This proposal will identify molecular changes that maintain the thymic microenvironment, deterioration of which is believed to cause age-related thymic involution. Thymic epithelial cells (TEC) constitute a major element of the thymic microenvironment that directs thymocyte development and controls key thymic functions that generate and maintain effective adaptive immunity to infection, and prevent development of autoimmunity. TEC differentiation requires the epithelial cell-autonomous gene forkhead box N1 (Foxn1). Several inborn Foxn1 mutations have provided information on mechanisms by which Foxn1 regulates TEC function during thymic organogenesis in the embryos. However, the function of Foxn1 in the mature thymus and in natural thymic aging is unclear. We have generated a Foxn1fx mouse, which permits conditional deletion of Foxn1 temporally and allows us to study the role of Foxn1 in functional maintenance of the postnatal thymus and age-related thymic involution. We will test the hypothesis that Foxn1-controlled TECs mediate critical changes of thymic aging, including T-lymphopoiesis and prevention of autoimmunity. To test this hypothesis, we propose four specific aims. 1) We will identify TEC subpopulations that are most sensitive to loss of Foxn1 by evaluating TEC phenotypes after rapid deletion of Foxn1 and during spontaneous and gradual excision of Foxn1, determine TEC-derived T-cell growth factors, including expression of Notch ligands. We will also determine if removal of Foxn1 affects thymocyte phenotypes, T-cell apoptosis. We will conditionally supply exogenous Foxn1 in the middle-aged mice to test if it delays age-related thymic insufficiency. 2) We will elucidate the mechanisms by which conditional Foxn1 deletion in the postnatal thymus causes the TEC phenotypes identified in aim 1, such as enhanced apoptosis and reduced expression of Notch ligands. 3) We will determine if the increased autoimmunity in aging is related to loss of Foxn1 function, and resulting reduced medullary TEC (mTEC) function. In the conditional Foxn1-deleted mice, we will evaluate mTEC differentiation and expression of genes associated with development of central tolerance in the postnatal thymus, as well as regulatory T-cell function. 4). We will map Foxn1-regulated molecules at transcriptional level by obtaining TECs from mice with deleted Foxn1, normal Foxn1, and normal-then-off Foxn1 expression, based on generation of a Foxn1geo gene-trap mouse model. RNA from TECs will be hybridized to microarrays to identify possible mechanisms and pathways by which Foxn1 regulates TEC development. These studies will provide critical new information on the molecular basis for age-related thymic involution and rejuvenation, and lay the groundwork for future studies to preserve or regenerate thymic function and to maintain T-cell immunity in the elderly. Molecular basis for age-related thymic involution and rejuvenation Narrative for the proposal: The thymic microenvironment is mainly controlled by the states of thymic epithelial cells, which are regulated by Foxn1 gene. We have developed a novel mouse model in which the Foxn1 gene can be temporally deleted, which provides a powerful tool to study the mechanisms of postnatal thymic involution, thymic insufficiency-derived immunosenescence, and the increased autoimmunity in aging. These studies will lay the groundwork for restoring thymic dysfunction and extending the functional lifespan of human cellular immunity.