Epstein-Barr Virus Infection of Naïve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Abstract

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD− CD27+ switched memory (SM) B cells, not in IgD+ CD27− naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event

“…This idea is supported by the observation that EBV reactivation is often delayed until 2 to 3 months after transplant, concomitant with the onset of lymphopoiesis. Whether EBV drives the differentiation of emerging naive B cells, either by germinal center transit 1 or by inducing a memory phenotype via a GC-independent pathway, 54 or through the direct infection of memory B cells, 55 remains to be tested. For personal use only.…”

Cytomegalovirus-Seropositive Children Show Inhibition of In Vitro EBV Infection That Is Associated with CD8+CD57+ T Cell Enrichment and IFN-γ

“…*p , 0.05, **p , 0.01. T cell signals are needed to alter the Ig phenotype of the parental population (naive or non-switched memory) (39). Furthermore, previous studies found that all B cell subsets irrespective of Ig class and memory status have the same ability to be infected in vitro and that the proliferation rate of infected switched memory B cells was equal to that of naive cells (39,40).…”

“…T cell signals are needed to alter the Ig phenotype of the parental population (naive or non-switched memory) (39). Furthermore, previous studies found that all B cell subsets irrespective of Ig class and memory status have the same ability to be infected in vitro and that the proliferation rate of infected switched memory B cells was equal to that of naive cells (39,40). Thus, the emergence of a large IgD 2 CD27 + B cell population in EBV-infected cultures of older children likely represents acquisition of a memory phenotype by infected naive cells along with class-switching/downregulation of IgD, rather than preferential expansion of pre-existing CD27 + B cells.…”

Repression of the Proapoptotic CellularBIK/NBKGene by Epstein-Barr Virus Antagonizes Transforming Growth Factor β1-Induced B-Cell Apoptosis

“…Virus titrations were carried out by quantitative PCR (qPCR) as described previously (59). Primary B cells were positively selected from apheresis cones (National Health Service Blood and Transplant [NHSBT], Birmingham, United Kingdom) by using CD19 Dynabeads (Invitrogen), followed by a detachment step and then assessment for purity as described elsewhere (60). Isolated resting B cells were incubated with virus preparations at a multiplicity of infection (MOI) of 100.…”