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

Summary

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.

Abstract

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'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 TFH and GC B cell subsets.

Introduction

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'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 intest....

Protocol

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

1. (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.

2. Surgical Excision and Tissue Preparation Steps:

1. Surgical removal of the Sma....

Results

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 (

Discussion

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 4–5 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: "total .......

Materials

List of materials used in this article

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