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Summary

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Abstract

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Introduction

The gastrointestinal (GI) tract has many folds and protrusions that represents the largest interface separating the internal body and the external environment. The intestinal immune system plays an essential role in maintaining the barrier function of the GI tract. It is constantly exposed to dietary antigens, commensal bacteria, and pathogenic microbes. As such, it must remain tolerant to food antigens and commensal bacteria while preserving the capacity to rapidly generate an effective immune response to enteropathogenic microbes¹. The intestinal immune system can be anatomically divided into inductive sites, where naïve lymphocytes are activated by antigen presenting cells carrying antigens from the intestinal mucosa, and effector sites, where activated lymphocytes exert specific effector functions². The inductive sites comprise the organized lymphoid structure of Peyer's patches (PP) that surveys the intestinal lumen directly through the action of specialized M cells and the regional draining mesenteric lymph nodes (MLN). The effector sites consist of the lamina propria (LP), which is the connective tissue below the basement membrane, and the intestinal epithelium, a single cell layer located above the basement membrane that contains intraepithelial lymphocytes (IEL). Lymphocytes are major players of adaptive immunity that mediate protection against infections and cancers and may also contribute to immunopathology in inflammatory diseases. It is important and highly relevant to study lymphocytes in these distinct anatomical mucosal compartments to better understand their induction and effector functions.

A relatively simple and unified protocol for the isolation of lymphocytes from these compartments is needed as the number of investigators exploring immune events occurring in the intestine are accelerating. Several research groups have published protocols that share several similar processes for isolating immune cells from the mouse small intestinal compartments^3,4,5,6,7. However, there are several technical differences among them depending on the focus of the individual protocol. For example, with the focus on isolating immune cells from the LP, one protocol examines the impact of various enzymatic digestions on cell viability, cell surface marker expression, and the composition of isolated immune cells⁵. Another protocol highlights a rapid and reproducible method for isolation of lymphocytes without density centrifugation⁶. Finally, specific protocols also exist for the purpose of isolating mononuclear phagocytes from different tissue layers of the small intestine⁷. Here, a highly reproducible protocol that allows sequential isolation of lymphocyte populations from the MLN, PP, LP, and IEL compartment of the small intestine is presented.

We focus on isolating highly purified populations from the LP and IEL compartments, which are largely free of contaminants from other intestinal compartments. This widely used protocol produces a high yield of maximally pure and viable lymphocytes within acceptable time constraints^{4,8,9,10,11,12}. This protocol also ensures the isolation of lymphocytes from the LP and IEL compartment with minimal cross compartmental contamination, allowing a bona fide opportunity to study lymphocytes in these distinct compartments. The isolated lymphocytes can be subjected to further manipulations like flow cytometric analysis or functional analysis. This protocol has been successfully applied to the isolation of lymphocytes from the mouse small intestine and colon during bacterial infections such as Listeria monocytogenes, Salmonella typhimurium, and Yersinia pseudotuberculosis infections and inflammatory conditions such as chemical- and pathogen-induced colitis. This protocol can also be used to isolate innate immune cells such as dendritic cells, macrophages, neutrophils, and monocytes from the mouse small intestine and colon.

Protocol

All animal experiments were conducted in accordance with National Institute of Health guidelines and approved by the Stony Brook University Institutional Animal Care and Use Committee. NOTE: Ensure that all approvals are granted prior to performing procedures. 1. Solution Preparation HGPG (HEPES, L-glutamine, penicillin/streptomycin, and gentamycin), 100× Mix 59.6 g HEPES (500 mM final), 14.6 g L-glutamine (200 mM final), 1×106 U penicillin (2,000 U/mL final), 1 g streptomycin (2 mg/mL final), and 2.5 mg gentamicin (5 µg/mL final). Add RPMI 1640 to 500 mL. Adjust pH to 7.5 using NaOH (before adjustment, the buffer is yellow/orange with a pH around 6.0). Filter sterilize using a 0.45 µm filter. Aliquot and store at -20 °C for up to 1 year or at 4 °C for up to 1 month. HEPES-bicarbonate buffer, 10x ​Mix 23.8 g HEPES (100 mM final), 21 g NaHCO3 (250 mM final) and add water to 1,000 mL. Adjust pH to 7.2 with HCl. Filter sterilize using a 0.45 μm filter. Store at room temperature. The pH changes over time. Re-adjust pH periodically. Harvest media (~ 1 bottle/2 mice) To 500 mL RPMI 1640, add 25 mL heat-inactivated fetal bovine serum (5% final), and 5 mL 100× HGPG. Store up to 6 months at 4 °C. Dithioerythritol (DTE) solution (40 mL/small intestine) Mix 50 mL 10× Hanks balanced salt solution, Ca2+- and Mg2+-free, 50 mL 10× HEPES-bicarbonate buffer, and 50 mL heat-inactivated fetal bovine serum (10% final). Add 350 mL H2O and 15.4 mg dithioerythritol/100 mL (1 mM final). Prepare this fresh before use. Ethylenediaminetetraacetic acid (EDTA) solution (50 mL/small intestine) Mix 50 mL 10× Hanks balanced salt solution, Ca2+- and Mg2+-free with 5 mL 100× HGPG. Add 445 mL H2O. Add 260 µL 0.5 M EDTA/100 mL (1.3 mM final). Prepare this fresh before use. Collagenase solution (30 mL/LP) To 500 mL RPMI, add 50 mL FBS (10% FBS final), 5 mL 100× HPGP, 1 mL 0.5 M MgCl2 (1 mM final), and 1 mL 0.5 M CaCl2 (1 mM final). Add collagenase, Type I (100 U/mL final). Prepare this fresh before use. Density gradient (DG) solutions Prepare 1× DG stock solution by mixing 90 mL DG stock solution (refer to the Table of Materials) with 10 mL of 10× PBS. Keep sterile at 4 °C. Prepare 44% DG solution (8 mL/IEL, 8 mL/LP) by mixing 44 mL of 1× DG stock solution and 56 mL RPMI 1640. Prepare this fresh before use. Prepare 67% DG solution (5 mL/IEL, 5 mL/LP) by mixing 67 mL of 1× DG stock solution and 33 mL RPMI 1640. Prepare this fresh before use. 2. Isolation of the Mesenteric Lymph Nodes, Intestines, and Peyer's Patches Euthanize the mouse using carbon dioxide narcosis and secondary cervical dislocation. Place the mouse on its back and spray with 70% ethanol. Use scissors to make a midline incision, and open the skin and abdominal wall to expose the peritoneal cavity. Locate the caecum and use forceps to gently pull the caecum down. Use forceps to carefully move the small intestine to the right, exposing the entire MLN chain, which is aligned with the colon. NOTE: A map of the mesenteric lymph nodes chain and lymphatic drainage of the intestines was previously depicted13. Identify the bottom node in the chain, located closest to the cecum. Use one set of forceps to grasp the mesentery/fat around it and gently remove the MLN chain from the bottom to the top by tweezing the fat from around the lymph node with another set of forceps. Lay the MLN chain on a paper towel moistened with harvest media. Remove mesentery/fat by rolling the chain on the paper tower and pulling the fat off with two sets of forceps. Place the MLN in cold harvest media for later isolation steps. NOTE: (i) Direct handling of the nodes is discouraged. Instead, grasp the mesentery/fat around them. (ii) It is critical to remove as much mesentery/fat as possible to improve cell viability. Cut the small intestine below the pyloric sphincter and above the cecum using scissors. Pull the intestine out slowly from the ileum to duodenum using one set of forceps while removing the attached mesentery/fat using another set of forceps. Place the intestine on a paper towel moistened with harvest media and tease any remaining mesentery/fat off with two sets of forceps. NOTE: (i) Be sure to remove as much mesentery/fat as possible for optimal cell yield and viability. (ii) Keep the intestine moistened throughout this and the following steps by applying harvest media periodically. Collect Peyer's patches by removing them from the intestine with curved scissors. Use a dissecting scope or magnifying glass if needed (beginners); most Peyer's patches should be visible to the trained eye. Collect 5-11 (typical) Peyer's patches from the small intestine. Place Peyer's patches in cold harvest media for later isolation steps. NOTE: Peyer's patches are small, primarily round bulges in the outer intestinal wall opposite the line of mesentery attachment. Use the flat side of forceps and gently slide from duodenum towards ileum to expel fecal content and mucus. Repeat once to remove most of the mucus. NOTE: Incomplete removal of mucus can decrease cell viability and yield. Nevertheless, be sure to slide the intestine gently to avoid stripping villi or damaging the basement membrane. Use fine scissors with a blunt end on one point, insert the blunt end into the intestine, and cut the intestine open longitudinally from duodenum to ileum. Laterally cut the opened intestine into ~2-cm pieces. Place the intestinal pieces in a 50-mL conical tube with 25 mL cold harvest media for later isolation steps. NOTE: Keeping the scissors moistened with media will help them slide effortlessly through the intestine. 3. Isolation of Lymphocytes from the Inductive Sites Isolate lymphocytes from MLN by gently dissociating through a 70-µm cell strainer using the plunger of a 3-mL syringe. Wash cell strainer with 5 mL of cold harvest media. Pellet MLN-cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion. NOTE: Red blood cell lysis buffer can be used to remove red blood cells if bleeding occurred during removal. Isolate lymphocytes from Peyer's patches by incubating Peyer's patches in a 15-mL conical tube containing 5 mL prewarmed collagenase solution at 37 °C and 220 rpm for 30 min. Vortex for 15 s at maximum setting and filter digested tissue and supernatant into a 50-mL conical tube through a 70-µm cell strainer. Gently dissociate the remaining tissue pieces using the plunger of a 3-mL syringe and wash the cell strainer with 5 mL of cold harvest media. Pellet Peyer's patch cells by centrifuging at 400 × g for 5 min at 4 °C and resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion. NOTE: Peyer's patches isolation may have some contaminating lamina propria lymphocytes and intraepithelial lymphocytes. 4. Isolation of Intraepithelial Lymphocytes from the Small Intestine Wash the pieces of small intestine three times with 25 mL of cold harvest media by inverting the tube 10 times, letting the intestinal pieces settle, and pouring off the supernatant. NOTE: Tissue pieces should readily settle to the bottom of tube. If they do not, it is an indication that too much mesentery/fat remains attached or they are associated with an air bubble. Add 20 mL of prewarmed DTE solution to the 50-mL conical tube containing intestine pieces and transfer to a 50-mL siliconized Erlenmeyer flask containing a stir-bar. Stir at 37 °C and 220 rpm on a magnetic stirrer for 20 min. Use a large magnet on the outside of the flask to hold the stir-bar and transfer the tissue and solution back to the 50-mL conical tube. Vortex the tube at maximum setting for 10 s. Transfer the supernatant into a new 50-mL conical tube through a 70-µm cell strainer being careful to keep the tissue pieces in the original tube. Do not discard the supernatant; the supernatant contains intraepithelial lymphocytes. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 10 mL cold harvest media and store on ice. Add 20 mL of prewarmed DTE solution to the 50-mL conical tube containing intestine pieces and transfer back to the 50-mL siliconized Erlenmeyer flask containing a stir-bar. Stir at 37 °C and 220 rpm on a magnetic stirrer for 20 min. Use a large magnet on the outside of the flask to hold the stir-bar and transfer the tissue and solution back to the 50-mL conical tube. NOTE: DTE is a reducing agent that enriches intraepithelial lymphocytes. Vortex the tube at maximum setting for 10 s. Transfer the supernatant through a 70-µm cell strainer into the previous tube containing cells from the supernatant of the first DTE treatment (from step 4.3). NOTE: The remaining intestinal pieces are used to isolate lymphocytes from the lamina propria. Quality Control: A piece of tissue can be removed and checked histologically to ensure that epithelial cells are present and the basement membrane is intact. Pellet cells by centrifuging the supernatant at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 8 mL of 44% DG solution at room temperature (RT). Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm ø x 100 mm style 14-mL polypropylene round-bottom tube. Underlay with 5 mL of RT 67% DG solution. Centrifuge at 1600 × g for 20 min at RT without using the brake. NOTE: Tubes may be pretreated with bovine serum to prevent cells from sticking to the tube walls. Note that viable cells form a band (buffy coat) at the 44% to 67% interface, dead cells and some epithelial cells are in a mucoid film at the top of the gradient, and the red blood cells are in the pellet. Remove the top layer of the gradient to within ~2 cm of the interface by vacuum aspiration. Use a Pasteur pipette to harvest the buffy coat at the interface and transfer them to a 50-mL conical tube containing 40 mL cold harvest media. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion. 5. Isolation of Lamina Propria Lymphocytes from the Small Intestine Remove epithelial cells from intestinal tissue pieces by adding 25 mL of prewarmed EDTA solution to the flask containing the remaining intestinal pieces. Stir flask at 37 °C and 220 rpm on a magnetic stirrer for 30 min. Use a large magnet on the outside of the flask to hold the stir-bar and carefully discard the supernatant while keeping intestinal pieces within the flask. NOTE: (i) EDTA is a calcium chelator that targets calcium-dependent junctions and promotes the detachment of intestinal epithelial cells. (ii) The supernatant should be cloudy and may be used to isolate IEC if their collection is desired. Repeat step 5.1. NOTE: The supernatant should be less cloudy following this incubation. Quality Control: A piece of tissue can be removed and checked histologically to ensure that intestinal epithelial cells have been removed and the basement membrane is intact. A sample from the supernatant can also be assessed by flow cytometry to confirm that leukocytes (CD45+ cells) are absent. Add 50 mL of RT harvest media to the flask. Stir briefly with a magnet. Let intestinal pieces settle and carefully discard the supernatant. NOTE: This step ensures the removal of EDTA prior to collagenase digestion as EDTA inactivates collagenase by chelating the calcium that is critical for collagenase function. Add 30 mL of prewarmed collagenase solution to the flask and stir intestinal pieces at 37 °C and 220 rpm on a magnetic stirrer for 45 min. Transfer digested tissue and supernatant to a 50-mL conical tube by filtering through a 70-µm cell strainer. Gently dissociate remaining tissue pieces by using the plunger of a 3-mL syringe to mash through the filter. Wash the filter with 10 mL of cold harvest media. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 8 mL of ambient temperature 44% DG solution. Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm ø x 100 mm style 14-mL polypropylene round-bottom tube. Underlay with 5 mL of ambient temperature 67% DG solution. Centrifuge the gradients at room temperature and 1600 × g for 20 min with no brake. Remove the fat layer on top by vacuum aspiration. Use a Pasteur pipette to harvest the buffy coat at the interface and transfer to a 50-mL conical tube containing 40 mL cold harvest media. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.

Representative Results

A schematic representation of the protocol is depicted (Figure 1). Lymphocytes within the intestinal mucosa inductive and effector sites are distinctly organized. Peyer's patches (PP) and mesenteric lymph nodes (MLN) contain lymphocytes in well-organized T-cell areas and B-cell follicles, whereas the intestinal epithelium contains lymphocytes that are more diffusely distributed. The lamina propria (LP) contains both diffusely distributed lymphocytes and l…

Discussion

A detailed protocol is presented for the isolation of lymphocytes from the intestinal mucosal inductive (MLN and PP) and effector (LP and IEL compartment) sites. The protocol has been developed to balance input (time) and output (viability and yield) to maximize productivity and results. The protocol also ensures minimal cross compartmental contamination between LP and IEL compartments.

Several protocols for the isolation of immune cells from the mouse small intestine have been published<sup c…

Materials

HEPES	Fisher Scientific	BP310-500
L-glutamine	Sigma-aldrich	G3126-100G
Penicillin-Streptomycin	Life Technologies	15140-122
Gentamicin	Life Technologies	15710-072
Sodium Hydroxide	Fisher Scientific	S318-500
RPMI 1640	Life Technologies	21870-076
Sodium bicarbonate	Fisher Scientific	S233-500
Fetal bovine serum	Life Technologies	26140-079
10x Hanks' balanced salt solution	Sigma-aldrich	H4641-500ML
1,4-Dithioerythritol	Sigma-aldrich	D9680-5G
0.5M EDTA, pH 8.0	Life Technologies	15575-020
Calsium chloride hexahydrate	Sigma-aldrich	21108-500G
Magnesium chloride hexahydrate	Sigma-aldrich	M2670-100G
Collagenase, Type I	Life Technologies	17100-017
DG gradient stock solution (Percoll)	GE Healthcare	17-0891-01
Red Blood Cell Lysis Buffer	Biolegend	420301
70-µm cell strainer	Corning	352350
14 mL Polypropylene Round-Bottom Tube	Corning	352059
Erlenmeyer flask	Kimble	26500R-50mL
Magnetic stirrer	Thermo Fisher	50094596
Stir bar	Fisher Scientific	14-512-148