A new quantitative (two-photon extracellular polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets

Cytotoxic T lymphocytes kill targets via secretion of lytic agents including perforin and granzymes. Recently, new methods have been developed to monitor cytotoxic T lymphocyte degranulation. These include detecting the appearance of lysosome-associated membrane protein on the cell's surface, and monitoring decreases in cellular perforin content. We have combined these methods with microscopy and flow cytometry to provide the first analysis of how single cytotoxic T cells degranulate. We used TALL-104 human leukaemic cytotoxic T cells as a model system, and stimulated them with thapsigargin and PMA, soluble agents that mimic the two major signalling pathways activated by T cell receptor cross-linking. Our results indicate that essentially every TALL-104 cell responds to maximal stimulation by releasing about half of its lytic granule complement. This reflects complete release of the contents of half the cell's granules, rather than partial release of the contents of all of the granules. Sub-maximal stimulation reduces both the fraction of cells that respond and the magnitude of single cell responses. We find that individual cells respond to maximal stimulation with a variable latency, and provide evidence that, once it starts, degranulation is a slow process taking tens of minutes.