Recent work in this laboratory has resulted in the development of a serum-free, chemically defined system for culturing human T lymphocytes, and has demonstrated that these cells reach the senescent phase of culture after precisely the same number of population doublings as fibroblasts. In the present project, T lymphocyte cultures will be used to differentiate between two potential mechanisms of cellular senescence. The first possibility, that aging is a random stochastic process in which a gradually increasing number of cells display a disarray of functional and phenotypic characteristics, will be assessed by functional assays, adapted to analyze individual cells and yield data in the proportion of cells showing a certain characteristic, such as activation-related events, mitotic activity, relative RNA/DNA content during different stages of the cell cycle, antigen-specific effector function, and membrane function. The alternative hypothesis, that cellular aging involves a discrete, genetically programmed event, will be tested largely by experiments aimed at identifying a T cell specific senescence antigen. The production of a monoclonal antibody specific for such an antigen will be of tremendous technical importance in the field of immunogerontology, since it will permit the isolation and further analysis of the subset of T cells which are """"""""old"""""""" within the functionally heterogeneous cell population of the aging organism. The final area of endeavor involves the rare spontaneous """"""""immortalized"""""""" T cell line which arises within the T cell cultures. Analysis of these cells, which appear to be fully transformed by several criteria, may help elucidate both the process of senescence and growth control in general. Although studies on the in vitro behavior of fibroblasts have been extremely productive in studying in vitro senescence, they are hampered by the fact that fibroblasts lack the precise cell markers and the wide variety of functional activities which can be measured in lymphocytes. In addition, they require fetal calf serum in the culture medium. Since one of the central phenomena of mammalian aging is the immune decline at the organismal level, T lymphocytes represent and eminently suitable model to further explore in vitro senescence.
