Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.     

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.     

Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.     

Serpins prevent granzyme-induced death in a species-specific manner

Expression of serine protease inhibitors (serpins) is one of the mechanisms used by tumour cells to escape immune surveillance. Previously, we have shown that expression of serpins SPI-6 and SPI-CI, respectively, renders tumour cells resistant to granzyme B (GrB)-mediated death and granzyme M (GrM)-mediated death. To obtain better insight into the interaction between serpins and their target proteases, we investigated the roles of protease inhibitor (PI)-9 and SPI-6 in the resistance to GrB-mediated and CD95-mediated death in further detail. Neither human PI-9 nor its murine orthologue SPI-6 was capable of preventing CD95-induced apoptosis in murine or human cells, indicating that these serpins do not inhibit the activation of apical caspases in this pathway. High expression of PI-9 or SPI-6 did prevent apoptosis induced by human GrB. Strikingly, only SPI-6, and not PI-9, was capable of inhibiting murine GrB, suggesting that a difference in enzymatic specificity exists between the mouse and the human granzymes. In agreement with this suggestion, murine GrB was clearly less effective in inducing apoptosis in human cells. Similar species specificity was also observed for SPI-CI and GrM when either their capacity to associate or the effectiveness of GrM-induced cytotoxicity was analysed. Our findings therefore indicate a species diversity that has a clear effect on mixed in vitro effector target settings.     

Expression of high levels of human proteinase inhibitor 9 blocks both perforin/granzyme and Fas/Fas ligand-mediated cytotoxicity

Proteinase inhibitor 9 (PI-9, SerpinB9) is the only known human intracellular granzyme B inhibitor. Whether expression of PI-9 is sufficient to block cytolysis induced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells remains controversial. To evaluate the roles of PI-9, we isolated and tested three lines of stably transfected HeLa cells expressing wild-type PI-9 and one line expressing an inactive mutant PI-9. Expressions of wild-type PI-9, but not the inactive mutant PI-9, inhibited cytolysis induced by human NK92 and NKL natural killer cells. Expression of high levels of PI-9 is therefore sufficient to protect human cells against NK cell-mediated cell death. Using two assays, we show that expressing wild-type PI-9, but not the inactive mutant PI-9, blocks Fas/Fas ligand (Fas/FasL)-mediated apoptosis. PI-9 expression has no effect on etoposide-induced apoptosis. HeLa cells exhibiting substantial resistance to Fas/FasL-mediated apoptosis contain 2- to 3-fold higher PI-9 levels than HCT116 human colon cancer cells and 2- to 3-fold lower PI-9 levels than MCF7/ERHA breast cancer cells, in which PI-9 is strongly induced by estrogens, and by tamoxifen. Expression of increasing levels of PI-9 in target cells may progressively inhibit immune surveillance by blocking NK and CTL-induced cytotoxicity through the perforin/granzyme pathway and then through the Fas/FasL pathway.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.     

Selective Regulation of Apoptosis: the Cytotoxic Lymphocyte Serpin Proteinase Inhibitor 9 Protects against Granzyme B-Mediated Apoptosis without Perturbing the Fas Cell Death Pathway

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P₁ mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P₁(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.     

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

Stably Transfected Antisense Granzyme B and Perforin Constructs Inhibit Human Granule-Mediated Lytic Ability

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV₂-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by >95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by >95%. Western analysis revealed a 30% decrease in the level of perlatin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.     

The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses

Proteinase inhibitor 9 (PI-9) is a 42-kDa human intracellular serpin present in cytotoxic lymphocytes (CLs). PI-9 is an extremely efficient inhibitor of the pro-apoptotic CL granule proteinase granzyme B and is thought to function in the cytosol of CLs to protect against apoptosis induced by endogenously expressed or released granzyme B, particularly during target cell killing. Here we show by immunohistochemistry that PI-9 is also present in endothelial cells, in every tissue examined. Cultured endothelial cells express functional PI-9 (as assessed by binding to recombinant granzyme B) localized to the cytoplasm and nucleus. Immunohistochemistry also showed PI-9 in mesothelial cells, and this was confirmed by analysis of primary cells cultured from pleural and serous effusions. Granzyme B expression was not detected in either endothelial or mesothelial cells. In both cell types, PI-9 is up-regulated at the mRNA and protein level by exposure to the phorbol ester PMA, consistent with a response to inflammatory stimuli. We postulate that PI-9 is present in these lining cell types to protect against misdirected, free granzyme B released during a local immune response.     

Proteinase Inhibitor 6 (PI-6) Expression in Human Skin: Induction of PI-6 and a PI-6/Proteinase Complex during Keratinocyte Differentiation

Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation.     

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.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis

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.     

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.     

Serpin-6 expression protects embryonic stem cells from lysis by antigen-specific CTL

The immune response to embryonic stem (ES) cells is still poorly understood. In this study, we addressed the adaptive cellular immune response to undifferentiated and differentiated ES cells infected with lymphocytic choriomeningitis virus (LCMV), a vertically transmitted pathogen in mice and humans. In contrast to the prevailing view, we found that undifferentiated and differentiated murine ES cells express MHC class I molecules, although at low levels. When cocultured with LCMV-infected ES cells, syngeneic but not allogeneic LCMV-specific CTL secrete IFN-gamma. Strikingly, LCMV-specific CTL do not efficiently kill LCMV-infected ES cells. ES cells showed high-level expression of the serine protease inhibitor 6, an endogenous inhibitor of the CTL-derived cytotoxic effector molecule granzyme B. Down-regulation of serpin-6 by RNA interference sensitized ES cells for CTL-induced cell death. The results of this study suggest that LCMV-infected murine ES cells present viral Ags and are recognized by LCMV-specific CTL in a MHC class I-restricted manner, yet resist CTL-mediated lysis through high-level expression of serine protease inhibitor 6.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.     

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.     

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.