Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue

Frontiers in Immunology

Golo Ahlenstiel²  Martijn P. Gosselink⁵  Angelina Di Re⁵  Geoffrey P. Collins⁵  Faizur Reza⁵  Grahame Ctercteko⁵  Nimalan Pathma-Nathan⁵  James W. T. Toh⁵  Andrew N. Harman⁶  Scott N. Byrne⁶  Jake W. Rhodes⁶  Chloe M. Doyle⁸  Heeva Baharlou⁸  Anthony L. Cunningham⁸  Erica E. Vine⁸  Kirstie M. Bertram⁸  Suat Dervish⁹ 

[1] Blacktown Clinical School, Western Sydney University, Blacktown, NSW, Australia;Blacktown Hospital, Western Sydney Local Area Health District (WSLHD), Blacktown, NSW, Australia;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;Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia;School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia;Storr Liver Centre, The Westmead Institute for Medical Research, Westmead, NSW, Australia;Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia;Westmead Cytometry, The Westmead Institute for Medical Research, Westmead, NSW, Australia;

关键词: dendritic cells (DC);    flow cytometry;    human tissue;    intestine;    enzymatic digestion;    macrophage – cell;   

DOI  :  10.3389/fimmu.2021.727952

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown