We previously developed an assay of cytotoxic T-lymphocyte lytic granule exocytosis based on externalization of LAMP-1/CD107A using nonphysiological stimuli to generate maximal levels of exocytosis. Here, we used polystyrene beads coated with anti-CD3 antibodies to stimulate cells. Light scatter let us distinguish cells that contacted beads from cells that had not, allowing comparison of signaling events and exocytosis from stimulated and unstimulated cells in one sample. Bead stimulation resulted in submaximal exocytosis, making it possible to detect compounds that either augment or inhibit lytic granule exocytosis. Coupled with the assay’s ability to distinguish responses in cells that have and have not contacted a stimulatory bead, it is possible to detect three kinds of compounds: inhibitors, stimulators, which cause exocytosis, and augmenters, which enhance receptor-stimulated exocytosis. To validate the assay, we screened a set of synthetic compounds identified using our previous assay and a library of 320 extracts prepared from tunicate-associated bacteria. One of the extracts augmented exocytosis threefold. Activity-guided fractionation and structure elucidation revealed that this compound is the known PKC activator teleocidin A-1. We conclude that our modified assay is suitable for screening synthetic compound plates and natural product collections, and will be useful for identifying immunologically active small molecules.
Calcineurin Activation Is Only One Calcium-dependent Step in Cytotoxic T Lymphocyte Granule Exocytosis
