Lion E, Smits E L J M, Berneman Z N, Van Tendeloo V F I
Vaccine and Infectious Disease Institute, Laboratory of Experimental Hematology, University of Antwerp, Wilrijkstraat 10, Antwerp B-2650, Belgium.
J Immunol Methods. 2009 Oct 31;350(1-2):89-96. doi: 10.1016/j.jim.2009.08.014. Epub 2009 Sep 4.
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.
鉴于自然杀伤（NK）细胞释放的干扰素（IFN）-γ在连接先天免疫系统和适应性免疫系统方面的重要性，它已成为主要的研究热点。有趣的是，关于肿瘤细胞刺激NK细胞的报道显示，在何种刺激可诱导IFN-γ产生方面存在不同的数据。在此，问题仍然是肿瘤细胞识别是否足以触发IFN-γ，或者是否需要第二个信号，如I型干扰素。虽然IFN-γ检测方法大量用于外周血单核细胞或纯化的T细胞组分作为反应细胞群体，但关于这些检测方法与纯化NK细胞比较的可用数据有限。在本研究中，我们通过使用三种不同检测方法测量IFN-γ，评估了用一种信号（肿瘤细胞或IFN-α）和两种信号（肿瘤细胞+IFN-α）刺激人纯化的静息外周血NK细胞之间的关系。我们对每个供体并行进行酶联免疫吸附测定（ELISA）、酶联免疫斑点（ELISPOT）测定和细胞内细胞因子染色（ICS）测定，并确定这些检测方法之间是否存在相关性。我们的结果表明，与单信号刺激相比，人静息NK细胞的双信号刺激诱导产生的IFN-γ明显更多，所有检测方法都能轻易检测到。此外，统计分析表明，在双信号刺激后产生的IFN-γ方面，这些检测方法之间存在正相关。重要的是，我们表明单独的肿瘤细胞刺激足以触发IFN-γ的分泌，但这一发现仅通过ELISPOT得到证实。这些结果表明，IFN-γ检测方法的选择可显著影响关于肿瘤细胞诱导NK细胞IFN-γ的结果。
