Division of Theoretical Bioinformatics at German Cancer Research Center (DKFZ), Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, BioQuant Center, Heidelberg University, Heidelberg, Germany.
Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany.
Front Immunol. 2018 Aug 8;9:1840. doi: 10.3389/fimmu.2018.01840. eCollection 2018.
Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes perforin pores or by activation of caspases death receptors. In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.
自然杀伤 (NK) 细胞通过递送颗粒酶和穿孔素孔或通过激活半胱天冬酶和死亡受体来消除感染和致瘤细胞。为了了解 NK 细胞如何结合不同的细胞死亡机制，在单细胞水平上定量靶细胞反应非常重要。然而，目前现有的报告器不允许在同一细胞内测量几种蛋白酶的活性。在这里，我们提出了一种在 NK 细胞与靶细胞相互作用时同时在单个靶细胞内比较两种不同蛋白酶的策略。我们开发了含有 RIEAD 或 VGPD 切割位点的单荧光蛋白报告器，用于测量颗粒酶 B 的活性。我们表明，这两种颗粒酶 B 报告器可与 caspase-8 或 caspase-3 报告器联合使用。虽然我们没有发现 caspase-8 被颗粒酶 B 激活，但我们的方法表明 caspase-3 活性滞后于颗粒酶 B 活性约 6 分钟。最后，我们说明了几种不同的颗粒酶 A、M、K 和 H 报告器的比较。这里提出的方法是研究细胞毒性淋巴细胞介导的细胞死亡的时间演变的一种有价值的手段。
