Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving bid

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the F as receptor or by the exocytosis of granzyme B and perforin. Caspase activa tion is an important component of both pathways. Granzyme B, a serine prote inase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an unders tanding of the contributions of caspases 8 and 3 during granule-induced apo ptosis in intact cells, we have used target cells that either stably expres s the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of casp ase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial di sruption during Fas-mediated cell death. In contrast, SPI-2 expression in t arget cells provided no protection against granzyme-mediated apoptosis, mit ochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibi ted caspases are not an essential requirement for the granzyme pathway. Cas pase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondr ial inner membrane potential. Furthermore, we demonstrate that granzyme B d irectly cleaves the proapoptotic molecule Bid, bypassing the need for caspa se 8 activation of Bid. These results provide evidence for a two-pronged st rategy for mediating target cell destruction and provide evidence of a dire ct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.