Pyroptosis in health and disease: mechanisms, regulation and clinical perspective.

Pyroptosis is a type of programmed cell death characterized by cell swelling and osmotic lysis, resulting in cytomembrane rupture and release of immunostimulatory components, which play a role in several pathological processes. Significant cellular responses to various stimuli involve the formation of inflammasomes, maturation of inflammatory caspases, and caspase-mediated cleavage of gasdermin. The function of pyroptosis in disease is complex but not a simple angelic or demonic role. While inflammatory diseases such as sepsis are associated with uncontrollable pyroptosis, the potent immune response induced by pyroptosis can be exploited as a therapeutic target for anti-tumor therapy. Thus, a comprehensive review of the role of pyroptosis in disease is crucial for further research and clinical translation from bench to bedside. In this review, we summarize the recent advancements in understanding the role of pyroptosis in disease, covering the related development history, molecular mechanisms including canonical, non-canonical, caspase 3/8, and granzyme-mediated pathways, and its regulatory function in health and multiple diseases. Moreover, this review also provides updates on promising therapeutic strategies by applying novel small molecule inhibitors and traditional medicines to regulate pyroptosis. The present dilemmas and future directions in the landscape of pyroptosis are also discussed from a clinical perspective, providing clues for scientists to develop novel drugs targeting pyroptosis.

Proteolysis of tau by granzyme A in tauopathies generates fragments that are aggregation prone.

Tauopathies, including Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy, are characterised by the aggregation of tau into insoluble neurofibrillary tangles in the brain. Tau is subject to a range of post-translational modifications, including proteolysis, that can promote its aggregation. Neuroinflammation is a hallmark of tauopathies and evidence is growing for a role of CD8+ T cells in disease pathogenesis. CD8+ T cells release granzyme proteases but what role these proteases play in neuronal dysfunction is currently lacking. Here, we identified that granzyme A (GzmA) is present in brain tissue and proteolytically cleaves tau. Mass spectrometric analysis of tau fragments produced on digestion of tau with GzmA identified three cleavage sites at R194-S195, R209-S210 and K240-S241. Mutation of the critical Arg or Lys residues at the cleavage sites in tau or chemical inhibition of GzmA blocked the proteolysis of tau by GzmA. Development of a semi-targeted mass spectrometry approach identified peptides in tauopathy brain tissue corresponding to proteolysis by GzmA at R209-S210 and K240-S241 in tau. When expressed in cells the GzmA-cleaved C-terminal fragments of tau were highly phosphorylated and aggregated upon incubation of the cells with tauopathy brain seed. The C-terminal fragment tau195-441 was able to transfer between cells and promote aggregation of tau in acceptor cells, indicating the propensity for such tau fragments to propagate between cells. Collectively, these results raise the possibility that GzmA, released from infiltrating cytotoxic CD8+ T cells, proteolytically cleaves tau into fragments that may contribute to its pathological properties in tauopathies.

Causal association of immune effector proteins with sepsis: A Mendelian randomization study.

Sepsis is an infection-induced systemic inflammatory response syndrome. Immune regulation plays a crucial role in sepsis. We looked into the link between immune effector-related proteins and sepsis in this study by using both univariate and multivariate Mendelian randomization (MR) analyses. We accessed and collected data from the Integrative Epidemiology Unit's Open About Sepsis genome-wide association study database. The 6 immune effector-associated proteins each contained 10,534,735 single-nucleotide polymorphisms from 3301 samples. Using the weighted median, MR-Egger, simplex, inverse-variance weighting, and weighted mode methods, univariate MR then investigated the link between complement factor H-related protein-5 (CFHR5), Fc epsilon receptor II (FCER2), granzyme B (GZMB), major histocompatibility complex, class II, DQ alpha (HLA-DQA2), mannose-binding lectin 2 (MBL2), or myeloperoxidase (MPO) and sepsis. In the inverse-variance weighted results, the P values of all 6 immune effector-related proteins were <0.05, suggesting a possible causal relationship between them and sepsis. MBL2 (odds ratio [OR] = 1.046) was a risk factor for sepsis, while the other proteins (FCER2: OR = 0.922; GZMB: OR = 0.908; CFHR5: OR = 0.858; HLA-DQA2: OR = 0.896; MPO: OR = 0.875) were safety factors. By revealing a causal link between sepsis and CFHR5, FCER2, GZMB, HLA-DQA2, MBL2, or MPO, our study offers an essential resource for additional investigations on the subject.

Prdm1 positively regulates liver Group 1 ILCs cancer immune surveillance and preserves functional heterogeneity.

Group 1 innate lymphoid cells (ILCs) comprise conventional natural killer (cNK) cells and type 1 innate lymphoid cells (ILC1s). The main functions of liver cNK cells and ILC1s not only include directly killing target cells but also regulating local immune microenvironment of the liver through the secretion of cytokines. Uncovering the intricate mechanisms by which transcriptional factors regulate and influence the functions of liver cNK cells and ILC1s, particularly within the context of liver tumors, presents a significant opportunity to amplify the effectiveness of immunotherapies against liver malignancies. Using Ncr1-drived conditional knockout mouse model, our study reveals the regulatory role of Prdm1 in shaping the composition and maturation of cNK cells. Although Prdm1 did not affect the killing function of cNK cells in an in vivo cytotoxicity model, a significant increase in cancer metastasis was observed in Prdm1 knockout mice. Interferon-gamma (IFN-γ), granzyme B, and perforin secretion decreased significantly in Prdm1-deficient cNK cells and liver ILC1s. Single-cell RNA sequencing (scRNA-seq) data also provided evidences that Prdm1 maintains functional subsets of cNK cells and liver ILC1s and facilitates communications between cNK cells, liver ILC1s, and macrophages. The present study unveiled a novel regulatory mechanism of Prdm1 in cNK cells and liver ILC1s, showing promising potential for developing innovative immune therapy strategies against liver cancer.

Regulatory B Cells Expressing Granzyme B from Tolerant Renal Transplant Patients: Highly Differentiated B Cells with a Unique Pathway with a Specific Regulatory Profile and Strong Interactions with Immune System Cells.

The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB+ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB+ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB+ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB+ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB+ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB+ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB+/- natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB+ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB+ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.

Distinct granzyme k expression in immune cells: a single-cell rna-seq meta-analysis.

BACKGROUND: Granzymes are essential serine proteases in cytotoxic T cells and natural killer (NK) cells, with GZMK's expression being less understood. This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. OBJECTIVE: This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. METHODS: We conducted a meta-analysis using cellxgene, an interactive data exploration platform developed by the Chan Zuckerberg Initiative. We focused on mature T cells, NK cells, B cells, and NKT cells. We also checked transcription factor binding sites at the granzyme gene promoter regions using JASPAR. Comparative analysis was also done using mouse single-cell RNA sequencing data. RESULTS: GZMK was the most lowly expressed in NK cells and mature NKT cells in most tissues except for colon and lymph nodes. In mature T cells, GZMK is similarly or more highly expressed than other granzymes. HBCA data revealed weak expression of GZMK in NK cells but strong expression in effector memory CD8-positive, alpha-beta T cells. Combined data shows no significant difference in GZMK expression between cell types. Subtype analysis shows that GZMK expression was higher in CD16-negative, CD56-bright NK cells when compared to CD16-positive, CD56-dim NK cells. We also identified unique transcription factor binding sites for GZMK. While this pattern in mouse data with low Gzmk expression in NK cells and higher T cells was repeated. CONCLUSION: GZMK expression is distinctively regulated among immune cells and tissues, with unique promoter regions and transcription factor binding sites contributing to this differential expression. These insights into GZMK's role in immune function and regulation offer potential therapeutic targets.

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

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

Cystatin F Depletion in Mycobacterium tuberculosis-Infected Macrophages Improves Cathepsin C/Granzyme B-Driven Cytotoxic Effects on HIV-Infected Cells during Coinfection.

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.

Single-Cell Analysis Reveals That CD47 mRNA Expression Correlates with Immune Cell Activation, Antiviral Isgs, and Cytotoxicity.

BACKGROUND/AIMS: Immune cells are reported to upregulate CD47 during infection, however, the role of CD47 in innate and adaptive immune cells remains unclear. METHODS: To bridge this knowledge gap, we analysed our single cell (sc)-RNA dataset along with other publicly available sc-RNA datasets from healthy controls, people with HIV-1 (PWH) and COVID-19 patients. We characterized each immune cell based on low, intermediate, and high expression of CD47 . RESULTS: Our analyses revealed that CD47 high pDCs and monocytes exhibited relatively higher expression of IFN-α regulatory genes, antiviral interferon-stimulated genes (ISGs) and MHC-I associated genes compared to CD47 inter. and CD47 low cells. Furthermore, CD47 high NK and CD8+ T cells showed higher expression of antiviral ISGs, as well as genes encoding for cytotoxic markers like granzyme B, perforin, granulysin, interferon gamma and NKG7. Additionally, CD47 high CD8+ T cells expressed higher levels of PD-1 and LAG-3 genes. Lastly, we found that CD47 high B cells had enriched expression of genes involved in cell activation and humoral responses. CONCLUSION: Overall, our analyses revealed that innate and adaptive immune cells expressing elevated activation and functional gene signatures also express higher CD47 levels.

HLA-B*35:01-mediated activation of emodin-specific T cells contributes to Polygonum multiflorum thunb. -induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: HLA-B*35:01 has been identified as a risk allele for Polygonum multiflorum Thunb.-induced liver injury (PMLI). However, the immune mechanism underlying HLA-B*35:01-mediated PMLI remains unknown. AIM OF THE STUDY: To characterize the immune mechanism of HLA-B*35:01-mediated PMLI. MATERIALS AND METHODS: Components of P. multiflorum (PM) bound to the HLA-B*35:01 molecule was screened by immunoaffinity chromatography. Both wild-type mice and HLA-B*35:01 transgenic (TG) mice were treated with emodin. The levels of transaminases, histological changes and T-cell response were assessed. Splenocytes from emodin-treated mice were isolated and cultured in vitro. Phenotypes and functions of T cells were characterized upon drug restimulation using flow cytometry or ELISA. Emodin-pulsed antigen-presenting cells (APCs) or glutaraldehyde-fixed APCs were co-cultured with splenocytes from emodin-treated transgenic mice to detect their effect on T-cell activation. RESULTS: Emodin, the main component of PM, could non-covalently bind to the HLA-B*35:01-peptide complexes. TG mice were more sensitive to emodin-induced immune hepatic injury, as manifested by elevated aminotransferase levels, infiltration of inflammatory cells, increased percentage of CD8+T cells and release of effector molecules in the liver. However, these effects were not observed in wild-type mice. An increase in percentage of T cells and the levels of interferon-γ, granzyme B, and perforin was detected in emodin-restimulated splenocytes from TG mice. Anti-HLA-I antibodies inhibited the secretion of these effector molecules induced by emodin. Mechanistically, emodin-pulsed APCs failed to stimulate T cells, while fixed APCs in the presence of emodin could elicit the secretion of T cell effector molecules. CONCLUSION: The HLA-B*35:01-mediated CD8+ T cell reaction to emodin through the P-I mechanism may contribute to P. multiflorum-induced liver injury.

Active juvenile systemic lupus erythematosus is associated with distinct NK cell transcriptional and phenotypic alterations.

While adaptive immune responses have been studied extensively in SLE (systemic lupus erythematosus), there is limited and contradictory evidence regarding the contribution of natural killer (NK) cells to disease pathogenesis. There is even less evidence about the role of NK cells in the more severe phenotype with juvenile-onset (J)SLE. In this study, analysis of the phenotype and function of NK cells in a large cohort of JSLE patients demonstrated that total NK cells, as well as perforin and granzyme A expressing NK cell populations, were significantly diminished in JSLE patients compared to age- and sex-matched healthy controls. The reduction in NK cell frequency was associated with increased disease activity, and transcriptomic analysis of NK populations from active and low disease activity JSLE patients versus healthy controls confirmed that disease activity was the main driver of differential NK cell gene expression. Pathway analysis of differentially expressed genes revealed an upregulation of interferon-α responses and a downregulation of exocytosis in active disease compared to healthy controls. Further gene set enrichment analysis also demonstrated an overrepresentation of the apoptosis pathway in active disease. This points to increased propensity for apoptosis as a potential factor contributing to NK cell deficiency in JSLE.

Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Neutrophil-mediated hypoxia drives pathogenic CD8+ T cell responses in cutaneous leishmaniasis.

Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8+ T cell responses mediate disease. Although these responses originate in the lymph node, we found that expression of the cytolytic effector molecule granzyme B was restricted to lesional CD8+ T cells in Leishmania-infected mice, suggesting that local cues within inflamed skin induced cytolytic function. Expression of Blimp-1 (Prdm1), a transcription factor necessary for cytolytic CD8+ T cell differentiation, was driven by hypoxia within the inflamed skin. Hypoxia was further enhanced by the recruitment of neutrophils that consumed oxygen to produce ROS and ultimately increased the hypoxic state and granzyme B expression in CD8+ T cells. Importantly, lesions from patients with cutaneous leishmaniasis exhibited hypoxia transcription signatures that correlated with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8+ T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as for other inflammatory skin diseases in which cytolytic CD8+ T cells contribute to pathogenesis.

Exploring GZMK as a prognostic marker and predictor of immunotherapy response in breast cancer: unveiling novel insights into treatment outcomes.

BACKGROUND: Granzyme K (GZMK) is a crucial mediator released by immune cells to eliminate tumor cells, playing significant roles in inflammation and tumorigenesis. Despite its importance, the specific role of GZMK in breast cancer and its mechanisms are not well understood. METHODS: We utilized data from the TCGA and GEO databases and employed a range of analytical methods including GO, KEGG, GSEA, ssGSEA, and PPI to investigate the impact of GZMK on breast cancer. In vitro studies, including RT-qPCR, CCK-8 assay, cell cycle experiments, apoptosis assays, Celigo scratch assays, Transwell assays, and immunohistochemical methods, were conducted to validate the effects of GZMK on breast cancer cells. Additionally, Cox regression analysis integrating TCGA and our clinical data was used to develop an overall survival (OS) prediction model. RESULTS: Analysis of clinical pathological features revealed significant correlations between GZMK expression and lymph node staging, differentiation grade, and molecular breast cancer subtypes. High GZMK expression was associated with improved OS, progression-free survival (PFS), and recurrence-free survival (RFS), as confirmed by multifactorial Cox regression analysis. Functional and pathway enrichment analyses of genes positively correlated with GZMK highlighted involvement in lymphocyte differentiation, T cell differentiation, and T cell receptor signaling pathways. A robust association between GZMK expression and T cell presence was noted in the breast cancer tumor microenvironment (TME), with strong correlations with ESTIMATEScore (Cor = 0.743, P < 0.001), ImmuneScore (Cor = 0.802, P < 0.001), and StromalScore (Cor = 0.516, P < 0.001). GZMK also showed significant correlations with immune checkpoint molecules, including CTLA4 (Cor = 0.856, P < 0.001), PD-1 (Cor = 0.82, P < 0.001), PD-L1 (Cor = 0.56, P < 0.001), CD48 (Cor = 0.75, P < 0.001), and CCR7 (Cor = 0.856, P < 0.001). Studies indicated that high GZMK expression enhances patient responsiveness to immunotherapy, with higher levels observed in responsive patients compared to non-responsive ones. In vitro experiments confirmed that GZMK promotes cell proliferation, cell division, apoptosis, cell migration, and invasiveness (P < 0.05). CONCLUSION: Our study provides insights into the differential expression of GZMK in breast cancer and its potential mechanisms in breast cancer pathogenesis. Elevated GZMK expression is associated with improved OS and RFS, suggesting its potential as a prognostic marker for breast cancer survival and as a predictor of the efficacy of immunotherapy.

Identifying the association between polymorphisms in the GZMB and IFIH1 genes and psoriasis in Egyptians.

OBJECTIVES: This study aims to examine whether the genetic variants in the genes for Granzyme B (GZMB) and Interferon Induced with Helicase C domain 1 (IFIH1) were associated with psoriasis. BACKGROUND: Psoriasis, a papulosquamous skin disease, was initially thought of as a disorder primarily of epidermal keratinocytes but is now recognized as one of the most common immune-mediated disorders. It is caused by the interplay between multiple genetic and environmental risk factors. SUBJECTS AND METHODS: This case-control study has 65 participants with psoriasis and 65 healthy controls. Real-time PCR was used to genotype GZMB (rs8192917) and IFIH1 (rs35667974). RESULTS: Genotype occurrence and allelic spreading for both SNPs are in Hardy - Weinberg equilibrium. The genotype and allele distributions of rs35667974 showed no differences between the studied groups. Regarding rs8192917, compared to Group II, there is a statistically significant rise in the CC genotype and C allele in Group I. Higher PASI scores are detected in the C/C and C/T genotypes more than the T/T genotype. Univariate and multivariate analyses revealed that BMI, catalase, MDA, and rs8192917 (C/C) are associated with psoriasis. CONCLUSION: GZMB rs8192917 was significantly related to psoriasis risk; its C allele is likewise associated with psoriasis vulnerability. However, our investigation found no link between rs35667974 and psoriasis.

The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis.

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Disruption of IFNγ, GZMB, PRF1, or LYST Results in Reduced Suppressive Function in Human CD8+ T Cells.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.

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.