Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Summary

Here, we describe a protocol to analyze the phenotype of regulatory T (T_reg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the T_reg cells.

Abstract

Regulatory T (T_reg) cells, which express Foxp3 as a transcription factor, are subsets of CD4⁺ T cells. T_reg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of T_reg cells is to suppress the proliferation of effector T (T_eff) cells and the production of cytokines such as IFN-γ, TNF-α, and IL-2. It has been demonstrated that T_reg cells' ability to inhibit the function of T_eff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of T_reg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human T_reg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated T_reg cells. To isolate activated T_reg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. T_reg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting T_reg cells isolated from naïve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated T_reg cells from resting T_reg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated T_reg cells.

Introduction

Regulatory T (T_reg) cells express forkhead box P3 (Foxp3) as a transcription factor for their development and function¹. Additionally, T_reg cells express various other molecules such as CD25², lymphocyte-activation gene 3 (LAG-3)³, glucocorticoid-induced tumor necrosis factor receptor⁴, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)⁵ on their surface or intracellular region. During chronic infection with various kinds of pathogens such as viruses^6,7, bacteria^8,9, and parasites^10-12, or in the course of cancer development^13,14, T_reg

Protocol

In this study, mice were maintained in a specific pathogen-free facility of the Yonsei Laboratory Animal Research Center of Yonsei University. All animal experiments were conducted in accordance with the Korean Food and Drug Administration guidelines using protocols approved by the International Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center at Yonsei University.

1. Preparation of Solutions

1. Prepare 2% RPMI media by diluting fetal bovine serum (FBS) to 2% and penicillin-streptomycin to 1% in RPMI
2. Prepare complete RPMI media. To RPMI media add 10% of FBS, 1% of penicillin-streptomycin, 1% o....

Results

We generated mice with persistent virus infection by injecting them with 2 x 10⁶ p.f.u. of LCMV CL13 intravenously. To investigate the phenotypic changes in T_reg cells and T_conv cells during chronic virus infection, splenic lymphocytes obtained from naïve and infected mice were stained with various antibodies and analyzed by flow cytometry. At 16 d p.i., PD-1 was upregulated in both Foxp3^-CD4⁺ T_conv (Figure 1A

Discussion

Although only a small number of T_reg cells exist in mice and humans, it is important to understand their function as they play a crucial role in regulating the immune response and maintaining immune tolerance. The number and suppressive functions of T_reg cells increases during a chronic virus infection^15-20 as well as cancer progression^13,14. This is probably due to continued antigen stimulation. To evaluate the T_reg cells function under antigen persistence and disea.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FITC Rat Anti-Mouse CD4	RM4-5	BD Biosciences	553047
Cytofix/Cytoperm		BD Biosciences	554714
U-Bottom Tissue Culture Plates		BD Biosciences	353077
Fixation buffer		BD Biosciences	554655
FITC Rat Anti-Mouse CD25	7D4	BD Biosciences	553072
Cell strainer, 70 mm		BD Biosciences	352350
Cell strainer, 40 mm		BD Biosciences	352340
Brilliant Violet 421 Anti-mouse CD279 (PD-1)	29F.1A12	BioLegend	135217
Brilliant Violet 605 Anti-Mouse CD4	RM4-5	Biolegend	100547
APC Anti-Mouse/Rat Foxp3	FJK-16s	eBioscience	17-5773
Foxp3/Transcription Factor Staining Buffer Set		eBioscience	00-5223
PerCP-Cyanine5.5 Anti-Mouse CD8a	53-6.7	eBiosicence	45-0081
Mouse IFN-gamma Platinum ELISA		eBiosicence	BMS606
RPMI 1640		GE Life Sciences	SH30027
PBS (1x)		GE Life Sciences	SH30256
ACK Lysing Buffer		Gibco	A10492-01
L-Glutamine, 200 mM solution		Gibco	25030
Penicillin-Streptomycin, 10,000 U/ml		Gibco	10378-016
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit		Life technologies	L-34975
CD8a+ T Cell Isolation Kit, mouse		Miltenyibiotec	130-104-075
CD4+ CD25+ Regulatory T Cell Isolation Kit, mouse		Miltenyibiotec	130-091-041
MACS Separation Columns, LD columns		Miltenyibiotec	130-042-901
MACS Separation Columns, LS columns		Miltenyibiotec	130-042-401
EDTA, 0.5 M (pH 8.0)		Promega	V4231
2-Mercaptoethanol		Sigma Life Science	M7522
Fetal Bovine Serum		Thermo Fisher Scientific	SH30919.03
CellTrace Violet Cell Proliferation Kit		Thermo Fisher Scientific	C34557
BD Canto II flowcytometer		BD Biosciences
Flowjo		TreeStar
Hematocytomer		Marienfeld superior