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Visualization and quantification of T cell–mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

Abstract

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

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Acknowledgements

We thank R. Ahmed for provision of reagents and helpful advice; P. Henkart, R. Mittler and S. Staprans for critical review of the manuscript; and T. Cinotte for technical assistance. This study was supported by NIH grant P01AI46007 and R2AI49089. M.B.F. is an Elizabeth Glaser Scientist of the Pediatric AIDS Foundation.

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Correspondence to Mark B. Feinberg.

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