The impact of microenvironmental factors on EBV carrying B cells

Epstein-Barr virus (EBV) is a human gamma-herpes virus that colonized more than 90% of the adult population. The virus is able to infect and immortalize B lymphocytes both in vitro and in vivo. Despite of the mostly harmless outcome of the EBV infection, EBV is associated with a number of malignancies, such as Burkitt lymphoma, classical Hodgkin lymphoma, and Diffuse large B-cell lymphoma (DLBCL).

DLBCLs, the most common group of malignant lymphomas, account for 30% of adult non-Hodgkin lymphomas (NHLs). EBV-positive DLBCL of the elderly is a newly recognized subtype of DLBCL which accounts for 8% to 10% of DLBCL in Asian countries, but seems to be less common in Western populations.

In this study we have characterized EBV-positive DLBCL cell lines by checking EBV latent gene and cellular gene expression. Then, we studied the modulation of EBV latent gene and cellular gene and the EBV modulated chemotaxis in the EBV-positive DLBCL lines upon cytokine treatment. We found that IL-4 and IL-21 upregulated LMP1 expression in EBV-positive DLBCL lines and IL-21 downregulated EBNA2 and EBNA1 expression in the type III line, Farage. IL-4 and IL-21 were found to induce different patterns of CXCR4 or CCR7 mediated chemotaxis in DLBCL lines. We also knocked out EBV from EBV-positive DLBCL lines and found that EBV provided survival factors to these lines. We further studied modulation of chemotaxis after downregulation of EBV encoded genes by dominant negative EBNA1 (dnEBNA1) in DLBCL cells upon cytokine treatment and observed decreased chemotaxis mediated by CXCR4 or CCR7 upon IL-4 or IL-21 treatment.

As IL-21was reported to induce apoptosis in DLBCL lines with unknown EBV status, we also examined the IL-21 sensitivity of the EBV positive type III DLBCL line, Farage, and found surprisingly that despite c-Myc upregulation, IL-21 induced cell proliferation rather than apoptosis. EBV knock-out counteracted the IL-21 induced proliferation of Farage and increased apoptosis. This finding reveals a previously unknown role of EBV in DLBCL that is of possible relevance for the current attempt to use IL-21 in therapy.

Studies on the EBV modulated chemotaxis after EBV infection on tonsillar B cells found downregulation of CXCR5 and CCR7 mediated chemotactic responses, which are important for migration into lymphoid tissue. These alterations may lead to retention of EBV-infected tonsillar B cells in the interfollicular region of the tonsil.

Further work on type I interferons (IFNs) identified their role in upregulation of LMP1 expression by direct activation of the ED-L1 promoter in several EBV-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulated LMP1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs.

Altogether our results not only provide evidence for the important roles of microenvironmental stimulation in EBV-carrying B cells but might also have future therapeutic implications.

List of scientific papers

I. Ehlin-Henriksson B, Wu L, Cagigi A, Mowafi F, Klein G, Nilsson A. Changes in chemokines and chemokine receptor expression on tonsillar B cells upon Epstein-Barr virus infection. Immunology. 2009 Aug; 127(4): 549-57.
https://doi.org/10.1111/j.1365-2567.2008.03029.x

II. Salamon D, Adori M, Ujvari D, Wu L, Kis LL, Madapura HS, Nagy N, Klein G, Klein E. Latency type-dependent modulation of Epstein-Barr virus-encoded latent membrane protein 1 expression by type I interferons in B cells. J Virol. 2012 Apr; 86(8): 4701-7.
https://doi.org/10.1128/JVI.06829-11

III. Wu L, Ehlin-Henriksson B, Zhu H, Ernberg I, Klein G. EBV counteracts IL-21-induced apoptosis in an EBV-positive diffuse large Bcell lymphoma cell line. Int J Cancer. 2013 Jan 30.

IV. Wu L, Ehlin-Henriksson B, Zhu H, Ernberg I, Kis LL and Klein G. EBV carrying DLBCL: what is the role of the virus? [Manuscript]