Granzyme B-deficient Mice Research Articles

Granzyme B prevents aberrant IL-17 production and intestinal pathogenicity in CD4+ T cells.

CD4+ T cell activation and differentiation are important events that set the stage for proper immune responses. Many factors are involved in the activation and differentiation of T cells, and these events are tightly controlled to prevent unwanted and/or exacerbated immune responses that may harm the host. It has been well documented that granzyme B, a potent serine protease involved in cell-mediated cytotoxicity, is readily expressed by certain CD4+ T cells, such as regulatory T cells and CD4+CD8αα+ intestinal intraepithelial lymphocytes, both of which display cytotoxicity associated with granzyme B. However, because not all CD4+ T cells expressing granzyme B are cytotoxic, additional roles for this protease in CD4+ T cell biology remain unknown. Here, using a combination of in vivo and in vitro approaches, we report that granzyme B-deficient CD4+ T cells display increased IL-17 production. In the adoptive transfer model of intestinal inflammation, granzyme B-deficient CD4+ T cells triggered a more rapid disease onset than their WT counterparts, and presented a differential transcription profile. Similar results were also observed in granzyme B-deficient mice infected with Citrobacter rodentium. Our results suggest that granzyme B modulates CD4+ T cell differentiation, providing a new perspective into the biology of this enzyme.

Perforin and Granzyme B Have Separate and Distinct Roles during Atherosclerotic Plaque Development in Apolipoprotein E Knockout Mice

The granzyme B/perforincytotoxic pathway is a well established mechanism of initiating target cell apoptosis. Previous studies have suggested a role for the granzyme B/perforin cytotoxic pathway in vulnerable atherosclerotic plaque formation. In the present study, granzyme B deficiency resulted in reduced atherosclerotic plaque development in the descending aortas of apolipoprotein E knockout mice fed a high fat diet for 30 weeks while perforindeficiency resulted in greater reduction in plaque development with significantly less plaque area than granzyme Bdeficient mice. In contrast to the descending aorta, no significant change in plaque size was observed in aortic roots from either granzyme Bdeficient or perforindeficient apolipoprotein E knockout mice. However, atherosclerotic plaques in the aortic roots did exhibit significantly more collagen in granzyme B, but not perforin deficient mice. Together these results suggest significant, yet separate roles for granzyme B and perforin in the pathogenesis of atherosclerosis that go beyond the traditional apoptotic pathway with additional implications in plaque development, stability and remodelling of extracellular matrix.

Granzyme B Is Dispensable in the Development of Diabetes in Non-Obese Diabetic Mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8+ T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8+ T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3+CD8+ T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.

Granzyme B Regulates Antiviral CD8+ T Cell Responses

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.

A Role for Granzyme M in TLR4-Driven Inflammation and Endotoxicosis

Lymphocyte perforin and serine protease granzymes are well-recognized extrinsic mediators of apoptosis. We now demonstrate that cytotoxic lymphocyte granule components profoundly augment the myeloid cell inflammatory cytokine cascade in response to TLR4 ligation. Whereas caspase-1-deficient mice were completely resistant to LPS, reduced serum cytokine production and resistance to lethal endotoxicosis were also obtained with perforin-deficient mice, indicating a role for granzymes. Consistently, a lack of granzyme M (GrzM) resulted in reduced serum IL-1alpha, IL-1beta, TNF, and IFN-gamma levels and significantly reduced susceptibility to lethal endotoxicosis. These altered responses were also observed in granzyme A-deficient but not granzyme B-deficient mice. A role for APC-NK cell cross-talk in the inflammatory cascade was highlighted, as GrzM was exclusively expressed by NK cells and resistance to LPS was also observed on a RAG-1/GrzM-double deficient background. Collectively, the data suggest that NK cell GrzM augments the inflammatory cascade downstream of LPS-TLR4 signaling, which ultimately results in lethal endotoxicosis. Most importantly, these data demonstrate that granzymes should no longer be considered solely as mediators of apoptosis, but additionally as potential key regulators of inflammation.

A Renaissance in Understanding the Multiple and Diverse Functions of Granzymes?

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 B and Perforin Are Important for Regulatory T Cell-Mediated Suppression of Tumor Clearance

Granzyme B is important for the ability of NK cells and CD8(+) T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.

Granzyme B Is Important for Treg-Mediated Suppression of NK-Dependent Tumor Clearance In Vivo.

NK-dependent clearance of RMAS lymphoma and B16 melanoma (MHC Ilow) has been demonstrated in NK cell-deficient mice (Kim S et al, PNAS. 2000 Mar; 97(6): 2731–6). We recently investigated the roles of granzyme (Gzm) A and B in NK-dependent clearance of RMAS and B16 cells. The survival curves following intravenous injection of 2x105 RMAS cells are shown in the Figure; similar results were found with B16 cells. All granzyme AxB-deficient mice died within 6 weeks. Survival of granzyme A-deficient mice was similar to that of WT mice. Surprisingly, granzyme B-deficient mice were more resistant than WT mice to these tumor challenges. Previously, our laboratory demonstrated that human regulatory T (Treg) cells can use the granule exocytosis pathway to kill a variety of autologous immune cells in vitro (Grossman WJ et al, Immunity. 2004 Oct; 21(4): 589–601). Based on these results, we hypothesized that these tumor cell lines may induce granzyme B expression in Tregs, which in turn suppress the function of the NK cells responsible for clearing the tumors. Indeed, flow cytometric studies revealed that granzyme B (but not granzyme A) was highly expressed in 10–30% of CD4+/FoxP3+ Tregs found in the tumor environment (i.e. ascites fluid, tumor-infiltrated livers, or lungs). In contrast, very few granzyme B-expressing cells (<1%) were detected in the Tregs found in the peripheral spleens or lymph nodes of tumor-bearing mice or in the spleens, livers and lungs of naive mice. Within the NK cell compartment, granzyme A expression was constitutive, and it was persistently expressed following tumor challenge. While granzyme B was detected in only 1–5% of NK cells in the spleens, livers and lungs of naive mice, 20–40% of NK cells in the tumor environment expressed granzyme B following tumor challenge. To determine whether Tregs require granzyme B to suppress NK cell function in vivo, we purified Tregs (95% pure CD4+/CD25+ cells) from the resting spleens of both WT and granzyme B-deficient mice, and co-injected them intraperitoneally with 2x106 luciferase-tagged RMAS cells into granzyme B-deficient mice. Tumor burden was assessed using in vivo bioluminescence imaging. Add-back of 2x106 WT Tregs into granzyme B-deficient mice restored tumor growth to 40% of that observed at day 7 in WT mice, whose tumor burden was approximately 50-fold higher than that of granzyme B-deficient mice. Add-back of equal doses of granzyme B-deficient Tregs had no effect on tumor burden. Our data suggest that these tumor cell lines induce the expression of granzyme B in Tregs, which subsequently suppress tumor-specific NK cells. These data demonstrate for the first time that granzyme B plays a crucial role in Treg-mediated suppression of NK cell function in vivo. By recruiting and activating Tregs that inhibit NK cell function, these tumors gain a survival advantage that is lost in the setting of granzyme B deficiency in the Tregs. This study therefore suggests that targeted inhibition of granzyme B in Tregs may represent a novel approach to break Treg-mediated tumor tolerance. [Display omitted]

Granzyme B is dispensable for immunologic tolerance to self in a murine model of systemic lupus erythematosus

Proteolytic autoantigen cleavage by the serine protease granzyme B has been implicated in the development of systemic autoimmune disease; however, there has been no conclusive demonstration of a pathogenic role for granzyme B in autoimmunity. In this study, we evaluated the role of granzyme B in a murine model of autoimmunity. To identify potential novel granzyme B substrates, complementary DNAs encoding nuclear factor 45 (NF45) and NF90 were used to generate (35)S-methionine-labeled proteins by coupled in vitro transcription/translation. Radiolabeled proteins were then incubated with purified recombinant granzyme B or caspases, and the cleavage products were analyzed by autoradiography. We also immunized granzyme B-deficient and granzyme B-intact mice with the mineral oil pristane. Production of autoantibodies directed against granzyme B substrates in response to pristane was evaluated by Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay. The double-stranded RNA-binding protein NF90 was identified as a novel substrate for caspases and granzyme B, both in vitro and in vivo. NF90 is uniquely cleaved by granzyme B in vitro; however, pristane immunization still induced anti-NF90 antibodies in granzyme B-deficient mice. Pristane-treated granzyme B-deficient mice also produced antibodies directed against the U1-70-kd antigen, a previously identified granzyme B substrate. Last, antibodies directed against U1-70 kd arose spontaneously in granzyme B-deficient mice. These results demonstrate that granzyme B is not required for the production of autoantibodies directed against antigens that are granzyme B substrates in vitro. The data also suggest a protective role for this proapoptotic protease in systemic autoimmunity.

Cleavage of CPP32 by Granzyme B Represents a Critical Role for Granzyme B in the Induction of Target Cell DNA Fragmentation

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.

Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.

Granzyme (Gzm) B-deficient mice obtained by gene targeting were used to assess the role of Gzm B in the mechanisms used by natural killer (NK) and lymphokine-activated killer (LAK) cells to destroy target cells. Gzm B-/- NK cells, LAK cells, and cytotoxic T lymphocytes (CTL) all are defective in their ability to rapidly induce DNA fragmentation/apoptosis in susceptible target cells. This defect can be partially corrected with long incubation times of effector and target cells. Moreover, Gzm B-/- NK cells (but not CTL or LAK cells) exhibit a defect in 51Cr release from susceptible target cells. This 51Cr release defect in Gzm B-deficient NK cells is also not overcome by prolonged incubation times or high effector-to-target cell ratios. We conclude that Gzm B plays a critical and nonredundant role in the rapid induction of DNA fragmentation/apoptosis by NK cells, LAK cells, and CTL. Gzm B may have an additional role in NK cells (but not in CTL or LAK cells) for mediating 51Cr release.