Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level

Eur J Immunol. 2005 Jun;35(6):1681-91. doi: 10.1002/eji.200526189.


The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Cross Reactions
  • DNA-Binding Proteins / analysis*
  • DNA-Binding Proteins / immunology
  • Forkhead Transcription Factors
  • Humans
  • Immune Tolerance
  • Lymphocyte Activation
  • Mice
  • Mice, Inbred BALB C
  • Receptors, Interleukin-2 / analysis*
  • T-Lymphocytes, Regulatory / chemistry*
  • T-Lymphocytes, Regulatory / physiology


  • Antibodies, Monoclonal
  • DNA-Binding Proteins
  • FOXP3 protein, human
  • Forkhead Transcription Factors
  • Foxp3 protein, mouse
  • Receptors, Interleukin-2