The thymus is essential for T cell development. Thymic epithelial cells (TECs) supply essential growth, differentiation and survival signals as thymocytes differentiate mature to nave T cells and emigrate to the periphery. Thymus involution reduces nave T cell output, which restricts the TCR repertoire and impairs immune responses to newly encountered antigens. Depletion of TECs is a major factor contributing to thymus involution during aging or after cytoablative therapy. We find that enforcing Cyclin D1 expression in keratin 5 (K5) expressing TECs results in a hyperplastic, yet functional thymus that fails to undergo involution during aging. These data suggest that sustained high levels of Cyclin D1 enhances TEC proliferation and differentiation, which, in turn, regulates thymus homeostasis and involution. A major goal of this proposal is to determine the mechanism(s) by which the K5.Cyclin D1 transgene prevents thymus involution. Although Cyclin D1 is a well-known cell cycle regulator, it also functions as a transcriptional regulator. Cyclin D1b is an alternatively spliced isoform in which the cyclin box is retained, but exon 5 is deleted. Exon 5 contains an LxxLL motif, which is a nuclear receptor interaction domain that enhances transcription by recruiting transcriptional coactivators. We find that the thymus involutes within normal time frame in K5.CyclinD1b transgenic mice. The distinct involution phenotypes in K5.Cyclin D1 and K5.Cyclin D1b mice implicate transcriptional regulation as a key mechanism by which enforced expression of Cyclin D1 prevents involution. Consistent with this notion, we find that the K5.Cyclin D1 transgene enhances expression of Foxn1, a transcription factor required for TEC development and maintenance. We will use the K5.CyclinD1 and K5.CyclinD1b transgenic lines to identify endogenous factors and pathways that regulate thymus involution and naive T cell output. Aims 1 and 2 will test the hypothesis that K5.CyclinD1 influences thymus homeostasis both by control of cell cycle progression and by transcriptional regulation. Aim 3 explores the consequences of preventing thymus involution on the composition of the peripheral T cell pool. Since we find an increased number of recent thymic emigrants (RTEs) in spleens of young K5.Cyclin D1 mice, we will test the hypothesis that preventing thymus involution sustains RTE output, which in turn maintains a diverse pool of naive peripheral T cells during aging or after cytoablative therapy. This investigation will provid a rational basis for developing therapeutic strategies to prevent or reverse thymus involution.