Cytotoxic T lymphocytes (CTL) are alerted to kill cancerous or virally infected cells when these cells present fragments of nonself proteins at the cell surface. We will probe the molecular mechanisms of antigen presentation and CTL killing by studying the behavior of specific molecules within individual, living CTL and target cells. We have synthesized fluorescent peptide antigens and demonstrated that upon injection in the cytoplasm of living cells, they are presented at the cell surface and induce genetically restricted killing. We have observed genetically restricted changes in the subcellular distribution of these peptides consistent with current models of antigen processing. The location of the peptides will be studied in single cells during processing and presentation, to determine the subcellular pathway of presentation and kinetics of transport, and to examine the basis for antigen selection. We will colocalize peptide with flourescent markers of the endoplasmic reticulum and golgi apparatus, and use transport inhibitors and mutant cells to induce accumulation in specific compartments thought to be involved in antigen processing. Mutant neuronal cells reversibly deficient in either TAP transporter function or MHC-I synthesis will be used to determine the role of these proteins in transport, and to probe for antigen selection on the basis of selective TAP transport or MHC binding. This system will provide a powerful assay to dissect the role of peptide structural features in these selection mechanisms. The location of peptide cleavage and selective transport of cleaved fragments will be studied through development of novel fluorescent peptide indicators whose fluorescence spectrum is altered by cleavage. Our preliminary studies have revealed distinct morphological changes in CTL and target cells during attachment and killing. These are rapid (2-3 minutes CTL attachment to target cell death) and include organized movements of cytotoxic granules in CTL, and contraction and blebbing of the target preceeding death. We have microinjected fluorescent analogs of actin and myosin II in living CTL and targets, and have observed CTL alter the target cell cytoskeleton during adhesion and killing. We will investigate the role of the microtubule and actin-myosin II cytoskeleton in directing granule movements and morphological changes in CTL. CTL behavior will be correlated with the timing and location of cytoskeletal changes within targets.
