Infection of Primary Human Tonsillar B Cell by KSHV

Kaposis sarcoma associated herpesvirus (KSHV/HHV8) latently infects B cells and, in immunocompromised persons, this infection can result in two distinct tumors of B cell origin: multicentric Castlemans disease (MCD) and primary effusion lymphoma (PEL). Identifying the original type of B cell infected by KSHV is critical to understanding not only viral transmission, but also the mechanisms by which infected cells become tumor cells. Based on evidence for salivary transmission of KSHV and the presence of KSHV transcripts in specific regions of tonsils isolated from infected individuals, we hypothesized that KSHV enters the host by infecting a subset of tonsillar B cells. We also predicted that if these cells were precursors of MCD and/or PEL, they might share some similarities with KSHV-associated B cell tumors. To test this idea, we exposed cultures of tonsillar mononuclear cells to KSHV and used multispectral imaging fluorescence cytometry (MIFC) to analyze the phenotype of rare latently infected B cells. While we found evidence that KSHV enters and initiates gene transcription in a variety of tonsil cell types, only lambda light chain expressing B cells were susceptible to latent KSHV infection. Furthermore, infected B cells expressed surface and cytoplasmic IgM, as well as CD27, remarkably similar to the MCD B cell phenotype. We also found that KSHV-infected B cells had increased nuclearcytoplasmic (N/C) ratio and that this ratio correlated positively with intracellular 5 viral load. The majority of de novo infected B cells were proliferating, evidenced by increased size, DNA content, and expression of Ki67, all of which positively correlated with intracellular viral load as well. Finally, KSHV-infected tonsillar B cells expressed the interleukin-6 receptor (IL-6R), with expression correlating with viral load, and exogenous IL-6 administration resulted in increased blasting transformation of KSHV-infected B cells. While multiple subsets of human tonsillar cells were permissive to KSHV entry, only IgM-lambda B cells displayed consistent LANA+ latent infection. In addition to revealing the subset of B cells that is susceptible to KSHV latent infection, as well as identifying the likely cell of origin of MCD, these findings demonstrate that in vitro analysis of KSHV-mediated B cell transformation is feasible.

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