title: Quantification of IFN-gamma produced by human purified NK cells following tumor cell stimulation: comparison of three IFN-gamma assays.
abstract: Interferon (IFN)-gamma 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-gamma production. Here, the question remains whether tumor cell recognition is sufficient to trigger IFN-gamma or whether a second signal is required such as type I IFN. While IFN-gamma 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-alpha) and two (tumor cell+IFN-alpha) signals by measuring IFN-gamma 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-gamma 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-gamma produced following two-signal stimulation. Importantly, we show that tumor cell stimulation alone is enough to trigger secretion of IFN-gamma, but this finding was only evidenced by ELISPOT. These results reveal that the choice of IFN-gamma detection method can markedly influence the outcome regarding induction of NK cell IFN-gamma by tumor cells.
authors.affiliation: Vaccine and Infectious Disease Institute, Laboratory of Experimental Hematology, University of Antwerp, Wilrijkstraat 10, Antwerp B-2650, Belgium. eva.lion@ua.ac.be
authors.forename: E
authors.initials: E
authors.lastname: Lion
authors.forename: E L J M
authors.initials: EL
authors.lastname: Smits
authors.forename: Z N
authors.initials: ZN
authors.lastname: Berneman
authors.forename: V F I
authors.initials: VF
authors.lastname: Van Tendeloo
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D007371:Interferon-gamma