Maquet Céline M, Gillet Laurent, Machiels Bénédicte D
Laboratory of Immunology and Vaccinology, Faculty of Veterinary Medicine, FARAH, University of Liège, Liège, Belgium.
Bio Protoc. 2023 Sep 20;13(18):e4815. doi: 10.21769/BioProtoc.4815.
Gammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4 T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4 T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4 lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4 T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4 T-cell regulation to prevent the development of virus-induced immunopathologies in the lung. Key features • High-resolution profiling of the functional properties of lung-infiltrating CD4 T cells after viral infection using conventional multiparametric flow cytometry. • Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining. • Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4 T cells. • Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation.
γ疱疹病毒，如爱泼斯坦-巴尔病毒（EBV），是其宿主免疫反应的主要调节因子。在相关研究（PMID：35857578）中，我们以鼠γ疱疹病毒感染（鼠γ疱疹病毒4型）作为人类EBV的模型，研究了Ly6Chi单核细胞在塑造效应性CD4 T细胞功能中的作用。为了解析CD4 T细胞亚群的多功能特性，我们使用多参数流式细胞术对肺细胞进行细胞内染色。因此，我们在此开发了一种细胞内染色工作流程，以在单细胞水平上识别同一样本中CD4淋巴细胞的细胞毒性和/或调节特性。简要地说，在对肺进行灌注、收集、消化和过滤以获得单细胞悬液后，将肺细胞与蛋白质转运抑制剂和特异性刺激培养基一起培养4小时，以积累感兴趣的细胞因子和/或细胞毒性颗粒。在进行多色表面标记、固定和轻度通透处理后，对肺细胞进行胞质内抗原染色，并用Fortessa 4激光细胞仪进行分析。这种通过流式细胞术定量细胞毒性介质以及促炎或抗炎细胞因子的方法，使我们能够高分辨率地解读病毒感染后募集的肺CD4 T细胞的功能异质性。因此，该分析有助于更好地理解CD4 T细胞调节在预防肺部病毒诱导的免疫病理发展中的重要性。关键特性• 使用传统多参数流式细胞术对病毒感染后肺浸润CD4 T细胞的功能特性进行高分辨率分析。• 小鼠肺解剖、单细胞悬液制备以及多色表面/细胞内染色设置的详细方案。• 用于研究肺浸润CD4 T细胞功能极化和细胞因子产生的最佳体外再刺激条件总结。• 确保可靠数据分析和解释的必要生物学和技术对照的综合汇编。
