Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.
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.
Liu
Luzheng
L
Emory Vaccine Center and Emory University School of Medicine, Atlanta, Georgia, USA.
Chahroudi
Ann
A
Silvestri
Guido
G
Wernett
Mary E
ME
Kaiser
William J
WJ
Safrit
Jeffrey T
JT
Komoriya
Akira
A
Altman
John D
JD
Packard
Beverly Z
BZ
Feinberg
Mark B
MB
D000818:Animals; D020169:Caspases / Q000378:metabolism*; D002860:Chromium Radioisotopes; D003602:Cytotoxicity, Immunologic / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D002860:Chromium Radioisotopes; D020169:Caspases