Induction of Interferon-Stimulated Genes on the IL-4 Response Axis by Epstein-Barr Virus Infected Human B Cells; Relevance to Cellular Transformation

Nikki Smith,Martin Rowe,Wenbin Wei,Paul G Murray,Rosemary Tierney,Ciaran B Woodman,Martina Vockerodt

doi:10.1371/journal.pone.0064868

Nikki Smith, Martin Rowe + Show 5 more

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https://doi.org/10.1371/journal.pone.0064868

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Journal: PloS one	Publication Date: May 27, 2013
Citations: 80	License type: CC BY 4.0

Affiliation: Cancer Research UK, University of Birmingham

Abstract

Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation lifespans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation. Of interest to us was a subset of 255 genes that were differentially expressed between EBV and CD40L/IL-4 blasts. Genes which were more highly expressed in EBV blasts were substantially and significantly enriched for a set of interferon-stimulated genes which on further in silico analyses were found to be repressed by IL-4 in other cell contexts and to be up-regulated in micro-dissected malignant cells from Hodgkin's lymphoma biopsies when compared to their normal germinal center cell counterparts. We hypothesized that EBV and IL-4 were targeting and discordantly regulating a common set of genes. This was supported experimentally in our B cell model where IL-4 stimulation partially reversed transcriptional changes which follow EBV infection and it impaired the efficiency of EBV-induced B cell transformation. Taken together, these data suggest that the discordant regulation of interferon and IL-4 pathway genes by EBV that occurs early following infection of B cells has relevance to the development or maintenance of an EBV-associated malignancy.

Highlights

The normal process of B lymphocyte activation in response to encountering antigen involves complex cellular and cytokine interactions in the germinal centers of peripheral lymphoid tissues, leading to clonal expansion followed by differentiation into effector or memory cells
Consistent with published data indicating the phenotypic similarities of Epstein-Barr virus (EBV)- and mitogen-induced B blasts [24], we found that there was considerable overlap in their transcriptomes and that mRNA changes relative to the original resting B cells largely reflected common changes associated with lymphocyte activation and cell proliferation
Of these overlapping 1410 significant transcriptional changes, 99.6% were altered in the same direction by EBV and by CD40L/IL-4; and only 6 genes were significantly up-regulated by EBV and significantly down-regulated by CD40L/IL-4, or vice versa (Figures 1B, 1C and Table 1)

Summary

Introduction

The normal process of B lymphocyte activation in response to encountering antigen involves complex cellular and cytokine interactions in the germinal centers of peripheral lymphoid tissues, leading to clonal expansion followed by differentiation into effector or memory cells. Some aspects of this self -limiting proliferation can be replicated in vitro through combinations of signals; one widely-used method of generating B cell blast involves costimulation with CD40L and IL-4. Showing no overall sequence homology with any cellular proteins, LMP1 functions as a constitutively-active member of the tumor necrosis factor receptor family which includes CD40 [19]. The cooperative functions of multiple viral genes are required, as demonstrated by studies with recombinant EBVs, which show that deletion of any one of EBNA1, EBNA2, EBNA3A, EBNA3C or LMP1 genes will abolish or substantially reduce transforming ability [22,23]

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