Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 µM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

Granzymes in health and diseases: the good, the bad and the ugly.

Granzymes are a family of serine proteases, composed of five human members: GA, B, H, M and K. They were first discovered in the 1980s within cytotoxic granules released during NK cell- and T cell-mediated killing. Through their various proteolytic activities, granzymes can trigger different pathways within cells, all of which ultimately lead to the same result, cell death. Over the years, the initial consideration of granzymes as mere cytotoxic mediators has changed due to surprising findings demonstrating their expression in cells other than immune effectors as well as new intracellular and extracellular activities. Additional roles have been identified in the extracellular milieu, following granzyme escape from the immunological synapse or their release by specific cell types. Outside the cell, granzyme activities mediate extracellular matrix alteration via the degradation of matrix proteins or surface receptors. In certain contexts, these processes are essential for tissue homeostasis; in others, excessive matrix degradation and extensive cell death contribute to the onset of chronic diseases, inflammation, and autoimmunity. Here, we provide an overview of both the physiological and pathological roles of granzymes, highlighting their utility while also recognizing how their unregulated presence can trigger the development and/or worsening of diseases.

Granzyme B May Act as an Effector Molecule to Control the Inflammatory Process in COPD.

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 FEV1%, suggesting an important role for Treg cells in COPD progression. Treg cells can regulate immunological responses via the granzyme B (GzmB) pathway. Immunohistochemical staining for GzmB in surgically resected lungs with centrilobular emphysema showed that the relationship between the amount of GzmB+ cells and FEV1% was comparable to that between Treg cell count and FEV1% in the COPD lung, suggesting that GzmB could be a functional marker for Treg cells. The volume fraction of GzmB+ cells in the small airways, the number of alveolar GzmB+ cells, and GzmB expression measured by enzyme-linked immunosorbent assay in the lung tissue of smokers were significantly correlated with FEV1%. These results suggest that the GzmB content in lung tissue may determine the progression of COPD by acting as an effector molecule to control inflammatory process. Interventions to augment GzmB-producing immunosuppressive cells in the early stages of COPD could help prevent or delay COPD progression.

Granzyme B is elevated in esophageal biopsies from children with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is an immune-mediated antigen-triggered inflammatory disease of the esophagus. Our aim was to investigate inflammatory responses by an ex vivo biopsy provocation-based method, stimulating biopsies with milk, wheat, and egg extracts. METHODS: An experimental study was conducted on esophageal biopsies from children who underwent esophagogastroduodenoscopy. Supernatants were collected before and after stimulation of the biopsies with food extracts and analyzed for 45 different inflammatory markers. Biopsies were also stained for histological analyzes. RESULTS: Study subjects included 13 controls, 9 active EoE, and 4 EoE in remission, median age 12 years. Of the 45 markers analyzed, three had significant differences between controls and patients with active EoE, Granzyme B, (GzmB), IL-1ra, and CXCL8 (p < .05). Levels of GzmB were higher, and levels of IL-1ra were lower in patients with active EoE compared with controls and EoE in remission both at baseline and after food extract stimulation. CXCL8 increased in active EoE compared with controls only after stimulation. The number of histologically detected GzmB-positive cells were significantly higher in patients with active EoE in contrast to control and EoE remission (p < .05). CONCLUSIONS: The levels of the barrier-damaging protease GzmB were higher in the supernatant both before and after stimulation with food extract ex vivo in patients with active EoE. GzmB was also observed histologically in biopsies from patients with active EoE. The presence of elevated serine protease GzmB in esophageal mucosa of children with active EoE suggests a role in the pathogenesis of this disorder.

Development of novel HPV therapeutic vaccine constructs based on engineered exosomes and tumor cell lysates.

AIMS: Human papillomavirus (HPV) infections are highly prevalent globally. While preventive HPV vaccines exist, therapeutic vaccines are needed to treat existing HPV lesions and malignancies. This study evaluated the immunostimulatory and anti-tumor effects of three therapeutic vaccine candidates based on the recombinant protein, tumor cell lysate (TCL), and engineered exosome (Exo) harboring the heat shock protein 27 (Hsp27)-E7 fusion construct in mouse model. MAIN METHODS: At first, the recombinant Hsp27-E7 protein was generated in E. coli expression system. Then, tumor cell lysates-based and engineered exosomes-based vaccine constructs harboring green fluorescent protein (GFP) and Hsp27-E7 were produced using lentiviral system. Finally, their immunological and antitumor effects were investigated in both prophylactic and therapeutic experiments. KEY FINDINGS: Our data showed that the recombinant Hsp27-E7 protein, TCL-Hsp27-E7 and Exo-Hsp27-E7 regimens can induce the highest level of IFN-γ, TNF-α and Granzyme B, respectively. The percentage of tumor-free mice was identical for three vaccine strategies (survival rate: 75 %) in both prophylactic and therapeutic experiments. Generally, the TCL-Hsp27-E7, Exo-Hsp27-E7 and recombinant Hsp27-E7 protein regimens induced effective immune responses toward Th1 and CTL activity, and subsequently antitumor effects in mouse model. SIGNIFICANCE: Regarding to higher Granzyme B secretion, lower tumor growth and more safety, the Exo-Hsp27-E7 regimen can be considered as the most promising HPV vaccination strategy.

A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

Compromised melanocyte survival due to decreased suppression of CD4+ & CD8+ resident memory T cells by impaired TRM-regulatory T cells in generalized vitiligo patients.

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-ß, 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-ß 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-ß & 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-ß) and increased production of TRM based IFN-γ, perforin and granzyme B production, thus compromising the melanocyte survival in GV.

Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity.

Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the CNS. CD4+ T cells are assumed to be the first to cross the blood-central nervous system (CNS) barrier and trigger local inflammation. Here, we explored how pathogenicity-associated effector programs define CD4+ T cell subsets with brain-homing ability in MS. Runx3- and Eomes-, but not T-bet-expressing CD4+ memory cells were diminished in the blood of MS patients. This decline reversed following natalizumab treatment and was supported by a Runx3+ Eomes+ T-bet- enrichment in cerebrospinal fluid samples of treatment-naïve MS patients. This transcription factor profile was associated with high granzyme K (GZMK) and CCR5 levels and was most prominent in Th17.1 cells (CCR6+ CXCR3+ CCR4-/dim ). Previously published CD28- CD4 T cells were characterized by a Runx3+ Eomes- T-bet+ phenotype that coincided with intermediate CCR5 and a higher granzyme B (GZMB) and perforin expression, indicating the presence of two separate subsets. Under steady-state conditions, granzyme Khigh Th17.1 cells spontaneously passed the blood-brain barrier in vitro. This was only found for other subsets including CD28- cells when using inflamed barriers. Altogether, CD4+ T cells contain small fractions with separate pathogenic features, of which Th17.1 seems to breach the blood-brain barrier as a possible early event in MS.

Detection of Granzyme B-associated Binding Targets in Peripheral Blood Samples of Hosts in Sickness and in Health Using a Granzyme B-like Peptide Fluorescent Conjugate (GP1R).

Granzyme B, mostly expressed by cytotoxic T lymphocytes in the fight against cancer and infection, is known to induce cell death based on its active enzymatic activity as a serine protease. Recent studies showed cytotoxicity of a non-enzymatic granzyme B-like peptide (also referred to as granzyme B-associated peptide or GP1 in this report) in tumor cells and presence of binding targets for GP1R (i.e., GP1 conjugated with rhodamine fluorochrome) in tumor cells, bacteria, and circulating platelets/neutrophils of healthy hosts. But there were no data on "sick" hosts to help substantiate any potential GP1 based medical applications. Thus, we adopted similar GP1R binding protocols to further study binding of GP1 in different biological samples (including different blood samples of hosts in sickness and in health, cancer cell lines, and trigeminal ganglia culture of infected hosts treated with and without GP1) and determine if any binding patterns might have any associations with different health conditions. The overall preliminary results appear to show certain GP1R + binding patterns in certain blood components (especially neutrophils) have potential correlations with certain health conditions of hosts at sampling times, indicating potential GP1R applications for diagnostic purposes. Findings of different GP1R binding patterns in different cancer cell lines, whole blood samples and trigeminal ganglia culture of experimental mice infected with HSV-1 virus (might cause neuropathy) within a week post-infection, and blood samples of GP1-treated mouse survivors on day 21 post-infection provided preliminary evidence of potential GP1-led tumor cell-specific cell death and treatment efficacy for greater survival.

Leptin/lipopolysaccharide-treated dendritic cell vaccine improved cellular immune responses in an animal model of breast cancer.

PURPOSE: In dendritic cells (DCs), leptin as an immune-regulating hormone, increases the IL-12 generation whereas it reduces the IL-10 production, thus contributing to TH1 cell differentiation. Using a murine model of breast cancer (BC), we evaluated the impacts of the Leptin and/or lipopolysaccharide (LPS)-treated DC vaccine on various T-cell-related immunological markers. MATERIALS AND METHODS: Tumors were established in mice by subcutaneously injecting 7 × 105 4T1 cells into the right flank. Mice received the DC vaccines pretreated with Leptin, LPS, and both Leptin/LPS, on days 12 and 19 following tumor induction. The animals were sacrificed on day 26 and after that the frequency of the splenic cytotoxic T lymphocytes (CTLs) and TH1 cells; interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor growth factor beta (TGF-ß) generation by tumor lysate-stimulated spleen cells, and the mRNA expression of T-bet, FOXP3 and Granzyme B in the tumors were measured with flow cytometry, ELISA and real-time PCR methods, respectively. RESULTS: Leptin/LPS-treated mDC group was more efficient in blunting tumor growth (p = .0002), increasing survival rate (p = .001), and preventing metastasis in comparison with the untreated tumor-bearing mice (UT-control). In comparison to the UT-control group, treatment with Leptin/LPS-treated mDC also significantly increased the splenic frequencies of CTLs (p < .001) and TH1 cells (p < .01); promoted the production of IFN-γ (p < .0001) and IL-12 (p < .001) by splenocytes; enhanced the T-bet (p < .05) and Granzyme B (p < .001) expression, whereas decreased the TGF-ß and FOXP3 expression (p < .05). CONCLUSION: Compared to the Leptin-treated mDC and LPS-treated mDC vaccines, the Leptin/LPS-treated mDC vaccine was more effective in inhibiting BC development and boosting immune responses against tumor.

Intraepithelial lymphocytes are associated with epithelial injury in feline intestinal T-cell lymphoma.

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

NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

Harnessing the Power of CAR-NK Cells: A Promising Off-the-Shelf Therapeutic Strategy for CD38-Positive Malignancies.

Background: CD38 is highly expressed on multiple myeloma (MM) cells and has been successfully targeted by different target therapy methods. This molecule is a critical prognostic marker in both diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Objective: We have designed and generated an anti-CD38 CAR-NK cell applying NK 92 cell line. The approach has potential application as an off-the-shelf strategy for treatment of CD38 positive malignancies. Methods: A second generation of anti-CD38 CAR-NK cell was designed and generated, and their efficacy against CD38-positive cell lines was assessed in vitro. The PE-Annexin V and 7-AAD methods were used to determine the percentage of apoptotic target cells. Flow cytometry was used to measure IFN-γ, Perforin, and Granzyme-B production following intracellular staining. Using in silico analyses, the binding capacity and interaction interface were evaluated. Results: Using Lentivirus, cells were transduced with anti-CD38 construct and were expanded. The expression of anti-CD38 CAR on the surface of NK 92 cells was approximately 25%. As we expected from in silico analysis, our designed CD38-chimeric antigen receptor was bound appropriately to the CD38 protein. NK 92 cells that transduced with the CD38 chimeric antigen receptor, generated significantly more IFN-γ, perforin, and granzyme than Mock cells, and successfully lysed Daudi and Jurkat malignant cells in a CD38-dependent manner. Conclusion: The in vitro findings indicated that the anti-CD38 CAR-NK cells have the potential to be used as an off-the-shelf therapeutic strategy against CD38-positive malignancies. It is recommended that the present engineered NK cells undergo additional preclinical investigations before they can be considered for subsequent clinical trial studies.

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

Administration of a glypican-3 peptide increases the infiltration and cytotoxicity of CD8+ T cells against testicular yolk sac tumor, associated with enhancing the intratumoral cGAS/STING signaling.

BACKGROUND: Glypican-3 (GPC3) is highly expressed in testicular yolk sac tumor (TYST). GPC3 has been evaluated as a cancer vaccine for some types of tumors, but little is known on the effects of GPC3 peptide-based therapy on TYST. Here, we evaluated the antitumor effect of GPC3144-152 on TYST and its potential mechanisms. METHODS: GPC3144-152 -specific CD8+ T cells were induced by vaccine immunization and examined by ELISPOT. The CD8+ T cells were purified for testing their cytotoxicity in vitro against TYST cells by CCK-8 and TUNEL assays and in vivo against tumor growth. The influence of GPC3144-152 loading and/or cGAS silencing on the tumor growth, apoptosis and cGAS/STING signaling was tested by immunohistochemistry, immunofluorescence, flow cytometry, and Western blot. RESULTS: Vaccination with GPC3144-152 induced tumor-specific CD8+ T cells that secreted high levels of IFN-γ and granzyme B, and had potent cytotoxicity against TYST in a dose-dependent manner. Adoptive transfer of CD8+ T cells and treatment with GPC3144-152 significantly inhibited the growth of TYST tumors, but less effective for cGAS-silenced TYST tumors in vivo. Treatment with GPC3144-152 enhanced the infiltration of CD8+ T cells into the tumor environment and their cytotoxicity against TYST tumors in vivo by up-regulating granzyme B and IFN-ß expression, but down-regulating GPC3 expression in the tumors. Co-culture of CD8+ T cells with TYST in the presence of exogenous GPC3144-152 enhanced peptide-specific CD8+ T-cell cytotoxicity in vitro, accompanied by enhancing cGAS, γH2AX, TBK1, and IRF3 phosphorylation in TYST cells, but less effective in cGAS-silenced TYST cells. CONCLUSIONS: These data indicated that GPC3 peptide-specific CD8+ T cells had potent antitumor activity against TYST tumor, particularly for combined treatment with the peptide, which was partially dependent on the intratumoral cGAS/STNG signaling. GPC3 peptide vaccine may be valuable for the combination treatment of TYST.

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

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

High-free Fatty Acid Treatment Induced Anti-inflammatory Changes in a Natural Killer (NK) Cell Line.

Background: Natural killer (NK) cells play a role in the pathogenesis of various metabolic diseases related to obesity. While our initial findings have indicated a potential involvement of NK cells in the pathogenesis of type 2 diabetes mellitus, the precise mechanism underlying NK cell-mediated development of this form of diabetes remains inadequately comprehended. Objective: To investigate the impact and the underlying mechanism of high glucose and elevated levels of free fatty acids (FFAs) on immune and inflammatory responses and oxidative stress in NK92 cells. Methods: In this experiment, the CCK8 cytotoxicity assay was used to select the 44.4 mM and 1.5 mM concentrations of high glucose and high FFAs, respectively, to treat NK92 cells for 4 days. The concentrations of superoxide dismutase (SOD) and glutathione (GSH) were determined using a biochemical analyzer. Intracellular reactive oxygen species (ROS) levels, cytokines concentrations (TNF-α, IFN-γ, IL-6, and IL-10), and the expression levels of intracellular molecules (perforin and granzyme B) were assessed by flow cytometry. Results: The number of NK92 cell clumps was significantly reduced in the high-FFA (HF) group. In addition, the production of ROS and levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-10) significantly decreased in the HF group but showed no significant change in the high-glucose (HG) group. This observation was consistent with the expression levels of perforin and granzyme B that decreased in the HF group. Conclusion: High FFAs induced morphological changes and serious damage to oxidative stress and inflammatory response in NK92 cells.

Expression of Tim-3/Galectin-9 pathway and CD8+T cells and related factors in patients with cystic echinococcosis.

OBJECTIVE: One of the primary reasons for the successful patriotization of Echinococcus multilocularis in patients is its ability to induce host immune tolerance. This study examined the expression of the immunosuppressive Tim-3/Galectin-9 pathway, CD8+T cells, and related factors in AE patients. The aim was to analyze the relationship between the Tim-3/Galectin-9 pathway and CD8+T cells in this disease and further understand the mechanism of immune tolerance induced by cystic echinococcosis. METHODS: Using flow cytometry, we evaluated the expression of CTL, CD8+CD28-T cells, CD8+CD28 + IFN-γ + T cells, CD8+CD28+perforin + T cells, CD8+CD28+granzyme B + T cells, CD8+CD28-IL-10 + T cells, CD8+CD28-TGF-ß+T cells, and Tim-3 expression on CD8+T cells in the peripheral blood of control (n = 30) and AE patients (n = 33). qRT-PCR was used to measure CD107a and Tim-3/Galectin-9 mRNA levels in PBMCs from the control and AE groups. Immunohistochemistry was employed to detect IL-10, TGF-ß, and Tim-3/Galectin-9 expressions in the infected livers of AE patients. RESULTS: AE patients exhibited a significant decrease in peripheral blood CTL ratio (P < 0.001) and an increase in CD8+CD28+IFN-γ+T cell ratio (P < 0.001). No significant changes were observed in the ratios of CD8+CD28+perforin + T cells (P = 0.720) and CD8+CD28+granzyme B + T cells (P = 0.051). The proportions of CD8+CD28-T cells (P < 0.001), CD8+CD28-IL-10 + T cells (P < 0.001), and CD8+CD28-TGF-ß+T cells (P < 0.001) were notably higher than in the control group. The expression of Tim-3 on CTL and CD8+CD28-T cells in AE patients was significantly upregulated (P < 0.001, P < 0.001). AE patients displayed a substantial decrease in peripheral blood PBMC CD107a mRNA levels (P < 0.001) and significant elevations in Tim-3/Galectin-9 mRNA levels (P < 0.001, P < 0.001). A negative correlation was observed between CD107a mRNA levels and both Tim-3 (r^2 = 0.411, P < 0.001) and Galectin-9 (r2 = 0.180, P = 0.019) mRNA levels. Expressions of IL-10 (P < 0.001), TGF-ß (P < 0.001), and Tim-3/Galectin-9 (P < 0.001, P < 0.001) in AE patient-infected livers were significantly higher than in uninfected regions. IL-10 and TGF-ß expressions showed a positive correlation with Tim-3/Galectin-9. CONCLUSION: This study suggests that the high expression of Tim-3 on CD8+T cell surfaces in AE patients might promote an increase in CD8+CD28-T cells and related factors, while suppressing CTL and related factor expressions. This potentially induces the onset of immune tolerance, which is unfavorable for the clearance of Echinococcus multilocularis in patients, leading to the exacerbation of persistent infections.

The cytotoxicity of γδT cells in non-small cell lung cancer mediated via coordination of the BCL-2 and AKT pathways.

The effectiveness and mechanisms of γδT-cell immunotherapy in lung cancer remain unclear. In this study, we assessed the effects of continuous, low-dose γδT-cell intervention on lung cancer cells. We cultured γδT cells with a lung cancer cell line (A549) and replaced the γδT-cell population every 48 hours. The killing effect of γδTcells on A549 cells and the Half-maximal inhibitory concentration (IC50) value were detected by the cholecystokinin octapeptide (CCK-8) method. The levels of perforin, granzyme B and the inflammatory factors interleukin-6 (IL-6), interferon (IFN)-γ, and tumor necrosis factor-alpha (TNF-a), in the supernatants of cocultured cells were measured by ELISA. The protein expression of Bcl-2, Bax, PI3K and Akt was detected by western blotting. Our results indicated that γδT-cell treatment decreased the protein expression of Bcl-2, PI3K, and AKT but upregulated that of Bax. Moreover, γδT-cell treatment increased perforin and granzyme B release related to the Bax/Bcl-2 signaling pathway. In addition, γδT-cell-mediated cytolysis for A549 cells involved the PI3K/AKT pathway. In vivo results were consistent with the in vitro results. γδT-cell immunotherapy integrated regulation of a signaling pathway network involving the mutual regulation of apoptosis and proliferation. γδT-cell immunotherapy could be used to enhance the cytotoxic killing of lung cancer cells.

Widespread and dynamic expression of granzyme C by skin-resident antiviral T cells.

After recognition of cognate antigen (Ag), effector CD8+ T cells secrete serine proteases called granzymes in conjunction with perforin, allowing granzymes to enter and kill target cells. While the roles for some granzymes during antiviral immune responses are well characterized, the function of others, such as granzyme C and its human ortholog granzyme H, is still unclear. Granzyme C is constitutively expressed by mature, cytolytic innate lymphoid 1 cells (ILC1s). Whether other antiviral effector cells also produce granzyme C and whether it is continually expressed or responsive to the environment is unknown. To explore this, we analyzed granzyme C expression in different murine skin-resident antiviral lymphocytes. At steady-state, dendritic epidermal T cells (DETCs) expressed granzyme C while dermal γδ T cells did not. CD8+ tissue-resident memory T cells (TRM) generated in response to cutaneous viral infection with the poxvirus vaccinia virus (VACV) also expressed granzyme C. Both DETCs and virus-specific CD8+ TRM upregulated granzyme C upon local VACV infection. Continual Ag exposure was not required for maintained TRM expression of granzyme C, although re-encounter with cognate Ag boosted expression. Additionally, IL-15 treatment increased granzyme C expression in both DETCs and TRM. Together, our data demonstrate that granzyme C is widely expressed by antiviral T cells in the skin and that expression is responsive to both environmental stimuli and TCR engagement. These data suggest that granzyme C may have functions other than killing in tissue-resident lymphocytes.