Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmA×B−/−) with those from single knockout mice deficient in gzmA (−/−), gzmB (−/−), or perforin (−/−) to induce nuclear damage and lysis in target cells. With the exception of perforin−/−, all in vitro– and ex vivo–derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmA×B−/− mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2–4 h), but not long-term (4–10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule– mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

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Granzyme A, a granule‐associated serine proteinase of activated cytotoxic T cells and natural killer cells, has been reported to play a critical role in DNA fragmentation of target cells. To address the question of the biological role of granzyme A, we have now generated a granzyme A‐deficient mouse mutant by homologous recombination. Western blot analysis, enzyme assays and reverse transcription‐PCR confirmed the absence of granzyme A in activated T cells. In addition, deletion of granzyme A does not alter the expression patterns of other granule components, such as granzymes B‐G and perforin. Granzyme A‐deficient mice are healthy and show normal hematopoietic development. Most notably, their in vitro‐ and ex vivo‐derived cytotoxic T cells and natural killer cells are indistinguishable from those of normal mice in causing membrane disruption, apoptosis and DNA fragmentation in target cells. Furthermore, granzyme A‐deficient mice readily recover from both lymphocytic choriomeningitis virus and Listeria monocytogenes infections and eradicate syngeneic tumors with kinetics similar to the wild‐type strain. These results demonstrate that granzyme A does not play a primary role in cell‐mediated cytotoxicity, as has been assumed previously.