KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation
Lynn M. Hassman, Thomas J. Ellison, Dean H. Kedes
Lynn M. Hassman, Thomas J. Ellison, Dean H. Kedes
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Research Article

KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation

Abstract

Kaposi sarcoma–associated herpesvirus (KSHV; also known as HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). However, little is known about the nature of the specific B cell subtype(s) most susceptible to infection. Identifying these cells would provide direct insight into KSHV transmission and virus-induced transformation. To identify this subset and to determine whether infection alters its cellular phenotype, we exposed human tonsillar cells to KSHV and characterized infected cells using high-throughput multispectral imaging flow cytometry (MIFC). Stable expression of the virally encoded latency-associated nuclear antigen (LANA), a marker of latent KSHV infection, was observed predominantly in cells expressing the l light chain of the B cell receptor. These LANA+ B cells proliferated and exhibited similarities to the cells characteristic of MCD (IgMl-expressing plasmablasts), including blasting morphology with elevated expression of Ki67, variable expression of CD27, and high levels of IgM and IL-6 receptor. Furthermore, the proportion of infected cells showing a blasting phenotype increased upon addition of exogenous IL-6. Our data lead us to propose that oral transmission of KSHV involves the latent infection of a subset of tonsillar IgMl-expressing B cells, which then proliferate as they acquire the plasmablast phenotype characteristic of MCD.

Authors

Lynn M. Hassman, Thomas J. Ellison, Dean H. Kedes

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Figure 4

Growth and proliferative effects of KSHV on latently infected B cells.

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Growth and proliferative effects of KSHV on latently infected B cells. T...
Tonsil cell suspensions were exposed to KSHV and analyzed for growth and proliferation by assessment of the area (of bright-field image) and DNA content (intensity of DAPI staining) of individual cells, respectively, at 48–84 hpi. (A) Representative scatter plot of area by DNA content (left), and images of representative cells from each gate (right). Three populations were evident, differing in size and/or DNA content: small (<110 μm2, gate R, resting), large (>110 μm2, gate B, blasting), and large with increased DNA content (gate D, dividing). (B) Distribution of these 3 cell types within LANA– (gray bars) and LANA+ (black bars) cells in cultures exposed to KSHV. Data represent mean ± SEM of 7 tonsil donors. (C) LANA+ cells were gated according to their intracellular viral load, with groups containing 1–2 (light gray), 3–4 (dark gray), or ≥5 (black) LANA dots/cell. Percentages of resting, blasting, and dividing cells were determined for 7 tonsil donors. P values refer to Spearman correlations, which were –0.937 for resting, 0.619 for blasting, and 0.37 for dividing. (D) Distribution of resting, blasting, and dividing cells within λ B cells unexposed (white bars) or exposed (gray bars) to KSHV for 7 tonsil donors. (E) Percentage of λ (squares), κ (diamonds), and non-B (circles) cells that incorporated the nucleoside EdU following exposure to increasing MOI of KSHV (left) or UV-inactivated KSHV (right). Results shown are representative of 3 different experiments.