Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Summary

The current study showcases protocols for assessing the early fate commitment of virus-specific T_FH cells and manipulating gene expression in these cells.

Abstract

Follicular Helper T (T_FH) cells are perceived as an independent CD4⁺ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific T_FH cells is determined in the early infection phase, and investigations of the early-differentiated T_FH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4⁺ T cells specifically recognizing LCMV glycoprotein epitope I-A^bGP_66-77, we described procedures to access the early fate commitment of virus-specific T_FH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4⁺ T cells, methods to manipulate gene expression in early-differentiated virus-specific T_FH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific T_FH cells.

Introduction

Encountering different pathogens or threats, naïve CD4⁺ T cells tailor their immune responses by differentiating into various helper T (T_H) cell subsets with specialized functions¹. In the scenario of acute viral infection, a large portion of naïve CD4⁺ T cells differentiate into follicular helper T (T_FH) cells that provide help to B cells^2,3. Distinct from other CD4⁺ T_H cell subsets (e.g., T_H1, T_H2, T_H9, and T_H17 cells), T_FH cells express a substantial leve....

Protocol

All animal experiments were conducted following procedures approved by the Institutional Animal Care and Use Committees of the Third Military Medical University. The following mouse strains were used in the present study: C57BL/6J (B6) mouse (both genders), aged 6 to 8 weeks, weighing 25-30 g; CD45.1⁺SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic), both genders, aged 6 to 8 weeks, weighing 25-30 g; and CXCR5-GFP CD45.1⁺SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic × CXCR5-GFP knock-in), both genders, aged 6 to 8 weeks, weighing 25-30 g. The transgenic species were generated following a previously published report

Results

Characteristics of early-differentiated virus-specific T_FH cells during acute LCMV infection
To probe the early fate commitment of virus-specific T_FH cells, naïve congenic SM CD4⁺ T cells that specifically recognize LCMV GP epitope I-A^bGP_66-77 were adoptively transferred into CD45.2⁺ C57BL/6 recipients. The next day, these recipients were intravenously infected with a high dosage of the acutely resolved LCMV Armstrong (

Discussion

The research in the field of T_FH cells has been highlighted since the discovery of the specialized function of T_FH cells in helping B cells. Accumulating studies indicated that T_FH cell differentiation is a multistage and multifactorial process³⁰, wherein the T_FH cell fate commitment is determined in the early stage⁵. Therefore, a better understanding of the mechanisms underlying early-differentiated T_FH cells is crucia.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Corning	25-052-CI
4% Paraformaldehyde Fix Solution, 4% PFA	Beyotime	P0099-500mL
70 μm cell strainer	Merck	CLS431751
Alexa Fluor 647 anti-mouse TCR Vα2 (clone B20.1)	Biolegend	127812	1:200 dilution
Alexa Fluor 700 anti-mouse CD45.1 (clone A20)	Biolegend	110724	1:200 dilution
APC anti-mouse CD25 (clone PC61)	Biolegend	101910	1:200 dilution
B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse	The Jackson Laboratory	002014
BeaverBeads Streptavidin	Beaver	22321-10
Biotin anti-mouse F4/80 Antibody (clone BM8)	Biolegend	123106	1:200 dilution
Biotin Rat anti-mouse CD11c (clone N418)	Biolegend	117304	1:200 dilution
Biotin Rat anti-Mouse CD19 (clone 6D5)	Biolegend	115504	1:200 dilution
Biotin Rat anti-Mouse CD8a (clone 53-6.7)	Biolegend	100704	1:200 dilution
Biotin Rat anti-mouse NK-1.1 (clone PK136)	Biolegend	108704	1:200 dilution
Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119)	Biolegend	116204	1:200 dilution
bovine serum albumin, BSA	Sigma	A7906
Brilliant Violet 421 anti-T-bet (clone 4B10)	Biolegend	644816	1:100 dilution
Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12)	Biolegend	135220	1:200 dilution
C57BL/6J (B6) mouse	The Jackson Laboratory	000664
CXCR5-GFP knock-in reporter mouse			In house; the CXCR5-GFP knock-in mouse line was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr5.
DMEM 10% medium			DMEM medium containing 10% FBS
DMEM medium	Gibco	11885092
EDTA	Sigma	E9884
FACSFortesa	BD Biosciences
Fetal bovine serum, FBS	Sigma	F8318
FlowJo (version 10.4.0)	BD Biosciences
Foxp3/Transcription Factor Staining Buffer Set	Invitrogen	00-5523-00	The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer.
Goat Anti-Rat IgG Antibody (H+L), Biotinylated	Vector laboratories	BA-9400-1.5	1:200 dilution
Invitrogen EVOS FL Auto Cell Imaging System	ThermoFisher Scientific
Isolation buffer			FACS buffer containing 0.5% BSA and 2mM EDTA
LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV)	Chinese Peptide Company
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation	Life Technologies	L10199	1:200 dilution
MigR1	addgene	#27490
NaN3	Sigma	S2002
Opti-MEM medium	Gibco	31985070
pCL-Eco	addgene	#12371
PE anti-mouse CD69 (clone H1.2F3)	Biolegend	104508	1:200 dilution
PE Mouse anti-Bcl-6 (clone K112-91)	BD Biosciences	561522	1:50 dilution
Phosphate buffered saline, PBS	Gibco	10010072
Polybrene	Solarbio	H8761
Purified Rat Anti-Mouse CXCR5 (clone 2G8)	BD Biosciences	551961	1:50 dilution
Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5)	Biolegend	100538	1:200 dilution
recombinant murine IL-2	Gibco	212-12-1MG
Red Blood Cell Lysis Buffer	Beyotime	C3702-500mL
RPMI 1640 medium	Sigma	R8758
RPMI 2%			RPMI 1640 medium containing 2% FBS
SMARTA (SM) TCR transgenic mouse			SM TCR transgenic line in our lab is a gift from Dr. Rafi Ahmed (Emory University). Additionally, this mouse line can also be obtained from The Jackson Laboratory (stain#: 030450).
Staining buffer			PBS containing 2% FBS and 0.01% NaN3
Streptavidin PE-Cyanine7	eBioscience	25-4317-82	1:200 dilution
TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate)	Cell signaling technology	6444S	1:400 dilution
TFH cell staining buffer			FACS buffer containing 1% BSA and 2% mouse serum
TransIT-293 reagent	Mirus Bio	MIRUMIR2700