Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, France.
Cytometry A. 2012 Apr;81(4):332-42. doi: 10.1002/cyto.a.22016. Epub 2012 Jan 25.
The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. © 2012 International Society for Advancement of Cytometry.
多色流式细胞术的发展为人类免疫学领域的重大进展做出了贡献。尽管使用这项技术对稀有细胞群进行了许多功能研究，但在这里我们使用多色流式细胞术来探索与先天免疫有关的复杂细胞系统的动态。我们使用活流感病毒刺激的 PBMC 作为实验模型。我们研究了 PBMC 的激活时间过程，其中包含 DC、单核细胞和 NK 细胞，所有这些细胞除了具有先天免疫特性外，还容易受到 Flu 感染。我们开发了 12 色面板，以同时研究这些细胞群中 IFN-α、TNF-α、IL-12、IL-6、IFN-γ、CD107 和流感病毒核蛋白的细胞内表达。这些面板允许在 DC 直接暴露于各种刺激或在 NK 细胞中通过间接 DC 介导的激活在复杂的细胞环境中，对诱导的激活标志物进行可重复的确定。使用少量细胞和减少的试剂数量的能力使我们能够进行动力学实验。多色流式细胞术与生物信息学工具相结合的能力使我们能够分析作为动态聚类图生成的多个功能数据。这些图谱提供了不同 PBMC 群体中诱导的激活事件的易于理解的视图。此外，它揭示了关于诱导的复杂途径的协调以及维持间接 DC 介导的 NK 细胞激活的细胞相互作用的新信息。我们的工作表明，多色流式细胞术是探索免疫和病毒学交叉点处未开发的复杂细胞系统的发现工具。 © 2012 国际细胞分析学会。
