Li Jinzhu, Figueira Sarah K, Vrazo Alexandra C A, Binkowski Brock F, Butler Braeden L, Tabata Yasuhiro, Filipovich Alexandra, Jordan Michael B, Risma Kimberly A
Division of Allergy/Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
Promega Corp., Madison, WI 53704;
J Immunol. 2014 Jul 15;193(2):519-28. doi: 10.4049/jimmunol.1301668. Epub 2014 Jun 13.
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.
据报道，半胱天冬酶介导的细胞凋亡激活是CTLs通过颗粒酶B和Fas介导的杀伤途径的共同特征；然而，由于无法实时监测靶细胞特异性凋亡，半胱天冬酶激活的动力学仍不明确。我们通过开发一种新型生物传感器检测方法克服了这一限制，该方法可检测靶细胞中连续的、蛋白酶特异性活性。生物传感器由环状排列的荧光素酶构建而成，内部通过半胱天冬酶3/7或颗粒酶B/半胱天冬酶8切割位点连接，因此在相应蛋白酶进行蛋白水解切割时会被激活。将小鼠CTLs与表达任一类型生物传感器的靶细胞共同孵育，在细胞接触后几分钟内就会产生强烈的发光信号。该信号受TCR信号强度、CTL/靶细胞比例以及所用生物传感器类型的调节。此外，如通过（51）Cr释放试验所测，30分钟时的荧光素酶信号与靶细胞死亡相关。与Fas介导的小鼠CTLs信号诱导相比，颗粒酶B诱导后半胱天冬酶3/7生物传感器激活速率出乎意料地快；在穿孔素或颗粒酶B缺陷的CTLs中，Fas介导的信号在延迟90分钟后逐渐出现。值得注意的是，当通过重定向杀伤测量时，穿孔素缺陷的人CTLs诱导的Fas依赖性半胱天冬酶3/7生物传感器信号在延迟90分钟后也可检测到。因此，我们使用一种新型实时检测方法证明了颗粒酶B与Fas在人和小鼠CTLs中诱导的半胱天冬酶激活模式不同。
