Name: unraveling key players of humoral immunity: advanced and optimized lymphocyte isolation protocol from murine peyer's patches
Uploaded: 2018-11-21T14:00:00
Description: 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

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 ...

<ol>
	<li>van den Berg, T. K., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+&#8216;self&#8217;+recognition:+a+role+for+CD47-SIRP&#945;+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune &#8216;self&#8217; recognition: a role for CD47-SIRP&#945; interactions in hematopoietic stem cell transplantation.</a> Trends in Immunology. 29 (5), 203-206 (2008).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews Immunology. 14 (10), 667-685 (2014).</li><li>Reboldi, A., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peyer&#8217;s+patches:+Organizing+B-cell+responses+at+the+intestinal+frontier.">Peyer&#8217;s patches: Organizing B-cell responses at the intestinal frontier.</a> Immunological Reviews. 271 (1), 230-245 (2016).</li><li>Heel, K. A., McCauley, R. D., Papadimitriou, J. M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Peyer&#8217;s+patches.">Review: Peyer&#8217;s patches.</a> Journal of Gastroenterology and Hepatology. 12 (2), 122-136 (1997).</li><li>Fagarasan, S., Kinoshita, K., Muramatsu, M., Ikuta, K., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+class+switching+and+differentiation+to+IgA-producing+cells+in+the+gut+lamina+propria.">In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.</a> Nature. 413 (6856), 639-643 (2001).</li><li>Hopkins, S. A., Niedergang, F., Corthesy-Theulaz, I. E., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+Salmonella+typhimurium+vaccine+strain+is+taken+up+and+survives+within+murine+Peyer&#8217;s+patch+dendritic+cells.">A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer&#8217;s patch dendritic cells.</a> Cellular Microbiology. 2 (1), 59-68 (2000).</li><li>Shreedhar, V. K., Kelsall, B. L., Neutra, M. 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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 &#38; 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 &#181;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...

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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 ...

This method can answer key questions about the phenotype of mouse Peyer's Patch isolated T and B cell subsets. In particular, follicular T helper and germinal center B cell populations. The main advantage of this technique is that it dispenses with several laborous preparation steps such as collagenase based digestion that compromise the quality and identity of the isolated lymphocytes.Begin by placing a mouse in the supine position and sterilizing the abdomen with 70%ethanol. Make a midline abdominal incision through the skin and peritoneum from the pubis to the rib cage to open the peritineal cavity and locate the cecum and the ileocecal junction. Make as distal as possible cut at the junction to separate the small intestine from the cecum.Cut the mesentery with scissors to carefully remove the entire small intestine up to the pyloric sphincter. Snip the junction between the pylorus and the duodenum to completely detach the small intestine from the abdominal cavity and place the isolated small intestine into one well of a 6-well plate containing cold RPMI medium supplemented with 10%fetal bovine serum, or FBS, on ice. Then, gently agitate the tissues manually until all of the segments are submerged in the cold medium.When all of the intestines have been harvested, use forceps to grasp the mesenteric fat of one edge of the intestine and place the sample, mesenteric side down, on a paper towel. Moisten the entire intestinal segment with RPMI 10%FBS to avoid tissue dehydration and stickiness and locate the Peyer's patches, which appear as white multilobulated aggregates with a cauliflower-like shape on the anti-mesenteric side of the intestinal wall. To harvest the Peyer's patches, use curved surgical scissors with the curve facing up to gently excise each patch from it's distal and proximal borders, taking care to exclude the surrounding intestinal tissue, and place the Peyer's patches into individual wells of a 12-well plate containing ice-cold RPMI 10%FBS on ice as they are collected.When all of the patches have been acquired, use scissors to cut the tip of a 1 millimeter micro pipette tip so that the diameter is large enough to aspirate individual Peyer's patches and transfer the Peyer's patches into 150 millimeter conical tubes per mouse containing 25 milliliters of 37 degrees Celsius RPMI 10%FBS. Then place the tube in an orbital shaker at 37 degrees Celsius with continuous agitation at 125-150 RPM for 10 minutes. At the end of the agitation, transfer the Peyer's patches onto 140 micrometer cell strainer per mouse, and use the rubber end of one 10 milliliter syringe plunger per animal to gently disrupt the Peyer's patches through the mesh into individual 50 milliliter conical tubes.Rinse the strainers with 15-20 milliliters of cold RPMI 10%FBS and collect the cells by centrifugation. After carefully discarding the supernatant, resuspend the cells at an approximately 1 times 10 to the 7th cells per milliliter of RPMI 10%FBS concentration for counting. Then transfer 2-2.5 times 10 to the 6th cells per 200 microliters of medium to each well of a 96-well round bottom plate and pellet the cells by centrifugation.After discarding the supernatant with gentle flicking, centrifuge wash the cells in 200 microliters of standing buffer. Label the cells in 100 microliters of fixable viability dye, diluted in protein-free buffer for 30 minutes at 4 degrees Celsius, protected from light, followed by 2 washes and fresh staining buffer. After discarding the supernatant, resuspend the pellets in 20 microliters of FC block for 15 minute incubation at 4 degrees Celsius, followed by the addition of 80 microliters of primary surface antibody cocktail of interest, for 30 minutes on ice, protected from light.At the end of the surface antibody incubation, centrifuge wash the cells two times in an excess volume of staining buffer, and resuspend the pellets in 100 microliters of secondary surface staining solution, supplemented with streptavidin. After 15 minutes on ice protected from light, centrifuge wash the cells 2 times in fresh staining buffer and resuspend the pellets in 200 microliters of fixation, permeablization working solution for a no more than 20 minute incubation on ice, protected from light. At the end of the incubation, centrifuge the plate immediately, followed by a centrifuge was in 200 microliters of permeablization buffer per well.Next, resuspend the cells in 20 microliters of FC block, diluted in permeablization buffer as demonstrated, followed by the addition of 80 microliters of the intracellular antibody cocktail of interest, for 30 minutes at room temperature, protected from light. Wash the cells with 100 microliters of permeablization buffer, followed by a second wash in 200 microliters of permeablization buffer. Then, resuspend the pellets in 200 microliters of staining buffer, and transfer the cells to the appropriate corresponding 5 milliliter flow cytometric analysis tubes, in a final volume of 400 microliters of staining buffer per tube.Peyer's patches are not evenly distributed throughout the small intestine, but are localized more densely towards the distal and proximal ends of the tissue. This protocol facilitates the isolation of a Peyer's patch lymphocyte population that demonstrates a forward-side scattered distribution, similar to that observed for splenocytes, with a greater than 95%cell viability. CD4 CD19 Double Positive cells constitute about 1%of the total Peyer's patch lymphocyte population.These express high levels of CXCR5 and GL7, which, when not excluded, can lead to false positive results in the gating of T-helper follicular and germinal center B cell, respectively. Compared to other secondary lymphoid organs, Peyer's patches exhibit a significantly higher ratio of germinal center B cells, at a range of 2-10%of the total B cell population under steady state conditions. Like germinal center B cells, the follicular T-helper cell fraction also varies in individual unimmunized mice, with a range of 10-20%of total, the CD4 Positive Peyer's patch T-lymphocyte population.Collagenase based enzymatic digestion leads to massive reduction of CXCR5 T-helper follicular cell expression, while leaving the expression of other surface molecules unaffected. After this development, this technique paved the way for the identification of key cellular factors within the Peyer's patches that are involved in mucosal and humeral immunity.

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