Differentiation of T lymphocytes takes place in the thymus in a process that involves programmed activation of multiple genetic loci. Some of these genes encode constitutive products of mature T cells, such as components of their antigen-binding receptors. Other genes of interest are inducible genes that mediate T-cell responses after the cells have been triggered by antigen, such as those encoding interleukin 2 (IL2) and the IL2 receptor. The goal of this proposal is to study the sequence of activation of T-cell-specific genes in the thymus, with a particular focus on the second (inducible) class. The experiments will be designed to determine separately when the genes first become inducible at the chromatin level, and when the cells develop the signal-transduction mechanism that can turn these genes on in response to stimuli such as antigen recognition. The basis for the work is recent evidence that the IL2 gene itself becomes inducible before the develpoing T cell expresses receptors that can trigger induction. In situ hybridization will be used to detect IL2 induction in individual cells, and flow cytometry using the Ca2+-sensitive dye indo-1 will be used to measure their mobilization of intracellular Ca2+ in response to mitogenic lectins. Expression of high-affinity IL2 receptors will be measured by binding of radiolabeled IL2, adapted to autoradiography for single-cell detection. These methods will be used to dissect heterogeneity in the immature precursor thymocyte class, and to analyze the apparent loss of competence in cells becoming committed to the sterile, major-cortical differentiation pathway. Finally, preliminary results suggest that immature precursor thymocytes can be maintained in culture without destroying their abiltiy to differentiate. A vital aim of the proposal is therefore to optimize these culture conditions so that thymic lineage relationships can be determined and differentiation pathways manipulated directly, under controlled conditions in vitro.
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