T-cell development endows thymocytes both with an appropriate range of recognition specificities and with the capacity to respond to antigen recognition functionally. Using the ability to express interleukin-2 (IL-2) as a model functional response, we have shown that acquisition of recognition specificity is separable from acquisition of function in developing thymocytes. The goal of this proposal is to elucidate the complex molecular mechanisms that make thymocytes competent to express IL-2. Based on our recent findings, we expect the events that are components of this mechanism to be extremely informative both about the earliest stages of T-cell differentiation and about the physiology of the later T-cell receptor (TcR)-dependent selection processes. We had previously established that the competence of thymocytes to express IL-2 upon induction undergoes several sharp changes as the cells progress through differentiation. Now we have analyzed thymocytes of different stages to explain their distinctive IL-2 gene expression patterns in terms of the availability of a set of seven, differentially regulated transcription factors. Our results have, first, shown that the molecular requirements for competence to make IL-2 are met very early in thymocyte development. In other words, the transcriptional machinery that will eventually mediate responses to TcR triggering is in place considerably earlier than the capacity to deliver these triggering signals through the TcR. However, a striking decrease then occurs in the availability of the critical factors, NF-AT and AP-1, spanning the important period when thymocytes become subject to positive and negative selection. Other factors remain inducible in cortical cells but are apparently insufficient to mediate activation of response genes like IL- 2. Only after positive selection do the cells regain use of NF-AT and AP-1. These findings are provocative in that they identify specific molecular targets that are altered by intrathymic processing. It is conceivable, based on these results, that these two factors, NF-AT and a form of AP-1, could form parts of a relatively simple """"""""switch"""""""" mechanism that reversibly controls whether thymocytes undergo activation or selection in response to TcR ligands. In this proposal, therefore, we plan to determine the biochemical basis of the alterations in AP-1 and NF-AT activity in thymocytes; to test their biological relationship with other known transitions such as onset of TcR gene rearrangement, TcR expression, or positive selection; to verify the roles of the full set of factors in IL-2 regulation and determine which ones become activated in thymocytes in response to TcR ligands and other linked stimuli; and to determine the earliest developmental stage in which the competence to express IL-2 regulatory factors distinguishes pre-T cells from other hematopoietic cells.
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