Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

In this report we questioned the current view that the two principal cytotoxic pathways, the exocytosis and the Fas ligand (FasL)/Fas-mediated pathway, have largely nonoverlapping biological roles. For this purpose we have analyzed the response of mice that lack Fas as well as granzyme A (gzmA) and gzmB (FasxgzmAxB ؊/؊ ) to infection with lymphocytic choriomeningitis virus (LCMV). We show that FasxgzmAxB ؊/؊ mice, in contrast to B6, Fas ؊/؊ , and gzmAxB ؊/؊ mice, do not recover from a primary infection with LCMV, in spite of the expression of comparable numbers of LCMV-immune and gamma interferon-producing cytotoxic T lymphocytes (CTL) in all mouse strains tested. Ex vivo-derived FasxgzmAxB ؊/؊ CTL lacked nucleolytic activity and expressed reduced cytolytic activity compared to B6 and Fas Studies with perforin (perf)-deficient mice have shown that perf is a key element in NK cell-and cytotoxic T-lymphocyte (CTL)-dependent recovery from viral infections (20,28,63). However, these studies did not address the question of whether NK cells and CTL can mediate their exocytosis-mediated function in vivo solely via perf or whether they depend on additional effector molecules for maximal effector function, in particular components of the exocytosis pathway, such as granzymes (gzm) (58), and/or those of other cytolytic processes, such as Fas ligand (FasL) or tumor necrosis factor (TNF) (15,21,58). A signature of all three effector pathways is induction of DNA fragmentation (nucleolysis) in vitro, a marker of apoptosis and target cell death (31, 64).In vitro studies using either purified perf (18, 38, 65) or mice lacking both gzmA and gzmB (45, 46) have shown that NK cells and CTL can lyse target cells-as monitored by the 51 Cr release assay-solely by means of perf. However, it is still unclear whether this in vitro cytotoxicity is of biological relevance for processes underlying NK cell-and CTL-mediated host defenses in vivo. In vitro induction of programmed cell death (termed apotosis) by the granule exocytosis pathway is strictly dependent on the concerted action of perf and functional active gzm (17,42,43,45,46). According to recent studies, gzm, in particular gzmA and gzmB, which are released by NK cells and CTL upon encountering antigens, are bound to and internalized by target cells, are subsequently delivered to the cytosol via perf, and initiate cell death by caspase-dependent (gzmB) and/or caspase-independent (gzmA, gzmB) pathways (7, 12-14, 27, 33, 40, 55, 58).Recent studies with mouse strains deficient in one or more components of the granule exocytosis pathway indicate that the concerted action of perf and gzm is essential for recovery from primary infection with a number of pathogens, such as ectromelia virus (EV) (29), mouse cytomegalovirus (39), herpes simplex virus (36), and Trypanosoma cruzi (30), as well as for the control of certain tumors (34). On the other hand, a number of related studies suggest that gzms and/or the FasL/ Fas pathway is dispensable for perf-mediated control of tumor growth (9,49,50,...