Search

Cytotoxic attack particles released by CTLs and NK cells include diverse phospholipid membrane and glycoprotein encapsulated entities that contribute to target cell killing. Supramolecular attack particles (SMAPs) are one type of particle characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a proteinaceous shell including thrombospondin (TSP)-1. TSP-4 was also detected in bulk analysis of SMAPs released by CTLs; however, it has not been investigated whether TSP-4 contributes to distinct SMAP types or the same SMAP type as TSP-1 and, if in the same type of SMAP, whether TSP-4 and TSP-1 cooperate or compete. Here, we observed that TSP-4 expression increased upon CD8 + T cell activation while, surprisingly, TSP-1 was down-regulated. Correlative Light and Electron Microscopy and Stimulated Emission Depletion microscopy localized TSP-4 and TSP-1 in SMAP-containing multicore granules. Superresolution dSTORM revealed that TSP-4 and TSP-1 are usually enriched in the same SMAPs while particles with single-positive shells are rare. Retention Using Selective Hooks assays showed that TSP-4 localizes to the lytic granules faster than TSP-1 and promotes its accumulation therein. TSP-4 contributed to direct CTL-mediated killing, as previously shown for TSP-1. TSP-4 and TSP-1 were both required for latent SMAP-mediated cell killing, in which released SMAPs kill targets after removal of the CTLs. Of note, we found that chronic lymphocytic leukemia (CLL) cell culture supernatants suppressed expression of TSP-4 in CTL and latent SMAP-mediated killing. These results identify TSP-4 as a functionally important component of SMAPs and suggest that SMAPs may be targeted for immune suppression by CLL., Competing Interests: Competing interests statement:M.L.D. is a co-founder and advisor of Granza Bio (https://www.granzabio.com). M.L.D. receives consulting fees. Granza Bio is a seed stage company and had no role in this paper—no funding was provided for this work and none of the co-authors are directly employed by Granza Bio. M.L.D. has 3% equity (founder’s shares) with a value of ~$20 and this will not be impacted by results of this paper. M.L.D. is a co-inventor on a patent application on proteinaceous particles-supramolecular attack particles. This paper will not impact this patent. We have made the paper available as a bioRxiv preprint with no new IP filed based on this work. https://www.granzabio.com/newsroom—This includes press releases mentioning M.L.D. as a founder. There are no public statements referring to the work in this paper other than the bioRxiv preprint—https://www.biorxiv.org/content/10.1101/2024.04.25.590546v1.

Type 1 diabetes (T1D) is an autoimmune disorder in which pancreatic β-cells are destroyed by CD8 + T cells. Anti-CD3 antibody effectively treats early-stage T1D when β-cell autoantibodies are detected but before symptoms appear. However, it impairs the immune system temporarily, exposing individuals to infection. A therapeutic that can reverse new-onset T1D without harming the immune system remains urgently needed. Herein, we have constructed cellular vesicles presenting granzyme B-responsive fusion proteins (designated aCD8-GrzBcs-IL2) composed of a single-chain variable fragment of anti-CD8 antibodies and a mutein interleukin-2 (IL2). aCD8-GrzBcs-IL2 is designed to simultaneously inhibit CD8 + T cells and promote Treg cells, especially when CD8 + T cells are attacking β-cells. In vitro , these cellular vesicles can inhibit the cell-killing effect of CD8 + T cells and enhance the expansion of Treg cells. Notably, intravenous administration of aCD8-GrzBcs-IL2-expressed cellular vesicles reversed newly onset diabetes in 77.8% of nonobese diabetic (NOD) mice without reducing blood CD3 + T cells and CD8 + T cells, indicating a favorable safety profile.

Mortality and recurrence rates of hepatocellular carcinoma (HCC) remain high despite the use of various treatment methods. Recently, cell-based immunotherapy using natural killer (NK) cells has attracted considerable attention in cancer immunotherapy. NK cells generated from induced pluripotent stem cells (iPSCs) are a new option for use as an NK cell resource. The eNK cells (HLCN061, developed by HEALIOS K.K.) are human iPSC-derived NK cells differentiated from clinical-grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced. In this study, we aimed to evaluate the potential of eNK cell therapy for HCC treatment. The analysis of eNK cells for cell surface and intracellular molecules revealed that antitumor-related surface molecules (TRAIL, CD226, and CD16) and intracellular cytotoxic factors (perforin, granzyme B, TNFα, and IFNγ) were highly expressed. In addition, eNK cells exhibited high cytotoxicity against HCC cell lines (HepG2, HuH7, and SNU-423), which are sensitive to NKG2D, TRAIL, and CD226. The TRAIL and perforin/granzyme B pathways are largely involved in this cytotoxic mechanism, as indicated by the reduction in cytotoxicity induced by TRAIL inhibitory antibodies and concanamycin A, which inhibits perforin/granzyme B-mediated cytotoxicity. Our data suggest that eNK cells, whose functions have been enhanced by genetic engineering, have the potential to improve HCC treatment., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

As a type of "cold tumor" with limited immune cell infiltration, ovarian cancer has historically shown limited efficacy in immunotherapy. In this study, we report that exosomes from ovarian cancer can specifically target omentum which is the predilection site for ovarian cancer to metastasize and combat subsequently implanted tumor. Furthermore, we found a substantial increase in the proportion of CD3 + T cells, particularly CD8 + T cells, within the omental tissue where exosomes homed. This increase was accompanied by a significant enhancement in granzyme B levels within CD8 + T cells. Additionally, there was a notable elevation in the concentration of interferon-gamma (IFN-γ) in peripheral blood. In vitro results indicated that exosomes could be internalized by dendritic cells (DCs), promote DC differentiation, and subsequently induce the production of granzyme B and IFN-γ in T cells. Surprisingly, we also observed high expression of programmed death ligand 1 (PD-L1) in the omentum. Therefore, we discovered whether combining PD-L1 blockade led to further tumor regression. However, although the combination group showed complete tumor regression, this difference did not reach statistical significance. But in general, we emphasize that in the case of pre-injection, exosomes have great potential to combat the famous "cold tumor", ovarian cancer, via targeting omentum and activating anti-tumor immunity, offering a novel avenue for overcoming ovarian cancer., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Tongji Hospital Ethics Committee for Research Involving Animals., (© 2025. The Author(s).)

Tumor-reactive CD4+ T cells often accumulate in the tumor microenvironment (TME) in human cancer, but their functions and roles in antitumor responses remain elusive. Here, we investigated the immunopeptidome of HLA class II-positive (HLA-II+) endometrial cancer with an inflamed TME using a proteogenomic approach. We identified HLA-II neoantigens, one of which induced polyclonal CD4+ tumor-infiltrating lymphocyte responses. We then experimentally demonstrated that neoantigen-specific CD4+ tumor-infiltrating lymphocytes lyse target cells in an HLA-II-dependent manner. Single-cell transcriptomic analysis of the TME coupled with T-cell receptor sequencing revealed the presence of CD4+ T-cell clusters characterized by CXCL13 expression. The CXCL13+ clusters contained two subclusters with distinct cytotoxic gene expression patterns. The identified neoantigen-specific CD4+ T cells were found exclusively in one of the CXCL13+ subclusters characterized by granzyme B and CCL5 expression. These results demonstrate the involvement of tumor-reactive CD4+ T cells with cytotoxic function in immune surveillance of endometrial cancer and reveal their transcriptomic signature., (©2024 American Association for Cancer Research.)

Background: Bronchiolitis obliterans syndrome (BOS) is one of the most devastating outcomes of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This remains an area of unmet clinical need for optimal therapy for BOS patients partly due to the limited understanding of pathogenic mechanisms., Methods: We collected blood samples from 22 patients with cGVHD and 11 patients without cGVHD following allo-HSCT. By applying a combination of mass cytometry (CyTOF), RNA-sequencing and the quantitative cytokine array, we discovered a new cellular hallmarker of patients with cGVHD-BOS. This finding was further validated in cGVHD-BOS murine models by using single-cell RNA sequencing (scRNA-seq) and paired single-cell V(D)J sequencing analyses., Findings: We revealed that circulating Granzyme K (GZMK)-expressing CD8 + T cells with increased expression of CCR5 were accumulated in cGVHD-BOS patients, and GZMK can induce the expression of fibrosis-essential proteins, collagen type I alpha 1 chain (COL1A1) and fibronectin (FN1), in human fibroblasts. As compared to those of control mice, GZMK + CD8 + T cells in the lungs of cGVHD-BOS mice were undergoing significant infiltration and clonal hyperexpansion, with more cytotoxic, pro-inflammatory, migratory and exhausted phenotypes. Moreover, we screened small-molecule drugs and revealed that Bosutinib, the second-generation BCR-ABL1-targeting tyrosine kinase inhibitor (TKI), could inhibit GZMK expression in CD8 + T cells and reduce lung stiffness and pulmonary fibrosis in cGVHD-BOS mice., Interpretation: This study provides proof-of-principle evidence for clonal GZMK + CD8 + T cells as an unexplored contributor to the pathogenesis of cGVHD-BOS, which can be an underlying biomarker for treatment., Funding: This work was supported by the National Natural Science Foundation of China (No. 82170141, 82100123, 81870136), and "Pioneer" and "Leading Goose" R&D Program of Zhejiang (grant No. 2022C03012)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Esophageal squamous cell carcinoma (ESCC) is a common malignancy. Programmed death ligand 1 of small extracellular vesicles (sEV-PDL1) induce immune evasion and enhance tumor progression. However, the role of ESCC derived sEV-PDL1 in modulating CD8 + T cell remains unclear. sEVs were isolated through differential centrifugation. CD8 + T cells were isolated, stimulated and cultured with sEVs to evaluate the proportions, phenotypes, and functions by flow cytometry. Lentivirus infection and Crisper-Cas9 were used to constructed stable transgenic cell lines: Eca109-PDL1 kd and mEC25-PDL1 ko . The proportions of CD8 + T cells in ESCC patients was lower than healthy donors (HD). Furthermore, a negative correlation between sEV-PDL1 and CD8 + T cell was observed. sEV-PDL1 induced CD8 + T cell exhaustion by reducing the expression levels of Ki67, Granzyme B (GrzmB), and interferon-γ (IFN-γ) both in vitro and in vivo. However, anti-PDL1 reversed the result. Our findings reveal that targeting sEV-PDL1 to rejuvenate CD8 + T cell functions is one of the mechnisms a promising therapeutic strategy for ESCC., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Inflammatory diseases are often chronic and recurrent, and current treatments do not typically remove underlying disease drivers 1 . T cells participate in a wide range of inflammatory diseases such as psoriasis 2 , Crohn's disease 3 , oesophagitis 4 and multiple sclerosis 5,6 , and clonally expanded antigen-specific T cells may contribute to disease chronicity and recurrence, in part by forming persistent pathogenic memory. Chronic rhinosinusitis and asthma are inflammatory airway diseases that often present as comorbidities 7 . Chronic rhinosinusitis affects more than 10% of the general population 8 . Among these patients, 20-25% would develop nasal polyps, which often require repeated surgical resections owing to a high incidence of recurrence 9 . Whereas abundant T cells infiltrate the nasal polyps tissue 10,11 , T cell subsets that drive the disease pathology and promote recurrence are not fully understood. By comparing T cell repertoires in nasal polyp tissues obtained from consecutive surgeries, here we report that persistent CD8 + T cell clones carrying effector memory-like features colonize the mucosal tissue during disease recurrence, and these cells characteristically express the tryptase Granzyme K (GZMK). We find that GZMK cleaves many complement components, including C2, C3, C4 and C5, that collectively contribute to the activation of the complement cascade. GZMK-expressing CD8 + T cells participate in organized tertiary lymphoid structures, and tissue GZMK levels predict the disease severity and comorbidities better than well-established biomarkers such as eosinophilia and tissue interleukin-5. Using a mouse asthma model, we further show that GZMK-expressing CD8 + T cells exacerbate the disease in a manner dependent on the proteolytic activity of GZMK and complements. Genetic ablation or pharmacological inhibition of GZMK after the disease onset markedly alleviates tissue pathology and restores lung function. Our work identifies a pathogenic CD8 + memory T cell subset that promotes tissue inflammation and recurrent airway diseases by the effector molecule GZMK and suggests GZMK as a potential therapeutic target., Competing Interests: Competing interests: H.Q. is a cofounder of Emergent Biomed Solutions, Ltd., (© 2025. The Author(s).)

Here we analyzed the relative contributions of CD4 + regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8 + regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4 + and CD8 + regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8 + regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8 + and CD4 + T cells. These findings highlight the distinct yet complementary roles of CD8 + and CD4 + regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity., Competing Interests: Competing interests: M.M.D., X.C., M.G. and V.M. are inventors on a patent application (PCT/US2023/010431) on the systems and methods incorporating modified T cells described in this work. The other authors declare no competing interests., (© 2025. The Author(s).)

Acute myocardial infarction (AMI), a critical cardiovascular condition, is often associated with serious health risks. Recent studies suggest a link between copper-induced apoptosis and immune cell infiltration. Specifically, abnormal accumulation of copper ions can lead to intracellular oxidative stress and apoptosis, while also affecting immune cell function and infiltration. Nevertheless, studies exploring this relationship in the context of AMI are notably scarce, underscoring the necessity of identifying biomarkers associated with cuproptosis in AMI. Consensus clustering analysis was employed to classify distinct subtypes of AMI in the GSE66360 dataset. Concurrently, differential expression analysis was performed to identify differentially expressed genes (DEGs) across subtypes and between AMI and control samples. We employed Venn diagrams to validate the selection of cuproptosis-related DEGs in patients with AMI. A protein-protein interaction network was constructed to pinpoint potential candidate genes. Receiver operating characteristic curves were generated to identify promising biomarkers. The immune infiltration milieu was analyzed using CIBERSORT algorithms. Finally, the expression levels of identified cuproptosis-related biomarkers were validated at the transcriptional level. We classified AMI into 2 distinct cuproptosis-related subtypes, leading to the identification of 157 cuproptosis-related DEGs. Further analysis refined this list to 10 potential candidate genes. Among these, 5 emerged as significant biomarkers for AMI: granzyme A (GZMA), GTPase immunity-associated proteins (GIMPAs) GIMAP7, GIMAP5, GIMAP6, and TRAF3 interacting protein 3 (TRAF3IP3). A comprehensive examination of immune infiltration in AMI samples revealed significant differences in the levels of 11 types of immune cells, with GZMA displaying the highest correlation with activated mast cells and CD8 + T cells. We observed markedly lower expression levels of GZMA, GIMAP6, and TRAF3IP3 in the AMI group compared to controls. This study identified 5 cuproptosis-related biomarkers (GZMA, GIMAP7, GIMAP5, GIMAP6, and TRAF3IP3) associated with AMI, laying a theoretical foundation for the treatment of AMI., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2025 the Author(s). Published by Wolters Kluwer Health, Inc.)

Protection against infections with intracellular bacteria requires the interaction of macrophages and T-lymphocytes, including CD8 + T cells. Recently, the expression of natural killer cell receptors NKG2A and NKG2C was introduced as markers of CD8 + T-cell subsets. The goal of this study was to functionally characterize human NKG2A and NKG2C-expressing T cells using the major pathogen Mycobacterium tuberculosis ( Mtb ) as a model organism. Sorted NKG2 populations were analyzed for their capacity to proliferate and degranulate and their intracellular expression of cytotoxic molecules. Cytokine release and the effect on bacterial growth were assessed after coculture of NKG2 populations with Mtb -infected macrophages. NKG2A + T cells released higher levels of IFN-γ and IL-10, whereas NKG2C + T cells released higher levels of IL-2, contained the greatest reservoir of intracellular granzyme B and showed a remarkable constitutive level of degranulation. Both subsets inhibited the intracellular growth of Mtb more efficiently than NKG2-negative CD8 + T cells. Antimicrobial activity of NKG2 + T cells was not associated with the release of cytokines or cytotoxic molecules. However, the frequency of polycytotoxic T cells (P-CTL), defined as CD8 + T cells co-expressing granzyme B, perforin, and granulysin, positively correlated with the ability of NKG2-expressing T cells to control Mtb -growth in macrophages. Our results highlight the potential of NKG2-expressing P-CTL to trigger the antibacterial activity of human macrophages. Targeting this population by preventive or therapeutic immune interventions could provide a novel strategy to combat severe infectious diseases such as tuberculosis., Competing Interests: The authors declare no conflict of interest.

Investigations on some innate immunity proteins can yield misleading information, as investigators often rely on static measurements and assume a direct correlation to function. As protein function is often not directly proportional to protein abundance, and mechanistic pathways are interconnected and under constant feedback regulatory control, functional analysis is required. In this study, we used functional mass spectrometry to measure anti-protease and complement activity in plasma obtained from coronavirus disease 2019 (COVID-19) patients. Our data suggests that within 48 h of hospital admission, COVID-19 patients undergo a protease storm with significantly elevated neutrophil elastase (p < 0.001) and lymphocyte granzyme B (p < 0.01), while, anti-protease activity is significantly increased, including alpha-1 antitrypsin (AAT; p < 0.001) and alpha-1-antichymotrypsin (ACT; p < 0.001). Concurrently, the ratio of C3a to C3beta activity significantly decreased with increasing COVID-19 severity, suggesting more complement activation (Mild COVID-19 p < 0.05; Severe COVID-19 p < 0.001). Activity levels of AAT, ACT and C3a/C3beta remained unchanged over 10 hospital days. Our data suggests that COVID-19 is associated with both a protease storm and complement activation, with the former somewhat balanced with increased anti-protease activity. Evaluation of the AAT/ACT ratio and C3a/C3beta ratio indicated that COVID-19 severity is associated with both neutrophil elastase neutralization and complement activation., Competing Interests: Some authors (SR and MK) are employees of Biotech Support Group LLC, who provide proteomic and genomic sample prep and enrichment products and services. The remaining authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2025 Fraser, Roy, Kuruc, Quintero, Van Nynatten, Cepinskas, Zheng, Soherwardy and Roy.)

Understanding donor-reactive T-cell behavior post-transplantation is challenging owing to the rarity and diversity of these cells. Here, we aimed to evaluate the relevance of an assay for rapidly detecting alloreactive T cells in a mouse transplantation model. After 18 h of one-way mixed lymphocyte reaction (MLR) culture with pre-activated donor-derived stimulators, CD4 + and CD8 + donor-reactive T cells were identified by CD154 and CD137 expression, respectively. Using full MHC mismatched mouse skin transplant models, we observed an increased donor-reactive T-cell proportion by direct presentation with elevated interferon gamma and granzyme B production 7 days post-transplantation, before graft rejection. Immunosuppression with CTLA-4 IgG and anti-CD154 antibody varied depending on donor-recipient strain combinations. On day 7, donor-reactive CD8 + T-cell proportions were lower in the tolerance model (BALB/c to C3H/HeJ) than in the rejection model (BALB/c to C57BL/6); conventional proliferation readout after 4 days of MLR could not distinguish these responses. Overall, although the conventional readout for evaluating T-cell proliferation following an MLR quantifies the precursor frequency of alloreactive T cells, the assay reported herein assesses T-cell activation markers after a short-term MLR to characterize immediate immune status. These findings offer a promising tool to elucidate immune responses post-transplantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Arata, Tanimine, Seidakhmetov, Ide, Tanaka and Ohdan.)

Coronary artery involvement (CAI) is a special but not rare manifestation of Takayasu arteritis (TAK). Granzyme B (GzmB) is a multifunctional protease associated with the immune system and coronary artery disease. However, its role in patients with TAK and CAI remains unclear. This study investigates the role of GzmB+ cell subsets in TAK. The study included 105 TAK patients and 58 healthy controls. The percentages of different GzmB+ cells in blood samples were analyzed by flow cytometry. We found that age, age at onset, body mass index, disease duration month, hypertension, and hyperlipidemia were significantly different between TAK patients with and without CAI (P = 0.000, P = 0.038, P = 0.003, P = 0.031, P = 0.039, P = 0.000). The proportions of CD3+CD8+cells (P = 0.001) and CD3+CD4+cells (P = 0.000) in GzmB+ cells were significantly increased, while the proportion of CD3-CD56+cells (P = 0.001) in GzmB+ cells was decreased in TAK patients. The proportions of three types of GzmB+ subsets in lymphocytes (CD3+CD4+GzmB+, CD3+CD8+GzmB+, CD3+CD56+ GzmB+) were higher in TAK patients with CAI compared with those without CAI (P = 0.021, P = 0.007, P = 0.007). The increased proportion of CD3+CD8+GzmB+cells/lymphocytes was an independent risk factor for coronary involvement in TAK (OR = 4.990 [1.766-14.098], P = 0.002). Additionally, patients with a high CD3+CD8+GzmB+cells/lymphocytes ratio had a higher major adverse cardiovascular events rate than those with a low ratio in TAK (P = 0.019). Our results indicate that CD8 cell-derived Gzm B may be a predictor for CAI and major adverse cardiovascular events in TAK patients. Targeting CD3+CD8+GzmB+ lymphocytes or using GzmB inhibitors could be a potential therapeutic approach for the treatment of CAI in TAK., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Keloid scars (KS) and hypertrophic scars (HS) are fibroproliferative wound healing defects characterized by excessive accumulation of extracellular matrix (ECM) in the dermis of affected individuals. Although transforming growth factor (TGF)-β is known to be involved in the formation of KS and HS, the molecular mechanisms responsible for its activation remain unclear. In this study we investigated Granzyme B (GzmB), a serine protease with established roles in fibrosis and scarring through the cleavage of ECM proteins, as a potential new mediator of TGF-β activation in KS and HS. Increased GzmB-positive mast cells were identified in the dermis of KS and HS but not healthy skin controls. Elevated levels of substance P, a neuropeptide involved in mast cell degranulation, suggest that GzmB is released extracellularly, as confirmed by the significant reduction of the established extracellular GzmB substrate decorin in KS and HS. Similarly, presence of latent TGF-β binding protein 1 (LTBP1), a protein involved in the extracellular tethering of latent TGF-β, was disrupted proximal to the dermal-epidermal junction (DEJ) of GzmB high KS and HS lesions. Using LTBP1-enriched medium as well as purified LTBP1, its cleavage by GzmB was confirmed in vitro . Increased TGF-β/Smad signaling pathway was observed in keratinocytes treated with GzmB-digested LTBP1 and was abolished by the addition of a pan-TGF-β inhibitor, suggesting that GzmB cleavage of LTBP1 contributes to TGF-β activation. In dermal fibroblasts, GzmB also cleaved cell-derived LTBP1 and induced TGF-β activation through the cleavage of one or more unidentified fibroblast-secreted proteins. Altogether, the present results suggest that GzmB contributes to KS and HS through ECM remodeling and TGF-β activation., Competing Interests: DG is a co-founder and Chief Officer for viDA Therapeutics Vancouver, Canada, BC which developed VTI-1002, a granzyme B inhibitor used in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Aubert, Goeres, Liu, Kao, Richardson, Jung, Hinz, Crawford and Granville.)

Objective: Oral lichen planus (OLP) is a chronic inflammatory disease characterized by a dense T-cell infiltration and the degeneration of basal keratinocytes. The potential functions of mucosal associated invariant T (MAIT) cells in OLP have been analyzed in our previous study. Keratinocytes under proinflammatory conditions have been demonstrated to activate T cells. This study was aimed to investigate how keratinocytes stimulate MAIT cells in OLP, and to explore the role of activated MAIT cells on keratinocytes., Methods and Results: Increased MAIT cells and higher activation marker CD69 were detected in OLP lesions by flow cytometry. The enhanced expression of MHC class I-like molecule (MR1) required for MAIT cell activation in the epithelial layer of OLP lesions was determined by immunohistochemistry. Keratinocytes treated by 5-A-RU prodrug and lipopolysaccharide, respectively, exhibited higher expression of MR1 and secretion of IL-18. In direct coculture systems consisting of keratinocytes and peripheral blood mononuclear cells, both 5-A-RU prodrug-pretreated keratinocytes and lipopolysaccharide-pretreated keratinocytes activated MAIT cells to secrete granzyme B, contributing to elevated keratinocyte apoptosis., Conclusions: Keratinocytes were capable to activate MAIT cells via MR1 and cytokines in OLP, and granzyme B produced by activated MAIT cells intensified keratinocyte apoptosis, engaging in the pathogenesis of OLP., (© 2024 Wiley Periodicals LLC.)

Background: Head and neck squamous cell carcinoma (HNSCC) is linked to human papillomavirus (HPV) infection. HPV-positive and HPV-negative HNSCC exhibit distinct molecular and clinical characteristics. Although checkpoint inhibitors have shown efficiency in recurrent/metastatic HNSCC, response variability persists regardless of HPV status. This study aimed to explore the CD8 + T-cell landscape in HPV-negative HNSCC., Methods: We performed simultaneous single-cell RNA and TCR sequencing of CD8 + tumor-infiltrating lymphocytes (TILs) from treatment-naïve HPV-negative HNSCC patients. Additionally, cells were stimulated ex vivo, which allowed for the tracking of clonal transcriptomic responses., Results: Our analysis identified a subset of CD8 + TILs highly enriched for interferon-stimulated genes (ISG). TCR analysis revealed ISG cells are clonally related to a population of granzyme K (GZMK)-expressing cells. However, unlike GZMK cells, which exhibited rapid effector-like phenotypes following stimulation, ISG cells were transcriptionally inert. Additionally, ISG cells showed specific enrichment within tumor and were found across multiple tumor entities., Conclusions: ISG-enriched CD8 + TILs are a consistent feature of various tumor entities. These cells are poorly understood but possess characteristics that may impact antitumor immunity. Understanding the unique properties and functionality of ISG cells could offer innovative treatment approaches to improve patient outcomes in HPV-negative HNSCC and other cancer types., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Cancer immunotherapies with antibodies blocking immune checkpoint molecules are clinically active across multiple cancer entities and have markedly improved cancer treatment 1 . Yet, response rates are still limited, and tumour progression commonly occurs 2 . Soluble and cell-bound factors in the tumour microenvironment negatively affect cancer immunity. Recently, growth differentiation factor 15 (GDF-15), a cytokine that is abundantly produced by many cancer types, was shown to interfere with antitumour immune response. In preclinical cancer models, GDF-15 blockade synergistically enhanced the efficacy of anti-PD-1-mediated checkpoint inhibition 3 . In a first-in-human phase 1-2a study (GDFATHER-1/2a trial, NCT04725474 ), patients with advanced cancers refractory to anti-PD-1 or anti-PD-L1 therapy (termed generally as anti-PD-1/PD-L1 refractoriness) were treated with the neutralizing anti-GDF-15 antibody visugromab (CTL-002) in combination with the anti-PD-1 antibody nivolumab. Here we show that durable and deep responses were achieved in some patients with non-squamous non-small cell lung cancer and urothelial cancer, two cancer entities identified as frequently immunosuppressed by GDF-15 in an in silico screening of approximately 10,000 tumour samples in The Cancer Genome Atlas database. Increased levels of tumour infiltration, proliferation, interferon-γ-related signalling and granzyme B expression by cytotoxic T cells were observed in response to treatment. Neutralizing GDF-15 holds promise in overcoming resistance to immune checkpoint inhibition in cancer., Competing Interests: Competing interests: M.H. and J.W. are co-founders and stock owners of CatalYm. A.d.S., M. Rüdiger, P.L’H., M.H., J.A., A.D., A.A., A.G., F.H., M.A., M.L., D.R., C.S.-W., F.S.L., P.F., K.K., F.H., B.R. and E.L. are current or former employees of and/or hold stock options in Catalym. R.D., M.H., J.W., K.K., E.L., F.H., M. Rüdiger and C.S.-W. are inventors on patents related to visugromab and anti-GDF-15 treatment (WO2014049087, WO2015144855, WO2017055613, WO2022101263, WO2024052532, WO2024126808 and unpublished). I. Melero, H.H., R.B., R.D., J.W., F.J.K., A.M.M.E., W.H.F., T.F.G., P.L., M.M., S.S., A.T. and J.R. have received research funding or consulting or advisory fees from CatalYm. Several authors and investigators declare potentially competing interests (grants or contracts and/or royalties or licences, stock or stock options, consulting fees, honoria, travel or meeting support, or advisory or data safety monitoring board participation) apart from their relationship with CatalYm: I. Melero (Agenus, Alligator, Allmiral, AstraZeneca, Biontech, BMS, Boehringer Ingelheim, Bright Peak, Crescendo Biologics, Curon, Dynamicure, F-Star, Genmab, Highlight Therapeutics, HotSpot, Merck Serono, MSD, Merus, Mestag, Numab, PharmaMar, Pieris, Pierre Fabre, Pioneering Medicines, Roche, Sanofi and Servier); G.d.V. (Astellas, AstraZeneca, Bayer, BMS, Ipsen, Janssen MSD, Merck Serono, Pfizer and Roche); E.G. (Anaveon, BeiGene, Boehringer Ingelheim, Ellipses Pharma, F-Star Therapeutics, Hengrui, Incyte, Janssen Global Services, MabDiscovery, Medscape, MSD, Novartis, Seattle Genetics, Sanofi, Roche, SeaGen, Taiho and Thermo Fisher); J.M.-L. (Astellas, BMS, Highlight Therapeutics, Merck Serono, MSD, Novartis, Ipsen, Roche, Sanofi and Pierre Fabre); M.J. (Adoram, AstraZeneca, Debiopharm, Novartis, Roche, Sanofi and Takeda); M.-E.G. (BMS, Janssen, Novartis and Roche); M.S. (Amgen, AstraZeneca, Blueprint Medicines, Boehringer Ingelheim, Bristol Myers Squibb, GlaxoSmithKline, Janssen, MSD, Novartis, Roche, Sanofi and Tacalyx); D.K. (AstraZeneca, Amgen, BMS, Merck Serono, MSD, Mirati, PharmaMar, Sanofi and Swiss Oncology in Motion); R.D. (Alligator, Amgen, BMS, MSD, Novartis, Pierre Fabre, Roche, Sun Pharma, Takeda, Sanofi, Second-Genome, Regeneron, T3 Pharma, MaviVax, Pfizer, Simcere and touchIME); M. Reig (AstraZeneca, Bayer, BMS, Boston Scientific, Biotoscana Pharma, Engitix Therapeutics, Eli Lilly, Geneos, Gilead, Ipsen, Merck, Roche and Universal DC); M.-E.R.R. (BMS, Imcore-Roche, Roche and Highlight Therapeutics); E.C. (Achilles, Adcendo, Alkermes, Amunix, Anaveon, Amcure, AstraZeneca, BeiGene, BMS, Boehringer, Chugai, Debiopharm, Diaccurate, Ellipses, Incyte, iTeos, Janssen, Merus, MonTa, MSD, Nanobiotix, Nouscom, Novartis, OncoDNA, PharmaMar, PsiOxus Therapeutics, Roche/Genentech, Sanofi, Servier, Shionogi, Syneos Health, TargImmune, T-knife and Tolremo); J.E.-V. (BMS, MSD and Pfizer); J.J.S.-C. (Eisai, Novartis, Pfizer and Seagen); K.-L.K. (Janssen and Takeda); O.S. (Affimed); C.S. (BMS, Eli Lilly and Boehringer Ingelheim); E.R. (BMS, Eli Lilly, Kyowa Kirin, Leo Pharma, MSD, Pierre Fabre and Sanofi); A.L.-M. (Incyte, Merck, PharmaMar, Pfizer, Roche, Rovi and Sanofi); I. Moreno (Digicore Initiative, Fondacion Intheos, Exafield, Guidepoint and Ellipses Pharma); A.H.-C. (BMS, Gilead, Kyowa Kirin, Merus and MSD); D.H. (Novartis and Roche); M.S.-Z. (Bayer, Instituto de Salud Carlos III (grant FI19/00222), BTG, MSD and Roche); A.V.-L. (AstraZeneca, BMS, Health in Code and Roche); J.T. (Amgen, AstraZeneca, Bristol Myers Squibb, Eisai, EXACT Therapeutics, Institut für Qualitätssicherung und Transparenz im Gesundheitswesen, Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, Ipsen, Lilly ImClone, medupdate, Merck Serono, Merck Sharp & Dome, Oncolytics Biotech, onkowissen.de, Roche, Servier and streamedup!); C.K. (AstraZenca, BMS, DGVS, Incyte, MCI Deutschland, MSD, Servier, Ipsen, Merck Serono and Roche); P.R.G. (AstraZeneca, Bayer, BMS, Boston Scientific, Eisai, Guerbet, Ipsen, MSD, Lilly, Roche and Sirtex); F.F. (AstraZeneca, BMS, Eisai, Merck Serono, Servier and Roche); Z.T. (Boehringer Ingelheim); H.H. (Secarna); F.J.K. (German Cancer Aid (Deutsche Krebshilfe)); H. Reis (AstraZeneca, BMS, Boehringer Ingelheim, Chop GmbH, Diaceutics, GSK, HUeG, Janssen, MCI, Merck, Novartis, Roche Pharma and Sanofi); M.M. (LYO-X AG and TATAA Biocenter); T.G. (Allogene, Bicara, BMS, Merck, Pyxis and Samyang); W.H.F. (Anaveon, Atreca, Incendia, Ichnos Sciences, Genenta, OSE Immunotherapeutics, Oxford Biotherapeutics, Mestag, Novartis, Roche and Tabby); A.M.M.E. (Acetra, Agenus, BMS, Boehringer Ingelheim, BioInvent, BioNTech, Ellipses, Galecto, GSK, IO Biotech, IQVIA, Isa Pharmaceuticals, Merck & Co, MSD, Pfizer, Pierre Fabre, Sairopa, Sellas, SkylineDX, TigaTx and Trained Immunity); R.B. (Amgen, Avencell); J.R. (Astellas, AstraZeneca, BMS, Daiichi Sankyo, GSK, MSD, Merck and Sanofi); F.H. (Thermosome); P.F. (Curevac); J.W. (Bayrische Forschungsstiftung (FORTiTher) and the German Ministry for Education & Research (Transcan-3)). The remaining authors declare no competing interests., (© 2024. The Author(s).)

Liver fibrosis is a well-established risk factor for liver cancer development. Despite extensive mechanistic studies on liver fibrosis, the role of the immune cell network in fibrotic disease remains poorly understood. In this study, we demonstrate that regulatory T cells (Tregs) are involved in preventing liver fibrosis by regulating the mevalonate pathway. Blocking the mevalonate pathway increased the granzyme B secretion from Tregs, while restoring the pathway reduced it. Statin treatment, which inhibits the mevalonate pathway, alleviated liver fibrosis progression and enhanced the immunosuppressive function of Tregs in vivo. Mechanistically, mevalonate products, including geranylgeranyl pyrophosphate, inhibited the phosphorylation and activation of LKB1, that is a key regulator of Treg homeostasis. Furthermore, these products disrupted the interaction between LKB1 and cAMP-dependent protein kinase (PKA), leading to further reduction of LKB1 phosphorylation. These findings suggest that targeting LKB1 in Tregs through statin treatment prevents the progression of liver fibrosis, offering a promising and safe therapeutic strategy for liver disease and liver cancer., Competing Interests: Declaration of competing interest All of authors in this study declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

HDAC3 has been demonstrated to play a crucial role in the progression of various tumors and the differentiation and development of T cells. However, its impact on peripheral T cells in the development of murine lung cancer remains unclear. In this experiment, a subcutaneous lung tumor model was established in C57BL/6 mice, and tumor-bearing mice were treated with the specific inhibitor of HDAC3, RGFP966, at different doses to observe changes in tumor size. Additionally, a lung tumor model was established using hdac3 fl/fl cd4cre +/+ mice to investigate its mechanism. Mice injected with 10 mg/kg RGFP966 had the smallest tumor volume, while those injected with 30 mg/kg RGFP966 had the largest tumors. Flow cytometry analysis revealed that the expression of HDAC3 in splenic T cells was reduced in all groups of mice, while IFN-γ and IL-17 A were increased. Moreover, the expression of granzyme B and perforin in splenic CD8 + T cells was increased in all groups of mice. Compared to the use of 30 mg/kg RGFP966 alone, the combination with anti-IL-17 A mAb reduced the infiltration of Neutrophils and exhausted T cells in mouse tumors, thereby impeding tumor development. These findings demonstrate that the use of RGFP966 or T cell-specific loss of hdac3 promotes the expression of IL-17 A in splenic T cells, leading to tumor resistance and providing insights for clinical treatment., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Accordance: We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. Arrive: All the procedure of the study is followed by the ARRIVE guidelines., (© 2024. The Author(s).)

The transcription factor T-bet is essential for the anti-tumor effector function of natural killer (NK) cells, but the mechanism regulating its expression in NK cells remains unclear. In this study, we aimed to identify an NK cell-intrinsic regulator that controls T-bet expression. Using T-bet-luciferase reporter assay screening, we identified a protein phosphatase inhibitor as a potential activator of T-bet expression. A series of protein phosphatase 2A (PP2A)-specific inhibitors (PP2Ai) or PP2A siRNA induced the expression of T-bet. In PP2Ai-treated mice, the expression of T-bet and its downstream effector molecules, granzyme B and IFN-γ, was also upregulated in NK cells. Mechanistically, PP2Ai increased the phosphorylation of mTOR and ribosomal protein S6 in NK cells, and mTOR inhibitor canceled the effects of PP2Ai in NK cells. Importantly, NK cells isolated from PP2Ai-treated mice showed higher cytotoxicity and IFN-γ production; therefore, they increased the anti-tumor effector function of NK cells. Accordingly, PP2Ai treatment inhibited lung metastasis of B16 melanoma by NK cell- and mTOR-dependent mechanisms. These results suggest that PP2A negatively regulates NK cell T-bet expression and effector function by an mTOR-dependent mechanism., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), and represents an attractive therapeutic target., Methods: We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. We assessed the construct's GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct's pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated., Results: GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with half-maximal cytotoxicity (IC 50 ) values below 100 nM for most positive lines. Ex vivo stability at 37°C indicated a half-life exceeding 96 hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (t 1/2 α=18.4 hours) and a much slower terminal clearance rate (t 1/2 β=73.1 hours). No toxicity was measured in a Chem16 panel between the control and the GrB-Fc-KS49. In vivo, the GrB-Fc-KS49 showed efficacy against a TNBC syngeneic (4T1/ FLuc ) mouse model, reducing tumor volume and cell proliferation and increasing cell death compared with controls. Treatment using an EMT6 mouse model confirmed these results. In addition to a significant impact on cell proliferation, GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor-infiltrating CD45+ cells and redistribution of tumor-associated macrophages. Transcriptomic analysis of tumors post-treatment confirmed the remodeling of the immune tumor microenvironment by the GrB-Fc-KS49 immunotoxin., Conclusions: GrB-Fc-KS49 showed high specificity and cytotoxicity towards EMP2-positive cells. In vivo, it reduced tumor burden and increased the recruitment of immune cells into the tumor, suggesting that GrB-Fc-KS49 is a promising therapeutic candidate against BC., Competing Interests: Competing interests: MW is an inventor on the University of California patents related to the anti-epithelial membrane protein-2 scFv sequence presented in this work. No other authors have potential conflicts of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Background: HIV-exposed uninfected (HEU) children are at increased risk of morbidity during the first years of life. Although the immune responses of HEU infants in early-life are relatively well described, studies of natural killer (NK) cells in older HEU children are lacking. NK cell subsets were analysed in HEU children and compared to those in HIV unexposed uninfected (HUU) children aged ~ five years., Methods: Multi-parametric flow cytometry was used to characterize peripheral blood-derived NK cell CD56, CD16, CD57, NKG2A and KIR3DL1/KIR2DL2/L3 expression, including intracellular perforin and granzyme B. NK cell subsets were compared between HEU children exposed to prenatal antiretroviral therapy (ART) from conception [long-term (HEULT)]; those exposed to ART during pregnancy [medium-term (HEUMT)] with continued exposure throughout the breastfeeding period and HUU peers. Furthermore, clinical data of the children, including sick clinic visits and hospitalizations documented in morbidity diaries from birth to 5 years were compared between HEU and HUU groups. Frequencies of CD56 bright and CD56 dim NK cell were correlated with these clinical parameters., Results: 139 children were enrolled however, 133 comprising 43 HEULT, 38 HEUMT and 52 HUU were included in the main analyses. Total NK cell, CD56 bright nor CD56 dim NK cell proportions differed between HEU and HUU children. However, HEULT children had lower frequencies of CD56 dim NK cells compared to HEUMT children, (p = 0.002) which maintained significance after controlling for preterm birth, p = 0.012. No differences were observed between HEULT and HUU. The expressions of NKG2A, KIR3DL1/KIR2DL2/L3 and CD57 on CD56 bright and CD56 dim NK cells were similar between the three groups. Furthermore, the frequencies of granzyme B and perforin double positive NK cells were similar between the HUU with HEULT and HEUMT children. CD56 dim NK cell counts had a significant moderate negative correlation with recurrent respiratory infections (rho=-0.38; p = 0.010) in HUU children and negatively correlated with total sick clinic visits in HEUMT (rho=-0.40, p = 0.064)., Conclusion: The proportions of total NK cell, CD56 bright and CD56 dim NK cells, NK cells inhibitory and differentiation surface marker expression and cytolytic granule-positive cells were similar between HEU and HUU children. These data suggest that early-life HIV/ART exposure may not result in major changes in NK cell subsets at 5 years of age., Competing Interests: Declarations. Ethics approval: This study was approved by the Joint Research Ethics Committee for the University of Zimbabwe College of Health Sciences and Parirenyatwa Group Hospitals (JREC/81/20) and the Medical Research Council of Zimbabwe (MRCZ/A/2662). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

BACKGROUNDCurrent diagnostic tools for tuberculous pleural effusion (TPE) are often inadequate, making accurate diagnosis challenging. Effective identification of TPE is critical for ensuring proper treatment and preventing tuberculosis relapse. This study explored the potential of granzyme A (GZMA) as a biomarker for TPE.METHODSPatients with TPE, malignant pleural effusion (MPE), and parapneumonic pleural effusion (PPE) were recruited into discovery and validation cohorts. The discovery cohort consisted of 200 patients with TPE and 100 patients with MPE, while the validation cohort included 167 patients with TPE, 84 patients with MPE, and 69 patients with PPE.RESULTSIn the discovery cohort, GZMA levels were significantly elevated in TPE compared with MPE, demonstrating 90% sensitivity and 91% specificity at a cutoff of 102.29 ng/mL for effectively distinguishing between the two conditions. In the validation cohort, GZMA maintained high diagnostic performance, distinguishing TPE from MPE with 87% sensitivity and 87% specificity and from PPE with 87% sensitivity and 84% specificity. Incorporating GZMA, lactate dehydrogenase (LDH), and adenosine deaminase (ADA) into a random forest model further improved diagnostic accuracy. In the discovery cohort, this model achieved 92% sensitivity and 100% specificity, and in the validation cohort, it distinguished TPE from MPE with 87% sensitivity and 94% specificity and from PPE with 87% sensitivity and 91% specificity.CONCLUSIONOverall, GZMA is a promising biomarker for diagnosing TPE, with improved accuracy when combined with LDH and ADA, providing a robust tool for timely identification and effective management of patients with TPE.FUNDINGThe study was supported by Science and Technology Project of Shenzhen (KCXFZ20211020163545004, KQTD20210811090219022, JCYJ20220818095610021, JSGG20220822095200001, JCYJ20210324094614038), Shenzhen Medical Research Funding (B2302035, A2302004), Provincial Natural Science Foundation of Guangdong (2022A1515220034), and Shenzhen Third People's Hospital Research Foundation (G2022155).

Local delivery of mRNA-based immunotherapy offers a promising avenue as it enables the production of specific immunomodulatory proteins that can stimulate the immune system to recognize and eliminate cancer cells while limiting systemic exposure and toxicities. Here, we develop and employ lipid-based nanoparticles (LNPs) to intratumorally deliver an mRNA mixture encoding the cytokines interleukin (IL)-21 and IL-7 and the immunostimulatory molecule 4-1BB ligand (Triplet LNP). IL-21 synergy with IL-7 and 4-1BBL leads to a profound increase in the frequency of tumor-infiltrating CD8 + T cells and their capacity to produce granzyme B and IFN-γ, leading to tumor eradication and the development of long-term immunological memory. Mechanistically, the efficacy of the Triplet LNP depends on tumor-draining lymph nodes to tumor CD8 + T-cell trafficking. Moreover, we highlight the therapeutic potential of the Triplet LNP in multiple tumor models in female mice and its superior therapeutic efficacy to immune checkpoint blockade. Ultimately, the expression of these immunomodulators is associated with better overall survival in patients with cancer., Competing Interests: Competing interests: J.F., E.B., S.D.K., and F.L. are employees of etherna. J.F., E.B., S.D.K., and F.L. have applied for a patent related to the study (Compositions and methods for delivery of agents to immune cells; WO2023118411A1). S.D.K. and B.G.D.G. have applied for a patent related to the ionizable lipids used in this work (Ionizable lipids; WO2022136641A1). All other authors declare no potential conflicts of interest., (© 2024. The Author(s).)

Chronic obstructive pulmonary disease (COPD) is caused by smoking, but only a small proportion of smokers have disease severe enough to develop COPD. COPD is not always progressive. The question then arises as to what explains the different trajectories of COPD. The role of autoimmunity and regulatory T (Treg) cells in the pathogenesis of COPD is increasingly being recognized. Nine published studies on Treg cells in the lung tissue or bronchoalveolar lavage fluid have shown that smokers with COPD have fewer Treg cells than smokers without COPD or nonsmokers. Three studies showed a positive correlation between Treg cell count and FEV 1 %, 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 FEV 1 % was comparable to that between Treg cell count and FEV 1 % 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 FEV 1 %. 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.

Cigarette smoking is associated with worse clinical outcomes in patients with chronic hepatitis B (CHB) infection, but the effects on hepatitis B surface antigen (HBsAg) seroclearance are unclear. This study aimed to investigate the effect of active smoking on HBsAg seroclearance (SC) and its impact on peripheral blood lymphocytes in patients with CHB infection. Longitudinal follow-up data was retrieved in 7833 antiviral-treated CHB subjects identified from a centralised electronic patient record database (Part 1). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs) from 27 CHB-infected patients (6 active smokers; 13 with SC) was performed by flow cytometry to assess programmed death-1 (PD-1) expression and proportion of regulatory T cells (CD4 + CD25 + CD127 lo ). Effector function of HBV-specific T cells was examined by comparing granzyme B (GZMB) and transforming growth factor beta (TGFβ) production in undepleted PBMCs and Treg-depleted PBMCs after 7 days in vitro stimulation with HBV envelope protein overlapping peptides (Part 2). Over a median follow-up of 5 years, smoking was associated with lower probability of SC (aHR 0.70, 95% CI 0.57-0.87). PD-1 expression was increased in CD4 + T cells, CD8 + T cells and CD20 + B cells among smokers compared to non-smokers and positively correlated with pack years (all p < 0.05). Treg depletion led to partial functional recovery of HBV-specific T cells, with significantly bigger magnitude in smokers (p = 0.0451, mean difference = 4.68%) than non-smokers (p = 0.012, mean difference = 4.2%). Cigarette smoking is associated with lower chance of HBsAg seroclearance, higher PD-1 expression on lymphocytes, and impairment of effector functions of HBV-specific T cells in CHB., (© 2024 The Author(s). Journal of Viral Hepatitis published by John Wiley & Sons Ltd.)

Background: Granzyme activity can affect the processing and stability of miRNAs within target cells. They also could induce changes in miRNA expression that impact apoptotic signaling. Granzyme-induced apoptosis might result in changes to the miRNA profile, which can further influence the apoptosis and inflammation processes., Objective: The aim of this study was to bioinformatically analyze which miRNAs and transcription factors bind to the CDS and UTR regions of the granzyme family to regulate gene expression in relation to granzyme evolution and their association with human cancer diseases., Methods: The expression patterns of granzyme genes were analyzed in various human tissues. MiRNAs binding to the CDS and UTR of the granzyme family were examined, and the transcription factors binding to these miRNAs binding sites were also analyzed. Cytoscape program was used to visualize and analyze the networks of interactions between granzyme mRNA and miRNAs. Additionally, the evolutionary patterns of the granzyme family in relation to miRNAs and transcription factors binding were investigated., Results: Analysis of the expression patterns of the granzyme family in various human tissues shows that GZMA and GZMK are strongly expressed in lymph nodes. GZMB exhibits strong expression in the bone marrow, while GZMA is prominently expressed in the spleen. Twenty-two miRNAs bind to both GZMK and GZMB mRNA, while six miRNAs bind to both GZMK and GZMM mRNA. The only miRNA that binds to GZMK, GZMB, GZMM, and GZMA mRNA is hsa-miR-146a-5p. Transcription factors JUND, FOS, and JUN are distinctly interconnected with has-miR-5696 and GZMK. Association data between the granzyme family and cancers showed that various miRNAs were consistently implicated and exhibited either upregulation or downregulation., Conclusion: Although the granzyme family possesses distinct genetic information, it shows relatively high expression levels in the lymph node, spleen, and bone marrow. Many miRNAs specifically regulate granzyme gene expression, and various transcription factors are involved. Analyzing the granzyme genes-miRNAs-transcription factors-related network will provide crucial insights into the mechanisms of cancer development and suppression., Competing Interests: Declarations. Conflict of interest: The authors have no conflict of interest to declare. Ethical approval: Not applicable. Informed consent: Not applicable., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Cellular senescence, recognized as a key hallmark of aging, leads to the accumulation of senescent cells in various tissues over time. While the detrimental effects of these cells on age-related pathological conditions are well-documented, there is still limited information about how senescent cells are distributed in normal tissues of both young and aged organs. Our research indicates that fully senescent p16 INK4A+ cells are rarely identified in the parenchyma of organic tissues and in the stromal cells crucial for structural maintenance, such as fibroblasts and smooth muscle cells. Instead, p16 INK4A+ cells are more commonly found in immune cells, whether they reside in the organ or are infiltrating. Notably, p16 INK4A+ senescent T cells have been observed to induce apoptosis and inflammation in colonic epithelial cells through Granzyme A-PARs signaling, compromising the integrity of the epithelial lining. This study showed that the senescence of immune cells could affect the phenotypical change of the parenchymal cells in the elderly and suggests that targeting immunosenescence might be a strategy to control functional decline in this population., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Aging is a major risk factor for pathologies that manifest later in life. Much attention is devoted towards elucidating how prolonged environmental exposures and inflammation promote biological (accelerated) tissue aging. Granzymes, a family of serine proteases, are increasingly recognized for their emerging roles in biological aging and disease. Widely recognized as intracellular mediators of cell death, granzymes, particularly granzyme B (GzmB), also accumulate in the extracellular milieu of tissues with age, contributing to chronic tissue injury, inflammation, and impaired healing. Consequently, this has prompted the field to reconsider how GzmB regulation, accumulation, and proteolysis impact health and disease with age. While GzmB is observed in numerous age-related conditions, the current review focuses on mechanistic studies where proof-of-concept has been forwarded., Competing Interests: Declaration of interests D.J.G. is a co-founder and chief scientific officer of viDA Therapeutics, which owns patents for and is developing inhibitors targeting granzymes as therapeutics. The remaining authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: To explore the changes in number and immune function of mucosal-associated invariant T (MAIT) cells in peripheral blood of patients with newly diagnosed acute myeloid leukemia (AML), and its correlation with the occurrence and development of AML., Methods: Seventy-five clinical samples of patients with newly diagnosed AML and 48 healthy control samples in our hospital from January 2022 to February 2023 were included. Multiparametric flow cytometry was used to detect the number of MAIT cells, membrane surface markers, effector phenotypes and functional indicators in the samples., Results: Compared with healthy controls, the percentage of MAIT cells in CD3 + T cells in peripheral blood of newly diagnosed AML patients was significantly reduced ( P < 0.001). The percentage of MAIT cells in all CD3 + T cells in bone marrow of AML patients was similar to that in peripheral blood ( P >0.05). Most of MAIT cells in peripheral blood of AML patients were effector memory T cells. Compared with healthy controls, the proportion of effector memory MAIT cells decreased ( P < 0.05), while the proportion of terminally differentiated effector memory MAIT cells and PD-1 + MAIT cells increased significantly (both P < 0.05). AML patients' peripheral blood MAIT cells expressed significantly higher levels of granzyme B and perforin than healthy controls (both P < 0.05), and secreted significantly lower levels of cytokines such as gamma interferon and tumor necrosis factor α than healthy controls (both P < 0.001)., Conclusion: Compared with healthy controls, the proportion of MAIT cells in AML patients is reduced and the expression of functional markers is abnormal, suggesting that their function is impaired and may be involved in the occurrence and development of AML.

LGALS9, also known as Galectin-9 and a member of the β-galactosidase family, plays a crucial role in immune regulation. However, its expression and function in CD8 T cells, as well as its association with cytotoxic T lymphocytes (CTL), remain unclear. This study aims to investigate LGALS9 expression patterns in human circulating CD8 T lymphocytes and elucidate its clinical significance in Systemic Lupus Erythematosus (SLE). Blood samples from 56 healthy controls and 50 new-onset SLE patients were collected. Flow cytometry was utilized to analyze LGALS9 expression in circulating CD8 T lymphocytes via intracellular staining. Compared to LGALS9 + CD8 + T cells, LGALS9-CD8 + T cells showed increased secretion of Granzyme B (GZMB) and Perforin, along with elevated expression levels of GPR56, CX3CR1, KLRD1, KLRF1, PD1, and CD29. A higher proportion of Tn (naive T cells) and TCM (central memory T cells) showed LGALS9 positivity, compared to TEM (effector memory T cells) and TEMRA (terminally differentiated effector memory T cells re-expressing CD45RA). Clinically, the downregulation of LGALS9 expression was significant in SLE patients. LGALS9 + CD8 + T cells exhibited an Area Under the Curve (AUC) of 0.6916, while CX3CR1 + in LGALS9 + CD8 + T cells had an AUC of 0.6478, and KLRF1 + had an AUC of 0.6419, for distinguishing SLE from healthy individuals. In conclusion, CD8 + LGALS9 + T cells display characteristics of low cytotoxicity, and their reduction is evident in SLE patients, potentially implicating them in SLE pathogenesis and providing diagnostic assistance., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Consent: All research protocols received approval from the Ethics Committee of Peking University People's Hospital and adhered strictly to the ethical standards outlined in the Helsinki Declaration. Furthermore, informed consent was obtained from all individuals participating in the study., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB -/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA -/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

GSDMB-mediated pyroptosis facilitates a pro-inflammatory immune microenvironment and needs to be tightly regulated to avoid excessive inflammation. Here, we provide evidence that itaconate and its cell-permeable derivative 4-octyl itaconate (4-OI) can significantly inhibit GSDMB-rendered pyroptotic activity independent of Nrf2. 4-OI interferes proteolytic process of GSDMB by directly modifying Cys54, Cys148 and Ser212 on granzyme A (GrzA), a serine protease that site-specifically cleaves the inter-domain linker of GSDMB, instead of interaction with GSDMB, thereby blocking pyroptosis and exerts anti-inflammatory effects. Moreover, 4-OI alleviates inflammation by suppressing GSDMB-induced pyroptotic cell death during acute colitis models in intestinal epithelial GSDMB conditional transgenic mice. Our data expand the role of 4-OI as a crucial immunometabolic derivative that regulates innate immunity and inflammation through a newly identified posttranslational modification, and targeting of pyroptosis by 4-OI therefore holds potent therapeutic potential for primarily inflammatory and/or autoimmune diseases., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Resident memory T cells (TRM cells) have been described in barrier tissues as having a "sensing and alarm" function where, upon sensing cognate Ag, they alarm the surrounding tissue and orchestrate local recruitment and activation of immune cells. In the immunologically unique and tightly restricted CNS, it remains unclear whether and how brain TRM cells, which express the inhibitory receptor programmed cell death protein 1 (PD-1), alarm the surrounding tissue during Ag re-encounter. Using mouse models, we reveal that TRM cells are sufficient to drive the rapid remodeling of the brain immune landscape through activation of microglia, dendritic cells, NK cells, and B cells, expansion of regulatory T cells, and recruitment of macrophages and monocytic dendritic cells. Moreover, we report that although PD-1 restrained granzyme B upregulation in brain TRM cells reactivated via viral peptide, we observed no apparent effect on cytotoxicity in vivo, or downstream alarm responses within 48 h of TRM reactivation. We conclude that TRM cells are sufficient to trigger rapid immune activation and recruitment in the CNS and may have an unappreciated role in driving neuroinflammation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Sophisticated interactions between stromal and immune cells play crucial roles in various biological and pathological processes. In chronic rhinosinusitis with nasal polyps (CRSwNP), the upper airway inflammation in many patients is driven by T H 2, ILC2, and eosinophils, thus being treated with glucocorticoids and anti-type 2 inflammation biologics. The resistance to these therapies is often associated with neutrophilic inflammation, which has also been widely identified in CRSwNP, but the underlying mechanisms remain unclear. Using single-cell analysis, spatial transcriptomics, and T-cell receptor sequencing, we identify an increased presence of granzyme K + (GZMK + ) CD8 + T cells in NPs, which possess a phenotype distinct from the cytotoxic GZMB + effector CD8 + T subset. GZMK + CD8 + T cells are found to express CXCR4 and interact with CXCL12-secreting fibroblasts, inducing the latter to produce neutrophil chemoattractants in a manner uniquely mediated by GZMK but not other granzymes. This GZMK + CD8 + T cell-fibroblast crosstalk is also observed in other inflammatory diseases. Furthermore, GZMK + CD8 + T cells exhibit a selective expansion of clones that recognize Epstein-Barr virus. Here, we show that GZMK marks a phenotypically distinct subset of effector CD8 + T cells that promote neutrophilic inflammation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Natural killer cells (NK cells) are essential in cancer immunosurveillance in the body as they can recognize cancer cells that lacking MHC class 1 on their surface. Regulatory cytokines, including interleukin (IL)-18, IL-12, IL-10, IL-8, interferon- γ (IFN- γ ), and secretory granules like perforin and granzyme are involved in NK cell-mediated cytotoxicity. Stimulating NK cells cytotoxicity towards cancer cells is an ideal strategy to combat cancer naturally. Medicinal plants have been reported to enhance immunity, with Pereskia bleo ( P. bleo ) particularly noteworthy due to its abundant bioactive compounds and ability to activate immune cells. This study aimed to evaluate the potential of methanol extract of P. bleo leaves (MEPB) for enhancing NK cell cytotoxicity against triple-negative human breast cancer cells (MDA-MB-231)., Methods: The optimal concentration of MEPB to activate NK cells was determined using healthy blood samples, assessing the expression of IL-12, IL-18, IL-10, IL-8, IFN- γ , perforin, and granzyme B via an enzyme-linked immunosorbent assay (ELISA). NK cell purity from healthy donors and breast cancer patients was determined using specific antibodies, and the number of NK cells was assessed using flow cytometry and a hemocytometer. A co-culture experiment, ELISA, and apoptosis assay were used to evaluate NK-mediated cytotoxicity pathways., Results: ELISA data indicated that MEPB at 7.5 µg/ml significantly increased the expression of IFN- γ , IL-12, IL-18, perforin, and granzyme B while decreasing IL-8 and IL-10 expression after 20 hrs of incubation. The average NK cell purity was 87.09 ± 0.043%. Breast cancer patients exhibited lower NK cell counts than healthy donors. Co-culture experiments demonstrated that NK cells induced apoptosis in MDA-MB-231 breast cancer cells in the presence of MEPB by increasing perforin, granzyme B, and IFN- γ expression in both healthy donors and breast cancer patients-experimental groups. P. bleo enhances NK cell activation, promoting the apoptosis of triple-negative human breast cancer cells (MDA-MB-231), suggesting the potential use of MEPB leaves as an anti-cancer immunostimulant., Competing Interests: The authors declare there are no competing interests., (©2024 Khalaf et al.)

Members of the vanin gene family include VNN1, VNN2, and VNN3 in humans. Although the functions of vanins have been widely examined in myeloid cells, their expression and functions have not been clarified in T lymphocytes. This study aimed to elucidate the significance of Vanin-2 (VNN2) on human peripheral blood T lymphocytes and study its expression in systemic lupus erythematosus (SLE). The differential expression of Vanins was analyzed by bioinformatics. VNN2 expressions in peripheral blood T-cell subsets were analyzed by single-cell RNA sequencing data and flow cytometry. Changes of VNN2 expression before and after T-cell activation were further clarified by western blot. The function of VNN2+ cells was studied by granzyme B (GZMB) and perforin detection. Changes in VNN2+ proportions in T-cell subsets of patients with SLE were further analyzed. In the present study, only VNN2 among vanins showed distinguishable expression in T cells. VNN2+ percentages were higher in CD8+ T cells those in CD4+ T cells. VNN2+ T cells were with a higher memory T-cell composition. VNN2 expression was significantly increased after T-cell stimulation. VNN2+ T cells had higher levels of GZMB and perforin secretion than VNN2- T cells. Clinically, VNN2+ percentages in T cells of patients with SLE were upregulated. Together, these data suggested that VNN2 is expressed in peripheral blood T cells characterized more GZMB and perforin secretion, and increased VNN2+ T cells in patients with SLE could reflect altered T-cell functions in vivo., Competing Interests: Conflict of interest statement. The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Simultaneous detection of different biomarkers related to the spatiotemporally dynamic immune events is of particular importance for the accurate evaluation of antitumor immune effects. Here, we have developed an AND-gate logic dual resonance energy transfer nanoprobe (named DRET) for dynamic monitoring of programmed CD8 + T cell activation and tumor cell apoptosis. Immunotherapy-induced granzyme B secretion from CD8 + T cells and the subsequent caspase-3 release from apoptotic tumor cells individually activate one of the tiers of the "AND-gate" logic DRET. The resulting fluorescence recovery and magnetic resonance T1 enhancement can be used for precise immunomodulatory drug screening, early efficacy prediction, and immune stratification. Particularly, not only "Responders" can be distinguished from "Non-responders", but also "Acquired resistance" can be identified from "Maintain responders", providing a novel approach to put forward the accurate evaluation of antitumor immunity.

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest regarding the publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Congenital heart disease (CHD) is the most common birth defect in newborns, often requiring cardiac surgery with concomitant thymectomy that is known to increase disease susceptibility later in life. Studies of γδ T cells, which are one of the dominant T cells in the early fetal human thymus, are rare. Here, we provide a comprehensive analysis of the γδ T cell compartment via flow cytometry and next-generation sequencing in children and infants with CHD, who underwent cardiac surgery shortly after birth. A perturbation of the γδ T cell repertoire is evident, and Vδ1 T cell numbers are reduced. However, those cells that are present, do retain cytotoxicity. In contrast, GZMA+CD28+CD161hi innate effector Vγ9Vδ2 T cells are found in higher proportions. TCR-seq identifies an increase in TRDJ3+ γδ T cell clones in children with CHD, but not in a confirmatory group of neonates prior to CHD surgery, which overall points to a persistence of fetal-derived effector γδ T cells in children with CHD., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

As one molecule related to cytotoxicity, surface expression of C-X3-C motif receptor 1 (CX3CR1) was highly correlated with intracellular granzyme B (GZMB) in natural killer and cytolytic T cells. However, the expression of CX3CR1 and GZMB in B cells has not been clarified, and their clinical significance in systemic lupus erythematosus (SLE) remains unclear. This study aimed to clarify the changes and clinical significance of peripheral blood B cells expressing GZMB and/or CX3CR1 in SLE. Peripheral blood was collected from 39 patients with SLE and 48 healthy controls. We found that GZMB and CX3CR1 expression varied in different B-cell subsets, with plasmablasts possessing the highest positive percentages, consistent with bioinformatics prediction. GZMB+ and CX3CR1+ percentages in circulating B cells and plasmablasts were increased in patients with SLE. CX3CR1 was upregulated on B cells after in vitro stimulation. Notch intracellular domain expression was significantly decreased in plasmablasts of patients with SLE, and CX3CR1 in plasmablasts was downregulated with the addition of JAG1. In conclusion, GZMB and CX3CR1 were increased in B cells and in plasmablasts of patients with SLE and CX3CR1 was negatively regulated by Notch signal in plasmablasts, which may be involved in SLE pathogenesis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In the competition between the pathogen infection and the host defense, infectious microorganisms may enter the host cells by evading host defense mechanisms and use the intracellular biomolecules as replication nutrient. Among them, intracellular Staphylococcus aureus relies on the host cells to protect itself from the attacks by antibiotics or immune system to achieve long-term colonization in the host, and the consequent clinical therapeutic failures and relapses after antibiotic treatment. Here, we demonstrate that intracellular S. aureus surviving well even in the presence of vancomycin can be effectively eliminated using an emerging cell-mimicking therapeutic strategy. These cell mimics with natural killer cell-like activity (NKMs) are composed of a redox-responsive degradable carrier, and perforin and granzyme B within the carrier. NKMs perform far more effectivly than clinical antibiotics in treating intracellular bacterial infections, providing a direct evidence of the NK cell-mimicking immune mechanism in the treatment of intracellular S. aureus .

Rheumatoid arthritis (RA) is a common autoimmune disorder characterized by exacerbated joint inflammation. Despite the well-documented accumulation of the serine protease granzyme B (GzmB) in RA patient biospecimens, little is understood pertaining to its role in pathobiology. In the present study, tenascin-C (TNC) - a large, pro-inflammatory extracellular matrix glycoprotein - was identified as a substrate for GzmB in RA. GzmB cleaves TNC to generate 3 fragments in vitro: a 130 kDa fragment that remains anchored to the matrix and 2 solubilized fragments of 70 and 30 kDa. Mass spectrometry results suggested that the 30 kDa fragment contained the pro-inflammatory TNC C-terminal fibrinogen-like domain. In the synovial fluids of patients with RA, soluble levels of GzmB and TNC were significantly elevated compared with healthy controls. Further, immunoblotting revealed soluble 70 and 30 kDa TNC fragments in the synovial fluids of patients with RA, matching TNC fragment sizes generated by GzmB cleavage in vitro. Granzyme K (GzmK), another serine protease of the granzyme family, also cleaves TNC in vitro; however, the molecular weights of GzmK-generated TNC fragments did not correspond to TNC fragment sizes detected in patients. Our data support that GzmB, but not GzmK, contributes to RA through the cleavage of TNC.

Cytotoxic activity is a hallmark of the immunopathogenesis in human cutaneous leishmaniasis (CL). In this study, we identified accumulation of CD4 + granzyme B producing T cells with increased cytotoxic capacity in CL lesions. These cells showed enhanced expression of activating NK receptors (NKG2D and NKG2C), diminished expression of inhibitory NKG2A, along with the upregulation of the senescence marker CD57. Notably, CD4 + T cells freshly isolated from CL lesions demonstrated remarkable capacity to mediate NL-like bystander cytolysis. Phenotypic analyses revealed that lesional CD4 + T cells are mainly composed of late-differentiated effector (CD27-CD45RA-) and terminally differentiated (senescent) TEMRA (CD27-CD45RA+) subsets. Interestingly, the TEMRA CD4 + T cells exhibited higher expression of granzyme B and CD107a. Collectively, our results provide the first evidence that senescent cytotoxic CD4 + T cells may support the skin pathology of human cutaneous leishmaniasis and, together with our previous findings, support the notion that multiple subsets of cytotoxic senescent cells may be involved in inducing the skin lesions in these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Covre, Fantecelle, Garcia de Moura, Oliveira Lopes, Sarmento, Freire-de-Lima, Decote-Ricardo, de Matos Guedes, da Fonsceca-Martins, de Carvalho, de Carvalho, Mosser, Falqueto, Akbar and Gomes.)

Chronic active Epstein-Barr virus infection (CAEBV) is a progressive and life-threatening disease characterized by persistent or recurrent EBV activation. It has been reported that, γδ T cells, a type of cytotoxic lymphocyte, play a critical role in restricting EBV. However, the functional status of γδ T cells in pediatric CAEBV patients has not yet been described. In this study, flow cytometry analysis was conducted to explore the cytokine production capacity of γδ T cells in CAEBV patients. A diminished frequency of γδ T cells and decreased expression of cytolytic molecule granzyme B were found in CAEBV patients, suggesting a dysfunction in the immune regulatory function of γδ T cells in this disease., (© 2024. The Author(s).)

To tackle the clinical challenge of noninvasively assessing immunotherapy efficacy in patients, here we used positron emission tomography (PET) with 68 Ga-grazytracer, which targets granzyme B, a crucial effector molecule secreted by activated CD8 + T cells. In this phase 1/2 clinical trial (NCT05000372) involving a diverse cohort of 24 patients with solid tumors and lymphomas who received immunotherapies, including immune checkpoint inhibitors (either alone or with chemotherapies) and chimeric antigen receptor-T cell therapy, we examined the in vivo behaviors of 68 Ga-grazytracer. Primary endpoints were safety, biodistribution, granzyme B specificity, and the predictive utility of 68 Ga-grazytracer, while secondary endpoint was the relationship between 68 Ga-grazytracer uptake and tumor immune phenotype. 68 Ga-grazytracer exhibited a safe profile and specifically targeted granzyme B in patients. 68 Ga-grazytracer PET showed superior predictive value for short-term prognosis and progression-free survival than those of conventional assessment criteria, including RECIST 1.1 and PERCIST. Moreover, the uptake of 68 Ga-grazytracer in tumors was significantly higher in those with a "non-desert" immune phenotype than those with an immune "desert" phenotype, thereby meeting the primary and secondary endpoints of this trial. Collectively, we successfully visualized CD8 + T cell effector function in humans using 68 Ga-grazytracer PET, offering insights for enhancing immunotherapy assessment, patient stratification and treatment planning., (© 2024. The Author(s).)

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8 + T cells. For unbiased assessment of cellular immunopathogenesis, here we perform single-cell (sc) transcriptome, surface proteome, and T cell receptor (TCR) sequencing on unaffected skin, affected skin, and blister fluid from 15 SJS/TEN patients. From 109,888 cells, we identify 15 scRNA-defined subsets. Keratinocytes express markers indicating HLA class I-restricted antigen presentation and appear to trigger the proliferation of and killing by cytotoxic CD8 + tissue-resident T cells that express granulysin, granzyme B, perforin, LAG3, CD27, and LINC01871, and signal through the PKM, MIF, TGFβ, and JAK-STAT pathways. In affected tissue, cytotoxic CD8 + T cells express private expanded and unexpanded TCRαβ that are absent or unexpanded in unaffected skin, and mixed populations of macrophages and fibroblasts express pro-inflammatory markers or those favoring repair. This data identifies putative cytotoxic TCRs and therapeutic targets., (© 2024. The Author(s).)

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

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., (Copyright © 2024. Published by Elsevier Ltd.)