Normal regulation of apoptosis is critical to longevity. This proposal will study the fas apoptosis gene as a candidate LAG in CD2-fas transgenic mice. The fas apoptosis gene has recently been found to be mutated in autoimmune lprcg mice, and defective apoptosis was proposed to lead to survival of abnormal autoreactive T cells. We have found that there is a dramatic decrease in the expression of the fas apoptosis gene in B cells, T cells and macrophages of aged mice compared to young mice. This proposal will evaluate the role of defective expression of the Fas apoptosis gene in loss of T cell self tolerance and immune senescence in aged mice compared to young mice. In order to understand the cellular and molecular basis of loss of self tolerance related to defective fas expression in aging, this proposal will analyze T-cell receptor (TCR) transgene encoding receptor molecules that are specific for the H-2Db+ male H-Y antigens. The presence of the transgene results in nearly 100% of the T cells in these TCR transgenic mice to be reactive with the H-2Db and male H-Y antigen. Therefore, in C57BL/6 (H-2Db) male mice, nearly all of the T-cells will be self-reactive. The T-cell receptor alpha/beta chains can be detected by the antibody KJ16 or the anti-clonotypic mAb M.33. This allows easy identification of the transgenic T-cell receptor by flow cytometry. The specific aims include: 1. Use of the M.33 antibody to precisely quantitate autoreactive T cells in the thymus and peripheral lymphoid organs during aging in young (2 mo) and aged (18 mo) TCR transgenic mice. Functional loss of self-tolerance by T cells will be assayed in aged mice after TCR crosslinking with the M.33 antibody and in cytotoxic assays using Db/HY target cells. 2. Determine the level of expression of co-stimulatory molecules and fas expression of non-anergic subpopulations from the thymus and spleen of aged TCR transgenic mice. 3. Determine if the age- related T cell tolerance defect is corrected in a CD2-Fas transgenic mouse, which restores high fas expression in T cells of aged mice. These experiments using transgenic mice to prevent a decline in fas expression in T cells with aging are the first attempt to correct defective apoptosis with aging by a direct genetic replacement of the defective apoptosis gene. These experiments will have a major impact on our knowledge of senescence of the immune system, and the mechanisms of cell aging in general. Failure to eliminate defective, abnormal cells due to defects in apoptosis would lead to accumulation of these abnormal cells and their molecular and function defects. The ability to restore normal apoptosis with aging in transgenic mice expressing the candidate LAG, fas, might also allow for elimination of such aged cells and regulated replacement with normal cells. Experiments outlined in this proposal should allow the identification of abnormalities of intrathymic and extrathymic differentiation of autoreactive T ...