Luminescence-based protease biosensor as a novel method to measure cytotoxic T lymphocyte function (P3386)

Abstract

Abstract Cytotoxic lymphocytes (CTL) induce target cell apoptosis through delivery of granzymes and subsequent activation of caspases. CTL function is usually assessed by either traditional Cr51 release assay or flow-based assay. A 96 well, non-radioactive assay is desirable to meet the need of the fast growth of the research and clinical application of CTL. We aimed to develop a new luminescence-based CTL assay, using protease biosensor technology. For this purpose we utilized a circularly-permuted, firefly luciferase to contain a caspase 3/7 proteolytic activation site, allowing luminescent activity upon cleavage during apoptosis. When we expressed the caspase biosensor in EL4 tumor cells and co-incubated with antigen-specific CD8 effector cells, the biosensor was triggered in minutes and allowed for evaluation of the kinetics of caspase induction in the target cells in real time. The biosensor signal correlates with the Cr51 release assay and was dependent upon perforin and granzyme B signal. The caspase-activated, luciferase biosensor has substantial advantage over the traditional methods to measure lymphocyte cytotoxicity, and allow, for the first time, measuring cytotoxic function in real time.