Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.     

Regulation of perforin activation and pre‐synaptic toxicity through C‐terminal glycosylation

Perforin is a highly cytotoxic pore‐forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca²⁺‐rich endoplasmic reticulum, remains unknown. Here, we show that N‐linked glycosylation of the perforin C‐terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C‐terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C‐terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post‐translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.     

The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal

Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumour cells by cytotoxic T lymphocytes and natural killer cells. After target-cell recognition, release of the cytotoxic granule contents into the immunological synapse formed between the killer cell and its target induces apoptosis. The granules contain two membrane-perturbing proteins, perforin and granulysin, and a family of serine proteases known as granzymes, complexed with the proteoglycan serglycin. In this review, I discuss recent insights into the mechanisms of granule-mediated cytotoxicity, focusing on how granzymes A, B and C and granulysin activate cell death through caspase-independent pathways.     

Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti-CD3-coated beads. However, rapid anti-CD3-induced granule secretion was drastically defective in both CD8(+) and CD4(+) T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor-stimulated interferon-gamma secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.     

Granzyme M mediates a novel form of perforin-dependent cell death

Cell death is mediated by cytotoxic lymphocytes through various granule serine proteases released with perforin. The unique protease activity, restricted expression, and distinct gene locus of granzyme M suggested this enzyme might have a novel biological function or trigger a novel form of cell death. Herein, we demonstrate that in the presence of perforin, the protease activity of granzyme M rapidly and effectively induces target cell death. In contrast to granzyme B, cell death induced by granzyme M does not feature obvious DNA fragmentation, occurs independently of caspases, caspase activation, and perturbation of mitochondria and is not inhibited by overexpression of Bcl-2. These data raise the likelihood that granzyme M represents a third major and specialized perforin-dependent cell death pathway that plays a significant role in death mediated by NK cells.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.     

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.     

A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene

To test the granule exocytosis model for lymphocyte cytotoxicity, we have expressed the gene for the cytotoxic lymphocyte granule protein cytolysin (perforin) in the noncytotoxic rat basophilic leukemia (RBL) cell line, which undergoes granule exocytosis when its high affinity IgE receptor is cross-linked. Homogenates of RBL-cytolysin (RBL-cy) transfectants showed a calcium-dependent hemolytic activity in dense granule fractions, demonstrating that the expressed cytolysin protein was correctly targeted to secretory granules. RBL-cy transfectants showed a potent and calcium-dependent cytolytic activity against IgE-coated RBCs, while the parental RBL line was not cytotoxic under these conditions. RBL-cy cells did not lyse non-IgE-coated RBCs copelleted with targets: this sparing of "innocent bystanders" parallels cytotoxic T lymphocyte lysis and suggests a polarized secretion of cytolysin. In contrast to RBC targets, IgE-coated tumor cells were much less sensitive to lysis by RBL-cy transfectants.     

Resistance of cytolytic lymphocytes to perforin-mediated killing. Murine cytotoxic T lymphocytes and human natural killer cells do not contain functional soluble homologous restriction factor or other specific soluble protective factors

CTL and NK cells produce a cytolytic pore-forming protein (perforin, cytolysin) localized in their cytoplasmic granules. These cytotoxic cells are resistant to killing mediated by other lymphocytes and by purified perforin. A membrane factor, known as homologous restriction factor (HRF), has been suggested to confer protection to different cell types against both C- and perforin-mediated lysis. The granules of human large granular lymphocytes have been reported to contain, in addition to perforin, a soluble HRF activity that can be eluted from anion-exchange columns at 115 mM NaCl. Here, we report that a soluble HRF activity is absent in the granules or the cytosol of murine CTL and human NK cells. Our data indicate that the inhibition attributed to HRF could be explained by exogenous EDTA added during granule fractionation. EDTA was shown to bind to Mono Q and to elute at 90 to 120 mM NaCl. A second perforin-inhibitory activity was also eluted from such a column. However, it was present in preparations obtained not only from CTL and NK cells, but also from some perforin-susceptible tumor cell lines, indicating that it has nonrestricted distribution and suggesting that it is probably irrelevant to the perforin-protection mechanism. Our results argue against a role for soluble granule HRF or other soluble factors in mediating resistance of cytotoxic lymphocytes against perforin-mediated lysis.     

LYST Controls the Biogenesis of the Endosomal Compartment Required for Secretory Lysosome Function

Chediak–Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome‐like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling‐late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal‐sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13‐4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin‐containing vesicles into secretory cytotoxic granules.      

Cutting edge: granzymes A and B are not essential for perforin-mediated tumor rejection

Controversy still exists regarding the biological function of granzyme serine proteases released with perforin from the cytotoxic granules of NK cells and CTLs. In particular, it is not clear whether the major granzymes, A and B, play an essential role in tumor rejection mediated by the perforin pathway. We have now examined the relative importance of perforin and granzyme A and B clusters in five different tumor models that stringently distinguish their importance. We conclude that granzyme A and B clusters are not essential for CTL- and NK cell-mediated rejection of spontaneous and experimental tumors, raising the likelihood that either perforin alone or in combination with an additional granzyme or granule component(s) mediates cytotoxicity of tumor cells in vivo.     

Poxvirus-encoded serpins do not prevent cytolytic T cell-mediated recovery from primary infections.

Previous observations that the highly conserved poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways was taken to indicate their involvement in immune evasion by poxviruses. We now show that interference with 51Cr release from target cells by ectromelia and cowpoxvirus is limited to alloreactive but not MHC-restricted CTL. The data are in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection and question the role of serpins in the evasion of poxviruses from killing by CTL. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression primarily affects the Fas-mediated, and to a lesser extent, the granule exocytosis pathway. Serpin-2 is the main contributor to suppression for both killing pathways. In addition, inhibition of lysis was shown to be both target cell type- and MHC allotype-dependent. We hypothesize that differences in TCR affinities and/or state of activation between alloreactive and MHC-restricted CTL as well as the quality (origin) of target cells are responsible for the observed phenomenon.     

Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A.

The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-cytolysin and RBL-cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%-60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.     

Functional significance of the perforin/granzyme cell death pathway

Perforin/granzyme-induced apoptosis is the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or transformed cells. Studies in gene-disrupted mice indicate that perforin is vital for cytotoxic effector function; it has an indispensable, but undefined, role in granzyme-mediated apoptosis. Despite its vital importance, the molecular and cellular functions of perforin and the basis of perforin and granzyme synergy remain poorly understood. The purpose of this review is to evaluate critically recent findings on cytotoxic granule-mediated cell death and to assess the functional significance of postulated cell-death pathways in appropriate pathophysiological contexts, including virus infection and susceptibility to experimental or spontaneous tumorigenesis.     

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.     

Structural and functional analysis of perforin mutations in association with clinical data of familial hemophagocytic lymphohistiocytosis type 2 (FHL2) patients

Perforin plays a key role in the immune system via pore formation at the target cell membrane in the elimination of virus‐infected and transformed cells. A vast number of observed mutations in perforin impair this mechanism resulting in a rare but fatal disease, familial hemophagocytic lymphohistiocytosis type 2 (FHL2). Here we report a comprehensive in silico structural analysis of a collection of 76 missense perforin mutations based on a proposed pore model. In our model, perforin monomers oligomerize having cyclic symmetry in consistent with previously found experimental constraints yet having flexibility in the size of the pore and the number of monomers involved. Clusters of the mutations on the model map to three distinct functional regions of the perforin. Calculated stability (free energy) changes show that the mutations mainly destabilize the protein structure, interestingly however, A91V polymorphism, leads to a more stable one. Structural characteristics of mutations help explain the severe functional consequences on perforin deficient patients. Our study provides a structural approach to the mutation effects on the perforin oligomerization and impaired cytotoxic function in FHL2 patients.     

Granzyme B translocates across the lipid membrane only in the presence of lytic agents

Granzyme B (GrB), a component of the cytotoxic cell granule secretion pathway, is designed to kill infected and transformed cells after intracellular delivery by the pore forming protein, perforin. The mechanism of the delivery remains speculative. In this study we tested the hypothesis that GrB possesses capacity to bind and disrupt lipid membranes. Here in comparison to previous studies that show GrB interacts with carbohydrate moieties, the protease does not bind membrane phospholipids nor has intrinsic membranolytic properties. To study the transmembrane movement of GrB, we developed a model membrane system consisting of a high-molecular weight GrB substrate encapsulated in unilamellar vesicles. Intra-vesicle proteolysis clearly requires concentrations of lytic agents (streptolysin O, perforin or Triton X-100) that disrupt unilamellar membranes.     

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.     

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.