: Naive CD8+ T lymphocytes lack effector function and undergo a differentiation process to exit the naive state and to acquire a primed effector phenotype. Preliminary results indicate that this transition is a complex and highly regulated process, involving receptor-mediated signal transduction, cell cycle progression, chromatin remodeling, and changes in gene expression. Evidence suggests that the naive state may be actively maintained, raising the possibility of a genetic program to place a brake on differentiation. In contrast, primed T-cells gain effector function, as well as altered signal transduction pathways and a more dynamic actin cytoskeleton, suggesting that new genes become expressed to mediate these processes. The overall goal of this application is to identify the molecular phenotype of naive and primed effector CD8+ T-cells, and to study the functional role of identified molecules in normal T-cell populations. The principal model will utilize 2C TCR transgenic mice crossed to a Rag2 background, to extinguish endogenous TCR gene rearrangement and ensure a naive phenotype. Mechanistic experiments in normal, post-thymic, T-cells will be enabled by a novel Coxsackie and adenovirus receptor (CAR) transgenic mouse and adenoviral transduction. The first specific aim is to identify genes and proteins that are differentially expressed between naive and primed effector CD8+ T-cells, by: I) examination of candidates by Western blotting, ii) analysis of expression of defined genes using Affymetrix microarrays, and iii) identification of unknown genes by subtractive library cloning. The second specific aim is to optimize the CAR transgenic mouse system for transduction of normal, post-thymic T-cells. Transgenic mice expressing CAR have been constructed using an LckICD2 promoter/enhancer cassette. Optimization of adenoviral transduction will be performed, and interbreeding with 2C/Rag2-/- mice will be carried out for studies of naive and primed CD8+ T-cells. The third specific aim is to analyze the function of differentially expressed signaling molecules and transcription factors linked to MAP kinase pathways in normal T-cells using CAR transgenic mice. Preliminary results have shown that several proteins involved in MAP kinase signaling become markedly up-regulated upon T-cell priming. Wildtype and mutant Ras, FosB, MKK7, JNK1, JNK2, and other candidates will be introduced into naive and primed CAR transgenic T-cells. Effects on early T-cell differentiation, as well as on apoptosis, TCR sensitivity, CD28 independence, and magnitude of cytokine production, will be examined. The fourth specific aim is to investigate the functional requirement for modulators of actin cytoskeleton dynamics in normal T-cells. Preliminary results have revealed that expression of several molecules associated with cytoskeletal dynamics is increased tremendously upon differentiation from the naive to the primed effector state. Wildtype and mutant a-Pak, Raci, and other candidates will be introduced into primed effector T-cells, and potential effects on TCR capping, conjugate formation, cell spreading, cytolysis, and cytokine production will be investigated. In addition, gelsolin4 -/- mice will be examined for functional defects in the T-cell compartment. The results of these experiments will provide invaluable information regarding the molecular and biochemical changes that accompany T-cell differentiation, pointing the way to new potential targets for manipulating immune responses in vivo.
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