402 T cell killing is facilitated by multiple cytotoxic pathways (original) (raw)
Related papers
European Journal of Immunology, 2002
Complementary approaches with purified molecules or transfected cytolytic effector cells have suggested that both, granzyme A (gzmA) and granzyme B (gzmB), similarly contribute to CTL-mediated and perforin (perf)-dependent apoptotic nuclear damage (DNA fragmentation) in target cells. Studies employing gzmA or gzmB single-knockout mice on the other hand indicated that gzmB is the prominent CTL effector molecule for the rapid induction of DNA fragmentation, with gzmA playing only a minor part. We have now taken ex vivo-derived virus-specific or in vitro generated alloreactive CTL from mice deficient in either gzmA or gzmB and a panel of three target cells to reinvestigate this unresolved issue. We show that rapid CTL-mediated DNA fragmentation of L1210.3 target cells is solely dependent on gzmB, whereas the DNA fragmentation of EL4.F15 target cells by the same CTL population is mainly induced by gzmA and only marginally by gzmB. Moreover, CTL-induced apoptosis of a third target cell, MC57G, was partially dependent on both gzmA and gzmB activities. The differential contribution of the two gzms to apoptosis was further verified by their distinct sensitivity to caspase inhibitors. The data suggest that both, gzmA and gzmB, have a similar potential to induce rapid perf-mediated apoptosis but that their individual contribution to the underlying intracellular processes is dictated by the quality of the target cell.
Journal of Cell Biology, 2004
urified 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 ϩ 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 Ϫ / Ϫ or gzmB Ϫ / Ϫ mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ⌬⌿ m loss, and reactive oxygen radical generation, though with distinct P kinetics; (b) CTL from gzmA Ϫ / Ϫ but not from gzmB Ϫ / Ϫ mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA Ϫ / Ϫ or gzmB Ϫ / Ϫ mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzminduced 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.
Chemistry & Biology, 2005
granzymes exhibit increased susceptibility to viral infections [8]. Additionally, far more is known about the 1 Chemistry and Chemical Biology Graduate Program molecular mechanism of action of these two gran-2 Department of Pharmaceutical Chemistry zymes than is known about the remaining "orphan" hu-University of California, San Francisco man granzymes, H, K, and M [9]. The substrate speci-San Francisco, California 94143 ficity of granzyme B is similar to that of apical caspases, and its function as an activator of apoptosis reflects this similarity. Granzyme B may also induce Summary death through caspase-independent pathways. Major hallmarks of granzyme B-induced cellular damage are The mechanism of target cell lysis in cytotoxic lymoligonucleosomal DNA fragmentation and mitochonphocyte-mediated death is not well understood, and drial damage [10]. An important pathway to granzyme the role of granzymes in this process is unclear. A-induced damage involves cleavage and inactivation Chemical functional probes were thus prepared for of SET (also known as PHAPII, TAF-Iβ, I 2 PP2A), which the major granzymes A and B to deconvolute their functions as an inhibitor of the DNase activity of the role in natural killer cell-mediated lysis of target cells. tumor metastasis suppressor NM23-H1. The resulting These biotinylated and substrate specificity-based hallmark of granzyme A-induced damage is singlediphenyl phosphonates allowed facile evaluation of stranded DNA nicks mediated by NM23-H1 [11]. selectivity through activity-based profiling in cell ly-Most information concerning the function of gransates and intact cells. Both inhibitors were found to zymes A and B has been derived from genetic studies, be extremely selective in vitro and in cells. Use of or from reconstituted systems in which target cells are these inhibitors in cell-based assays revealed grankilled by exogenous addition of purified granzymes and zyme A to be a minor effector and granzyme B to be perforin. Although genetic deletions in mice offer a high a major effector of target cell lysis by natural killer level of physiological relevance, compensation by recells. These studies indicate that the proapoptotic lated genes can sometimes obscure effects [12]. Comgranzyme B functions also as a pronecrotic effector pensation may be a particularly valid consideration in of target cell death. the case of the granzymes because several family members are highly homologous. Use of a reconstitu
Cell Death and Differentiation, 2008
Granzyme B (gzmB) of cytotoxic T lymphocytes (CTL) is essential for recovery from intracellular pathogens, but the molecular basis of its action is still unresolved. Here, we analyzed gzmB-mediated death pathways under physiological conditions using ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB þ CTL). We show that gzmB þ CTL abrogate target cell proliferation most likely by inducing cell death, independent of caspases and mitochondrial signaling. In addition, the data reveal that gzmB þ CTL independently induce pro-apoptotic processes either via caspase-3/-7, leading to plasma membrane perturbance and ROS production or via Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit loss of DW m . Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB presumably to counteract evasion strategies of pathogens and to control tumors.
The Journal of Immunology
The perforin-facilitated entry of granzymes in target cells is a major mechanism used by CTL to induce cell death. It has been reported that granzyme B can cleave and activate the apoptotic cysteine protease p32 (CPP32)/Yama and its homologues in vitro. However, the mechanism for granzyme-based cytolysis exerted by intact CTL remains unclear. In the present work, we have used anti-CD3 mAb-redirected lysis of Fas-negative L1210 cells by CTL clones as a model to study perforin/granzymebased cytotoxicity separately from the contribution of the Fas/Fas ligand system. N-acetyl-Asp-Clu-Val-Asp aldehyde (Ac-DEVD-CHO), a specific inhibitor of CPP32-like proteases, completely prevented the former type of lysis in 3-h assays, but not in long-term (1 6-h) assays. A combination of Ac-DEVD-CHO and the granzyme A inhibitor IGA (7-(phenyl-ureido)-4-chloro-3-(2-isothioureidoethoxy)-isocoumarin) inhibited long-term cytolysis. 3,4-Dichloroisocoumarin, a serine-protease inhibitor that efficiently inhibits granzyme B and poorly inhibits granzyme A, had similar effects as Ac-DEVD-CHO on anti-CD3 mAb-redirected lysis of L1210 cells. On the other hand, Fas-based cytolysis exerted by the same CTL clones on Fas-transfected L1210 cells (L1210Fas) was inhibited completely by Ac-DEVD-CHO, irrespective of the incubation time. These results suggest that granzyme Band Fas-based cytotoxicity exerted by CTL clones converge at the level of CPP32-like protease activation, while granzyme A acts via a different, still undefined, pathway. We also demonstrate that perforin/granzyme-based cytolysis occurs without increase in the cellular ceramide content, ruling out the contribution of the sphingomyelinase pathway to this mechanism of cell death.
Cytotoxic T-cell-derived granzyme B activates the apoptotic protease ICE-LAP3
Current Biology, 1996
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells provide immune surveillance against viruses and neoplasms, and play a central role in the pathogenesis of autoimmune disease, AIDS and graft rejection [1,2]. Thus, it is important to understand the precise molecular mechanism(s) whereby cytotoxic lymphocytes destroy susceptible target cells. Granule-mediated cytotoxicity requires a combination of both perforin and granzyme B [3]. Perforin polymerizes to form transmembrane channels and presumably allows granzyme B access to target cell substrates, which until recently, were unknown. One clue to the identity of the physiological substrate(s) activated by granzyme B comes from its unusual specificity for cleaving synthetic substrates after aspartate residues [4]. Members of the ICE/CED-3 family of cysteine proteases are prime candidates as they are important apoptotic effectors [5,6] and are expressed as zymogens, which can be processed to form active heterodimeric enzymes after cleavage at specific aspartate residues. Previous studies have shown that granzyme B proteolytically activates the cell death effector Yama/CPP32/apopain [7,8] (referred to here as Yama). Here we report that granzyme B also activates ICE-LAP3/Mch3/CMH-1 (referred to here as ICE-LAP3), which, along with Yama and Mch2, forms a subset of the ICE/CED-3 family of cysteine proteases most closely related to the Caenorhabditis elegans cell death gene, CED-3 [6,9]. Importantly, Jurkat T cells incubated with granzyme B and a sublytic concentration of perforin undergo apoptosis, which is preceded by the activation of endogenous ICE-LAP3. Thus, we propose that granzyme B mediates apoptosis by directly engaging the target cell's death effector machinery, which is probably composed of an arsenal of intracellular, CED-3-like cysteine proteases.