FIGURES AND TABLES
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Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs
J Pardo, R Wallich, P Martin, C Urban, A Rongvaux, R A Flavell, A Müllbacher, C Borner and M M Simon
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Figure 1.
gzmB+CTLs induce membrane and mitochondrial perturbation in the absence of either Bid or Bak and Bax. Gp33-pulsed MEF.wt, MEF.Bid-/- (a, b) and MEF.Bak 
Bax-/- (c, d) cells were incubated with ex vivo virus-immune gzmB+CTL (MACS selected, 95% CD8+ cells) (1 h and/or 4 h, 10:1 effector/target ratio). Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (a, c, e) and, in parallel, 
m loss (DiOC6(3)) and ROS generation (2-HE; b, d, f) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression as described in Materials and Methods. A representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the right panels are represented as the mean
S.E.M. of six (a, b), five (c) and four (d) independent experiments, respectively. (a, c) Histograms corresponding to the annexin-V staining in the dot plots are shown
Figure 2.
gzmB+CTLs induce cell death independently of Bid, Bak, Bax and caspase activity. Gp33-pulsed MEF.wt, MEF.Bid-/- and MEF.Bak Bax-/- (a) or MEF.wt, MEF.Casp 3 7-/- and MEF.Casp 9-/- and (b) cells were incubated with ex vivo virus-immune gzmB+CTL (4 h, 10:1 effector/target ratio) in the presence or absence of the general caspase inhibitor Ac-ZVAD-fmk (80 
M) or the caspase-3/-7 inhibitor Ac-DEVD-fmk (not shown) (a, 80 M both). Target cell death was monitored by survival assay as described in Materials and Methods. Data are given as meanS.E.M. of five (MEF.wt, MEF.Bid-/-; a), four (MEF.Bak Bax-/-; a) and three (MEF.wt, MEF.Casp 3 7-/-, MEF.Casp 9-/-, b) independent experiments, respectively. (c) Gp33-pulsed MEF.wt and MEF.Bak Bax-/- cells were incubated with ex vivo virus-immune gzmB+CTL at different effector/target ratio (4 h) in the presence or absence of the general caspase inhibitor Ac-ZVAD-fmk (80 M). Target cell death was monitored by survival assay as described in Materials and Methods. Representative of two independent experiments. Ctr: only target cells; ctr ZVAD: target cells+ZVAD-fmk
Figure 3.
gzmB+CTL-induced conformational change of Bak and Bax depends on Bid and is critical for cytochrome c release independent of caspases. Gp33-pulsed MEF.wt (see Materials and Methods), MEF.Bid-/- (a, c) and MEF.Bak Bax-/- (a) or MEF.wt (see Materials and Methods), MEF.Casp 3 7-/- and MEF.Casp 9-/- (b) cells were incubated with ex vivo virus-immune gzmB+CTL (4 h, 10:1 effector/target ratio). Cytochrome c release from mitochondria (a, b) and Bak and Bax conformational change (c) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and Methods. Histograms show a representative experiment of two independent experiments. Ctr: only target cells. (d) Gp33-pulsed MEF.wt cells were incubated with ex vivo virus-immune B6 or gzmB-/-CTL (1 h, 10:1 effector/target ratio). Subsequently, lysates were prepared and Bid cleavage was analyzed by western blot as described in Materials and Methods. Bid=22 kDa, tBid=16 kDa
Figure 4.
gzmB+CTL-induced activation of caspase-3 is independent of Bid or Bak and Bax and is crucial for PS exposure but not for mitochondrial perturbation. (a) Gp33-pulsed MEF.wt, MEF.Bid-/- and MEF.Bak Bax-/- were incubated with ex vivo virus-immune gzmB+CTL (1 and 4 h as indicated, 10:1 effector/target ratio). Activation of caspase-3 was monitored with an FITC-labeled mAb against the active form of the enzyme. Histograms show a representative experiment. Numbers correspond to the percentage of cells as indicated by the horizontal bars. (b) gp33-pulsed MEF.wt was incubated with ex vivo virus-immune gzmB+CTL (4 h, 10:1 effector/target ratio) in the presence or absence of the general caspase inhibitor Ac-ZVAD-fmk (80 M). Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (b) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and Methods. A representative experiment is shown in the upper panels. Numbers correspond to the percentage of cells in each quadrant. Data in the graph (PS/PI) are represented as meanS.E.M. of three independent experiments. Ctr: only target cells; ZVAD: target cells+ZVAD-fmk
Figure 5.
Role of caspase-3, -7 and -9 in gzmB+CTL-induced membrane and mitochondrial perturbation. Gp33-pulsed MEF.wt, MEF.Casp 3 7-/- and MEF.Casp 9-/- (a, b) or MEF.wt, MEF.Casp 3-/- and MEF.Casp 7-/- (c) cells were incubated with ex vivo virus-immune gzmB+CTL (4 h, 10:1 effector/target ratio). Subsequently, PS exposure and PI uptake (a, c) and, in parallel, suppression of m and ROS generation (b, c) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression as described in Materials and Methods. (a, b) A representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the graphs (a, b, right panels) are represented as meanS.E.M. of five (MEF.wt, MEF.Casp 3 7-/-) and three (MEF.Casp 9-/-) independent experiments, respectively. (c) Data in the graphs are represented as meanS.E.M. of five (MEF.wt, MEF.Casp 3-/-) and three (MEF.Casp 7-/-) independent experiments, respectively. Ctr: only target cells
Figure 6.
gzmB+CTL-induced mitochondrial damage is blocked in the absence of Bak and Bax and simultaneous inhibition of caspase-3 and -7. Gp33-pulsed MEF.Bid-/- and MEF.Bak Bax-/- cells were incubated with ex vivo virus-immune gzmB+CTL (4 h, 10:1 effector/target ratio) in the presence or absence of the general caspase inhibitor Ac-ZVAD-fmk or the caspase-3/-7 inhibitor Ac-DEVD-fmk (80 M both). m loss and ROS generation were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and Methods. A representative experiment is shown in the upper panels. Numbers correspond to the percentage of cells in each quadrant. Data in the graphs are represented as meanS.E.M. of three (ZVAD) and two (DEVD) independent experiments, respectively. Ctr: only target cells; ZVAD: target cells+ZVAD-fmk; DEVD: target cells+ZVAD-fmk
Figure 7.
Model for gzmB-mediated pro-apoptotic pathways and cell death elicited by ex vivo CTL. gzmB exerts pleiotropic pro-apoptotic potential in gzmB+CTL-induced cell death. First, gzmB directly activates caspase-3 and -7, independently of mitochondrial events such as Bid-induced Bak/Bax activation and cytochrome c release. Caspase-7 can replace caspase-3 in the induction of plasma membrane perturbance (PS exposure and PI permeability), and both caspases are critical in gzmB-mediated ROS production. However, both processes are dispensable for gzmB+CTL-mediated cell death. Second, gzmB-induced activation of Bid and Bak/Bax is independent of caspase-3 and -7 and leads to cytochrome c release. The absence of Bid or Bak/Bax does not interfere with gzmB+CTL-induced cell death. Third, gzmB-mediated mitochondrial perturbation is triggered by two distinct pathways, one involving Bid/Bax/Bak, independent of caspases, to induce m loss and cytochrome c, and one triggering caspase-3/-7 activation without the implication of Bid/Bax/Bak to induce m loss and ROS production. Only when both pathways are simultaneously blocked, mitochondrial depolarization is prevented. Finally, gzmB kills target cells independent of caspases and mitochondrial signals, including PS exposure, mitochondrial depolarization, ROS production and cytochrome c release
