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Immunology and Infection

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

Published: June 22nd, 2016

DOI:

10.3791/54138

1Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University

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

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

Regulatory T (Treg) cells express forkhead box P3 (Foxp3) as a transcription factor for their development and function1. Additionally, Treg 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 viruses6,7, bacteria8,9, and parasites10-12, or in the course of cancer development13,14, Treg

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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.......

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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 Treg cells and Tconv 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+ Tconv (Figure 1A<.......

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Although only a small number of Treg 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 Treg cells increases during a chronic virus infection15-20 as well as cancer progression13,14. This is probably due to continued antigen stimulation. To evaluate the Treg cells function under antigen persistence and disea.......

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This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

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Name Company Catalog Number Comments
FITC Rat Anti-Mouse CD4 RM4-5 BD Biosciences 553047 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Cytofix/Cytoperm BD Biosciences 554714 Use this reagent for cell surface staining.
U-Bottom Tissue Culture Plates BD Biosciences 353077
Fixation buffer BD Biosciences 554655 Use this reagent for cell surface staining.
FITC Rat Anti-Mouse CD25 7D4 BD Biosciences 553072 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Cell strainer, 70mm BD Biosciences 352350 Use this strainer for grinding the whole spleen.
Cell strainer, 40mm BD Biosciences 352340 Use this strainer for filtering the cells before column enrichment.
Brilliant Violet 421 Anti-mouse CD279 (PD-1) 29F.1A12 BioLegend 135217 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Brilliant Violet 605 Anti-Mouse CD4 RM4-5 Biolegend 100547 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
APC Anti-Mouse/Rat Foxp3  FJK-16s eBioscience 17-5773 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Foxp3 / Transcription Factor Staining Buffer Set eBioscience 00-5223
PerCP-Cyanine5.5 Anti-Mouse CD8a 53-6.7 eBiosicence 45-0081 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
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, 200mM solution Gibco  25030
Penicillin-Streptomycin, 10,000U/mL Gibco  10378-016
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit Life technologies L-34975 Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.
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 Use this column for Treg cell isolation
MACS Separation Columns, LS columns Miltenyibiotec 130-042-401 Use this column for CD8+ T cell and Treg cell isolation
EDTA, 0.5M (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 Flow cytometer*
Flowjo TreeStar Flow cytometry software†
Hematocytomer Marienfeld superior

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