We propose a series of studies of T lymphocyte activation in old and young mice aimed at identifying in biochemical terms of defect(s) that prevent many T cells from old mice from responding to mitogens. We have previously shown age-related derangements in the generation of cytoplasmic calcium signals in T cells from old donors, and now propose to test two explanatory hypotheses: (a) that aged T cells have over-active calcium extrusion systems; and (b) that the low resting membrane potential of T cells from old mice interferes with calcium signal degeneration. Our studies of early signal transduction have hinted that pathways dependent on protein kinase C may be less impaired by aging than other kinase- dependent paths; we now propose more direct and systematic tests of protein kinase function and substrate specificity in T cells from old mice. We have previously documented age-related defects in Con A-induced expression of c-myc mRNA. We now propose to test for age-related changes in expression of selected cell cycle related genes (c-fos, c-jun IL-2, and IL-2R) in responses to mitogenic stimuli (Con A, anti-CD3). Nonmitogenic activators (PMA, ionomycin) will be used in studies of gene expression and protein kinase function to identify pathways to particularly susceptible to age-related dysfunction. Antibodies to c-myc and c-fos proteins will be used to look for altered gene expression at the single cell level and in T cell subsets thought to be particularly susceptible to senescent change. Finally we propose to look for evidence of age-related defects in splicing of primary RNA transcripts, defects that we have previously suggested may be responsible for age-related loss of myc mRNA accumulation. We hope to identify specific age-related defects in calcium signals, protein kinase function, and gene expression that might contribute to poor T lymphocyte function in old mice.
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