Figure 5. Caspase inhibition confers protec- tion from mouse, but not human, GzmB. CEM cells were incubated for 45 min either alone, with 100 nM hGzmB, or with 120 nM mGzmB, all in the presence of 1.25 μg/ml SLO. Cells were then cultured for the remainder of the experiment with or without 100 μM zVADfmk as indicated. Note that different amounts of GzmB were used to achieve approximately the same amount of cell death within 6 h. Cell cultures were then assessed at the indicated time points by phase-contrast microscopy (A), and annexin V/PI staining by ﬂow cytometry (B and C). (D) Cell proliferation under the same conditions was also assessed by direct hemo- cytometer counting. Results are representative of three independent experiments. Error bars represent the mean ± the SD. have suggested that BID proteolysis is indirect and is mediated by caspase-3, which is also activated by GzmB (Metkar et al., 2003). The contention that BID is a preferential substrate for GzmB is supported by studies that have shown that Bcl-2 can block GzmB-initiated apoptosis, whereas caspase inhibitors fail to do so (Pinkoski et al., 2001). The latter observations would support an upstream role for BID, independent of cas- pase activity, in promoting mitochondrial outer membrane per- meabilization as a result of Bax/Bak activation. In this scenario, inhibition of caspase activity would fail to block mitochondrial outer membrane permeabilization, and cells would die in a caspase-independent manner. Bcl-2 would be expected to block Figure 6. Inhibition of caspase activity by zVADfmk confers clonogenic protection from mouse GzmB in a human cell line. Human Jur- kat cells were incubated for 45 min either alone, with 120 nM hGzmB, or 133 nM mGzmB, all in the presence of 1 μg/ml SLO. Cells were then cultured for the remainder of the experiment with or without 100 μM zVAD- fmk as indicated. Note that different amounts of GzmB were used to achieve approximately the same amount of cell death within 3 h. Cell cul- tures were then assessed at the indicated time points by phase-contrast microscopy (A), and annexin V/PI staining by ﬂow cytometry (B). (C and D) Jurkat cells were treated as described above and were then plated into soft agar after 48 h. Colony formation in soft agar was as- sessed four weeks after plating. Results are rep- resentative of three independent experiments. Error bars represent the mean ± the SD. HUMAN AND MURINE GRANZYME B ARE DISTINCT ENZYMES • CULLEN ET AL. 441