Mouse Granzyme Research Articles

Synthesis and evaluation of 111 In-labeled tetrapeptide-based compounds as single-photon emission computed tomography imaging probes targeting granzyme B.

Granzyme B is an attractive target as a biomarker for contributing to improve the treatment with immune checkpoint inhibitor (ICI). In this study, we designed novel 111 In-labeled granzyme B-targeting single-photon emission computed tomography (SPECT) imaging probes, [111 In]IDT and [111 In]IDAT. Nonradioactive In-labeled granzyme B-targeting compounds ([nat In]IDT, [nat In]IDAT) showed the affinity for recombinant mouse granzyme B. [111 In]IDT and [111 In]IDAT were obtained with moderate radiochemical yield and high stability in mouse plasma (>95%). In a biodistribution experiment using tumor-bearing mice, [111 In]IDT and [111 In]IDAT showed moderate accumulation in tumor. Ex vivo autoradiography (ARG) indicated that the accumulation of radioactivity in tumor was correlated to expression of granzyme B confirmed by the immunohistochemical staining. These results indicated that [111 In]IDT and [111 In]IDAT showed the basic properties as granzyme B-targeting SPECT probes.

In vivo bioluminescence imaging of granzyme B activity in tumor response to cancer immunotherapy

Cancer immunotherapy has revolutionized the treatment of cancer, but only a small subset of patients benefits from this new treatment regime. Imaging tools are useful for early detection of tumor response to immunotherapy and probing the dynamic and complex immune system. Here, we report a bioluminescence probe (GBLI-2) for non-invasive, real-time, longitudinal imaging of granzyme B activity in tumors receiving immune checkpoint inhibitors. GBLI-2 is made of the mouse granzyme B tetrapeptide IEFD substrate conjugated to D-luciferin through a self-immolative group. GBLI-2 was evaluated for imaging the dynamics of the granzyme B activity and predicting therapeutic efficacy in a syngeneic mouse model of CT26 murine colorectal carcinoma. The GBLI-2 signal correlated with the change in the population of PD-1- and granzyme B-expressing CD8+ Tcells in tumors.

Upregulation of torso-like protein (perforin) and granzymes B and G in non-adherent, lymphocyte-like haemocytes during a WSSV infection in shrimp

Invertebrates only have an innate immunity in which haemocytes play an important role. In our lab, 5 subpopulations of haemocytes were identified in the past by an iodixanol density gradient: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. For the two latter subpopulations, the haemocytes have small cytoplasm rims, do not adhere to the bottom of plastic cell-culture grade wells and present folds in the nucleus. These characteristics are similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity. First, a fast isolation technique was developed to separate the non-adherent haemocytes from the adherent haemocytes. After 60 min incubation on cell culture plates, the non-adherent haemocytes were collected. The purity reached 93% as demonstrated by flow cytometry and light microscopy upon a Hematoxylin and Eosin (H&E) staining. Cytotoxicity by lymphocytes is mediated by molecules such as perforin and granzymes and therefore, we searched for their genes in the shrimp genome. Genes coding for a torso-like protein, granzyme B and granzyme G were identified. Primers were designed and RT-PCR/RT-qPCR assays were developed. The results demonstrated that torso-like protein, granzyme B and granzyme G were mainly expressed in non-adherent haemocytes. The shrimp torso-like protein gene was most related to that of the crab torso-like protein; granzyme B gene was most related to that of mouse granzyme B and granzyme G gene was most related to that of zebrafish granzyme G. In a 72-hour in vivo WSSV infection challenge, the mRNA expression of shrimp torso-like protein, granzyme B and granzyme G in haemocytes was increasing over time, which indicated that torso-like protein, granzyme B and granzyme G of shrimp haemocytes are involved in the immune response during a viral infection. In the future, antibodies will be raised against these proteins for more in-depth functional analyses.

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of ∼ 5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-γ), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

Substrate specificities of the granzyme tryptases A and K.

The physiological roles of the granzymes A and K have been debated, especially concerning their involvement in cytotoxic and inflammatory processes. By performing N-terminal COFRADIC assisted N-terminomics on the homologous human granzymes A and K, we here provide detailed data on their substrate repertoires, their specificities, and differences in efficiency by which they cleave their substrates, all of which may aid in elucidating their key substrates. In addition, the so far uncharacterized mouse granzyme K was profiled alongside its human orthologue. While the global primary specificity profiles of these granzymes appear quite similar as they revealed only subtle differences and pointed to substrate occupancies in the P1, P1', and P2' position as the main determinants for substrate recognition, differential analyses unveiled distinguishing substrate subsite features, some of which were confirmed by the more selective cleavage of specifically designed probes.

Elucidating Sources and Roles of Granzymes A and B during Bacterial Infection and Sepsis

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.

Identification and characterization of novel granzyme in fish (971.1)

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Granzymes induce apoptosis within virus‐infected and transformed cells. Information on the function of fish granzymes is limited, although granzyme‐like genes have been reported in several fishes. Here, we report the identification and characterization of a granzyme in ginbuna crucian carp, Carassius auratus langsdorfii, focusing on substrate specificity and gene expression after allo‐sensitization and bacterial infection.A full‐length granzyme cDNA, termed gcGranzyme was identified from ginbuna crucian carp. gcGranzyme molecule has trypsin‐like catalytic triad: His66, Asp109, and Ser201 residues, and active center: Ser201 residue. Phylogenetic analysis of deduced amino acids sequence revealed that gcGranzyme formed a cluster with human and mouse granzyme B (separate from the other members of the granzyme). gcGranzyme was secreted from the HeLa cells transfected with gcGranzyme as in mammals. Enzyme assay using recombinant gcGranzyme indicated that the enzyme hydrolyzed Bz‐Arg‐4Methyl‐Coumaryl‐7‐amide, suggesting that the gcGranzyme was different from mammalian granzyme B in substrate specificity.To investigate the role of gcGranzyme in cell‐mediated immunity in fish, we analyzed the gcGranzyme mRNA expression in allo‐sensitized fish. Expression level of gcGranzyme mRNA from CD8+ T cells was greatly elevated by allo‐sensitization. Furthermore, gcGranzyme mRNA expression in CD8+ T cells was also enhanced by the in vivo infection with intracellular bacteria, Edwardsiella tarda.These results suggest that newly found gcGranzyme is a novel secretory serine protease involved in cell‐mediated immunity in fish with similar structure to human granzyme B but different substrate specificity.

Identification of Serpinb6b as a Species-specific Mouse Granzyme A Inhibitor Suggests Functional Divergence between Human and Mouse Granzyme A

The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10(5) M(-1) s(-1) and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC50 value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B.

BackgroundPrevious screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).ResultsIn vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3′ position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1′ position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5′ leader sequence (5′UTR) of BNIP-2.ConclusionsDespite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

Human and mouse granzyme M display divergent and species-specific substrate specificities

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

Mouse granzyme K has pro-inflammatory potential

Granzymes (gzms) are key components of T-killer (Tc) cells believed to mediate pro-apoptotic activities. Recent evidence suggests that gzms also possess non-cytotoxic activities that contribute to host defense. In this study, we show that Tc cells from lymphocytic choriomeningitis virus (LCMV)-infected wild-type (wt) and gzm A/B-deficient mice express similar levels of gzmK protein, with both mouse strains efficiently controlling infection. GzmK, in recombinant form or secreted by ex vivo-derived LCMV-immune gzmAxB(-/-) Tc cells, lacks pro-apoptotic activity. Instead, gzmK induces primary mouse macrophages to process and secrete interleukin-1β, independent of the ATP receptor P2X(7). Together with the finding that IL-1Ra (Anakinra) treatment inhibits virus elimination but not generation of cytotoxic Tc cells in wt mice, the data suggest that Tc cells control LCMV through non-cytotoxic processes that involve gzmK.

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.

Human and Mouse Granzyme A Induce a Proinflammatory Cytokine Response

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Analysis of Protein Processing by N-terminal Proteomics Reveals Novel Species-specific Substrate Determinants of Granzyme B Orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.

Differential Regulation of Granzyme B and C Expression in Murine Cytotoxic T and NK Cells.

Cytotoxic lymphocytes, which include CD4+ and CD8+ T cells as well as NK cells, use the granule exocytosis pathway to kill virus-infected and tumor cells. Previous studies have characterized the expression of many genes in this pathway (e.g. perforin, granzyme A, granzyme B, etc.), although no reagent has been developed to measure granzyme C protein at the single-cell level. The murine granzyme C gene, orthologous to human granzyme H, lies 24.2Kb directly downstream from granzyme B in the granzyme B gene cluster. Recombinant granzyme C rapidly induces target cell death in a manner distinct from granzyme A- or B-induced death. To further understand the regulation and function of murine granzyme C, we developed a granzyme C-specific monoclonal antibody and used flow cytometry to examine the expression of granzyme C in resting and activated murine cytotoxic lymphocyte compartments. Naive CD4+ and CD8+ T cells express little or no granzyme C (0.1 + 0.1% of cells are positive). After activation of splenocytes with plate-bound CD3 and CD28 agonistic antibodies for 4 days, a small percentage of CD4+ (2.3 + 1.0% positive) and CD8+ (6.6 + 0.3% positive) T cells express granzyme C. However, only 24 hours later, almost all CD4+ (96.7 + 2.7%) and CD8+ (98.7 + 1.4%) T cells express granzyme C. Granzyme B co-staining revealed that granzyme B is detectable in both CD4+ and CD8+ T cells at least 48 hours before granzyme C is expressed. Furthermore, we employed a fully mismatched GVHD mouse model in order to examine T cell expression of granzymes B and C in vivo; similarly, granzyme B expression preceded granzyme C by at least 48 hours. In addition, CD4+Foxp3+ regulatory T cells also expressed granzyme C in this model. Murine NK cell granzyme C mRNA expression was assessed using Affymetrix microarrays (MOE430v2) at rest and following IL-15 activation. Resting NK cells express minimal granzyme C mRNA (mean gzmC probe set signal intensity + SD [n=3]): 356 + 202, which increased after IL-15 activation as follows: 4,180 + 1,106 (day 1), 60,788 + 8,455 (day 2), 167,448 + 26,398 (day 4), and 178,451+14,037 (day 6). Utilizing our granzyme C-specific mAb, we showed that very few resting murine NK cells express granzyme C protein (2.3 + 0.4% positive). Consistent with the mRNA data, the percentage of granzyme C-expressing NK cells was substantially increased after 3 days of IL-15 activation (88.8 + 5.2% positive). In contrast, granzyme B mRNA levels are high in resting NK cells (41,208 + 2,534), and increase only incrementally with IL-15 treatment (Fehniger et al., Immunity 2007 Jun;26(6):798–811). Granzyme B protein expression is controlled at a post-transcriptional level in NK cells, whereas granzyme C expression is transcriptionally regulated. Taken together, our findings show that the mouse granzyme B and C genes, despite being only 24.2Kb apart, are activated in cytotoxic lymphocytes with different kinetics; moreover, granzyme B and C protein abundance is controlled by completely different mechanisms in NK cells. These data suggest that the granzyme genes are differentially regulated in lymphocyte compartments, and that this regulation may be relevant for how cytotoxic lymphocytes function.

The granzyme B gene is highly polymorphic in wild mice but essentially invariant in common inbred laboratory strains

Granzyme B is a 247 amino acid pro-apoptotic protease secreted by effector lymphocytes for the purpose of killing virus-infected cells. While the capacity of granzyme B to potently induce caspase-dependent apoptosis has long been recognized, it has only recently been found that human and mouse granzyme B activate overlapping but distinct apoptotic pathways. To investigate a possible evolutionary basis for this observation, we sequenced the exons and flanking intronic sequences of the mouse Gzmb gene from a variety of inbred laboratory strains and wild mice. The sequences of 12/13 inbred strains encoded identical proteins, the exception being DBA/2, whose sequence varied at two amino acids. By contrast with the laboratory strains, there was extensive polymorphism in the Gzmb gene of 54 wild mice and 28 wild-derived inbred mice examined, resulting in 2-18 amino acid differences in the predicted proteins, a discrepancy rate of up to 7.3%. Many of these amino acid variations were found in rat and/or human granzyme B. The granzyme B allotype of inbred laboratory strains could be identified in only one of three geographically dispersed clans of wild mice and was absent from all 28 wild-derived inbred strains. The Gzmb gene of Mus musculus castaneus, a close relative of laboratory mice, encoded six amino acid differences compared with the laboratory strains, all of which were also found in corresponding positions in the granzyme B molecules of wild mice. Unlike the protease, the extended granzyme B recognition and cleavage site in Bid, a key pro-apoptotic substrate, was invariant.