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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Starr TK, Jameson SC, Hogquist KA. (2003) Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176.
Josefowicz SZ, Rudensky A. (2009) Control of regulatory T cell lineage commitment and maintenance. Immunity 30, 616–625.
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.
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.
Lio CW, Hsieh CS. (2008) A two-step process for thymic regulatory T cell development. Immunity 28, 100–111.
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.
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.
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.
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.
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.
Acknowledgments
The authors would like to thank Malinda Aitken, Christina Mergenthaler, Abigail Liebow, Alissa Basehoar, and Lori Mroz for their invaluable help in maintaining the transgenic mouse lineages described here. This work was supported by R01-AI59166 and by the Commonwealth Universal Research Enhancement Program, Pennsylvania department of Health. DMS is supported by T32 CA09171.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Simons, D.M., Caton, A.J. (2011). Flow Cytometric Profiling of Mature and Developing Regulatory T Cells in the Thymus. In: Kassiotis, G., Liston, A. (eds) Regulatory T Cells. Methods in Molecular Biology, vol 707. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61737-979-6_5
Download citation
DOI: https://doi.org/10.1007/978-1-61737-979-6_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61737-978-9
Online ISBN: 978-1-61737-979-6
eBook Packages: Springer Protocols