A real-time killing assay to follow viral epitope presentation to CD8 T cells.
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.
Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Harvard Medical School, Cambridge, MA, USA.
Pauline
P
Gourdain
Julie
J
Boucau
Georgio
G
Kourjian
Nicole Y
NY
Lai
Ellen
E
Duong
Sylvie
S
Le Gall
D017951:Antigen Presentation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D002461:Cell Line, Transformed; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005260:Female; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000276:immunology; D015658:HIV Infections / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D008297:Male
D018984:Epitopes, T-Lymphocyte; D005954:Glucosephosphate Dehydrogenase
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