Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

Summary

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

Abstract

T Follicular Helper (Tfh) cells are an independent CD4⁺ T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

Introduction

In the recent decade, numerous studies have been focused on the newly identified CD4⁺ T cell subset, Tfh cell subset, for its essential roles in germinal center (GC) B development. B cell lymphoma 6 (Bcl6), which is mainly considered as a gene repressor, is the lineage-defining factor of Tfh cells for the evidence that ectopic expression of Bcl6 is sufficient to drive Tfh differentiation while deficiency of Bcl6 results in vanished Tfh differentiation^1,2,3. Unlike other CD4⁺ T helper subsets performing their effector function by migration to the sites of....

Protocol

Animal experiments were approved by the Institutional Animal Care and Use Committee of Institut Pasteur of Shanghai, China. All the experiments were performed based on the Institutional Animal Care and Use Committee-approved animal protocols.

NOTE: Virus infection of mice and isolation of organs should be performed under ABSL2 condition.

1. Inoculation of PR8 influenza virus and recording of mice weight

1. Prepare 8-week-old male C57BL/6 mice for infection at ABSL2 room.
   NOTE: This protocol is also suitable in experiments with female mice.
2. Dilution of PR8 virus

Results

Characterization of mouse morbidity in influenza virus infection
After influenza virus infection, mice are less active and anorexic due to illness, which is reflected by severe weight loss, a commonly used symptom to monitor the mouse morbidity¹⁹. As shown in Figure 1a, PR8 virus-infected mice started to lose weight on day 6, reached the highest loss level on day 8 and returned to the initial level on day 10. As expected, weight l.......

Discussion

Due to specialized roles in providing B-cell help for generating high-affinity antibodies, Tfh cells have been extensively studied in the mechanisms of differentiation and manipulation to provide new strategies for vaccine design. Influenza virus infection induces vigorous Tfh and GC B cells responses, thus being an appropriate model for this field of research. In this paper, we described protocols of influenza virus infection by intranasal inoculation, evaluation of Tfh-associated response by flow cytometry, immunofluor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Immunostaining of Tfh cells, NP-specific Tfh cells and Bcl-6
37% formaldehyde	Sigma	F1635
Anti-CD16/32 mouse	Thermo Fisher Scientific	14-0161-86
APC-conjugated-IAbNP311-325 MHC class II tetramer	NIH
Bcl-6 PE	Biolegend	358504	clone:7D1
Biotin-CXCR5	Thermo Fisher Scientific	13-7185-82	clone: SPRCL5
CD4 Percp-eFluor 710	Thermo Fisher Scientific	46-0041-82	clone:GK1.5
CD44 eVolve 605	Thermo Fisher Scientifi	83-0441-42	clone:IM7
CD44 FITC	Thermo Fisher Scientifi	11-0441-82	clone:IM7
CD62L FITC	BD Pharmingen	553150	clone:MEL-14
ICOS BV421	Biolegend	564070	clone:7E.17G9
PD1 PE/Cy7	Biolegend	135216	clone:29F.1A12
Streptavidin BV421	BD Pharmingen	563259
Streptavidin PE	BD Pharmingen	554081
Intracelluar staining of IL21
37% formaldehyde	Sigma	F1635
anti-human IgG	Jackson ImmunoResearch Laboratories	109-605-098
Brefeldin A	Sigma	B6542
human FCc IL-21 receptor	R&D System
ionomycin	Sigma	I0634
Live/Dead Fixable Aqua Dead Cell staining kit	Thermo Fisher Scientific	L34966
PMA	Sigma	P1585
Saponin	MP	102855
GC B and plasma cells staining
B220 APC	Thermo Fisher Scientific	17-0452-81	clone:RA3-6B2
CD138 PE	BD Pharmingen	561070	clone:281-2
CD95 (FAS) PE/Cy7	BD Pharmingen	557653	clone:Jo2
IgD eFluor 450	Thermo Fisher Scientific	48-5993-82	clone:11-26c
PNA FITC	Sigma	L7381
Assay of HA-specific antibody titer with ELISA
PR8-HA	Sino Biological	11684-V08H
BSA	SSBC
Goat anti mouse Ig (SBA Clonotyping System-HRP)	SouthernBiotech	5300-05
Goat anti mouse IgM (SBA Clonotyping System-HRP)	SouthernBiotech	5300-05
Goat anti mouse IgG1 (SBA Clonotyping System-HRP)	SouthernBiotech	5300-05
Goat anti mouse IgG2b (SBA Clonotyping System-HRP)	SouthernBiotech	5300-05
Goat anti mouse IgG2c (SBA Clonotyping System-HRP)	SouthernBiotech	5300-05
TMB Substrate Reagent Set	BD Pharmingen	555214
Histology
Alexa Fluor 555-Goat-anti rat IgG	Life Technology	A21434
anti-mouse IgD	Biolegend	405702
biotinylated PNA	Vector laboratories	B-1075
dilute Alexa Fluor 488-streptavidin	Life Technology	S11223
normal goat serum	SouthernBiotech	0060-01
Pro-long gold antifade reagent	Thermo Fisher Scientific	P3630
STREPTAVIDIN/BIOTIN blocking kit	Vector laboratories	SP-2002