We would like to thank Laura Strauss and Peter Sage for helpful discussions and support with flow cytometry analyses.

All studies and experiments described in this protocol were conducted under guidelines according to Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center.
1. Designing Experimental Set-up and Mouse Groups
<ol>
	<li>(Optional) Co-house the experimental mice to facilitate horizontal transmission of gut microbiota between experimental mice and to reduce non-specific variability within PP lymphocytes. Additionally, use littermate controls of the same gender to minimize variability.</li>
</ol>
2. Surgical Excision and Tissue Preparation Steps:
<ol>
	<li>Surgical removal of the Small Intestine (SI)
	<ol>
		<li>Euthanize mice using CO2 asphyxiation or any equivalent method approved by the institutional animal ethics committee.</li>
		<li>Transfer the mouse to a dedicated area for surgical excision. Place backside down and sanitize the abdomen with 70% ethanol. Perform a laparotomy by cutting the abdominal skin and peritoneum along the midline from pubis to the rib cage thus opening the peritoneal cavity. 
		NOTE: Perform the first incision on a relatively small area of the skin to avoid penetrating the peritoneal cavity and damaging intestinal tissue. Continue the excision until desired anatomical border.</li>
		<li>Identify the caecum, which is an ideal landmark for the detection of the terminal ileum, which constitutes the distal segment of the small intestine. 
		NOTE: Caecum is usually located on the lower left side of the mouse abdomen.</li>
		<li>Pinpoint ileocaecal junction and make a cut at this level as distal as possible to separate the small intestine from the caecum. Throughout the next steps, avoid excessive physical contact with the intestinal wall because fragile PPs collapse easily upon touch.</li>
		<li>Gently remove the entire small intestine until the pyloric sphincter by cutting the mesentery using scissors. Identify the junction between pylorus and duodenum, and snip the duodenum at this level, which will lead to complete detachment of small intestine from the abdominal cavity. 
		NOTE: (i) Avoid hyperextension as this might cause the rupture of the intestinal wall. (ii) If LP lymphocyte isolation is desired in addition to PP lymphocytes, complete removal of the mesenteric fat is necessary. However, for PP isolation only, remaining mesenteric fat could provide some benefits during the further isolation steps; therefore, it should be preserved.</li>
		<li>Place detached small intestines in a 6-well plate filled with cold RPMI + 10% fetal bovine serum (FBS) and gently agitate the tissues manually until all intestinal segments are submerged in the cold media. Maintain the tissues on ice throughout the next steps.</li>
		<li>After dissecting the desired number of mouse small intestines, proceed to PP excision from collected small intestines.</li>
	</ol>
	</li>
	<li>Surgical Excision of PPs and Preparation of Single Cell Suspension:
	<ol>
		<li>Gently transfer the small intestine on a paper towel by gripping the mesenteric fat using forceps and place the mesenteric side facing the paper towel. Moisten the entire intestinal segment with cold RPMI + 10% FBS to avoid tissue dehydration and stickiness. 
		NOTE: Remaining mesenteric fat can be helpful at this stage because fat tissue segments on the mesenteric site of SI will stick to the paper towel keeping the anti-mesenteric site facing up.</li>
		<li>Identify the PPs, which appear as white multi-lobulated aggregates in a &#8220;cauliflower-like&#8221; shape on the anti-mesenteric side of the intestinal wall. 
		NOTE: Flushing out the luminal content is not recommended until all PPs are excised. Emptying the luminal content might cause the collapse of the PPs and will prevent the color contrast between PPs and the intestinal wall, which is very helpful for visual identification of PPs.</li>
		<li>After identifying PPs on the anti-mesenteric side, place the surgical curved-end scissor on PPs (curve should face up) restraining the PP from its distal and proximal border. 
		Optional:&#160;Push the PP gently toward the blades of scissor using a fingertip. This maneuver will lead to the better exclusion of surrounding non-PP tissue. Excise the PPs gently, excluding the surrounding intestinal tissue. 
		NOTE: (i) This step is crucial to obtain maximal PP cell yield while minimizing the cell contamination from neighboring intestinal compartments such as LP and intestinal epithelium, which are also rich in T cells. (ii) From one SI excised from C57BL/6 mouse, 5-10 PPs (average size, multi-lobulated) can be collected. By aiming even smaller PPs (not multi-lobulated), collection of up to 12-13 PPs per mouse (C57BL/6) is possible using this protocol.</li>
		<li>Transfer the excised PPs to a 12-well tissue culture plate filled with ice-cold RPMI + 10% FBS and maintained on ice using forceps or curved surgical scissors. 
		NOTE: (i) Immediately after excision of PPs, mucus and intestinal content on the PP surface can be cleaned by rubbing the tissue gently on a paper towel. This step will help improve the viability of PP lymphocytes. (ii) Instead of transferring the PPs to a plate, transferring to separated tubes can also be considered depending on total sample number.</li>
		<li>Optional: When PP excision and subsequent placement into the well plate are completed, the number and size of PPs collected from different experimental/mouse groups can be documented by taking a picture of the tissue culture plate containing PPs.</li>
		<li>Prepare a set of 50 mL conical tubes filled with 25 mL of RPMI + 10% FBS (pre-warmed at 37 &#176;C). Using a pair of scissors, cut the edge of a 1,000 &#181;L tip from the distance that will allow the aspiration of the PPs with 1 mL pipette. Aspirate the PPs with 1 mL pipette and transfer them from the 12-well tissue culture plate to the prepared 50 mL conical tubes. 
		NOTE: Use a new tip for each mouse sample to avoid cross-contamination among the samples.</li>
		<li>Secure the lid and place the tubes vertically in an orbital shaker at 37 &#176;C, with continuous agitation at 125&#8211;150 rpm for 10 min. Meanwhile, prepare a new set of 50 mL tubes and place a 40 &#181;m cell strainer on the top of each tube, through which single cell suspension will be prepared. 
		NOTE: (i) The agitation step will remove the remaining intestinal content, mucus and cell debris, which decrease the cell viability and recovery of PP lymphocytes if not removed. (ii) Do not apply any kind of digestive enzymes on PP tissue because the digestion process causes a dramatic loss of CXCR5 expression from the cell surface.</li>
		<li>After the agitation, transfer the PPs to the 40 &#181;m cell strainer placed on the top of the newly prepared conical 50 mL tubes. Using the rounded side of a 10 mL syringe plunger, gently disrupt the PPs through the cell strainer to generate single cell suspension. Rinse the strainer with 15&#8211;20 mL of cold RPMI + 10 % FBS. 
		NOTE: (i) Before filtering, shake the tubes containing the PPs horizontally. This short shake will facilitate the transfer of PPs into cell strainers. (ii) Using a 70 &#181;m cell strainer for the isolation of non-lymphoid immune cells (e.g., monocytes, macrophages, DCs) is recommended.</li>
		<li>Centrifuge the single cell suspensions at 350&#8211;400 x g for 10 min at 4 &#176;C.</li>
		<li>Carefully discard the supernatant and resuspend the cells at a concentration of 10 x 106 cells/mL. Count the cells using a hemocytometer. 
		NOTE: (i) Prior to cell counting, the total cell number for each mouse PP group can be approximately estimated using the following formula: &#8220;0.5-1 x 106 cells x (number of PPs) = total cell count&#8221;. Further dilutions with trypan blue for cell counting might be necessary. (ii) As an alternative to manual counting, automated cell counters can be used.</li>
		<li>Transfer 2-2.5 x 106 cells in appropriate volume (e.g., 200 &#181;L) into a 96-well round-bottom plate. 
		NOTE: For single color and negative control samples, 0.5-1 x 106 cells might be sufficient.</li>
		<li>Centrifuge the plate at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
		<li>Wash the cells in 200 &#181;L of Staining Buffer.</li>
	</ol>
	</li>
</ol>
3. Surface Antibody Staining
<ol>
	<li>Viability Staining:
	<ol>
		<li>After the final wash, resuspend the cells in 100 &#956;L of fixable viability dye diluted in PBS (1:1,000). Incubate for 30 min on ice or at 4 &#176;C in the dark. 
		NOTE: (i) Intracellular staining for the detection of key transcription factors such as Foxp3 or Bcl-6 requires fixation of the cells. In that case, non-fixable viability dyes (e.g., 7-AAD, DAPI) cannot be used. (ii) To dilute fixable viability dye, do not use any staining buffer that contains protein. The media used in this step must be protein-free. (iii) Exclusion of dead cells by using viability staining is crucial because dead cells can cause serious technical difficulties in flow cytometry analysis by emitting autofluorescence and by binding surface antibodies nonspecifically, which might lead to false positive results.</li>
		<li>Wash the cells twice with staining buffer. Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
	</ol>
	</li>
	<li>Fc Block and Surface -Layer I:
	<ol>
		<li>Prepare the Fc-block solution by diluting anti-CD16/32 antibody (1:200) in staining&#160;buffer.</li>
		<li>Resuspend the cells in 20 &#956;L of prepared Fc-block solution. Incubate for 15 min on ice.</li>
		<li>Without washing, add 80 &#956;L of surface antibody cocktail (see Table 1 for the antibody summary) prepared at appropriate dilutions. Incubate on ice for at least 30 min.</li>
		<li>Wash twice by adding excessive staining&#160;buffer. Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate.</li>
	</ol>
	</li>
	<li>Surface -Layer II:
	<ol>
		<li>Prepare Streptavidin staining solution by diluting fluorochrome-conjugated Streptavidin in staining buffer.</li>
		<li>After the final wash, resuspend the cells with 100 &#956;L of pre-diluted Streptavidin (1:100) staining solution. Incubate for at least 15 min on ice in the dark.</li>
		<li>Wash twice with staining buffer. Centrifuge at 350 x g for 5 min at 4&#176;C. Flick the plate. 
		Optional: If intracellular staining for follicular&#160;regulatory T cell detection is not desired, after the last wash, resuspend the cells in 200 &#956;L of staining buffer and transfer into appropriate tubes to acquire data in flow cytometer. When the samples are not fixed, acquisition of data by flow cytometry should be performed within 3&#8211;4 h to obtain accurate results.</li>
	</ol>
	</li>
</ol>
4. Cell Fixation
<ol>
	<li>Prepare fixation/permeabilization (Fix/Perm) working solution using the reagents from Foxp3/Transcription Factor Staining Buffer Set. Mix one-part of fixation/permeabilization concentrate with three parts of fixation/permeabilization diluent to the desired final volume.</li>
	<li>After the final wash, resuspend the cells in 200 &#956;L of Fix/Perm working solution.</li>
	<li>Incubate the plate on ice or at 4 &#176;C for 20 min. Do not exceed 20 min for this step. Longer incubation time might severely decrease the cell recovery.</li>
	<li>Centrifuge at 350 x g for 5 min at 4 &#176;C. Flick the plate. (Optional) After this step, fixed cells can be stored for several days at 4 &#176;C in staining buffer containing bovine serum albumin (BSA) or FBS until subsequent intracellular staining or flow cytometry acquisition.</li>
	<li>Resuspend the cells in 200 &#956;L of permeabilization Buffer (x1) freshly pre-diluted in purified deionized water.</li>
	<li>Centrifuge at 350 x g for 5 min at room temperature&#160;(RT).</li>
	<li>Wash once with 200 &#956;L of permeabilization buffer and centrifuge at 350 x g for 5 min at RT.</li>
</ol>
5. Intracellular Staining
<ol>
	<li>Prepare the Fc-block solution by diluting anti-CD16/32 antibody (1:200) in permeabilization buffer. 
	NOTE: After the fixation step, the cells must be maintained in permeabilization buffer until the end of the intracellular staining process.</li>
	<li>After the final wash, resuspend the cells in 20 &#956;L of Fc-block solution. Incubate for 10&#8211;15 min at RT in the dark.</li>
	<li>Without washing, add 80 &#956;L of intracellular antibody cocktail (100 &#956;L final volume) pre-diluted in permeabilization buffer. Incubate for 30 min at RT.</li>
	<li>Add 100 &#956;L of Perm Buffer, and centrifuge at 350 x g for 5 min at RT.</li>
	<li>Wash once with 200 &#956;L of permeabilization buffer, centrifuge at 350 x g for 5 min at RT.</li>
	<li>After the final wash, resuspend the cells in 200 &#956;L of staining buffer and transfer the cells into appropriate tubes (final volume of 400 &#956;L in staining buffer) and acquire data by flow cytometry. Samples stained in 96-well plate may also be&#160;run without transferring into tubes if a flow cytometer with plate reader option is available. This step minimizes cell loss during cell transferring.</li>
	<li>Acquire a minimum of 5 x 105 total cells on the flow cytometer to be able to perform reproducible analysis of TFH, TFR as well as GC B cells.</li>
</ol>

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T., 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=Interleukin+(IL)+-21+promotes+intestinal+IgA+response+to+microbiota.">Interleukin (IL) -21 promotes intestinal IgA response to microbiota.</a> Mucosal Immunology. 8 (5), 1072-1082 (2015).</li><li>Wei, J., 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=Autophagy+enforces+functional+integrity+of+regulatory+T+cells+by+coupling+environmental+cues+and+metabolic+homeostasis.">Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis.</a> Nature Immunology. 17 (3), 277-285 (2016).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> European Journal of Microbiology &amp; Immunology. 2 (2), 112-120 (2012).</li><li>Trapecar, M., 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=An+Optimized+and+Validated+Method+for+Isolation+and+Characterization+of+Lymphocytes+from+HIV++Human+Gut+Biopsies.">An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies.</a> AIDS Research and Human Retroviruses. 33 (S1), (2017).</li><li>Bergqvist, P., Gardby, E., Stensson, A., Bemark, M., Lycke, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+IgA+Class+Switch+Recombination+in+the+Absence+of+CD40+Does+Not+Occur+in+the+Lamina+Propria+and+Is+Independent+of+Germinal+Centers.">Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers.</a> Journal of Immunology. 177 (11), 7772-7783 (2006).</li><li>Keil, B., Gilles, A. M., Lecroisey, A., Hurion, N., Tong, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+collagenase+from+Achromobacter+iophagus.">Specificity of collagenase from Achromobacter iophagus.</a> FEBS Letters. 56 (2), 292-296 (1975).</li><li>Mora, J. R., 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=Selective+imprinting+of+gut-homing+T+cells+by+Peyer&#8217;s+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer&#8217;s patch dendritic cells.</a> Nature. 424 (6944), 88-93 (2003).</li><li>Reboldi, A., 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=Mucosal+immunology:+IgA+production+requires+B+cell+interaction+with+subepithelial+dendritic+cells+in+Peyer&#8217;s+patches.">Mucosal immunology: IgA production requires B cell interaction with subepithelial dendritic cells in Peyer&#8217;s patches.</a> Science. 352 (6287), (2016).</li></ol>

No conflicts of interest declared.

Here, we describe a protocol optimized for flow cytometric characterization of TFH and GC B cells. One of the major advantages of our protocol is that it enables the isolation of up to 107 (average 4&#8211;5 x 106 cells) total PP cells from a single mouse (C57BL/6 strain) without any digestive process. We observed that the total cell yield was positively correlated with the number of PPs and could be estimated from the following simple equation which is helpful for experimental planning: &#34;total ...

The entire gastrointestinal tract from the beginning to the end is decked with an extensive lymphoid network that contains immune cells more than any other organ in human and mouse1. Peyer&#39;s patches (PPs) constitute a major component of the intestinal branch of this cellular immune organization, so-called gut-associated lymphoid tissue (GALT)2,3. Within PPs, thousands of millions of antigens derived from dietary materials, commensal microbiota and pathogens are being sampled continuously, and when necessary appropriate immune responses toward them are mounted thus maintaining intestinal immune homeostasis. In that sense, PPs could be named as &#34;tonsils of the small intestine&#34;. PPs consist of major sub-compartments: subepithelial dome (SED), large B-cell follicle zones; the overlying follicle-associated epithelium (FAE) and interfollicular region (IFR) where T cells are located4. This unique compartmentalization of PPs enables different effector cell subsets to cooperate, thus, confers immunocompetence in the gut.
PPs lack afferent lymphatics, and due to this reason, antigens transported to PPs from small intestine are not being conducted through lymphatic vessels in contrast to most of the other lymphoid organs. Instead, specialized epithelial cells located in FAE, so-called M cells, are responsible for the transfer of luminal antigens into the PPs5. Subsequently, the transported antigens are picked up by the dendritic cells (DCs) and phagocytes that are located in the subepithelial dome (SED) region beneath the FAE6,7. This antigen sorting process by DCs in the PP is crucial to initiate adaptive immune response8 and subsequent generation of IgA secreting cells9.
Due to the heavy antigenic burden from commensal flora and dietary material, PPs host endogenously activated effector T and B cell subsets in great abundances such as TFH and IgA+ GC B cells10, suggesting that PPs represent a site of active immune response11. Detection of up to 20&#8211;25% TFH cells within total CD4+ T cell compartment and up to 10&#8211;15% GC B cells within total B cells is possible in PPs collected from unimmunized young C57BL/6 mice12. In contrast to other T helper cell types (i.e., Th1, Th2, Th17 cells), TFH cells show unique tropism into B cell follicles primarily owing to CXCR5 expression, which promotes TFH cell homing along CXCL13 gradient13. In the B cell follicle zones of PPs, TFH cells induce IgA class switch recombination and somatic hypermutation in activated B cells from which high-affinity IgA producing cells differentiate14. Subsequently, these antibody-secreting plasma cells migrate to the lamina propria (LP) and regulate immune homeostasis in the gut10.
Identification and characterization of TFH and GC B cell populations within PPs might enable researchers to investigate the dynamics of humoral immune responses under steady-state conditions without the need of time-consuming immunization models traditionally used in TFH-GC B cell studies15,16,17,18. Analyzing TFH cells within PPs is not as straightforward as other cell subsets. Technical challenges include identifying ideal tissue preparation conditions, surface antibody-marker combination, as well as selecting appropriate positive and negative controls. Both TFH and PP research fields exhibit great variability in terms of experimental procedures and are far from conferring a consensus to establish standardized protocols due to several reasons. First, each cell subset within PPs tends to be affected differentially by tissue preparation conditions requiring further modifications in a cell subset-specific manner. Second, there is a significant discrepancy among the reported methods regarding the details of cell preparation from PPs. Third, the number of protocol-based comparative studies investigating ideal tissue preparation techniques and experimental conditions for PP and TFH research is rather limited.
Current protocol-based studies suggested for PP cell preparation19,20,21,22 were not TFH- or GC B cell-oriented. Moreover, some tissue preparation conditions recommended for PPs19,20 such as collagenase-based digestion were found to affect the outcome of TFH identification by flow cytometry negatively18. On this basis, we reasoned that an optimized, standardized and reproducible protocol that can be used to study TFH and GC B cell dynamics within PPs would be valuable to investigators working on this topic. This need gave us the impetus to generate an improved and up-to-date protocol for the isolation and characterization of PP lymphocytes that is finely optimized for cellular recovery, viability, and efficiency for flow cytometric characterization of several T and B cell subsets. We also aimed to exclude several laborious preparation steps suggested in previous protocols, thereby, reducing the required manipulations and time for tissue and cell preparation from PPs.

<table><tbody><tr><td>anti-mouse CD4 antibody</td><td>eBioscience, Biolegend*</td><td>17-0041-81 ,10054*</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse CD19 antibody</td><td>eBioscience</td><td>MA5-16536</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse PD-1 antibody</td><td>eBioscience</td><td>61-9985-82</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse ICOS antibody</td><td>eBioscience</td><td>12-9942-82</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse GL7 antibody</td><td>Biolegend</td><td>144610</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse CXCR5 antibody</td><td>Biolegend*, BD Bioscience</td><td>145512*, 551960</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse BCL-6 antibody</td><td>Biolegend</td><td>358512</td><td>For detailed information see Table 1</td></tr><tr><td>anti-mouse Foxp3 antibody</td><td>eBioscience</td><td>17-5773-82</td><td>For detailed information see Table 1</td></tr><tr><td>Streptavidin-BV421</td><td>BD Bioscience</td><td>563259</td><td>For detailed information see Table 1</td></tr><tr><td>FixableViability Dye</td><td>eBioscience</td><td>L34957</td><td>For detailed information see Table 1</td></tr><tr><td>7AAD</td><td>Biolegend</td><td>420404</td><td>For detailed information see Table 1</td></tr><tr><td>FcBlock (CD16/32)</td><td>BD Bioscience</td><td>553141</td><td>For detailed information see Table 1</td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>LS004188</td><td></td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td>eBioscience</td><td>00-5523-00</td><td></td></tr><tr><td>6-well,12-well &amp;amp; 96-well plates</td><td>Falcon/Corning</td><td>353046,353043/3596</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td>3520</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>10 mL syringe-plunger</td><td>Exel INT</td><td>26265</td><td></td></tr><tr><td>RPMI</td><td>Corning</td><td>15-040-CV</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>Model 200</td><td></td></tr><tr><td>Curved-end scissor</td><td></td><td></td><td></td></tr><tr><td>Fine Serrated Forceps</td><td></td><td></td><td></td></tr><tr><td>Small curved scissor</td><td></td><td></td><td></td></tr></tbody></table>

unraveling key players of humoral immunity: advanced and optimized lymphocyte isolation protocol from murine peyer's patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In contrast to a previous protocol20, we have observed that PPs are not evenly distributed throughout the SI but are localized more densely towards the distal and proximal ends of the SI as shown in Figure 1A. Flow cytometric analysis showed that, if followed correctly, our protocol gives a PP lymphocyte population that demonstrates forward-side scatter distribution similar to splenocytes (<strong clas...

Watch this Scientific Journal Video about Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches at JoVE.com

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

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Cancer Research

13.5K Views.  Harvard Medical School. In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

Video: Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

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HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches