The study of EBV’s ability to convert resting B cells into immortalized lymphoblastoid cell lines (LCLs) is an important in vitro model for understanding EBV-driven lymphomagenesis. A long-standing mystery is that the EBV LMP2A oncogene, which mimics an activated B cell receptor (BCR), appears to be important for EBV lymphomas in vivo but not for LCL transformation in vitro. Using an EBV mutant that lacks the LMP2A gene (∆LMP2A-EBV), we are able to investigate the role of LMP2A in EBV transformed B cells. We are pursuing these aims through both biological mechanistic studies and bioinformatic analyses using Next Generation Sequencing (NGS) techniques.
In our biological studies, we aim to identify ways in which LMP2A or BCR isotype affect transformation, growth, and survivability of LCLs. A CRISPR screen by Ma Y., et al. (2014) indicated that BLNK and BTK, two downstream mediators of LMP2A and BCR antigen signaling are required for LCL outgrowth. Subsequent studies in our lab have showed that BLNK and BTK are essential for WT-LCLs but can be inhibited in ∆LMP2A-LCLs and still allow outgrowth.
Furthermore, we have identified a key phenotypic difference between our WT- and ∆LMP2A-LCLs. B cells transformed by wildtype EBV resemble an Activated B-cell DLBCL (ABC-DLBCL) and tend to express the IgM BCR, whereas B cell transformed by ∆LMP2A-EBV resemble a Germinal Center DLBCL (GC-DLBCL) and tend to express the IgG BCR.
Additional studies continue to elucidate the perceived preference for BCR isotype by EBV transformation and proliferation. Preliminary studies indicate that IgM expressing B cells are capable of being transformed by ∆LMP2A-EBV, but the demands of proliferation outweigh the production capacity of the cell resulting in a largely apoptotic population.
Our results suggest that LMP2A, while not absolutely essential for transformation, makes crucial qualitative contribution to the lymphoma phenotype.
~ Makoto Ohashi
In our transcriptomic analysis, we identify ways in which LMP2A and BCR isotype associated affect differ from each other. Previous transcriptomic studies of WT and ∆LMP2A-LCLs failed to consider the difference in BCR isotype expression of their samples. Through a reanalysis of previously published data, we confirmed that other labs were experiencing the same trend where ΔLMP2A-LCLs express the IgG-BCR instead of the IgM-BCR which is typical of LCLs transformed by wildtype EBV. This suggests that some of the activities previously ascribed to LMP2A may be due to BCR isotype differences.
To distinguish the direct effects of LMP2A on LCL gene expression from those mediated by BCR-isotype, we performed RNA-seq on three populations of LCLs: WT-LCLs (with either IgM-BCR or IgG-BCR) and ΔLMP2A-LCLs (IgG-BCR). Our results showed that nearly 40% of putative LMP2A upregulated genes and 36% of downregulated genes are in fact differentially expressed due to differences associated with BCR isotype.
This analysis allowed the identification of genes regulated by LMP2A regardless of BCR isotype, which are candidates for mediators of LMP2A effects in EBV+ lymphomas. We have identified a strong plasma-like signature in the LMP2A expressing cells regardless of BCR isotype indicating that LMP2A may be overriding the natural tendencies of memory B cells with specific BCR isotypes. Additionally, the analysis of BCR-isotype associated effects in WT-LCLs has shown that IgM expressing B cells appear to be poised in a pro-apoptotic state with BIM as the effector, evidence that is further supported by our phenotypic observations of IgM expressing LCLs.
These findings indicate that LMP2A effects and BCR isotype associated effects are independent and such differences should be considered when assessing LMP2A’s role in lymphomagenesis.
~ Mariah Riel
