TY - JOUR
T1 - Optimal isolation protocols for examining and interrogating mononuclear phagocytes from human intestinal tissue
AU - Doyle, Chloe M.
AU - Vine, Erica E.
AU - Bertram, Kirstie M.
AU - Baharlou, Heeva
AU - Rhodes, Jake W.
AU - Dervish, Suat
AU - Gosselink, Martijn P.
AU - Di Re, Angelina
AU - Collins, Geoffrey P.
AU - Reza, Faizur
AU - Toh, James W. T.
AU - Pathma-Nathan, Nimalan
AU - Ahlenstiel, Golo
AU - Ctercteko, Grahame
AU - Cunningham, Anthony L.
AU - Harman, Andrew N.
AU - Byrne, Scott N.
N1 - Publisher Copyright:
© Copyright © 2021 Doyle, Vine, Bertram, Baharlou, Rhodes, Dervish, Gosselink, Di Re, Collins, Reza, Toh, Pathma-Nathan, Ahlenstiel, Ctercteko, Cunningham, Harman and Byrne.
PY - 2021/9/1
Y1 - 2021/9/1
N2 - 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.
AB - 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.
UR - https://hdl.handle.net/1959.7/uws:78068
U2 - 10.3389/fimmu.2021.727952
DO - 10.3389/fimmu.2021.727952
M3 - Article
C2 - 34566985
SN - 1664-3224
VL - 12
JO - Frontiers in Immunology
JF - Frontiers in Immunology
M1 - 727952
ER -