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Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.

Cytotoxic T lymphocyte (CTL)-induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB(-/-) CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB(-/-) CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB(-/-) mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.