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

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.

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,

INTRODUCTION
Intestinal mononuclear phagocytes (MNPs), specifically dendritic cells (DC), macrophages and monocytes, play a major role in maintaining tolerance to food-derived antigens and resident microbiota without compromising the hosts ability to respond to invading pathogens (1). DCs are professional antigen presenting cells (APC) capable of antigen uptake, processing and migration out of the tissue to draining lymph nodes where they present antigens and activate naïve T cells (2,3). In contrast, macrophages perform this antigen presenting function weakly and do not readily migrate out of the tissue. Their primary function is to phagocytose and destroy invading pathogens as well as secrete a variety of immune-modulating cytokines (2,3). Monocytes migrate into tissue from blood and differentiate into monocyte-derived macrophages or DCs. Embedded within these broad cell types are a diverse range of DC and macrophage subsets that each possess their own tissuespecific phenotype and function. Accurate identification of these subsets in the human intestine is required before we can understand their role in homeostasis and disease settings. Indeed, the misidentification of MNP subsets has caused much confusion in the literature (4)(5)(6)(7)(8).
Isolating MNPs from human tissues is a challenging task as the techniques used to liberate them can alter their phenotype and affect their viability (9). We have previously published optimised methods for isolating and interrogating immature DCs and macrophages from human abdominal skin via enzymatic digestion (9). However, skin and intestinal tissue have marked phenotypic, functional and structural differences which necessitates a modified approach to isolating MNPs. Recent literature has described a suite of consistent markers used to define all currently known subsets of human tissue DCs and macrophages (8)(9)(10)(11)(12)(13). In human abdominal skin, these comprise XCR1 + cDC1 (conventional DC), CD1c + cDC2 that includes langerin expressing and langerin negative populations and CD14-expressing cells including tissue-resident autofluorescent (AF) macrophages and monocyte-derived macrophages (13). In human intestinal tissue, three populations of DCs have been identified using CD103 and SIRPa (11,12) and four intestinal CD14 + macrophage populations (Mf1-4) have been identified using HLA-DR, CD14, CD11c and CD11b (10). More recently, high-resolution analyses have revealed a subpopulation within human blood cDC2s (CD1c + DCs) that expresses CD14 and a monocyte-like gene signature termed DC3s (14,15). As defined by the literature, DC3s are CD88 -CD1c + CD163 + and express varying levels of CD14 (14,15).
In this study, we present an intestinal tissue specific MNP isolation protocol to liberate high yields of viable, immature and biologically active MNPs from human intestinal jejunum, ileum and colon as well as terminal ileum biopsies. We also present techniques to anatomically separate the mucosa and submucosa, including their associated lymphoid follicles being Peyer's Patches in the small bowel and lymphoid aggregates in the large bowel, to better understand these distinct immune compartments. We emphasize the importance of carefully selecting antibodies that target the appropriate epitope postdigestion as well as markers that accurately define intestinalderived MNPs according to the most recent and reliable literature. Further, we present a high-parameter flow cytometry gating strategy to identify all currently known human MNPs in human tissues. We also include a method for correcting AF spillover from tissue-resident macrophages which considerably improves the accuracy of measuring cell surface expression levels and correct MNP definition.

Human Specimens
This study was approved by the Western Sydney Local Area Health District (WSLHD) Human Research Ethics Committee (HREC); reference number (4192) AU RED HREC/15 WMEAD/ 11. Large human intestinal specimens were taken with informed consent from patients undergoing surgery for intestinal cancer, 10-20 cm away from tumours, where present. Samples were processed within 2 hours of collection except for samples destined for cell sorting which were covered in Roswell Park Memorial Institute (RPMI) (Lonza, Switzerland) 1640 supplemented with 0.25% gentamycin and stored overnight at 4°C for processing the following morning.

Tissue Processing
Typical whole tissue intestinal specimens ranged in size from 5-40cm 2 , with all data obtained with whole tissue specimens unless biopsies are stated. The muscularis externa was mechanically removed from the submucosa using curved surgical scissors and forceps. The tissue was then cut into approximately 25mm 2 pieces and incubated for 15 minutes twice in RPMI-1640 (Lonza) supplemented with 10% Foetal Bovine Serum (FBS) (Sigma-Aldrich, Missouri, USA), 0.3% Dithiothreitol (DTT) (Sigma-Aldrich) and 2mM EDTA at 37°C (herein referred to as DTT treatment). The tissue was washed in Dulbecco's Phosphate Buffered Saline Mg ++ /Ca ++ free (DPBS) (Lonza) prior to incubation on a MACSmix Tube Rotator (Miltenyi Biotec, California, USA) 2-3 times in RPMI-1640 supplemented with 5µL/mL DNase I (Worthington Industries, New Jersey, USA) and 3mg/mL collagenase type IV (Worthington) for 30 min each time at 37°C. The supernatant was separated from undigested intestinal tissue using a tea strainer. The cell suspension was passed through a 100µM cell strainer (Greiner Bio-One, North Carolina, USA) before being washed twice in DPBS. The singlecell suspension in RPMI-1640 was spun on a Ficoll-Paque PLUS (GE Healthcare, Illinois, USA) gradient and mononuclear cells were harvested from the RPMI-Ficoll interface. When required, the mucosa was anatomically separated from the submucosa using a dissection microscope and forceps. Lymphoid follicles present in the mucosa and submucosa were then dissected out using a scalpel. The mucosa was digested as above, with the submucosa not requiring the DTT treatment. For terminal ileum biopsies, a 10 min DTT treatment at 37°C was required. A single cell suspension from terminal ileum biopsies and isolated lymphoid follicles was generated by shaking them at 170rpm in a 37°C incubator in RPMI-1640 supplemented with 5µL/mL DNase I and 3mg/mL collagenase type IV for 45 min.

Flow Cytometry
Cells were labelled in aliquots of 1-3 x 10 6 cells per 100µL of DPBS. Non-viable cells were excluded by staining with Fixable Viability Stain 700 (BD Biosciences, New Jersey, USA).
Cells were washed with FACS wash (1% FBS (v/v), 2 mM EDTA, 0.1% sodium azide (w/v) in PBS) and surface stained with the antibodies indicated in Table 1 in 10µL Brilliant stain buffer plus (BD Biosciences) for 30 mins on ice. Cells were washed twice with FACS wash prior to resuspension in 100µL of CytoFix (BD Biosciences) for 15 mins at 4°C. Cells were washed and resuspended in 100µL of FACS wash. Flow cytometry was performed on an LSR Fortessa or FACSymphony flow cytometers (BD Biosciences) with BD Diva Software (V8.0) and data analysed on FlowJo (TreeStar V10.7.1).

Culturing of Ex Vivo Mononuclear Phagocytes
Following dead cell depletion (STEMCELL Technologies, Vancouver, Canada) as per the manufacturer's instructions and CD45 + enrichment, 5 x 10 5 cells per condition were taken as a time 0-hour aliquot. All remaining cells were cultured at 1 x 10 6 cells/mL for 14 hours in DC culture media. Cells were then washed once in DPBS and stained with Fixable Viability Stain 700. Cells were washed with FACS wash and surface stained with antibodies indicated in

Optimisation of Enzymatic Digestion Protocols for the Isolation and Interrogation of Intestinal Mononuclear Cells
We optimised a protocol for the mechanical separation and enzymatic digestion of human intestinal tissue to liberate MNPs. Enzymatic access to the mucosal tissue was increased by removing the underlying muscle layer and fine dicing the tissue with a scalpel. The epithelium and any remnant mucus were removed with DTT treatment prior to digestion with collagenase type IV at 37°C which we previously showed to the best collagenase blend for MNP tissue isolation (9) ( Figure 1A). Using this method, we compared the yield and proportions of MNPs (CD3 -CD19 -HLA-DR + CD45 + ) from sequential 30-minute collagenase digestions ( Figure 1B). Digestions were performed in sequence rather than one extended digestion to both limit the exposure of the liberated cells to the enzyme, and to replenish the enzyme for optimal activity. The first digestion resulted in significantly lower yield and proportion of MNPs than the subsequent digestions, suggesting that 30 minutes was not long enough to liberate our cells of interest from whole tissue specimens. The second digestion provided the highest proportion of MNPs as well as the highest yield, in numbers suitable for phenotyping assays. (Figures 1C,  D). A third digestion liberated additional cells which increased the total yield ( Figure 1C), without compromising the proportion of HLA-DR + CD45 + cells ( Figure 1D) or their viability ( Figure 1E). Two digestions were performed for phenotyping purposes, while three digestions were utilised when higher numbers of MNPs were required for cell sorting and functional assays.

Manual Separation of Intestinal Tissue Compartments
Intestinal mucosal tissue consists of multiple compartments that require separation to investigate their unique immune cell profiles (16). To achieve this, the mucosa and submucosa were mechanically separated along the biological border of the two layers, the muscularis mucosa, and lymphoid follicles were isolated prior to digestion (Figure 2A). This was performed under light microscopy without the need for tissue staining, with lymphoid follicles being visually identified and removed with a scalpel. The mucosa was digested as per the protocol for whole tissue ( Figure 1A), with the submucosa digested without DTT treatment as it does not have an epithelial layer. Lymphoid follicles (<2mm 2 ), like biopsies (<5mm 2 ), are smaller than the diced pieces of whole tissue (25mm 2 ) and as such were fully digested in a shorter, single digestion. The lymphoid follicles as well as terminal ileum biopsies underwent collagenase type IV digestion for 45 min at 37°, with biopsies receiving a 10 min DTT treatment at 37°C before digestion as described in Figure 2A. Using tissue from 3 individual donors and a minimal staining panel we were able to show by flow cytometry that of the CD45 + cells liberated, CD3 + T cells, CD19 + B cells and HLA-DR + MNPs were represented in different proportions in each compartment ( Figure 2B), confirming that we can manually dissect intestinal tissue to enrich for specific tissue compartments using visual confirmation.

Accurate Identification of Tissue Dendritic Cell Subsets via Flow Cytometry
With the ever-changing classification of DCs (11,12,14,17,18), their identification by flow cytometry has required an increasing number of discriminatory markers. For tissue DCs this task is further complicated by the cleavage of surface identification markers that often occurs during enzymatic liberation. A subset of cDC2 that express FCeR1a have recently been described (14,18).  We confirmed that FCeR1a was expressed on some cDC2s using the clone AER-37, though it was not until the same cells were stained with the clone 9E1 that we could confirm that the epitope recognised by AER-37 was being partially cleaved by collagenase type IV ( Figure 3A). In addition to epitope cleavage, a further consideration is whether markers found on the surface of circulating cells correspond to tissue DCs. Blood cDC1s have been classified as CD141 + and XCR1 + (19), however, this does not appear to be the case in intestinal tissues where only a small portion of XCR1 + cells express CD141, the expression of which does not correlate to XCR1 expression ( Figure 3B). Therefore, CD141 is not a reliable marker of cDC1s in the intestine. Taken together, these results emphasise the importance of marker and antibody clone selection for the proper identification of enzyme-liberated intestinal DCs.

Autofluorescent Correction of Tissue-Resident Macrophage Subsets via Flow Cytometry
Tissue-resident yolk sac derived macrophages are characteristically autofluorescent (AF) (20). This unique characteristic allows for their identification by flow cytometry as AF CD14 + (13,20). However, as more subsets of macrophages are defined in the intestine (10), their AF properties can mask the expression of key defining markers as well as presenting false positives. Roca, Burton (21) recently described a method for AF correction using AutoSpill, an algorithm for calculating spillover coefficients, by assigning AF as an additional endogenous dye using an unstained sample. We acquired an unstained sample of intestinal-liberated cells in addition to single-colour controls on beads to facilitate AF correction for manual compensation. Using the compensation wizard in FlowJo, the unstained sample was allocated to an unused detector channel where autofluorescence spills naturally, for example B710_50 on the FACSymphony, treating the AF signal from tissue cells as a single-colour control. By setting positive and negative gates for AF ( Figure 3C) and re-calculating the compensation matrix with AF as a measurable parameter,  AF spillover into other detectors was minimised, as shown with the correction of false signal using Fluorescence Minus One (FMO) ( Figure 3D) and on CD45 + HLA-DR + CD3 -CD19cells ( Figure 3E). Furthermore, not using this method leads to inaccurate determination of surface marker expression as indicated for key DC expression marker CD11c in Figure 3E.
Here we show that the CD14 + CD11c -AF macrophages can be corrected to display as CD11c -, instead of spilling false signal into the detector allocated to CD11c.

Identification of Intestinal-Derived Mononuclear Phagocytes
MNPs share several common markers in tissue making their definitive identification and characterisation challenging (8,10).

Mononuclear Phagocytes Are Phenotypically Immature at the Time of Liberation
A complication of the digestion process can be the inadvertent activation or maturation of cells (9,23,24). We investigated the maturation status of MNPs isolated by our digestion protocol, by assessing the surface expression of adhesion (CD54) and costimulatory molecules (CD80, CD83 and CD86), which are upregulated upon maturation (25). Immediately after isolation, all MNP subsets expressed low levels of CD54, CD80, CD83 and CD86 ( Figure 5A). In two of three donors, Mf1s, Mf3s and cDC1s did not survive the 14-hour culture and therefore their maturation status at this time was not determined. However, significant upregulation of CD54 was seen on Mf2s, CD54, CD83 and CD86 on langerin +/-cDC2s, with langerin -cDC2 also upregulating CD80 post-culture (n=3) (Figures 5B, C). This culture-induced upregulation of maturation markers suggests that not only were the tissue-liberated MNPs viable, but they also remained immature throughout the enzymatic digestion.

Liberated Intestinal Mononuclear Phagocytes Can Be Used for Functional Assays Ex Vivo
Having isolated and identified immature MNPs, we next confirmed that their biological functionality had been maintained. Sorted MNPs were stimulated and cultured for 16 hrs to determine their cytokine response. We observed, via intracellular staining, that AF macrophages, cDC1s and cDC2s could produce TNF, IL-23p40 and IL-6 in response to R848 ( Figure 6). This cytokine response suggests that the intestinalderived MNPs were liberated in a functional state.

DISCUSSION
The ability to interrogate immune cells from fresh human tissue is critical for advancing our understanding of the role these cells play in human disease settings. This is especially the case with intestinal MNPs, which are integral components of the mucosal innate immune system. Here, they play a crucial role in maintaining tolerance of the commensal microbiota while also remaining poised to respond to invading pathogens. While sophisticated animal models have greatly expanded our knowledge of intestinal MNPs, they are not able to completely recapitulate human tissue with many cells differing phenotypically and functionally (12,26). Translating the data generated from these animal models requires the development of isolation methods to interrogate human immune cells ex vivo while maintaining their biological state. We previously described tissue digestion protocols for the isolation of MNPs from human abdominal skin (9) and anogenital tissues (8,13). However, given the distinct phenotypic, functional and structural differences between skin and mucosal tissue, we present an isolation protocol to extract functionally immature MNPs from human intestinal tissue. We have carefully selected the optimal antibody clones and corrected AF spillover to develop highparameter flow cytometry gating strategies to accurately identify all currently known subsets of MNPs in fresh human intestinal tissue. An important consideration when isolating cells via enzymatic digestion is the delicate balance between cell yield and viability. Many groups perform one round of enzymatic digestion (10,11), however, Lefrancois and Lycke (27) showed the advantages of performing a second round of digestion in murine mucosal tissues to enhance cell yield and viability. Our findings show that we can digest human intestinal tissue for a total of 90 minutes without compromising cell viability or proportions. As a result, we can liberate a higher quantity of  Table 1). All mononuclear phagocytes were gated within the live, single, CD45 + CD3 -CD19population. Intestinal mononuclear phagocytes (MNP) were gated in sequential order with percentage of total mononuclear phagocytes in brackets. Macrophage (Mf) 1 were defined as CD14 + HLA-DR low , with all sequential MNPs gated as HLA-DR + . Cells were then divided by their expression of CD11c. CD11ccells included CD14 -CD123 + plasmacytoid DCs (pDCs), CD14 + Calprotectin + CD88 + monocytes and CD14 + autofluorescent + Mf3s which could be further subdivided into CD11b + Mf4 detectable only in the jejunum. CD11c + cells were divided as follows: CD88 -CD163 + CD1c + DC3s, CD14 + Mf2s, CD14 -CD1c -XCR1 + cDC1s, CD14 -XCR1 -CD1c + cDC2s divided by their expression of langerin. (B) Alternative gating for CD11c + CD14cells, characterised by SIRPa and CD103, overlaid with cDC1 and cDC2s from the main gating strategy for comparison. (C) Pie cart representation of proportion of mononuclear phagocyte subsets as part of whole of all mononuclear phagocytes (D) Cells isolated from intestinal biopsies were positively selected for CD45 and stained with FACS Sort Panel ( Table 2). Live, single CD45 + HLADR + cells were divided by CD14 expression. CD14 + cells were sorted as CD11cautofluorescent + macrophages and CD11c + macrophages. CD14cells were sorted as CD123 + plasmacytoid DCs, XCR1 + cDC1s, CD1c + cDC2s langerin +/-. total cells for downstream functional assays, which can be a limiting factor when working with human tissue-derived cells (13,28). Most of the data we have presented here has taken advantage of our privileged access to whole pieces of intestinal tissue. While these larger tissue samples are often required to perform sorting for functional assays ex vivo, it is far more common to receive surgical biopsies from patients. To that end, we have shown that using our protocols it is possible to liberate, phenotype and even sort MNPs from human punch biopsies. Due to punch biopsies being smaller, a shorter digestion protocol is sufficient to liberate the cells which will make the interrogation of DC and macrophages from the intestine easier and far more accessible.
In the intestine, there are several different anatomical compartments containing varying ratios of functionally specialised immune cells each with their own unique interactions with the local intestinal environment (29). To interrogate these immunological compartments and the unique role each plays in disease settings it is crucial to isolate and interrogate them separately. The four primary compartments investigated in this study are the mucosal and submucosal layers, and their associated lymphoid follicles. Posing the greatest obstacle to deepening our knowledge of these immune compartments has been the difficultly in delicately separating the tissue layers and correctly identifying and removing the small lymphoid follicles. Other groups have attempted to expedite this task by using dyes to better visualise the lymphoid follicles (16), however, here we present an alternative method that removes this step and the risk it may pose in altering their function (30). Based on the method of Fenton, Jørgensen (16,31), we were able to separate these compartments and show their unique immune profiles.
A known complication of enzymatic digestion is the cleavage of cell surface proteins, which can lead to the misidentification of cell subsets. We have previously shown that type IV collagenase best preserved the cell surface proteins used to identify tissue MNPs which led to the discovery of previously unidentified epidermal DC subset (8,9). However, even with this collagenase blend, surface proteins can be partially cleaved, leading to underrepresentation of cell subsets within the tissue or their  Table 2). Autofluorescent (AF) macrophages (Macs), conventional DC (cDC) 1 and cDC2s were sorted based on gating in Figure 4C. Sorted cells were cultured at 1x10 6 cells/mL in DC Culture Media with 1µg/mL R848 for 2 hours. 2.5µg/mL Brefeldin A was added, and the cells cultured for a further 14 hours. Cells were then stained with Fixable Viability Stain 700 and intracellularly stained with anti-TNF, anti-IL-23p40 and anti-IL-6. Cells were acquired on a FACSymphony. loss of function. It has proven particularly challenging to identify tissue-specific cDC2s as their markers overlap with monocytes and macrophages. Dutertre, Becht (18) revealed that FCϵR1a was one of two exclusive markers used to identify tissue cDC2s. Upon reviewing the literature, we selected FCϵR1a clone AER-37 as this was the most widely used clone. By using this specific antibody clone and type IV enzyme combination, we observed that FCϵR1a was partially sensitive to enzymatic cleavage as an unusually low proportion of cDC2s were FCϵR1a + . It wasn't until we selected a new FCϵR1a antibody clone: 9E1, that we were able to detect a much higher proportion of FCϵR1a expression on cDC2s in the intestine. Hence, FCϵR1a clone 9E1 with collagenase type IV is the superior combination.
Another consideration is the change in the pattern of marker expression on MNPs from different tissue compartments. In human blood and cutaneous abdominal tissue, cDC1s have been shown to express both CD141 and XCR1 (32). However, in human intestinal tissue we found that the expression of CD141 on cDC1s is extremely low and does not correlate with expression of XCR1. In addition, tissue cDC2s can upregulate their expression of CD141 (33). Peripheral blood cDC1s are defined by their CD141 and CADM1 expression, although CADM1 is not expressed by human skin cDC1s (9). We show here that CD141 is an unreliable marker in human intestine, therefore, we recommend XCR1 as the optimal marker for identifying cDC1, with the S154046E clone, in our experience as the only reproducibly reliable antibody that currently works on tissue-derived cells. This highlights the importance of carefully selecting antibodies that not only target preserved receptor epitopes post-digestion but reflect the expression of cell surface receptors in the tissue of interest.
The classification of MNPs is a rapidly evolving field of research and identifying them by flow cytometry can be challenging as subsets of MNPs share common markers (8,10). This is further complicated by the AF properties of tissueresident macrophages. Here we were able to correct AF by using an unstained tissue-derived control sample, allowing us to differentiate between real and false signal and improve resolution of surface markers used to identify MNPs. We could identify all currently known subsets of intestinal DCs using CD103 and SIRPa (11,12) as well as the newly published DC3s (14,15) in the same tissue. Furthermore, the SIRPa -CD103 + cells could be identified as XCR1 + cDC1 and SIRPa + CD103 +/cells as CD1c + (langerin +/-) cDC2, which is more relevant across different tissue types.
Following enzymatic extraction of MNPs from human intestinal tissue we have optimised methods to interrogate the function of these cells. This is important as extraction of MNPs from the tissue by enzymatic digestion can alter their biological function. Mature tissue MNPs have been shown to express elevated levels of leukocyte adhesion molecule CD54 (ICAM-1) and co-stimulatory molecules such as CD80, CD83 and CD86 (25). Conversely, immature MNPs freshly isolated from healthy human intestinal tissue should express low levels of these receptors on their surface. Following overnight culture, we showed that Mf2s and cDC2s had a substantial increase in maturation marker expression confirming that the cells were in an immature state upon liberation. Interestingly, we observed that Mf2s and cDC2s survived the culture, whereas Mf1s, Mf3s and cDC1 did not survive in two out of three donors. This suggests that cDC2 and Mf2, which are phenotypically very similar (10), share a propensity for survival outside of their tissue environment. We also showed that tissue liberated MNPs could produce cytokines in response to a TLR7/8 ligand. These findings confirm that intestinal MNPs liberated by enzymatic digestion following our optimised mucosal tissue digestion protocol are in an immature and biologically functional state. This is advantageous over other isolation methods such as spontaneous migration of DCs out of tissue during culture, which triggers the maturation of DCs limiting their use in functional assays (9).
In summary, our study demonstrates an enzymatic digestion protocol for the isolation of human intestinal MNPs in an immature state from tissue and biopsies while maintaining their biological function. We provide a method for the anatomical separation of the different gut-associated lymphoid tissues without the use of dyes. We also demonstrate the need for careful selection of antibodies that target preserved receptor epitopes and reflect their unique tissue-specific phenotype. These optimised protocols will greatly enhance our ability to interrogate APCs in human tissue, which will be important to understanding their role in homeostasis and diseases of the intestine.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Western Sydney Local Area Health District (WSLHD) Human Research Ethics Committee (HREC); reference number (4192) AU RED HREC/15 WMEAD/11. The patients/participants provided their written informed consent to participate in this study.