Vu Manh Thien-Phong, Dalod Marc
Centre d'Immunologie de Marseille-Luminy, UNIV UM2, Aix Marseille Université, 163 Avenue de Luminy, 13288, Marseille, France.
U1104, INSERM, Marseille, France.
Methods Mol Biol. 2016;1423:211-43. doi: 10.1007/978-1-4939-3606-9_16.
Dendritic cells (DCs) are immune sentinels of the body and play a key role in the orchestration of the communication between the innate and the adaptive immune systems. DCs can polarize innate and adaptive immunity toward a variety of functions, sometimes with opposite roles in the overall control of immune responses (e.g., tolerance or immunosuppression versus immunity) or in the balance between various defense mechanisms promoting the control of different types of pathogens (e.g., antiviral versus antibacterial versus anti-worm immunity). These multiple DC functions result both from the plasticity of individual DC to exert different activities and from the existence of various DC subsets specialized in distinct functions. Functional genomics represents a powerful, unbiased, approach to better characterize these two levels of DC plasticity and to decipher its molecular regulation. Indeed, more and more experimental immunologists are generating high-throughput data in order to better characterize different states of DC based, for example, on their belonging to a specific subpopulation and/or on their exposure to specific stimuli and/or on their ability to exert a specific function. However, the interpretation of this wealth of data is severely hampered by the bottleneck of their bioinformatics analysis. Indeed, most experimental immunologists lack advanced computational or bioinformatics expertise and do not know how to translate raw gene expression data into potential biological meaning. Moreover, subcontracting such analyses is generally disappointing or financially not sustainable, since companies generally propose canonical analysis pipelines that are often unadapted for the structure of the data to analyze or for the precise type of questions asked. Hence, there is an important need of democratization of the bioinformatics analyses of gene expression profiling studies, in order to accelerate interpretation of the results by the researchers at the origin of the research project, of the data and who know best the underlying biology. This chapter will focus on the analysis of DC subset transcriptomes as measured by microarrays. We will show that simple bioinformatics procedures, applied one after the other in the framework of a pipeline, can lead to the characterization of DC subsets. We will develop two tutorials based on the reanalysis of public gene expression data. The first tutorial aims at illustrating a strategy for establishing the identity of DC subsets studied in a novel context, here their in vitro generation in cultures of human CD34(+) hematopoietic progenitors. The second tutorial aims at illustrating how to perform a posteriori bioinformatics analyses in order to evaluate the risk of contamination or of improper identification of DC subsets during preparation of biological samples, such that this information is taken into account in the final interpretation of the data and can eventually help to redesign the sampling strategy.
树突状细胞(DCs)是机体的免疫哨兵,在协调固有免疫系统和适应性免疫系统之间的通讯中发挥关键作用。DCs可使固有免疫和适应性免疫向多种功能极化,有时在免疫反应的整体控制中发挥相反作用(例如,耐受性或免疫抑制与免疫),或在促进控制不同类型病原体的各种防御机制之间的平衡中发挥相反作用(例如,抗病毒免疫与抗菌免疫与抗蠕虫免疫)。DCs的这些多种功能既源于单个DC发挥不同活性的可塑性,也源于存在专门执行不同功能的各种DC亚群。功能基因组学是一种强大的、无偏倚的方法,可更好地表征DC可塑性的这两个层面并破译其分子调控机制。事实上,越来越多的实验免疫学家正在生成高通量数据,以便例如基于DC属于特定亚群、暴露于特定刺激或发挥特定功能的能力,更好地表征DC的不同状态。然而,这些大量数据的解读因生物信息学分析的瓶颈而受到严重阻碍。实际上,大多数实验免疫学家缺乏先进的计算或生物信息学专业知识,不知道如何将原始基因表达数据转化为潜在的生物学意义。此外,将此类分析外包通常令人失望或在经济上不可持续,因为公司通常提供的标准分析流程往往不适用于要分析的数据结构或所提问题的精确类型。因此,迫切需要使基因表达谱研究的生物信息学分析民主化,以便加速研究项目的发起者、数据的研究者以及最了解基础生物学的人员对结果的解读。本章将重点分析通过微阵列测量的DC亚群转录组。我们将表明,在一个流程框架内依次应用简单的生物信息学程序,可实现对DC亚群的表征。我们将基于对公共基因表达数据的重新分析开发两个教程。第一个教程旨在说明一种策略,用于确定在新背景下研究的DC亚群的身份,这里指在人CD34(+)造血祖细胞培养物中的体外生成。第二个教程旨在说明如何进行事后生物信息学分析,以评估在生物样品制备过程中DC亚群受到污染或鉴定不当的风险,以便在数据的最终解读中考虑这些信息,并最终有助于重新设计采样策略。
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