The long-term goals of this project are: i) to understand the spatial restrictions on cytokine gene expression - and, consequently, on cell function - within the CD4+T cell population of healthy individuals, and ii) to identify, within, the CD4+ T cell compartment of elderly individuals, alterations which may underlie the phenomenon of immunosenescence. It is proposed herein to use the mouse system to model phenotypic and functional heterogeneity among human CD4+ T cells. Flow cytofluorometric techniques will be used to analyze splenic CD4+ cells from young adult, middle-aged, and old mice for the simultaneous expression of three cell surface determinants - 3G11, CD45RB, and CD44 - thought to demarcate CD4+ cell subsets with restricted patterns of cytokine production and cell function. Subsequently, CD4+ cells, isolated from mice of each age group, will be stimulated with anti-CD3epsilon monoclonal antibody in vitro and then evaluated for: i) extent of cell cycle entry and progression and ii) patterns of production of several T cell-associated cytokines, monitored at the level of either steady-state mRNA accumulation or protein secretion. In addition, CD4+ cells from young Thy-1 allotype congenic mice and from old mice of the appropriate background strain will be used in cell mixing experiments to study age-related alterations in CD4+-CD4+ cell communications. In order to correlate patterns of 3G11, CD45RB, and CD44 expression with CD4+ cell function, cell subsets will be isolated based on the expressed levels of these markers and then evaluated for stimulation-dependent cell cycle progression and patterns of cytokine gene expression. Comparative analyses will be performed both with distinct subsets within the young group and with subset-matched preparations between age groups. Finally, the cell-mixing experiments described above will be extended to include evaluations of subset-subset communications among CD4+ cells from young mice, and potential alterations in these communications with advancing mouse age. Results from these studies should further our understanding of functional heterogeneity in the CD4+ cell compartment of healthy individuals, and of the consequences of aging on the representation and competence of CD4+ cell subsets.
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