Browse Articles

High Resolution Single-Cell Epigenetic Atlas of Immune Cell Types Reveals Gene Regulatory Circuits in Healthy Humans 2308992

Abstract Introduction Single-cell chromatin accessibility (scATAC-seq) profiles genome-wide regulatory elements that shape immune cell identity and function, but its interpretation is currently limited by low cell type resolution and small reference datasets. Existing datasets annotate fewer than 20 immune cell types and are too coarse to resolve heterogeneity and characterize cell type-specific gene regulatory programs and functions. Here, we present a large-scale scATAC-seq resource that substantially improves immune cell annotation and regulatory inference. Methods By integrating matched-donor scRNA-seq and scATAC-seq data from human peripheral blood mononuclear cells (PBMCs) with trimodal TEA-seq (single-cell ATAC, RNA, and surface protein), we classified 36 immune cell types, including 4 myeloid, 6 B cell, 5 NK cell, 6 CD4 T cell, and 15 CD8 T cell subtypes. Cell frequencies from published scRNA-seq and new scATAC-seq labels were highly correlated (median ρ = 0.84). Labels were applied to our longitudinal multi-modal dataset of 206 samples spanning over 3 million PBMCs from 78 healthy human donors. Results We used these annotations to define baseline epigenetic states, age-associated differences, and epigenetic changes following influenza vaccination. Our analysis revealed extensive sets of differentially accessible tiles and enriched transcription factor motifs that define cell type-specific regulatory identities. Linking these chromatin regions and transcription factors to differentially expressed target genes enabled the construction of gene regulatory circuits associated with cell type, aging, and vaccination. Additionally, we trained a classification model for high resolution cell type labeling and doublet detection in new scATAC-seq datasets. Conclusion Together, this multi-modal atlas and associated cell type-labeling model provide an unprecedented reference for immune cell gene regulatory circuits and a valuable resource for exploring the epigenome of human immune cells. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

Cyclodextrin-Based Nano-Immunotherapy Enhances Dendritic Cell Activation to Drive Anti-Tumor Immunity in Bladder Cancer 2334395

Abstract Introduction High risk non muscle invasive bladder cancer is commonly treated with intravesical BCG therapy, yet many patients fail treatment or develop recurrent disease. Effective anti tumor immunity in bladder cancer depends on dendritic cell activation and IL-12 production, which promotes T cell priming and effector function. We developed CANDI, a modular nanoparticle immunotherapy platform designed to deliver small molecule innate immune agonists directly to tumor antigen-presenting cells and enhance IL-12-driven immunity. Using optical screening, we identified a combination of innate stimulants that robustly induce IL-12. Unexpectedly, inclusion of the JAK inhibitor ruxolitinib further enhanced IL-12 production when co-delivered with innate agonists. Methods Bone marrow-derived dendritic cells from IL-12 reporter mice were used to quantify cytokine production following CANDI treatment. Therapeutic efficacy and immune responses were evaluated in orthotopic and metastatic MB49 bladder cancer models using intravesical and intravenous delivery. Tumor burden, dendritic cell migration, and T cell responses were assessed by imaging and flow cytometry. Results Triple combination CANDI significantly increased IL-12 production and dendritic cell activation compared to dual formulations. In tumor-bearing mice, CANDI reduced tumor burden, enhanced dendritic cell trafficking to tumor-draining lymph nodes, and improved antigen presentation and T cell priming. Comparable immune activation and tumor control were observed following intravesical and systemic delivery. In metastatic disease, CANDI reduced lung tumor burden and improved survival. Therapeutic efficacy was largely dependent on CD4 T cells, which amplified local IL-12 production by tumor resident dendritic cells. Conclusion Triple combination CANDI is a potent dendritic cell activating nano immunotherapy that drives durable anti tumor immunity and provides a rational strategy to overcome limitations of prior innate immune agonist therapies. Funding Source NIH, DOD, BICAN Topic Categories Vaccines and Immunotherapy (VAC)

Discovery and validation of novel IgA proteases for the treatment of autoimmune and inflammatory diseases 2328257

Abstract Introduction IgA is a multifunctional immunoglobulin essential for maintaining tolerance to commensal bacteria in the gut lumen and for detecting and neutralizing harmful pathogens both systemically and within the gut. When IgA activity becomes dysregulated, it can trigger immune activation and inflammation through autoimmune responses. Galactose-deficient IgA1 (Gd-IgA1) acts as an antigen for autoantibodies, leading to immune complex (IC) formation that contributes to kidney dysfunction in IgA nephropathy (IgAN), one of the most common forms of glomerulonephritis and a frequent cause of end-stage renal disease. Approaches that clear IgA from serum represent promising strategies for treating IgA-mediated autoimmune and inflammatory diseases. Methods N/A Results N/A Conclusion Previously, bacteria-derived serine proteases have been shown to clear ICs in animal models of IgAN and human tissue samples. However, their large molecular weight has made them difficult to develop as therapeutic agents. In contrast, a 60 kDasmaller cysteine protease isolated from commensal bacteria has been described, though never sequenced. To address this, we searched bacterial genomes for secreted proteins and identified several protease candidates capable of cleaving IgA1. Using our IMPACT Platform and evolutionary screening, we discovered additional novel cysteine IgA proteases from various human commensal bacteria. Our findings show these proteases are potent in cleaving IgA1 from healthy donor serum and remain active on both biochemically generated Gd-IgA1 and mixed IgA1 from IgAN patient serum samples, highlighting a promising new approach to ablate IgA1treat IgAN patients. Further protein engineering and optimization with the IMPACT Platform are needed for multi-objective refinement of our candidates to reduce immunogenicity, increase selectivity, and improve drug-like properties. Funding Source Research performed at for-profit company (Seismic Therapeutic) Topic Categories Immune Mechanisms of Human Disease (HUM)

Investigating immune checkpoint blockade of LY-6A protein as cancer immunotherapy in mice 2330710

Abstract Introduction Ly-6A is a potential immune checkpoint on T cells. Evidence suggests that overexpression of Ly-6A diminishes T cell responses; ligand interaction induces the intrinsic apoptotic pathway, whereas the loss of this expression leads to moderate hyperresponsiveness. Notably, Ly-6A-/- mice exhibit reduced B16-F10 tumor growth compared with wild-type controls. This study evaluated the therapeutic efficacy of four anti-Ly-6A monoclonal antibodies in the B16-F10 mouse model using wild-type C57Bl/6 mice. Methods A B16-F10 melanoma model was established in C57BL/6 mice. 100 µg of each antibody was injected intraperitoneally. 3 days post initial treatment, tumor cells were injected subcutaneously. Tumor volume was measured starting at palpability and calculated as V = ½ x {(Length) x (Width²)}. Efficacy was assessed using three parameters: overall survival, terminal tumor weight, and day 12 tumor volume. Following euthanasia, tumors were cryopreserved in liquid nitrogen. Statistical analysis of volume and weight was performed via one-way ANOVA. Harvested tumor and spleen sections were processed for immunohistochemical analysis of T-cell markers. Results While most mice exhibited progressive tumor kinetics, one antibody clone demonstrated superior therapeutic efficacy. In this cohort, 80% of mice had no palpable tumors by day 12. Although mean growth rates did not reach statistical significance across all time points, the reduction in early tumor burden and the increase in overall survival (p < 0.05) highlight the efficacy of this specific Ly-6A clone. Conclusion This study established the therapeutic efficacy of anti-Ly-6A antibodies and identified one candidate with superior tumor growth suppression. Ongoing research will evaluate the synergy of a dual Ly-6A and PD-1 blockade. To elucidate the underlying mechanisms, we will utilize multi-parameter FACS and RNA-seq of tumor-infiltrating lymphocytes to identify cellular changes associated with this novel checkpoint inhibition. Funding Source Funded by Villanova University College of Liberal Arts and Sciences Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Tumor-innervating nociceptors drive immuno-suppressive monocyte differentiation to prevent T cell-mediated anti-cancer immunity 2259756

Abstract Introduction Nociceptors — peripheral nervous system neurons that trigger the sensation of pain or itch in response to noxious stimuli — can communicate with leukocytes and modulate immune responses in a context-dependent manner. Indeed, recent work has indicated that nociceptors may also play important roles in tumor immunity. Nevertheless, much remains unknown about which types of tumors may be affected by nociceptors and what the effects of nociceptors are. Results Here, using The Cancer Genome Atlas (TCGA), we found that in human patients with urinary bladder carcinoma (BC), high expression of neurotrophic factors promoting sensory innervation correlated with significantly worse prognosis. Accordingly, in a murine orthotopic model of BC, incipient tumors promoted dense innervation by nociceptors. Strikingly, nociceptor ablation significantly decreased tumor incidence in WT but not RAG-deficient animals, indicating that nociceptors may prevent immune-mediated eradication of tumors. Nevertheless, nociceptors did not directly affect the development of tumor antigen-specific T cells in tumor-draining lymph nodes; rather, their activity was essential during the early stages of tumor formation to establish an immunosuppressive microenvironment, which protected the tumor cells from T-lymphocyte-mediated killing. As a result, chemogenetic inhibition of nociceptors during the early stages of tumorigenesis markedly increased the rate of BC rejection. Mechanistically, we observed that nociceptors induce a phenotype reminiscent of myeloid-derived suppressor cells (MDSC) in tumor-infiltrating monocytes, imbuing them with the ability to inhibit T-cell responses. Accordingly, monocyte depletion in nociceptor-sufficient mice also inhibited BC formation. Conclusion Collectively, these findings demonstrate that solid tumors can co-opt an immunosuppressive neuro-immune axis to evade anti-tumor immunity, and identify nociceptors as potential targets for bladder cancer treatment. Funding Source n/a Topic Categories Neuroimmunology (NEUR)

Multiphoton Imaging of Regulatory T Cell and Oligodendrocyte Interactions During Neural Stem Cell Transplantation in Autoimmune Encephalitis 2308362

Abstract Introduction Multiple Sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by demyelination and neuroinflammation. Regulatory T cells (Tregs) maintain self-tolerance, resolve inflammation, and are important for tissue repair in the CNS. Using experimental autoimmune encephalomyelitis (EAE), a model of immune-mediated demyelination, we have shown that transplantation of human neural stem cells (NSCs) in situ elevates Treg numbers and promotes tissue repair. In this study, we image cellular interactions among Tregs and oligodendrocytes (ODCs), the cells that produce myelin, to uncover the mechanisms of myelin repair. Methods We used quantitative multiphoton imaging (MPI) with NSC transplantation and fate-mapped reporter mice in the EAE model to elucidate the spatial organization of Treg cells relative to ODCs in the spinal cord. Treg cell therapy and flow cytometry were used to evaluate ex vivo expansion of Tregs using an NSC-derived antigen and homing of adoptively transferred Tregs into the CNS to promote functional recovery during EAE. Results During EAE, NSC transplantation resulted in a two-fold higher Treg density in the spinal cord. Time-lapse MPI revealed that NSC-expanded Treg cells migrate in slightly straighter trajectories while maintaining their velocities, contributing to their local spread in the spinal cord. Tissue-wide imaging revealed EAE-specific loss of ODCs in the lumbar region, which was restored by NSC transplantation. Moreover, NSC-expanded Treg cells were observed in proximity to ODCs. Finally, NFM (neurofilament medium), an NSC-derived antigen, expanded Tregs ex vivo, and adoptive transfer of NFM-expanded Tregs reduced EAE severity. Conclusion Our results show that NSC-expanded Treg cells promote tissue repair during EAE, in part by restoring regional ODC numbers. Our study highlights the strength of direct imaging of neuro-immune interactions and the therapeutic potential of Treg expansion strategies for autoimmune demyelinating diseases of the CNS. Funding Source Supported by NIH/NIAID grants R01AI168063 and U01AI160397 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

A novel hydrophilic nanoparticle mRNA-based platform to deliver antigen-specific immune therapies in autoimmune diseases 2334959

Abstract Introduction We developed NanoGalaxy™ a hydrophilic nanoparticle (HNP) platform, to deliver mRNA to spleen antigen-presenting cells (APC) and induce antigen-specific immune tolerance (ASIT) in autoimmune diseases. Methods mRNA delivery to spleen APC and T cell activation were assessed by intravenous (i.v.) injection of HNPs encapsulating GFP and Ovalbumin (Ova) mRNA and flow cytometry. ASIT was assessed in a delayed-type hypersensitivity (DTH) model induced with Ova protein immunization and in the experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein peptide (MOGp) immunization. For ASIT therapies, mice were injected i.v. with HNP encapsulating Ova or MOGp mRNAs plus/minus tolerogenic immune modulators. Therapeutic responses were assessed by measuring footpad inflammation (DTH) or clinical scores (EAE), and immune correlates were assessed by cytometric bead array (CBA) and flow cytometry in splenocytes. To assess translatability, Cynomolgus macaques received two i.v. doses of HNPs encapsulating Ova mRNA administered three weeks apart. T cell responses were assessed by FluoroSpot and safety was assessed by analyzing blood parameters. Results In mice, HNPs transfected APCs in spleen with minimal hepatic uptake, and induced antigen-specific T cell responses. HNPs encapsulating Ova or MOGp mRNAs plus tolerogenic immune modulators efficiently induced ASIT in the DTH and EAE models, respectively, while HNPs without tolerogenic co-payloads did not lead to optimal immune tolerance. In Cynomolgus macaques, immunization with HNPs delivering Ova mRNA was well tolerated and induced antigen-specific T cell responses, without evidence of hepatic toxicity or changes in key blood parameters. Conclusion Thus, NanoGalaxy™ HNP is a versatile platform to deliver mRNA-encoded antigens plus immune modulators to spleen APCs and induce ASIT in autoimmune diseases, while showing translational potential and safety in non-human primates. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

NIR‐II Protein‐Escaping Dyes Enable High‐Contrast and Long‐Term Prognosis Evaluation of Flap Transplantation

Heterogeneity of mechanical responses of αβ T-cell receptors 2257683

Abstract Introduction Alpha beta T cell receptors (TCRs) recognize target ligands, antigenic peptide-loaded major histocompatibility complex molecules (pMHCs). The recognition occurs under variegated physical conditions including distinct local mechanical environments and target ligand densities in the background of self-pMHC molecules. A key TCR feature is mechanosensing, a process whereby picoNewton-level forces applied to the TCRαβ-pMHC complexes during immune surveillance elicit a catch bond response. Our recent computational and experimental studies indicate that TCRs exhibit a diverse range of mechanical behaviors including differences in catch bond profiles and stiffness. The basis of this divergence is yet to be elucidated. Methods We perform all-atom molecular dynamics simulations where picoNewton-level forces are applied to three structures of TCRαβ-pMHC (NP366/Db) complexes bearing an identical peptide from influenza A virus. While those TCRs differ by only 1-3 amino acids in their CDR3 loops, the corresponding T cells selectively differentiate into different CD8 memory cell lineages. In simulations, we apply loads in various ways, including tension, shear, and differential loading on α and β chains, which cover various scenarios of loading on the holo-TCR during immune surveillance. Results Despite the structural similarity of the NP TCRs, we first find that the stability of each TCRαβ-pMHC interface varies without load. Application of shear forces in different directions relative to pMHC reveals that the increase in interfacial stability depends on the loading direction. Conclusion These results indicate that the differences in response to load may control conformational motion of the holo-TCR, leading to distinct signaling outcomes. Funding Source US National Institutes of Health (Grant number P01AI143565) Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Decoding the Antibody Reactome at Scale with Novel Antigen Display Technology to Decipher Immune History 2335216

Abstract Introduction The human immune system serves as a ledger of environmental exposures, infections, and self-reactivity and there is a there is a critical need for high-throughput tools capable of mapping the entire human antibody reactome (all the antibody-antigen interactions). However, capturing the entire “immune history” can be difficult as conventional assays are limited to a narrow set of defined antigens. Methods Molecular Indexing of Proteins by Self-Assembly (MIPSA) technology employs a single-pot process to create libraries with antigens are covalently linked to unique DNA barcodes and decoded via Next-Generation Sequencing. This multiplexed, sample-sparing approach enables the simultaneous profiling of tens of thousands of interactions via core libraries: (1) HuSIGHT, covering the human proteome with 353,034 peptides and 16,516 full-length proteins; (2) VirSIGHT, covering all viruses known to infect humans with 285,669 peptides; and (3) EnviroSIGHT, covering 1,207 genera of allergens, microbial antigens, and toxins with 242,884 peptides. Results Using commercial monoclonal antibodies and serum from patients with confirmed autoimmune diseases, the inclusion of both peptides and full-length proteins increases the detection of autoantibody targets on HuSIGHT. Profiling with VirSIGHT effectively distinguished “common” viral signatures from unique exposure histories. Furthermore, EnviroSIGHT allowed for the discrimination of distinct allergic profiles and geographical locations based on endemic pathogen exposure of the participants. We validated the platform’s robustness against technical and clinical confounders. Conclusion The MIPSA technology offers a scalable, high-resolution solution for uncovering immune insights offering a comprehensive view of immune history, this platform facilitates the discovery of novel biomarkers, infectious triggers of chronic conditions, the characterization of host-pathogen interactions, and the development of more effective therapeutic interventions for human health. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Single cell profiling identifies effector CD8+ T cells and peripheral helper T cells as drivers of recurrent immunotherapy mediated arthritis 2328826

Abstract Introduction Immune checkpoint inhibitor (ICI) therapy is often associated with inflammatory arthritis (ICI-IA). However, the mechanisms underlying ICI-IA, especially its recurrence, have not been fully elucidated. The purpose of this study is to elucidate mechanisms of recurrent ICI-IA by analyzing longitudinal synovial fluid (SF) samples. Methods SF samples were collected from ICI-IA patients at the first and second occurrences of ICI-IA and analyzed with single-cell RNA sequencing (scRNAseq), scTCRseq, scBCRseq, and flow cytometry. SF samples from cancer-naïve osteoarthritis patients were used as negative controls. Results Effector CD8+ T cells and PD-1hi CXCL13hi CD4+ T cells were enriched in the SF of ICI-IA patients. Ninety three percent and fifty percent of the top ten expanded clones of effector CD8+ T cells and PD-1hi CXCL13hi CD4+ T cells, respectively, were shared between the first and second ICI-IA flare. They were more clonally expanded and characterized by the production of pro-inflammatory cytokines including IFNγ, TNFα, and IL-21, especially in the second flare, suggesting immune memory responses to cognate antigen. Cell-cell communication analysis suggests that effector CD8+ T cells and PD-1hi CXCL13hi CD4+ T cells interact with each other and with myeloid cells and B cells through chemokines (CXCL9/10/11/13, CCL5) and cytokines (MIF, IL-2/7/15/21). Conclusion Longitudinal SF analysis from ICI-IA patients for first time revealed that expansion of effector CD8+ T cells and PD-1hi CXCL13hi CD4+ T cells with proinflammatory characteristics Funding Source n/a Topic Categories Basic Autoimmunity (BA)

Investigating Inflammasome Responses to Pathogenic Fungi 2331094

Abstract Introduction Fungal pathogens are increasingly recognized as important activators of the innate immune system, particularly through engagement of inflammasomes. These are large protein complexes that trigger pyroptosis, an inflammatory cell death resulting in the release of IL-1 family cytokines. Although a large body of evidence demonstrates that diverse fungal species activate inflammasomes, they employ various evasion mechanisms that can limit the level of cell death and IL-1 release. Methods Among the inflammasomes, NLRP3 has emerged as a central mediator of antifungal immunity. However, NLRP3 is not regarded as a direct pathogen sensor, but rather integrates signals related to potassium efflux, reactive oxygen species, and lysosomal damage. Given that many cytosolic sensors have defined ligands, a question that remains largely unexplored in the field of fungal innate immunity is whether specific cytosolic detection pathways exist for fungal cell wall components. Results Our work aims to understand the molecular mechanisms governing fungal intracellular sensing and inflammasome activation, to provide critical insights into host—fungi interactions and inform the development of therapies for the increasing emergence of invasive and inflammatory fungal diseases. Conclusion   Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Amphiregulin production from regulatory T cells does not require autocrine or paracrine feedback signaling 2260280

Abstract Introduction Pneumonia triggers inflammatory responses followed by recovery and tissue repair. T regulatory (Treg) cells play a necessary role in coordinating the recovery process. Amphiregulin (Areg) is an epidermal growth factor receptor ligand that mediates the lung tissue-reparative function of Treg cells in mice. While IL-18 receptor and ST2 signaling promote Areg secretion from Treg cells, the signals required for optimal Areg production are unclear. Methods Mice were inoculated with influenza A with examination of lung and spleen Treg cells during the recovery phase of pneumonia. Results Here, we show in a model of influenza A pneumonia that Areg expression in lung and spleen Treg cells does not require paracrine or autocrine signaling following restimulation. Furthermore, optimal Areg detection required nuclear permeabilization, indicating that Areg may be localized within the nucleus. Conclusion These findings suggest that Treg cells are intrinsically programmed to produce Areg upon activation, independent of autocrine and paracrine signaling cues. Additionally, Areg may function to regulate transcriptional programs within the nucleus of Treg cells. Funding Source R01HL149883, P01HL154998 Topic Categories Cytokines and Chemokines and their Receptors (CCR)

Discovery of potent, selective and insurmountable MRGPRX2 antagonists for mast cell-driven disorders 2308475

Abstract Introduction Mas-related G protein—coupled receptor X2 (MRGPRX2) is an important mediator of non-IgE—dependent mast cell activation and has emerged as a key target in pseudo-allergic reactions and chronic inflammatory diseases. Yet, drugs targeting this mechanism remain limited. We report the discovery and characterization of novel series of potent, selective, and insurmountable MRGPRX2 oral small-molecule antagonists. Methods Compounds were evaluated in vitro using CHO-K1 and HEK293 cells overexpressing human MRGPRX2, LAD2 human mast cells, and mast cells differentiated from mobilized CD34+ hematopoietic progenitor cells. Functional activity was assessed across multiple assay platforms – including calcium flux, β-arrestin recruitment, inositol monophosphate (IP1) accumulation and mast cell degranulation assays – using different MRGPRX2 agonists. Selectivity against the closest human homologs MRGPRX1 and MRGPRX4, dog and monkey MRGPRX2 orthologs, and a broad panel of G protein-coupled receptor proteins were tested to ensure high target specificity. Lead molecules were further evaluated in vivo in a humanized MRGPRX2 mouse model of cutaneous anaphylaxis for efficacy. Results Lead antagonists demonstrated potent and insurmountable inhibition of MRGPRX2-mediated responses across all cellular models and were active against all MRGPRX2 agonists tested. They exhibited minimal off-target activity, no cross-reactivity with dog and monkey orthologs, and no activity against MRGPRX1 and MRGPRX4, underscoring their high target selectivity. In vivo, lead compounds showed favorable pharmacokinetics and significantly reduced mast cell—dependent vascular leakage in a dose-dependent manner. Conclusion These findings highlight the therapeutic promise of those insurmountable MRGPRX2 antagonists for the treatment of mast cell—driven disorders. Funding Source Blueprint Medicines Corporation Topic Categories Translational and Interventional Immunology (TI)

Understanding long term consequences of respiratory viral infection 2260810

Abstract Introduction In the wake of the SARS-CoV-2 pandemic, most people have experienced at least one respiratory viral infection and many endured severe disease, yet the long term immune consequences remain unclear. We hypothesized that severe infection episode durably reshapes circulating immunity and accelerates age-like change. Methods To enable within-animal longitudinal inference, we focused on blood so the same mice could be profiled over time. Wild type mice received sublethal influenza A virus (IAV) or mock, and whole blood single-cell RNA-seq of PBMCs was performed at 2, 4, 8, and 14 months post infection from the same animals. Results Severe IAV was associated with a progressive rise in the neutrophil-to-lymphoid ratio (NLR) from 2-14 months versus mock, with concomitant declines in naïve B and T cells. A GZMK+ CD8 T-cell subset increased in frequency and activation state in infected mice. These features paralleled aging like myeloid bias programs, consistent with infection accelerated immunosenescence. Conclusion Ongoing analyses use batch aware mixed effects models and gene set scoring to define cell-state programs linked to NLR and GZMK+ CD8 expansion. Our findings suggest that severe respiratory viral insult can imprint durable remodeling of circulating immunity, identifying the specific transcriptional signatures and upstream mediators may reveal targets to restore immune balance and resilience during age. Funding Source n/a Topic Categories Viral Immunology (VIR)

Entropy‐Driven Conformational Disorder Enables Outstanding High‐Temperature Energy Storage in Dielectric Polymers

ABSTRACT The pursuit of high‐temperature polymer dielectrics is consistently hindered by the intrinsic tradeoff between ensuring robust electrical insulation and maintaining thermal stability. While aromatic polyimide (PI) has superior thermal resistance, its dense π–π stacking facilitates the formation of charge transfer complexes, causing significant leakage and capacitive failure at elevated temperatures. In this study, we developed an entropy‐driven conformational disorder strategy to maximize the conformational entropy of our designed ternary random copolymerized PI (R‐PI, Δ S conf = 5.76 J/(mol·K)). The π‐conjugation decoupling and electron localization of the R‐PI were achieved by dynamic conformational flipping. Density functional theory and molecular dynamics calculations indicate that the structural randomized state generates a highly fluctuating electrostatic potential field. This field creates high‐density deep energy traps that effectively suppress the long‐range hopping transport of charge carriers. As a result, the optimal R‐PI‐0.5 delivers a discharged energy density of 6.12 J/cm 3 ( η = 91.1%) under an applied field of 650 MV/m at 200°C. This molecular‐level design paradigm leverages conformational entropy to exceed traditional dielectric limits, offering a robust pathway for next‐generation harsh‐environment energy storage.

The ascorbate-Tet axis is a checkpoint regulator of naive T-cell quiescence 2257733

Abstract Introduction T-cell peripheral tolerance is crucial for maintaining immune homeostasis and preventing autoimmunity, which is characterized by limited response of conventional T cells to antigen stimulation. The mechanisms controlling this process remain to be fully elucidated. Methods To investigate the role of nutritional factors and related epigenetic mechanisms, we conducted in vivo CRISPR screening and identified ascorbate transporter Slc23a2. Results Ablation of Slc23a2 in T cells decreases intracellular ascorbate levels, leading to DNA hypermethylation in specific regions. This results in increased differentiation of naive T (Tn) cells into effector and memory T cells, accompanied by low-grade autoimmune inflammation, which is comparable to ascorbate deprivation, Tet dioxygenase deletion, and aged T cells. Mechanistically, Slc23a2 through ascorbate activates Tet methylcytosine dioxygenases to restrict Tn cell activation and differentiation into effector or memory T cells by attenuating TCR signaling, reducing helper T-cell determinants, and enhancing Tcf1 expression and chromatin binding. Tcf1 ablation partially mimics Slc23a2 deficiency, while Tcf1 overexpression suppresses its effect. Conclusion Therefore, the ascorbate-Tet axis functions as a checkpoint regulator for Tn quiescence, ensuring peripheral tolerance, particularly during aging. Funding Source N/A Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Interferon lambda 4 genotype determines baseline expression of interferon-stimulated genes and lipid metabolism-related genes in the healthy liver 2304507

Abstract Introduction Interferon lambda 4 is an ancestral protein produced in the presence of the rs368234815-delta-G allele. This haplotype has been associated with decreased inflammation and disease progression in chronic hepatitis C and metabolic dysfunction-associated steatohepatitis. Methods To investigate the underlying mechanism, we performed single-cell RNA sequencing of liver biopsies from 14 subjects who were cured of chronic hepatitis C five years earlier. Results Twelve patients encoded IFN-lambda4 (rs368234815 delta-G/delta-G or delta-G/TT). Two of these patients encoded a functionally attenuated IFN-lambda4 protein, due to a P70S substitution (rs117648444-A/G). Two patients did not encode IFN-lambda4 (rs368234815-TT/TT). Interferon-stimulated gene expression was significantly higher in hepatocytes and liver sinusoidal endothelial cells of patients who produced IFN-lambda4 than in those who did not. Patients with a functionally attenuated IFN-lambda4 had significantly lower expression of interferon-stimulated genes than those with conserved IFN-lambda4 but higher expression than those without IFN-lambda4. These results extended to all intrahepatic immune cell populations, including T and NK cells that do not express IFNLR1. This suggests the induction of secondary type I and II IFN responses downstream of IFN-lambda4, which was confirmed by Hallmark gene enrichment analysis. Clinical data from a larger cohort of genotyped patients that had been cured of chronic hepatitis C (n = 44, male:28) revealed higher serum ALT activity in healthy male patients with IFN-lambda4 expression than those without, which was associated with an enrichment of the fatty acid degradation pathway and depletion of the steroid biosynthesis pathway in hepatocytes (KEGG database). Conclusion IFN-lambda4 induces interferon-stimulated gene expression in immune cells, endothelial cells, and hepatocytes in the liver, with a secondary effect on lipid metabolism in hepatocytes. Funding Source Intramural Research Program of NIDDK, NIH Topic Categories Immune Mechanisms of Human Disease (HUM)

Impact of Pre-Existing Adenovirus Immunity on Vaccine Immunity Induced by ChAdOx1 nCoV-19 in Immunodeficient Patients 2330644

Abstract Introduction Chimpanzee adenovirus—vectored COVID-19 vaccines, including ChAdOx1 nCoV-19 (nCoV-19), were selected in part due to low seroprevalence in humans. However, the impact of pre-existing immunity to human adenoviruses on immune responses to these vaccines remains incompletely understood. We investigated immunological cross-reactivity between ChAdOx1 and human adenovirus type 5 (HuAd5) and tested the hypothesis that pre-existing HuAd5-specific T cells may enhance SARS-CoV-2 spike-specific T cell responses following nCoV-19 vaccination in antibody-deficient individuals. Methods We analyzed samples from healthcare workers and immunodeficient patients vaccinated with either nCoV-19 or BNT162b2. HuAd5 hexon-specific T cells responses were assessed using IFN-γ ELISpot and flow cytometry. Anti-HuAd5 heron antibodies were measured using Luminex for IgG and a GFP-expressing HuAd5 neutralization assay for neutralizing activity. Findings from the current study were compared with previously published COVID-19 vaccine response data in these cohorts. Results We observed significant T cell and antibody cross-reactivity between ChAdOx1 and HuAd5. In immunodeficient individuals vaccinated with nCoV-19 HuAd5 hexon-specific T cell frequencies were positively associated with SARS-CoV-2 spike-specific T cell responses, an association not observed in BNT162b2 recipients. Conversely, higher anti-HuAd5 hexon antibody levels were inversely correlated with spike-specific T cell responses in the nCoV-19 cohort. Conclusion These findings support a model in which heterologous pre-existing adenovirus-specific T cell immunity contributes to enhanced spike-specific T cell responses following adenovirus-vectored vaccination in antibody deficient individuals. This bystander T cell effect provides an explanation for the observation in several studies of higher T cells response to adenovirus-vectored COVID-19 vaccines compared to mRNA vaccines in immunodeficient or elderly patients. Funding Source NIHR/UKRI grant (COV0170) Humoral Immune Correlates of COVID-19 (HICC), Cambridge Commonwealth, European & International Trust (Cambridge Trust) Topic Categories Vaccines and Immunotherapy (VAC)

Leveraging Advances In Long-Read Sequencing to Improve Analysis of Host Immune Responses 2332674

Abstract Introduction Short-read based technologies have dominated high-throughput sequencing workflows for years. However, many complex sequences such as immune genes exhibit repetitive regions, duplications, or splicing variations and it is challenging to assemble or align them accurately using short reads. Since long-read sequencing has traditionally been lower-throughput and higher in cost, recent advances conferred by Pacbio’s Kinnex platform may help address these limitations, but new protocol developments are needed to enable the use of existing material prepared for standard short-read platforms. Methods To establish new Kinnex based workflows that are compatible with commonly used single cell platforms, we processed cDNAs generated from various rhesus tissue types using the 10x Genomics technology according to the MAS-ISO-seq protocol through TSO artifact removal and then enriched IG, TCR, or MHC transcripts using custom designed primers. Separately, existing Qiaseq based bulk human TCR libraries, containing an Illumina Nextera backbone, were amplified using construct specific primers. In both cases, we introduced compatibility to the PacBio Kinnex 16S rRNA workflow through custom primers? and incorporated amplicons into the Kinnex workflow at the point of array PCR. Resulting libraries were sequenced on PacBio’s Revio platform. Results Successful gene-targeted single cell library constructions were accomplished for the 10x Genomics cDNAs, and existing Qiagen bulk TCR libraries were converted to SMRTbell libraries. Increased mapping efficiencies were observed across the entire transcripts, particularly concerning full-length VDJ transcripts. Conclusion Adaptation of existing library materials into a Kinnex-based protocol allows for streamlining of multiple constructs into a single workflow while expanding accessibility to PacBio’s long-read sequencing platform, resulting in a more affordable and productive approach for improving data quality and facilitating the study of immune system expression. Funding Source NIH U19 AI131471, NIH R01 AI175411, CHHE grant # ES025128 Topic Categories Technological Innovations in Immunology (TECH)