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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. [ABSTRACT FROM AUTHOR]

Murine cytokine-induced killer (CIK) cells are heterologous cells that kill various allogeneic and isogenic tumors and have functional and phenotypic characteristics of natural killer cells and T lymphocytes. However, the effect of CIK cells alone on solid tumor therapy is only limited. To enhance the therapeutic effect, it is vital to discover a mix of several therapy approaches. Immune cell function is inhibited by abnormal tumor vessels and the tumor microenvironment, which block lymphocyte entry into tumor tissue. To increase the effectiveness of CIK cells' antitumor activity, antivascular therapy and CIK cell therapy can be combined. Furthermore, anlotinib is a tiny drug with multitarget tyrosine kinase inhibitors that can block cell migration, delay angiogenesis, and decrease blood vessel density. Compared with other antiangiogenesis drugs, anlotinib stands out due to the wider target of action and lower effective dose. In this work, anlotinib and murine CIK cells were coupled to boost CD3+ T cell infiltration, CD3+CD4+ T cell infiltration, and expression of granzyme B and interferon γ from CD3+CD8+ T cells, which increased the antitumor activity. Through the generation of cytotoxic cytokines by T lymphocytes, the therapeutic group using anti-PD-1 monoclonal antibodies in conjunction with anlotinib and CIK cells was more successful than the group receiving dual therapy. The preclinical study contributes to exploring the therapeutic alternatives for patients with lung adenocarcinoma, thus prolonging their lives. [ABSTRACT FROM AUTHOR]

Objective To investigate the feasibility of granzyme B (GB) promoter-controlled ferritin heavy chain (FTH1) reporter gene expression for monitoring the activation status of chimeric antigen receptor T cells (CAR-T) by magnetic resonance imaging (MRI). Methods Cytotoxic T lymphocytes (CTLs) were screened by Ficoll density gradient centrifugation and flow sorting. The GB promoter and FTH1 gene were ligated together with disialoganglioside 2 (GD2) CAR, and lentiviral vectors were transfected into CTLs to construct GD2-CAR-T/pGB-FTH1 cells. GD2-CAR-T/pCMV-FTH1, GD2-CAR-T, and T cells served as control cells. CytoTox96@non-radioactive cytotoxicity was used to detect the killing effect of each group of cells after co-culture with human neuroblastoma cells (SK-N-SH). ELISA was employed to detect the coincubation factor as well as the amount of GB secretion. Western blotting, Prussian blue staining and cellular MRI were applied to detect the expression of the FTH1 gene after co-culture. Results CTLs were successfully obtained, and then GD2-CAR-T/pGB-FTH1, GD2-CAR-T/pCMV-FTH1 and GD2-CAR-T cells were constructed. The killing effect, co-incubation factor and GB secretion of the above 3 groups of cells were significantly higher than those of the T cells, and the level of GB expression was highest at day 1, and then decreased in order at day 3 and day 7 after co-culturing with SK-N-SH cells. The relative expression of FTH1 and iron content of the GD2-CAR-T/pGB-FTH1 cells showed the same trend as GB expression, and the MRI signals were gradually increased. There were no significant differences in the relative expression of FTH1, iron content and MRI signals in the GD2-CAR-T/pCMV-FTH1 cells at all time points. No FTH1 expression or iron aggregation was observed in the GD2-CAR-T and T cells groups. Conclusion MRI based on the FTH1 reporter gene driven by the granzyme B promoter can reflect the GB expression level and tumor killing effect of CAR-T cells, which provides a potential real-time visual means to monitor the cell activation status for CAR-T therapy.

AbstractChronic 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.

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. [ABSTRACT FROM AUTHOR]

Heterogenous immune responses to checkpoint blockade therapy remain a major challenge to early prediction of treatment efficacy. Current methods of evaluating treatment efficacy in tumors are ineffective in accurately monitoring the disease status after immunotherapy early on. This study reports an immune response monitoring strategy that longitudinally reports on two crucial protease activities involved in T cell‐mediated target cell death, granzyme B (GrzB) and downstream caspase 3 (Casp3), during and after immune checkpoint antibody treatment. Specifically, activatable nanoreporters are engineered to sensitively and selectively capture the activity of GrzB and Casp3 enzymes in tumor cells triggered by cytotoxic T cells in real‐time, providing a direct readout of the tumor response to immune checkpoint inhibitors (ICIs). Furthermore, incorporating 3D tumor‐derived organoids and ex vivo cultures on microfluidic devices with nanoreporters enables high‐throughput screening of various ICIs and their combinations in immunocompetent tumor models. These findings suggest that efficient monitoring of dynamic immunological processes in the tumor microenvironment could help uncover mechanisms associated with ICI response or resistance. It is further anticipated that combining the dual‐sensing nanoreporter with tumor‐derived organoid‐on‐chip technology could lead to an early prediction of treatment outcomes with high fidelity and accelerate the development of optimal treatment in immuno‐oncology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD), a prevalent and progressive degenerative disease of the macula, is the leading cause of blindness in elderly individuals in developed countries. The advanced stages include neovascular AMD (nAMD), characterized by choroidal neovascularization (CNV), leading to subretinal fibrosis and permanent vision loss. Despite the efficacy of anti-vascular endothelial growth factor (VEGF) therapy in stabilizing or improving vision in nAMD, the development of subretinal fibrosis following CNV remains a significant concern. In this review, we explore multifaceted aspects of subretinal fibrosis in nAMD, focusing on its clinical manifestations, risk factors, and underlying pathophysiology. We also outline the potential sources of myofibroblast precursors and inflammatory mechanisms underlying their recruitment and transdifferentiation. Special attention is given to the potential role of mast cells in CNV and subretinal fibrosis, with a focus on putative mast cell mediators, tryptase and granzyme B. We summarize our findings on the role of GzmB in CNV and speculate how GzmB may be involved in the pathological transition from CNV to subretinal fibrosis in nAMD. Finally, we discuss the advantages and drawbacks of animal models of subretinal fibrosis and pinpoint potential therapeutic targets for subretinal fibrosis. [ABSTRACT FROM AUTHOR]

Lymphoma is the most common tumour of domestic cats, developing most frequently in the small intestine. Feline small intestinal lymphoma predominantly demonstrates a T-cell immunophenotype identified by standard immunopositivity for T cells with CD3 or immunopositivity for B cells with CD20. In contrast, a wide spectrum of immunohistochemical antibodies are applied in humans to diagnose the various specific lymphoma subtypes according to the WHO classification. Our aim was to augment our knowledge of immunophenotypes in feline non-B-cell lymphomas forming macroscopic masses in the intestinal tract. We evaluated the combined immunohistochemistry and flow cytometry findings from 15 cases. Neoplastic lymphoid cells were immunopositive for CD3 in 93% (14/15), granzyme B in 87% (13/15), CD5 in 20% (3/15), CD8 in 13% (2/15), CD4 in 7% (1/15) and CD56 in 7% (1/15) of cases. Cytotoxic granules indicating a cytotoxic origin of the neoplastic cells were identified by histopathology only in 13% (2/15) and by cytology in 47% (7/15) of the cases. Without immunohistochemical labelling of the cytotoxic protein granzyme B, the cytotoxic status would have been missed in 46% (6/13) of the cytological and in 85% (11/13) of the histopathological slides. These findings suggest that more complex immunophenotyping may advance our understanding and help prognosticate small intestinal T-cell lymphoma in cats. [ABSTRACT FROM AUTHOR]

Recent breakthroughs in discovering novel immune signaling pathways have revolutionized different disease treatments. SERPINB9 (Sb9), also known as Proteinase Inhibitor 9 (PI-9), is a well-known endogenous inhibitor of Granzyme B (GzmB). GzmB is a potent cytotoxic molecule secreted by cytotoxic T lymphocytes and natural killer cells, which plays a crucial role in inducing apoptosis in target cells during immune responses. Sb9 acts as a protective mechanism against the potentially harmful effects of GzmB within the cells of the immune system itself. On the other hand, overexpression of Sb9 is an important mechanism of immune evasion in diseases like cancers and viral infections. The intricate functions of Sb9 in different cell types represent a finetuned regulatory mechanism for preventing immunopathology, protection against autoimmune diseases, and the regulation of cell death, all of which are essential for maintaining health and responding effectively to disease challenges. Dysregulation of the Sb9 will disrupt human normal physiological condition, potentially leading to a range of diseases, including cancers, inflammatory conditions, viral infections or other pathological disorders. Deepening our understanding of the role of Sb9 will aid in the discovery of innovative and effective treatments for various medical conditions. Therefore, the objective of this review is to consolidate current knowledge regarding the biological role of Sb9. It aims to offer insights into its discovery, structure, functions, distribution, its association with various diseases, and the potential of nanoparticle-based therapies targeting Sb9. [ABSTRACT FROM AUTHOR]

Objective Methods and Results Conclusions 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.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.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. [ABSTRACT FROM AUTHOR]

The pivotal role of Granzyme B (GzmB) in immune responses, initially tied to cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, has extended across diverse cell types and disease models. A number of studies have challenged conventional notions, revealing GzmB activity beyond apoptosis, impacting autoimmune diseases, inflammatory disorders, cancer, and neurotoxicity. Notably, the diverse functions of GzmB unfold through Perforin-dependent and Perforin-independent mechanisms, offering clinical implications and therapeutic insights. This review underscores the multifaceted roles of GzmB, spanning immunological and pathological contexts, which call for further investigations to pave the way for innovative targeted therapies. [ABSTRACT FROM AUTHOR]

Peripheral blood mononuclear cells contain secretory granules with Perforin and Granzyme B for defense against pathogens. The objective of the present study was to compare the effects of immunosuppressive induction therapies on Perforin and Granzyme B transcripts in kidney transplant recipients. Transcripts were determined in 408 incident kidney transplant recipients eight days posttransplant using quantitative real-time PCR. Compared to 90 healthy subjects, the median Perforin transcripts were lower in kidney transplant recipients with blood-group ABO-incompatible donors (N = 52), compatible living donors (N = 130), and deceased donors (N = 226) (25.7%; IQR, 6.5% to 46.0%; 31.5%; IQR, 10.9% to 57.7%; and 35.6%; IQR, 20.6% to 60.2%; respectively; p = 0.015 by the Kruskal–Wallis test). Kidney transplant recipients who were treated with thymoglobulin (N = 64) had significantly lower Perforin as well as Granzyme B compared to all other induction therapies (N = 344) (each p < 0.001). Receiver operator characteristics analysis showed that both Perforin (area under curve, 0.919) and Granzyme B (area under curve, 0.915) indicated thyroglobulin-containing induction therapies. Regression analysis showed that both reduction in plasma creatinine and human leukocyte antigen mismatches were positively associated with elevated Perforin/Granzyme B transcript ratio posttransplant. We conclude clinical parameters and therapies affect Perforin and Granzyme B transcripts posttransplant. [ABSTRACT FROM AUTHOR]

Abstract Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

Introduction: Anti-PD-1 immunotherapies enhance T-cell responses against tumor cells by blocking the interaction between PD-1 and its ligand, PD-L1. While these therapies offer significant benefits in treating various malignancies, they can also lead to several immune-related adverse events (irAEs), most notably manifesting in the skin. Lichenoid reactions, eczema, and vitiligo are the three most prevalent forms of cutaneous irAE. Case Presentation: Here, we report a rare case of a pityriasis lichenoides et varioliformis acuta (PLEVA) that developed during pembrolizumab treatment for invasive bladder cancer. A 53-year-old man, receiving pembrolizumab for invasive bladder cancer, developed erythematous papules on his legs after his 11th infusion. The skin lesions gradually spread to his entire trunk and extremities. A punch biopsy revealed several apoptotic keratinocytes and spongiosis, along with perivascular and lichenoid lymphocytic infiltration with vacuolar alteration. Immunohistochemistry showed infiltration of CD4+ and CD8+ T cells in both the epidermis and dermis. Granzyme B-positive inflammatory cells were also slightly present. From these results, he was diagnosed with PLEVA, which might be classified as a lichenoid eruption, especially based on the histological findings. Conclusion: We hypothesize that the anti-PD-1 antibody might lead to epidermal necrosis by amplifying the expression of cytolytic molecules such as granzyme B in CD8+ T cells.

Abstract 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.

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)–Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)–Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs. [ABSTRACT FROM AUTHOR]

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1β, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1β, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1β and GzmB, IL-17 participates in the pathology of CL. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD), a prevalent and progressive degenerative disease of the macula, is the leading cause of blindness in elderly individuals in developed countries. The advanced stages include neovascular AMD (nAMD), characterized by choroidal neovascularization (CNV), leading to subretinal fibrosis and permanent vision loss. Despite the efficacy of anti-vascular endothelial growth factor (VEGF) therapy in stabilizing or improving vision in nAMD, the development of subretinal fibrosis following CNV remains a significant concern. In this review, we explore multifaceted aspects of subretinal fibrosis in nAMD, focusing on its clinical manifestations, risk factors, and underlying pathophysiology. We also outline the potential sources of myofibroblast precursors and inflammatory mechanisms underlying their recruitment and transdifferentiation. Special attention is given to the potential role of mast cells in CNV and subretinal fibrosis, with a focus on putative mast cell mediators, tryptase and granzyme B. We summarize our findings on the role of GzmB in CNV and speculate how GzmB may be involved in the pathological transition from CNV to subretinal fibrosis in nAMD. Finally, we discuss the advantages and drawbacks of animal models of subretinal fibrosis and pinpoint potential therapeutic targets for subretinal fibrosis.

Abstract Background Cytotoxic lymphocytes (CLs) express potent toxins, including perforin (P) and granzyme-B (G), which brings about target cell death. The purpose of this study was to evaluate the killing capacity of tumor-infiltrating CLs by means of P and G analysis, and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto’s thyroiditis (HT). Methods Infiltration of lymphocytes in PTC was observed in frozen sections. Both fresh tumor tissues and paracancerous tissues with lymphocyte infiltration were collected and prepared into a single cell suspension. Flow cytometry was used to detect the percentages of CD3+P+, CD3+G+, CD8+P+, and CD8+G+ T lymphocytes (TLs) and CD16-CD56+P+ and CD16-CD56+G+ natural killer (NK) cells. Finally, we investigated differential expression of P and G in NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T, n = 44) and paracancerous tissues (group N, n = 44) from patients with PTC with the BRAF V600E mutation. Furthermore, patients were divided into two groups according to whether cervical central lymph node metastasis (CCLNM) existed: group A (with lymph node metastases, n = 27) and group B (with nonlymph node metastases, n = 17). Patients were also divided into three groups according to the total number of positive CCLNM: group B, group C (with low-level lymph node metastases, less than 5, n = 17) and group D (with high-level lymph node metastases, no less than 5, n = 10). Results The percentage of CD3+P+ CTLs was significantly higher in group N than in group T (P 0.05). The percentages of CD3+P+ CTLs in group A and group C were significantly higher in the paracancerous tissue than in the tumor tissue (P

Abstract 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.

Atherosclerosis is accompanied by damage to the vascular endothelium of arteries followed by development of inflammatory response and formation of atherosclerotic plaques. Innate immunity is an important component of this response being the earliest non-specific key mechanism. Our objective was to perform a comprehensive assessment of the cellular link of innate immunity, and to compare the results obtained at various terms after coronary stenting.The study involved 50 patients with coronary atherosclerosis (Group 1), who had clinical indications for stenting of coronary arteries, and 20 volunteers (Group 2), who have no signs of coronary artery disease. The study of immune parameters was carried out before surgery, at 4-5, 9-10 and 28-30 days after operation (during early postoperative period), as well as 6 and 12 months after stenting, i.e. over the late post-surgical period. Phenotyping of peripheral blood monocytes and lymphocytes was performed by flow cytometry using monoclonal antibodies (Beckman Coulter, USA). Intracellular content of Granzyme B was carried out with an FC500 flow laser cytofluorimeter. Metabolic activity of neutrophils was assessed by the NBT test. Alpha defensin was determined in blood plasma by ELISA technique (Hycult Biotech, USA). Statistical analysis was performed using the Statistica 12.0 program (StatSoft, USA). Statistical significance was considered significant at p ≤ 0.05.The numbers of natural killer cells and their activity, as well as those of monocytes, were increased in patients with coronary atherosclerosis. We have also shown a suppression of antigen presentation processes, an imbalance in microbicidal activity of neutrophils, with predominant secretion of antimicrobial peptides. Over the early post-surgical period, significant changes included only decreased content of intracellular Granzyme B on days 4-5, and expression of TLR4 and HLA-DR on days 4-5 and 9-10. During the late period, the patients with coronary artery disease exhibited a significant decrease in the content of some lymphocyte subsets: CD3+CD16+, CD16+Gr+ as well as amounts of monocytes: CD14+CD282+, CD14+CD284+, CD14+CD289+, along with HBT-test activity and α-defensin contents, and increased numbers of HLA-DR-expressing monocytes.There are changes in cellular component of innate immunity, indicating persistent inflammation in patients with coronary heart disease. The dynamics of revealed changes following coronary artery stenting may reflect a lability of assessed indicators mostly over the late postoperative period, thus serving a basis for predicting the outcome of coronary stenting.

The aim of the following review is to shed light on the putative role of regulatory B cells (Bregs) in various human diseases and highlight their potential prognostic and therapeutic relevance in humans. Regulatory B cells are a heterogeneous group of B lymphocytes capable of suppressing inflammatory immune reactions. In this way, Bregs contribute to the maintenance of tolerance and immune homeostasis by limiting ongoing immune reactions temporally and spatially. Bregs play an important role in attenuating pathological inflammatory reactions that can be associated with transplant rejection, graft-versus-host disease, autoimmune diseases and allergies but also with infectious, neoplastic and metabolic diseases. Early studies of Bregs identified IL-10 as an important functional molecule, so the IL-10-secreting murine B10 cell is still considered a prototype Breg, and IL-10 has long been central to the search for human Breg equivalents. However, over the past two decades, other molecules that may contribute to the immunosuppressive function of Bregs have been discovered, some of which are only present in human Bregs. This expanded arsenal includes several anti-inflammatory cytokines, such as IL-35 and TGF-β, but also enzymes such as CD39/CD73, granzyme B and IDO as well as cell surface proteins including PD-L1, CD1d and CD25. In summary, the present review illustrates in a concise and comprehensive manner that although human Bregs share common functional immunosuppressive features leading to a prominent role in various human immunpathologies, they are composed of a pool of different B cell types with rather heterogeneous phenotypic and transcriptional properties. [ABSTRACT FROM AUTHOR]

Background: Cytotoxic lymphocytes (CLs) express potent toxins, including perforin (P) and granzyme-B (G), which brings about target cell death. The purpose of this study was to evaluate the killing capacity of tumor-infiltrating CLs by means of P and G analysis, and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto's thyroiditis (HT). Methods: Infiltration of lymphocytes in PTC was observed in frozen sections. Both fresh tumor tissues and paracancerous tissues with lymphocyte infiltration were collected and prepared into a single cell suspension. Flow cytometry was used to detect the percentages of CD3+P+, CD3+G+, CD8+P+, and CD8+G+ T lymphocytes (TLs) and CD16-CD56+P+ and CD16-CD56+G+ natural killer (NK) cells. Finally, we investigated differential expression of P and G in NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T, n = 44) and paracancerous tissues (group N, n = 44) from patients with PTC with the BRAF V600E mutation. Furthermore, patients were divided into two groups according to whether cervical central lymph node metastasis (CCLNM) existed: group A (with lymph node metastases, n = 27) and group B (with nonlymph node metastases, n = 17). Patients were also divided into three groups according to the total number of positive CCLNM: group B, group C (with low-level lymph node metastases, less than 5, n = 17) and group D (with high-level lymph node metastases, no less than 5, n = 10). Results: The percentage of CD3+P+ CTLs was significantly higher in group N than in group T (P < 0.05). The percentage of CD8+G+ CTLs was significantly higher in group T than in group N (P < 0.05). The percentages of CD3+G+, CD16-CD56+P+and CD16-CD56+G+ NK cells showed no significant difference in either group T or group N (P > 0.05). The percentages of CD3+P+ CTLs in group A and group C were significantly higher in the paracancerous tissue than in the tumor tissue (P < 0.05). The percentages of CD8+G+ CTLs in group A and group C were significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). The percentage of CD16-CD56+G+ NK cells in group D was significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). Conclusions: The killing capacity of infiltrating CLs in PTC differed between tumor tissues and paracancerous tissues. In cases with CCLNM, higher expression of CD16-CD56+G+ NK cells in tumor tissues may be associated with a high risk of lymph node metastasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are innate lymphocytes possessing potent tumor surveillance and elimination activity. Increasing attention is being focused on the role of NK cells in integral antitumor strategies (especially immunotherapy). Of note, therapeutic efficacy is considerable dependent on two parameters: the infiltration and cytotoxicity of NK cells in tumor microenvironment (TME), both of which are impaired by several obstacles (e.g., chemokines, hypoxia). Strategies to overcome such barriers are needed. Radiotherapy is a conventional modality employed to cure solid tumors. Recent studies suggest that radiotherapy not only damages tumor cells directly, but also enhances tumor recognition by immune cells through altering molecular expression of tumor or immune cells via the in situ or abscopal effect. Thus, radiotherapy may rebuild a NK cellsfavored TME, and thus provide a cost-effective approach to improve the infiltration of NK cells into solid tumors, as well as elevate immune-activity. Moreover, the radioresistance of tumor always hampers the response to radiotherapy. Noteworthy, the puissant cytotoxic activity of NK cells not only kills tumor cells directly, but also increases the response of tumors to radiation via activating several radiosensitization pathways. Herein, we review the mechanisms by which NK cells and radiotherapy mutually promote their killing function against solid malignancies. We also discuss potential strategies harnessing such features in combined anticancer care. [ABSTRACT FROM AUTHOR]

Introduction: Anti-PD-1 immunotherapies enhance T-cell responses against tumor cells by blocking the interaction between PD-1 and its ligand, PD-L1. While these therapies offer significant benefits in treating various malignancies, they can also lead to several immune-related adverse events (irAEs), most notably manifesting in the skin. Lichenoid reactions, eczema, and vitiligo are the three most prevalent forms of cutaneous irAE. Case Presentation: Here, we report a rare case of a pityriasis lichenoides et varioliformis acuta (PLEVA) that developed during pembrolizumab treatment for invasive bladder cancer. A 53-year-old man, receiving pembrolizumab for invasive bladder cancer, developed erythematous papules on his legs after his 11th infusion. The skin lesions gradually spread to his entire trunk and extremities. A punch biopsy revealed several apoptotic keratinocytes and spongiosis, along with perivascular and lichenoid lymphocytic infiltration with vacuolar alteration. Immunohistochemistry showed infiltration of CD4+ and CD8+ T cells in both the epidermis and dermis. Granzyme B-positive inflammatory cells were also slightly present. From these results, he was diagnosed with PLEVA, which might be classified as a lichenoid eruption, especially based on the histological findings. Conclusion: We hypothesize that the anti-PD-1 antibody might lead to epidermal necrosis by amplifying the expression of cytolytic molecules such as granzyme B in CD8+ T cells. [ABSTRACT FROM AUTHOR]

Echinococcus granulosus (sensu lato), a cestode that is endemic in Egypt, causes cystic echinococcosis (CE), a significant but neglected zoonotic disease that is prevalent throughout the world. Infected hydatid cysts are classified as fertile or non-fertile based on the presence of protoscoleces; nevertheless, the mechanism of non-fertile CE cysts remains unknown. The study aimed to assess whether granzyme B (GrB) expression and CD4 + /CD8 + could be related to the induction of non-fertile CE cysts. A total of fifty-eight individuals diagnosed with visceral hydatid cysts were selected, and they were further divided according to cyst fertility into fertile and non-fertile. Immunohistochemistry for CD4, CD8, and GrB was done. According to the results, hydatid cysts are common in adults and have no gender preference. The same clinical and laboratory data were shared by patients with fertile and non-fertile cysts (p = 0.186). GrB expression was not impacted by the fibrous deposition inside the hydatid cyst wall (p = 0.85); however, GrB was significantly correlated with the inflammatory density (p = 0.005). GrB expression was also found to be significantly higher in non-fertile cysts (p = 0.04). GrB expression is positively correlated with CD4 and CD8 expression. In conclusion, the expression of GrB in hydatid cysts may exacerbate the inflammatory response and impede cyst fertility while not affecting the fibrous deposition in the cyst wall. [ABSTRACT FROM AUTHOR]

Our previous study indicated that cytotoxicity of intraepithelial lymphocytes is a poor prognostic factor in feline intestinal T-cell lymphoma (FITL), but the effect of cytotoxic lymphocytes on mucosal epithelium is still unknown. Thus, we investigated the association between cytotoxic lymphocytes and mucosal epithelium in 71 cases of feline intestinal T-cell lymphoma (FITL): epithelial injury, basement membrane injury, cleaved-caspase-3 positivity of epithelial cells, and the number and Ki67 positivity of intraepithelial lymphocytes in granzyme B (GRB)+ and GRB- FITLs were evaluated. Epithelial injury score and the number of intraepithelial lymphocytes in granzyme B (GRB)+ FITL were significantly higher than those of GRB- FITL (P<0.05, P<0.05), but no significant differences were found in the basement membrane injury score, the percentage of cleaved-caspase-3+ epithelial cells, and the percentage of Ki67+ intraepithelial lymphocytes. There was a significant correlation between the epithelial injury score and the number of intraepithelial lymphocytes (P<0.05), but no significant correlation was observed between the epithelial injury score and Ki67+ percentage of intraepithelial lymphocytes. Because epithelial cell cleaved-caspase-3 positivity was observed in FITL, regardless of GRB expression in lymphocytes, GRB-mediated apoptosis may not contribute to epithelial injury in FITL. The association between increased number of intraepithelial lymphocytes and epithelial injury suggests that intraepithelial lymphocytes infiltration may contribute to epithelial injury in FITL. [ABSTRACT FROM AUTHOR]

Significant progress has been made in tumor immunotherapy that uses the human immune response to kill and remove tumor cells. However, overreactive immune response could lead to various autoimmune diseases and acute rejection. Accurate and specific monitoring of immune responses in these processes could help select appropriate therapies and regimens for the patient and could reduce the risk of side effects. Granzyme B (GzmB) is an ideal biomarker for immune response, and its peptide substrate could be coupled with fluorescent dyes or contrast agents for the synthesis of imaging probes activated by GzmB. These small molecules and nanoprobes based on PET, bioluminescence imaging, or fluorescence imaging have proved to be highly GzmB specific and accuracy. This review summarizes the design of different GzmB‐responsive imaging probes and their applications in monitoring of tumor immunotherapy and overreactive immune response. This article is categorized under:Diagnostic Tools > In Vivo Nanodiagnostics and Imaging [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are gaining popularity in the field of cancer immunotherapy. The present study was designed to investigate the effect of a natural flavonol compound limocitrin in increasing cytotoxicity of a permanent NK leukemia cell line KHYG‐1 against an aggressive leukemia cell line K562. The findings revealed that limocitrin increased the expressions of cytolytic molecules perforin, granzymes A and B, and granulysin in KHYG‐1 cells by inducing phosphorylation of transcription factor CREB, leading to increased lysis of K562 cells. Mechanistically, limocitrin was found to increase the expressions of t‐Bid, cleaved caspase 3, and cleaved PARP to induce K562 cell apoptosis. Moreover, limocitrin reduced the expressions of SET and Ape1 to inhibit DNA repair mechanism, leading to caspase‐independent K562 cell death. At the molecular level, limocitrin was found to increase the phosphorylation of ERK, p38, and JNK to increase granzyme B expression in KHYG‐1 cells. Taken together, the study indicates that limocitrin increases cytotoxicity of NK cells against a range of cancer cells. [ABSTRACT FROM AUTHOR]

Chemotherapy remains one of the irreplaceable treatments for cancer therapy. The use of immunogenic cell death (ICD)-inducing chemotherapeutic drugs offers a practical strategy for killing cancer cells, simultaneously eliciting an antitumor immune response by promoting the recruitment of cytotoxic immune cells and production of granzyme B (GrB). However, numerous malignant cancers adaptively acquired the capacity of secreting serpinb9 (Sb9), a physiological inhibitor of GrB, which can reversibly inhibit the biological activity of GrB. To circumvent this dilemma, in this study, an integrated tailor-made nanomedicine composed of tumor-targeting peptide (Arg-Gly-Asp, RGD) decorated liposome, doxorubicin (DOX, an effective ICD inducer), and the compound 3034 (an inhibitor of Sb9), is developed (termed as D3RL) for breast cancer chemo-immunotherapy. In vitro and in vivo studies show that D3RL can directly kill tumor cells and trigger the host immune response by inducing ICD. Meanwhile, D3RL can competitively relieve the inhibition of Sb9 to GrB. The restored GrB can not only effectively induce tumor immunotherapy, but also degrade matrix components in the tumor microenvironment, consequently improving the infiltration of immune cells and the penetration of nanomedicines, which in return enhance the combined antitumor effect. Taken together, this work develops an integrated therapeutic solution for targeted production and restoration of GrB to achieve a combined chemoimmunotherapy for breast cancer. [ABSTRACT FROM AUTHOR]

The pivotal role of Granzyme B (GzmB) in immune responses, initially tied to cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, has extended across diverse cell types and disease models. A number of studies have challenged conventional notions, revealing GzmB activity beyond apoptosis, impacting autoimmune diseases, inflammatory disorders, cancer, and neurotoxicity. Notably, the diverse functions of GzmB unfold through Perforin-dependent and Perforin-independent mechanisms, offering clinical implications and therapeutic insights. This review underscores the multifaceted roles of GzmB, spanning immunological and pathological contexts, which call for further investigations to pave the way for innovative targeted therapies.

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.

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.

Peripheral blood mononuclear cells contain secretory granules with Perforin and Granzyme B for defense against pathogens. The objective of the present study was to compare the effects of immunosuppressive induction therapies on Perforin and Granzyme B transcripts in kidney transplant recipients. Transcripts were determined in 408 incident kidney transplant recipients eight days posttransplant using quantitative real-time PCR. Compared to 90 healthy subjects, the median Perforin transcripts were lower in kidney transplant recipients with blood-group ABO-incompatible donors (N = 52), compatible living donors (N = 130), and deceased donors (N = 226) (25.7%; IQR, 6.5% to 46.0%; 31.5%; IQR, 10.9% to 57.7%; and 35.6%; IQR, 20.6% to 60.2%; respectively; p = 0.015 by the Kruskal–Wallis test). Kidney transplant recipients who were treated with thymoglobulin (N = 64) had significantly lower Perforin as well as Granzyme B compared to all other induction therapies (N = 344) (each p < 0.001). Receiver operator characteristics analysis showed that both Perforin (area under curve, 0.919) and Granzyme B (area under curve, 0.915) indicated thyroglobulin-containing induction therapies. Regression analysis showed that both reduction in plasma creatinine and human leukocyte antigen mismatches were positively associated with elevated Perforin/Granzyme B transcript ratio posttransplant. We conclude clinical parameters and therapies affect Perforin and Granzyme B transcripts posttransplant.

Abstract Introduction Cytotoxic (CD8+) and natural killer (NK) cells play critical roles in anti-tumor immunity. Dysfunction in these cells is considered as one of the extrinsic mechanisms for tumor relapse. Aim We aimed in this study to assess cytotoxic activities of CD8 + T and NK cells in the peripheral blood from lung cancer patients before and after induction of chemotherapy. Subjects and methods Healthy (n = 5) volunteers and lung cancer patients (n = 15:5 before, 5 during, and 5 after induction of chemotherapy) were recruited. Flow cytometry was used to analyze the numbers of CD8 + T cells, NK and CD56+T cells and their intracellular expression of granzyme B (GzB) in fresh peripheral blood mononuclear cells (PBMCs) and after 72 h of their culture in vitro and stimulation with 5 µg/ml Concanavalin A (Con A) and 50ng/ml IL-2). In addition, the plasma levels of inflammatory cytokines were measured using luminex. Results After culture, significant increases in the number of GzB expressing cells gated on CD3+, CD4+, CD8 + and NKCD8 + T cells in the PBMCs from lung cancer patients before induction of chemotherapy as compared to control individuals as well as patients during and after induction of chemotherapy. Serum levels of IL-1 and CXCL8 in patients before induction of chemotherapy showed 37- and 40-fold increases, respectively, as compared to control individuals. Both GzB expression and cytokines levels in patients during and after chemotherapy were similar. Conclusion Polyclonal stimulation of PBMCs can restore the cytolytic activities of cytotoxic CD8 and NK cells from lung cancer patients even after chemotherapy.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

ABSTRACTTailored treatment for patients with rectal cancer requires clinically available markers to predict their response to neoadjuvant treatment. The quantity of tumor-infiltrating lymphocytes (TILs) in pre-operative tumor biopsies has been suggested to predict a favorable response, but opposing results exist. A biopsy-adapted Immunoscore (ISB) based on TILs has recently emerged as a promising predictor of tumor regression and prognosis in (colo)rectal cancer. We aimed to refine the ISB for prediction of response using multiplex immunofluorescence (mIF) on pre-operative rectal cancer biopsies. We combined the distribution and density of conventional T cell subsets and γδT cells with a type I Interferon (IFN)-driven response assessed using Myxovirus resistance protein A (MxA) expression. We found that pathological complete response (pCR) following neoadjuvant treatment was associated with type I IFN. Stratification of patients according to the density of CD8+ in the entire tumor tissue and MxA+ cells in tumor stroma, where equal weight was assigned to both parameters, resulted in improved predictive quality compared to the ISB. This novel stratification approach using these two independent parameters in pre-operative biopsies could potentially aid in identifying patients with a good chance of achieving a pCR following neoadjuvant treatment.

Objective To analyze the diagnostic value of the expression of Granzyme B and Perforin in peripheral blood T lymphocytes and NK cells in patients with breast cancer （BC）, to investigate the correlation between the expression of granzyme B and perforin and the clinicopathological characteristics of BC, including molecular typing and tumor stage, and the evaluation of the therapeutic effect on BC. Methods Flow cytometry was used to detect the expression of granzyme B and perforin in CD3+ T cells, CD8+ T cells and NK cells in 108 patients with BC and 70 benign breast disease controls, and to evaluate the diagnostic efficiency of the receiver operating characteristic （ROC） curve of each indicator in BC； to compare the expression of granzyme B and perforin in patients with different clinicopathological characteristics and the changes of various indicators before and after treatment. Results The expressions of granzyme B and perforin in T lymphocyte subsets and NK cells in BC patients were significantly higher than those of the control group （P < 0.05）. ROC analysis showed that the area under the curve （AUC） of granzyme B and perforin positive rates in CD3+ T cells, CD8+ T cells and NK cells was greater than 0.5. The expression of granzyme B and perforin in CD3+ T cells was positively correlated with the tumor size and clinical stage of BC （P < 0.05）, and granzyme B in CD8+ T cells was also positively correlated with the tumor size and clinical stage of BC （P < 0.05）. The expression of granzyme B and perforin in CD3+ T cells in BC patients with lymph node metastasis was significantly greater than that in BC patients without lymph node metastasis （P < 0.05）. The expression of granzyme B and perforin in peripheral blood T lymphocytes in BC patients after treatment was greater than that before treatment （P < 0.05）. Conclusion The expression of granzyme B and perforin in CD3+ T cells, CD8+ T cells and NK cells in peripheral blood has certain reference for the diagnosis of BC, and has correla⁃ tion with tumor size, lymph node metastasis and clinical stage, which can assist in the early screening, diagnosis and treatment evaluation of BC. [ABSTRACT FROM AUTHOR]

Background: Granzyme B (GZMB) is a serine protease produced by cytotoxic lymphocytes that reflects the activity of anti-tumor immune responses in tumor-infiltrating lymphocytes (TILs); however, the prognostic significance of GZMB+ TILs in lung adenocarcinoma is poorly understood. Methods: We analyzed 273 patients with pathological stage (pStage) I–IIIA lung adenocarcinoma who underwent surgery at Kyushu University from 2003 to 2012. We evaluated GZMB+ TIL counts by immunohistochemistry. We set the cut-off values at 12 cells/0.04 mm2 for GZMB+ TILs and divided the patients into GZMB-High (n = 171) and GZMB-Low (n = 102) groups. Then, we compared the clinicopathological characteristics of the two groups and clinical outcomes. Programmed cell death ligand-1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) expression in tumor cells was also evaluated, and combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 were performed. Results: GZMB-Low was significantly associated with pStage II–III, PD-L1 positivity, and IDO1 positivity. Disease-free survival (DFS) and overall survival (OS) in the GZMB-Low group were significantly worse than in the GZMB-High group. In multivariable analysis, GZMB-Low was an independent prognostic factor for both DFS and OS. Furthermore, combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 showed that GZMB-Low with high expression of these immunosuppressive proteins had the worst prognosis. Conclusions: We analyzed GZMB+ TIL counts in lung adenocarcinoma and elucidated its prognostic significance and association with PD-L1 and IDO1. GZMB+ TIL counts might reflect the patient's immunity against cancer cells and could be a useful prognostic marker of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Ischemic heart disease (IHD) is also known as coronary artery disease (CAD) or coronary heart disease (CHD). It remains a leading cause of morbidity and mortality worldwide. It occurs when there is not enough blood supply to the heart muscle, usually as a result of narrowing or blockage of the coronary arteries. IHD is a multifactorial condition with a complex pathophysiology, and various factors contribute to its development and progression. Among these factors, apoptosis, a form of programmed cell death, plays an important role. Granzyme b, a serine protease mainly found in cytotoxic T-lymphocytes and natural killer cells, has been implicated in cardiac cell apoptosis and the pathogenesis of IHD. This article examines the role of granzyme b as an apoptosis factor in the development of ischemic heart disease, focusing on its mechanisms, regulation, and potential therapeutic implications. [ABSTRACT FROM AUTHOR]