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

✍ Scribed by R. Mendes; K.V. Bromelow; M. Westby; J. Galea-Lauri; I.E. Smith; M.E.R. O'Brien; B.E. Souberbielle

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 206 KB
• Volume: 39
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(20000101)39:1<72::aid-cyto10>3.0.co;2-r

✦ Synopsis

Background: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We 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 cytokine 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/ CD56ϩve NK population or on the CD3ϩ/CD56ϩ NK-T-cell population. Results: Detectable levels of tumour necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-␥ and TNF-␣ production in NK cells to a lesser extent than did PMA and ionomycin 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 CD3ϩCD56ϩ NK-T lymphocytes in patients with relevant diseases.