Regulatory T Cells pp 55-69

Part of the Methods in Molecular Biology book series (MIMB, volume 707)

Flow Cytometric Profiling of Mature and Developing Regulatory T Cells in the Thymus

  • Donald M. Simons
  • Andrew J. Caton
Protocol

Abstract

Natural Regulatory T (Treg) cells are a subset of CD4+ T cells characterized by expression of the transcription factor Foxp3 and the ability to suppress immune responses. Treg cells develop in the thymus in response to highly specific interactions between the T cell receptor (TCR) and self-antigens. These processes can be recapitulated in antigen-specific systems using transgenic mice that coexpress a TCR with its cognate peptide as a neoself-antigen. Here, we describe a method for using such a system to establish a flow cytometric profile of phenotype markers expressed by developing and mature Treg cells in the thymus. Our approach is to compare antigen-specific thymocytes developing in the presence or absence of Treg cell-selecting ligands to identify phenotypic changes that characterize thymocytes undergoing selection into the Treg cell lineage.

Key words

Thymocyte Foxp3 Immune regulation Treg progenitor cell Immunophenotyping 

References

  1. 1.
    Starr TK, Jameson SC, Hogquist KA. (2003) Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176.PubMedCrossRefGoogle Scholar
  2. 2.
    Josefowicz SZ, Rudensky A. (2009) Control of regulatory T cell lineage commitment and maintenance. Immunity 30, 616–625.PubMedCrossRefGoogle Scholar
  3. 3.
    Jordan MS, Boesteanu A, Reed AJ et al. (2001) Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2, 301–306.PubMedCrossRefGoogle Scholar
  4. 4.
    Apostolou I, Sarukhan A, Klein L, von Boehmer H. (2002). Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3, 756–763.PubMedGoogle Scholar
  5. 5.
    Lio CW, Hsieh CS. (2008) A two-step process for thymic regulatory T cell development. Immunity 28, 100–111.PubMedCrossRefGoogle Scholar
  6. 6.
    Aschenbrenner K, D’Cruz LM, Vollmann EH et al. (2007) Selection of Foxp3(+) regulatory T cells specific for self antigen expressed and presented by Aire(+) medullary thymic epithelial cells. Nat. Immunol. 8, 351–358.PubMedCrossRefGoogle Scholar
  7. 7.
    Picca CC, Oh S, Panarey L, Aitken M, Basehoar A, Caton AJ. (2009) Thymocyte deletion can bias Treg formation toward low-abundance self-peptide. Eur. J. Immunol. 39, 3301–3306.PubMedCrossRefGoogle Scholar
  8. 8.
    Walker LS, Chodos A, Eggena M, Dooms H, Abbas AK. (2003) Antigen-dependent proliferation of CD4+ CD25+ regulatory T cells in vivo. J. Exp. Med. 198, 249–258.PubMedCrossRefGoogle Scholar
  9. 9.
    Kawahata K, Misaki Y, Yamauchi M et al. (2002) Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression. J. Immunol. 168, 4399–4405.PubMedGoogle Scholar
  10. 10.
    Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K, von Boehmer H. (1994) Thymic selection of CD8+ single positive cells with a class II major histocompatibility complex-restricted receptor. J. Exp. Med. 180, 25–34.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Donald M. Simons
    • 1
  • Andrew J. Caton
    • 1
  1. 1.The Wistar InstitutePhiladelphiaUSA

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