title: Real-time detection of CTL function reveals distinct patterns of caspase activation mediated by Fas versus granzyme B.
abstract: Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a (51)Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B- compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B-deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs.
authors.affiliation: Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Li
authors.affiliation: Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Figueira
authors.affiliation: Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Alexandra C A
authors.initials: AC
authors.lastname: Vrazo
authors.affiliation: Promega Corp., Madison, WI 53704;
authors.forename: Brock F
authors.initials: BF
authors.lastname: Binkowski
authors.affiliation: Promega Corp., Madison, WI 53704;
authors.forename: Braeden L
authors.initials: BL
authors.lastname: Butler
authors.affiliation: Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Tabata
authors.affiliation: Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Filipovich
authors.affiliation: Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Jordan
authors.affiliation: Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and Kimberly.Risma@cchmc.org.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Risma
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D003602:Cytotoxicity, Immunologic / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology; D053804:Granzymes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D054353:Perforin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D019014:fas Receptor / Q000276:immunology* / Q000378:metabolism
chemical_list: D019014:fas Receptor; D054353:Perforin; D008156:Luciferases; D053804:Granzymes; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases