The CD4 T helper subset has recently been subdivided in to two groups on the basis of their ability to provide help to B cells and on the patterns of their cytokine secretion. T helper 1 (TH1) type cells produce IL-2, TNF, LT and IFN-gamma, and participate in delayed type hypersensitivity responses, while TH2 cells in vivo has recently been implicated in controlling responses to pathogens such as the human immunodeficiency virus. However, the molecular mechanisms controlling the development of TH1 and TH2 cells are not fully understood. Recently, IL-4 has been shown to be important in regulating this development. As a first approach to understanding TH1/TH2 development, our first Specific Aim will focus on the regulation of the IL-4 gene.
In Specific Aim 1 we propose to purify, clone and functionally characterize a novel DNA binding protein termed PCC. We present preliminary data, both by functional analysis and binding studies, that suggest an important role for this factor in the expression of the IL- 4 gene after T cell activation. In this Aim, we will further characterize the binding site of this factor by DNase1 footprint analysis, select the appropriate source of cells for purification, and then purify the PCC complex by standard techniques and clone the gene encoding it. The PCC cDNA and antibodies prepared to it will be used to study the function of this transcription factor in Il-4 regulation in TH2 cells.
In Specific Aim 2, we will examine the role of NF-AT protein, key transcriptional factors in tissue-specific transcription of the IL-2 and IL-4 genes in TH1 and TH2 cells. Since T helper cell subsets produce different cytokines, yet appear to share NF-AT factors, we will address the possibility that differential expression of multiple NF-AT genes or that different combinations of NF-AT homodimers or heterodimers determine tissue-specific expression in TH1 vs. TH2 cells. Disruption of nF-AT genes in vivo is an important part of this aim. In additional experiments, we propose to search for novel tissue- specific coactivators present in TH1 vs. TH2 cells that bind to NF-AT proteins. We suspect that there are other genes in addition to nF-AT whose expression may be differentially controlled in TH1 vs. TH2 cells and may contribute to their development. Therefore, in Specific Aim 3, we propose to identify genes induced or suppressed in the transition from the THp to the TH1 or TH2 stage of differentiation. A bipotential CD4+ IL-2-producing precursor cell (THp) can be driven along either a TH1 or TH-2-like lineage, given the appropriate culture conditions. This affords us the unique opportunity to identify genes specifically expressed or repressed in the transition from the THp to the TH1 or TH2 cell lineages. In this Aim, we propose to identify such genes, some of which may represent the novel transcription factors sought after in Aims 1 and 2, by the differential display technique using pCR. Once identified, the expression and function of such genes will be characterized as appropriate. The ability to manipulate ratios of TH1 and TH2 cells in vivo possibly through transcriptional regulation of the IL-4 gene has therapeutic implications for the treatment of infectious diseases, such as AIDS.
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