Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
Front Immunol. 2021 Sep 1;12:727952. doi: 10.3389/fimmu.2021.727952. eCollection 2021.
The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin and langerin), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.
人类肠道内含有大量单核吞噬细胞（MNP），包括常规树突状细胞（cDC）、巨噬细胞（Mf）和单核细胞等亚群，它们在肠道免疫系统和稳态中各自发挥独特的作用。如果我们要了解这些细胞在稳态、疾病状态和免疫治疗中的作用，那么从新鲜人体组织中分离和检测 MNP 的能力至关重要。然而，从组织中释放这些细胞存在问题，因为它们表达的许多关键表面识别标记物容易受到酶切的影响，而且它们非常容易死亡。此外，提取过程会引发免疫激活/成熟，从而改变其功能表型。因此，通过流式细胞术鉴定 MNP 的不断变化、复杂和高度异质的组合需要仔细选择释放活细胞并保留涉及抗体克隆选择的识别表位的酶混合物，以实现分析和分选用于功能测定。在这里，我们描述了一种从人空肠、回肠和结肠中分离粘膜和粘膜下层以及分离淋巴滤泡的解剖分离方法。我们还详细描述了获得功能不成熟和具有生物学功能的肠道 MNP 所需的优化酶消化方法。还提供了经过筛选的抗体克隆的综合列表，这些抗体克隆可用于开发高通量流式细胞术面板，以区分所有当前鉴定的人类组织 MNP 亚群，包括 pDC、cDC1、cDC2（朗格汉斯和朗格汉斯）、新描述的 DC3、单核细胞、Mf1、Mf2、Mf3 和 Mf4。我们还提出了一种新方法来解释组织巨噬细胞的自发荧光信号。最后，我们证明这些方法可以成功用于分选功能性、未成熟的肠道 DC，可用于功能测定，如细胞因子产生测定。
