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

Abstract

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.

Identification of CX3CR1+ mononuclear phagocyte subsets involved in HIV-1 and SIV colorectal transmission

“…Conflicting results have been reported concerning CD14 expression by intestinal Mφs, which may possibly reflect varying conditions under which tissues were obtained. A number of studies described colonic Mφs being mostly CD14 − cells ( Caër and Wick, 2020 ; P. Smith et al., 1997a , 1997b ; Smythies et al., 2005 ), whereas other groups detected an abundant population of CD14 + Mφs ( Bernardo et al., 2016 , 2018 ; Bujko et al., 2018 ), which include tissue resident autofluorescent Mφs ( Doyle et al., 2021 ; Haniffa et al., 2009 ). Here we show that CD14 − Mφs outnumbered the CD14 + ones in the macaque colonic mucosa at steady-state.…”

OMIP 082: A 25‐color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal‐associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue

“…Gating strategy of the 25‐color measurement of innate lymphoid cells (ILC), mucosal associated invariant T (MAIT) cells, natural killer (NK) cells, and γδ T cells. (A) Cells from fresh healthy human intestinal tissue were extracted using our optimized isolation protocol [20], stained, and then acquired on a BD FACSymphony A5 instrument 1‐day post‐staining. Cells were gated on live (FVS 700 − ) single cells (FSC‐area vs. FSC‐height).…”

“…Identification of the optimal enzyme for the isolation of innate lymphoid cells from human tissue. Healthy human colon was split into roughly seven equal parts and digested with a range of different enzymes: Blend F, collagenase type I, type II, type IV, type D, P and the tumor dissociation kit (TDK), human (Miltenyi Biotec) as per our optimized isolation protocol [20]. The cell suspension was stained with the 25‐color phenotyping panel and acquired on a BD FACSymphony A5 instrument 1‐day post‐staining.…”

“…The main barrier to identifying tissue‐resident ILCs is obtaining a viable single‐cell suspension with high yield while minimizing cell surface receptor cleavage to detect key surface antigens. Our lab has developed isolation methods to obtain immune cell suspensions from human intestine and skin to interrogate for flow cytometry [20–24]. The first critical step when isolating cells by enzymatic digestion is to determine whether the enzyme used cleaves the cell surface markers needed for immunophenotyping and identify antibody clones that work following enzyme digestion (Figure 1).…”

“…The main barrier to identifying tissue-resident ILCs is obtaining a viable single-cell suspension with high yield while minimizing cell surface receptor cleavage to detect key surface antigens. Our lab has developed isolation methods to obtain immune cell suspensions from human intestine and skin to interrogate for flow cytometry [20][21][22][23][24].…”