Spontaneous EBV-reactivation during in vitro B-cell differentiation: a model for polymorphic EBV-driven lymphoproliferations studied with targeted single cell expression analysis

Epstein Barr Virus (EBV) driven B-cell neoplasms arise from reactivation of latently infected B-cells. In a subset of patients EBV drives a polymorphous lymphoproliferative disorder (LPD) in which B-cell differentiation is retained. Spontaneous EBV reactivation following B-cell mitogen stimulation provides a potential model of polymorphic EBV-driven LPD. We have developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B-cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation we analyzed 8 differentiations at the PC stage, with a targeted single cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B-cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency ranging from 3-28% of cells. Most EBV-associated cells expressed PC genes such as XBP1 or MZB1 and in all samples these included a quiescent PC fraction lacking cell cycle gene expression. With increasing EBV-associated cells, populations with B-cell features became prominent, co-expressing germinal centre (GC) and activated B-cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained MS4A1/CD20 expression and IGHM and IGHD co-expression, while IGHM only and class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation are consistent with recently proposed models relating EBV-mediated transformation in lymphoblastoid cell lines to features of GC B-cells, and suggest a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B-cells. Overall design: Single cell experiments were carried out using the BD Rhapsody Express Single-cell Analysis System. Cryopreserved cells were thawed and dead cells removed by lymphoprep density gradient centrifugation. Cells were enumerated on a hemocytometer. The cells from individual donors were labelled with a unique sample tag identifier using the BD Single-Cell Multiplexing Kit. Donors (n=8) were pooled in equal ratios, with the aim of loading 4000-5000 cells per donor onto a cartridge. Donors 1-4 and 5-8 were run on independent cartridges using a custom designed 701 gene panel.