This work was supported by the Agence National de la Recherche sur le SIDA et les H&#233;patites ANRS (J-P.H) for the experiments and N.B. fellowship (AAP 2017 166). N.S. acknowledges support from the ANRS for fellowship (AAP 2016 1), the European Molecular Biology Organization EMBO for Fellowship (LT 834 2017), the startup funding program &#34;Baustein&#34; of the Medical Faculty of Ulm University (LSBN.0147) and the Deutsche Forschungsgemeinschaft DFG (SM 544/1 1).

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 Comit&#233; de Protection des Personnes (IDRCB/EUDRACT: 2018A0135847). Furthermore, consent of each patient or legal representative is requested to obtain donors&#39; personal data (e.g., sex, age, history of ENT infections) that can help interpret experimental results.
1. Handling of the Human Tonsillar Tissue
<ol>
	<li>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&#39;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.</li>
	<li>Clean all tools in between each use.
	<ol>
		<li>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.</li>
		<li>Brush each utensil to remove the tissue and wash with clear water.</li>
		<li>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.</li>
		<li>Place all the tools in sterile bags and autoclave.</li>
	</ol>
	</li>
</ol>
2. Dissection of the Human Tonsillar Tissue and Isolation of the Tonsillar Mononuclear Cells (TMCs)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Remove cauterized, bloody, and fibroid tissue using forceps and a scalpel. 
	NOTE: Tonsils removed from children do not need this step.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place a sterile 70 &#181;m 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.</li>
	<li>Centrifuge the cells at 250 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet by gently mixing the vial, then resuspend the cells in 35 mL of PBS.</li>
	<li>In a new vial, place a new 70 &#181;m 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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Centrifuge the solution at 1,000 x g for 30 min at RT with acceleration and break off.</li>
	<li>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.</li>
	<li>Remove the TMCs with a sterile 1 mL pipette tip and place in a new 50 mL vial.</li>
	<li>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 &#176;C to remove the remaining platelets.</li>
	<li>Repeat step 3.5, but centrifuge the tube at 400 x g for 10 min at 4 &#176;C.</li>
	<li>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 &#181;g/mL streptomycin).</li>
	<li>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.).</li>
	<li>If needed, freeze the cells for further use using standard techniques for primary cell freezing.
	<ol>
		<li>Count the number of cells.</li>
		<li>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 &#176;C and the FBS and DMSO solution should be kept at 4 &#176;C before using.</li>
		<li>Distribute 1 mL of the cell solution into cryotubes and put them in a slow freezing container that has been at 4 &#176;C overnight to control the rate of cell freezing. Place it at -80 &#176;C.</li>
		<li>To thaw the cells, place the cryovials in a warm bath at 37 &#176;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 &#181;m sieve before using it.</li>
	</ol>
	</li>
</ol>
4. Phenotyping of TMCs by Flow Cytometry
<ol>
	<li>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.</li>
	<li>Wash the cells in PBS and centrifuge at 400 x g for 5 min at 4 &#176;C. Resuspend them in 500 &#181;L of PBS (1 x 106 cells/mL) and incubate with a viability stain for 30 min at 4 &#176;C in the dark.</li>
	<li>Add 5 &#181;L heat inactivated human AB serum per 100 &#181;L of cell suspension and incubate for 15 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells in PBS + 2% FBS + 2 mM EDTA (Wash Buffer) and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the TMCs in 500 &#181;L of Wash Buffer containing the antibodies as detailed in Table 1. Incubate the cells for 30 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells in Wash Buffer and centrifuge at 400 x g for 5 min at 4 &#176;C. Resuspend the TMCs in 500 &#181;L of PBS containing 0.5% of paraformaldehyde (PFA). Keep in the dark at 4 &#176;C until acquisition by flow cytometry. 
	NOTE: PFA is hazardous. Use a commercial ready-to-use solution to prepare the 0.5% PFA solution.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Clone</td>
			<td>Fluorochrome</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>Live-Dead</td>
			<td></td>
			<td>BV405</td>
			<td>ThermoFisher Scientific</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>SP34-2</td>
			<td>V500</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>SK1</td>
			<td>Amcyan</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>BW135/80</td>
			<td>VioBlue</td>
			<td>Miltenyi Biotec</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>RPA-T4</td>
			<td>PE-Cy7</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>HI30</td>
			<td>PerCP-cy5.5</td>
			<td>BD</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>HD237</td>
			<td>ECD</td>
			<td>Beckman Coulter</td>
		</tr>
		<tr>
			<td>CD20</td>
			<td>2H7</td>
			<td>Alexa Fluor 700</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>M5E2</td>
			<td>PE-cy7</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>M5E2</td>
			<td>APC</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>3G8</td>
			<td>APC-H7</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>MEM188</td>
			<td>PE</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD123</td>
			<td>7G3</td>
			<td>PE</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>BDCA-1</td>
			<td>L161</td>
			<td>Pacific Blue</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>BDCA-3</td>
			<td>AD5-14H12</td>
			<td>FITC</td>
			<td>Miltenyi Biotec</td>
		</tr>
		<tr>
			<td>BDCA-4</td>
			<td>AD5-17F6</td>
			<td>APC</td>
			<td>Miltenyi Biotec</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>G646-6</td>
			<td>PerCP-cy5.5</td>
			<td>BD Pharmingen</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>3.9</td>
			<td>Alexa Fluor 700</td>
			<td>ebiosciences</td>
		</tr>
	</tbody>
</table>
Table 1: List of antibodies for cell characterization by flow cytometry.
5. Example of Activation of TMCs and Measurement of Cytokine Production
<ol>
	<li>Dilute the TMCs in R10 medium to get a concentration of 2 x 106 cells/mL.</li>
	<li>Distribute 400,000 TMCs in 200 &#181;L of R10 in a 96 well round bottom plate.</li>
	<li>To activate the cells, add 5 &#181;g/mL of the TLR7/8 agonist resiquimod (R848) overnight.</li>
	<li>Centrifuge the plates and retrieve the TMCs&#39; 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&#39;s protocol.</li>
	<li>Assess the viability of the cells using a luminescent cell viability assay following the manufacturer&#39;s protocol. Briefly, add 60 &#181;L of cell viability solution to the wells and measure luminescence within 10 min (acquisition for 1 s/well).</li>
</ol>

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The authors have nothing to disclose.

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 secondary lymphoid organs) and not in blood.
The use of human tissue explants as well as the use of cell culture or blood cells each have their advantages. However, for the of study cytokine secretion and cell activation, tissue explants were not the best model. In fact, we could not detect any IFN production in the supernatant of tissue explants (Figure 2B). We speculate that it is directly consumed by the surrounding cells. On the other hand, stimulation of blood cells (PBMCs) can mimic the primary response to infection but do not mimic what is happening in the tissues, where most of the cytokines are produced and where viruses replicate. Therefore, we set up a protocol to isolate and purify cells arising out of a secondary lymphoid tissue, the tonsil. We purified TMCs from healthy human tonsils, which allows us to investigate immune cell activation upon stimulation. However, one of the limitations of this model is that TMCs do not produce as many pro-inflammatory cytokines as PBMCs ex vivo upon TLR7/8 stimulation, although levels of IFN&#945;, the major antiviral cytokine, is similar in both cultures.
The study of compound toxicity on TMCs versus PBMCs revealed that TMCs are more sensitive to a toxic drug (Figure 3). Thus, the toxicity of new drugs and future treatments should be tested in cells from tissues and not only on cell lines, PBMCs, or tissue explants. Therefore, the use of TMCs for drug testing seems to be a future application of the method described.
Tonsils from adults or children can be obtained and processed as described. However, we recommend obtaining tonsils from children for two main reasons: 1) Children&#39;s tonsils contain more cells than the adults&#39;20. Indeed, tonsillar hypertrophy is particularly common in children, because they fight more childhood viruses. Thus, these large tonsils can become obstructive tonsils and need to be removed by a partial tonsillectomy to avoid complications like sleep apnea13; 2) Because adults&#39; tonsils are usually removed after several episodes of ear-nose-throat (ENT) infections, these tonsils are less na&#239;ve and contain fewer cells.
It is critical to work on the tonsils as soon as possible after the removal surgery, ideally &#60;3 h after collection. Indeed, right after removal and until dissection, the tonsils from each side are kept together in a 50 mL sterile vial containing around 20 mL of PBS, so that they are totally submerged.
Interdonor variability can represent a major issue for reproducibility. Indeed, variability in cellular composition between donors can be significant. Our protocol, using TMCs and not tissue explants, limits this variability because the cells from the whole tonsils are mixed before being plated and placed in culture, while tissue explants bring intradonor variability in cellular composition because the pieces come from different areas within the same specimen. Partial tonsillectomy can only be performed on noninflamed tonsils, which limits the inflammatory status variability between patients. We also recommend collecting tonsils surgically removed on different days. We have clearly noticed that inter-surgeon and inter-day variabilities are higher than interdonor variability (data not shown).

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 complex4,5,6. 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&#39;s tonsillar ring and is the most accessible MALT8,9. 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 cells11,12. 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 circulation14,15. 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.

<table><tbody><tr><td>10 meshes steel grid - 1910 &amp;micro;m</td><td>Dutscher</td><td>198586</td><td>To put in the cell strainer Cellector</td></tr><tr><td>60 meshes steel grid - 230 &amp;micro;m</td><td>Dutscher</td><td>198591</td><td>To put in the cell strainer Cellector</td></tr><tr><td>70 &amp;micro;m white ClearLine cell strainers</td><td>Dutscher</td><td>141379C</td><td></td></tr><tr><td>Anios Excell D detergent</td><td>Dutscher</td><td>59852</td><td>Detergent</td></tr><tr><td>Antibiotic solution, 100x</td><td>Thermo Fisher</td><td>15140122</td><td>100 U/mL Penicilium and 100 &amp;mu;g/mL Streptomycin - to add to culture media</td></tr><tr><td>BD FalconTM Round-Bottom Tubes, 5 mL</td><td>BD Biosciences</td><td>352063</td><td>FACS Tubes</td></tr><tr><td>Cell strainer Cellector, 85 mL and 37 mm diameter</td><td>Dutscher</td><td>198585</td><td></td></tr><tr><td>CellTiter-Glo (CTG) Luminescent Cell Viability Assay</td><td>Promega</td><td>G7572</td><td>Viability assay</td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Conical tubes Falcon 50 mL</td><td>Dutscher</td><td>352070</td><td></td></tr><tr><td>Curved tweezers</td><td>Dutscher</td><td>711200</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td>Without calcium and magnesium</td></tr><tr><td>EnVision</td><td>PerkinElmer</td><td></td><td>Measures the luminescence</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td></td><td></td><td>To add to culture media</td></tr><tr><td>Fluorescence labeles antibodies</td><td>See Table 1</td><td></td><td></td></tr><tr><td>Glass Pestle</td><td>Dutscher</td><td>198599</td><td></td></tr><tr><td>Hepes (1 M)</td><td>Thermo Fisher</td><td>15630056</td><td>Use at 20 mM</td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>LEGENDplex Human Anti-Virus Response Panel</td><td>BioLegend</td><td>740390</td><td>Bead-based immunoassay</td></tr><tr><td>Lymphoprep</td><td>StemCell</td><td>7801</td><td>Density gradient medium</td></tr><tr><td>Mr. Frosty container</td><td>Thermo Fisher</td><td>5100-0001</td><td>Slow freezing container</td></tr><tr><td>Pierce 16% Formaldehyde (w/v), Methanol-free</td><td>Thermo Fisher</td><td>28908</td><td></td></tr><tr><td>Resiquimod (R848)</td><td>InvivoGen</td><td>tlrl-r848</td><td>TLR7/8 agonist</td></tr><tr><td>RPMI-1640 Medium</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>SPL Cell Culture Dish, 150 mm x 25 mm (SPL150)</td><td>Dutscher</td><td>330009</td><td></td></tr><tr><td>Surgical blade sterile N&amp;deg;23</td><td>Dutscher</td><td>132523</td><td></td></tr><tr><td>UltraComp eBeads Compensation Beads</td><td>Thermo Fisher</td><td>01-2222-41</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Thermo Fisher</td><td>15575020</td><td>To make wash buffer in PBS</td></tr></tbody></table>

isolation of tonsillar mononuclear cells to study ex vivo innate immune responses in a human mucosal lymphoid tissue

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60914/60914fig1.jpg" /> 
Figure 1: Phenotyping of TMCs in tonsils. TMCs from healthy donors were obtained after dissection of human tonsillar tissue and isolation of cells. (A) Cells were stained, and data were acquired by flow cytometry. Data represent a representative experiment. (B) The table summarizes the percentage of each cell type in live TMCs in tonsils from six different healthy donors and compares each to the percentage of each cell type in PBMCs from blood, as defined in the literature18,19. Data are shown as the percentage of live cells SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60914/60914fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We then tested the immunological responses of the TMCs. One way to reproduce a cell&#39;s activation against a pathogen is to stimulate the innate immune sensors from the Toll-Like Receptors (TLRs) family. TLRs are mainly present in monocytes/macrophages, all dendritic cell (DC) subtypes, plasmacytoid dendritic cells (pDCs), and also in B cells. In this example, we studied TLR7 and TLR8, because they specialize in antiviral responses and most cases of tonsillitis (i.e., tonsil infections) are caused by a viral infection. Thus, TMCs from tonsils (graph in blue) from seven healthy donors were stimulated overnight with the TLR7/8 ligand resiquimod (R848). As in PBMCs (graph in green), activation through TLR7/8 signaling triggered production of type I interferons (IFN) and pro-inflammatory cytokines. In fact, the cytokine most highly produced in TMCs upon activation was IFN&#945;, a member of the type I IFN family that is mainly involved in innate immunity against viral infection and is mostly produced by the pDCs. All cytokines tested, except IFN2/3 and IL-8, were produced by TMCs, although at lower levels than in PBMCs. Interestingly, some cytokines (mainly IL-8, but also IL-6, IL-10, and TNF&#945;) were present in larger amount at basal levels. Furthermore, we compared the cytokine produced by isolated TMCs or by tonsillar tissue blocs prepared as described previously6. We measured all types of IFN produced in the supernatant of both cell cultures using the STING-37 cell line reporter assay as described previously17 and showed that we could only measure IFN levels in the isolated TMCs.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60914/60914fig2.jpg" /> 
Figure 2: TMCs produced cytokines upon stimulation. (A) Purified and isolated TMCs (blue) and PBMCs (green) were stimulated overnight with R848 (5 &#181;g/mL). Supernatants were retrieved and frozen until use. A bead-based immunoassay was performed to quantify multiple soluble cytokines simultaneously using a flow cytometer. The graphs represent the concentration in picograms per milliliter of each measured cytokine. The fold increases are annotated in red on the graphs. Mann-Whitney U test. (B) Purified and isolated TMCs from tonsils (blue) and tonsillar tissue blocs (orange) were stimulated for 24 h with Influenza A virus (IAV). A STING-37 reporter cell line assay was used to measure IFN production in the supernatant. Box and whisker plots with median &#177; minimum to maximum. Mann-Whitney U test. ***P &#60; 0.001, **P &#60; 0.01, *P &#60; 0.05. NS = non-stimulated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60914/60914fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, we tested the viability of the TMCs. Several techniques are available to measure cell viability. Here, we used a luminescent cell viability assay, an easy and quick two-step assay. As shown in Figure 3, we clearly detected the cytotoxicity induced by compound 1 in TMCs, which was not toxic to PBMCs. Thus, TMCs showed a higher sensitivity to the drug tested.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60914/60914fig3.jpg" /> 
Figure 3: Compound 1 was toxic to TMCs. Purified and isolated TMCs and PBMCs were preincubated with compound 1 (C1) at various concentrations for 1 h and then stimulated overnight with R848 (5 &#181;g/mL). Supernatants were retrieved, 60 &#181;L of cell viability solution was added, and luminescence was measured. The * represents the statistical analysis comparing C1 to NS. Box and whisker plots with median &#177; minimum to maximum. Kruskal-Wallis test with Dunn&#39;s post hoc correction. ****P &#60; 0.0001, ***P &#60; 0.001, **P &#60; 0.01, *P &#60; 0.05. NS = non-stimulated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60914/60914fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue at JoVE.com

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

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.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

isolation-tonsillar-mononuclear-cells-to-study-ex-vivo-innate-immune

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.4K Views.  Ulm University Medical Center. Tonsillaire mononucleaire cellen zijn relevanter en complexer dan bloedcellen, en hun isolatie maakt de studie van immuuncelrespons in pathologieën met betrekking tot mucosale immuniteit mogelijk. De techniek die we hebben vastgesteld maakt het herstel van een groot aantal immuuncellen uit een slijmvliesweefsel mogelijk terwijl ze hun integriteit behouden voor ex vivo-studies. De methode is vrij eenvoudig, maar de amandelen moeten snel en met zorg worden behandeld om de opbrengst te maximali...

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

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Biology

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

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

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