IL-6 Signal Research Articles

Screening of potential key pathogenic and intervention targets of low-grade glioma based on bioinformatics.

Sialic acid-binding immunoglobulin-like lectin 8 (SIGLEC8) is involved in the progression of numerous diseases. This study aimed to examine the relationship between SIGLEC8 and the prognosis of patients with low-grade glioma (LGG) and the related mechanisms. First, screening of the differentially expressed genes (DEGs) SIGLEC8 in The Cancer Genome Atlas (TCGA) database was performed. The expression was then correlated with the prognosis of patients with LGG and then verified using the Tumor Immune Estimation Resource (TIMER) and TCGA databases. Cox regression was employed to conduct multifactorial analysis and was followed by the construction of an internally validated nomogram based on these results. To investigate the possible mechanisms, we used gene set enrichment analysis (GSEA). We conducted a retrospective analysis of the clinical information of patients with LGG who were treated at Longgang Central Hospital of Shenzhen from January 2018 to December 2020 and from whom tumor and peritumoral tissues were taken during surgery. Expression of essential genes was identified by employing quantitative real-time polymerase chain reaction (qRT-PCR). Multivariate analysis, via Cox regression, was employed to determine the prognostic factors for patients with LGG. The transcriptional activity of SIGLEC8 was found to be elevated in LGG neoplastic tissues compared to neighboring nonneoplastic tissues. Overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were improved in patients with LGG with reduced expression of SIGLEC8 as compared to those with increased expression of SIGLEC8. The nomogram's C-index is 0.804 (0.781-0.827). indicating good predictive accuracy. GSEA revealed that SIGLEC8 might influence LGG biological events by participating in the PD-1, IL3, JAK/STAT, and PI3KCI signal transduction pathways, as well as cytokine and inflammatory response, cell cycle, homeostasis, and extracellular matrix. This study included 72 patients with LGG. qRT-PCR showed upregulated SIGLEC8 expression in LGG tumor tissues, which was significantly associated with tumor number and metastasis to the lymph nodes (P<0.05). Multivariate analysis using Cox regression identified the high expression of SIGLEC8 as an independent risk factor in LGG prognosis (P<0.05). For the prognosis of patients with LGG, the transcriptional activity of SIGLEC8 is increased in LGG tissues and is an independent risk factor. Interference with SIGLEC8 could promote tumor progression by regulating the JAK/STAT signaling pathway, indicating that SIGLEC8 may function as a distinctive predictive biomarker for patients with LGG.

Identification of susceptibility loci and relevant cell type for IgA nephropathy in Han Chinese by integrative genome-wide analysis.

Although many susceptibility loci for IgA nephropathy (IgAN) have been identified, they only account for 11.0% of the overall IgAN variance. We performed a large genome-wide meta-analysis of IgAN in Han Chinese with 3616 cases and 10 417 controls to identify additional genetic loci of IgAN. Considering that inflammatory bowel disease (IBD) and asthma might share an etiology of dysregulated mucosal immunity with IgAN, we performed cross-trait integrative analysis by leveraging functional annotations of relevant cell type and the pleiotropic information from IBD and asthma. Among 8 669 456 imputed variants, we identified a novel locus at 4p14 containing the long noncoding RNA LOC101060498. Cell type enrichment analysis based on annotations suggested that PMA-I-stimulated CD4+CD25-IL17+ Th17 cell was the most relevant cell type for IgAN, which highlights the essential role of Th17 pathway in the pathogenesis of IgAN. Furthermore, we identified six more novel loci associated with IgAN, which included three loci showing pleiotropic effects with IBD or asthma (2q35/PNKD, 6q25.2/SCAF8, and 22q11.21/UBE2L3) and three loci specific to IgAN (14q32.32/TRAF3, 16q22.2/TXNL4B, and 21q21.3/LINC00113) in the pleiotropic analysis. Our findings support the involvement of mucosal immunity, especially T cell immune response and IL-17 signal pathway, in the development of IgAN and shed light on further investigation of IgAN.

Highly Efficient Synergistic Chemotherapy and Magnetic Resonance Imaging for Targeted Ovarian Cancer Therapy Using Hyaluronic Acid-Coated Coordination Polymer Nanoparticles.

The diagnosis and treatment of ovarian cancer (OC) are still a grand challenge, more than 70% of patients are diagnosed at an advanced stage with a dismal prognosis. Magnetic resonance imaging (MRI) has shown superior results to other examinations in preoperative assessment, while cisplatin-based chemotherapy is the first-line treatment for OC. However, few previous studies have brought together the two rapidly expanding fields. Here a technique is presented using cisplatin prodrug (Pt-COOH), Fe3+, and natural polyphenols (Gossypol) to construct the nanoparticles (HA@PFG NPs) that have a stable structure, controllable drug release behavior, and high drug loading capacity. The acidic pH values in tumor sites facilitate the release of Fe3+, Pt-COOH, and Gossypol from HA@PFG NPs. Pt-COOH with GSH consumption and cisplatin-basedchemotherapy plus Gossypol with pro-apoptotic effects displays asynergisticeffect forkillingtumorcells. Furthermore, the release of Fe3+ at the tumor sites promotes ferroptosis and enables MRI imaging of OC. Inthe patient-derived tumor xenograft (PDX)model, HA@PFG NPs alleviate the tumor activity. RNA sequencing analysis reveals that HA@PFG NPs ameliorate OC symptoms mainly through IL-6 signal pathways. This work combines MRI imaging with cisplatin-based chemotherapy, which holds great promise for OC diagnosis and synergistic therapy.

The pro-inflammatory cytokines IL-1β and IL-6 promote upregulation of the ST6GAL1 sialyltransferase in pancreatic cancer cells

The ST6GAL1 sialyltransferase is overexpressed in multiple cancers including pancreatic ductal adenocarcinoma (PDAC). ST6GAL1 adds an α2-6-linked sialic acid to N-glycosylated membrane receptors, which consequently modulates receptor structure and function. While many studies have investigated the effects of ST6GAL1 on cell phenotype, there is a dearth of knowledge regarding mechanisms that regulate ST6GAL1 expression. In the current study, we evaluated the regulation of ST6GAL1 by two pro-inflammatory cytokines, IL-1β and IL-6, that are abundant within the PDAC tumor microenvironment. Cytokine activity was monitored using the Suit-2 PDAC cell line and two Suit-2-derived metastatic subclones, S2-013 and S2-LM7AA. For all three cell models, treatment with IL-1β or IL-6 increased the expression of ST6GAL1 protein and mRNA. Specifically, IL-1β and IL-6 induced expression of the ST6GAL1 YZ mRNA isoform, which is driven by the P3 promoter. The ST6GAL1 H and X isoforms were not detected. Promoter reporter assays confirmed that IL-1β and IL-6 activated transcription from the P3 promoter. We then examined downstream signaling mechanisms. IL-1β is known to signal through the NFκB transcription factor, whereas IL-6 signals through the STAT3 transcription factor. CUT&RUN experiments revealed that IL-1β promoted the binding of NFκB to the ST6GAL1 P3 promoter, and IL-6 induced the binding of STAT3 to the P3 promoter. Finally, we determined that inhibitors of NFκB and STAT3 blocked the upregulation of ST6GAL1 stimulated by IL-1β and IL-6, respectively. Together, these results highlight a novel molecular pathway by which cytokines within the tumor microenvironment stimulate the upregulation of ST6GAL1 in PDAC cells.

Investigation of the pharmacological mechanisms of Shenfu injection in acute pancreatitis through network pharmacology and experimental validation

BackgroundShenfu Injection (SFI) has emerged as a prevalent therapeutic intervention in clinical practice for the management of acute pancreatitis (AP). The purpose of this research was to investigate and validate the potential mechanisms of SFI in the treatment of AP through network pharmacology. MethodsNetwork pharmacology was adopted to investigate the potential targets and mechanisms of SFI in the treatment of AP. Molecular docking was employed to evaluate the binding affinity between active components and targets. Single-cell transcriptome analysis was conducted to explore the cell types associated with SFI treatment in AP. In vitro and in vivo models of AP were induced by caerulein. The histopathological changes were observed by HE staining. Cell apoptosis was detected using flow cytometry and Tunel staining. Cell viability was assessed using CCK-8 assay. Western blot and ELISA were used to detect the protein expression and inflammatory cytokines, respectively. ResultsA total of 104 SFI active components were obtained, of which 29 targeted 76 genes. After intersecting with 3370 AP-related genes, 42 SFI treatment AP potential targets were identified. Enrichment analysis revealed that these targets were associated with cell apoptosis, necroptosis, and multiple signal transduction pathways, such as p53, IL-17 and TNF signal pathways, etc. Molecular docking demonstrated that the active components of SFI had good binding affinity with the corresponding targets and the binding ability of NGF and aromadendrene was the strongest. Bioinformatics analysis revealed that SFI treatment in AP is associated with various cell types, including acinar cells, endothelial cells, T cells, dendritic cells, ductal cells, and mesenchymal cells. Furthermore, in vitro experiments demonstrated that SFI induces acinar cell apoptosis in a dose-dependent manner, accompanied by increased expression of cleaved-caspase3/caspase3 and cleaved-caspase8/caspase8 proteins, and inhibition of inflammatory cytokine (TNF-ɑ, IL-1β, and PTGS2) expression. In vivo experiments demonstrated that SFI improved histopathological alterations, reduces inflammation, and promotes apoptosis and the expression of cleaved-casp3 and cleaved-casp8 in AP rats. ConclusionsThis study elucidated the multi-component, multi-target, and multi-cellular characteristics of SFI in the treatment of AP, and confirmed its mechanism of promoting acinar cell apoptosis.

Identification of key genes and functions in lung metastasis of osteosarcoma based on bioinformatics

To screen the differentially expressed genes of lung metastasis of osteosarcoma by bioinformatics, and explore their functions and regulatory networks. The data set of GSE14359 was screened from GEO database(http://www.ncbi.nlm.nih.gov/gds) and the differentially expressed gene(DEG) was identified using GEO2R online tool. Download osteosarcoma disease related miRNAs from the online HMMD database(http://www.cuilab.cn/hmdd) and then FunRich software was used to predict the target gene, intersects with DEG to obtains the target gene. The miRNA-mRNA relationship pairs were formed according to the targeted joints, then the data was imported into Cytoscape for visualization, DAVID was used to performe GO and KEGG analysis on target genes, STRING was used to construct PPI network, Cytoscape visualization, CytoHubba plug-in screening central genes and online website for expression and survival analysis. Total 704 DEGs were identified, consisting of 477 up-regulated genes and 227 down regulated genes. FunRich predicted 7 888 mRNAs and 343 target genes were obtained through intersection of the two. KEGG analysis showed that it was mainly involved in focal adhesion, ECM receptor interaction, TNF signal pathway, PI3K-Akt signal pathway, IL-17 signal pathway and MAPK signal pathway. Ten central genes (CCNB1, CHEK1, AURKA, DTL, RRM2, MELK, CEP55, FEN1, KPNA2, TYMS) were identified as potential key genes. Among them, CCNB1, DTL, MELK were highly correlated with poor prognosis. The key genes and functional pathways identified in this study may be helpful to understand the molecular mechanism of the occurrence and progression of lung metastases from osteosarcoma, and provide potential therapeutic targets.

STAT4 Mediates IL-6 Trans-Signaling Arrhythmias in High Fat Diet Guinea Pig Heart.

Obesity is a major risk factor for the development of life-threatening malignant ventricular tachyarrhythmias (VT) and sudden cardiac death (SCD). Risks may be highest for patients with high levels of the proinflammatory cytokine interleukin (IL)-6. We used our guinea pig model of high-fat diet (HFD)-induced arrhythmias that exhibit a heightened proinflammatory-like pathology, which is also observed in human obesity arrhythmias, as well as immunofluorescence and confocal microscopy approaches to evaluate the pathological IL-6 trans-signaling function and explore the underlying mechanisms. Using blind-stick and electrocardiogram (ECG) techniques, we tested the hypothesis that heightened IL-6 trans-signaling would exhibit increased ventricular arrhythmia/SCD incidence and underlying arrhythmia substrates. Remarkably, compared to low-fat diet (LFD)-fed controls, HFD promoted phosphorylation of the IL-6 signal transducer and activator of transcription 4 (STAT4), leading to its activation and enhanced nuclear translocation of pSTAT4/STAT4 compared to LFD controls and pSTAT3/STAT3 nuclear expression. Overactivation of IL-6 trans-signaling in guinea pigs prolonged the QT interval, which resulted in greater susceptibility to arrhythmias/SCD with isoproterenol challenge, as also observed with the downstream Janus kinase (JAK) 2 activator. These findings may have potentially profound implications for more effective arrhythmia therapy in the vulnerable obese patient population.

Effects of IL-6R Expression on IL-6/STAT3/HIF-1α Signaling Pathway in a Mouse Model of Parkinson's Disease with Type 2 Diabetes Co-Morbidity.

More and more evidence has shown the process of Parkinson's disease (PD). Probably, inflammation exerts a crucial role between them. Therefore, the aim of this study was to analyze the impact of interleukin-6 receptor (IL-6R) expression on the IL-6/signal transducer and activator of transcription 3 (STAT3)/hypoxia-inducible factor-1α (HIF-1α) inflammatory signaling pathway within a mouse model of PD with type 2 diabetes mellitus (T2DM) as co-morbidity. We chose healthy wild-type C57BL/6J male mice at the age of 10 weeks to prepare a mouse model of PD with T2DM co-morbidity. Adeno-associated virus (AAV) overexpressing IL-6R or AAV IL-6R-shRNA genes were injected into the substantia nigra (SN) of the mice. The behavioral indices of the pole test were used for examining the motor function of the mice. Using immunofluorescence analysis, the impacts of IL-6R on the level of tyrosine hydroxylase (TH) and anti-ionized calcium-binding adaptor molecule 1 (IBA-1) on dopaminergic neurons and microglia were examined. Additionally, enzyme-linked immunosorbent assay (ELISA) was adopted for determining the expressions of HIF-1α and inflammatory cytokines like tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-4 in the serum. In this study, the protein expression levels of TH, α-Synuclein (α-Syn), IBA-1, IL-6, IL-6R, phosphorylated and total signal transducer and activator of transcription 3 (p-STAT3 (Tyr705) and STAT3) and HIF-1α in the SN were tested via western blotting. To ascertain the mRNA expressions of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α, we used quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). IL-6R-shRNA treatment could markedly shorten the total time of PD in the T2DM co-morbidity mouse model based on the pole test results, reverse the decrease in TH-positive neurons stimulated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), and lower the activation of microglia (all p < 0.05). Further, IL-6R-shRNA treatment hindered the expression of IL-6, p-STAT3 (Tyr705), and HIF-1α in the SN, lowered the levels of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α in the serum, and mRNA expressions of TNF-α, IL-1β, IL-6, and HIF-1α in the SN (all p < 0.05). In contrast, IL-6R overexpression reduced TH levels, upregulated the level of IBA-1, IL-6, p-STAT3 (Tyr705), and HIF-1α, increased the level of IL-1β, TNF-α, IL-6, IL-4, and HIF-1α (all p < 0.05) in the serum and SN in the PD mouse model with T2DM as a co-morbidity. PD progression with T2DM as a co-morbidity can be boosted by AAV IL-6R-overexpression through upregulation of the IL-6/STAT3/HIF-1α axis. Conversely, AAV IL-6R-shRNA treatment suppressed the IL-6/STAT3/HIF-1α pathway and alleviated neuroinflammation, thus weakening the development of PD with T2DM as a co-morbidity.

Integrating network pharmacology and experimental verification to reveal the anti-inflammatory ingredients and molecular mechanism of pycnogenol.

Introduction: Pycnogenol (PYC), a standardized extract from French maritime pine, has traditionally been used to treat inflammation. However, its primary active components and their mechanisms of action have not yet been determined. Methods: This study employed UPLC-MS/MS (Ultra-high performance liquid chromatography-tandem mass spectrometry) and network pharmacology to identify the potential active components of PYC and elucidate their anti-inflammatory mechanisms by cell experiments. Results: 768 PYC compounds were identified and 19 anti-inflammatory compounds were screened with 85 target proteins directly involved in the inflammation. PPI (protein-protein interaction) analysis identified IL6, TNF, MMP9, IL1B, AKT1, IFNG, CXCL8, NFKB1, CCL2, IL10, and PTGS2 as core targets. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis suggested that the compound in PYC might exert anti-inflammatory effects through the IL17 and TNF signal pathways. Cell experiments determined that PYC treatment can reduce the expression of IL6 and IL1β to relieve inflammation in LPS (lipopolysaccharide)-induced BV2 cells. Conclusion: PYC could affect inflammation via multi-components, -targets, and -mechanisms.

Analysis and Validation of the Network Pharmacology of the Mechanism of Glycyrrhetinic Acid for the Treatment of High-Altitude Pulmonary Hypertension

Glycyrrhetinic acid (GA) is a pentacyclic triterpene component in Glycyrrhize glabra L, it has demonstrated an inhibitive effect on high-altitude pulmonary hypertension (HAPH), but the molecular action is still not known. We aimed to explore the mechanism of GA for the treatment of HAPH based on network pharmacology and molecular docking method. Cell experiment validation was also conducted. The targets for GA were screened using the Swiss Target Prediction and Batman databases. The HAPH-related targets were obtained using the GeneCards and OMIM databases. The common targets for diseases and drugs were obtained using a Venn diagram. The core targets were screened using the String database. Then, a component-target-disease diagram and protein–protein interaction (PPI) network mutual assistance diagram were developed using Cytoscape3.9.1 software. GO functional and KEGG pathway enrichment analyses were conducted using the Metascape database. Finally, molecular docking of the target and its corresponding active components were performed using Autodock software. A total of 68 common targets for glycyrrhetinic acid high-altitude pulmonary hypertension were screened out. The core targets include PTGS1, MMP1, SERPINA6, and nitric oxide synthase (NOS2), involving PPAR signal pathway, human cytomegalovirus infection, IL-17 signal pathway, proteoglycans in cancer, and other pathways. The molecular docking affinity was −8.4 kcal·mol−1 in average, indicating that GA has a good binding stability with key target proteins. In the PDGF-BB-induced PASMC proliferation model, PASMC proliferation and the p-p38, p38, p-ERK1/2, and ERK1/2 protein expression were inhibited. The pharmacological mechanism of GA for the treatment of HAPH was characterized by multi-target and multi pathway. GA may serve as a promising therapeutic candidate for HAPH but still needs further in vivo/in vitro experiment.

Arid5a-dependent RNA translation underlies pathology in IL-17-driven autoantibody-induced glomerulonephritis

Abstract Autoantibody-mediated glomerulonephritis (AGN) results from kidney-damaging autoimmune responses, and there is urgent need for better treatment. Emerging studies implicate Th17 cells in AGN. IL-17 signals in part through the noncanonical RNA binding protein Arid5a, which we show is induced in renal epithelial cells (RTECs) in an IL-17-dependent manner during experimental AGN. Arid5a is undetectable in healthy kidney but elevated in human glomerulonephritis biopsies. Arid5a-/- mice were refractory to AGN. Mice lacking C/EBPd but not IkBz were protected from AGN. Although Arid5a bound to Cebpd mRNA, Arid5a-deficiency did not alter cellular Cebpd mRNA concentrations, whereas C/EBPd protein levels were substantially suppressed. In line with this, global protein synthesis was suppressed in RTECs lacking Arid5a. Transcriptome-wide RIP-Seq analysis in RTECs and stromal cells showed that IL-17 prompts Arid5a to bind to inflammatory mRNAs. Additionally and unexpectedly, Arid5a interacted with ribosomal RNAs (rRNAs) and other transcripts involved in protein translation. After IL-17 treatment, Arid5a was enriched in the cytoplasm and associated with the ribosome through interactions with 40S and 60S rRNAs. Accordingly, Arid5a drives IL-17-induced renal autoimmunity through post-transcriptional control of RNA at the level of translation and ribosome interactions, providing mechanistic insight into how cytokines mediate inflammation.

Factors of Interleukin-6 Signaling in COVID-19 Patients with Lung Damage of Varying Degrees: A Pilot Study.

Specific features of IL-6 signal transduction were studied in 89 patients with lung damage of varying degrees during the first COVID-19 pandemic wave. The levels of IL-6 signaling components (IL-6, sIL-6R, and sgp130) and highly sensitive C-reactive protein (hsCRP) were examined in patients with intact lungs (CT-0), mild (CT-1), moderate (CT-2), moderate to severe (CT-3), and severe (CT-4) lung damage. Seventy patients were re-examined 3-7 months after discharge from the hospital. The IL-6 and hsCRP levels increased several times with severing lung damage severity. In patients with CT-3, sIL6-R increased statistically significantly and remained high in CT-4 patients. sgp130 levels were lower in CT-1 and CT-2 patients and higher in CT-3 and CT-4 patients compared to CT-0 patients. We revealed a positive correlation between IL-6 and hsCRP levels in CT-1, CT-2, and CT-3 patients. In CT-3 patients, sIL-6R levels positively correlated with IL-6 concentration. The studied parameters decreased considerably in all patients 3-7 months after discharge. It can be suggested that IL-6 classic-signaling is predominant in CT-1 and CT-2, while trans-signaling prevails in CT-3. Disorders in regulatory mechanisms of IL-6 signaling occur in CT-4, which prevents physiological elimination of IL-6 hyperactivity. The results obtained are preliminary and require a broader study.

Effects of interleukin-6 signal inhibition on Treg subpopulations and association of Tregs with clinical outcomes in rheumatoid arthritis.

Anti-IL-6 receptor antibodies are clinically efficacious in the management of RA with an associated increase in Tregs; however, the role of functional Treg subsets has yet to be clarified. This study aimed to evaluate how functional Treg subsets are altered by IL-6 receptor blockade and to analyse the relationship between these Treg subsets and the clinical outcome of RA. We collected frozen peripheral blood mononuclear cells (PBMCs) from 40 patients with RA who started tocilizumab (TCZ) with or without MTX and 11 healthy controls (HCs). We fractionated Tregs with flow cytometry based on markers of phenotype and function and measured the proportions of detailed Treg subsets sequentially from baseline to week 52. The proportions of resting Tregs (rTregs) and rTregs+activated Tregs (aTregs) were significantly lower in RA patients at baseline than in HCs. The proportions of all those CD127low Tregs, rTregs, aTregs and rTregs+aTregs were significantly increased with TCZ treatment. In patients treated with TCZ without MTX, rTreg were increased. Patients with an increase in the proportion of rTregs at week 12 had significantly less arthritis flares during the observation period. Blocking the IL-6 receptor with TCZ increased the proportion of rTregs, a functional Treg subpopulation. Patients with an early increase in rTregs showed a favourable treatment course and this increase in rTregs may reflect molecular remission induced by IL-6 signal inhibition.

Abstract 6312: Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against blood malignancies. However, prominent barriers, including limited proliferation, CAR T cell persistence, and poor T cell effector function, hinder its potential, especially in solid tumors. Various engineering approaches attempt to address these issues by armoring CAR T cells with factors that enhance the function and maintenance of T cells in the anti-tumor response. We previously described a human orthogonal IL-2/IL-2R system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (oRβ) that does not significantly respond to its native ligand, wild type IL-2. This system provides a “private IL-2 signal” in engineered oRβ-expressing cells while avoiding signaling in bystander immune cells, reducing toxicities. We have demonstrated STK-009/oRβ’s ability to selectively expand oRβ-expressing CAR T cells (SYNCAR T cells) targeting heme (SYNCAR-001, CD19) and solid tumor (SYNCAR-002, GPC3) targets at will. Importantly, the system enhanced anti-tumor control driving deep and durable responses in both bulky lymphoma and hepatocellular carcinoma (HCC) tumor models. Additionally, in a non-human primate model, STK-009 did not potentiate wild type T or NK cells and exhibited no evidence of toxicity. Currently, the combination of STK-009 + SYNCAR-001 is undergoing Phase I clinical trials (NCT05665062). Here, we conducted a direct comparison of several armoring approaches including the SYNCAR platform, the overexpression of c-Jun, an IL-15/IL-15Rα fusion, and IL-18 in human CAR T cells targeting GPC3. To assess their effects in overcoming CAR T cell dysfunction, cells were subjected to continuous antigen exposure in vitro. STK-009 treated SYNCAR-002 T cells displayed an enhanced proliferative capacity, sustained effector cytokine secretion, and an increased activation state when compared to other armoring technologies and PBS treatment. In a HCC tumor model, treatment with STK-009/SYNCAR-002 demonstrated superior tumor control with minimal toxicities. Interestingly, IL-18 overexpression also controlled tumors, but was accompanied by uncontrolled CAR T cell expansion and toxicity resembling severe GVHD. scRNAseq analyses revealed STK-009-treated SYNCAR-002 T cells simultaneously activated effector genes and repressed an exhaustion gene signature. This analysis also identified a novel mechanism by which STK-009/oRβ signaling induces a superior synthetic effector state via transcriptional repression of the stress response/proteosome. These findings support the advantages of an orthogonal platform that selectively drives and sustains potent T cell effector function without safety concerns associated with constitutively expressed armored approaches. Citation Format: Ethan Jung, Helena Silva, Marie Semana, Ivan Cheng, Somya Singh, Michele Bauer, Mohammed Ali, Henry Rosas, Ryan Burgess, George Zeng, Woei Chang, Navneet Ratti, Deepti Rokkam, Patrick J. Lupardus, Martin Oft, Paul-Joseph Aspuria. Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6312.

Exploring the Mechanisms of Action of Quercetin for the Treatment of Cervical High-Risk-Human Papilloma Virus Using Network Pharmacology and Molecular Docking

Abstract Objective Our objective was to investigate the therapeutic mechanism of quercetin in the management of cervical HR-HPV through the integration of network pharmacology and molecular docking techniques. Methods The GeneCard database was utilized to analyze and identify potential therapeutic targets in HR-HPV. Subsequently, a protein–protein interaction (PPI) network was constructed by employing the String database. The visualization and construction of PPI networks were accomplished using Cytoscape. The R language was utilized to conduct Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results A total of 154 active constituents of quercetin were identified through screening. Additionally, 139 target genes associated with the effects of quercetin on cervical HR-HPV were predicted. PPI analyses revealed that threonine kinase Akt1, mitogen-activated protein kinase1, human IL-6 protein, signal transducer and activator of transcription 3, and epidermal growth factor receptor (EGFR) may serve as potential targets for quercetin in the treatment of cervical HR-HPV. Furthermore, GO and KEGG analyses demonstrated that quercetin is involved in various functional pathways, biological processes, molecular categories, including the Th17 signaling pathway, tumor necrotizing factor (TNF) signaling pathway, EGFR signaling pathway, PI3K-Akt signaling pathway, among others. Conclusion Quercetin exhibits multifaceted characteristics, targeting multiple components, pathways, and targets, in the therapeutic intervention of HR-HPV, primarily by modulating inflammatory responses, oxidation reactions, and apoptotic processes.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

Longitudinal plasma proteomic analysis identifies biomarkers and combinational targets for anti-PD1-resistant cancer patients

The response rate of anti-PD1 therapy is limited, and the influence of anti-PD1 therapy on cancer patients is unclear. To address these challenges, we conducted a longitudinal analysis of plasma proteomic changes with anti-PD1 therapy in non-small cell lung cancer (NSCLC), alveolar soft part sarcoma (ASPS), and lymphoma patients. We included 339 plasma samples before and after anti-PD1 therapy from 193 patients with NSCLC, ASPS, or lymphoma. The plasma proteins were detected using data-independent acquisition-mass spectrometry and customable antibody microarrays. Differential proteomic characteristics in responders (R) and non-responders (NR) before and after anti-PD1 therapy were elucidated. A total of 1019 proteins were detected using our in-depth proteomics platform and distributed across 10–12 orders of abundance. By comparing the differential plasma proteome expression between R and NR groups, 50, 206, and 268 proteins were identified in NSCLC, ASPS, and lymphoma patients, respectively. Th17, IL-17, and JAK-STAT signal pathways were identified upregulated in NR group, while cellular senescence and transcriptional misregulation pathways were activated in R group. Longitudinal proteomics analysis revealed the IL-17 signaling pathway was downregulated after treatment. Consistently, many proteins were identified as potential combinatorial therapeutic targets (e.g., IL-17A and CD22). Five noninvasive biomarkers (FLT4, SFTPB, GNPTG, F5, and IL-17A) were further validated in an independent lymphoma cohort (n = 39), and another three noninvasive biomarkers (KIT, CCL3, and TNFSF1) were validated in NSCLC cohort (n = 76). Our results provide molecular insights into the anti-PD1 therapy in cancer patients and identify new therapeutic strategies for anti-PD1-resistant patients.

P176 TYK2 inhibition attenuates the stimulation of sensory neurons with hyper-IL-6

Abstract Background Abdominal pain and diarrhoea are a cause of significant morbidity for people with colitis. Recent single cell RNA sequencing of sensory neurons innervating the gut highlights a likely role for sensory nerves in the pathophysiology of these symptoms based on the marked expression of cytokine receptors from the IL-6 receptor family in gut projecting sensory neurons. In keeping with the reported downstream signalling pathway for these receptors marked co-expression of TYK2 transcripts were also found along with IL-6 signal transducer (GP130) in colonic nociceptors characterised by expression of the capsaicin receptor TRPV1. These observations suggest that TYK2 inhibitors may ameliorate symptoms of pain and diarrhoea in people with colitis by preventing neurogenically mediated gut contractility and nociceptor activation. Methods TYK2 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to the hyper-IL-6 fusion protein of the soluble IL-6 receptor and IL-6, which transactivates the GP130 receptor subunit to mimic IL-6 family receptor signalling following pre-treatment with vehicle, the TYK2 inhibitor Deucravacitinib (100nM) or the pan-JAK inhibitor Pyridone-6 (2 μM). Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of hyper-IL-6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons which was significantly attenuated by pre-treatment with the TYK2 specific inhibitor Deucravacitinib (p&amp;lt;0.01) or the pan JAK inhibitor Pyridone-6 (p = &amp;lt;0.05). Conclusion Findings demonstrate that inhibition of TYK2 inhibitor prevents sensory neuron activation in response to IL-6 trans-signalling. This data suggests that TYK2 inhibitors may have utility for the treatment of abdominal pain in people with colitis.

A Self-Activating IL-15 Chimeric Cytokine Receptor to Empower Cancer Immunotherapy.

Enhancing NK cells' antitumor activity requires sustained cytokine signaling. Interleukin-15 (IL-15) is a potent immunostimulatory cytokine used to armor CAR-NK and CAR-T cell immunotherapies. However, strategies to increase IL-15 expression and antitumor effect may trigger systemic toxicity with the potential to promote oncogenesis and autoimmune diseases. To overcome these limitations, we developed a new platform (IL15RB) whereby IL-15 with IL-2 signal peptide is tethered to its receptor, IL2Rβ. NK92-expressing IL15RB (NK92IL15RB) cells expand indefinitely without exogenous cytokines and have significantly higher anticancer activity than NK-92 stimulated by IL-15, IL-2, or expressing tethered IL-2. NK92IL5RB showed resistance to irradiation and IL-4. However, TGFβ1 substantially reduced NK92IL5RB killing, suggesting the need to inhibit TGFβ1 in IL-15-mediated immunotherapies. IL15RB induced strong STAT3 but weaker STAT5 and STAT1 activation compared to IL-2. Chronic exposure of NK92IL15RB cells to cancer cells reduced STAT3 and STAT1 activation irreversibly, suggesting a role in exhaustion. Combination with CAR-CD19 enhanced NK92IL15RB antitumor activity against leukemia and increased its STAT5 activation. NK92IL15RB anti-tumors activity was further enhanced by combination with anti-PD1. Our data suggest that the tethering of IL-15 to its receptor IL2Rβ empowers NK cell cytolytic activity. Additionally, the tethering of IL-15 will prevent any systemic risk of toxicity.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.