The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells--which does not account for epitope processing--or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 min after addition of CTL. It has comparable sensitivity to (51)chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination.
Keywords: 7-AAD; 7-amino-actinomycin D; Antigen processing; CAM; CD8 T cells; CFSE; Cytotoxicity; G6PDH; HIV; Kinetics; MTG; MitoTrackerGreen; Real-time killing assay; calceinacetoxymethylester; carboxyfluorescein succinimidyl ester; glucose 6-phosphate dehydrogenase.
© 2013.