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.

Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition.

Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.

Investigating the effects of PTEN mutations on cGAS-STING pathway in glioblastoma tumours.

BACKGROUND: PTEN is a tumour suppressor gene and well-known for being frequently mutated in several cancer types. Loss of immunogenicity can also be attributed to PTEN loss, because of its role in establishing the tumour microenvironment. Therefore, this study aimed to represent the link between PTEN and cGAS-STING activity, a key mediator of inflammation, in tumour samples of glioblastoma patients. METHODS: Tumour samples of 36 glioblastoma patients were collected. After DNA isolation, all coding regions of PTEN were sequenced and analysed. PTEN expression status was also evaluated by qRT-PCR, western blot, and immunohistochemical methods. Interferon-stimulated gene expressions, cGAMP activity, CD8 infiltration, and Granzyme B expression levels were determined especially for the evaluation of cGAS-STING activity and immunogenicity. RESULTS: Mutant PTEN patients had significantly lower PTEN expression, both at mRNA and protein levels. Decreased STING, IRF3, NF-KB1, and RELA mRNA expressions were also found in patients with mutant PTEN. Immunohistochemistry staining of PTEN displayed expressional loss in 38.1% of the patients. Besides, patients with PTEN loss had considerably lower amounts of IFNB and IFIT2 mRNA expressions. Furthermore, CD8 infiltration, cGAMP, and Granzyme B levels were reduced in the PTEN loss group. CONCLUSION: This study reveals the immunosuppressive effects of PTEN loss in glioblastoma tumours via the cGAS-STING pathway. Therefore, determining the PTEN status in tumours is of great importance, like in situations when considering the treatment of glioblastoma patients with immunotherapeutic agents.

Single-cell transcriptomics reveals granzyme K-expressing cytotoxic Tfh cells in tertiary lymphoid structures in IgG4-RD.

BACKGROUND: Germinal center (GC) responses controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells are crucial for the generation of high-affinity antibodies. Acquired immune responses to tissue-released antigens might be mainly induced in tertiary lymphoid organs (TLOs) with GCs in affected tissues. IgG4-related disease (IgG4-RD) demonstrates polarized isotype switching and TLOs in affected tissues. We performed single-cell transcriptomics of tissue-infiltrating T cells from these TLOs to obtain a comprehensive, unbiased view of tissue-infiltrating GC-Tfh cells. OBJECTIVE: To identify GC-Tfh-cell subsets in TLOs in patients with IgG4-RD using single-cell transcriptomics. METHODS: Single-cell RNA sequencing of sorted CD3+ T cells and multicolor immunofluorescence analysis were used to investigate CD4+CXCR5+Bcl6+ GC-Tfh cells in affected lesions from patients with IgG4-RD. RESULTS: Infiltrating CD4+CXCR5+Bcl6+ Tfh cells were divided into 5 main clusters. We detected HLA+ granzyme K+ (GZMK+) Tfh cells with cytotoxicity-associated features in patients with IgG4-RD. We also observed abundant infiltrating Tfr cells with suppressor-associated features in patients with IgG4-RD. These GZMK+ Tfh cells and Tfr cells clustered together in affected tissues from patients with IgG4-RD. CONCLUSIONS: This single-cell data set revealed a novel subset of HLA+GZMK+ cytotoxic Tfh cells infiltrating affected organs in patients with IgG4-RD, suggesting that infiltrating Tfr cells might suppress cytotoxic Tfh cells.

Molecular and clinicopathological features of granzyme B-negative extranodal NK/T-cell lymphoma.

Extranodal NK/T-cell lymphoma (ENKTL) generally expresses cytotoxic molecules, including granzyme B (GZMB), T-cell-restricted intracellular antigen-1 (TIA-1), and perforin; however, the expression of these molecules varies across cases. We performed gene expression profiling and identified unique biological and clinicopathological features of GZMB-negative ENKTL. We reviewed the clinicopathological characteristics of 71 ENKTL samples. Gene expression profiling on nine ENKTLs using multiplexed, direct, and digital mRNA quantification divided ENKTLs into Groups A (n = 7) and B (n = 2) through hierarchical clustering and t-distributed stochastic neighbor embedding. Group B was characterized by downregulation of genes associated with IL6-JAK-STAT3 signaling and inflammatory responses. GZMB mRNA expression was significantly downregulated in Group B. GZMB protein expression was evaluated with immunohistochemistry in all 71 ENKTLs, and expression data of Tyr705-phosphorylated STAT3 (pSTAT3) and MYC from our previous study was utilized. T-cell receptor gamma (TRG) gene rearrangement in the selected samples was also assessed using PCR. GZMB expression was higher in pSTAT3-positive (p = 0.028) and MYC-positive (p = 0.014) ENKTLs. Eighteen percent (13/71) of all ENKTLs were negative for GZMB (defined by positivity <10 %); patients with GZMB-negative ENKTLs were often in a higher clinical stage (p = 0.016). We observed no other correlations with clinical parameters or TRG rearrangement and no significant association between GZMB expression and survival. In conclusion, GZMB expression is highly heterogeneous in ENKTLs and is associated with the activation of the JAK-STAT3 pathway and higher MYC expression. GZMB-negative ENKTLs correlate with an advanced clinical stage, suggesting the potential utility of GZMB immunohistochemistry as a biomarker of ENKTL.

Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

[Formononetin enhances the antitumor effect of H22 hepatoma transplanted mice].

Objective To explore the effect of formononetin on immunity of mice with transplanted H22 hepatocarcinoma. Methods Male C57BL/6 mice were subcutaneously inoculated with H22 cells (4×105) to establish a tumor-bearing mouse model. The mice were treated with formononetin [10 mg/(kg.d)] or [50 mg/(kg.d)] for 28 days, and then the tumor inhibition rate was calculated. Carrilizumab was used as a positive control drug. The expressions of CD8, granzyme B and forkbox transcription factor 3 (FOXP3) in HCC tissues were analyzed by immunohistochemical staining. The mRNA and protein expression of programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) in HCC tissues were detected by real-time PCR or Western blot analysis, respectively. The serum levels of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) were detected by ELISA. Results Formononetin increased the tumor inhibition rate and the positive rate of CD8 and granzyme B staining in tumor-bearing mice. There was no significant difference in the positive rate of FOXP3 staining in tumor tissues of mice in each group. Formononetin decreased the levels of IL-10 and TGF-ß in serum of tumor-bearing mice, and decreased the relative expression of mRNA and protein of PD-1 and PD-L1 in tumor tissue of tumor-bearing mice. Conclusion Formononetin can activate CD8+ T cells and reduce the release of immunosuppressive factors in regulatory T cells by blocking PD-1/PD-L1 pathway and play an antitumor role.

The USP7-STAT3-granzyme-Par-1 axis regulates allergic inflammation by promoting differentiation of IL-5-producing Th2 cells.

Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.

CAR-NK Cells Generated with mRNA-LNPs Kill Tumor Target Cells In Vitro and In Vivo.

Natural killer (NK) cells are cytotoxic lymphocytes that are critical for the innate immune system. Engineering NK cells with chimeric antigen receptors (CARs) allows CAR-NK cells to target tumor antigens more effectively. In this report, we present novel CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect NK cells expanded from primary PBMCs and to generate functional CAR-NK cells. CD19-CAR mRNA and BCMA-CAR mRNA were embedded into LNPs that resulted in 78% and 95% CAR expression in NK cells, respectively. BCMA-CAR-NK cells after transfection with CAR mRNA-LNPs killed multiple myeloma RPMI8226 and MM1S cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner in vitro. In addition, CD19-CAR-NK cells generated with CAR mRNA-LNPs killed Daudi and Nalm-6 cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner. Both BCMA-CAR-NK and CD19-CAR-NK cells showed significantly higher cytotoxicity, IFN-gamma, and Granzyme B secretion compared with normal NK cells. Moreover, CD19-CAR-NK cells significantly blocked Nalm-6 tumor growth in vivo. Thus, non-viral delivery of CAR mRNA-LNPs can be used to generate functional CAR-NK cells with high anti-tumor activity.

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.

Two granzyme A/K homologs in Zebra mbuna have different specificities, one classical tryptase and one with chymase activity.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna, granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species.

Heterogeneity of T cells in periapical lesions and in vitro validation of the proangiogenic effect of GZMA on HUVECs.

AIM: T cells are key immunomodulatory cells in periapical lesions. This study aimed to explore the roles of T cells in chronic apical periodontitis (CAP) using single-cell RNA sequencing and to further investigate Granzyme A (GZMA) in angiogenesis regulation. METHODOLOGY: A total of five CAP samples were collected for single-cell RNA sequencing. We performed subcluster and lineage-tracing analyses for T cells. According to differential gene expression, distinct biological functions enriched in T cells of CAP were presented by gene set enrichment analysis (GSEA) and compared with healthy gingiva (data obtained from the GEO database). CellChat was used to explore potential ligand-receptor interactions between T cells and endothelial cells in CAP. The coculture of primary human umbilical vein endothelial cells (HUVECs) and Jurkat T cells, as well as the addition of GZMA recombinant protein, was used to validate the predicted pair of GZMA and coagulation factor II thrombin receptor (F2R) by RT-PCR, angiogenesis and migration assays. RESULTS: A transcriptomic atlas of 44 746 individual cells was constructed from the periapical lesions of five patients with CAP by single-cell RNA-seq, and eight cell types were identified. We identified nine subsets of T cells and deciphered the cellular heterogeneity of T cells in CAP at the functional level by subclustering and GSEA. Lineage tracing revealed a distinct lineage of T cells in CAP and predicted the transition of the T cellular state upon CAP. GSEA revealed multiple biological processes and relevant angiogenesis genes upregulated in CAP T cells. GZMA-F2R pairs were predicted by cell-cell interactions in CAP. High expression of GZMA and F2R was observed in the coculture of HUVECs and Jurkat T cells, and the proangiogenic capacity of the GZMA recombinant protein was emphasized by in vitro experiments. CONCLUSIONS: Our study provides novel insights into the heterogeneity of T cells in periapical lesions and reveals the potential role of GZMA in T cells in regulating angiogenesis in HUVECs.

Granzyme-A deficiency attenuates experimental osteoarthritis in mice, but perforin deficiency does not.

OBJECTIVES: This study aims to assess the development of osteoarthritis (OA) in granzyme A- (gzmA) and B- (gzmB) and perforin- (perf) knockout mice. MATERIALS AND METHODS: A total of 75 male and female C57BL/6 (eight to nine-week-old) mice were allocated to: gzmA-deficient (gzmA-/-) (11 females, 8 males), gzmB-deficient (gzmB-/-) (9 females, 8 males), perf-deficient (perf-/-) (10 females, 9 males), and control group (10 females, 10 males). Osteoarthritis was induced in the right knee by instability of the meniscus medial ligament. Sham surgery was practiced in the left knee. Knee samples obtained eight weeks after surgery were stained (Safranin-O) and blindly scored in lateral and medial femur and tibia using the Osteoarthritis Research Society International scale (OARSI) (from Grade 0, cartilage intact to 6, deformation), (five stages from 0, no OA to 4, >50% surface involvement); OARSI score (Grade x Stage); and a semi-quantitative scale from Grade 0 (normal) to 6 (cartilage erosion >80%). RESULTS: Significantly higher values in all scales in the right knees compared to the left knees in male and female mice were observed (p<0.05). Males of all strains showed in the right knee higher values than females on all scales. Deficiency of perforin did not modify OA severity in any sex. The gzmA-/- females presented less degenerative changes than the other groups. CONCLUSION: Our study results show that sex plays an important role in the development of experimental OA in mice. Deficiency of gzmA can protect from the development of OA in female mice.

GSK-3 Inhibitor Elraglusib Enhances Tumor-Infiltrating Immune Cell Activation in Tumor Biopsies and Synergizes with Anti-PD-L1 in a Murine Model of Colorectal Cancer.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFß signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells.

INTRODUCTION: Granzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine. METHODS: Naïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker. RESULTS: By RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells. CONCLUSION: Our findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.

Inhibitory Effect of Zinc on Colorectal Cancer by Granzyme B Transcriptional Regulation in Cytotoxic T Cells.

Zinc is one of the essential trace elements and is involved in various functions in the body. Zinc deficiency is known to cause immune abnormalities, but the mechanism is not fully understood. Therefore, we focused our research on tumor immunity to elucidate the effect of zinc on colorectal cancer and its mechanisms. Mice were treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) to develop colorectal cancer, and the relationship between zinc content in the diet and the number and area of tumors in the colon was observed. The number of tumors in the colon was significantly higher in the no-zinc-added group than in the normal zinc intake group, and about half as many in the high-zinc-intake group as in the normal-zinc-intake group. In T-cell-deficient mice, the number of tumors in the high-zinc-intake group was similar to that in the normal-zinc-intake group, suggesting that the inhibitory effect of zinc was dependent on T cells. Furthermore, we found that the amount of granzyme B transcript released by cytotoxic T cells upon antigen stimulation was significantly increased by the addition of zinc. We also showed that granzyme B transcriptional activation by zinc addition was dependent on calcineurin activity. In this study, we have shown that zinc exerts its tumor-suppressive effect by acting on cytotoxic T cells, the center of cellular immunity, and increases the transcription of granzyme B, one of the key molecules in tumor immunity.

Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires.

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Single-cell RNA sequencing highlights the role of PVR/PVRL2 in the immunosuppressive tumour microenvironment in hepatocellular carcinoma.

Introduction: The conflict between cancer cells and the host immune system shapes the immune tumour microenvironment (TME) in hepatocellular carcinoma (HCC). A deep understanding of the heterogeneity and intercellular communication network in the TME of HCC will provide promising strategies to orchestrate the immune system to target and eradicate cancers. Methods: Here, we performed single-cell RNA sequencing (scRNA-seq) and computational analysis of 35786 unselected single cells from 3 human HCC tumour and 3 matched adjacent samples to elucidate the heterogeneity and intercellular communication network of the TME. The specific lysis of HCC cell lines was examined in vitro using cytotoxicity assays. Granzyme B concentration in supernatants of cytotoxicity assays was measured by ELISA. Results: We found that VCAN+ tumour-associated macrophages (TAMs) might undergo M2-like polarization and differentiate in the tumour region. Regulatory dendritic cells (DCs) exhibited immune regulatory and tolerogenic phenotypes in the TME. Furthermore, we observed intensive potential intercellular crosstalk among C1QC+ TAMs, regulatory DCs, regulator T (Treg) cells, and exhausted CD8+ T cells that fostered an immunosuppressive niche in the HCC TME. Moreover, we identified that the TIGIT-PVR/PVRL2 axis provides a prominent coinhibitory signal in the immunosuppressive TME. In vitro, antibody blockade of PVR or PVRL2 on HCC cell lines or TIGIT blockade on immune cells increased immune cell-mediated lysis of tumour cell. This enhanced immune response is paralleled by the increased secretion of Granzyme B by immune cells. Discussion: Collectively, our study revealed the functional state, clinical significance, and intercellular communication of immunosuppressive cells in HCC at single-cell resolution. Moreover, PVR/PVRL2, interact with TIGIT act as prominent coinhibitory signals and might represent a promising, efficacious immunotherapy strategy in HCC.

Modulation of granzymes mRNA expression in neural tissue of BoAHV-1 and BoAHV-5-infected cattle.

Bovine alphaherpesviruses (BoAHV)-1 and 5 are neurotropic viruses of cattle which differ in their neuropathogenic potential. BoAHV-5 is responsible for non-suppurative meningoencephalitis in calves while BoAHV-1 can occasionally cause encephalitis. Granzymes (GZMs) are serine-proteases that participate in CD8+ T cells-mediated killing of virally-infected cells upon release through perforin (PFN)-formed pores in the cell membrane. Recently, six GZMs have been identified in cattle (A, B, K, H, M and O). However, their expression in bovine tissues has not been evaluated. In this study, the mRNA expression levels of PFN and GZMs A, B, K, H and M in the nervous system of calves experimentally-inoculated with BoAHV-1 or BoAHV-5 were analyzed at the three distinct stages of the infectious cycle of alphaherpesviruses: acute infection, latency and reactivation. This is the first report describing the expression of GZMs in bovine neural tissue and the first analysis of GZM expression in the context of bovine alphaherpesviruses neuropathogenesis. The findings revealed that PFN and GZM K are upregulated during BoAHV-1 or BoAHV-5 acute infection. In contrast to BoAHV-1, during BoAHV-5 latency a significant up-regulation of PFN, GZM K and GZM H was detected. PFN and GZM A, K and H expression was also up-regulated during BoAHV-5 reactivation. Therefore, a distinct pattern of PFN and GZM expression is evident along the infectious cycle of each alphaherpesvirus and this might contribute to the differences in BoAHV-1 and BoAHV-5 neuropathogenesis.

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies.