Pro-inflammatory Cells Research Articles

Staphylococcus aureus–specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis–like flares in mice

BackgroundThe contribution of Staphylococcus aureus (S. aureus) to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. ObjectiveTo reappraise the main bacterial factors and underlying immune mechanisms by which S. aureus triggers AD-like inflammation. MethodsWe capitalized on a pre-clinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. ResultsWe report that the development of S. aureus-induced dermatitis depended on the nature of the S. aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and non-secreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor protein ASC- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S. aureus, and an accumulation of S. aureus-specific γδ and CD4+ tissue resident memory T (Trm) cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis upon new bacteria exposures, but also, protected the mice from persistent bacterial colonization. ConclusionThese data highlight the induction of unique AD-like inflammation, with the generation of pro-inflammatory but protective Trm cells in a context of natural exposure to pathogenic S. aureus strains.

Microglia and macrophages in glioblastoma: landscapes and treatment directions.

Glioblastoma is the most common primary malignant tumour of the central nervous system and remains uniformly and rapidly fatal. The tumour-associated macrophage (TAM) compartment comprises brain-resident microglia and bone marrow-derived macrophages (BMDMs) recruited from the periphery. Immune-suppressive and tumour-supportive TAM cell states predominate in glioblastoma, and immunotherapies, which have achieved striking success in other solid tumourshave consistently failed to improve survival in this 'immune-cold' niche context. Hypoxic and necrotic regions in the tumour core are found to enrich, especially in anti-inflammatory and immune-suppressive TAM cell states. Microglia predominate at the invasive tumour margin and express pro-inflammatory and interferon TAM cell signatures. Depletion of TAMs, or repolarisation towards a pro-inflammatory state, are appealing therapeutic strategies and will depend on effective understanding and classification of TAM cell ontogeny and state based on new single-cell and spatial multi-omic in situ profiling. Here, we explore the application of these datasets to expand and refine TAM characterisation, to inform improved modelling approaches, and ultimately underpin the effective manipulation of function.

Spatial Characterization of the Immune Microenvironment within HIV+ Lymph Nodes

Abstract Background: Persistent HIV replication is hypothesized to induce chronic inflammation in ART-suppressed people living with HIV (PLWH), however defining this chronic inflammation within tissues is limited. Molecular and spatial characterization of chronic inflammation within human lymph nodes from PLWH provide valuable information on the immunological microenvironment that limits viral eradication. Methods: We spatially characterized the transcriptomic and proteomic profiles of the lymph node microenvironment from 6 human lymph nodes: three from ART-suppressed PLWH, two from chronically infected PLWH, and one from a male HIV- control donor. For spatial transcriptomics analysis, lymph nodes were either from fresh-frozen or FFPE tissue. Lymph node sections (10μm) were placed onto the 10X Visium slides, stained with H&E, and imaged prior to RNA-Seq library prep. Following sequencing cluster identification was done with single cell RNA-seq datasets and downstream bioinformatics analyses were performed. Results: Following the bioinformatic integration of spatial transcriptomic datasets, cluster analysis identified pro-inflammatory T cells within the HIV+ tissues compared to the HIV- tissue within the T cell zone. Additionally, two sets of proinflammatory myeloid cells were identified that were spatially located within the afferent and efferent lymphatics in the HIV+ tissues. These proinflammatory clusters further characterize chronic inflammation that occurs in PLWH.

IL-18 promotes pro-inflammatory cell death in cytotoxic T-cell responses

Abstract Hyperinflammation is a life-threatening systemic inflammatory state that complicates 11% of all SIRS cases. Clinical observations have identified several genetic and/or acquired suseptibility factors. Though Perforin-deficiency is the best-studied mechanism of hyperinflammatory susceptibility, several genetic and biomarker discoveries support excess IL-18 is another. However, it remains unclear how IL-18 drives hyperinflammation. We hypothesized that excess IL-18 leads to excess IFN-y that operates, at least in part, at the critical point of T-cell activation known as the immunological synapse (IS). Methods. We observed the effects of IL-18 and IFN-y on three IS parameters: IS duration, cytokine production, and the mode of target cell death. We measured IS duration by live-cell microscopy, and measured cytokine levels in supernatants. We performed experiments in the presence of several cell death inhibitors. In vitro findings were further analyzed in Il18tg and Prf1-/- mice infected with LCMV. Results . Prf deficiency has been shown to increase IS duration and IFN-y production, however, excess IL-18 shortened IS duration but increased IFN-y production. Moreover, excess IL-18 or the use of BMDCs that were pre-exposed to high levels of IFN-y induced RIPK-1 dependent cell death. These data suggest that T-cell IFN-y, amplified by IL-18, acts on target BMDC to promote necroptosis. Correspondingly, in vivo Nec-1 (RIPK-1 inhibitor) dampened inflammation during LCMV infection.

In-depth immune profiling of peripheral blood mononuclear cells in patients with pancreatic ductal adenocarcinoma reveals discriminative immune subpopulations.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6-CXCR3- memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

Oncolytic virus V937 in combination with PD-1 blockade therapy to target immunologically quiescent liver and colorectal cancer

V937 is an investigational, genetically unmodified Kuykendall strain of coxsackievirus A21, which has been evaluated in the clinic for advanced solid tumor malignancies. V937 specifically infects and lyses tumor cells that overexpress intercellular adhesion molecule-1 (ICAM-1). Intratumoral V937 as a monotherapy and in combination with anti-PD-1 antibody pembrolizumab has shown clinical response in patients with metastatic melanoma, which overexpresses ICAM-1. Here, we investigate in pre-clinical studies the potential bidirectional cross-talk between hepatocellular carcinomas (HCC) or colorectal carcinomas (CRC) and immune cells when treated with V937 alone or in combination with pembrolizumab. We show that while V937 treatment of tumor cell lines or organoids or peripheral blood mononuclear cells (PBMCs) alone induced a minimal immunological response, V937 treatment of non-contact co-cultures of tumor cell lines or CRC organoids with PBMCs led to robust production of pro-inflammatory cytokines and immune cell activation. In addition, both recombinant interferon-gamma (IFN-γ) and pembrolizumab increased ICAM-1 on tumor cell lines or organoids and, in turn, amplified V937-mediated oncolysis and immunogenicity. These findings provide critical mechanistic insights on the cross-talk between V937-mediated oncolysis and immune responses, demonstrating the therapeutic potential of V937 in combination with PD-1 blockade to treat immunologically quiescent cancers.

Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Design, synthesis, molecular docking, and molecular dynamic studies of novel quinazoline derivatives as phosphodiesterase 7 inhibitors.

Introduction: Phosphodiesterase 7 (PDE7) is a high-affinity cyclic AMP (cAMP)-specific PDE that is expressed in immune and proinflammatory cells. In this work, we explore the possibility that selective small molecule inhibitors of this enzyme family could provide a novel approach to alleviate the inflammation that is associated with many inflammatory diseases. Methods: A series of novel substituted 4-hydrazinoquinazoline derivatives and fused triazoloquinazolines were designed, synthesized, and evaluated in vitro for their PDE7A inhibition activities, in comparison with Theophylline, a non-selective PDE inhibitor, and BRL50481, a selective PDE7A inhibitor. This series of novel quinazoline derivatives were synthesized via multi-step reactions. The reaction sequence began with selective monohydrazinolysis of compounds 2a,b to give 3a,b. Schiff bases 4a-h were synthesized by the reaction of the quinazolylhydrazines 3a,b with various substituted aromatic aldehydes. The reaction of 4a-h with bromine in acetic acid, in turn, gave fused triazoloquinazolines 5a-h. These compounds were characterized by satisfied spectrum analyses mainly including 1HNMR, 13CNMR, and MS together with elemental analyses. Results and discussion: The results of in vitro PDE7A inhibition activity clearly indicated that compounds 4b, 4g, 5c, and 5f exhibited good potency. Molecular docking and molecular dynamic simulation studies further supported our findings and provided the basis of interaction in terms of conventional hydrogen bonds and π-π stacking patterns. The present results lay the groundwork for developing lead compounds with improved phosphodiesterase seven inhibitory activities.

Synovium microenvironment-responsive injectable hydrogel inducing modulation of macrophages and elimination of synovial fibroblasts for enhanced treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a progressive autoimmune disease accompanied by joint swelling, cartilage erosion and bone damage. Drug therapy for RA has been restricted due to poor therapeutic effect, recurrence and adverse effects. Macrophages and synovial fibroblasts both play important roles in the pathology of RA. Macrophages secrete large amount of pro-inflammatory cytokines, while synovial fibroblasts are tightly correlated with hypoxia synovium microenvironment, cytokine release, recruitment of pro-inflammatory cells, bone and cartilage erosion. Therefore, in this timely research, an injectable and pH-sensitive peptide hydrogel loading methotrexate (MTX) and bismuthene nanosheet/polyethyleneimine (BiNS/PEI) has been developed to reduce the activity of macrophages and eliminate over-proliferated synovial fibroblasts simultaneously. MTX can reduce the cytokine secretion of macrophages/anti-apoptosis property of synovial fibroblasts and BiNS/PEI can eliminate synovial fibroblasts via photodynamic therapy (PDT) and photothermal therapy (PTT) routes. The hydrogel was injected into the acidic inflammatory synovium for precise targeting and served as a drug reservoir for pH responsive and sustained drug release, while improving the bioavailability and reducing the toxicity of MTX. Excellent therapeutic efficacy has been achieved in both in vivo and in vitro studies, and this unique drug delivery system provides a new and robust strategy to eliminate synovial fibroblasts and modulate immune system for RA treatment in clinical.Graphic

595 Unremitting Pro-inflammatory Cell Phenotypes and Macrophage Activity, Following Paediatric Burn Injury

595 Unremitting Pro-inflammatory Cell Phenotypes and Macrophage Activity, Following Paediatric Burn Injury

Defining mesenchymal stem/stromal cell-induced myeloid-derived suppressor cells using single-cell transcriptomics

Mesenchymal stem/stromal cells (MSCs) modulate the immune response through interactions with innate immune cells. We previously demonstrated that MSCs alleviate ocular autoimmune inflammation by directing BM cell differentiation from pro-inflammatory CD11bhiLy6ChiLy6Glo cells into immunosuppressive CD11bmidLy6CmidLy6Glo cells. Herein, we analyzed MSC-induced CD11bmidLy6Cmid cells using single-cell RNA sequencing and compared them with CD11bhiLy6Chi cells. Our investigation revealed 7 distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11bmidLy6Cmid cells, while CD11bhiLy6Chi cells included mostly monocytes/macrophages with a small cluster of neutrophils. These MSC-induced MDSCs highly expressed Retnlg, Cxcl3, Cxcl2, Mmp8, Cd14 and Csf1r as well as Arg1. Comparative analyses of CSF-1RhiCD11bmidLy6Cmid and CSF-1RloCD11bmidLy6Cmid cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of IL-10. Functionally, these CSF-1RhiCD11bmidLy6Cmid cells, compared with the CSF-1RloCD11bmidLy6Cmid cells, inhibited CD4+ T cell proliferation and promoted CD4+CD25+Foxp3+ Treg expansion in culture and in a mouse model of experimental autoimmune uveoretinitis. RELM-γ encoded by Retnlg, one of the highly-upregulated genes in MSC-induced MDSCs, had no direct effects on T cell proliferation, Treg expansion or splenocyte activation. Together, our study revealed a distinct transcriptional profile of MSC-induced MDSCs and identified CSF-1R as a key cell-surface marker for detection and therapeutic enrichment of MDSCs.

Characterization of Biomarkers of Thrombo-Inflammation in Patients with First-Diagnosed Atrial Fibrillation.

Patients with first-diagnosed atrial fibrillation (FDAF) exhibit major adverse cardiovascular events (MACEs) during follow-up. Preclinical models have demonstrated that thrombo-inflammation mediates adverse cardiac remodeling and atherothrombotic events. We have hypothesized that thrombin activity (FIIa) links coagulation with inflammation and cardiac fibrosis/dysfunction. Surrogate markers of the thrombo-inflammatory response in plasma have not been characterized in FDAF. In this prospective longitudinal study, patients presenting with FDAF (n = 80), and 20 matched controls, were included. FIIa generation and activity in plasma were increased in the patients with early AF compared to the patients with chronic cardiovascular disease without AF (controls; p < 0.0001). This increase was accompanied by elevated biomarkers (ELISA) of platelet and endothelial activation in plasma. Pro-inflammatory peripheral immune cells (TNF-α+ or IL-6+) that expressed FIIa-activated protease-activated receptor 1 (PAR1) (flow cytometry) circulated more frequently in patients with FDAF compared to the controls (p < 0.0001). FIIa activity correlated with cardiac fibrosis (collagen turnover) and cardiac dysfunction (NT-pro ANP/NT-pro BNP) surrogate markers. FIIa activity in plasma was higher in patients with FDAF who experienced MACE. Signaling via FIIa might be a presumed link between the coagulation system (tissue factor-FXa/FIIa-PAR1 axis), inflammation, and pro-fibrotic pathways (thrombo-inflammation) in FDAF.

Acidovorax temperans skews neutrophil maturation and polarizes Th17 cells to promote lung adenocarcinoma development

Change within the intratumoral microbiome is a common feature in lung and other cancers and may influence inflammation and immunity in the tumor microenvironment, affecting growth and metastases. We previously characterized the lung cancer microbiome in patients and identified Acidovorax temperans as enriched in tumors. Here, we instilled A. temperans in an animal model driven by mutant K-ras and Tp53. This revealed A. temperans accelerates tumor development and burden through infiltration of proinflammatory cells. Neutrophils exposed to A. temperans displayed a mature, pro-tumorigenic phenotype with increased cytokine signaling, with a global shift away from IL-1β signaling. Neutrophil to monocyte and macrophage signaling upregulated MHC II to activate CD4+ T cells, polarizing them to an IL-17A+ phenotype detectable in CD4+ and γδ populations (T17). These T17 cells shared a common gene expression program predictive of poor survival in human LUAD. These data indicate bacterial exposure promotes tumor growth by modulating inflammation.

Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

Pyrogallol protects against influenza A virus-triggered lethal lung injury by activating the Nrf2-PPAR-γ-HO-1 signaling axis.

Pyrogallol, a natural polyphenol compound (1,2,3-trihydroxybenzene), has shown efficacy in the therapeutic treatment of disorders associated with inflammation. Nevertheless, the mechanisms underlying the protective properties of pyrogallol against influenza A virus infection are not yet established. We established in this study that pyrogallol effectively alleviated H1N1 influenza A virus-induced lung injury and reduced mortality. Treatment with pyrogallol was found to promote the expression and nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor gamma (PPAR-γ). Notably, the activation of Nrf2 by pyrogallol was involved in elevating the expression of PPAR-γ, both of which act synergistically to enhance heme oxygenase-1 (HO-1) synthesis. Blocking HO-1 by zinc protoporphyrin (ZnPP) reduced the suppressive impact of pyrogallol on H1N1 virus-mediated aberrant retinoic acid-inducible gene-I-nuclear factor kappa B (RIG-I-NF-κB) signaling, which thus abolished the dampening effects of pyrogallol on excessive proinflammatory mediators and cell death (including apoptosis, necrosis, and ferroptosis). Furthermore, the HO-1-independent inactivationof janus kinase 1/signal transducers and activators of transcription (JAK1/STATs) and the HO-1-dependent RIG-I-augmented STAT1/2 activation were both abrogatedby pyrogallol, resulting in suppression of the enhanced transcriptional activity of interferon-stimulated gene factor 3 (ISGF3) complexes, thus prominently inhibiting the amplification of the H1N1 virus-induced proinflammatory reaction and apoptosis in interferon-beta (IFN-β)-sensitized cells. The study provides evidence that pyrogallol alleviates excessive proinflammatory responses and abnormal cell death via HO-1 induction, suggesting it could be a potential agent for treating influenza.

Impacts of immune microenvironment on musculoskeletal health

AbstractUnderstanding the interplay between immune cells and the bone marrow microenvironment is crucial for elucidating age‐related musculoskeletal changes. In this commentary manuscript, we summarized that studies have shown that proinflammatory immune cells in the bone marrow, such as macrophages and neutrophils, can inhibit bone formation by secreting grancalcin. Research on skeletal stem cells (SSCs) in aged mice reveals a shift towards pro‐inflammatory gene expression, affecting their osteogenic potential, while another study maps age‐related changes in cranial SSC niches, emphasizing the role of the CXCL12–CXCR4 axis in stem cell‐immune cell communication, Additionally, the immune system influences hematopoietic stem cells (HSCs); Niche ageing and accumulation of mutations with age lead to HSC exhaustion and a bias towards myeloid differentiation, with toll‐like receptors playing a key role in maintaining hematopoiesis and bone metabolism balance.

Screening, Deconvolution and Parallel Synthesis of Trisubstituted Piperazine and Trisubstituted 2,3-diketopierazine Libraries for the Rapid Identification of Antagonists of the Nuclear Retinoic Acid Receptor-related Orphan Receptor Gamma (RORγ)

Background: Genetic studies support a key role for RORγ and RORα in the differentiation of proinflammatory Th17 cells, and a growing body of evidence suggests a pathogenic role for Th17 in several autoimmune diseases, including MS, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis. RORγ antagonists have been shown to suppress Th17 differentiation and delay the onset of disease in an experimental autoimmune encephalomyelitis mouse model of MS. Objective: Given the high therapeutic interest of RORγ antagonists and the promising activity of currently known ligands, small molecules with higher potency and receptor selectivity (in particular within the ROR family) are highly desirable. We used our small molecule compound library to discover, characterize, and optimize novel RORγ antagonists for the treatment of autoimmune diseases from Mixture-based Combinatorial Chemical Libraries. Methods: We screened the FIU collection of small molecule libraries (&gt;30 million compounds) composed of 75 molecular scaffolds systematically arranged in positional scanning and scaffold ranking formats. We identified scaffolds that selectively inhibit the binding of RORγ, RORγ, and RORβ but not RORα, and others that function as antagonists of all three receptors. Results: The deconvolution of selected PS-SCL mixtures led to the identification of novel chemical entities, trisubstituted piperazine and diketopiperazine that function as RORγ antagonists. Conclusion: The screening of a large complex library led to the rapid identification of novel trisubstituted piperazine and diketopiperazine antagonists of the nuclear retinoic acid receptor-related orphan receptor gamma (RORγ).

Protective Effect of Turmeric against Aluminium-induced Prostate Damage in Rats

This study investigates the potential protective properties of turmeric (Curcuma longa L.) against aluminium (Al) toxicity-induced prostate damage in male rats. The administration of AlCl3 to rats resulted in decreased body weight, increased oxidative stress markers, hormonal depletion, apoptosis induction, and pro-inflammatory cell infiltration in the prostate tissue. Additionally, histopathological examination revealed various abnormalities. However, co-treatment with turmeric extract improved body weight, increased testosterone levels, reduced apoptosis, and mast cell infiltration. C. longa L. exhibited strong antioxidant and anti-inflammatory effects, highlighting its potential as a phytomedicine against reproductive disorders like prostatitis induced by Al exposure.

Abstract 2673: Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy

Abstract Neuroblastoma is the most frequent extra-cranial pediatric cancer with high-risk disease possessing a dismal five-year survival rate &amp;lt;50%. Antibody-based therapies targeting the surface disialoganglioside GD2 have shown promising anti-tumor activity in patients however efficacy is strongly hampered by immunosuppressive mechanisms present in the tumor microenvironment. Neuroblastomas typically feature low mutational burdens, limited major histocompatibility complex class I expression, increased immune checkpoint markers, sparse immune infiltration, and soluble mediators which drastically limit anti-tumor immunity. Given the emerging link between elevated copper levels and tumoral immune evasion, we explored copper chelation as a therapeutic strategy using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA). Using the immunocompetent TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling of resected tumors to assess cellular and molecular changes occurring after one week of TEPA treatment. Therapy was observed to successfully deplete intratumoral copper and reinvigorate anti-tumor immunity with increased infiltration and activity of pro-inflammatory immune cells, and was specifically found to induce egress and N1-polarisation of neutrophils. Further mechanistic studies in vitro revealed the sequestration of copper by neuroblastoma cells attenuated neutrophil function, which could be successfully reversed upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Using the TH-MYCN model, TEPA was employed as a priming agent to effectively potentiate anti-GD2 antibody therapy and achieve durable tumor control. Importantly, this was associated with increased Fc-receptor-bearing natural killer and myeloid cells, which elicit tumor clearance via antibody-dependent cellular cytotoxicity. Currently indicated for the treatment of copper overload disorder Wilson’s disease, TETA was evaluated for potential repurposing as a novel immunomodulatory agent. Using the syngeneic NXS2 model, TETA exhibited an exceptional safety profile and combination therapy achieved durable tumor control with no relapses occurring after treatment cessation. This result was similarly associated with the infiltration of pro-inflammatory immune cells, including N1 neutrophils. Collectively, this study credentials copper chelation as a non-toxic strategy to disrupt the immunosuppressive tumor microenvironment and reinvigorate anti-tumour immunity. Findings provide crucial translational evidence for repurposing TETA to potentiate anti-GD2 antibody therapy and improve neuroblastoma patient responses. Citation Format: Jourdin R. Rouaen, Antonella Salerno, Fabio Luciani, Jayne Murray, Tyler Shai-Hee, Nicodemus Tedla, Michelle Haber, Toby N. Trahair, Orazio Vittorio. Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy [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 2673.

Abstract 6938: Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [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 6938.