Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion

Abstract

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

Keywords: Apoptosis; B Cell Lymphoma 2 (Bcl-2) Family; Cancer; Cancer Therapy; Immunotherapy; T Cell.

FIGURE 1.

Cell death induced by ex vivo-derived LCMV-specific Tc cells from gzmA^−/−, gzmAxB^−/−, and perfxgzmAxB^−/− (PAB^−/−) mouse strains. 3T9 MEF WT (A) and SV40 MEFWT (B) cells were incubated with ex vivo virus immune CD8⁺ cells from either gzmA^−/−, gzmAxB^−/−, and PAB^−/− mice in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and 7-AAD uptake were analyzed by three-color flow cytometry in the cell population negative for CD8 expression as described under “Experimental Procedures.” A representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the right panel are represented as the mean ± S.D. of three independent experiments. *, p < 0,05; **, p < 0,01; ***, p < 0,001. No symbol means not significant. C, 3T9 MEF WT cells were incubated with ex vivo virus immune CD8⁺ cells from gzmA^−/−mice in the presence of the LCMV peptide gp33, annexin V-FITC, and PI for 3 h. Sequential images were taken every 2.5 min as indicated under “Experimental Procedures.” DIC, differential interference contrast.

FIGURE 2.

gzmB⁺ Tc cells induce cell death in 3T9 MEF.WT but neither in 3T9 MEF.Bim^−/− nor in 3T9 MEF.BakxBax^−/−. MEF.WT, MEF.Bim^−/−, and MEF.BakxBax^−/− cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and 7-AAD uptake (A) and, in parallel, ΔΨm loss (TMRE, B) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression as described under “Experimental Procedures.” In some cases MEF cells were also preincubated with the caspase inhibitor Ac-DEVD-fmk 100 μm. A, representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the right panel are represented as the mean ± S.D. of three independent experiments. B, histograms show a representative experiment of three independent experiments. Numbers correspond to the percentage of cells in regions marked by the horizontal bar. Data in the left panel are represented as the mean ± S.D. of three independent experiments. C, MEF.WT, MEF.Bim^−/−, and MEF.BakxBax^−/− cells were incubated with sorafenib (Sorf, 40 mm) and bortezomib (Bort, 20 mm) for 18 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) was analyzed. Data in this panel are represented as the mean ± S.D. of two independent experiments. *, p < 0,05; **, p < 0,01; ***, p < 0,001. No symbol means not significant. D, MEF.WT, MEF.Bim^−/−, or MEF.WT cells treated with siRNA against Bim (siBim) were incubated with ex vivo virus immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and 7-AAD uptake were analyzed. Bim expression in WT, Bim^−/−, and siBim-treated cells was analyzed by immunoblot (BimEL isoform). Actin served as loading control.

FIGURE 3.

Caspase-3 activation and cytochrome c release induced by gzmB⁺ Tc cell in 3T9 MEF. MEF.WT, MEF.Bim^−/−, and MEF.BakxBax^−/− cells or MEF.WT cells treated with a siRNA against Bim (siBim) or Bid (siBid) were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h (A) or 2 h (B). A, caspase-3 activation was analyzed by flow cytometry in the target cell population with a mAb against the active form of caspase-3. Histograms show a representative experiment of three independent experiments. Numbers correspond to the percentage of cells with activated caspase-3, limited by regions marked by the horizontal bar. Data in the right panel are represented as the mean ± S.D. of three independent experiments. B, cytochrome c release from mitochondria was analyzed by flow cytometry in the cell population negative for CD8 expression as described under “Experimental Procedures.” Histograms show a representative experiment of two independent experiments. Data in the right panel are represented as the mean ± S.D. of two independent experiments. *, p < 0.05; **, p < 0.01. No symbol means not significant. C, MEF.WT cells were treated with a siRNA against Bim (siBim) or Bid (siBid) and incubated with ex vivo virus immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 2 h. Cytochrome c release from mitochondria was analyzed by flow cytometry in the cell population negative for CD8 expression as described under “Experimental Procedures.”

FIGURE 4.

Mcl-1 and Bcl-X_L degradation, transcriptional regulation of Bim and survival assay. A–C, 3T9 or SV40 MEF.WT and MEF.Casp3x7^−/− (B) cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Mcl-1 and Bcl-X_L degradation was analyzed by flow cytometry in the target cell population with a mAb against a specific Mcl-1 epitope. Histograms show a representative experiment of three independent experiments. Numbers correspond to the percentage of cells with Mcl-1 degraded, limited by regions marked by the horizontal bar. In the second histogram (A and C), the filled histograms represent cells without gp33 (−gp), black line represents cells plus gp33 (+gp33). and broken line represents cells with gp33 plus DEVD (+gp-DEVD). B, black and blue histograms correspond to the isotype antibody control and Mcl-1 specific mAb, respectively. Numbers in gray corresponds to the % of annexin V⁺ cells. Data in the right panel are represented as the mean ± S.D. of three independent experiments. D and E, gp33-pulsed 3T9 MEF.WT and SV40 MEF.WT cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice in the presence of salubrinal, 10 μm (D), or cycloheximide (chx) (E) for 2 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) was analyzed. Data in this panel are represented as the mean ± S.D. of two independent experiments. *, p < 0,05; **, p < 0,01; ns, not significant. F, for survival assay, MEF.WT, MEF.Bim^−/−, and MEF.BakxBax^−/− cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33. 3 h after incubation, cells were washed, trypsinized, and counted. 100 cells per well were seeded in 6-well plates and incubated in fresh medium for 7 days. After incubation, cells were stained with crystal violet, and colonies were counted. Survival was calculated as percentage of colonies relative to the colonies in the controls. Values were represented as the mean ± S.D. of four independent experiments. *, p < 0,05; **, p < 0,01; ***, p < 0,001. No symbol means not significant.

FIGURE 5.

Implication of mitochondrial apoptotic pathway in cell death induced by gzmB⁺ Tc in MEF 3T9 and MEF SV40. 3T9 MEF.WT, 3T9 MEF.BakxBax^−/−, SV40 MEF.WT, and SV40 MEF.BakxBax^−/− cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and 7-AAD uptake (A) and, in parallel, ΔΨm loss (TMRE) and caspase-3 activation (B) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression. A, representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the right panel are represented as the mean ± S.D. of six independent experiments. B, histograms show a representative experiment of three independent experiments. Numbers correspond to the percentage of cells in regions marked by the horizontal bar. C, SV40 MEF.WT and SV40 MEF.Bim^−/− cells were incubated with ex vivo virus-immune CD8⁺ cells from gzmA^−/− mice, in the presence (+gp) or absence (−gp) of the LCMV peptide gp33 for 3 h. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and 7-AAD uptake was analyzed by three-color flow cytometry in the cell population negative for CD8 expression. A representative experiment is shown in the left panels. Numbers correspond to the percentage of cells in each quadrant. Data in the right panel are represented as the mean ± S.D. of two independent experiments. *, p < 0,05; **, p < 0,01; ***, p < 0,001. No symbol means not significant. Data in the right panels are represented as the mean ± S.D. of three independent experiments. D, lysates of MEF 3T9 WT, MEF 3T9 Bim^−/−, MEF 3T9 BakxBax^−/−, MEF SV40 WT, and MEF SV40 BakxBax^−/− cells were prepared and Bim (BimEL isoform), Bid, Bak, Bax, Bcl-X_L, Mcl-1, XIAP, Smac/DIABLO, caspase-3, and caspase-9 expression were analyzed by Western blot as described under “Experimental Procedures.” Actin was used as loading control.

FIGURE 6.

Role of Bim during gzmB-induced apoptosis in 3T9 MEF cells. gzmB cleaves a small amount of Mcl-1 and Bcl-X_L, which enhances the pro-apoptotic activity of Bim and activates the mitochondrial apoptotic pathway leading to caspase-3 activation. Active caspase-3 would then increase Mcl-1 and Bcl-X_L degradation, Bim release, and heightened caspase-3 activation, an amplification loop that would be required to maximize PS translocation and apoptosis.