In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells

Nat Protoc. 2007;2(7):1789-94. doi: 10.1038/nprot.2007.258.


CD4+ CD25+ regulatory T cells (Tregs) are crucial for the maintenance of immunological tolerance. Recent data indicate that Tregs not only develop in the thymus during ontogeny but can also differentiate from naive T cells in the periphery. The following protocol describes a method by which Tregs are generated in vitro by stimulation of naive T cells in the presence of transforming growth factor beta (Ti-Tregs). In vitro-induced regulatory T cells express markers of conventional Treg such as CD25 and the genetic program committing transcription factor FoxP3. Functionally the in vitro-generated Ti-Tregs suppress T-cell activation and proliferation while in vivo these cells have been proven to control inflammation in different animal models, suggesting a potential use of these cells for immunotherapy. The protocol can be completed within 5 days.

MeSH terms

  • Animals
  • Biomarkers / analysis
  • CD28 Antigens / analysis
  • CD3 Complex / analysis
  • CD4 Antigens / genetics
  • CD4-Positive T-Lymphocytes / immunology*
  • Interleukin-2 Receptor alpha Subunit / genetics
  • Interleukin-2 Receptor alpha Subunit / immunology*
  • Mice
  • Mice, Inbred BALB C
  • T-Lymphocytes / immunology*
  • T-Lymphocytes, Regulatory / immunology*


  • Biomarkers
  • CD28 Antigens
  • CD3 Complex
  • CD4 Antigens
  • Interleukin-2 Receptor alpha Subunit