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Granzymes are a family of small (∼32 kDa) serine proteases with a range of substrate specificities that are stored in, and released from, the cytoplasmic secretory vesicles ('granules') of cytotoxic T lymphocytes and natural killer cells. Granzymes are not digestive proteases but finely tuned processing enzymes that target their substrates in specific ways to activate various signalling pathways, or to inactivate viral proteins and other targets. Great emphasis has been placed on studying the pro-apoptotic functions of granzymes, which largely depend on their synergy with the pore-forming protein perforin, on which they rely for penetration into the target cell cytosol to access their substrates. While a critical role for granzyme B in target cell apoptosis is undisputed, both it and the remaining granzymes also influence a variety of other biological processes (including important immunoregulatory functions), which are discussed in this review. This includes the targeting of many extracellular as well as intracellular substrates, and can also lead to deleterious outcomes for the host if granzyme expression or function are dysregulated or abrogated. A final important consideration is that granzyme repertoire, biochemistry and function vary considerably across species, probably resulting from the pressures applied by viruses and other pathogens across evolutionary time. This has implications for the interpretation of granzyme function in preclinical models of disease., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8 + T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8 + T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation., (©2020 American Association for Cancer Research.)

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.

The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11 , or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2 . Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means., (© 2016 Federation of European Biochemical Societies.)

A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin–granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology.

Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal "cytokine storm" resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells., (© 2015 Jenkins et al.)

Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.

Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.

Granzymes A and B (GrAB) are known principally for their role in mediating perforin-dependent death of virus-infected or malignant cells targeted by CTL. In this study, we show that granzymes also play a critical role as inducers of Ag cross-presentation by dendritic cells (DC). This was demonstrated by the markedly reduced priming of naive CD8(+) T cells specific for the model Ag OVA both in vitro and in vivo in response to tumor cells killed in the absence of granzymes. Reduced cross-priming was due to impairment of phagocytosis of tumor cell corpses by CD8α(+) DC but not CD8α(-) DC, demonstrating the importance of granzymes in inducing the exposure of prophagocytic "eat-me" signals on the dying target cell. Our data reveal a critical and previously unsuspected role for granzymes A and B in dictating immunogenicity by influencing the mode of tumor cell death and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in apoptosis induction.

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Destruction of target cells by cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells requires the coordinated action of the pore forming protein perforin (Pfp) and the granzyme (Gzm) family of serine proteases. The activation of a number of serine proteases, including GzmA and B, is predominately mediated by cathepsin C (CatC). Deficiencies in CatC-null mice were therefore expected to replicate the defects observed in GzmAB-deficient mice. We have previously determined that GzmAB-deficient mice exhibit increased susceptibility to murine cytomegalovirus (MCMV) infection. Here, we have compared the ability of CatC(-/-) mice to control MCMV infection with that of GzmAB-deficient animals. We found that CatC(-/-) mice have organ-specific defects in the ability to control MCMV replication, a phenotype that is distinct to that observed in GzmAB(-/-) mice. Significantly, the cytolytic function of CatC-deficient NK cells and CTLs elicited during infection was indistinguishable from that of wild-type cells. Hence, CatC is involved in limiting MCMV replication; however, this effect is independent of its role in promoting effector cytolytic activity. These data provide evidence for a novel and unexpected role of CatC during viral infection.

Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore-forming protein perforin (pfp) and a family of serine proteases known as granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene-targeted mice have allowed investigators to ask why granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non-apoptotic functions of granzymes.

The secretory granule-mediated cell death pathway is the key mechanism for elimination of virus-infected and transformed target cells by cytotoxic lymphocytes. The formation of the immunological synapse between an effector and a target cell leads to exocytic trafficking of the secretory granules and the release of their contents, which include pro-apoptotic serine proteases, granzymes, and pore-forming perforin into the synapse. There, perforin polymerizes and forms a transmembrane pore that allows the delivery of granzymes into the cytosol, where they initiate various apoptotic death pathways. Unlike relatively redundant individual granzymes, functional perforin is absolutely essential for cytotoxic lymphocyte function and immune regulation in the host. Nevertheless, perforin is still the least studied and understood cytotoxic molecule in the immune system. In this review, we discuss the current state of affairs in the perforin field: the protein's structure and function as well as its role in immune-mediated diseases.

Type 1 diabetes results from autoimmune destruction of pancreatic beta-cells by CD8(+) T cells. The requirement for CD8(+) T cells implicates perforin and granzymes as effectors of tissue destruction. Diabetogenic cytotoxic T cells kill beta-cells by the perforin/granzyme pathway in vitro. In the non-obese diabetic mouse model of type I diabetes, perforin deficiency results in a highly significant reduction in disease, indicating a direct role for perforin in beta-cell death in vivo, although other cell death pathways must account for the residual diabetes in perforin-deficient mice. Perforin and granzyme B are also important in allogeneic destruction of islets. The dominant role of the perforin/granzyme pathway in beta-cell destruction in type I diabetes and allogeneic islet graft rejection make this pathway an important target for blockade in future therapies for type I diabetes. In addition, granzymes have a newly recognized role in inflammation, a feature of both type I and II diabetes, suggesting their role should be further explored in both the common forms of diabetes.

Cytotoxic lymphocytes (CLs) are the killer cells that destroy intracellular pathogen-infected and transformed cells, predominantly through the cytotoxic granule-mediated death pathway. Soluble cytotoxic granule components, including pore-forming perforin and pro-apoptotic serine proteases, granzymes, synergize to induce unscheduled apoptosis of the target cell. A complete loss of CL function results in an aggressive immunoregulatory disorder, familial hemophagocytic lymphohistiocytosis, whereas a partial loss of function seems to be a factor strongly predisposing to hematological malignancies. This review discusses the pathological manifestations of CL deficiencies due to impaired perforin function and describes novel aspects of perforin biology.

A pore-forming protein, PRF, and serine proteases, Grz, are key effector molecules of CL. These toxins are stored within secretory granules, which exocytose their contents in response to immune synapse formation between the CL and virus-infected or transformed target cell. There, PRF and Grz synergize to induce various apoptotic death pathways and to maintain immune homeostasis. Mechanistic aspects of the synergy and apoptotic mechanisms are still not fully understood, and the current review will address some of the hotly debated controversies in the field.

Cytotoxic T (Tc) cells lyse target cells via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and in the absence of gzm A and B the induction of apoptosis is severely impaired. However, using in vivo Tc cell killing assays, we find that virus-immune, gzm A x B-deficient (gzmAxB(-/-)) mice are competent to eliminate adoptively transferred target cells pulsed with an immunodominant Tc cell determinant as rapidly and completely as their wild-type counterparts. Specific target cell elimination occurred with similar kinetics in both spleen and lymph nodes. Thus, neither gzmA nor gzmB are required for rapid and efficient in vivo cytotoxicity by Tc cells.

Time-lapse video microscopy allows analysis of the interaction between individual CTLs and adherent peptide-pulsed targets, from contact, to lymphocyte detachment, APC rounding, phosphatidylserine exposure and finally loss of plasma membrane integrity characteristic of end-stage apoptosis. Using in vitro-stimulated effectors specific for the ovalbumin K(b)OVA(257) (OT-I) and influenza A virus D(b)NP(366) and D(b)PA(224) epitopes, no significant correlation was found between the duration of CTL contact and the time to phosphatidylserine exposure or loss of membrane integrity. Furthermore, there were minimal indications that transgenic T cells specific for the K(b)OVA(257) epitope (TCR) diversity had any effect. However, when the analysis was repeated with D(b)NP(366) and D(b)PA(224)-specific CTLs recovered directly from the lungs of mice with influenza pneumonia, the lower avidity D(b)NP(366)-specific set was found to elute much more quickly. Shorter contact time may allow individual CTLs to lyse more targets, suggesting that lower TCR/epitope avidity may be more beneficial than higher epitope avidity for cell-mediated immunity.

In this issue of Immunity, Metkar et al. (2008) present evidence that granzyme A plays a role in inflammatory signaling and that contrary to previous studies, it is incapable of inducing target cell death. The work challenges us to reconsider the broader biological roles of all the granzymes.

Granzyme (grz) AB(-/-) H2(b) mice generate numerically normal cytotoxic T lymphocyte (CTL) responses to the prominent influenza A virus D(b) NP(366) and D(b) PA(224) epitopes and terminate the infectious process in the pneumonic lung with the same kinetics as the WT controls. Though grz B protein expression is fully compromised, there is only a partial effect on the level of CTL activity measured in a classical, short-term (51)Cr release assay. Single-cell polymerase chain reaction (PCR) analysis of both highly activated effector and "resting" memory CD8(+) T cells from influenza A virus-infected grzAB(-/-) mice showed a high prevalence of grzK mRNA(+) expression in tetramer (tet)(+) CTLs as was found in WT mice. However, a marked reduction in cytotoxicity present in the primary splenic CTLs of grzAB(-/-) mice correlated with decreased grzK expression, as measured by real-time PCR. This suggests that grzK plays an important role in CD8(+) T-cell cytotoxicity both in the presence and absence of grzA and B.

Cytotoxic lymphocytes (CL) are highly motile cells that utilize granule exocytosis to kill virus-infected or transformed targets. Isolated CL and purified granule proteins have been used to investigate the molecular processes that CL use to kill their targets and to investigate the basis of human disease. We have set out various methods that are routinely used to isolate CL and characterize the cell death pathways they induce. As cell death mediated through TNF-superfamily members and their respective receptors is covered elsewhere, this manuscript will deal specifically with cytotoxic granule-mediated cell death.

Perforin (PRF), a pore-forming protein expressed in cytotoxic lymphocytes, plays a key role in immune surveillance and immune homeostasis. The A91V substitution has a prevalence of 8% to 9% in population studies. While this variant has been suspected of predisposing to various disorders of immune homeostasis, its effect on perforin's function has not been elucidated. Here we complemented, for the first time, the cytotoxic function of perforin-deficient primary cytotoxic T lymphocytes (CTLs) with wild-type (hPRF-WT) and A91V mutant (hPRF-A91V) perforin. The cytotoxicity of hPRF-A91V-expressing cells was about half that of hPRF-WT-expressing counterparts and coincided with a moderate reduction in hPRF-A91V expression. By contrast, the reduction in cytotoxic function was far more pronounced (more than 10-fold) when purified proteins were tested directly on target cells. The A91V substitution can therefore be manifested by abnormalities at both the lymphocyte (presynaptic) and target cell (postsynaptic) levels. However, the severe intrinsic defect in activity can be partly rescued by expression in the physiological setting of an intact CTL. These findings provide the first direct evidence that hPRF-A91V is functionally abnormal and provides a rationale for why it may be responsible for disordered immune homeostasis if inherited with another dysfunctional perforin allele.

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.

Perforin, a pore-forming protein toxin synthesized and stored in the cytoplasmic vesicles of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, is secreted when these effector lymphocytes encounter virus-infected or neoplastic cells. Perforin is encoded by a single-copy gene and is critical for immune homeostasis and defense of the organism against intracellular sepsis. A complete deficiency of perforin expression in either mice or humans is associated with a syndrome of immune insufficiency and severely deregulated lymphoid homeostasis. Humans who inherit inactivating mutations of perforin or defects in various parts of the cellular machinery that delivers perforin to the target cell suffer from familial hemophagocytic lymphohistiocytosis (FHL), a fatal condition necessitating bone marrow transplantation, usually in infancy. In mice, a high incidence of spontaneous B cell lymphoma has also been noted as the animals age. Across human populations, a number of polymorphisms that result in measurable, but suboptimal CTL activity have been noted, and some of these predispose to attenuated FHL or susceptibility to infectious disease, but in many cases, to no discernible disease predisposition. This chapter discusses the significance of human perforin polymorphisms, particularly those associated with diseases other than FHL, and recent advances in our understanding of perforin biology and function.

The lysosomal protease cathepsin B has been proposed to protect cytotoxic T lymphocytes from the membrane-disruptive effects of perforin secreted during the execution phase of target cell death. Accordingly, cathepsin B that translocates to the lymphocyte surface upon degranulation has been postulated to cleave and inactivate perforin molecules that diffuse back to the killer cell. We have found that recombinant perforin is cleaved inefficiently by cathepsin B and shows no significant reduction in its lytic activity following co-incubation. Furthermore, purified CD8+ cytotoxic T lymphocytes of cathepsin B-null gene-targeted mice were able to induce normal death of target cells both in vitro and in vivo and to survive the encounter with target cells as efficiently as cathepsin B-expressing killer cells. We conclude that cathepsin B is not essential for protection of cytotoxic lymphocytes from the toxic effects of their secreted perforin.

Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition.

Direct interaction between auto-reactive CTL and specific peptide-MHC class I complexes on pancreatic beta cells is critical in mediating beta cell destruction in type I diabetes. We used mice with genetic modifications in three major pathways used by CTL, perforin, Fas and pro-inflammatory cytokines to assess the relative contribution of these mechanisms to beta cell death. In vitro-activated ovalbumin (OVA)-specific CTL, from OT-I TCR-transgenic mice, specifically killed transgenic beta cells expressing OVA (from RIP-mOVA mice) in a 16-h cytotoxicity assay. Perforin-deficient CTL had a reduced ability to kill OVA-expressing islets in vitro (22.1 +/- 3.8%) compared with wild-type CTL (71.4 +/- 4.6%). Fas-deficient islets were only slightly protected from wild-type CTL but were completely protected from the residual killing observed with perforin-deficient CTL. Residual cytotoxicity in perforin-deficient CTL was also prevented by overexpression of SOCS-1, which blocks multiple cytokine signaling pathways. It was also prevented by pre-incubation with anti-tumor necrosis factor-alpha (anti-TNFalpha) antibody or by blocking IFNgamma responsiveness through expressing a dominant negative IFNgamma receptor. Perforin-deficient CTL produced IFNgamma and TNFalpha that was shown to directly induce islet Fas expression during the assays. This suggests that Fas-deficiency, SOCS-1 overexpression and blockade of IFNgamma and TNFalpha all protect beta cells from residual cytotoxicity of perforin-deficient CTL by blocking Fas upregulation. These findings indicate that wild-type CTL destroy antigen-expressing islets via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFNgamma and TNFalpha.

Cytotoxic T lymphocyte (CTL)-induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB(-/-) CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB(-/-) CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB(-/-) mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.

Cytotoxic lymphocytes (CL) induce death of their targets by granule exocytosis. During this process, enzymes contained within cytotoxic granules (granzymes) are delivered to the target cell where the enzymes trigger the cell death by cleaving specific substrates. Granzyme B is the only granzyme that has been shown to induce cell death by apoptosis, but the exact pathway by which this is achieved has been the subject of hot debate. Furthermore, several other death-inducing granzymes have been identified; therefore, the exact contribution of granzyme B to CL-induced death is unclear. In this study, we discuss our recent findings on granzyme B-induced cell death and discuss the potential relevance of this pathway to CL-induced death of viral-infected and transformed cells.

Background: The precise effector mechanisms and molecular mediators used by alloreactive cytotoxic T lymphocytes to kill transplanted pancreatic beta cells are poorly defined. We have used mouse (H2b-anti-d) CTLs raised in strains deficient in various key cytotoxic effector molecules to assess the importance of the various signaling pathways mobilized to kill primary mouse pancreatic islet cells, the beta cell line NIT-1, and NIT-1 cells overexpressing dominant-negative FADD and Bcl-2., Methods: Death of target cells was assessed using 51Cr release assays., Results: In short-term assays (<5 hours) beta cell death did not require a functional FasL/Fas pathway, and was not inhibited by Bcl-2. However, the absence of either perforin or granzyme B resulted in cell survival. By contrast, a crucial role for granzyme B was not seen when hematopoietic P815 cells were used as targets, indicating differential regulation of apoptosis. Interestingly, coincubation with CTL for 24 hours revealed an additional but less potent "late phase" of beta cell death that did not require perforin. This delayed death was blocked by dominant-negative FADD, but not by Bcl-2, and was likely to be due to TNFalpha secretion., Conclusions: This study suggests that strategies to protect beta cells from allogeneic CTL attack will need to inhibit the perforin/granzyme and probably also the TNFalpha pathway. As there are no known pharmacological approaches to blocking perforin, therapeutic approaches based on overexpressing both dominant negative FADD and an inhibitor of granzyme B may hold promise in prolonging beta cell survival in the allogeneic setting.

When a cancer escapes the growth-inhibitory effects of TGF-beta secreted by cancer cells themselves or by cells in the local stroma, a further adverse outcome for the host is the associated TGF-beta-induced suppression of anticancer T cell immunity. In addition to the previously described dampening of T cell activation and proliferation, TGF-beta markedly and directly suppresses the transcription of genes encoding multiple key proteins of the "cytotoxic program" of CD8+ CTL, such as perforin and granzymes, cytotoxins that act through the granule exocytosis pathway. The findings described below suggest that TGF-beta and its signaling pathways will be major targets for novel cancer therapeutics.

Most mammalian cells are constantly threatened by viral infection and oncogenic transformation. To maintain healthy function of organs and tissues it is critical that afflicted cells are efficiently detected and removed. Cytotoxic lymphocytes (CL) are chiefly responsible for efficiently seeking out and eliminating damaged or infected cells. It is known that CLs must specifically recognize and bind to their targets, but the molecular events that occur within the target cell that lead to its death are still poorly understood. The two main processes initiated by CLs to induce target cell death are mediated by ligation of surface receptors or release of toxic proteins from secretory granules (granule exocytosis) of the CL. Here we review some of the key findings that have defined our knowledge of the granule exocytosis-mediated pathways to CL-mediated killing and discuss recent insights that challenge conventional views in the important area of CL effector function.

Cytotoxic lymphocytes protect their host from viral infection and cellular transformation by delivering a range of toxins stored within intracellular granules. One of the most potent of these toxins is the serine protease granzyme B. This review will discuss mechanisms used by granzyme B to enter target cells and the ways in which it synergizes with other granule toxins to cause cell death.

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.

Tumor cells are usually weakly immunogenic as they largely express self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The challenge for immunotherapies has been to provide vigorous immune effector cells that circumvent these tumor escape mechanisms and eradicate established tumors. One promising approach is to engineer T cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the therapeutic efficacy of erbB2-reactive chimeric receptors that contain either T-cell receptor zeta (TCR-zeta) or CD28/TCR-zeta signaling domains. We have demonstrated that primary mouse CD8(+) T lymphocytes expressing the single-chain Fv (scFv)-CD28-zeta receptor have a greater capacity to secrete Tc1 cytokines, induce T-cell proliferation, and inhibit established tumor growth and metastases in vivo. The suppression of established tumor burden by cytotoxic T cells expressing the CD28/TCR-zeta chimera was critically dependent upon their interferon gamma (IFN-gamma) secretion. Our study has illustrated the practical advantage of engineering a T-cell signaling complex that codelivers CD28 activation, dependent only upon the tumor's expression of the appropriate tumor associated antigen.

Recent studies have provided evidence that some cancers can aberrantly express molecules capable of inducing apoptosis of tumor-reactive lymphocytes. Several other potential tumor escape mechanisms that can block the cytotoxic pathways activated by killer T cells have also been proposed.

The induction of cell death by cytotoxic T-lymphocytes (CTL) or natural killer (NK) cells is one of the main ways by which higher organisms protect themselves from rogue cells, including those infected by a virus, or posing a risk of cancer. Considering the rapidity of viral replication and spread to uninfected cells, CTL and NK are extremely efficient killers. This is at least partly due to the variety of pathways that these cytolytic lymphocytes (CL) can use to ensure the death of a cell. Primarily, CL utilize two independently initiated pathways involving either ligation of death receptors or perforin mediated trafficking of granzyme B to the target cell cytosol to activate a family of death proteases (caspases) in the target cell. The caspases then orchestrate the orderly dismantling of that cell by cleavage of a set of critical substrates. If caspases are inactivated, due either to mutations in proteins that signal their activation or direct inhibition by a viral gene product, CL can utilize a caspase-independent pathway to ensure the death of the target cell. Here we will discuss the mechanisms by which these stellar killers achieve their goal.

The structurally related TCR-zeta and Fc receptor for IgE (Fc epsilon RI)-gamma are critical signaling components of the TCR and Fc epsilon RI, respectively. Although chimeric Ab receptors containing zeta and gamma signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the zeta and gamma subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-zeta and scFv-gamma receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-zeta chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-gamma receptors. T cells expressing scFv-zeta chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the zeta signaling pathway.

Summary: Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.

Recent studies have demonstrated that granzymes A and B make an important contribution to the clearance of the orthopoxvirus ectromelia, and in graft versus host disease. To test whether granzymes are generally necessary for lymphocyte-mediated cytotoxicity in vivo, we assessed the cytotoxic capacity of granzyme A and/or B-deficient lymphocytes in several perforin-dependent settings. Splenocytes and allogeneic CTL of granzyme A and/or B-deficient mice were defective for induction of DNA fragmentation, but induced significant membrane damage and target cell death. These results correlated well with the behavior of granzyme A/B-deficient CTL and NK cells in three different perforin-dependent tumor models. In a classical assay of NK cell-mediated rejection, granzyme A and/or B-deficient mice inoculated with RMA-S cells were as susceptible to tumor as wild-type mice. Perforin-deficient mice were also considerably more susceptible to tumor initiation by methylcholanthrene than granzyme A and/or B-deficient mice. Furthermore, rejection of the K1735-melanoma expressing MHC class I and II molecules was mediated by adoptively transferred H-2b anti-k CTL from immunized granzyme A and/or B-deficient mice. In summary, these data suggest that granzymes A and B are not critical for most anti-tumor effector functions of NK cells and CTL that are perforin mediated.

We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme B-mediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly145-->Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme B-independent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death.

Recent advances in our understanding of cytolytic effector mechanisms include the partial characterization of caspase-independent apoptotic pathways triggered by granzymes, a realization of the vital importance of perforin and granzymes in the defence against certain virus infections in vivo and the first description of hereditary immunodeficiency due to disordered perforin expression in humans.

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.