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Lung Tfh13 Differentiate from Lymph Node-derived Progenitor Tfh Cells to Sustain Allergic Antibody Responses 2266239

Abstract Introduction Allergic inflammation is driven by high-affinity IgE antibody production. We previously reported that during acute allergy, IL-13—expressing Tfh13 cells promote IgE responses in lung draining lymph nodes. However, as allergy progresses to a chronic state, IgE responses can be compartmentalized, with locally produced IgE maintaining inflammation in lung tissue. This led us to hypothesize that local interactions among lung-resident Tfh, Tfh13, and B cells contribute to chronic allergic inflammation. Methods To test this, we used genetically engineered mice and a house dust mite (HDM) sensitization—challenge model. Lung Tfh subsets were characterized by flow cytometry, single-cell RNA-seq, TCR-seq, spatial transcriptomics, and adoptive cell transfer. Conditional deletion of Tfh populations was used to define functional requirements for lung Tfh populations. Results Tfh and Tfh13 cells accumulated in allergic lungs, with Tfh13 comprising 5—25% of CD4+CXCR5+PD-1+ T cells and producing large amounts of type 2 cytokines. Single-cell RNA-seq showed that lung Tfh13 cells possess a distinct transcriptional program compared to LN Tfh13 or lung effector Tfh. However, TCR-seq linked Tfh13 cells to Tfh progenitor (Tfh prog) populations in lung draining lymph nodes. Adoptive transfer assays further confirmed that Tfh prog cells travel from draining lymph nodes to inflamed lungs, where they differentiate into IL-21—producing eTfh and Tfh13 cells. Spatial transcriptomics further show that lung Tfh populations have distinct positioning indicating exposure to unique environmental factors. Finally, conditional deletion of effector Tfh cells reduced GC B cell and HDM-specific IgG/IgE responses, and attenuated eosinophilic inflammation. Conclusion These findings define in situ differentiation of effector Tfh populations from LN Tfh prog precursor cells and that these cells contribute to local lung inflammation in the context of allergic airway disease. Funding Source N/A Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Effects of biological therapy with anti-TNF and anti-IL-12/IL-23 antibodies on parodontitis in patients with inflammatory bowel diseases 2309583

Abstract Introduction The prevalence of periodontitis is elevated in patients with inflammatory bowel disease (IBD). The neutralisation of antibodies against the cytokines TNF and interleukin-12/23 (IL-12/23) is a fundamental component of the clinical management of IBD. However, the extent to which such cytokine blockade in IBD influences the clinical picture of periodontitis remains to be elucidated. Methods In this exploratory study, 45 patients suffering from IBD who were undergoing biological therapy with cytokine blockers were examined for the presence of periodontitis. The prospective study identified three distinct groups: anti-TNF therapy (n = 15), anti-IL-12/IL-23 therapy (n = 15), no anti-TNF or anti-IL-23 therapy (control group; n = 15). The depth of the gum pockets in all sextants was determined using the Periodontal Screening Index (PSI). Furthermore, a comprehensive medical history pertaining to IBD and oral hygiene was obtained. IL-6 levels were determined by ELISA in gingival crevicular fluid (GCF). Results Of the 45 patients with IBD, 14 had known or evidence of previously unknown periodontitis. The investigation revealed that the mean PSI index in the anti-IL-12/IL-23 group was significantly lower than in the other groups. Within the anti-IL-12/IL-23 group, two individuals with a history of periodontitis exhibited a PSI index of 0, while no subjects demonstrated indications of latent periodontitis. In contrast, three individuals in the anti-TNF group and six individuals in the control group had previously been diagnosed with periodontitis, and two and three additional individuals from these groups exhibited signs of previously unknown periodontitis. IL-6 levels in GCF were significantly lower in the anti-IL-12/IL-23 group as compared to the control group. Conclusion This exploratory study suggested that anti-IL-12/IL-23 therapy in IBD may have protective effects against periodontitis. Possible links between biologic therapy and periodontitis should be investigated prospectively in larger cohorts. Funding Source N/A Topic Categories Immune Mechanisms of Human Disease (HUM)

TLR2 sensing of the SARS-CoV-2 envelope protein drives NLRP3-dependent pyroptosis in post-COVID-19 systemic lupus erythematosus 2300032

Abstract Introduction In systemic lupus erythematosus (SLE), accelerated cell death promotes autoantigen release and immune complex formation, leading to visceral deposition and alveolar hemorrhage (AH) and lupus nephritis (LN). Viral infection has been implicated in autoimmunity, and post-COVID-19 disease flares are increasingly reported in SLE. Elevated pro-inflammatory cytokines are observed in SARS-CoV-2—infected individuals and patients with active SLE; however, the mechanistic role of Toll-like receptor 2 (TLR2) sensing of the SARS-CoV-2 envelope (Env) protein in post-COVID-19 SLE flares remains unclear. Methods Peripheral blood, AH lung tissues, and LN kidney tissues were obtained from SLE experiencing post-COVID-19 flares. THP-1 and RAW264.7 cells were stimulated with Env protein, followed by TLR2 inhibition. In vivo, pristane-induced AH or LN mouse models received intratracheal Env pseudovirus infusion in TLR2-deficient mice or in wild-type mice with concomitant sh-TLR2 delivery. Results Molecular docking predicted TLR2—Env interaction, which was validated by co-immunoprecipitation and solid-phase binding assays. Env stimulation activated NF-κB/MAPK signaling, increased ROS production and pro-inflammatory cytokines in RAW264.7 cells, which were attenuated by TLR2 inhibition or knockdown. In patients with post-COVID-19 SLE flares, plasma IL-1β levels were elevated with increased NLRP3, caspase-1, and N-gasdermin D expression in peripheral blood mononuclear cells, lung, and kidney tissues. Env-induced pyroptosis in THP-1 cells increased LDH and IL-1β release and was suppressed by TLR2 inhibition. In vivo, Env pseudovirus infusion exacerbated pristane-induced alveolar hemorrhage and lupus nephritis via enhanced pyroptosis, whereas TLR2 suppression markedly alleviated disease severity. Conclusion Together, these findings indicate that TLR2 sensing of the SARS-CoV-2 envelope protein promotes NLRP3-dependent pyroptosis and contributes to post-COVID-19 disease flares in systemic lupus erythematosus. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

Beyond Wisdom in III‐V Optoelectronics: Physics‐Enhanced Neural Networks Unveil the Role of Cubic Boron Arsenide

ABSTRACT Cubic boron arsenide (BAs), an emerging III‐V semiconductor renowned for its exceptional transport properties, is gaining recognition as a promising material for next‐generation electronics and optoelectronics. Despite advances exploiting BAs as an individual semiconductor, its broader role within the III‐V semiconductor family remains underappreciated. Here we propose the BAs‐III‐V material platform as active layers to advance beyond conventional wisdom in III‐V optoelectronics since the crystal and band structure of BAs endow the platform with high bandgap tunability. To accelerate the generation of materials for target substrates and bandgaps (TGTMatGen), we extracted structural information from synthetic BAs crystals to explore lattice‐matched BAs‐III‐V and developed physics‐enhanced deep neural networks (PhysenNet) to access bandgaps. PhysenNet excels in bandgap prediction, achieving a mean absolute error of 0.0251 eV between predicted and experimental values. Guided by TGTMatGen, we designed two previously unattainable devices—a 1.55 µm surface‐emitting laser on GaAs and a 3 µm photodetector on InP—both demonstrating commendable performance. We believe our findings contribute not only to the advancement of optoelectronics but also to the broader semiconductor landscape through interdisciplinary innovation.

Behavioural and social drivers of COVID-19 vaccination among front-line health care workers in South-west Ethiopia: a cross-sectional study

B Cells Undergo Transcriptional Changes Following Autoimmune Sicca Development in Melanoma Patients Treated with Immune Checkpoint Inhibitors 2256997

Abstract Introduction Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of melanoma and other cancers but can cause immune-related adverse events (irAEs) and lasting autoimmune tissue damage. One such irAE (ICI-sicca) shares some features with the rheumatic disease, Sjögren’s syndrome, with dry mouth being the symptom typically reported by ICI-sicca patients. A fraction of ICI-sicca patients have Sjögren’s-associated autoantibodies, signifying autoreactive B cell activity in this disease. Methods To identify B cell gene dysregulation that arises with ICI-sicca onset, we used single-cell RNA-sequencing to compare CD19+ cells isolated from peripheral blood collected 3-4 weeks after ICI therapy initiation (pre-ICI sicca) or following ICI-sicca symptom onset (post-ICI sicca) in n = 6 melanoma patients. Transcriptome-based clustering using Seurat identified n = 14 clusters, which we manually collapsed into five major B cell subsets (transitional, naïve, activated, memory, and plasmablasts). Results We observed upregulation of genes following ICI-sicca development in naïve (n = 1296 genes), activated (n = 42 genes), and memory (n = 775 genes) B cell subsets, relative to the pre-ICI sicca timepoint, based on adjusted p-value < 0.05 and fold change > 1.2 cutoffs. These upregulated genes were related to BCR signaling, actin cytoskeleton rearrangement, antigen processing and presentation, and interferon responses. Conclusion Some of the ICI-sicca upregulated genes were also noted to be increased in rheumatic disease patients relative to healthy controls in other single-cell RNA-seq studies performed by our lab, suggesting the potential for shared B cell liability across spontaneous and ICI-induced autoimmunity. Future studies will validate alteration of these key B cell functions and explore pharmacologic methods to restore this aberrant B cell rewiring as a means to generate new hypotheses that can be tested with the ultimate goal of limiting ICI sicca, and potentially other irAE development. Funding Source National Institutes of Health Grants R03 AI163835 (RHB), Luke and Susan Simons Directorship (DBJ), and K12 HD043483 (RHB), as well as the Vanderbilt University Summer Research Program (HB), MARC/START (HB) supported this work. Studies were performed using Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

CD36-mediated metabolic changes in Tbet+CD11c+ B cell during obesity 2309773

Abstract Introduction CD21- CD23- CD11c+ Tbet+ B cells expand in primary immune and peripheral tissues during chronic conditions such as aging, autoimmune diseases, and obesity. CD11c+ Tbet+ B cells are a class-switched, antigen-specific memory population that exhibit a distinct transcriptomic profile from other B cell subsets characterized primarily by enrichment of metabolism and cytokine pathways. Our previous study demonstrated that transfer of T-bet + B cells exacerbates metabolic syndrome during obesity. In addition, CD36 is a scavenger receptor expressed highly in CD11c+ T-bet+ B cells which also mediates oxLDL-induced metabolic reprogramming from OXPHOS toward glycolysis in macrophages. Methods This project uses cutting-edge approaches, including SCENITH and single-cell RNA sequencing, combine with flow cytometry based metabolic analyses to comprehensively characterize metabolic pathways in CD11c+ T-bet+ B cells at the molecular level. In parallel, a high-fat diet in a unique mouse model with B cell specific CD36 deletion, allows us to define the role of CD36 in mediating metabolic reprogramming in CD11c+ T-bet+ B cells during obesity. Results Our preliminary data show that CD36 knockdown in B cells markedly reduces CD11c+Tbet+ B expansion and lowers circulating anti-oxLDL autoantibody levels in both HFD fed and R848-immunized mice. Assessment of alterations in glycolytic and oxidative phosphorylation pathways, mitochondrial morphology, mass, and function, help us to define the transcriptomic profiles of CD11c+ T-bet+ B cells during obesity, while determining how B cell specific CD36 deletion modulates these metabolic programs. Conclusion Overall, defining the role of CD36 in driving metabolic reprogramming of CD11c+ T-bet+ B cells during disease progression will provide critical insights for the development of therapies that selectively target defined cellular pathways. Funding Source The Flow Cytometry Shared Resource at UT Health San Antonio is supported by the National Cancer Institute (P30CA054174) through the Mays Cancer Center, the Cancer Prevention and Research Institute of Texas (CPRIT) (RP210126), the National Institutes of He Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

SLC7A5-mediated glutamine export is required for humoral immunity 2308652

Abstract Introduction Germinal center (GC) B cells and plasma cells strongly upregulate gene expression of SLC7A5, which is an amino acid antiporter that simultaneously mediates leucine uptake and glutamine export. While the importance of leucine uptake is established, the significance of glutamine export and its role in humoral immunity is unknown. Methods B-cell-specific Slc7a5-KO mice Results SLC7A5 was dispensable for B cell development. However, Slc7a5-/- GC-B cells failed to undergo IgG1 class switch and somatic hypermutation upon immunization. Moreover, Slc7a5-/- GC-B cells showed lower dark/light zone (DZ/LZ) cell ratio, cell cycle arrest in DZ, reduced AID expression, and impaired plasma cell differentiation and antibody production. In collagen induced arthritis model, Slc7a5 deletion decreased disease severity and reduced serum levels of anti-collagen IgG2a, IgG3, IgM, as well as IL6. To test whether impaired humoral immunity was caused by defective glutamine export, we created an alternative glutamine disposal route using 4-phenylbutyric acid (4-PBA). 4-PBA treatment significantly rescued IgG1 class switch of Slc7a5-/- cells ex vivo, supporting the critical role of glutamine export in GC-B cells. Metabolomics and isotope tracing revealed that Slc7a5-/- B-cells redirected glutamine from glutaminolysis to nucleotide salvage, prioritizing its role as nitrogen donor over carbon donor. We thus hypothesized that defective glutamine export led to cell defects by nitrogen (ammonia) accumulation. While only hepatic cells can use the urea cycle to detoxify ammonia, non-hepatic cells are known to transport ammonia to liver in the form of glutamine. To test if SLC7A5-mediated glutamine export indeed enables ammonia detoxification, we introduced the urea cycle enzyme CPS1 into Slc7a5-/- cells, which restored cell fitness, confirming the key role of SLC7A5-mediated glutamine export in ammonia detoxification. Conclusion SLC7A5-mediated glutamine export maintains humoral immunity by enabling ammonia detoxification. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

ExTh17 Cells Arising from Th17 Support Chronic RA-Like Inflammation Independent of IL-17 2260860

Abstract Introduction T helper 17 (Th17) cells are present in both peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA), yet IL-17-targeted therapies have shown limited efficacy in established disease. The role of IL-17 and Th17 cells in RA has not been completely clarified. Although inflammation can drive Th17 cells to transdifferentiate into IL-17-negative Th1- or Tr1-like so-called exTh17 cells, the role of these cells in arthritis remains unclear. Here, we investigate whether exTh17 cells contribute to joint inflammation in autoimmune arthritis. Methods Using Th17 lineage tracing in the SKG mouse model of RA, we demonstrate that synovial Th17 cells transdifferentiate into IFNγ- and IL-10-producing exTh17 cells, which become the dominant CD4+ T cell population in chronic arthritis. These exTh17 cells are more arthritogenic than Th17 cells and maintain synovial inflammation through IL-17-independent mechanisms involving elevated IL-6 and TNF, two key cytokines in RA pathogenesis. Synovial exTh17 cells analyzed by bulk, single-cell RNASeq, and Spatial transcriptomics exhibit a distinct gene expression profile, including upregulation of S1PR4, in CD4+ T cell profiles from mouse and human RA synovial tissue. We further show, using co-culture assays, that interaction between Th17 cells and fibroblast-like synoviocytes (FLS) via S1P signaling promotes Th17-to-exTh17 conversion. Results The pro-inflammatory environment of arthritis induces a population of exTh17 cells, promoting chronicity of arthritis without re-expression of IL-17. Blocking S1P—S1PR4 signaling inhibits their formation independently of lymph node T cell trafficking. Fibroblast-like synoviocytes (FLS) promote exTh17 generation via S1P production, and RA FLS are major S1P sources in both humans and mice. Conclusion These new insights open the door to more targeted therapeutic strategies beyond IL-17 inhibition. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Membrane Shedding Through T Cell Microvilli Enhances Clonal Expansion via mTOR Pathway Activation 2266804

Abstract Introduction When T cells establish an immunological synapse with antigen-presenting cells (APCs), they release surface membrane fragments enriched with TCR micro-clusters. This membrane shedding, induced by T cell-target cell contact, delivers materials and information from T cells to other cells in particle form. Recent studies have revealed that T cells enhance their clonal expansion via this membrane shedding. However, the mechanism by which membrane release upregulates T cell proliferation remains unclear. Methods Using various adhesion matrices and chemical compounds, we mimicked membrane shedding and observed that release of microvilli from the T cell surface during activation affects the mTOR pathway. Results Membrane shedding through surface microvilli altered the lipid composition of T cells, promoted phosphorylation of phosphoinositide 3-kinase (PI3K), and upregulated mTOR-related signaling pathways, driving T cell clonal expansion. Conclusion Conversely, suppression of T cell microvilli shedding led to weakened mTOR signaling and impaired clonal expansion. Our findings demonstrate that contact between T cells and APCs induces T cell surface membrane release, which provides essential signals for clonal expansion. Furthermore, we propose that membrane shedding is not a disadvantageous loss of cellular components but rather an active mechanism intrinsic to T cell proliferation. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Genetic Depletion of Tumor-cell Derived Complement Factor H Suppresses Glioblastoma Development 2334891

Abstract Introduction Complement factor H (CFH) is a pivotal inhibitory modulator in complement alternative activation pathway. Our previous study has shown that tumor cell-derived CFH enhances both regulatory T cells and myeloid derived suppressor cells in the glioblastoma (GBM) microenvironment. Overexpression of CFH in GBM cells resulted in significantly decreased survival in murine models of GBM. Based on these findings, here we aim to test the hypothesis whether inhibition of tumor cell originated CFH could suppress tumor growth, providing further rationale for CFH-targeted GBM therapy. Methods Human glioma cell line U87 cells were transduced with lentiviral particles encoding either CFH-specific CRISPR-CAS9 or scramble control. CFH depleted U87 cells were selected and verified by Western Blot. In vitro cell proliferation rate was measured by the Cell Counting Kit 8. Nude mice (B6.Cg-Foxn1/nu/J) were intracranially injected with the parental (parental, n = 8), scramble control (scramble, n = 8) and the CFH-depleted (KO, n = 8) U87 cells, respectively and monitored for survival. Comparison between multiple experimental groups was conducted using one-way ANNOVA and Kaplan-Meier curve was used to compare the survival rate between multiple groups with Bonferroni correction. Results Depletion of CFH resulted in significant decrease of in vitro proliferation of U87 cells (parental vs. KO, P < 0.001, scramble vs. KO, P < 0.001, parental vs. scramble, ns). Mice intracranially injected with CFH-depleted U87 cells had a dramatically increased survival rate with median overall survival (mOS) at 41.5 days (P < 0.0001) in comparison to mOS=29 days for the parental group and mOS=25.5 days for the scramble group. Conclusion Data from current study demonstrates that genetic depletion of tumor cell-derived CFH represents a promising approach for GBM targeted therapy. Further mechanistic investigation is warranted to delineate the molecular underpinning of CFH-mediated tumorigenesis and immunosuppression in GBM. Funding Source Philanthropic donation Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Δ8-Tetrahydrocannabinol (Δ8-THC) attenuates LPS-induced acute respiratory distress syndrome (ARDS) through immune-endothelial reprogramming 2300199

Abstract Introduction Acute Respiratory Distress Syndrome (ARDS) is a severe inflammatory lung disorder associated with high mortality. Currently, there are no FDA-approved pharmaceuticals approved for treatment of this disorder. This study evaluated the therapeutic potential of Δ8-Tetrahydrocannabinol (Δ8-THC), a cannabinoid that is less psychoactive than Δ9-THC, in a murine model of LPS-induced ARDS. Methods Female C57BL/6 mice received LPS (10 mg/kg, intratracheally) to induce ARDS, followed by Δ8-THC or vehicle treatment (i.p.). After 48h, lung tissue was collected for scRNA-seq analysis. Results scRNA-seq analysis of vehicle & Δ8-THC treated lungs identified sixteen transcriptionally dis-tinct clusters across stromal, endothelial, myeloid, and lymphoid lineages. Δ8-THC significantly reduced infiltration of neutrophils, while increasing the populations of polymorphonuclear MDSCs, immune endothelial cells and macrophages compared to vehicle. Neutrophil clusters from vehicle group showed enrichment of ribosomal and translational activity reflecting inflammatory stress which were significantly downregulated in Δ8-THC treated group, and instead shifted gene expression toward interferon-responsive, immunoregulatory programs. Δ8-THC-enriched clusters exhibited increased interactions via COLLAGEN, APP, CypA and CDH5 pathways, consistent with enhanced endothelial stabilization. Conclusion Δ8-THC alleviated lung inflammation in LPS-induced ARDS by reprogramming immune and endothelial responses toward interferon-driven, regulatory states. These findings suggest a potential therapeutic role for Δ8-THC in restoring lung homeostasis during ARDS. Funding Source This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Ligand Engineering for Precise Control of Ultrathin CsPbI ₃ Nanoplatelet Superlattices for Efficient Light‐Emitting Diodes

ABSTRACT Strongly‐confined perovskite nanoplatelets (PeNPLs) offer opportunities not found in conventional isotropic nanocubes, especially in producing linearly polarized light, as well as enhancing outcoupling through control over the transition dipole moment. But this requires ultrathin nanoplatelets with three or fewer monolayers of PbI 6 octahedra across the thickness, which are challenging to synthesise uniformly, and their luminescence is strongly affected by surface defects. Together, these limit the performance of ultrathin PeNPLs in light‐emitting diodes (LEDs). Here, we address these challenges with an ancillary ligand engineering strategy. We demonstrate that ligands with phosphoryl functional groups strongly bind to the perovskite surface, while having an organic backbone that is not sterically bulky ensures high ligand density. By modulating nucleation and growth, these ancillary ligands lead to monodisperse PeNPLs that stack more uniformly when self‐assembled into superlattices, with suppressed agglomeration. As a result, from edge‐up PeNPL superlattices, we achieve an enhanced degree of polarization, while from face‐down PeNPL superlattices, we achieve enhanced outcoupling that results in LEDs with 13.1% external quantum efficiency, the highest reported for ultrathin PeNPL LEDs. This work establishes ancillary ligand‐induced synthesis as a decisive route to achieve uniform nanoplatelets with robust orientation control, enabling full utilization of the multifunctionality of anisotropic PeNPLs.

Validity and reliability of the Turkish version of the Diabetes Acceptance and Action Scale (DAAS-R)

Antibody Recruiting Small Molecules as Immune Probes 2254059

Abstract Introduction Immunotherapies that enhance the immune response may offer a novel approach for eliminating mycobacteria infections. Our collaborative team synthesized and characterized the first antibody-recruiting small molecule (ARM) for mycobacteria. ARMs are bispecific molecules that attach haptens to pathogenic cells and recruit endogenous, anti-hapten antibodies that promote immunity. The core component to our ARMs is trehalose, which integrates into the bacterial membrane via a conserved pathway and displays the hapten, DNP, for antibody binding. Tre-DNP incorporation increases antibody recruitment, phagocytosis, and killing by macrophages. Methods We hypothesized that antibody-dependent Fc receptor (FcR) signaling promotes macrophage phagolysosome (PL) fusion, which we tested by measuring colocalization of Lysotracker-labeled lysosomes and CMFDA-stained bacteria. To further enhance ARM incorporation and antibody recruitment, we developed second-generation ARMs using a two-step click conjugation approach. Results Macrophages that ingested ARM-labeled and opsonized mycobacteria exhibited greater PL fusion compared to the controls. Preliminary data indicated that these ARMs achieved improved incorporation and antibody recruitment compared with the one-step approach. Conclusion Ongoing studies are testing whether greater antibody recruitment or different haptens promote a stronger immune response. A mechanistic understanding of how Tre-DNP enhances the immune response toward mycobacteria could lead to further refinement of this immunotherapeutic strategy. Funding Source William and Linda Frost Fund, NIH 2R15AI117670-03 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Itaconate alleviates autoimmune uveitis by boosting FOXP3 and blocking succinate-driven inflammation 2309786

Abstract Introduction We recently reported the activation of the Irg1/itaconate axis in promoting inflammation resolution during ocular infectious disease. This study aims to determine the immunomodulatory role of itaconate in the pathogenesis of experimental autoimmune uveitis (EAU). Methods EAU was induced in wild-type (WT) or Irg1 KO (knock out) mice by immunization with IRBP. Disease severity was assessed based on fundus examination and retinal function testing. Ocular tissues (retina and cornea), splenocytes, and cervical lymph nodes were harvested at 21 days post-immunization. Flow cytometry was used to determine the frequencies of myeloid and lymphoid cells. In vitro studies were performed using mouse splenocytes, and challenged with IRBP in the presence or absence of succinate and 4-octyl itaconate. ELISA and qPCR assays were performed to measure inflammatory mediators. Results Our data show that Irg1 KO mice developed severe EAU. The exacerbated EAU in Irg1 KO mice is accompanied by increased frequencies of myeloid cells, specifically macrophages, as well as pathogenic Th17 cells. The elevated frequency of these immune cell infiltrations coincided with a decrease in regulatory T cells. Additionally, Irg1 KO mice exhibited increased levels of succinate and its receptor. The in vitro experiments using splenocytes from Irg1 KO mice or succinate treatment in WT mice revealed decreased regulatory T cells and Foxp3 expression. However, in splenocytes (Irg1 KO) treated with 4-OI, the regulatory T cell numbers and Foxp3 expression were restored. Moreover, the 4-OI treatment significantly suppressed the levels of inflammatory mediators and succinate. Conclusion Our study demonstrates that Krebs cycle metabolites regulate innate and adaptive immune responses during EAU. Itaconate derivative supplementation exerts anti-inflammatory effects, inhibits succinate expression, and promotes the regulatory cells, indicating its therapeutic potential in treating ocular inflammation during autoimmune uveitis. Funding Source U.S. Department of Defense grant HT94252510634, NEI grants R01EY026964, R01EY027381, R01EY035499 Topic Categories Basic Autoimmunity (BA)

Glycolysis Dependence of Lupus Antigen-Specific B cells Linked to Mitochondrial Dysfunction and Age-associated B Cell Differentiation 2309226

Abstract Introduction Different metabolic requirements between autoreactive and non-autoreactive B cells are potential targets of therapeutics to selectively inhibit autoantibody (autoAb) production in systemic lupus erythematosus. Pharmacological inhibition of glycolysis blocks autoAb production in lupus mouse models, but not that of immunization-induced Ab. The goal of this study was to elucidate the mechanisms behind the glycolysis requirements of lupus antigen (Ag) specific B cells. Methods Rheumatoid factor (RF)-producing AM14 BCR Tg B cells transferred into BALB/c mice were stimulated with either cognate Ag PL2-3 or TLR7/8 agonist R848. In parallel, NP-specific B1-8 BCR Tg B cells were transferred into BALB/c mice immunized with NP-OVA. Recipient mice were treated or not with 2-deoxy-D-glucose (2DG) to inhibit glycolysis. Bulk RNA-seq of FACS-sorted AM14 and NP+ B cells was performed followed by Ingenuity Pathway Analysis (IPA). Results 2DG decreased the production of RF in PL2-3-, but not R848-stimulated AM14 recipient mice. 2DG also decreased anti-NP IgM and IgG in B1-8 recipient mice. However, 2DG induced major transcriptional changes only in PL2-3-stimulated AM14 B cells, in which it downregulated mitochondrial dysfunction, cytokine signaling, phagosome formation, and Inositol Phosphate Compounds pathways, while upregulating the oxidative phosphorylation (OXPHOS) pathway. IPA Upstream Regulator Analyses predicted activation of metabolism-related transcription regulators, MYC and NFAT5, and inhibition of extrafollicular (EF) age-associated B cell (ABC) differentiation regulators, IFN-γ, IRF7, and IL-21. Expression of ABC markers also decreased, consistent with the EF pathway differentiation of AM14 B cells. Conclusion 2DG treatment shifts the transcriptional profile of autoreactive AM14 B cells to favor OXPHOS and inhibit pathways related to BCR and endosomal TLR signaling. These results suggest that glycolysis inhibitors may curb the EF differentiation lupus-antigen specific B cells. Funding Source NIH R37AI128901 Topic Categories Basic Autoimmunity (BA)

Macrophage Priming Against Glyco-Antigens in the Host Microbiome 2260858

Abstract Introduction The mammalian immune system recognizes and responds to a broad variety of glycan antigens, most notoriously including lipopolysaccharides (LPS). LPS lacking O-antigens, known as lipooligosaccharides (LOS) , can also cause strong immune responses, including Guillain-Barré syndrome (GBS), the leading cause of autoimmune flaccid paralysis in the USA. This condition can develop following resolution of food-borne illness and is commonly associated with infection by Campylobacter jejuni, which produces LOS structures that mimic neuronal gangliosides. Methods Previously, we showed that macrophage-like THP-1-derived monocytes can be tolerized against the GM1 ganglioside antigen. Pre-incubation with GM1-mimicking C. jejuni (or purified LOS) results in dampened pro-inflammatory cytokine responses following challenge with GM1-mimicking C. jejuni. Here, we continue utilizing this model, complemented with transcriptomics, to probe macrophage responses to C. jejuni challenge following pre-incubation with opportunistic Enterococcus species originating from guts of multiple animals. Results We show that cholera toxin B subunit (GM1 ganglioside probe) cross-reacts to glyco-antigens produced by opportunistic Enterococcus species found within the gut microbiome. Further, a subset of these species can prime THP-1 cells towards a pro-inflammatory cytokine response upon subsequent exposure to C. jejuni. Conclusion Together, these data further our understanding of how the host immune system recognizes and responds to microbial glycans within the gut microbiome and how this can impact subsequent autoimmune development. These data suggest that colonization with certain Enterococcus species may serve as a potential risk factor for GBS development. Funding Source NIAID R21AI175925, DoD CDMRP PR181475 Topic Categories Mucosal and Regional Immunology (MUC)

Antigen clade matching enhances protective efficacy of H5N1 DNA vaccines delivered by electroporation or lipid nanoparticles 2267127

Abstract Introduction Highly pathogenic avian influenza viruses (HPAIs) continue to threaten both agriculture and human health. Recent H5N1 clades have caused zoonotic infections in humans with mortality rates approaching 50%. However, currently licensed H5 vaccines are based on ancestral strains and may provide suboptimal protection against circulating variants. Rapidly adaptable DNA vaccine platforms offer a promising approach for clade-specific protection. Methods Codon-optimized plasmid DNA vaccines expressing hemagglutinin (HA) from two recently circulating H5N1 clades (2.3.2.1c and 2.3.4.4b) were generated and delivered by either intramuscular electroporation (EP) or a lipid nanoparticle (LNP) formulation. Cellular and humoral responses were evaluated by multiparameter flow cytometry and ELISpot and by ELISA and pseudovirus neutralization, respectively. Protective efficacy was evaluated in lethal H5N1 murine challenge models. Results EP delivery of clade 2.3.2.1c HA (pCamb) elicited strong humoral and cellular responses and achieved complete protection against homologous viral challenge, but only partial protection against heterologous 2.3.4.4b challenge. In contrast, vaccination with clade 2.3.4.4b HA (pMich) DNA supported robust immune responses and full protection against contemporary clade challenge. Co-immunization with both plasmids via EP induced broad binding and neutralizing antibodies and conferred complete protection from clade 2.3.4.4b challenge. Moreover, formulation of the pMich plasmid optimized LNPs generated durable, protective immunity following a single dose, effective at both acute and memory timepoints. Conclusion These studies demonstrate that antigenic clade-matching is likely critical for protection against H5N1 and suggest that currently stockpiled H5N1 vaccines may not protect against contemporary viruses. Further this data suggests that DNA vaccine platforms including EP or LNP formulations can provide a flexible approach for rapid adaptation to evolving influenza strains. Funding Source NIH NIAID CIVICs Topic Categories Vaccines and Immunotherapy (VAC)

Immunotherapy Resistant Triple Negative Breast Cancer Utilizes Interferon Gamma Signaling to Evade NK Responses during Metastasis 2335047

Abstract Introduction Combination immunotherapy is an approved treatment for stage II-III triple negative breast cancer (TNBC). However, partial responders to immunotherapy develop rapid recurrence and lethal metastatic disease. The mechanisms underlying this relapse and subsequent metastasis are largely unknown. Methods To address this problem we used mouse models to investigate how cancer cells in the primary tumor mass that survive immunotherapy contribute to subsequent recurrence. We found that immunotherapy-resistant cancer cells are more metastatic and more efficiently colonize distal organs. Molecular characterization of these cells revealed both transcriptional upregulation of interferon stimulated genes (ISGs) and increased chromatin accessibility and occupancy of STAT and IRF factors, indicating augmented interferon signaling and sensitivity. Results Exposure to interferon gamma (IFNg) alone resulted in a durable increase in metastatic burden both in naïve and tumor experienced mice but not in immunodeficient hosts. Individual metastatic foci from IFNg treated cells were similarly sized to those from untreated cells and neither obtained a proliferative advantage, nor maintained significantly higher PD-L1 or MHC-I expression. Antibody depletion studies showed that the IFNg mediated evasion was through escape of NK cell interactions. Specifically, these interactions occurred within the first day of metastatic seeding and were mediated by the upregulation of both canonic and novel ISGs Conclusion Combined, these data reveal a possible mechanism by which patients with partial responses to immunotherapy undergo metastatic recurrence and highlight the need for close monitoring of these patients for metastatic spread. Funding Source 1F32CA298459-01A1 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)