Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes.
Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes.
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DOI：
10.1016/j.bbrc.2017.08.045
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发表时间：
2017-10
影响因子：
3.1
通讯作者：
A. Takagi;Yutaka Horiuchi;M. Matsui
中科院分区：
文献类型：
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作者：
A. Takagi;Yutaka Horiuchi;M. Matsui
几种非放射性方法已被广泛用于检测抗原特异性细胞毒性T淋巴细胞（CTL）反应，而不是经典的51cr释放法。这些方法包括细胞内细胞因子染色、主要组织相容性复合体I类四聚体和CD107a动员试验。然而，它们不能直接测量靶细胞的死亡。相比之下，已经有几次尝试开发流式细胞术CTL （FC-CTL）检测来评估细胞毒性。然而，要使其标准化，还需要进一步的改进。在这里，我们利用表达绿色荧光蛋白（GFP）的靶细胞系评估了基于碘化丙啶（PI）摄取的FC-CTL测定的特性。发现FC-CTL检测足够敏感，可以检测原发性CTL反应。使用预先建立的表达gfp的靶细胞系简化了分析过程，并能够明确区分靶细胞和效应细胞。时间过程分析表明，在抗原刺激后，pi染色的靶细胞早在表面CD107a表达时就被检测到。因此，基于PI/ gfp的FC-CTL检测具有足够的灵敏度，可以实际检测靶细胞死亡的早期阶段，并且可能有很大的潜力成为测量CTL活性的标准工具。
Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical51Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.