Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Single-cell multiomics reveals persistence of HIV-1 in expanded cytotoxic T cell clones.

Understanding the drivers and markers of clonally expanding HIV-1-infected CD4+ T cells is essential for HIV-1 eradication. We used single-cell ECCITE-seq, which captures surface protein expression, cellular transcriptome, HIV-1 RNA, and TCR sequences within the same single cell to track clonal expansion dynamics in longitudinally archived samples from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. Despite antiretroviral therapy, persistent antigen and TNF responses shaped T cell clonal expansion. HIV-1 resided in Th1-polarized, antigen-responding T cells expressing BCL2 and SERPINB9 that may resist cell death. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides another direction for HIV-1 eradication.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

Granzyme B-activated IL18 potentiates αß and γδ CAR T cell immunotherapy in a tumor-dependent manner.

Interleukin (IL)18 is a potent pro-inflammatory cytokine that is activated upon caspase 1 cleavage of the latent precursor, pro-IL18. Therapeutic T cell armoring with IL18 promotes autocrine stimulation and positive modulation of the tumor microenvironment (TME). However, existing strategies are imperfect since they involve constitutive/poorly regulated activity or fail to modify the TME. Here, we have substituted the caspase 1 cleavage site within pro-IL18 with that preferred by granzyme B, yielding GzB-IL18. We demonstrate that GzB-IL18 is constitutively released but remains functionally latent unless chimeric antigen receptor (CAR) T cells are activated, owing to concomitant granzyme B release. Armoring with GzB-IL18 enhances cytolytic activity, proliferation, interferon (IFN)-γ release, and anti-tumor efficacy by a similar magnitude to constitutively active IL18. We also demonstrate that GzB-IL18 provides a highly effective armoring strategy for γδ CAR T cells, leading to enhanced metabolic fitness and significant potentiation of therapeutic activity. Finally, we show that constitutively active IL18 can unmask CAR T cell-mediated cytokine release syndrome in immunocompetent mice. By contrast, GzB-IL18 promotes anti-tumor activity and myeloid cell re-programming without inducing such toxicity. Using this stringent system, we have tightly coupled the biological activity of IL18 to the activation state of the host CAR T cell, favoring safer clinical implementation of this technology.

Modeling antibody drug conjugate potential using a granzyme B antibody fusion protein.

BACKGROUND: Antibody drug conjugates (ADCs) constitute a promising class of targeted anti-tumor therapeutics that harness the selectivity of monoclonal antibodies with the potency of cytotoxic drugs. ADC development is best suited to initially screening antibody candidates for desired properties that potentiate target cell cytotoxicity. However, validating and producing an optimally designed ADC requires expertise and resources not readily available to certain laboratories. RESULTS: In this study, we propose a novel approach to help streamline the identification of potential ADC candidates by utilizing a granzyme B (GrB)-based antibody fusion protein (AFP) for preliminary screening. GrB is a non-immunogenic serine protease expressed by immune effector cells such as CD8 + T cells that induces apoptotic activity and can be leveraged for targeted cell killing. CONCLUSIONS: Our innovative model allows critical antibody parameters (including target cell binding, internalization, and cytotoxic potential) to be more reliably evaluated in vitro through the creation of an ADC surrogate. Successful incorporation of this AFP could also significantly expand and enhance ADC development pre-clinically, ultimately leading to the accelerated translation of ADC therapies for patients.

Raddeanin A augments the cytotoxicity of natural killer cells against chronic myeloid leukaemia cells by modulating MAPK and Ras/Raf signalling pathways.

Natural killer (NK) cell therapy, a developing approach in cancer immunotherapy, involves isolating NK cells from peripheral blood. However, due to their limited number and activity, it is essential to significantly expand these primary NK cells and enhance their cytotoxicity. In this study, we investigated how Raddeanin A potentiate NK activity using KHYG-1 cells. The results indicated that Raddeanin A increased the expression levels of cytolytic molecules such as perforin, granzymes A and granzymes B, granulysin and FasL in KHYG-1 cells. Raddeanin A treatment increased CREB phosphorylation, p65 phosphorylation, NFAT1 and acetyl-histone H3 expression. Raddeanin A elevated caspase 3 and PARP cleavage, increased t-Bid expression, promoting apoptosis in K562 cells. Furthermore, it reduced the expression of HMGB2, SET and Ape1, impairing the DNA repair process and causing K562 cells to die caspase-independently. Additionally, Raddeanin A increased ERK, p38 and JNK phosphorylation at the molecular level, which increased granzyme B production in KHYG-1 cells. Raddeanin A treatment increased Ras, Raf phosphorylation, MEK phosphorylation, NKG2D, NKp44 and NKp30 expression in KHYG-1 cells. Collectively, our data indicate that Raddeanin A enhances the cytotoxic activity of NK cells against different cancer cells.

Granzyme B degrades extracellular matrix and promotes inflammation and choroidal neovascularization.

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.

Extracellular CIRP induces CD4CD8αα intraepithelial lymphocyte cytotoxicity in sepsis.

BACKGROUND: In sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4+ T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis. METHODS: We subjected wild-type (WT) and CIRP-/- mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4-/- mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry. RESULTS: We found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP-/- mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4-/- mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP-/- mice were protected from tissue injury and cell death in the small intestines compared to WT mice. CONCLUSION: In sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.

Granzyme B+ B cells detected by single-cell sequencing are associated with prognosis in patients with intrahepatic cholangiocarcinoma following liver transplantation.

B cells possess anti-tumor functions mediated by granzyme B, in addition to their role in antigen presentation and antibody production. However, the variations in granzyme B+ B cells between tumor and non-tumor tissues have been largely unexplored. Therefore, we integrated 25 samples from the Gene Expression Omnibus database and analyzed the tumor immune microenvironment. The findings uncovered significant inter- and intra-tumoral heterogeneity. Notably, single-cell data showed higher proportions of granzyme B+ B cells in tumor samples compared to control samples, and these levels were positively associated with disease-free survival. The elevated levels of granzyme B+ B cells in tumor samples resulted from tumor cell chemotaxis through the MIF- (CD74 + CXCR4) signaling pathway. Furthermore, the anti-tumor function of granzyme B+ B cells in tumor samples was adversely affected, potentially providing an explanation for tumor progression. These findings regarding granzyme B+ B cells were further validated in an independent clinic cohort of 40 liver transplant recipients with intrahepatic cholangiocarcinoma. Our study unveils an interaction between granzyme B+ B cells and intrahepatic cholangiocarcinoma, opening up potential avenues for the development of novel therapeutic strategies against this disease.

Granzyme B inhibition reduces autoantibody-induced dermal-epidermal separation in an ex vivo model of epidermolysis bullosa acquisita.

The pemphigoid disease epidermolysis bullosa acquisita (EBA) is an autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7), immune cell infiltrates at the dermal-epidermal junction and subepidermal blistering. Proteases, particularly granzyme B (GzmB), have been established as therapeutic targets for the treatment of EBA and other pemphigoid diseases. We investigated the impact of the novel GzmB inhibitor SNT-6935 on anti-COL7 IgG-induced subepidermal blistering in a well-established EBA ex vivo model. Our findings demonstrate that pharmacological targeting of GzmB with its selective inhibitor SNT-6935 significantly reduced autoantibody-induced dermal-epidermal separation in human skin cryosections. Interestingly, treatment of skin cryosections with recombinant human GzmB alone did not cause dermal-epidermal separation, suggesting that additional mechanisms alongside GzmB are required for tissue damage in EBA. In conclusion, our study highlights the significant contribution of GzmB to the pathogenesis of EBA and supports the notion of GzmB as a therapeutic target in EBA and other pemphigoid diseases.

Granzyme B PET imaging inflammation and remodeling in myocardial infarction.

PURPOSE: This study aimed to evaluate whether granzyme B (GzmB)-targeted positron emission tomography (PET) imaging agent (68 Ga-grazytracer) can characterize cardiac inflammation and remodeling in myocardial infarction (MI). METHODS: Rats with MI were subjected to GzmB-targeted PET/CT on post-operative days 1, 3, 6, 14, and 28. Autoradiography, Masson staining, immunohistochemistry, and ELISA were performed to verify the inflammatory response and remodeling after MI in vitro. Rats were treated with GzmB inhibitor Z-IETD-FMK to improve cardiac remodeling. Cardiac function tests were performed by echocardiography at 6 weeks after MI. RESULTS: The highest uptake of 68 Ga-grazytracer was observed on day 3 after MI compared with the values obtained on the other days (0.294 ± 0.03% ID/g at 3 days vs. 0.122 ± 0.01% ID/g in the sham group, P < 0.001). Immunohistochemistry showed significantly high expression of GzmB and CD8, in line with the PET/CT imaging results. Autoradiography revealed 68 Ga-grazytracer accumulation in the infarcted myocardium. The 68 Ga-grazytracer uptake of treated rats was significantly reduced compared with that in the MI groups (0.184 ± 0.03%ID/g vs. 0.286 ± 0.03%ID/g; P < 0.001). Echocardiography showed that the left ventricular ejection fraction was lower in the MI groups than in the ischemia reperfusion group. GzmB inhibitor treatment was shown to be effective in improving cardiac function without significantly shortening infarct size. CONCLUSIONS: This study demonstrated the potential of 68 Ga-grazytracer imaging to delineate adverse inflammatory responses and pathological cardiac remodeling, which can help predict heart function. PET/CT imaging-guided therapy may reduce myocardial injury and improve heart function in MI.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

Granzyme B secreted by T cells is involved in anti-bacterial immune response of tilapia.

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB + T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Keratinocytes stimulate MAIT cells to produce granzyme B via MR1 and cytokines in oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease characterized by a dense T-cell infiltration and the degeneration of basal keratinocytes. The potential functions of mucosal associated invariant T (MAIT) cells in OLP have been analyzed in our previous study. Keratinocytes under proinflammatory conditions have been demonstrated to activate T cells. This study was aimed to investigate how keratinocytes stimulate MAIT cells in OLP, and to explore the role of activated MAIT cells on keratinocytes. METHODS AND RESULTS: Increased MAIT cells and higher activation marker CD69 were detected in OLP lesions by flow cytometry. The enhanced expression of MHC class I-like molecule (MR1) required for MAIT cell activation in the epithelial layer of OLP lesions was determined by immunohistochemistry. Keratinocytes treated by 5-A-RU prodrug and lipopolysaccharide, respectively, exhibited higher expression of MR1 and secretion of IL-18. In direct coculture systems consisting of keratinocytes and peripheral blood mononuclear cells, both 5-A-RU prodrug-pretreated keratinocytes and lipopolysaccharide-pretreated keratinocytes activated MAIT cells to secrete granzyme B, contributing to elevated keratinocyte apoptosis. CONCLUSIONS: Keratinocytes were capable to activate MAIT cells via MR1 and cytokines in OLP, and granzyme B produced by activated MAIT cells intensified keratinocyte apoptosis, engaging in the pathogenesis of OLP.

Granzyme B Promotes Proliferation, Migration and EMT Process in Gastric Cancer.

Granzyme B (GZMB), a critical member of the Gr gene family, is known to play an essential role in diverse physiological and pathological processes such as inflammation, acute and chronic inflammatory diseases, and cancer progression. In this study, we delve deeper into the role of GZMB within the context of gastric cancer (GC) to examine its expression patterns and functional implications. To accomplish this, we applied a combination of quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry techniques. These methodologies allowed us to accurately gauge GZMB expression levels in GC tissues and investigate their correlation with various clinical-pathological variables. Our secondary focus was to discern the regulatory influence of GZMB on GC cell biology. We used an array of assays including cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine, and migration assays. The effect of GZMB on gastric cancer progression was further validated through a subcutaneous xenograft mouse model. Our findings underscored that GZMB mRNA and protein levels were upregulated in GC tissues, a feature that showed a significant correlation with GC staging. We also discovered that a decrease in GZMB expression via knockdown experiments suppressed the proliferation and migration capabilities of GC cells. This effect was manifested through diminished expression levels of epithelial-mesenchymal transition (EMT) markers. In stark contrast, the overexpression of GZMB through plasmid transfection appeared to enhance the proliferation and migration abilities of GC cells. This was coupled with an upregulation in EMT expression. Our study concludes by emphasizing that GZMB promotes the growth, migration, and EMT processes in gastric cancer. In vitro, cell-based experiments and in vivo xenograft mouse models confirm this. Our findings provide a more comprehensive understanding of GZMB's role in gastric cancer pathogenesis, potentially opening doors for novel therapeutic strategies targeting this molecular pathway.

Granzyme B May Act as an Effector Molecule to Control the Inflammatory Process in COPD.

Chronic obstructive pulmonary disease (COPD) is caused by smoking, but only a small proportion of smokers have disease severe enough to develop COPD. COPD is not always progressive. The question then arises as to what explains the different trajectories of COPD. The role of autoimmunity and regulatory T (Treg) cells in the pathogenesis of COPD is increasingly being recognized. Nine published studies on Treg cells in the lung tissue or bronchoalveolar lavage fluid have shown that smokers with COPD have fewer Treg cells than smokers without COPD or nonsmokers. Three studies showed a positive correlation between Treg cell count and FEV1%, suggesting an important role for Treg cells in COPD progression. Treg cells can regulate immunological responses via the granzyme B (GzmB) pathway. Immunohistochemical staining for GzmB in surgically resected lungs with centrilobular emphysema showed that the relationship between the amount of GzmB+ cells and FEV1% was comparable to that between Treg cell count and FEV1% in the COPD lung, suggesting that GzmB could be a functional marker for Treg cells. The volume fraction of GzmB+ cells in the small airways, the number of alveolar GzmB+ cells, and GzmB expression measured by enzyme-linked immunosorbent assay in the lung tissue of smokers were significantly correlated with FEV1%. These results suggest that the GzmB content in lung tissue may determine the progression of COPD by acting as an effector molecule to control inflammatory process. Interventions to augment GzmB-producing immunosuppressive cells in the early stages of COPD could help prevent or delay COPD progression.

Monensin induces secretory granule-mediated cell death in eosinophils.

BACKGROUND: Eosinophils contribute to the pathology of several types of disorders, in particular of allergic nature, and strategies to limit their actions are therefore warranted. OBJECTIVE: We sought to evaluate the possibility of targeting the acidic, lysosome-like eosinophil granules as a potential means of inducing eosinophil cell death. METHODS: To this end, we used monensin, an ionophoric drug that has previously been shown to permeabilize the secretory granules of mast cells, thereby inducing cell death. RESULTS: Our findings reveal that monensin induces cell death in human eosinophils, whereas neutrophils were less affected. Blockade of granule acidification reduced the effect of monensin on the eosinophils, demonstrating that granule acidity is an important factor in the mechanism of cell death. Furthermore, monensin caused an elevation of the granule pH, which was accompanied by a decrease of the cytosolic pH, hence indicating that monensin caused leakage of acidic contents from the granules into the cytosol. In agreement with a granule-targeting mechanism, transmission electron microscopy analysis revealed that monensin caused extensive morphological alterations of the eosinophil granules, as manifested by a marked loss of electron density. Eosinophil cell death in response to monensin was caspase-independent, but dependent on granzyme B, a pro-apoptotic serine protease known to be expressed by eosinophils. CONCLUSIONS: We conclude that monensin causes cell death of human eosinophils through a granule-mediated mechanism dependent on granzyme B.

Granzyme B Expression in Conjunctiva of Patients with Pterygium.

Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.