Quantification of IFN-γ produced by human purified NK cells following tumor cell stimulation: Comparison of three IFN-γ assays

• Published in: Journal of immunological methods Vol. 350; no. 1; pp. 89 - 96
• Main Authors: Lion, E., Smits, E.L.J.M., Berneman, Z.N., Van Tendeloo, V.F.I.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 31.10.2009; Elsevier
• Subjects: Antiviral Agents - pharmacology; Biological and medical sciences; Biological Assay - methods; Coculture Techniques; ELISA; ELISPOT; Enzyme-Linked Immunosorbent Assay - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IFN-γ; Interferon-alpha - pharmacology; Interferon-gamma - analysis; Interferon-gamma - immunology; Intracellular cytokine staining; K562; K562 Cells; Killer Cells, Natural - cytology; Killer Cells, Natural - immunology; Molecular immunology; NK cells; Sensitivity and Specificity; Signal Transduction - drug effects; Signal Transduction - immunology; Techniques; Intracellular cytokine staining; NK cells; IFN-γ; K562; ELISPOT; ELISA; Human; Cytokine; Staining; Immunological method; Natural killer cell; ELISPOT assay; Intracellular; ELISA assay; Tumor cell; Quantitative analysis; Gamma interferon
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2009.08.014

Interferon (IFN)-γ released by natural killer (NK) cells has become a subject of major interest, given its importance in bridging the innate and adaptive immune system. Interestingly, reports concerning tumor cell stimulation of NK cells show divergent data on which stimuli induce IFN-γ production. Here, the question remains whether tumor cell recognition is sufficient to trigger IFN-γ or whether a second signal is required such as type I IFN. While IFN-γ detection methods are abundantly used with peripheral blood mononuclear cells or purified T cell fractions as responder populations, only limited data is available about comparison of these assays with purified NK cells. In this study, we assessed the relationship between stimulation of human purified resting peripheral blood NK cells with one (tumor cell or IFN-α) and two (tumor cell + IFN-α) signals by measuring IFN-γ using three different assays. We performed the enzyme-linked immunosorbent assay (ELISA), the enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining (ICS) assay in parallel per donor and determined whether there was a correlation between these assays. Our results show that two-signal stimulation of human resting NK cells induces significantly more IFN-γ as compared to one-signal stimulation, readily picked up by all assays. Moreover, statistical analysis points towards a positive correlation between these assays for IFN-γ produced following two-signal stimulation. Importantly, we show that tumor cell stimulation alone is enough to trigger secretion of IFN-γ, but this finding was only evidenced by ELISPOT. These results reveal that the choice of IFN-γ detection method can markedly influence the outcome regarding induction of NK cell IFN-γ by tumor cells.