Flow cytometric visualisation of cytokine production by CD3-CD56+NK cells and CD3+CD56+NK-T cells in whole blood

Background: Natural killer (NK) cells produce multiple cytokines with poten tial immune regulatory roles. Wie standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. Methods: With a three-colour fluorescent labelling technique, specific cyto kine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK populat ion or on the CD3+/CD56+ NK-T-cell population. Results: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were e asily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Bre feldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibo dy and incubation with K562, the NK-sensitive cell Line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and i onomycin stimulation. Conclusions: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3CD56+ NK-T lymphocytes in patients with relevant diseases. (C) 2000 Wiley-L iss, Inc.