Granzyme B plays a critical role in cytotoxic lymphocyte-induced apoptosis.

Cytotoxic lymphocytes lacking granzyme B have a striking defect in their ability to rapidly induce target cell DNA fragmentation, as measured in lytic assays using 51 Cr or 125 IUdR-labeled target cells (Table I). Using a gene-targeting approach, we (Heusel et al. 1994) and others (Kagi et al. 1994, Kojima et al. 1994, Lowin et al. 1994, Walsh et al. 1994) have confirmed the importance of the PFP/ granzyme pathway of cellular cytotoxicity. Our studies have clearly shown that granzyme B is one of the molecules required for the induction of DNA fragmention in target cells. After investigating the function of granzyme B in the 'rapid' pathway of cytotoxicity, we examined the role of this protease in extended lytic assays. The null mutation of granzyme B did not completely abrogate 125 IUdR release in extended assays with CTL or LAK effectors, indicating the presence of intact granzyme B-independent mechanism(s) of cytotoxicity in these effector cells. NK cells, in contrast, appear to be deficient in one or more of these granzyme B-independent cytotoxic pathways, since granzyme B-/- NK cells remained severely defective in their ability to induce either 51 Cr or 125 IUdR release from their targets despite extended incubation times. Granzyme A, Fas, or other molecule(s) may play a role in the granzyme B-independent pathway. Shi et al. (1992b) have demonstrated that granzyme A is 'slower' than granzyme B in causing DNA fragmentation of permeabilized target cells. In some systems, Fas/Fas ligand-mediated cytotoxicity is also slower because of the requirement for fas ligand induction upon TCR-stimulation (Anel et al. 1994). Therefore, analysis of mice doubly deficient for granzymes A and B or for granzyme B and Fas ligand should help to define any role that these molecules may play in granzyme B-independent cytotoxicity. Our data raise questions about the role(s) of the other granzymes (A, and C-G) in mice. Clearly none of these granzymes can compensate for the role of granzyme B in rapid induction of target cell apoptosis. Since only granzyme B has been shown to be an Asp'ase, its unique specificity may define its role in apoptosis. However, since granzyme A (a tryptase) can also induce DNA fragmentation in permeabilized cells, perhaps multiple proteolytic pathways can converge onto final common pathway(s) for the induction of cell death. Finally, the variable activation profiles of other granzymes (C-G) with various T cell activation strategies (Prendergast et al. 1992) suggests that these granzymes may have specialized roles in cytotoxic lymphocytes; these roles may be further defined by additional knockout studies. The separation of granzyme B-dependent and -independent pathways of cytotoxicity in granzyme B -/- mice will allow us to assess the physiological relevance of these pathways in intact animals. Specifically, we have now begun to use the granzyme B-deficient mice to determine the importance of the granzyme B-dependent pathway for various in vivo activities of cytolytic lymphocytes, including viral clearance, tumor surveillance and allogeneic transplantation rejection. Besides distinguishing the roles of granzyme B-dependent and -independent pathways of cytotoxicity, the granzyme B-deficient mouse model has allowed us to investigate the roles of DNA fragmentation versus pore formation for cytotoxicity. We have shown that an intact 51 Cr release pathway does not necessarily permit rapid DNA fragmentation, suggesting that the PFP-formed pores are not sufficient to induce the apoptotic death of target cells. Since 51 Cr release is intact in granzyme B-/- CTL effector cells, we hope to further assess the role of necrosis versus apoptosis for the in vivo functions of cytolytic lymphocytes. All of these in vivo studies with granzyme B-/- mice should yield information regarding the potential application of specific inhibitors of granzyme B to control cell-mediated cytotoxicity.