This application is to pursue two lines of inquiry arising from our long- term investigation of the mechanisms that confer functional competence on developing thymocytes. One is to utilize in vivo perturbations of T-cell development to test whether critical changes in the ability to make interleukin-2 (IL-2) depend on expression of a selectable T-cell receptor (TcR). The other is to resolve whether immature pre-T cells express IL-2 within the thymus, by generating transgenic mice in which descendants of any such IL-2 producers can be identified. In the last project period, we found that cells in the postnatal thymus apparently do not express IL-2 during their maturation, although they become competent to respond to pharmacological stimuli with IL-2 induction. Inducibility of IL-2 was found even in cells too immature to express TcR of any kind. Only in TcR+ thymocyte subsets, however, was IL-2 induced in response to phosphoinositide pathway mediators alone. TcR- cells did not make IL-2 unless IL-1 was present as well. Therefore, the mature-type, IL- 1 independent ability to express IL-2 depends on some event temporally linked to TcR expression, possibly in alteration in intracellular signal transduction pathways. Such an event could play a major role in the regulation of commitment to an IL-2 producer lineage. Our first and second Specific Aims will test the relationship between the """"""""maturation"""""""" of IL-2 induction requirements and expression of TcR. Radiation chimeras, in which IL-2 expression matures late relative to TcR expression; SCID mice, in which TcR are not expressed; and transgenic mice with the alpha beta chains of a defined TcR, which are expressed precociously, will be examined by phenotypic analysis, in vitro stimulation, and in situ hybridization to quantitate and identify IL-2 producers. In alpha beta-TcR transgenic mice, response maturation will be correlated with positive and negative selection. The third Specific Aim is to develop a new approach to T-cell response maturation, using transgenic mice in which the """"""""response"""""""" gene regulatory sequence drives inducible expression of a gene that marks or functionally perturbs responding cells in vivo. Using IL-2 regulatory sequences to control the diphtheria toxin A subunit or a nontoxic recorder gene, we will optimize strategies for producing mice in which IL-2 induction leads to selective cell suicide. Such mice would provide a sensitive trace of the activation histories of cells capable of making IL-2. They will initially define any subsets derived from intrathymic IL-2 producers and ultimately allow sophisticated analyses of the lineage relationships of peripheral helper T cells.
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