Although many cell surface markers are available to delineate the stages of T cell development in the thymus, we know very little about the underlying mechanisms that regulate these events. This is particularly true for positive selection, a process in which developing thymocytes are d riven to differentiate by the T cell antigen receptor (TCR)-mediated recognition of major histocompatibility complex (MHC) encoded molecules on the surface of thymic epithelial cells. We propose to characterize early changes in gene expression in the responding thymocyte. In order to accomplish this, we will take advantage of a novel system we have developed involving an immature CD4+8+ T cell line, designated DPK, that maintains the ability to differentiate into CD4+8- cells in vitro or in vivo. In addition, we have recently isolated a conditionally immortalized cortical thymic epithelial cell line that can induce the TCR-mediated differentiation of DPK cells in the absence of an exogenous source of antigen. Together, this system represents the only model for positive selection that utilizes cultured long term cell lines, allowing many new avenues of investigation. We have used this system to identify a potential candidate protein as a mediator between cell surface signals and changes in gene expression during positive selection. Using representational difference analysis, we have found that the egr-1 zinc finger transcription factor gene is induced during DPK cell differentiation, and that this protein is expressed in a subset of cortical and medullary normal thymocytes. Using the DPK system and normal and TCR transgenic mice, we will determine the expression of egr-1 during T cell development and its role in positive and negative selection. In addition, by expression of this transcription factor, and a truncated version that contains the DNA binding domain, in DPK cells and in transgenic animals we will assess its function in the context of T cell development. Finally, we propose to use the egr-1 promoter to create transgenic mice that express a cell surface marker for egr-1 expression. Together, these experiments will address some of the critical molecular and cellular events that regulate the production of a mature T cell repertoire.
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