Real-Time Detection of CTL Function Reveals Distinct Patterns of Caspase Activation Mediated by Fas versus Granzyme B

摘要: Activation of caspase-mediated apoptosis is reported to be a hallmark both granzyme B- and Fas-mediated pathways killing by CTLs; however, the kinetics caspase activation remain undefined owing an inability monitor target cell-specific in real time. We have overcome this limitation developing novel biosensor assay that detects continuous, protease-specific activity cells. Biosensors were engineered from circularly permuted luciferase, linked internally either 3/7 or B/caspase 8 cleavage sites, thus allowing upon proteolytic respective proteases. Coincubation murine CTLs with cells expressing type led robust luminescent signal within minutes cell contact. The was modulated strength TCR signaling, ratio CTL/target cells, used. Additionally, luciferase at 30 min correlated death, as measured (51)Cr-release assay. rate unexpectedly rapid following compared induction latter appeared gradually after 90-min delay perforin- B-deficient CTLs. Remarkably, Fas-dependent, induced perforin-deficient human also detectable when redirected killing. Thus, we used novel, real-time demonstrate distinct pattern B versus Fas