Emily Heath

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.

Epstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD(-)CD27(+)) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD(+)CD27(-)) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non-isotype-switched (IgD(+)CD27(+)) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.