IL2 deficiency and reduced responsiveness to IL2 contribute to the diminished T-cell immunity at advanced age. However, it is not clear if natural killer (NK) cells, the non-specific effectors of anti-tumor and anti-viral immunity are also affected during senescence. By virtue of their ability to secrete cytokines, e.g. interferon gamma (IFN-gamma) rapidly, NK cells may play an important role in the early phases of infection/ malignancy/immunoregulation. We have observed a senescence- related decline in in vitro sensitivity of purified human NK cells to lL2, i.e., release of IFN-gamma or LAK activity. Our long term goal is to investigate the cellular and molecular mechanisms involved in the preferential decline in IL2 inducible cytokine secretory functions of NK cells during senescence. We hypothesize that during senescence, there may be a reduced frequency of IL2 responding, IFN-gamma secreting NK cells and/or a defect in IL2- NK cell interaction at the level of IL2 receptors or the expression of IL2 receptor gamma chain or IFN-gamma genes, resulting in impaired IFN- gamma secretion per cell. To test this hypothesis, the amount of lFN-gamma released by sorted, IL2 activated NK subsets will be measured. The CD56(bright) and CD56(dim), cells represent the two distinct NK subsets. The frequency of IFN-gamma secretors among NK cells from healthy young and elderly will be quantitated by ELISPOT. Quantitative flow cytometric analysis of CD56(bright) (""""""""immature"""""""") and CD56(dim) (""""""""mature"""""""") NK subsets will be done. To investigate a potentially defective IL2-NK cell interaction, the expression of alpha (CD25) and beta (CD122) lL2 receptor chains (flow cytometry) and their affinity to IL2 or density per NK cell (equilibrium binding studies with (125)I-lL2) will be measured. To rule out a delayed translocation of IL2, the kinetics of internalization of cell surface bound lL2 will be followed. To locate a potential defect in IFN-gamma gene expression, the level of IFN-gamma mRNA in lL2 activated, sorted NK cells will be analyzed by quantitative RT-PCR by using PCR MIMICS. The level of IL2R-gamma mRNA will also be measured by PCR. Since a senescence-related decline in T- & B-cell DNA synthesis was noted by us before, the proliferative potential of IL2 induced NK cells will be assessed. These results will have theoretical implications in malignancy, infection and immune regulation and therapeutic applications in immunopharmacological interventions and cytokine gene therapies, especially in elderly patients.
