Flow Cytometric Detection of Human Regulatory T Cells

  • Barbara Fazekas de St Groth
  • Erhua Zhu
  • Suzanne Asad
  • Loretta Lee
Part of the Methods in Molecular Biology book series (MIMB, volume 707)


Tregs are absolutely required for the maintenance of self tolerance in mouse and man. Major abnormalities in Treg number or function cause rare but fatal syndromes with autoimmune, allergic and inflammatory features. Whether subtle Treg abnormalities contribute to the pathogenesis of sporadic autoimmune, allergic and immunoinflammatory diseases in man remains controversial. Robust methods for identifying and isolating human Tregs in patients and healthy controls are essential if we are to understand their role in these increasingly common diseases. We have outlined below a flow cytometric technique to detect and isolate the entire human Treg population based on expression of CD4, CD25, and CD127. Use of a number of additional antibodies for defining subsets within the Treg compartment is described. For analysis, anti-Foxp3 can be added to the cocktail, but the necessity for fixation and permeabilisation may reduce the signal from other antibodies.

Key words

Treg Human Flow cytometry CD127 Foxp3 


  1. 1.
    Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains (CD25); breakdown of a single mechanism. J Immunol 155:1151–1164.PubMedGoogle Scholar
  2. 2.
    Baecher-Allan, C., J.A. Brown, G.J. Freeman, and D.A. Hafler. 2001. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 167:1245–1253.PubMedGoogle Scholar
  3. 3.
    Valmori, D., A. Merlo, N. Souleimania, C. Hesdorffer, and M. Ayyoub. 2005. A peripheral circulating compartment of natural naive CD4 Tregs. J Clin Invest 115:1953–1962.PubMedCrossRefGoogle Scholar
  4. 4.
    Seddiki, N., B. Santner-Nanan, S.G. Tangye, S.I. Alexander, M. Solomon, S. Lee, R. Nanan, and B. Fazekas de St Groth. 2006. Persistence of naïve CD45RA+ regulatory T cells in adult life. Blood 107:2830–2838.Google Scholar
  5. 5.
    Seddiki, N., B. Santner-Nanan, J. Martinson, J. Zaunders, S. Sasson, A. Landay, M. Solomon, W. Selby, S.I. Alexander, R. Nanan, A. Kelleher, and B. Fazekas de St Groth. 2006. Expression of IL-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 203:1693–1700.Google Scholar
  6. 6.
    Liu, W., A.L. Putnam, Z. Xu-Yu, G.L. Szot, M.R. Lee, S. Zhu, P.A. Gottlieb, P. Kapranov, T.R. Gingeras, B. Fazekas de St Groth, C. Clayberger, D.M. Soper, S.F. Ziegler, and J.A. Bluestone. 2006. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 203:1701–1711.Google Scholar
  7. 7.
    Morgan, M.E., J.H. van Bilsen, A.M. Bakker, B. Heemskerk, M.W. Schilham, F.C. Hartgers, B.G. Elferink, L. van der Zanden, R.R. de Vries, T.W. Huizinga, T.H. Ottenhoff, and R.E. Toes. 2005. Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans. Hum Immunol 66:13–20.PubMedCrossRefGoogle Scholar
  8. 8.
    Cao, D., V. Malmstrom, C. Baecher-Allan, D. Hafler, L. Klareskog, and C. Trollmo. 2003. Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis. Eur J Immunol 33:215–223.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Barbara Fazekas de St Groth
    • 1
  • Erhua Zhu
    • 1
  • Suzanne Asad
    • 1
  • Loretta Lee
    • 1
  1. 1.T Cell Biology Research Program, Centenary Institute and Faculty of MedicineUniversity of SydneySydneyAustralia

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