Upon antigen recognition, effector T lymphocytes secrete a variety of mediators that influence, creating a network of responsive cells. Most of these mediators are newly synthesized proteins that are immediately exported from the T cells. However, cytotoxic lymphocytes have a hallmark "regulated" secretory pathway in which pre-formed cytotoxic mediators (perforin and granzymes) are stored in cytoplasmic granules until antigen signaling triggers their rapid exocytosis. Another laboratory studying cloned cytotoxic T cells reported that the chemokine RANTES was also stored in cytotoxic granules. When we examined activation-induced RANTES secretion from purified memory and effector subpopulations of CD8+ human T cells from blood as well as short term cultured blasts, we found an early burst of preformed RANTES secretion with more rapid kinetics than the exocytosis of the lysosomal/cytotoxic granules. Deconvolution fluorescence microscopy revealed that intracellular RANTES is present in vesicles that do not colocalize with lysosomal markers, perforin or granzymes, a result confirmed by immunogold staining of ultrathin EM sections. Like these CD8+ lymphocytes, CD4+ blasts and NK cells also contain preformed RANTES in highly mobilizable non-lysosomal cytoplasmic vesicles, providing a rapid burst of local chemokine release that can recruit other inflammatory cells after antigen recognition. As a follow-up project, we have examined the intracellular localization and surface expression of the negative regulator CTLA-4 in T cell blasts and in the CD4+CD25+ "T regulatory" cells. This protein binds avidly to co-stimulatory molecules on antigen-presenting cells and negatively signals T cells, allowing for a termination of antigen-induced activation. In both types of T cells, deconvolution microscopy shows that CTLA-4 is present in vesicles that do not significantly colocalize with lysosomal or endosomal markers. In mouse lymph node CD4+CD25+ cells, activation-induced CTLA-4 surface expression is rapid, and resistant to protein synthesis inhibitors for the first hour. Using Rab27a-defective ashen mice that fail to exocytose lysosomal/cytotoxic granules after TcR crosslinking, this stimulation triggers normal CTLA-4 surface expression in both CD8+ T cell blasts and in the CD4+CD25+ "T regulatory" cells. The Rab27a-independent exocytosis of CTLA-4 indicates that intracellular CTLA-4 in T cells is stored in yet another distinct T cell compartment that undergoes rapid TcR-induced exocytosis.