JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Immunology and Infection

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

Published: June 22nd, 2016



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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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<.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Hori, S., Nomura, T., Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 299 (5609), 1057-1061 (2003).
  2. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 155 (3), 1151-1164 (1995).
  3. Huang, C. T., et al. Role of LAG-3 in regulatory T cells. Immunity. 21 (4), 503-513 (2004).
  4. McHugh, R. S., et al. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity. 16 (2), 311-323 (2002).
  5. Takahashi, T., et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 192 (2), 303-310 (2000).
  6. Manigold, T., et al. Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis. C. Blood. 107 (11), 4424-4432 (2006).
  7. Andersson, J., et al. The prevalence of regulatory T cells in lymphoid tissue is correlated with viral load in HIV-infected patients. J Immunol. 174 (6), 3143-3147 (2005).
  8. Chen, X., et al. CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin Immunol. 123 (1), 50-59 (2007).
  9. Shafiani, S., Tucker-Heard, G., Kariyone, A., Takatsu, K., Urdahl, K. B. Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis. J Exp Med. 207 (7), 1409-1420 (2010).
  10. Belkaid, Y., Piccirillo, C. A., Mendez, S., Shevach, E. M., Sacks, D. L. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature. 420 (6915), 502-507 (2002).
  11. Grainger, J. R., et al. Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway. J Exp Med. 207 (11), 2331-2341 (2010).
  12. Taylor, M. D., van der Werf, N., Maizels, R. M. cells in helminth infection: the regulators and the regulated. Trends Immunol. 33 (4), 181-189 (2012).
  13. You, Z. Tumor regulatory T cells potently abrogate antitumor immunity. J Immunol. 182 (10), 6160-6167 (2009).
  14. Curiel, T. J., et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 10 (9), 942-949 (2004).
  15. Dittmer, U., et al. Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection. Immunity. 20 (3), 293-303 (2004).
  16. Robertson, S. J., Messer, R. J., Carmody, A. B., Hasenkrug, K. J. In vitro suppression of CD8+ T cell function by Friend virus-induced regulatory T cells. J Immunol. 176 (6), 3342-3349 (2006).
  17. Iwashiro, M., et al. Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proc Natl Acad Sci U S A. 98 (16), 9226-9230 (2001).
  18. Suvas, S., Kumaraguru, U., Pack, C. D., Lee, S., Rouse, B. T. CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J Exp Med. 198 (6), 889-901 (2003).
  19. Suvas, S., Azkur, A. K., Kim, B. S., Kumaraguru, U., Rouse, B. T. CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions. J Immunol. 172 (7), 4123-4132 (2004).
  20. Veiga-Parga, T., et al. On the role of regulatory T cells during viral-induced inflammatory lesions. J Immunol. 189 (12), 5924-5933 (2012).
  21. Wherry, E. J., et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 27 (4), 670-684 (2007).
  22. Jin, H. T., et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci U S A. 107 (33), 14733-14738 (2010).
  23. Punkosdy, G. A., et al. Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens. Proc Natl Acad Sci U S A. 108 (9), 3677-3682 (2011).
  24. Blackburn, S. D., et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol. 10 (1), 29-37 (2009).
  25. Park, H. J., et al. PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells. J Immunol. 194 (12), 5801-5811 (2015).
  26. Virgin, H. W., Wherry, E. J., Ahmed, R. Redefining chronic viral infection. Cell. 138 (1), 30-50 (2009).
  27. Penaloza-MacMaster, P., et al. Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection. J Exp Med. 211 (9), 1905-1918 (2014).
  28. Chang, M., et al. The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity. Nat Immunol. 12 (10), 1002-1009 (2011).
  29. Krishnamoorthy, N., et al. Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma. Nat Med. 18 (10), 1525-1530 (2012).
  30. Yadav, M., et al. Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J Exp Med. 209 (10), 1713-1722 (2012).
  31. Tai, X., et al. Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells. Blood. 119 (22), 5155-5163 (2012).
  32. Rushbrook, S. M., et al. Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection. J Virol. 79 (12), 7852-7859 (2005).
  33. Sekiya, T., et al. The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4. T cells. Nat Commun. 2 (269), (2011).
  34. Merianos, D. J., et al. Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice. J Clin Invest. 119 (9), 2590-2600 (2009).
  35. Allakhverdi, Z., et al. Expression of CD103 identifies human regulatory T-cell subsets. J Allergy Clin Immunol. 118 (6), 1342-1349 (2006).
  36. Camisaschi, C., et al. LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites. J Immunol. 184 (11), 6545-6551 (2010).
  37. Wang, R., et al. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells. Proc Natl Acad Sci U S A. 106 (32), 13439-13444 (2009).
  38. Myers, L., et al. IL-2-independent and TNF-alpha-dependent expansion of Vbeta5+ natural regulatory T cells during retrovirus infection. J Immunol. 190 (11), 5485-5495 (2013).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved