membrane protein 2A (LMP2A) functions as a B-cell receptor (BCR) mimic known to provide survival signals to infected B cells. Co-expression of human MYC and LMP2A in mice (LMP2A/?-MYC) accelerates B lymphoma onset compared with mice expressing human MYC alone (?-MYC mice). Here we show a novel role of LMP2A in potentiating MYC to promote G1-S transition and hyperproliferation by downregulating cyclin-dependent kinase inhibitor p27(kip1) in a proteasome-dependent manner. Expressing a gain-of-function S10A mutant of p27(kip1) has minor effect on tumor latency. However, pretumor B cells from ?-MYC mice expressing homozygous S10A mutant show a significant decrease in the percentage of S-phase cells. Interestingly, LMP2A is able to counteract the antiproliferative effect of the S10A mutant to promote S-phase entry. Finally, we show that LMP2A expression correlates with higher levels of MYC expression and suppression of p27(kip1) before lymphoma onset. Our study demonstrates a novel function of EBV LMP2A in maximizing MYC expression, resulting in hyperproliferation and cellular transformation into cancer cells in vivo.