FoxP3 gene expression and regulatory Tcells

The biological and medical relevance of regulatory T-cells to modulate immune responses was discovered recently due to cell-transfer experiments in mice. Based on the initial study, a number of reports confirm now that regulatory T cells play an important role in induction and regulation of immunological tolerance. However, at the present there is 1) no clear marker available for this type of cells, 2) little is known how they recognize own and extrinsic antigens and transmit the immune dampening information to other cells of the immune system, and 3) there is also completely uncovered yet the molecular mechanism responsible for their development and differentiation. The very recent discovery that the transcription factor Foxp3 (Forkhead box protein 3) is selectively upregulated on regulatory T cells was an essential step in their characterization. Thus, to date Foxp3 is the most specific marker to discriminate regulatory T-cells. Furthermore, upon over-expression of Foxp3 naive T cells acquire the negative regulating phenotype indicating that Foxp3 is not a mere marker but plays a critical role in this negative regulating immune process. Despite the relevance of Foxp3, there is still less knowledge about its own regulation. Based on bioinformatic data we will select candidates for the human FOXP3 promoter. Cloned fragments will be checked in reporter-gene assays to investigate regulatory elements. Furthermore, we will examine transcription factors involved in Foxp3 gene expression by electro-mobility shift assays. Finally, we intend to uncover Foxp3 downstream targets by the chromatin-immuno-precipitation method and an ultra-sensitive advanced imaging system. Examination of the promoter, upstream genes involved in its activation as well as potential Foxp3 targets will lead to further knowledge concerning T-cell polarization and accessibility to this important cell type of the adaptive immune system.

A small population of T-lymphocytes is described as regulatory T cells (Tregs) that maintain peripheral immune tolerance and thus play a major role in immune responses and prevention of autoimmunity. At present the best- characterized Tregs are described amid the CD4+CD25+ T cell population. The discovery of the transcription factor Forkhead box protein 3 (Foxp3) was a hallmark for the characterization of these Tregs. Mutations in the human FOXP3 gene cause X-linked human IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X- linked syndrome) and XLAAD (X-linked autoimmunity-allergic dysregulation syndrome). In this project we analyzed three prominent evolutionary conserved regions (ECRs) upstream of the human Foxp3 transcription start site. We cloned these ECRs into a modified vector that allowed us to measure the expression of destabilized green fluorescence protein (dEGFP) in cell lines and human T cells. By using this tool we screened for dominant transactivator and repressor candidates of the FOXP3 promoter. Our data revealed that proximal FOXP3 promoter regulation is maintained via T-cell receptor (TCR) induced signalling cascades.