Ex Vivo Generation of Regulatory T Cells: Characterization and Therapeutic Evaluation in a Model of Chronic Colitis

  • Fridrik Karlsson
  • Sherry A. Robinson-Jackson
  • Laura Gray
  • Songlin Zhang
  • Matthew B. Grisham
Part of the Methods in Molecular Biology book series (MIMB, volume 677)


Naturally occurring regulatory T cells (nTregs; CD4+CD25+Foxp3+) are capable of suppressing the chronic inflammation observed in a variety of different animal models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, and arthritis. A major limitation in exploring how and where nTregs exert their suppression in vivo is the relative paucity of these regulatory cells. Although several laboratories have described different methods to expand flow-purified nTregs or convert conventional/naïve T cells (CD4+Foxp3) to Foxp3-expressing “induced” Tregs (iTregs; CD4+Foxp3+) ex vivo, we have found that many of these approaches are encumbered with their own limitations. Therefore, we sought to develop a relatively simple ex vivo method to generate large numbers of Foxp3-expressing iTregs that can be used to evaluate their trafficking properties, suppressive activity, and therapeutic efficacy in a mouse model of chronic gut inflammation in vivo. We present a detailed protocol demonstrating that polyclonal activation of conventional CD4+ T cells in the presence of IL-2, TGFβ, and all trans retinoic acid induces >90% conversion of these T cells to Foxp3-expressing iTregs as well as promotes a three- to fourfold increase in proliferation following a 4-day incubation period in vitro. This protocol enhances modestly the surface expression of the gut-homing adhesion molecule CCR9 but not α4β7. Furthermore, we provide preliminary data demonstrating that these iTregs are significantly more potent at suppressing T-cell activation in vitro and are equally effective as freshly isolated nTregs at attenuating chronic colitis in vivo. Finally, we report that this protocol has the potential to generate 30–40 million iTregs from one healthy mouse spleen.

Key words

iTregs TGFβ Retinoic acid Colitis Inflammatory bowel disease 


  1. 1.
    Sakaguchi, S. et al. (2006) Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212, 8–27.PubMedCrossRefGoogle Scholar
  2. 2.
    Scheffold, A., Murphy, K.M. & Hofer, T. (2007) Competition for cytokines: T(reg) cells take all. Nat. Immunol. 8, 1285–1287.PubMedCrossRefGoogle Scholar
  3. 3.
    Uhlig, H.H. et al. (2006) Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis. J. Immunol. 177, 5852–5860.PubMedGoogle Scholar
  4. 4.
    Vignali, D.A., Collison, L.W. & Workman, C.J. (2008) How regulatory T cells work. Nat. Rev. Immunol. 8, 523–532.PubMedCrossRefGoogle Scholar
  5. 5.
    Earle, K.E. et al. (2005) In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation. Clin. Immunol. 115, 3–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Tang, Q. et al. (2004) In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199, 1455–1465.PubMedCrossRefGoogle Scholar
  7. 7.
    Battaglia, M., Stabilini, A. & Roncarolo, M.G. (2005) Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743–4748.PubMedCrossRefGoogle Scholar
  8. 8.
    Taylor, P.A., Lees, C.J. & Blazar, B.R. (2002) The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99, 3493–3499.PubMedCrossRefGoogle Scholar
  9. 9.
    Chen, W. et al. (2003) Conversion of peripheral CD4+. J. Exp. Med. 198, 1875–1886.PubMedCrossRefGoogle Scholar
  10. 10.
    Benson, M.J., Pino-Lagos, K., Rosemblatt, M. & Noelle, R.J. (2007) All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation. J. Exp. Med. 204, 1765–1774.PubMedCrossRefGoogle Scholar
  11. 11.
    Fantini, M.C., Dominitzki, S., Rizzo, A., Neurath, M.F. & Becker, C. (2007) In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat. Protoc. 2, 1789–1794.PubMedCrossRefGoogle Scholar
  12. 12.
    Kang, S.G., Lim, H.W., Andrisani, O.M., Broxmeyer, H.E. & Kim, C.H. (2007) Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells. J. Immunol. 179, 3724–3733.PubMedGoogle Scholar
  13. 13.
    Mucida, D. et al. (2007) Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 317, 256–260.PubMedCrossRefGoogle Scholar
  14. 14.
    Fontenot, J.D. et al. (2005) Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329–341.PubMedCrossRefGoogle Scholar
  15. 15.
    Zheng, S.G., Wang, J.H., Wang, P., Gray, J.D. & Horwitz, D.A. (2007) IL-2 is essential for TGF-beta to convert naive CD4(+)CD25(−) cells to CD25(+)Foxp3(+) regulatory T cells and for expansion of these cells. J. Immunol. 178, 2018–2027.PubMedGoogle Scholar
  16. 16.
    Sun, C.M. et al. (2007) Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J. Exp. Med. 204, 1775–1785.PubMedCrossRefGoogle Scholar
  17. 17.
    Hill, J.A. et al. (2008) Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+CD44hi Cells. Immunity 29, 758–770.PubMedCrossRefGoogle Scholar
  18. 18.
    Mucida, D. et al. (2009) Retinoic acid can directly promote TGF-beta-mediated Foxp3(+) Treg cell conversion of naive T cells. Immunity 30, 471–472.PubMedCrossRefGoogle Scholar
  19. 19.
    Thornton, A.M. & Shevach, E.M. (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296.PubMedCrossRefGoogle Scholar
  20. 20.
    Ostanin, D.V. et al. (2009) T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G135–G146.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2010

Authors and Affiliations

  • Fridrik Karlsson
    • 1
  • Sherry A. Robinson-Jackson
    • 1
  • Laura Gray
    • 1
  • Songlin Zhang
    • 2
  • Matthew B. Grisham
    • 1
  1. 1.Immunology and Inflammation Research Group, Department of Molecular and Cellular PhysiologyLSU Health Sciences CenterShreveportUSA
  2. 2.Immunology and Inflammation Research Group, Department of PathologyLSU Health Sciences CenterShreveportUSA

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