Polyclonal Expansion of Human CD4+CD25+ Regulatory T Cells

  • Petra Hoffmann
  • Ruediger Eder
  • Matthias EdingerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 677)


Based on results from experimental animal models, the adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg) is expected to be efficacious in treating autoimmune and inflammatory diseases, as well as in preventing alloresponses after solid organ or stem-cell transplantation. For potential clinical applications, large numbers of Treg cells in maximum purity will be required to avoid the risk of disease exacerbation by contaminating effector T cells. We have recently described methods for the efficient in vitro expansion of human Treg cells and identified CD4+CD25highCD45RA+ T cells as the ideal starting population for the generation of homogeneous and stable Treg cell products. Here, we provide detailed instructions for their identification, isolation, expansion, and functional characterization.

Key words

Immunotherapy Tolerance Suppressor T cell Transplantation T-cell therapy 


  1. 1.
    Josefowicz SZ, and Rudensky A (2009) Control of regulatory T cell lineage commitment and maintenance. Immunity 30:616–25.PubMedCrossRefGoogle Scholar
  2. 2.
    Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6:295–307.PubMedCrossRefGoogle Scholar
  3. 3.
    Belkaid Y, and Tarbell K (2009) Regulatory T cells in the control of host–microorganism interactions. Annu Rev Immunol 27:551–89.PubMedCrossRefGoogle Scholar
  4. 4.
    Sakaguchi S, Yamaguchi T, Nomura T, and Ono M (2008) Regulatory T cells and immune tolerance. Cell 133:775–87.PubMedCrossRefGoogle Scholar
  5. 5.
    Mottet C, Uhlig HH, and Powrie F (2003) Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 170:3939–43.PubMedGoogle Scholar
  6. 6.
    Feng G, Chan T, Wood KJ, and Bushell A (2009) Donor reactive regulatory T cells. Curr Opin Organ Transplant 14:432–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Hoffmann P, and Edinger M (2006) CD4+CD25+ regulatory T cells and graft-versus-host disease. Semin Hematol 43:62–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Edinger M, Hoffmann P, Ermann J, Drago K, Fathman CG, Strober S, and Negrin RS (2003) CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 9:1144–50.PubMedCrossRefGoogle Scholar
  9. 9.
    Hoffmann P, Ermann J, Edinger M, Fathman CG, and Strober S (2002) Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 196:389–99.PubMedCrossRefGoogle Scholar
  10. 10.
    Hoffmann P, Boeld TJ, Eder R, Albrecht J, Doser K, Piseshka B, Dada A, Niemand C, Assenmacher M, Orso E, Andreesen R, Holler E, and Edinger M (2006) Isolation of CD4(+)CD25(+) regulatory T cells for clinical trials. Biol Blood Marrow Transplant 12:267–74.PubMedCrossRefGoogle Scholar
  11. 11.
    Trzonkowski P, Bieniaszewska M, Juscinska J, Dobyszuk A, Krzystyniak A, Marek N, Mysliwska J, and Hellmann A (2009) First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells. Clin Immunol 133:22–6.PubMedCrossRefGoogle Scholar
  12. 12.
    Hoffmann P, Eder R, Kunz-Schughart LA, Andreesen R, and Edinger M (2004) Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood 104:895–903.PubMedCrossRefGoogle Scholar
  13. 13.
    Thornton AM, and Shevach EM (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 188:287–96.PubMedCrossRefGoogle Scholar
  14. 14.
    Hoffmann P, Eder R, Boeld TJ, Doser K, Piseshka B, Andreesen R, and Edinger M (2006) Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood 108:4260–7.PubMedCrossRefGoogle Scholar
  15. 15.
    Sallusto F, Geginat J, and Lanzavecchia A (2004) Central memory and effector memory T cell subsets: function, generation and maintenance. Annu Rev Immunol 22: 745–63.PubMedCrossRefGoogle Scholar
  16. 16.
    Kim HP, and Leonard WJ (2007) CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation. J Exp Med 204:1543–51.PubMedGoogle Scholar
  17. 17.
    Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, and Huehn J (2007) Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 5:e38.PubMedCrossRefGoogle Scholar
  18. 18.
    Baron U, Floess S, Wieczorek G, Baumann K, Grutzkau A, Dong J, Thiel A, Boeld TJ, Hoffmann P, Edinger M, Turbachova I, Hamann A, Olek S, and Huehn J (2007) DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol 37:2378–89.PubMedCrossRefGoogle Scholar
  19. 19.
    Schmidl C, Klug M, Boeld TJ, Andreesen R, Hoffmann P, Edinger M, and Rehli M (2009) Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity. Genome Res 19:1165–74.PubMedCrossRefGoogle Scholar
  20. 20.
    Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, de St Groth BF, Clayberger C, Soper DM, Ziegler SF, and Bluestone JA (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 203:1701–11.PubMedCrossRefGoogle Scholar
  21. 21.
    Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, Kelleher A, and Fazekas de St Groth B (2006) Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 203:1693–700.PubMedCrossRefGoogle Scholar
  22. 22.
    Kleinewietfeld M, Starke M, Di Mitri D, Borsellino G, Battistini L, Rotzschke O, and Falk K (2009) CD49d provides access to “untouched” human Foxp3+ Treg free of contaminating effector cells. Blood 113:827–36.PubMedCrossRefGoogle Scholar
  23. 23.
    Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G, Olek S, Dietmaier W, Andreesen R, and Edinger M (2009) Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol 39:1088–97.PubMedCrossRefGoogle Scholar
  24. 24.
    Peltz GA, Trounstine ML, and Moore KW (1988) Cloned and expressed human Fc receptor for IgG mediates anti-CD3-dependent lymphoproliferation. J Immunol 141:1891–6.PubMedGoogle Scholar

Copyright information

© Humana Press 2010

Authors and Affiliations

  • Petra Hoffmann
    • 1
  • Ruediger Eder
    • 1
  • Matthias Edinger
    • 1
    Email author
  1. 1.Department of Hematology and OncologyUniversity Hospital RegensburgRegensburgGermany

Personalised recommendations