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Aegerolysin proteins are involved in various interactions by recognising a molecular receptor in the target organism. The formation of pores in combination with larger, non‐aegerolysin‐like protein partners (such as membrane attack complex/perforin proteins [MACPFs]) is one of the possible responses in the presumed competitive exclusion of other organisms from the ecological niche. Bicomponent pairs are already observed at the gene level. Fungi growing under extreme conditions can be divided into ubiquitous and extremotolerant generalists which can compete with mesophilic species and rare, isolated extremophilic and extremotolerant specialists with narrow ecological amplitude that cannot compete. Under extreme conditions, there are fewer competitors, so fungal specialists generally produce less diverse and complicated profiles of specialised molecules. Since extremotolerant and extremophilic fungi have evolved in numerous branches of the fungal tree of life and aegerolysins are unevenly distributed across fungal genomes, we investigated whether aegerolysins, together with their partner proteins, contribute to the extreme survival ecology of generalists and specialists. We compiled a list of 109 thermo‐, psihro‐, acido‐, alkali‐, halo‐, metallo‐ and polyextremo‐tolerant/‐philic fungal species. Several challenges were identified that affected the outcome: renaming fungal species, defining extremotolerant/extremophilic traits, identifying extremotolerant/extremophilic traits as metadata in databases and linking fungal isolates to fungal genomes. The yield of genomes coding aegerolysins or MACPFs appears to be lower in extremotolerant/extremophilic fungi compared to all fungal genomes. No candidates for pore‐forming gene pairs were identified in the genomes of extremophilic fungi. Aegerolysin and MACPFs partner pairs were identified in only two of 69 species with sequenced genomes, namely in the ubiquitous metallotolerant generalists Aspergillus niger and A. foetidus. These results support the hypothesised role of these pore‐forming proteins in competitive exclusion. [ABSTRACT FROM AUTHOR]

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide--MHC-I complex by the T-cell receptor (TCR) and costimulation. This cognate interaction promotes the organisation of intimate cell-- cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell--cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 55S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. [ABSTRACT FROM AUTHOR]

The "Supplementary Material" from Frontiers in Immunology delves into the regulation of cell asymmetry and function in human CD8+ T lymphocytes through cytosolic protein translation. The document includes figures and tables displaying experiments on cell viability, protein translation, mitochondrial staining, and effector function analysis. Statistical analyses using linear mixed models are utilized to analyze data from Seahorse experiments, comparing control groups with various treatments and conditions to reveal significant differences in cellular values. The data sheds light on the effects of interventions on cellular functions. [Extracted from the article]

Background: Adoptive cell cancer therapies aim to re-engineer a patient's immune cells to mount an anti-cancer response. Chimeric antigen receptor T and natural killer cells have been engineered and proved successful in treating some cancers; however, the genetic methods for engineering are laborious, expensive, and inefficient and can cause severe toxicities when they over-proliferate. Results: We examined whether the cell-killing capacity of activated T and NK cells could be targeted to cancer cells by anchoring antibodies to their cell surface. Using metabolic glycoengineering to introduce azide moieties to the cellular surface, we covalently attached a dibenzocyclooctyne-modified antibody using the strain-promoted alkyne azide cycloaddition reaction, creating antibody-conjugated T and NK cells. We targeted the immune cells to tumors possessing the xenoantigen, N-glycolyl neuraminic acid GM3 ganglioside, using the 14F7hT antibody. These activated T and NK cells are "armed" with tumour-homing capabilities that specifically lyses antigen-positive cancer cells without off-target toxicities. Moreover, when exposed to target cells, 14F7hT-conjugated T cells that are not preactivated exhibit increased perforin, granzyme, CD69, and CD25 expression and specific cell killing. Conclusions: This research shows the potential for a non-genetic method for redirecting cytotoxic immune cells as a feasible and effective approach for tumor-targeted cell immunotherapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG. Methods: Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells. Results: Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56dimCD16-NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-g). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NKcell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function. Discussion: Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NKcell ability to inhibit autoimmunity may be a new direction in the treatment of MG. [ABSTRACT FROM AUTHOR]

Paulownin, a natural compound derived from Paulownia tomentosa wood, exhibits various physiological functions, including anti-bacterial and anti-fungal effects. However, the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study, we investigated the effect of paulownin on NK cell activity both in vitro and in vivo, and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia, human colon, and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules, including granzyme B and perforin in NK-92 cells. Moreover, these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice, resulting in enhanced cytotoxicity against YAC-1 cells. Moreover, the antitumor effects of paulownin were reduced upon the depletion of NK cells. Therefore, these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: FMF is the most common autoinflammatory disease. The activation of the pyrin inflammasome is the mainstay of the pathogenesis, which might lead to a specific cell-death mechanism, pyroptosis. Pyroptosis is a programmed inflammatory cell death mediated by gasdermin proteins, featuring cell swelling, membrane rupture, and release of inflammatory contents Aim: In this study we aimed to analyze the cell-death mechanisms in the pathogenesis of FMF attacks. Methods: Twenty-five FMF patients were included, and PFAPA patients (n = 10) and healthy controls (HC, n = 10) served as controls. We collected plasma samples from FMF and PFAPA patients during the attack and the attack-free period. We measured the soluble plasma levels of sFas, sFasL, granzyme A, granzyme B, perforin, granulysin, IL-2, IL-4, IL-10, IL-6, IL-17A, TNF-α, and IFN-γ by commercial pre-defined cytometric bead array kits. Results: There was no significant difference between groups in terms of sex and age between FMF patients and HCs, but PFAPA patients were younger than other groups due to the nature of the disease. We then analyzed the components of apoptosis and pyroptosis. The levels of sFasL (p = 0.035) and granzyme A (p = 0.038) in FMF patients were significantly increased during the attack period and decreased to levels comparable to HCs during the attack-free period. This increase was not seen in the PFAPA patients, with comparable levels with the HC group both during attack period and attack-free period. During the attack period of FMF patients, granzyme B (p = 0.145) and perforin (p = 0.203) levels were also increased; however, the differences were not statistically significant. The levels of sFasL, granzyme A, granzyme B, and perforin were closely correlated with each other during the attack period of FMF patients. Conclusions: Our study on death pathways during an FMF attack, suggests an upregulation in both pyroptosis through the granzyme-gasdermin pathway and apoptosis with the increased FasL and perforin levels, which was different from PFAPA patients. These findings might shed light on the reason for the nature of self-limited attacks, but further studies are needed to prove this hypothesis. [ABSTRACT FROM AUTHOR]

This article discusses the association between cytotoxic effector memory CD8+ T cells and sustained unresponsiveness (SU) after peanut oral immunotherapy (OIT). The study found that a higher frequency of effector memory CD8+ T cells was associated with a higher likelihood of achieving SU. The researchers also identified differentially expressed immune genes and observed that cytolytic molecules such as Granulysin (Gnly) and Granzyme B (GzB) may play a supportive role in the development of SU. However, the study acknowledges the need for further validation using larger cohorts. [Extracted from the article]

Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the expression of GPR56 in the T cells of early-stage lung adenocarcinoma (LUAD) patients and clarify its diagnostic significance. Methods: Blood samples were collected from 32 patients with stage IA LUAD and 31 healthy controls. GPR56 and perforin were analysed in circulating T-cell subsets by flow cytometry. In addition, a correlation between perforin and GPR56 expression was detected. Changes in GPR56+ cells in early LUAD patients were analysed, and the diagnostic significance of GPR56+ T cells for early LUAD was studied by receiver operating characteristic (ROC) curve analysis. Results: The expression of GPR56 in CD8+ T cells from early-stage LUAD patients was significantly greater than that in CD4+ T cells. The percentage of perforin-positive GPR56+ cells in early-stage LUAD patients was high. GPR56 levels in the T cells of LUAD patients were significantly lower than those in healthy controls. ROC analysis revealed that the area under the curve for the percentage of GPR56-positive CD8+ TEMRA cells to distinguish early-stage LUAD patients from healthy individuals- reached 0.7978. Conclusion: The decreased expression of GPR56 in the peripheral blood of early-stage LUAD patients correlated with perforin levels, reflecting compromised antitumor immunity and aiding early-stage LUAD screening. [ABSTRACT FROM AUTHOR]

The advancement of cell‐mimic materials, which can forge sophisticated physicochemical dialogues with living cells, has unlocked a realm of intriguing prospects within the fields of synthetic biology and biomedical engineering. Inspired by the evolutionarily acquired ability of T lymphocytes to release perforin and generate transmembrane channels on targeted cells for killing, herein we present a pioneering DNA‐encoded artificial T cell mimic model (ARTC) that accurately mimics T‐cell‐like behavior. ARTC responds to acidic conditions similar to those found in the tumor microenvironment and then selectively releases a G‐rich DNA strand (LG4) embedded with C12 lipid and cholesterol molecules. Once released, LG4 effectively integrates into the membranes of neighboring live cells, behaving as an artificial transmembrane channel that selectively transports K+ ions and disrupts cellular homeostasis, ultimately inducing apoptosis. We hope that the emergence of ARTC will usher in new perspectives for revolutionizing future disease treatment and catalyzing the development of advanced biomedical technologies. [ABSTRACT FROM AUTHOR]

Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA. Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty's syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis, produced minimal citrullination. We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA. [ABSTRACT FROM AUTHOR]

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 CD47high pDCs and monocytes exhibited relatively higher expression of IFN-α regulatory genes, antiviral interferon-stimulated genes (ISGs) and MHC-I associated genes compared to CD47inter. and CD47low cells. Furthermore, CD47high 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, CD47high CD8+ T cells expressed higher levels of PD-1 and LAG-3 genes. Lastly, we found that CD47high 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. [ABSTRACT FROM AUTHOR]

In previous studies, we developed a novel fusion protein named "melittin‐MIL‐2" which exhibited more anti‐tumor activity. However, it remains unclear whether melittin‐MIL‐2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin‐MIL‐2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin‐MIL‐2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN‐γ secretion in PBMCs. After melittin‐MIL‐2 stimulation, perforin expression and LAK/NK‐like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin‐MIL‐2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM‐1 and Fas expression in A549 cells exposed to melittin‐MIL‐2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin‐MIL‐2 stimulation. In vivo, melittin‐MIL‐2 substantially impeded the growth of lung adenocarcinoma and formed an immune‐stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin‐MIL‐2 exhibits strong anti‐tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA‐1/ICAM‐1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN‐γ and perforin, and boosting LAK/NK‐like killing activities. Immuno‐effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue. [ABSTRACT FROM AUTHOR]

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow‐derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC‐mediated CTL priming, and further investigate the role of PI3K‐Akt engaged in K33O‐increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC‐based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R‐mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K‐Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC‐based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme‐linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K‐Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K‐Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC‐mediated CTL priming in bone marrow‐derived dendritic cells via PI3K‐Akt signalling. [ABSTRACT FROM AUTHOR]

Background: The impact of chronic hepatic infection on antigen non-specific immune cells in circulation remains poorly understood. We reported lasting global hyperfunction of peripheral CD8 T cells in HCV-infected individuals with cirrhosis. Whether gene expression patterns in bulk CD8 T cells are associated with the severity of liver fibrosis in HCV infection is not known. Methods: RNA sequencing of blood CD8 T cells from treatment naïve, HCV infected individuals with minimal (Metavir F0-1 ≤ 7.0 kPa) or advanced fibrosis or cirrhosis (F4 ≥ 12.5 kPa), before and after direct-acting antiviral therapy, was performed. CD8 T cell function was assessed by flow cytometry. Results: In CD8 T cells from pre-DAA patients with advanced compared to minimal fibrosis, Gene Ontology analysis and Gene Set Enrichment Analysis identified differential gene expression related to cellular function and metabolism, including upregulated Hedgehog (Hh) signaling, IFN-a, -g, TGF-b response genes, apoptosis, apical surface pathways, phospholipase signaling, phosphatidyl-choline/inositol activity, and second-messenger-mediated signaling. In contrast, genes in pathways associated with nuclear processes, RNA transport, cytoskeletal dynamics, cMyc/E2F regulation, oxidative phosphorylation, and mTOR signaling, were reduced. Hh signaling pathway was the top featured gene set upregulated in cirrhotics, wherein hallmark genes GLI1 and PTCH1 ranked highly. Inhibition of Smo-dependent Hh signaling ablated the expression of IFN-g and perforin in stimulated CD8 T cells from chronic HCV-infected patients with advanced compared to minimal fibrosis. CD8 T cell gene expression profiles post-DAA remained clustered with pre-DAA profiles and disparately between advanced and minimal fibrosis, suggesting a persistent perturbation of gene expression long after viral clearance. Conclusions: This analysis of bulk CD8 T cell gene expression in chronic HCV infection suggests considerable reprogramming of the CD8 T cell pool in the cirrhotic state. Increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease. [ABSTRACT FROM AUTHOR]

Objectives: Gout is characterized by hyperuricemia and recurrent inflammatory episodes caused by intra-articular crystal deposition of monosodium urate (MSU). There is a clear relationship between gout and metabolic syndrome. Recent evidence indicates that perforin plays a role in regulating glucose homeostasis and provides protection in diet-induced non-alcoholic steatohepatitis models. However, the impact of perforin on immune inflammation in gout remains unclear. Methods: We induced acute gout models in both wild-type (WT) mice and Prf1null mice by administering intra-articular injections of MSU crystals. We compared the ankle joint swelling and the histological score between the two groups. Furthermore, we investigated underlying mechanisms through in vitro co-culture experiments involving CD8 T cells and macrophages. Results: In this study, Prf1null mice showed significantly more pronounced ankle swelling with increased inflammatory cell infiltrations compared with WT mice 24 h after local MSU injection. Moreover, MSU-induced Prf1null mice exhibited increased accumulation of CD8 T cells but not NK cells. Perforin-deficient CD8 T cells displayed reduced cytotoxicity towards bone marrow–derived M0 and M1 macrophages and promoted TNF-α secretion from macrophage. Conclusions: Perforin from CD8 T cells limits joint inflammation in mice with acute gout by downregulating macrophage-mediated inflammation. Key Points • Perforin deficiency increased swelling in the ankle joints of mice upon MSU injection. • Perforin deficiency is associated with increased immune cell recruitment and severe joint damage in gout. • Perforin regulated CD8 T cell accumulation in gout and promoted CD8 T cell cytotoxicity towards M0 and M1 macrophages. • CD8 T cell-derived perforin regulated pro-inflammatory cytokine secretion of macrophage. [ABSTRACT FROM AUTHOR]

Objective Unravel the potential mechanism(s) of the on- and off-target actions of dopamine agonist therapy in both human prolactinoma tumors and neighboring stromal and immune cells. Design and Methods Five surgically resected prolactinomas (PRLomas) from 3 cabergoline (CBG)-treated patients and 2 treatment-naive patients were analyzed by using single-cell RNA sequencing (scRNA-seq) to compare the cellular composition and transcriptional landscape. Results Six major cell populations, namely tumor (88.2%), immune (5.6%), stromal (4.9%), progenitor cells (0.6%), proliferating cells (0.4%), and erythrocytes (0.2%), were observed. Tumor cells from CBG-treated patients expressed lower levels of genes that regulated hormone secretion, such as SCG2, VGF, TIMP1, NNAT, and CALD1, consistent with the inhibitory effects of CBG on hormone processing and secretion. Interestingly, we also observed an increased number of CD8+ T cells in the CBG-treated tissues. These cytotoxic CD8+ T cells expressed killing granule components such as perforin and the granzymes GZMB, GNLY, and KLRD1 as well as the inflammatory cytokine CCL5. Immune cell activation of these CD8+ T cells was further analyzed in a compartment-specific manner, and increased CD25 (IL2R) expression was noted in the CD8+ T cells from the CBG-treated samples. Additionally, and confirming prior reports, we noted a higher stromal cell population in the CBG-treated samples. Conclusions Our scRNA-seq studies revealed key differences in the transcriptomic features of CBG-treated and CBG-untreated PRLomas in both tumor and microenvironment cellular constituents, and for the first time, describe the previously unknown activation of CD8+ T cells following CBG treatment, which may play a role in the tumoricidal actions of CBG. [ABSTRACT FROM AUTHOR]

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

Objective: To investigate mechanisms of IL-17D on cytotoxic function of CD93+CTL in lung tumor microenviron ment (TME) and promotion of Platycodon grandiflorum (PG). Methods: Lung of B16 melanoma model mice and control mice were treated with PG or IL-17D, and lung tumor clone formation was observed. IL-17D expression change in lung was detected by single cell sequencing, immunofluorescence staining and flow cytometry. Single cell sequencing and flow cytometry were used to detect CD93+CTL content, cytotoxic phenotype and functional factors changes, including CD107a, perforin, granzyme B, chemokine CCL2, CXCL9 and their ligand CCR2 and CXCR3. Western blot and RT-PCR were used to detect effect of Platycodonopsis saponin D on IL-17D and its transcriptional regulator NRF2 expressions in EL4 cells. Results: Pulmonary CD93+CTL highly expressed cytotoxic effectors such as perforin and CD107a and chemokine receptors CXCR3 and CCR2 than CD93-CTL, while there was no significant difference in secretion of granzyme B. In mouse lung tumor model, pulmonary IL-17D and CD93+CTL were significantly decreased (P< 0.001) ; in tumor-bearing mice after IL-17D backfill assay, or after 10 days of treatment with PG, proportion and absolute number of IL-17D and CD93+CTL in lungs were significantly incr eased (P<0.05), and tumor clones were significantly reduced; meanwhile, tumor-local expressions of cytokines CCL2, CXCL9, which are related to recruitment and function of CD93+CTL, and IL-17D were significantly upregulated. Up-regulation of IL-17D and its transcriptional regulator NRF2 by PG was verified in vitro experiments on EL4 cell line by PD. Conclusion: Traditional Chinese medicine PG and its extracts can up-regulate expression of IL-17D in lungs, improve infiltration and cytotoxic function of CD93+CTL and antagonize malignant progression of lung tumors, this is an important pharmacological mechanism of PG in improving immune TME of tumors in lung. [ABSTRACT FROM AUTHOR]

Granzyme B (GrB)-based immunotherapy is of interest for cancer treatment. However, insufficient cellular uptake and a lack of targeting remain challenges to make use of GrB for solid tumour therapy. As GrB induced cell death requires the help of perforin (PFN), we designed a system (nGPM) for the co-delivery of GrB and PFN. Therefore, GrB and PFN were loaded in a porous polymeric nanocapsule rich in acetylcholine analogues and matrix metalloproteinase-2 (MMP-2) responsive peptides. The neutrally charged nGPM nanocapsules showed as long circulating time and accumulated at the tumour sites. Once in the tumour the outside shell of nanocapsules became degraded by overexpressed MMP-2 proteases, resulting in the release of GrB and PFN. We found that the PFN complex formed small pores on the surface of tumour cells which allow GrB to enter the cytoplasm of tumour cells inducing cell apoptosis and tumour suppression significantly. [Display omitted] [ABSTRACT FROM AUTHOR]

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella -specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella , we provide the first proof for reverse recruitment from venom to organismal development. [ABSTRACT FROM AUTHOR]

The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10melanoma syngeneic tumormodel demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes. [ABSTRACT FROM AUTHOR]

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6–pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid. [ABSTRACT FROM AUTHOR]

Mechanical force contributes to perforin pore formation at immune synapses, thus facilitating the cytotoxic T lymphocytes (CTL)-mediated killing of tumor cells in a unidirectional fashion. How such mechanical cues affect CTL evasion of perforin-mediated autolysis remains unclear. Here we show that activated CTLs use their softness to evade perforin-mediated autolysis, which, however, is shared by T leukemic cells to evade CTL killing. Downregulation of filamin A is identified to induce softness via ZAP70-mediated YAP Y357 phosphorylation and activation. Despite the requirements of YAP in both cell types for softness induction, CTLs are more resistant to YAP inhibitors than malignant T cells, potentially due to the higher expression of the drug-resistant transporter, MDR1, in CTLs. As a result, moderate inhibition of YAP stiffens malignant T cells but spares CTLs, thus allowing CTLs to cytolyze malignant cells without autolysis. Our findings thus hint a mechanical force-based immunotherapeutic strategy against T cell leukemia. Cell softness protects cytotoxic T lymphocytes (CTL) from autolysis by own soluble factors such as perforin secreted for killing target cells. Here the authors show that softness can be induced by YAP activation, and that T leukemic cells are more sensitive to YAP inhibition than CTLs, thereby hinting YAP inhibitors as a potential therapy for T leukemia. [ABSTRACT FROM AUTHOR]

We investigated the polarisation of CD68+ macrophages and perforin and granulysin distributions in kidney lymphocyte subsets of children with IgA vasculitis nephritis (IgAVN). Pro-inflammatory macrophage (M)1 (CD68/iNOS) or regulatory M2 (CD68/arginase-1) polarisation; spatial arrangement of macrophages and lymphocytes; and perforin and granulysin distribution in CD3+ and CD56+ cells were visulaised using double-labelled immunofluorescence. In contrast to the tubules, iNOS+ cells were more abundant than the arginase-1+ cells in the glomeruli. CD68+ macrophage numbers fluctuated in the glomeruli and were mostly labelled with iNOS. CD68+/arginase-1+ cells are abundant in the tubules. CD56+ cells, enclosed by CD68+ cells, were more abundant in the glomeruli than in the tubuli, and co-expressed NKp44. The glomerular and interstitial/intratubular CD56+ cells express perforin and granulysin, respectively. The CD3+ cells did not express perforin, while a minority expressed granulysin. Innate immunity, represented by M1 macrophages and CD56+ cells rich in perforin and granulysin, plays a pivotal role in the acute phase of IgAVN. [ABSTRACT FROM AUTHOR]

Despite effective antiretroviral therapies, 20–30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with β-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

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

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell–cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Interleukin-36 (IL-36) signaling plays an important role in promoting CD8+ T cell-mediated antitumor immune responses. The role of IL-36 signaling in CD8+ T cells that are involved in host immune responses during human immunodeficiency virus-1 (HIV-1) infection has not been characterized. Sixty-one patients living with chronic HIV-1 infection and 23 controls were enrolled in this study. The levels of IL-36 cytokine family members were measured by enzyme-linked immunosorbent assay. Purified CD8+ T cells were stimulated with recombinant IL-36gamma (1 or 10 ng/mL). The expression of inhibitory receptors, the secretion of cytotoxic molecules and interferon-gamma, and the mRNA levels of apoptosis-related ligands were assessed to evaluate the effect of IL-36gamma on CD8+ T cell function in vitro. There were no significant differences in IL-36alpha, IL-36beta, or IL-36 receptor antagonist levels between patients living with chronic HIV-1 infection and controls. Plasma IL-36gamma levels were reduced in patients living with chronic HIV-1 infection. Perforin, granzyme B, and granulysin secretion, as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) mRNA expression, but not programmed death-1 (PD-1) or cytotoxic T lymphocyte-associated protein-4 (CTLA-4) expression was downregulated in CD8+ T cells from patients living with chronic HIV-1 infection. The addition of both 1 and 10 ng/mL IL-36gamma enhanced perforin, granzyme B, granulysin, and interferon-gamma secretion by CD8+ T cells without affecting PD-1/CTLA-4 or TRAIL/FasL mRNA expression in CD8+ T cells from patients living with chronic HIV-1 infection. The addition of 1 ng/mL IL-36gamma also promoted perforin and granzyme B secretion by HIV-1-specific CD8+ T cells from patients living with chronic HIV-1 infection. The reduced IL-36gamma levels in patients living with chronic HIV-1 infection might be insufficient for the activation of CD8+ T cells, leading to CD8+ T cell exhaustion. [ABSTRACT FROM AUTHOR]

Objective: To study the bone marrow (BM) immunohistomorphological characteristics in adult systemic lupus erythematosus (SLE) associated macrophage activation syndrome (SLE-MAS). Materials and methods: Immunohistochemical (IHC) expression of CD3, CD8, perforin (PFN), and CD163 was studied on BM trephine biopsies from 30 cytopenic adult SLE cases (male: female = 1:5, age; 24 years, range; 19–32) and compared them with ten age matched controls. Clinicopathological parameters were compared among the cases likely (L) or unlikely (U) to have MAS using probability scoring criteria. The best cut off laboratory parameters to discriminate between the two were obtained through receiver operator curve (ROC) analysis. Results: MAS occurred in 12/30 (40%) cases and was more commonly associated with prior immunosuppressive therapy (p =.07), ≥ 3 system involvement (p =.09), lower fibrinogen (p <.01), increased triglyceride (p =.002), increased BM hemophagocytosis (p =.002), and higher MAS score [185 (176–203) vs. 105 (77–119), p <.01] than MAS-U subgroup. Although PFN+CD8+ T lymphocytes significantly decreased among cases than controls (p <.05), it was comparable between MAS-L and MAS-U subgroups. Fibrinogen (< 2.4 g/L, AUC; 0.93, p <.01), hemophagocytosis score (> 1.5, AUC; 0.71, p =.03), and an MAS probability score of ≥ 164 (AUC; 1, p <.01) discriminated MAS from those without MAS. Conclusion: We noted a decrease in perforin mediated CD8 + T cell cytotoxicity in SLE. Immunohistochemical demonstration of the same along with histiocytic hemophagocytosis on BM biopsy may be useful adjunct in early diagnosis and management of MAS in SLE. [ABSTRACT FROM AUTHOR]

CCI increases C3b deposition as a response to nerve injury. C3 convertase (C3bBb) is regulated by the inhibitors CR1, CD55 or CFH. In the absence of inhibitors, C3b cleaves C5 into C5a and C5b. C5b along with C6, C7, C8 and poly-C9 combine to form the terminal component, the membrane attack complex (MAC). MAC targeting myelin leads to myelin degradation, which is cleared by phagocytes. MAC can be regulated by the binding of inhibitors Clusterin and CD59. Voluntary wheel running appears to reduce MAC deposition by increasing the inhibitor CD59, thereby preserving myelin. Running did not alter Clusterin or C3b inhibitors in a manner that would result in decreased MAC. [Display omitted] • Six weeks of wheel running prior to nerve injury reduced MAC at the injury site. • Loss of myelin in the injury site after CCI was decreased with prior running. • Complement C3, CD55, CR1, CFH, Clusterin and CD59 were altered by CCI. • CD59, an inhibitor of MAC was increased with prior running. • Treatment with exogenous CD59 reduced mechanical allodynia and MAC deposition. Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are involved in the suppression of activated T cells in generalized vitiligo (GV). The study was aimed to investigate resident memory (TRM)‐Tregs and antigen‐specific Tregs' numbers and functional defects in 25 GV patients and 20 controls. CD4+ & CD8+ TRM cell proliferation was assessed by BrDU assay; production of IL‐10, TGF‐β, IFN‐γ, perforin and granzyme B were assessed by ELISA and enumeration of TRM cells was done by flowcytometry. GV patients showed significantly increased frequency and absolute count of CD4+ & CD8+ TRM cells in lesional (L), perilesional (PL) and non‐lesional (NL) skin compared to controls (p = 0.0003, p = 0.0029 & p = 0.0115, respectively & p = 0.0003, p = 0.003 & p = 0.086, respectively). Whereas, TRM‐Treg (p < 0.0001 & p = 0.0015) and antigen‐specific Tregs (p = 0.0014 & p = 0.003) exhibited significantly decreased frequency and absolute counts in L & PL skin. GV patients showed reduced suppression of CD8+ & CD4+ TRM cells (with increased IFN‐γ, perforin & granzyme B) and decreased TRM‐Tregs and antigen‐specific Tregs (with decreased IL‐10 & TGF‐β production) and reduced proliferation of SK‐Mel‐28 cells in co‐culture systems. Immunohistochemistry revealed increased expression of TRM stimulating cytokines: IL‐15 & IL‐17A and reduced expression of TGF‐β & IL‐10 in L, PL, NL skins compared to controls. These results for the first time suggest that decreased and impaired TRM‐Tregs and antigen‐specific Tregs are unable to suppress CD4+ & CD8+ TRMs' cytotoxic function and their proliferation due to decrease production of immunosuppressive cytokines (IL‐10 & TGF‐β) and increased production of TRM based IFN‐γ, perforin and granzyme B production, thus compromising the melanocyte survival in GV. [ABSTRACT FROM AUTHOR]

There is evidence that the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in exhausted CD8 + T cells and regulates PD-L1 in tumors. This study investigated the effects of potent bis-indole-derived NR4A1 antagonists on reversing T-cell exhaustion and downregulating PD-L1 in colon tumors/cells. NR4A1 antagonists inhibited colon tumor growth and downregulated expression of PD-L1 in mouse colon MC-38-derived tumors and cells. TILs from MC-38 cell-derived colon tumors and splenic lymphocytes exhibited high levels of the T-cell exhaustion markers including PD-1, 2B4, TIM3+ and TIGIT and similar results were observed in the spleen, and these were inhibited by NR4A1 antagonists. In addition, treatment with NR4A1 antagonists induced cytokine activation markers interferon γ, granzyme B and perforin mRNAs and decreased TOX, TOX2 and NFAT in TIL-derived CD8 + T cells. Thus, NR4A1 antagonists decrease NR4A1-dependent pro-oncogenic activity and PD-L1 expression in colon tumors and inhibit NR4A1-dependent T-cell exhaustion in TILs and spleen and represent a novel class of mechanism-based drugs that enhance immune surveillance in tumors. [ABSTRACT FROM AUTHOR]

This experiment will explore the role of TIGIT/PVR signaling pathway in the pathogenesis of MDS immune tolerance through in vitro co-culture of NK cells and MDSC cells. Flow cytometry was used to detect the expression percentage of MDSCs and CD155 on MDSCs in the bone marrow of MDS patients and controls. The expression of NK cell surface receptors (NKG2D, NKp30, NKp46), secreted cytokines (perforin, granzyme B, CD107a, IFN-γ) and NK cell apoptosis rate were detected by flow cytometry to evaluate the effect of MDSCs on NK cell function. The number of MDSCs in bone marrow of MDS patients was notably higher than that of the control group (8.39 ± 7.01 vs 2.31 ± 1.65, P = 0.0001). Compared with the control group, the expression of CD155 on MDSCs in MDS group was increased (31.81 ± 21.33 vs. 10.49 ± 6.53, P < 0.0001). After NK cells were co-cultured with MDSCs, NKG2D, NKp30, NKp46, CD107a, IFN-γ, perforin and granzyme B were decreased, and the NK function partially recovered after the addition of inhibitors. Compared with the normal control, MDSCs and CD155 on MDSCs were highly expressed in MDS patients. After co-culture with MDSCs, the expression of NK cells' surface receptors decreased, the secretion of cytokines decreased and the apoptosis rate increased. After blocking TIGIT/CD155 pathway, NK cell function was reversed, but NK cell apoptosis was not reduced. [ABSTRACT FROM AUTHOR]

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

Background: The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. Methods: A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. Results: MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT- positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. Conclusion: This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells. [ABSTRACT FROM AUTHOR]

• T cells in the chronically infected brain express metabotropic glutamate receptors. • Using flow cytometry and scRNAseq analysis, mGluR expressing T cells are a defined subset of CD8+ T cells with a memory phenotype. • Production of IFN-g depends on mGluR stimulation. Within the brain, a pro-inflammatory response is essential to prevent clinical disease due to Toxoplasma gondii reactivation. Infection in the immunocompromised leads to lethal Toxoplasmic encephalitis while in the immunocompetent, there is persistent low-grade inflammation which is devoid of clinical symptoms. This signifies that there is a well-balanced and regulated inflammatory response to T. gondii in the brain. T cells are the dominant immune cells that prevent clinical disease, and this is mediated through the secretion of effector molecules such as perforins and IFN-γ. The presence of cognate antigen, the expression of survival cytokines, and the alteration of the epigenetic landscape drive the development of memory T cells. However, specific extrinsic signals that promote the formation and maintenance of memory T cells within tissue are poorly understood. During chronic infection, there is an increase in extracellular glutamate that, due to its function as an excitatory neurotransmitter, is normally tightly controlled in the CNS. Here we demonstrate that CD8+ T cells from the T. gondii -infected brain parenchyma are enriched for metabotropic glutamate receptors (mGluR's). Characterization studies determined that mGluR+ expression by CD8+ T cells defines a distinct memory population at the transcriptional and protein level. Finally, using receptor antagonists and agonists we demonstrate mGluR signaling is required for optimal CD8+ T cell production of the effector cytokine IFNγ. This work suggests that glutamate is an important environmental signal of inflammation that promotes T cell function. Understanding glutamate's influence on T cells in the brain can provide insights into the mechanisms that govern protective immunity against CNS-infiltrating pathogens and neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: Inflammasome activation and the subsequent release of pro-inflammatory cytokines including Interleukin 1β (IL-1β) have been widely reported to contribute to the progression of retinal degenerations, including age-related macular degeneration (AMD), the leading cause of blindness in the Western World. The role of Gasdermin D (GSDMD), a key executioner of pyroptosis following inflammasome activation, however, is less well-established. In this study we aimed to characterise the role of GSDMD in the healthy and degenerating retina, and uncover its role as a conduit for IL-1β release, including via extracellular vesicle (EV)-mediated release. Methods: GSDMD mutant and knockout mice, in vitro models of inflammation and a well-established in vivo model of retinal degeneration (photo-oxidative damage; PD) were utilised to explore the role and pathological contribution of GSDMD in regulating IL-1β release and propagating retinal inflammation. RNA sequencing of whole retinas was used to investigate GSDMD-mediated inflammation during degeneration. The role of EVs in GSDMD-mediated IL-1β release was investigated using nanoparticle tracking analysis, ELISA and EV inhibition paradigms. Finally, the therapeutic efficacy of targeting GSDMD was examined using GSDMD-specific siRNA. Results: We identified in this work that mice deficient in GSDMD had better-preserved retinal function, increased photoreceptor survivability and reduced inflammation. RNA-Seq analysis revealed that GSDMD may propagate inflammation in the retina via NF-κB signalling cascades and release of pro-inflammatory cytokines. We also showed that IL-1β was packaged and released via EV in a GSDMD-dependent manner. Finally, we demonstrated that impairing GSDMD function using RNAi or blocking EV release was able to reduce IL-1β content in cell-free supernatant and EV. Conclusions: Taken together, these results suggest that pyroptotic pore-forming protein GSDMD plays a key role in the propagation of retinal inflammation, in particular via the release of EV-encapsulated IL-1β. Targeting GSDMD using genetic or pharmacological inhibitors may pose a therapeutic opportunity to dampen inflammatory cascades and delay the progression of retinal degeneration. [ABSTRACT FROM AUTHOR]

Background: The diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed. Methods: LAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry. Results: According to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SUDHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis. Conclusion: High LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL. [ABSTRACT FROM AUTHOR]

Background: In kidney transplant recipients (KTRs) who are immunosuppressed, human BK polyomavirus (BKPyV) infection can be reactivated, resulting in BKPyV-associated nephropathy (BKPyVN). Considering that BKPyV inhibits CD4+ T cell differentiation, we investigated the effect of BKPyV large T antigen (LT-Ag) on the maturation of CD4+ T cell subsets during active BKPyV infection. Methods: In this cross-sectional study, we examined the following groups: 1) five KTRs with active viral infection (BKPyV+ KTRs), 2) five KTRs without active viral infection (BKPyV-KTRs), and 3) five healthy controls. We measured the frequency of CD4+ T cells and their different subsets, such as naive T cells, central memory T cells (Tcm), and effector memory T cells (Tem). All these subsets were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs) stimulated with the overlapping BKPyV LT-Ag peptide pool. In addition, CD4+ T cell subsets were analyzed by flow cytometry for the presence of CD4, CCR7, CD45RO, CD107a, and granzyme B (GB). In addition, mRNA expression of transcription factors (TFs) such as T-bet, GATA-3, STAT-3, and STAT-6 was examined. The probability of inflammation with perforin protein was examined by SYBR Green real-time PCR. Results: After stimulation of PBMCs, naive T cells (CD4+CCR7+CD45RO-) (p = 0.9) and CD4+ T cells which release CD107a+ (CD4+CD107a+Geranzyme B-) (p = 0.9) T cells were more abundant in BKPyV+ KTRs than in BKPyV- KTRs. In contrast, central memory T cells (CD4+CCR7+CD45RO+) (p = 0.1) and effector memory T cells (CD4+CCR7-CD45RO+) (p = 0.1) were more abundant in BKPyV- KTRs than in BKPyV+ KTRs. The mRNA expression levels of T-bet, GATA-3, STAT-3, and STAT-6 were significantly higher (p < 0.05) in BKPyV- KTRs than in BKPyV+ KTRs which may be due to a higher differentiation level of CD4+ T cells. Due to inflammation, the mRNA expression level of perforin was higher in BKPyV+ KTRs, than in BKPyV- KTRs, but the difference was not significant (p = 0.175). Conclusions: The high number of naive T cells after PBMC stimulation with the LT-Ag peptide pool was observed in BKPyV+ KTRs due to the interaction of LT-Ag with T cells. This means that BKPyV by using its LT-Ag can inhibit the naive T cell differentiation to other T cell subsets like central and effector memory T cells. However, the frequency of CD4+ T cell subsets and the combination of the activities of these cells with the expression profile of the target genes in this study may be efficient in treating and diagnosing BKPyV infections in kidney recipients. [ABSTRACT FROM AUTHOR]

Problem: Pyruvate dehydrogenase kinase 1 (PDK1) is an important enzyme for immune cell development. However, PDK1's role in human decidual natural killer (dNK) cells remains largely unknown. Methods of study: PDK1 expression in dNK cells from patients with recurrent spontaneous abortions (RSA) and age‐matched healthy controls was analyzed by qRT‐PCR, western bolt and flow cytometry. Moreover, dNK cells were treated with PDK1 inhibitor or the PDK1 siRNA followed by functional assays. Results: The dNK cells from patients who underwent RSAs had higher mRNA expression and increased protein of PDK1, perforin (PRF1), Granzyme B (GZMB), IFN‐γ (IFNG), and CD107a expression compared to dNK cells from age‐matched healthy controls. Perforin, Granzyme B, IFN‐γ and CD107a expression levels in dNK cells were down‐regulated when dNK cells were treated with a PDK1 inhibitor. As measured by the 51Cr release assay, the killing activity of dNK cells was found to be decreased. We also demonstrated that PDK1 blockade could up‐regulate the migration and adhesion of dNK cells. Furthermore, PDK1 inhibition reduced the glycolysis of dNK cells. Conclusion: This study suggested that PDK1 plays an important role in regulating dNK cell functions and human RSA. [ABSTRACT FROM AUTHOR]

Nitrogen from ammonia is one of the most common pollutants toxics to aquatic species in aquatic environment. The intestinal mucosa is one of the key mucosal defenses of aquatic species, and the accumulation of ammonia nitrogen in water environment will cause irreversible damage to intestinal function. In this study, histology, immunohistochemistry, ultrastructural pathology, enzyme activity analysis and qRT-PCR were performed to reveal the toxic effect of ammonia nitrogen stress on the intestine of Pelteobagrus fulvidraco. According to histological findings, ammonia nitrogen stress caused structural damage to the intestine and reduced the number of mucous cells. Enzyme activity analysis revealed that the activity of bactericidal substances (Lysozyme, alkaline phosphatase, and ACP) had decreased. The ultrastructure revealed sparse and shortened microvilli as well as badly degraded tight junctions. Immunohistochemistry for ZO-1 demonstrated an impaired intestinal mucosal barrier. Furthermore, qRT-PCR revealed that tight junction related genes (ZO-1, Occludin, Claudin-1) were downregulated, while the pore-forming protein Claudin-2 was upregulated. Furthermore, as ammonia nitrogen concentration grew, so did the positive signal of Zap-70 (T/NK cell) and the expression of inflammation-related genes (TNF, IL-1β, IL-8, IL-10). In light of the above findings, we conclude that ammonia nitrogen stress damages intestinal mucosal barrier of Pelteobagrus fulvidraco and induces intestinal inflammation. [ABSTRACT FROM AUTHOR]

Background: Systemic and local profiles have each been associated with asthma, but parsing causal relationships between system-wide and airway-specific processes can be challenging. We sought to investigate systemic and airway processes in asthma and their causal relationships. Methods: Three hundred forty-one participants with persistent asthma and non-asthmatic controls were recruited and underwent peripheral blood mononuclear cell (PBMC) collection and nasal brushing. Transcriptome-wide RNA sequencing of the PBMC and nasal samples and a series of analyses were then performed using a discovery and independent test set approach at each step to ensure rigor. Analytic steps included differential expression analyses, coexpression and probabilistic causal (Bayesian) network constructions, key driver analyses, and causal mediation models. Results: Among the 341 participants, the median age was 13 years (IQR = 10–16), 164 (48%) were female, and 200 (58.7%) had persistent asthma with mean Asthma Control Test (ACT) score 16.6 (SD = 4.2). PBMC genes associated with asthma were enriched in co-expression modules for NK cell-mediated cytotoxicity (fold enrichment = 4.5, FDR = 6.47 × 10−32) and interleukin production (fold enrichment = 2.0, FDR = 1.01 × 10−15). Probabilistic causal network and key driver analyses identified NK cell granule protein (NKG7, fold change = 22.7, FDR = 1.02 × 10−31) and perforin (PRF1, fold change = 14.9, FDR = 1.31 × 10−22) as key drivers predicted to causally regulate PBMC asthma modules. Nasal genes associated with asthma were enriched in the tricarboxylic acid (TCA) cycle module (fold enrichment = 7.5 FDR = 5.09 × 10−107), with network analyses identifying G3BP stress granule assembly factor 1 (G3BP1, fold change = 9.1 FDR = 2.77 × 10−5) and InaD-like protein (INADL, fold change = 5.3 FDR = 2.98 × 10−9) as nasal key drivers. Causal mediation analyses revealed that associations between PBMC key drivers and asthma are causally mediated by nasal key drivers (FDR = 0.0076 to 0.015). Conclusions: Integrated study of the systemic and airway transcriptomes in a well-phenotyped asthma cohort identified causal key drivers of asthma among PBMC and nasal transcripts. Associations between PBMC key drivers and asthma are causally mediated by nasal key drivers. [ABSTRACT FROM AUTHOR]

Background: Intraepithelial lymphocytes (IELs) are the first immune cells to contact and fight intestinal pathogens such as Cryptosporidium, a widespread parasite which infects the gut epithelium. IFN-γ producing CD4+ T IELs provide an efficient and a long-term protection against cryptosporidiosis while intraepithelial type 1 innate lymphoid cells limits pathogen spreading during early stages of infection in immunodeficient individuals. Yet, the role of T-cell like innate IELs, the most frequent subset of innate lymphocytes in the gut, remains unknown. Methods: To better define functions of innate IELs in cryptosporidiosis, we developed a co-culture model with innate IELs isolated from Rag2-/- mice and 3D intestinal organoids infected with C. parvum using microinjection. Results: Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IELs secrete IFN-g in response to C. parvum, the cytokine was not sufficient to inhibit parasite proliferation at early stages of the infection. The rapid protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis of the Cryptosporidium-infected organoids revealed that epithelial cells down regulated Serpinb9b, a granzyme inhibitor, which may increase their sensitivity to cytolytic attack by innate IELs. Conclusion: Based on these data we conclude that innate IELs, most likely T-cell-like innate IELs, provide a rapid protection against C. parvum infection through a perforin/granzymes-dependent mechanism. C. parvum infection. The infection may also increase the sensitivity of intestinal epithelial cells to the innate IEL-mediated cytotoxic attack by decreasing the expression of Serpin genes. [ABSTRACT FROM AUTHOR]

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