15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity

Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa granulysin are largely distinct. Nine kilodalton granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.     

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.     

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.     

Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1

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.     

Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle

Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.     

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.     

Perforin and Fas act together in the induction of apoptosis, and both are critical in the clearance of lymphocytic choriomeningitis virus infection

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(-/-) CTL. Furthermore, virus-immune CTL with functional FasL and perforin (gzmAxB(-/-)) are more potent in causing target cell apoptosis in vitro than those expressing FasL alone (perfxgzmAxB(-/-)). This synergistic effect of perforin on Fas-mediated nucleolysis of target cells is indicated by the fact that, compared to perfxgzmAxB(-/-) CTL, gzmAxB(-/-) CTL induced (i) an accelerated decrease in mitochondrial transmembrane potential, (ii) increased generation of reactive oxygen species, and (iii) accelerated phosphatidylserine exposure on plasma membranes. We conclude that perforin does not mediate recovery from LCMV by itself but plays a vital role in both gzmA/B and FasL/Fas-mediated CTL activities, including apoptosis and control of viral infections.     

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced 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.     

Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help

Recently, CD4(+) T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL-)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4(+) T cell-derived IL-21 induces differentiation of B cells into granzyme B (GzmB)-secreting cytotoxic cells. Using fluorescence-activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4(+) T cells, activated via their T-cell receptor without co-stimulation, can produce IL-21, but do not express CD40L and rapidly induce GzmB in co-cultured B cells in an IL-21 receptor-dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL-21-induced GzmB induction in B cells in a dose-dependent manner. Surprisingly, although GzmB-secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL-2 instead of IL-21 or when target cells were cultured with IL-21 alone. Our findings suggest GzmB(+) cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen-specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL-21 induces differentiation of B cells into plasma cells or into granzyme B-secreting cytotoxic cells.     

Nuclear translocation of granzyme B in target cell apoptosis

Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.     

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

Background

Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.

Methods and Findings

Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB⁺ Tc cells) or granzyme A and granzyme B deficient (GzmA×B^−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.

Significance

Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.     

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.     

Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.     

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.     

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells

 Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase. Received: 20 November 2000 / Accepted: 8 March 2001     

Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression

Hyperthermia, which is used as an adjunctive therapy for cancer, is known to modulate the activity of natural killer (NK) cells in vitro, but its effect in vivo is unclear. In the present study, we used a whole body hyperthermia (WBH) device heated by infrared rays to evaluate the effect of WBH on mice models. We demonstrate here that wild type C57BL/6J mice exposed to 42 degrees C for 60min had reduced NK cell cytolytic activity against YAC-1 target cells as determined by cytolytic assay. This result was confirmed using Rag-2 knockout mice, which possess functional NK but not cytolytic T or NK-T cells. Moreover, WBH decreased the mRNA expression of perforin and granzyme B in spleens of mice. But the expression of TNF cytokines (Fas ligand and TRAIL) was unchanged. These data suggest that the suppression of NK cell activity induced by WBH could be mediated through the perforin/granzyme pathway.     

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB⁺Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB⁺Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-X_L, the anti-apoptotic counterparts of Bim. In contrast, gzmB⁺Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB⁺Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB⁺Tc-induced death pathways.     

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls ( P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.     

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

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.