CD28-mediated costimulatory signal plays a pivotal role in the outcome of many immune responses including cytolytic responses in tumor and autoimmune diseases. Depending on the primary stimulation, CD28 can initiate multiple intracellular signaling pathways including a pathway that is insensitive to immunosuppressive drug, Cyclosporin A (CsA). This CsA- insensitive pathway is believed to be involved in graft-vs-host disease (GVHD) during allogeneic bone marrow transplantation. Our current objectives focus on three areas: (1) characterization of the CsA-resistant rapamycin-sensitive pathway of T cell activation; (2) examination of the physiological significance of this pathway; and (3) the effect of aging on the rapamycin-sensitive pathway. Our recent work has demonstrated that the immunosuppressive drug rapamycin selectively affects the CsA-resistant pathway. Our initial studies have focused on the mechanism of activation of the IL-2 gene in a CsA-resistant manner. We found that the effects of rapamycin on the IL-2 expression was due to alteration in IL-2 mRNA stability. More recently, we have also shown that activation of T cells by IL-12 is resistant to CsA, but sensitive to rapamycin. As the intracellular target of rapamycin is mTOR, we are investigating the mechanism of activation of mTOR, particularly the role p85S6K1, an isoform of p70S6K1, in turning on mTOR downstream signaling leading to protein translation. Regarding the physiological role of the resistant pathway, we have observed the effect of cytokine signaling, particularly the combination of IL-12 and IL-18 but not individual cytokine alone, in activating resting human peripheral blood T cells in producing IFN- in a CsA-resistant, but rapamycin-sensitive manner. This cytokine-mediated activation of resting T cells was independent of antigen. We are currently investigating the in vivo physiological role of this cytokine signaling pathway.