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Interleukin-9 Receptor Signaling Promotes an Immunosuppressive Tumor Immune Microenvironment in Oncogene-Driven Non-Small Cell Lung Cancer 2264775
Abstract Introduction Non-small cell lung cancer (NSCLC), particularly oncogene-driven subtypes, remains a leading cause of cancer-related mortality, underscoring the urgent need for novel immunotherapies that effectively target the tumor immune microenvironment (TIME). The interleukin-9 receptor (IL-9R) signaling pathway is understudied but emerging evidence, including our previous work, suggests a pro-tumoral role in NSCLC by promoting an immunosuppressive TIME. We hypothesize that IL-9R signaling fosters immunosuppression within the lung TIME of NSCLC and that its blockade represents a viable therapeutic strategy to restrain tumor progression. Methods Using a K-rasLSL-G12D/+; p53fl/fl (KP) mouse model of NSCLC, we compared tumor development between IL-9R knockout (Il9r—/—) and wild-type (WT) mice. Flow cytometry was performed to analyze lung-infiltrating immune cell populations. Bone marrow chimera experiments were conducted to assess the contribution of IL-9R signaling in hematopoietic versus non-hematopoietic compartments. Results Il9r—/— tumor-bearing mice exhibited significantly reduced lung tumor burden compared to WT controls. Flow cytometric analysis revealed that IL-9R deficiency decreased the accumulation of immunosuppressive myeloid cells, including neutrophils/granulocytic MDSCs and arginase 1+ macrophages, while enhancing pro-inflammatory cytokine production (IFN-γ, TNF-α, and IL-17) by T cells. Bone marrow chimera studies showed that WTmice reconstituted with Il9r—/— bone marrow exhibited reduced tumor burden, whereas Il9r—/— recipients reconstituted with WTbone marrow displayed tumor progression comparable to WT controls, indicating that IL-9R signaling in hematopoietic cells drives tumor-promoting immunosuppression. Conclusion Our findings identify IL-9R signaling as a critical regulator of the immunosuppressive lung TIME and suggest that targeting the IL-9R pathway may represent a promising immunotherapeutic strategy for oncogene-driven NSCLC. Funding Source The Indiana Clinical & Translational Sciences Institute (CTSI) Biomedical Research Grant Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Interferon signaling primes the lung epithelium for macrophage-mediated repair 2261178
Abstract Introduction The lung is continuously exposed to the external environment and is therefore highly susceptible to infection and injury. Successful recovery following pathogen encounter requires coordinated responses between epithelial and immune compartments to restore homeostasis. Alveolar type II epithelial cells (ATIIs) act as progenitors for the alveolar epithelium, while alveolar macrophages (AMs) regulate inflammation and repair. Type I interferon (IFN-I), produced in response to viral stimuli, is essential for antiviral defense but has been traditionally viewed as antagonistic to tissue regeneration. However, whether early IFN-I sensing influences epithelial repair programs remains unclear. Methods We used in vivo models of infection (influenza) and viral stimuli (poly(I:C)) to investigate how IFN signaling influences ATII behavior. Transcriptional profiling, flow cytometry, immunofluorescence microscopy, lineage tracing, and ex vivo alveolar organoids were employed to assess ATII proliferation, phenotype, and interactions with macrophages. Results Acute IFN-I signaling in the lung transiently induced a Sca-1+ ATII population that constituted the primary proliferative subset in vivo. IFN-I exposure heightened ATII sensitivity to macrophage-derived growth factors, enhancing their responsiveness to reparative cues. Ex vivo, Sca-1+ ATIIs displayed increased organoid-forming efficiency compared with Sca-1⁻ ATIIs, consistent with elevated progenitor potential and regenerative capacity. Conclusion Together, these findings suggest that IFN-I sensing primes ATIIs for subsequent repair. This work uncovers a previously unrecognized role for IFN-I in preparing the lung epithelium for regeneration following inflammation and injury. Funding Source NIH R35GM150816, NIH F31HL172650 Topic Categories Mucosal and Regional Immunology (MUC)
Resistin-Like Molecules Modulate Lung Immunity during Influenza 2266488
Abstract Introduction Resistin-like molecules (RELMs) are cysteine-rich epithelial secreted proteins implicated in mucosal immunity. RELMα is broadly immunoregulatory, whereas RELMβ is associated with pro-inflammatory activity. We previously showed that acute allergic asthma protects against influenza morbidity and observed markedly increased RELMα and RELMβ in the lungs during asthma and influenza comorbidity, leading us to hypothesize that RELMs contribute to enhanced antiviral defense. Methods Both loss- (knockout mice) and gain-of-function (recombinant proteins) approaches were used. Results Allergic Retnla and Retnlb deficient mice infected with pandemic A/CA/04/2009 influenza A virus exhibited altered disease kinetics relative to wild-type allergic controls, with knockout mice showing distinct viral clearance patterns, indicating that endogenous RELMs influence influenza pathogenesis during allergic inflammation. Our scRNA sequencing data revealed that Retnl gene expression in immune and structural cells were significantly different between the asthma- and influenza-only controls. RELM-deficiency was associated with worsened airway physiology after infection, supporting a functional role for these proteins in preserving lung integrity during viral challenge. In complementary prophylactic studies, recombinant RELMα, RELMβ, or both administered intranasally to wild-type mice significantly reduced lung viral burden and improved airway mechanics. RELM treatment remodeled the airway immune landscape, increasing macrophages, neutrophils, and CD8+ T cells, with corresponding shifts in CD4+ T cell frequency and humoral responses. Conclusion Together, these data identify RELMs as epithelial-derived modulators of antiviral immunity that contribute to the protection observed in the asthma and influenza comorbidity setting. Ongoing studies are aimed at testing post-infection therapeutic administration and combined delivery to determine whether RELMs can be harnessed to enhance host resistance to respiratory viral disease. Funding Source R01 AI125481; American Lung Association Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
High Dimensional Profiling of T cells in ME/CFS Uncovers Sex Specific Dysregulation 2309017
Abstract Introduction Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME) is a debilitating and poorly understood chronic disease. ME affects more women than men and studies have shown sex differences in disease presentation. Patients experience symptoms indicative of immune dysregulation including sore throat and tender lymph nodes. Transcriptomic and functional analyses have identified T cell dysfunction in ME (Vu et al 2024; Gil A et al 2024; Visser et al 1998). ME T cell research has largely been done on bulk lineage populations and fails to discriminate dysregulation within different functional or differentiation states. Analysis has often combined data from both sexes, even though sex differences in immune response could mask sex-specific disease presentation. Given the complexity and importance of the T cell compartment and the role sex plays in immune function, it is critical to rigorously identify sex and subset-specific dysregulation in ME. Methods Cytometry by Time of Flight identifies cell surface protein expression at the single cell level. We used a panel tailored to T cells to characterize peripheral blood mononuclear cells from 53 ME patients (11 M and 42 F) and 36 (10 M and 26 F) healthy controls and identified cell types using unsupervised clustering. Results Only in ME males is there an increase in the percent of CD27+ γδ T cells, which produce IFNγ. IFNγ targets intracellular pathogens and the increase could implicate an increased response to viral pathogens in ME. Female ME CD8+ T cells show a chronically stimulated phenotype across T memory subsets, while male ME memory cells show a subset specific activation phenotype. Conclusion This work identifies functionally relevant changes in T cell subsets in ME and highlights the importance of examining sex differences when studying chronic disease. Understanding T cell dysfunction in ME on a sex and subtype level will aid in determining the mechanism of immune dysfunction and reveal potential therapeutic targets. Funding Source NIH NIAID U54AI178855 Topic Categories Translational and Interventional Immunology (TI)
The roles of Type I interferon subtypes and signaling in mRNA lipid nanoparticle vaccine-induced CD8 T-cell protective immunity 2266569
Abstract Introduction The modification of RNA by the incorporation of N-methyl pseudo-uridine (N1mψ) renders the mRNA less inflammatory, resulting in durable cellular and humoral responses. Interestingly, mRNA-LNP vaccines still elicit strong innate immune activation and stimulate the production of type I interferons (IFN-I) by antigen-presenting cells (APCs). IFN-I is critical in the induction of CD8 T-cell responses to mRNA vaccines; however, the mechanisms underlying this process are poorly understood. Methods We determined protection from lethal infection with ectromelia virus in mice with cell-specific deletion of IFNAR1, as well as in mice deficient in specific IFN-I, following vaccination with a previously described EVM158 mRNA-LNP vaccine containing an immunodominant CD8 T-cell epitope. Results We demonstrate that the optimal induction of CD8 T-cells requires IFN-I signaling in multiple myeloid cell types, not just in CD8 T-cells, dendritic cells, or monocytes. We also show that at optimal vaccine doses, IFN-β- and IFN-α-deficient mice generate protective CD8 T-cell responses. However, at suboptimal doses, male mice deficient in IFN-β and in IFN-α4, which are regulated by constitutive transcription factors, do not generate completely protective CD8 T cells. Moreover, mice deficient in the NF-κB binding site in the IFN-β promoter and the transcription factor IRF7 generate poorly protective responses. Conclusion Our results indicate that optimal induction of protective CD8 T-cell responses by mRNA LNP vaccines requires IFN-I signaling in myeloid cells but not in CD8 T-cells, that IFN-β and IFN-α can individually provide the signals required for protection, and that regulation of IFN-β by NF-κB and all IFN-I subtypes by IRF is required to induce protective CD8 T-cells. Funding Source NIAID, NIH Topic Categories Vaccines and Immunotherapy (VAC)
Bioinspired Subnanowire‐Based Optical Devices With Programmable Polarization Irisations Through Photocuring 3D Printing
ABSTRACT The fabrication of photocrosslinkable subnanowires (SNWs) suitable for bioinspired photocurable 3D printing has not been realized. Moreover, constructing SNW‐based optical devices that integrate optical rotation (OR) and birefringence (BF) remains a significant challenge. Here, the photocrosslinkable Tri‐MPT@GdOOH SNW ( 1 ) with tunable chirality was synthesized via in‐situ ligand exchange and covalent modification. Inspired by Bouligand architecture and optical properties of scarab cuticles, the 1 ‐based levorotatory assembly was employed to fabricate a SNW‐based hybrid gel ( 1a ) through photocuring 3D printing. The resulting material exhibits a well‐defined layered structure, an ultrahigh compressive strength of 155.89 MPa, excellent stability, and pronounced left‐handed chirality, with a 1.03 × 10 −3 g‐factor. The 1a ‐based waveplate demonstrates a strong synergistic effect between left‐handed OR and stress‐induced BF. Specifically, it achieves remarkable specific rotations of 2042, 1545, and 1410 mL/(g·dm) under 405, 520, and 650 nm lasers, respectively, and a BF index of 2.28 × 10 −4 . Furthermore, a series of 1a ‐based meta‐optical arrays and bioinspired 3D optical devices ( 2‐12 ) with tunable architectures and polarization irisations were successfully fabricated, which exhibit significant application potential in optical information security. This work presents a representative case for the fabrication of photocrosslinkable SNWs and highlights the promising application prospect of bioinspired SNW‐based optical devices.
Engineering Layered Magnetic Hydrogels for Cell Placement via Shear and Magnetic Field‐Induced Assembly
ABSTRACT The design of hydrogel‐based artificial tissues capable of reversible, programmed, and complex motions requires both stimuli‐responsiveness and structural anisotropy. In this work, non‐unidirectional anisotropies are generated in biocompatible hydrogels by structuring magnetic particle suspensions into lamellar architectures through two distinct routes: the application of an unsteady magnetic field to a quiescent sample, and the superposition of a steady magnetic field with shear flow. In both approaches, magnetic particles undergo directed self‐assembly within a polymer matrix that subsequently gels, thereby preserving the formed structures. We analyze the assembly kinetics, characterize the resulting lamellar patterns, and construct phase diagrams for each method. The morphology and periodicity of the lamellae are shown to depend strongly on geometric confinement, enabling tunable interlamellar spacing from tens to hundreds of microns. Crucially, it is demonstrated that the resulting layered hydrogels can confine human fibroblasts between adjacent particle‐rich lamellae, maintain cell viability above 95% over 7 days of culture, and promote preferential cell alignment parallel to the layered structures. These findings establish magnetic field‐directed lamellar structuring as a versatile route to anisotropic hydrogels with programmable internal architecture, opening new opportunities in tissue engineering, bioactuation, and soft robotics.
E3 Ubiquitin Ligase Peli1 Regulates TLR7-Driven Spontaneous Germinal Center and Plasma Cell Development 2309783
Abstract Introduction TLR7 signaling is indispensable for the development of spontaneous germinal center (GC) and plasma cell (PC); however, the mechanism that restrains GC and PC formation remain incompletely defined. Here, our goal is to identify the role of E3 ubiquitin ligase Peli1 in the regulation of TLR7-driven spontaneous GC and PC responses. Methods Peli1 expression was assessed in activated B cell subsets from SLE-prone Sle1b.Yaa mice, which overexpress TLR7, and in wild-type B cells stimulated with TLR7 agonist R848 in vitro. B cell—intrinsic functions of Peli1 were assessed using global and conditional Peli1-deficient mice. GC and PC responses were assessed by flow cytometry and immunofluorescence imaging of GC architecture. Antibody production was measured by ELISA and metabolic activity was determined by Seahorse analysis, at baseline and following imiquimod treatment. Results Peli1 was upregulated in B cells from Sle1b.Yaa mice and in R848-stimulated wild-type B cells. Peli1 deficiency led to increased Tlr7 expression and elevated levels of the NF-kB member, c-Rel. Functionally, loss of Peli1 resulted in enhanced GC B cell and PC responses, increased IgG2c antibody production, and heightened metabolic activity, all of which were further amplified by imiquimod treatment. Conditional deletion of Peli1 in peripheral B cells recapitulated these phenotypes, indicating that Peli1 restrains B cell—intrinsic TLR7-driven immune Conclusion We identify the role of Peli-1 in negative regulation of TLR7 expression and TLR7-driven spontaneous GC, PC and IgG2c antibody responses. Funding Source NIH Topic Categories Basic Autoimmunity (BA)
Machine learning-driven stratification of Immune disorders using serum cytokine autoantibody profiles detected by cell-based multiplex flow cytometry 2259074
Abstract Introduction Impaired immune tolerance triggers aberrant or sometimes overshooting immune responses, contributing to disease manifestations and/or exacerbations. Notably, the prevalence of serum cytokine-specific autoantibodies (c-AAbs) has been documented in ‘healthy’ individuals as well as in association with acute and chronic states of immune disorders. Precise detection of c-AAbs, which can either neutralize, stabilize, or modulate the function of cytokines, requires sensitive assays that present the antigen in its native conformation. Methods A novel multiplex flow cytometry-based cellular assay (mFCCA) was devised based on a collection of stable HEK-293T single-cell clones expressing functional interferons and interleukins, for the assessment of c-AAbs in patients’ serum (n = 400), comprising controls, HIV-infected, and those with suspected hyperinflammatory, autoimmune, or malignant disorders characterized by laboratory parameters. The IgG immunoreactivity against 15 cytokine-expressing cell lines was evaluated in a multiplex format, alongside the parental cell line, with specific binding quantified as delta channel geometric mean fluorescence intensity. Subsequently, a cross-validated Random Forest algorithm was applied to discriminate between the five disease phenotypes and the controls. Model performance was gauged by the receiver operating characteristic curves. Results Distinct disease-specific c-AAb signatures were identified across patient groups. For instance, HIV patients exhibited significantly higher frequencies of anti-IFN-α2a, IL-1α, IL-2, and IL-5 autoantibodies compared with controls. The classification model demonstrated high accuracy, with area under the curve values ranging from 0.91 to 0.99, and correct assignment of 331 of 400 samples (83%) to their respective groups. Conclusion The high-throughput approach for screening c-AAb reactivities against surface-expressed cytokines via multiplexed flow cytometry enables rapid immunopathology classification and may assist targeted therapy. Funding Source The work was supported by the Federal State of Lower Austria under the Danube Allergy Research Cluster 2.0 (Danube-ARC 2.0) [grant no. FA624A0404 —Project#13] and the Medical University of Vienna. Topic Categories Basic Autoimmunity (BA)
Uncovering novel regulators of immune response in rhesus macaque single-cell RNA-seq data 2260001
Abstract Introduction Single-cell RNA-seq (scRNA-seq) analyses rely on accurate gene annotations, a challenge for many species with less completely curated genomes. Rhesus macaque (RM), a widely used model for human biomedical research, is one such case where missing gene annotations have hindered the study of immune responses. We aim to develop a computational framework to identify and reintegrate missing gene features, improving immune response characterization in RM. Methods In a preliminary analysis, we computationally searched in a RM peripheral blood mononuclear cell (PBMC) scRNA-seq dataset from a kidney allograft study for unannotated but transcriptionally active regions (uTARs). We then performed cell clustering twice, once on annotated-gene expression and again on uTAR expression, and assessed uTAR expression for cell-type specificity and association with immune-related pathways. Results We identified >5,500 uTARs, indicating that numerous features–e.g., long non-coding RNAs or alternative transcripts of existing genes–are missing from current RM annotations. uTARs exhibit cell-type-specific expression and, when used to group cells, separate major cell types, paralleling cell clustering using annotated rhesus genes. These findings illustrate substantial gaps in the RM genome annotation and highlight the biological relevance of these missing genes or transcripts. Conclusion uTARs likely harbor many previously unannotated genes or transcripts that are involved in immune regulation in RM. Ongoing work will denoise the signals in scRNA-seq data and prioritize a subset of uTARs as candidate transcriptional regulators. We will also infer regulatory relationships between candidate regulators and downstream targets. Our immediate goal is to identify drivers of transplant rejection. However, this framework is broadly applicable to scRNA-seq datasets across species and experimental contexts. Funding Source NIAID U19 AI131471 Topic Categories Technological Innovations in Immunology (TECH)
Type I Interferon priming in Macrophages is associated with poor responses in Patients with Advanced Prostate Cancer 2267089
Abstract Introduction Prostate cancer patients acquire resistance to standard-of-care strategies resulting in 350,000 yearly deaths. As acquired resistance is mediated by increased androgen receptor (AR) expression, “Bipolar Androgen” therapy (BAT) is being developed to cycle serum testosterone from supraphysiological to near-castrate levels, maximizing toxicity to high and low AR-expressing cells respectively. BAT is a clinically effective and safe approach that improves quality of life, produces biochemical and objective responses, and re-sensitizes tumors to AR inhibitors. We previously demonstrated that despite its conception as a “targeted” therapy, BAT’s effects on the immune system appear critical for success. Methods We examined immune cells from tumor biopsies and peripheral blood mononuclear cells (PBMCs) before and after BAT, using spectral flow cytometry, single cell RNA sequencing and spatial transcriptomics. Results We found that macrophages in tumors of non-responding patients have increased Type I IFN gene expression, compared to responding patients. As macrophages in tumors arise from circulating monocytes, we examined how monocyte tumor engraftment was impacted by BAT. Intriguingly, classical monocytes display enhanced expression of Type I IFN response genes only in patients with poor responses, mimicking what we observed in tumors. Moreover, we discovered a subset of Type I IFN primed monocytes that are increased in frequency in these patients and demonstrated their altered response to IFN alpha and ability to generate macrophages with an altered phenotype. Conclusion BAT reprograms circulating monocytes, conferring a Type I IFN signature that is mirrored by macrophages in tumors from patients with poor therapeutic response. Type I IFN has been associated with resistance to other cancer therapies and could play an important role in limiting immune control of prostate cancer following BAT. Critically, BAT reprograms tumor immunity and our results provide an attractive target to improve this therapy. Funding Source Prostate Cancer Foundation Young Investigator Award, Prostate Cancer Foundation (PCF) Maryland Moonshot Scholar, FY24 JHI Maryland Cancer Moonshot Initiative Supplemental Research Grant, Maryland Department of Health and Mental Hygiene National Institutes of Health — National Cancer Institute, Award ID: 1R21CA273501-01A1 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Kolliphor EL Alters IgE-Mediated Immune Functions 2266473
Abstract Introduction Food allergies (FA) are becoming increasingly prevalent worldwide, affecting more than 220 million people globally. Anaphylaxis is the most rapid and severe allergic reaction resulting in a rapid drop in blood pressure, constriction of airways, hives and swelling, gastrointestinal symptoms, and can be deadly. IgE-mediated anaphylaxis occurs when mast cells (MCs) with IgE bound to its high affinity receptor become crosslinked by allergen, leading to degranulation and the release of mediators such as histamine and tryptase, causing allergic symptoms. While food allergens are the most well-known trigger, drug-induced anaphylaxis is often linked to the solubilizing agents in pharmaceutical formulations; however, this is poorly understood. Kolliphor EL (KE) also known as PEG-35 castor oil, is a commonly used non-ionic surfactant and drug solubilizer. KE has been reported to trigger MC activation leading to hypersensitivity and anaphylactic reactions, although it is unclear if these are IgE dependent or not. Methods IgE sensitized bone marrow derived mast cells (BMMCs) from wild-type C57B/6 mice were pre-treated with KE for 1 hour before antigen IgE crosslinking. Beta hexosaminidase was measured using an enzymatic reaction 30 minutes after crosslinking and IL-13 was measured by ELISA 16 hours after crosslinking. For in vivo experiments C57B/6 mice were injected with DNP specific IgE and KE. The next day, mice received another dose of KE 1 hour before DNP induced anaphylaxis. Results Surprisingly, we demonstrate that KE decreased IgE-mediated degranulation and IL-13 production in a dose-dependent manner in vitro. In vivo, mice treated with KE were protected against IgE-mediated passive systemic anaphylaxis. Conclusion Collectively, these findings suggest that KE modifies immune responses that require further investigation. This research aims to determine whether KE’s immune effects can be beneficial for treatment of anaphylaxis or whether it should be more carefully restricted in clinical formulations. Funding Source N/A Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)
Covid19 Effects on V-ATPase Subunit Gene Expression in Ionocytes that Regulate Ion and pH Homeostasis in the Oral Cavity 2267195
Abstract Introduction Understanding innate immune responses to SARS-CoV-2 in oral minor salivary glands provides important insight into how the virus establishes infection and contributes to disease. Ionocytes in the oral cavity are still poorly defined, particularly in relation to ion transport and pH. An RNAseq UMAP of human minor salivary glands (https://www.covid19cellatlas.org/warner20/) identified buccal ionocytes expressing FoxI1, a specific biomarker, along with FoxI2, hBD-1, CFTR, ASCl3, NKCC1, and V-ATPase subunits ATP6V1G3 and ATP6V0D2. Here, mRNA expression of these V-ATPase genes was examined to understand the molecular identity of ionocytes during viral infection. We hypothesize that gene expression of ATP6V0D2 and ATP6V1G3 will be modulated by SARS-CoV-2 infection. Methods Buccal swabs were collected from September 2021 to February 2022. qRT-PCR was performed on total RNA extracted (Qiagen RNeasy Kit) from 47 COVID19(+) adults, 10 COVID19(-) patients and 16 recently recovered COVID-19(-) patients using an iScript and SYBR-green protocol, ATP6V1G3, ATP6V0D2, and β-actin primers. Results A significant (p < 0.05, Mann-Whitney U) increase of ATP6V0D2 mRNA in COVID19 patients occurred compared with both non-COVID19 and recovered COVID19 patients. ATP6V1G3 mRNA was unchanged between all groups. Conclusion Buccal cells contain epithelial cells and ionocytes, explaining the increase of ATP6V0D2 in the Covid19 cohort. This suggests that Covid19 influences the ionocyte and may affect pH in oral minor salivary glands. Funding Source NIH/NIDCR, Summer Dental Research Program, University of Louisville School of Dentistry Topic Categories Viral Immunology (VIR)
Progressive Development of Ectopic Lymphoid Structures as Mediators of Autoimmunity in a Sjögren’s Disease Mice Model 2309026
Abstract Introduction Ectopic lymphoid structures (ELS) are dynamic immune assemblies that acquire phenotypic and functional features of secondary lymphoid organs (SLO). They develop and persist in inflammatory conditions such as Sjögren’s Disease (SjD), a chronic autoimmune disease characterized by lymphocytic infiltration of lacrimal (LG) and salivary (SG) gland. The presence of ELS in patient SG biopsies is linked to poor prognosis but less is known about ELS formation in the LG in SjD in human disease or murine models. Methods Development of ELS was studied in LG from male NOR mice, the diabetes free sub-strain of the NOD (n = 6 LG from 6 mice/group) at early (8 weeks), intermediate (16 weeks) and established (24 weeks) disease. ELS were identified based on immunofluorescence labeling of B/T cells, and the presence of follicular dendritic cells (FDC) and high endothelial venules (HEV). Gene expression of essential markers associated with the initiation and development of ELS were explored as well as those involved in glandular health in terms of secretory function and apoptosis. Results ELS were identified by identification of regions with distinct segregation of B and T cell zones and further validated by demonstrating the presence of HEV, the FDC network, GL7+ germinal center B cells, and the presence of IgG-producing plasma cells. Analysis of gene expression showed significant early upregulation of Ltb, Glycam1, Cxcl13, Cxcr5, Ccl19 and Ccr7 (P-value< 0.05) while other markers of ELS were elevated later in disease (Acida and Il21) (P-value< 0.05). In parallel, compromised glandular epithelial health was indicated by increased gene expression of FasL (P-value< 0.05), suggestive of apoptosis, and decreased expression of Gpx4 (P-value< 0.05), suggestive of ferroptosis, in intermediate disease. Conclusion Male NOR mice spontaneously develop ELS in LG with features of SLO. These structures may function as key local drivers of autoimmunity and autoantibody production. Funding Source NIH grant RO1 EY026635, NIH RO1 EY011386, P30EY029220 to the Department of Ophthalmology at USC Keck School of Medicine. Topic Categories Basic Autoimmunity (BA)
The Fungal Connection: Gut Mycobiome and Multiple Sclerosis 2267567
Abstract Introduction Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system with unclear etiology, influenced by genetic and environmental factors. Recent studies have highlighted a potential role of the gut microbiome in MS, with a specific focus on the bacterial components. However, the impact of the gut mycobiota- the fungal community, on immune function and disease remains underexplored. Methods We investigated human samples and different experimental murine model systems by using various microbial, immunological, bioinformatics and molecular biology state-of-the-art techniques. Results Our results show distinct fungal community profiles in patients with MS compared to healthy controls and the manipulation of the mycobiota in experimental autoimmune encephalomyelitis (EAE), a mouse model mimicking many aspects of MS, indicated an important role on gut microbiome, immune cells and disease activity. Conclusion Based on these findings, we hypothesize that gut fungi play an important role on microbiome-immune interactions and thereby could impact the pathogenesis of MS. Thus, the further exploration of our research may have the potential to lead to the discovery of unique biomarkers and to the future development of novel treatment or prevention strategies for MS. Funding Source FWO Topic Categories Neuroimmunology (NEUR)
True Closed‐Loop Recyclable Hydrogels Enabled by Imine Boronic Ester Crosslinking
ABSTRACT The relationship between molecular structure and macroscopic function is a foundational principle in materials science, in which subtle molecular variations produce pronounced differences in strength, stiffness, and elasticity of macromolecular solids. In hydrogels, replacing static covalent bonds with dynamic covalent bonds (DCBs) creates newfound capabilities, including self‐healing and recyclability. Herein, substitutional differences in dual DCB imine boronic ester crosslinkers, together with matrix pH, influencing hydrogel properties are investigated. A comparison of ortho ‐ and para ‐imine boronic esters showed the formation of 3‐amino‐benzoxaborole heterocycles in hydrogels derived from 2‐formylphenylboronic acid. Tautomerization to the heterocycle significantly enhanced hydrogel elasticity, despite a lower crosslinking density than in hydrogels formed with 4‐formylphenylboronic acid. Two closed‐loop end‐of‐life (EOL) management pathways are also demonstrated. Reprocessing through self‐healing is accomplished, with hydrogels regaining at least 90% of their original rheological properties. A fully circular recycling pathway is also established, recovering all starting materials for reuse, with recycled hydrogels achieving over 100% recovery of rheological properties. Overall, the presence of previously undisclosed 3‐amino‐benzoxaborole structures is demonstrated, expanding the understanding of formylphenylboronic acids in polymeric materials, and complete closed‐loop EOL pathways are designed to inspire greater focus on full EOL processes in materials circularity.
Immunometabolic Dichotomy Underlying Monocyte-Driven Kawasaki Disease and Lymphocyte-Driven MIS-C Pathogenesis 2309868
Abstract Introduction Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) share overlapping clinical features but differ markedly in severity and cardiac outcomes. Both conditions involve systemic hyperinflammation, yet the cellular and metabolic mechanisms underlying their divergent pathogenesis remain unclear. Because immune cell function is tightly regulated by metabolic programs, we hypothesized that KD and MIS-C are driven by distinct cell-type—specific immunometabolic alterations. Methods Publicly available single-cell RNA sequencing datasets of PBMCs from KD and MIS-C patients (GSE184330, GSE168732) were analyzed. Metabolic pathway activity was quantified using KEGG and Reactome gene sets. Pathway scores were compared between both diseases, and Random Forest analysis was used to identify pathways and cell types that best discriminated disease states. Results Both KD and MIS-C showed shared metabolic activation in classical monocytes, including increased glycolysis, fatty acid synthesis, and cholesterol metabolism. However, a clear immunometabolic divergence was observed. KD was characterized by metabolically hyperactive monocytes with reduced itaconate-associated anti-inflammatory signaling, while adaptive immune cells retained higher itaconate pathway activity. In contrast, MIS-C exhibited relative metabolic restraint in monocytes but marked suppression of itaconate-linked regulatory pathways in lymphoid populations, including NK cells, γδ T cells, Th1 cells and regulatory T cells. Random Forest analysis identified lymphocyte-specific loss of itaconate regulation as the strongest feature distinguishing MIS-C from KD. Conclusion KD is driven by innate myeloid metabolic overactivation, whereas MIS-C reflects loss of metabolic regulation in adaptive lymphoid cells. This immunometabolic divergence highlights cell-type—specific itaconate pathway activity as a potential diagnostic biomarker. Funding Source IIT Palakkad Topic Categories Immune Mechanisms of Human Disease (HUM)
Structural valency of APRIL identifies and controls B cell intrinsic class switch recombination 2259580
Abstract Introduction CSR is an intrachromosomal DNA rearrangement of the IgH locus in which constant regions are replaced by another isotype, diversifying antibody effector function but not antigen specificity. Conventionally, CSR is triggered and directed by T cell help but can also be initiated by T independent cytokines, including APRIL. APRIL is a homotrimer produced primarily in myeloid cells that initiates T cell independent CSR by engaging its receptor TACI on B cells. In this project I define how the valency of APRIL controls the T independent CSR. Methods Recombinant APRIL (Ala96-Leu 240) was expressed in Expi293 cells and purified using size exclusion chromatography where trimeric and monomeric fractions were collected separately. Naïve splenic B cells from C57BL/6 mice (1 × 10^6 cells/mL) were stimulated with APRIL trimer or monomer (2ug/mL)±IL-4 (20ng/mL); media served as control. CSR was assessed by sterile transcripts at 48 hours (RT-qPCR) and surface IgG and IgM at 96 hours (flow cytometry). Results After 48-hour culture, sterile transcripts, formed by the opening of a transcription bubble at the switch region, were detected using qPCR, and after a 96-hour culture surface Ig isotype was detected using flow cytometry. At the protein level, APRIL trimer induced significantly more CSR to IgG than APRIL monomer. Monomer CSR activity to IgG can be rescued with the addition of IL4 to the level of trimer. At the transcript level, trimer did not induce significantly more sterile transcripts to any IgG subtype than monomer, pointing to trimeric ligation of TACI by APRIL as a requirement for generating productive spliced DNA at the IgM to IgG switch region. In the monomeric state, CSR can be induced as evidenced by sterile transcripts, but intrachromosomal DNA rearrangements to form productive IgG transcripts cannot. Conclusion We present a model for T independent CSR, where trimeric structure of APRIL is needed to push CSR beyond initiation steps to a fully spliced DNA and productive mRNA IgG transcript. Funding Source NIH Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
Impact of Thymus Removal in Altering CD8 T Cell Properties 2260157
Abstract Introduction The thymus supplies naïve T cells to sustain T cell populations, but it gradually shrinks with age, leading to a decline in thymic output. Consequently, cessation of thymic output reduces naïve T cell numbers, raising the question of whether the remaining cells adapt to maintain immune competence. Methods Here, we examined the quantity and quality of antigen-specific CD8 T cells in mice after thymectomy. Results Our experiments showed that thymectomy reduced the number of antigen-specific CD8 T cells. Interestingly, following acute lymphocytic choriomeningitis virus (LCMV) infection, antigen-specific CD8 T cells derived from thymectomized mice exhibited greater expansion and generated more memory cells than those from controls. To explore why CD8 T cells from thymectomized mice possess such augmented expansion capacity, we analyzed these CD8 T cells prior to infection and found significant phenotypic remodeling after thymectomy, thereby altering their subset composition. Virtual memory (VM) cells, which exhibits memory phenotypes yet remain antigen inexperienced, increased within the antigen-specific CD8 T cell pool after thymectomy. In addition, thymectomy altered the phenotypes of antigen-specific naïve CD8 T cells, greatly enriching the IL-18Rα+ CD73+ CXCR3+ subset. Importantly, this subset contributed to the enhanced expansion of antigen-specific CD8 T cells after LCMV infection. Conclusion Together, our findings suggest that upon loss of thymic output during thymic involution or thymectomy, antigen-inexperienced CD8 T cells undergo qualitative remodeling, compensating for reduced cell numbers by increasing functionally potent subsets that help preserve antiviral immunity. Funding Source n/a Topic Categories Viral Immunology (VIR)
Siglec15 and Siglec14 are new immune check point receptors during Mycobacterium tuberculosis infection of human macrophages 2259532
Abstract Introduction Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) killing a million people each year. Mtb persists in macrophages and tissues from where reactivation of TB can occur. We found that Mtb upregulated several Siglec receptors in human macrophages (MDMs) and we hypothesized that they are immune check point receptors (ICPRs) Methods MDMs were infected with Mtb, and treated with specific MABs or IgG isotype and Mtb survival was measured along with cellular oxide responses and transcriptional responses using RNAseq post antibody treatment. Results 1) Blockade using specific MABs enhanced the ability of MDMs to kil intracellular Mtb; blockade of Siglec15 and Sigec14 were most potent followed by Siglec1,7,9 and 10. 2) MABs induced reactive oxygen species (ROS) in MDMs and their blockade enhanced the growth of Mtb suggesting that Siglec15 and Sigec14 regulate ROS response in MDMs. 3) Transcriptome analysis was done using KEGG, Gene Ontology and Reactome work flow. Unlike others, MAB-Siglec14/MAB-Siglec15 signal through DAP12 and enhanced gene expression of multiple modules of innate immunity in MDMs including cytokine-cytokine receptor, chemokines, TNF, TLR, CLR, NF-kB and JAK-STAT pathways. 4) MABs to Siglec1,7,9 and 10 also upregulated similar modules and in addition, genes of the IFN responsiveness. Conclusion Siglecs are novel ICPRs and targets for host directed therapy of TB Funding Source AI-161015 NIAID, NIH. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)