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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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+Blood+Mononuclear+Cells.">Peripheral Blood Mononuclear Cells.</a> The Impact of Food Bioactives on Health. , 161-167 (2015).</li><li>Ban, Y. L., Kong, B. H., Qu, X., Yang, Q. F., Ma, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDCA-1+,+BDCA-2++and+BDCA-3++dendritic+cells+in+early+human+pregnancy+decidua.">BDCA-1+, BDCA-2+ and BDCA-3+ dendritic cells in early human pregnancy decidua.</a> Clinical and Experimental Immunology. 151 (3), 399-406 (2008).</li><li>Papaioannou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Dependent+Changes+in+the+Size+of+Adenotonsillar+Tissue+in+Childhood:+Implications+for+Sleep-Disordered+Breathing.">Age-Dependent Changes in the Size of Adenotonsillar Tissue in Childhood: Implications for Sleep-Disordered Breathing.</a> The Journal of Pediatrics. 162 (2), 269-274 (2013).</li></ol>

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 seconda...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 meshes steel grid - 1910 &#181;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 &#181;m</td>
			<td>Dutscher</td>
			<td>198591</td>
			<td>To put in the cell strainer Cellector</td>
		</tr>
		<tr>
			<td>70 &#181;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 &#956;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&#39;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 (1M)</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 x 25 mm (SPL150)</td>
			<td>Dutscher</td>
			<td>330009</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade sterile N&#176;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 0.5M 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="...

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 ...

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Immunology and Infection

7.4K Views.  Ulm University Medical Center. 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...