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

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+CD25high T cells in peripheral blood. By contrast, less than half of the CD4+CD25int population were FOXP3+, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4+CD25high 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. See accompanying Commentary http://dx.doi.org/10.1002/eji.200526303