Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Summary

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Abstract

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

Introduction

Studies on human organs are restricted due to accessibility as well as obvious ethical reasons. However, they are essential to fully understand the complexity of human biology. Cultures of isolated cells (primary cultures or cell lines) are a standard system in cell biology studies due to their availability. While isolated cell cultures have allowed outstanding discoveries, the use of cell lines has come upon closer scrutiny because they do not fully mimic in vivo organ biology. However, the culture of three-dimensional cells or tissue explants is highly complex^4,5,6. Indeed, a piece of tissue or organ is highly heterogenous because its cell composition differs depending on the localization in the tissue. Thus, using tissue blocks requires the analysis of many technical and biological replicates, leading to the need of a large number of donors or patients.

The mucosa-associated lymphoid tissues (MALT) are structurally similar to the lymph nodes but have unique functions, because their main role is to regulate mucosal immunity⁷. Unlike the lymph nodes, which are usually located at some distance from the tissues, MALT are generally located immediately below the epithelium of the mucosal tissue. Histologically, they are mainly composed of high concentrations of B and T cells, but also antigen-presenting cells such as macrophages and dendritic cells. MALT constitute about 50% of the lymphoid tissue in the human body. MALT are subdivided into nine groups depending on their location: GALT (gut-), BALT (bronchus-), NALT (nasal-), CALT (conjunctival), LALT (larynx-), SALT (skin-), VALT (vulvo-), O-MALT (organized), and D-MALT (diffused). The O-MALT is mainly composed of the tonsils of Waldeyer's tonsillar ring and is the most accessible MALT^8,9. Indeed, tonsils located in the oropharynx constitute the major barrier protecting the digestive and respiratory tracts from (potential) invasive microorganisms¹⁰. In addition, the tonsils are covered by a fine stratified squamous non-keratinizing epithelium, supported by a capsule of connective tissue containing blood vessels, nerves, and lymphatics, providing easy access to the immune cells^11,12. Furthermore, tonsillectomy, the surgical act of removing tonsils, is a common procedure performed on children having sleep-disordered breathing, making tonsils an easily available tissue¹³ in physiological settings.

Tonsils allow the study of immune cell response in pathologies involving mucosal immunity. Indeed, in HIV infection, because tonsils are composed of a high concentration of immune cells, they are the main target of viral replication but also produce a large amount of cytokines that are not detected in the circulation^14,15. At steady states, rare populations of innate-like cells are present in various mucosal tissues, including the tonsils, but are essentially absent from blood.

Thus, mononuclear cells from tonsils (TMCs) are a more relevant and complex model than PBMCs and can answer more profound questions. On the other hand, the use of tissue explants can be complex and not always relevant to innate immune studies. Thus, we established a model to study mucosal immune activation using TMCs¹⁶. Here, we describe a method for efficient isolation of TMCs from fresh human tonsils. This method allows the recovery of a large number of immune cells while keeping their integrity for ex vivo studies.

Protocol

The specimens are not collected specifically for research purposes and the study is not considered invasive. However, human tonsils collection requires ethical approval by the local relevant authorities. In our case, it was approved by the Comité de Protection des Personnes (IDRCB/EUDRACT: 2018A0135847). Furthermore, consent of each patient or legal representative is requested to obtain donors' personal data (e.g., sex, age, history of ENT infections) that can help interpret experimental results.

1. Handling of the Human Tonsillar Tissue

1. Put tonsils from every donor in one sterile 50 mL vial containing 25 mL PBS 1x and transport at room temperature (RT) to the laboratory according to the authority's recommendations (use three levels of protection: vials, a safety box, and a bag).
   NOTE: The entire procedure should be carried out in a biological safety level 2 laboratory. All human specimens should be handled with care as they have not been previously qualified and may contain infectious agents.
2. Clean all tools in between each use.
   1. After use, disassemble the cell strainer and keep with all the other instruments (e.g., pestle, forceps) in a bath containing a detergent solution (1/10 volume detergent in H₂O) overnight.
   2. Brush each utensil to remove the tissue and wash with clear water.
   3. Dry all parts well, then prepare the cell strainer (85 mL, 37 mm diameter) by placing a 60 mesh steel grid on top of a 10 mesh steel grid and tightly close the ring.
   4. Place all the tools in sterile bags and autoclave.

2. Dissection of the Human Tonsillar Tissue and Isolation of the Tonsillar Mononuclear Cells (TMCs)

1. Place the cell strainer onto one 150 x 25 mm² (SPL150) cell culture dish and all the instruments in another SPL150 to keep them sterile.
2. Transfer the tonsils from the vial into the cell strainer using sterile forceps. Also pour in all of the PBS, which contains some cells that have egressed the tonsils. If necessary, add more PBS to immerse the grid.
   NOTE: The tissue should be immersed at all times to avoid desiccation.
3. Remove cauterized, bloody, and fibroid tissue using forceps and a scalpel.
   NOTE: Tonsils removed from children do not need this step.
4. Cut the tissues into small pieces of less than 0.5 cm in diameter using the scalpel and the curved tweezers. Cut all the tissue so that the small pieces can be immersed at all times.
5. Place a few pieces of tissue in the cell strainer and scrape them onto the grid with a glass pestle until only a really thin layer of white stroma remains. Remove and discard the stroma to avoid clogging the grid.
6. Once all the tissues have been squeezed through the grid, use a 10 mL pipette to transfer all the cell suspension onto the grid and scrape it one last time.
7. Transfer the cell suspension with a 10 mL pipette into a sterile vial. Wash the grid and the cell strainer with PBS 1x. Let the cell suspension rest for 5 min at RT on the bench. This step allows the precipitation and agglomeration of the leftover stroma, dead cells, and any released DNA, and will facilitate the next step.
8. Place a sterile 70 µm sieve on top of a new 50 mL vial (remove carefully from the envelope) and gently transfer the cell suspension onto it with a 10 mL pipette. Do not mix the suspension, because a pellet is frequently present. If the sieve is clogged, use the back of a sterile 1 mL pipette tip to scratch the cells trough the sieve. Change the sieve as often as necessary.
9. Centrifuge the cells at 250 x g for 10 min at 4 °C.
10. Discard the supernatant and resuspend the pellet by gently mixing the vial, then resuspend the cells in 35 mL of PBS.
11. In a new vial, place a new 70 µm sieve on top and transfer the cell suspension onto it with a 10 mL pipette. If the sieve is clogged, use the technique detailed in step 2.7.

3. Isolation of the TMCs by Cell Density Gradient

NOTE: The TMCs can be used after step 2.10. However, to obtain a clearer cell solution and to remove other cell debris and any red cells, it is advised to perform a cell density gradient isolation of the mononuclear cells.

1. Add 15 mL of the density gradient medium (d = 1.076 g/mL) in a new 50 mL vial. Pour the TMCs solution on top of the density gradient medium, being careful to minimize mixing of the suspension with the density gradient medium.
2. Centrifuge the solution at 1,000 x g for 30 min at RT with acceleration and break off.
3. Remove with a 10 mL pipette and discard the upper layer (containing mostly PBS 1x) without disturbing the interface between the TMCs and the density gradient medium.
   NOTE: The TMCs contains a low number of erythrocytes, therefore the solution is clear. Thus, the interface between the solution of TMCs in PBS and the density gradient medium can be difficult to visualize. Accordingly, cells can be resuspended in RPMI 1640 supplemented with 20 mM HEPES (without FBS) before the density gradient is performed.
4. Remove the TMCs with a sterile 1 mL pipette tip and place in a new 50 mL vial.
5. Wash the cells by adding 50 mL of PBS containing 2% of fetal bovine serum (FBS) and 2 mM EDTA and centrifuge the tube at 250 x g for 10 min at 4 °C to remove the remaining platelets.
6. Repeat step 3.5, but centrifuge the tube at 400 x g for 10 min at 4 °C.
7. Prepare culture medium (R10) by supplementing RPMI 1640 with 10% heat inactivated FBS, 2 mM L-glutamine, and the antibiotic solution (100 U/mL penicillium and 100 µg/mL streptomycin).
8. Resuspend the TMCs in 10 mL of R10 and count the cells. On average, this technique yields 5 x 10⁸-2 x 10⁹ TMCs per pair of tonsils. The cells can now be used for investigations like PBMCs from whole blood (e.g., purification of specific cell type, cell culture, flow cytometry, RT-qPCR, freezing, etc.).
9. If needed, freeze the cells for further use using standard techniques for primary cell freezing.
   1. Count the number of cells.
   2. Centrifuge the cells and remove the supernatant. Dissolve the pellet in enough 100% FBS for a final concentration of 1 x 10⁸ cells/mL, then add the same volume of an 80% FBS + 20% DMSO solution. The cells will then be at 5 x 10⁷ cells/mL. This step should be done at 4 °C and the FBS and DMSO solution should be kept at 4 °C before using.
   3. Distribute 1 mL of the cell solution into cryotubes and put them in a slow freezing container that has been at 4 °C overnight to control the rate of cell freezing. Place it at -80 °C.
   4. To thaw the cells, place the cryovials in a warm bath at 37 °C for a few seconds with constant agitation. As soon as the cells start to thaw, transfer them to 49 mL of R10 and centrifuge to remove the DMSO. Count the cells and use them as PBMCs.
      NOTE: Although freezing and thawing the TMCs will remove debris, it will also damage some rare cell types. If clumps or debris are present after thawing, it is best to pass the cell suspension through a sterile 70 µm sieve before using it.

4. Phenotyping of TMCs by Flow Cytometry

1. Retrieve 5 x 10⁶ cells from the previous solution for each antibody mixture panel that needs to be tested and place in a 5 mL cytometry tube. Retrieve 1 x 10⁶ cells for the unstained control.
2. Wash the cells in PBS and centrifuge at 400 x g for 5 min at 4 °C. Resuspend them in 500 µL of PBS (1 x 10⁶ cells/mL) and incubate with a viability stain for 30 min at 4 °C in the dark.
3. Add 5 µL heat inactivated human AB serum per 100 µL of cell suspension and incubate for 15 min at 4 °C in the dark.
4. Wash the cells in PBS + 2% FBS + 2 mM EDTA (Wash Buffer) and centrifuge at 400 x g for 5 min at 4 °C.
5. Resuspend the TMCs in 500 µL of Wash Buffer containing the antibodies as detailed in Table 1. Incubate the cells for 30 min at 4 °C in the dark.
6. Wash the cells in Wash Buffer and centrifuge at 400 x g for 5 min at 4 °C. Resuspend the TMCs in 500 µL of PBS containing 0.5% of paraformaldehyde (PFA). Keep in the dark at 4 °C until acquisition by flow cytometry.
   NOTE: PFA is hazardous. Use a commercial ready-to-use solution to prepare the 0.5% PFA solution.

Antibody	Clone	Fluorochrome	Company
Live-Dead		BV405	ThermoFisher Scientific
CD3	SP34-2	V500	BD Pharmingen
CD8	SK1	Amcyan	BD Pharmingen
CD8	BW135/80	VioBlue	Miltenyi Biotec
CD4	RPA-T4	PE-Cy7	BD Pharmingen
CD45	HI30	PerCP-cy5.5	BD
CD19	HD237	ECD	Beckman Coulter
CD20	2H7	Alexa Fluor 700	BD Pharmingen
CD14	M5E2	PE-cy7	BD Pharmingen
CD14	M5E2	APC	BD Pharmingen
CD16	3G8	APC-H7	BD Pharmingen
CD56	MEM188	PE	BD Pharmingen
CD123	7G3	PE	BD Pharmingen
BDCA-1	L161	Pacific Blue	BD Pharmingen
BDCA-3	AD5-14H12	FITC	Miltenyi Biotec
BDCA-4	AD5-17F6	APC	Miltenyi Biotec
HLA-DR	G646-6	PerCP-cy5.5	BD Pharmingen
CD11c	3.9	Alexa Fluor 700	ebiosciences

Table 1: List of antibodies for cell characterization by flow cytometry.

5. Example of Activation of TMCs and Measurement of Cytokine Production

1. Dilute the TMCs in R10 medium to get a concentration of 2 x 10⁶ cells/mL.
2. Distribute 400,000 TMCs in 200 µL of R10 in a 96 well round bottom plate.
3. To activate the cells, add 5 µg/mL of the TLR7/8 agonist resiquimod (R848) overnight.
4. Centrifuge the plates and retrieve the TMCs' supernatant and freeze it for further analysis. Cytokine production will be assessed using bead-based immunoassays to quantify multiple soluble cytokines simultaneously using a flow cytometer following the manufacturer's protocol.
5. Assess the viability of the cells using a luminescent cell viability assay following the manufacturer's protocol. Briefly, add 60 µL of cell viability solution to the wells and measure luminescence within 10 min (acquisition for 1 s/well).

Results

We first characterized the immune profile of cells present in the culture and analyzed the amount of TMCs. We phenotyped the TMCs from tonsils with flow cytometry. As shown in Figure 1, all major immune cell types present in PBMCs from blood were represent in the TMCs from tonsils. However, in TMCs the frequency of all cell types, except B cells, were lower than in PBMCs.

Discussion

Human tonsils represent an integrative and physiological ex vivo model to study innate immune responses at the mucosal interface, because they mimic the role of a secondary lymphoid organ. Interestingly, the cellular composition of the TMCs is similar to the PBMCs and includes all the major cell populations, although their percentage can be different from PBMCs from blood (Figure 1). Additional populations can also be found, as all immune responses are initiated in tissues (mucosa or seconda...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 meshes steel grid - 1910 µm	Dutscher	198586	To put in the cell strainer Cellector
60 meshes steel grid - 230 µm	Dutscher	198591	To put in the cell strainer Cellector
70 µm white ClearLine cell strainers	Dutscher	141379C
Anios Excell D detergent	Dutscher	59852	Detergent
Antibiotic solution, 100x	Thermo Fisher	15140122	100 U/mL Penicilium and 100 μg/mL Streptomycin - to add to culture media
BD FalconTM Round-Bottom Tubes, 5 mL	BD Biosciences	352063	FACS Tubes
Cell strainer Cellector, 85 mL and 37 mm diameter	Dutscher	198585
CellTiter-Glo (CTG) Luminescent Cell Viability Assay	Promega	G7572	Viability assay
Centrifuge 5810 R	Eppendorf
Conical tubes Falcon 50 mL	Dutscher	352070
Curved tweezers	Dutscher	711200
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
Dulbecco's Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537	Without calcium and magnesium
EnVision	PerkinElmer		Measures the luminescence
Fetal Bovine Serum (FBS)			To add to culture media
Fluorescence labeles antibodies	See Table 1
Glass Pestle	Dutscher	198599
Hepes (1 M)	Thermo Fisher	15630056	Use at 20 mM
Incubator
LEGENDplex Human Anti-Virus Response Panel	BioLegend	740390	Bead-based immunoassay
Lymphoprep	StemCell	7801	Density gradient medium
Mr. Frosty container	Thermo Fisher	5100-0001	Slow freezing container
Pierce 16% Formaldehyde (w/v), Methanol-free	Thermo Fisher	28908
Resiquimod (R848)	InvivoGen	tlrl-r848	TLR7/8 agonist
RPMI-1640 Medium	Sigma-Aldrich	R8758
SPL Cell Culture Dish, 150 mm x 25 mm (SPL150)	Dutscher	330009
Surgical blade sterile N°23	Dutscher	132523
UltraComp eBeads Compensation Beads	Thermo Fisher	01-2222-41
UltraPure 0.5 M EDTA, pH 8.0	Thermo Fisher	15575020	To make wash buffer in PBS