Granzyme B (GzmB) is a serine protease best known for inducing target cell apoptosis when released by cytotoxic T lymphocytes (CTLs) or natural killer cells with pore-forming perforin. As a result, GzmB detected in the serum of virus-infected individuals has typically been attributed to these sources. Here, we show that patients with recently diagnosed infectious mononucleosis caused by Epstein-Barr virus (EBV) have high circulating levels of GzmB that may be derived from infected B cells early in course of disease. We recently reported that human B cells from healthy donors secrete active GzmB when stimulated in vitro through B-cell receptor (BCR) ligation and interleukin (IL)-21. We found that infecting B cells with EBV greatly amplified GzmB secretion in response to the same stimuli, but the expression was terminated once the infection had become latent. Our results represent a rare instance of GzmB expression by non-CTL/natural killer cells in the context of infection with a human pathogen.
Bibliographical noteFunding Information:
This project had ethics approval of the Peter MacCallum Cancer Centre (No. 12/73, age range 18–40 years), the QIMR Berghofer Medical Research and Uniting Care Health Human Research Ethics Committees, and by the Research Subjects Protection Program of the University of Minnesota. After obtaining informed consent, peripheral blood was taken from healthy donors or patients with recently diagnosed IM, as determined by detection of anti-VCA-IgM, or age-matched EBV+ healthy controls. Serum was collected and peripheral blood mononuclear cells were isolated by Ficoll density gradient. Primary B cells were cultured in RPMI-1640 supplemented with 10% FBS in 48-well plates at 1 × 106 cells per well per ml, if not stated otherwise. For infection with EBV, B cells were incubated for 2 h at 37 °C with B95.8 virus (kindly provided by Joanne Davis, Royal Melbourne Hospital, Melbourne, Australia), washed and plated as indicated in the figure legends. Human recombinant IL-21 (Invitrogen) was added at 50 ng ml−1. Polyclonal B-cell stimulation was achieved with affinity purified rabbit F(ab’)2 against human IgA+IgG+IgM (H+L) at 6.5 μgml−1 (anti-BCR; Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
We thank Vicki Milovac, Sophie Kotsakidis and Ralph Rossi for flow cytometry support, and Amanda Ross for help with recruiting blood donation volunteers at Peter MacCallum Cancer Centre. We thank Dr Joanne Davis (Royal Melbourne Hospital, Australia) for B95.8 virus. This work was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grant HA 6136/1-1 to MH, and by Program and Project Grants from the National Health and Medical Research Council of Australia to JAT and IV.
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