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.

Early onset post-transplant lymphoproliferative disease (PTLD) tumours resemble Epstein-Barr virus (EBV)-positive in vitro transformed lymphoblastoid cell lines (LCLs) both in cellular phenotype and in EBV latent gene expression. Here viral transformation appears to be sufficient for tumour growth. However, in cases of late onset PTLD and Hodgkin’s disease (HD) where viral gene expression is more restricted and tumour evolution has involved additional cellular genetic changes, the role of EBV in pathogenesis is not certain. Many cases of PTLD and HD lack surface Ig and have functionally inactivated immunoglobulin (Ig) sequences. They appear to arise from atypical post-germinal centre (GC) B cells which have acquired crippling mutations in the Ig genes during GC transit. Such crippled GC cells normally die by apoptosis but if rescued may form a pool of cells particularly prone to tumour development. In the present work we investigated if EBV infection in vitro can transform crippled GC cells. GC cells isolated by FACS sorting CD10+ tonsillar B cells were infected with EBV (B95.8 strain) in vitro and seeded at limiting dilutions onto fibroblast feeders to establish cell lines. In parallel experiments, naïve and memory B cells, isolated from peripheral blood by FACS sorting of IgD+, CD27− and IgD−, CD27+ subsets respectively, were infected with EBV in vitro to establish LCLs. Over 100 GC-derived cell lines (GC-LCLs) and a similar number of peripheral blood-derived LCLs were established. All peripheral blood-derived LCLs consistently expressed surface Ig (predominantly IgM+, IgD+ co-expression in naïve and IgG+ or IgA+ expression in memory transformants). In comparison, whilst most GC-LCLs expressed surface Ig, we identified six lines that lacked surface Ig expression. Sequencing the variable regions of the IgH genes of the six surface Ig-negative GC-LCLs confirmed that three lines carried hypermutated but non-functional IgH genes. There was a 7 nucleotide insertion resulting in a frameshift of the downstream IgH sequence in one cell line, while the other two carried mutated Ig genes containing stop codons in the CDR1 and/or the CDR2 regions of the IgH sequence. In the three other surface Ig-negative GC-LCLs, the IgH reading frame was mutated but still intact. Sequence inactivation was not seen in any of the surface Ig-positive GC-LCLs analysed in parallel nor, in any of the peripheral blood derived LCLs. Interestingly, GC-derived transformants lost their original GC phenotype and acquired activation markers typical of conventional LCLs. Thus they were CD10−, CD23+, CD27+ and CD38+. Analysis of EBV gene expression showed that GC-LCLs including the six surface Ig-negative lines expressed EBNA1, EBNA2, LMP1 and LMP2 as seen in conventional LCLs. The present work provides strong evidence that EBV infection in vitro can rescue crippled GC cells, leading to the outgrowth of surface Ig-negative LCLs with functionally inactivated IgH alleles. These lines appear to be in vitro correlates of PTLD both in their cellular phenotype and in the expression of EBV latent proteins. Though the cellular phenotype and EBV latent antigen expression pattern is different in HD, it is possible that these tumours arise by different pathways from the same pool of atypical post-GC survivors.