Browse Articles

Bivalent vaccination fails to enhance conserved spike T cell responses across BetaCoronaviruses 2260452

Abstract Introduction The COVID-19 pandemic highlighted the need for vaccines strategies that elicit broad T cell-mediated immunity against emerging viral families. BetaCoronaviruses - including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) and SARS-CoV-2 — pose significant pandemic risks due to their zoonotic potential and genetic diversity. In previous work, we identified conserved Spike T cell epitope regions (S-CTERs) within the ancestral Wuhan S protein sequence that demonstrated strong cross-reactive potential across diverse BetaCoronaviruses. Methods Here, we investigated whether bivalent vaccination (Beta + Omicron) preferentially enhances T cell responses targeting CTERs and improves cross-reactivity across Betacoronavirus subgenera. PBMC samples were collected at baseline (day 0) and post-vaccination (day 90) from a cohort of 60 adults receiving either the Pfizer or Moderna bivalent vaccine. Antigen-specific CD4+ and CD8+ T cell responses were assessed using a combined activation-induced marker (AIM) and intracellular cytokine (ICS) assay in a 25-colour-flow cytometry panel. Results Overall, bivalent vaccination did not increase the response magnitude to spike or the relative fraction of S-CTER responses within total spike responses. A trend toward increased cytokine polyfunctionality was observed post-vaccination, but no significant differences were observed between pre- and post-vaccination samples across CTER pools derived from multiple BetaCoV isolates. Conclusion These findings suggest that spike-based bivalent vaccination alone cannot direct a T-cell focused response to achieve broad immunity across the BetaCoronaviruses family. Incorporation of additional protein sequences will likely be required to shift response toward the CTER approach. Understanding how bivalent vaccinations shape T cell recognition of CTER pools will be critical for advancing universal BetaCoronavirus vaccine design. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)

Age-Linked Neutrophilic Inflammation Signatures Associate with Disease Severity in Primary Ciliary Dyskinesia 2309093

Abstract Introduction Primary ciliary dyskinesia (PCD) is characterized by chronic neutrophil-predominant airway inflammation, yet spirometric measures alone incompletely capture disease heterogeneity and progression. Quantitative inflammatory signatures may help identify biologically meaningful disease phenotypes beyond spirometry alone. Methods Plasma samples were collected from genetically confirmed PCD patients at clinical baseline, defined by the absence of acute respiratory exacerbation. Using high-sensitivity NULISA™ proteomic profiling, we quantified ten neutrophil-associated inflammatory and remodeling markers (MPO, CXCL8, S100A9, LCN2, MMP8, MMP9, CCL3, CCL4, CCL20, and CCL2). A composite Neutrophil Inflammation Score (NIS) was defined as the mean z-score of these markers. A Neutrophil-to-Lung Function Index (NLFI) was calculated by normalizing NIS to FEV1 % predicted, integrating inflammatory burden with airway function. Patients were stratified into High- and Low/Moderate-inflammation groups. Associations with age and lung function were examined. Results NIS was significantly higher in PCD patients compared with healthy controls (p = 0.0020). Within the PCD cohort, NIS showed no significant association with lung function (FEV1 % predicted; r = —0.232, p = 0.4244), but demonstrated a significant positive association with age (r = 0.552, p = 0.0438). Patients in the High-inflammation group exhibited markedly reduced lung function compared with the Low/Moderate group (Cohen’s d = —1.25). The NLFI increased across age strata, with the highest values observed in older patients. This shows that with aging and disease progression, neutrophilic inflammation increases while lung function declines. Conclusion Quantitative neutrophil-inflammatory profiling showed an age-linked, high-risk inflammatory endotype in PCD that is not apparent from spirometry alone. Integrated inflammatory indices may improve patient stratification and inform current and future studies of neutrophil-targeted therapeutic strategies. Funding Source Research Centers in Minority Institutions (RCMI) Center for Research Resources Grant and the Molecular and Genomics Core (#U54MD007579), PR-INBRE Developmental Research Project Program (DRPP) (#5P20GM103475-21), Graham Grant. Topic Categories Immune Mechanisms of Human Disease (HUM)

Limosilactobacillus reuteri-educated T cells protect against activation of liver inflammation in immunocompromised mice 2229185

Abstract Introduction Oral administration of probiotic Limosilactobacillus reuteri DSM 17938 prolongs survival and reduces Th1- and Th2-associated inflammation in Treg-deficient scurfy (SF) mice, which model human IPEX syndrome. It is unclear how DSM 17938 educated SF-CD4+T cells can facilitate T cell-liver communication. Methods We adoptively transferred by intraperitoneal injection cells isolated from SF spleens into lymphocyte-deficient-RAG1KO mice and compared the CD4+T cells from SF mice gavage-fed with DSM 17938 with the CD4+T cells from untreated SF mice (Prob-SF-CD4+T cells versus SF-CD4+T cells). Liver inflammatory histology and transcriptomes in RAG1KO mice were analyzed. Results We observed that Prob-SF-CD4+T cells reduced the incidence and severity of liver inflammation caused by transferring SF-CD4+T cells. SF-CD4+T cells up-regulated expression of liver genes involved in TLR cascades, inflammatory cytokine and death receptor signals, while down-regulated genes linked to mitochondrial respiratory chain complexes, TCA cycle, liver detoxification and lipid metabolism. However, Prob-SF-CD4+T cell transfer reversed SF-CD4+T cell-induced transcriptomic changes in inflammatory and metabolic clusters by modulating distinct genes participating in TLR regulation, mitochondrial function and cell cycle. Conclusion In conclusion, inflammatory CD4+T cells can perpetuate an exaggerated immune response in the immunologically naïve host. DSM17938 has the capacity to reprogram inflammatory CD4+T cells and allowed them to benefit the recipient. Probiotic-modulated T cells can be further explored as a therapeutic option for autoimmune liver diseases. Funding Source National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) R03AI153725 Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Multi‐Legged Magnetic Soft Robot for On‐Demand Targeted Delivery in Pulmonary Environments

ABSTRACT Intrabronchial drug delivery is an important method for treating respiratory diseases. Precise and selective deep‐lung delivery of drugs is still challenging through inhalation administration and bronchoscopes, due to the passive transport and insufficient maneuverability in narrow and tortuous distal airways. Herein, a magnetic‐driven soft robot with a multi‐legged structure is developed for minimally invasive and precise targeted drug delivery in the lung. The robot exhibits multimodal and adaptive locomotion in complex airways, relying on its soft nature. A magnetothermal‐triggered and on‐demand liquid drug release mechanism is integrated onto the robot. Rolling, stick‐slip and crawling motion modes can be achieved, and the mode transition is realized to enhance the adaptivity. Precise navigation in complex environments and the distal bronchial tree of a phantom is realized. The magnetothermal‐induced drug release is also demonstrated. Ex vivo distal navigation, targeted drug delivery and retrieval of the robot in a porcine lung are validated.

Immunomodulatory Effects of Lactococcus lactis on the Gut—Pancreas Axis 2257747

Abstract Introduction Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of pancreatic beta cells by T lymphocytes, resulting in insulin deficiency. Our group has shown unique alterations in the gut mucosa and microbiome dysbiosis in pre-diabetic mice that occur prior to insulin deficiency in Non-Obese Diabetic (NOD) mice. These findings suggest that the gut microbiome may influence the gut mucosa and modulate disease progression. Lymph node sharing between the intestine and the pancreas may play a critical role in determining disease onset. This study investigated the immunomodulatory effects of Lactococcus lactis NCDO 2118 supplementation on 4 weeks female NOD mice treated for 28 days. Methods Major gut microbiota phyla were analyzed by qPCR of fecal DNA, and short-chain fatty acids were determined by liquid chromatography. Secretory IgA (SIgA) was quantified by ELISA, and IgA-bound bacteria (Bac-IgA) and cell populations were analyzed by flow cytometry. Results L. lactis administration significantly reduced (p = 0.0095) the frequency and severity of pancreatic insulitis, without affecting food or water intake, blood glucose, body weight, intestine size, or SIgA levels. Bac-IgA levels were decreased in treated mice. Innate Lymphoid Cells, Intraepithelial and T lymphocytes profiles were unchanged in the gut, while B cells and plasmocytes were decreased in the Peyer’s patches of treated mice. L. lactis-treated mice displayed a lower proportion of tolerogenic dendritic cells in the spleen and gut-pancreas shared lymph nodes. An increase in total bacteria and Firmicutes, and a reduction in Proteobacteria, were observed in the colon of the treated group, along with higher levels of acetate and lactate. Conclusion In conclusion, L. lactis NCDO 2118 reduced insulitis in NOD mice and promoted an immunomodulatory profile during T1DM development, likely mediated by alterations in the gut microbiota and the gut—pancreas axis. Funding Source CNPq, CAPES and FAPEMIG (#RED-00096-22, #RED-00067-23) Topic Categories Mucosal and Regional Immunology (MUC)

Meningeal lymphoid hubs support autoreactive immune responses 2306190

Abstract Introduction Immune cells within the meninges are increasingly recognized as key regulators of CNS homeostasis and disease. The dura mater, which contains diverse innate and adaptive immune populations, also supports B cell development. We recently identified organized lymphoid architecture surrounding a fenestrated venous plexus at the rostral—rhinal confluence of sinuses, termed the rostral-rhinal venolymphatic hub (hub). Following intranasal viral infection, this hub mounts robust germinal center (GC) responses composed of T follicular helper (Tfh) cells and GC B cells undergoing proliferation, class-switch recombination, and somatic hypermutation. Here we evaluate the role of the hub in the development of autoimmunity. Methods Using complementary structural and functional approaches as molecular biology, imaging and flow cytometric analysis. Results We show that this same hub also supports autoimmune responses to CNS antigens, particularly myelin debris that preferentially accumulates at this site. This deposition appears to result from a loose glial limitans over the olfactory bulbs and the proximity of fenestrated vasculature that drains into the hub. B cells act as key “housekeepers,” as their depletion markedly increases myelin accumulation. Beyond debris clearance, hub-resident B cells express PDL-1 and TGFβ, suggesting a regulatory program that may reinforce tolerance to self-antigens. During autoimmune priming, myelin oligodendrocyte glycoprotein (MOG)—reactive CD4+ T cells preferentially localize to the hub, engage B cells, proliferate, and differentiate into Tfh cells. B cell depletion augments the accumulation of these autoreactive T cells in the hub and accelerates onset of EAE. Conclusion Collectively, these findings reveal how dural lymphoid architecture positioned around a specialized vascular plexus can sample foreign and self-antigens, generating responses that either protect against infection or drive autoimmune pathology in the adjacent CNS. Funding Source National Institutes of Health Topic Categories Neuroimmunology (NEUR)

Canonical WNT pathway utilizes LEF1 to coordinate innate immune memory 2335059

Abstract Introduction Wnt signaling is a fundamental and evolutionarily conserved pathway that governs cell fate, proliferation, and tissue homeostasis. While canonical Wnt/β-catenin signaling has been extensively characterized in stem and stromal cells, its function in lymphocytes remains context dependent and poorly understood. Natural killer (NK) cells provide a powerful system to interrogate this pathway, as they bridge innate and adaptive immunity and undergo antigen-specific clonal expansion and memory formation during antiviral responses such as cytomegalovirus (CMV) infection. Given the capacity of canonical Wnt/β-Catenin signaling to orchestrate core cellular processes, defining how NK cells access and interpret this conserved pathway to shape the transcriptional architecture of antiviral immunity is of particular interest. Methods We used a well-established model of mouse CMV (MCMV) infection, in which Ly49H+ NK cells recognize the viral glycoprotein m157 on infected targets. Molecular, genomic, and epigenomic approaches were applied to dissect how NK cells utilize Wnt signaling during viral infection. Results We identify a cytokine-independent mechanism of NK cell expansion mediated by canonical Wnt signaling. We found that NK cells uniquely express the Wnt ligand receptor Frizzled-5 (Fzd5), which is essential for regulating β-catenin-dependent canonical Wnt signaling. During MCMV, we demonstrate that the Wnt-Fzd5 signaling pathway requires β-catenin as a co-factor and LEF1 as a downstream transcription factor to activate key regulators of cell cycle entry and proliferation. Conclusion These findings establish Fzd5-mediated canonical Wnt signaling and LEF1 activity as critical drivers of the adaptive NK cell response during viral infection, offering new insights into the regulation of innate immunity. Funding Source T32 AI134632-05; F31AI178958 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Characterization of Immune Cell Populations in Lupus Nephritis Using a SignalStar® Multiplex Immunohistochemistry Assay 2332985

Abstract Introduction Systemic Lupus Erythamatosis (SLE) is a chronic autoimmune disease characterized by dysregulated immune response, production of autoantibodies, and tissue damage. Lupus nephritis (LN) is a kidney disease caused by SLE. Many immune cell types are implicated in LN. In contrast to healthy kidney tissue, LN tissues have increased presence of B cells, macrophages, regulatory T cells (Tregs) and dendritic cells (DCs). Additional insights into these heterogenous immune cells in LN will be valuable for understanding disease progression and therapeutic strategies. Methods We interrogated normal and LN formalin-fixed, paraffin-embedded (FFPE) tissues using an 8-plex SignalStar® multiplex immunohistochemistry (mIHC) assay to compare total (CD68+), M2c (CD68+CD163+), and M2a (CD68+CD206+) macrophages, CD19+ and CD20+ B cells, and total (CD3+) and FoxP3+ regulatory T cells (Tregs). CD11c was used to mark dendritic cells (DCs). All stains were performed using Leica Biosystems BOND RX. Slide images were collected at 20X magnification using Akoya PhenoImager HT. Phenochart 1.1 software was used to define regions of interest and inForm 3.0 software was used to perform population analyses on regions of interest from tissue sections. Results We observed increased immune cell presence in LN tissues. B cells, identified with CD19 and CD20, were increased; the number of CD19+ compared to CD20+ B cells was higher in the LN samples. We also observed increased levels of subtypes of macrophages, M2c (CD68+CD163+) and M2a macrophages (CD68+CD206+), and increased CD3+ T cells. Conclusion The SignalStar® mIHC assay enables robust spatial analysis of SLE LN tissue samples. LN tissues show increased immune cell presence, with increased levels of CD19+ B cells, M2a and M2c macrophages, and CD3+ T cells. These cell types play roles in autoantibody production, debris clearance, and inflammation. Further studies of these cell types in LN will help with understanding disease progression and design of more effective therapeutics. Funding Source N/A Topic Categories Basic Autoimmunity (BA)

Flow Cytometric Antigen-Specific Immune Responses in Latent Tuberculosis Infection: A Comparison Between Low- and High-Endemic TB Settings 2260608

Abstract Introduction Screening individuals with latent tuberculosis infection (LTBI) from regions with varying TB endemicity is critical for global TB control. Immune responses to TB differ due to variations in exposure, immune adaptations, and environmental factors. This study evaluated antigen-specific immune responses in untreated LTBI individuals from low- and high-TB-endemic settings. Methods Peripheral blood mononuclear cells from untreated LTBI individuals from low-endemic (USA) and high-TB-endemic settings (Mexico) were stimulated with PPD, MTB300, ESAT-6/CFP-10 (RD1) peptides, mycobacterial glycopeptidolipids (GPL), and controls. We compare flow cytometric immune responses of T-cell intracellular cytokines and activation markers using non-parametric statistical methods. Results We studied age-matched, untreated LTBI individuals who tested IGRA(+)/TST(+) from low-TB-endemic (n = 18) and 18 healthcare workers from a high-TB-endemic settings. IGRA results showed no statistical differences across groups. The frequency of CD3+CD8+IFN-γ+HLA-DR+ and total CD3+TNF-α+ T cells after RD1 stimulation was significantly higher in the high-TB-endemic group (P < 0.05). Additionally, we observed significant increased frequencies in CD3/CD4+TNF-α+HLA-DR+ and CD8+TNF-α+HLA-DR+ T cells stimulated by GPL in the same group, probably associated with nontuberculous mycobacteria (NTM) exposures. Our findings suggest possible recent TB and NTM exposures in healthcare workers from a high-TB-endemic setting, resulted in increased antigen-specific adaptive TNF-α and IFN-γ producing T cells, which are important for pathogen control and immunity. Conclusion We found significant differences in antigen-specific cell immunity, including HLA-DR activated T-cells, a marker of recent antigen encounter, in individuals from a high-TB-endemic settings. These findings underscore the importance of settings-specific immune biomarkers for improving LTBI diagnostics, clinical management, and vaccination trials. Funding Source National Institute of Allergy and Infectious Diseases at the National Institutes of Health Topic Categories Immune Mechanisms of Human Disease (HUM)

Pre-clinical Development of a Novel CAR-T Therapy Targeting TIM-1 for Treating Solid Tumors 2309293

Abstract Introduction Chimeric antigen receptor (CAR)-based therapy represents a major advancement in cancer treatment, especially in hematopoietic malignancies. However, it faces many challenges in treating solid tumors, including the lack of tumor-specific antigens and on-target, off-tumor effects. We have developed a novel CAR-T cell therapy, designed to target a specific tumor antigen (TIM-1) in renal cell carcinoma (RCC) and ovarian clear cell carcinoma (OCCC). Expression of TIM-1 in healthy tissues is limited to absent. This tumor-specific expression makes TIM-1 an ideal target for CAR-based approaches. Methods TIM-1-specfic response of the CAR-T cells was validated by IFN-γ production upon recombinant TIM-1 stimulation and in vitro cytotoxicity against TIM-1-expressing tumor cells. Therapeutic efficacy of TIM-1 CAR-T cells was evaluated in xenograft models. Both tumor growth and survival of the mice were monitored. Moreover, possible toxicity of TIM-1 CAR-T cells in treating RCC was examined in a human TIM-1 knock-in mouse kidney ischemia/reperfusion model. Results TIM-1 CAR-T cells had significantly increased IFN-γ production in response to recombinant TIM-1 stimulation and TIM-1-expressing tumor cells. TIM-1-specific cytotoxicity was observed with TIM-1 CAR-T cells against TIM-1-expressing RCC and OCCC tumor cells. TIM-1 CAR-T cell treatment showed 100% survival in an RCC model, and 90% survival in an OCCC model. Tumor growth was significantly suppressed by TIM-1 CAR-T cell treatment in both models. Minimal toxicity was observed with up to 5*107 CAR-T cells in a human TIM-1 knock in mouse kidney ischemia/reperfusion model. There was no increase in kidney and liver toxicity markers with TIM-1 CAR-T treatment after ischemia/reperfusion injury. Conclusion TIM-1 CAR-T cell therapy is efficacious and safe in treating RCC and OCCC in pre-clinical models. The pre-IND development is underway in advancing this novel therapy to the clinic. Funding Source SBIR Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Erythroid progenitors exhibit non-canonical cellular response to double-stranded DNA 2243743

Abstract Introduction Erythroid cells, like classical immune cells, arise from hematopoietic stem cells, yet their immune potential remains largely unexplored. Methods To investigate their role in immunity, we analyzed single cell RNA-seq data from human umbilical cord blood and discovered that erythroid progenitors express key components of the cytosolic DNA-sensing cGAS-STING pathway. We confirmed constitutive expression and canonical localization of these genes in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells. Results HUDEP-2 cells are a unique model to probe the DNA-sensing capacity of erythroid progenitors due to their doxycycline-inducible expression of HPV-E7, which degrades STING. Surprisingly, despite degradation of STING, HUDEP-2 cells mounted a robust transcriptional response to transfection with dsDNA. These changes did not align with a type I interferon response, but instead pointed to a non-canonical NFκB-dependent activation. Upregulated genes were enriched for immune cell differentiation pathways, including myeloid-specific transcription factors. Conclusion Our results suggest that erythroid progenitors are capable of sensing exogenous DNA independently of STING and subsequently initiating a non-canonical, interferon-independent immune program that may influence lineage fate decisions. These findings challenge the assumption that erythroid progenitors are immunologically inert and instead suggest a role for erythroid cells in mediating innate antiviral immunity and hematopoiesis. Funding Source NIH R15AI188361, NIH K12GM081259, American Heart Association 23POST1013585, NIH 5T32DK007780, Burroughs Wellcome Fund 2022 Next Gen Pregnancy Initiative Award Topic Categories Hematopoiesis and Immune System Development (HEM)

Beyond the Active Site: Hydrophobic Microenvironment Engineering for Single‐Atom Catalysts

ABSTRACT Single‐atom catalysts (SACs) offer near‐unity atomic utilization and uniform active sites, yet their aqueous‐phase performance is constrained by competitive water adsorption, parasitic side reactions, and mass transfer limitations. This review systematically examines hydrophobic microenvironment engineering as a strategy to overcome these challenges, proposing a unified framework integrating wettability regulation with reaction‐transport coupling. We comprehensively discuss construction strategies including surface modification, intrinsically hydrophobic supports, and biomimetic hierarchical structures, establishing a complete synthetic‐to‐wettability framework. Mechanistically, we elucidate how hydrophobic microenvironments optimize catalysis through mass transport regulation, active site protection, and electronic modulation, revealing multi‐scale coupling from macroscopic contact angles to atomic dynamics. Drawing on advances in organic synthesis, energy conversion, and environmental catalysis, we outline core design principles such as moderate hydrophobicity and outline future directions, including stimuli‐responsive catalysts. This framework guides the rational design and industrial translation of hydrophobic SACs.

Functional Analysis of GAPDHP65 Suggests a Role in Triple-Negative Breast Cancer Viability 2257990

Abstract Introduction Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. Dysregulated pseudogene expression is increasingly recognized in cancer progression, though many remain poorly characterized. This study examines GAPDHP65, a pseudogene involved in TNBC growth and survival. Methods Computational analysis of gene expression profiles identified GAPDHP65 as highly expressed in TNBC compared to normal breast tissue. Experimental validation was conducted using the HCC-1937 TNBC cell line, where GAPDHP65 knockout was generated through CRISPR-Cas9 and editing efficiency was confirmed by indel detection and ligation sequencing. Cell viability and proliferation were measured in wild-type and knockout HCC-1937 cells using standard assays, forming the basis of our previously presented work. Current experiments are focused on expanding validation to the HTB-26 TNBC cell line and conducting detailed apoptosis and cell cycle analyses to further define GAPDHP65 function. Results GAPDHP65 expression was elevated in TNBC cells, and its knockout (validated by sequencing) reduced proliferation in HCC-1937 cells, suggesting a role in maintaining cell survival. Preliminary observations indicate similar trends in the HTB-26 line. Ongoing cell cycle and apoptosis analyses are aimed at determining whether GAPDHP65 loss alters S-phase progression or increases programmed cell death, which will further clarify its contribution to TNBC growth dynamics. Conclusion Our previous work demonstrated that GAPDHP65 knockout impairs TNBC cell survival and progression, revealing its potential as a therapeutic target. These findings suggested that GAPDHP65 supports tumor growth by influencing apoptosis and cell cycle progression. Building on this foundation, the current study expands the analysis to additional TNBC lines and investigates downstream molecular changes following GAPDHP65 loss. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Excess Collagen Accumulation in Microglia-Deficient Rats Mediated by Border-Associated Macrophages 2306686

Abstract Introduction There has been recent interest in investigating microglial dysfunction as a contributor to neurodegenerative conditions. Notably, recent studies have observed an accumulation of extracellular matrix (ECM) proteins in the brain of microglia-deficient rodents, potentially contributing to neurodegeneration by impairing removal of CNS waste products. The mechanism by which excess ECM deposits occur in the absence of microglia has not been elucidated. Recent work from the Pridans Lab at the University of Edinburgh has identified clusters of ECM-related and complement cascade proteins upregulated by border-associated macrophages (BAMs). This has led to the hypothesis that, in microglia absence, BAMs adopt microglial-like functions and overexpress ECM and complement cascade proteins resulting in excess ECM deposits. Methods This project intends to test this hypothesis by conducting immunoassays and histological analysis of collagen and complement proteins in brain tissue taken from microglia-deficient rats. FIRE-knockout rats will be used, which lack microglia but retain mostly unaffected BAM populations. Results Collagen deposition and complement protein expression is expected to increase in rats lacking microglia relative to wildtype controls. Conclusion This is the first study to explore microglia and collagen accumulation using a model that retains an almost normal BAM population. The results from this project may challenge the current consensus that microglia are solely responsible for maintaining ECM homeostasis, and propose BAM activity as a novel mechanism by which excess collagen accumulates in the CNS. Besides neurodegenerative diseases, this project may contribute to understanding the pathology of other conditions associated with dysfunctional microglia, including autism. The results could suggest a novel mechanism for these conditions and indicate future therapeutic targets. Funding Source Simons Institute for the Developing Brain (https://sidb.org.uk/) Topic Categories Neuroimmunology (NEUR)

Oxidative Stress as a Mechanistic Link Between Subclinical Food Allergy and Behavioral Alterations 2334366

Abstract Introduction Mood and cognitive alterations are often reported in individuals with subclinical food allergies who are tolerant of offending foods. Using a mouse model of subclinical cow’s milk allergy (CMA), we previously demonstrated that CMA mice exhibited anxiety-like and/or depression-like behavior and cognitive decline after allergen consumption. While the adverse effects of peripheral inflammation on brain function are increasingly recognized, how mood and cognition are affected remain unclear. Because CMA mice also showed impaired blood-brain barrier (BBB) and elevated systemic and brain histamine levels, which can disrupt cerebrovascular homeostasis and induce inflammatory responses, we examined whether chronic food allergen exposure would lead to oxidative stress and alter neuronal activities in the brain. Methods C57BL/6J male mice were orally sensitized to a bovine whey allergen, β-lactoglobulin (BLG; Bos d 5), or vehicle alone and then placed on a whey-free diet for five weeks, followed by exposure to a whey-containing diet for two weeks. After behavioral testing, brains were collected for immunohistochemical analyses for oxidative stress and neuronal activation using 4-hydroxynonenal (4HNE) and c-Fos staining, respectively. Results BLG-sensitized mice showed increased 4HNE staining and decreased c-Fos expression compared to naïve and sham mice. These changes occurred in brain regions involved in mood and cognition, including the dorsal raphe nucleus, lateral septal nucleus, hippocampus, thalamus, and inferior colliculus, indicating that allergen consumption induced lipid peroxidation and reduced neuronal activities in select brain regions in CMA mice. Conclusion Our results support our hypothesis that consumption of offending foods causes oxidative stress in the brain, providing a possible mechanism that links peripheral immune sensitization with altered brain function. Controlling oxidative stress may be a beneficial intervention for mitigating allergy-associated mood and cognitive disorders. Funding Source National Institute of Health grant number R01AI168563 Topic Categories Neuroimmunology (NEUR)

Decoding the role of CD4 T cells in driving autoimmune type 1 diabetes 2333351

Abstract Introduction Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease where CD8 T cells eliminate insulin-producing β cells in the pancreatic islets. Yet, MHC class II haplotypes confer the greatest genetic risk for the development of T1D, suggesting a critical role for CD4 T cells. Employing the non-obese diabetic (NOD) mouse model, our lab identified in the pancreatic lymph node (pLN) a stem-like β cell-specific CD8 T cell pool required to initiate and sustain disease: pLN β cell-specific stem-CD8 T cells self-renew and continuously give rise to differentiated progenies which migrate to the pancreas (PA) and eliminate β cells; the pLN stem-CD8 T cell pool is absolutely required to sustain β cell destruction. Given the importance of autoimmune stem-CD8 T cells and the association of MHC class II in T1D pathogenesis, we wanted to understand the role of CD4 T cells in autoimmune CD8 T cell stemness and differentiation. Methods We employed the NOD model and longitudinally assessed the phenotypic and functional characteristics of β cell-specific CD4 T cells using flow cytometry, serial transplantation, CRISPR/Cas9-mediated gene editing, and transcriptomic studies. Results Our studies reveal, for the first time, how CD4 T cells drive autoimmune CD8 T cell stemness, differentiation, and pathogenicity. We find β cell-specific CD4 T cells in pLN and PA reveal two distinct populations based on their expression of TCF1, a transcription factor critical for stemness and self-renewal. Functional studies identify pLN TCF1hi CD4 T cells as stem-T cell subset needed to drive the generation and maintenance of autoimmune stem-CD8 T cells in pLN, their differentiation into β cell-destroying cytotoxic effector cells, and ultimately T1D. Conclusion A unique population of β cell-specific CD4 T cells in pLN is critical for autoimmune CD8 T cell stemness, differentiation and disease. Identifying therapeutic strategies that target autoimmune stem-CD4 T cells could emerge as powerful approaches for the treatment of T1D. Funding Source NIH grant 1F31DK145180, NIH grant R01AI173249, Juvenile Diabetes Research Foundation grant JDRF SRA-2023-1410-S-B, Topic Categories Basic Autoimmunity (BA)

Characterization of H1N1 and interferon-β-responsive long non-coding RNAs using single-cell CRISPRi in human lung epithelial and immune cell lines 2260688

Abstract Introduction Despite 36,000 long non-coding RNAs (lncRNAs) annotated in the human genome, functional characterization remains limited, and the roles of lncRNAs in the interferon (IFN) response are largely unknown. We previously identified VILMIR as an IFN-stimulated lncRNA that enhances the host IFN response. Through analysis of public bulk RNA-seq datasets, we identified two additional lncRNAs that are consistently upregulated following H1N1 influenza infection and interferon-β (IFN-β) treatment. This study aims to identify immune genes that are perturbed by candidate lncRNA knockdowns (KDs) and explore the relationship between KD efficiency of lncRNA-targeting guide RNAs and the scale of transcriptomic perturbations. Methods To investigate the roles of these lncRNAs, we used CRISPRi to generate stable KD cell lines in human lung epithelial cells, monocytes, and T-lymphoblasts. These cell lines were treated with IFN-β for 6 hours, and mock versus IFN-treated samples were analyzed using 10x CRISPRi scRNA-seq analysis. Results Transcriptome analysis of each lncRNA KD line compared to controls revealed a set of differentially expressed immune genes in response to IFN-β stimulation. Conclusion In summary, we have identified two novel interferon-responsive human lncRNAs that may play important roles in the regulation of the host interferon response across different cell types. The results provide mechanistic targets for future studies and serve as preliminary data for future large-scale screens of candidate lncRNAs. Funding Source National Institutes of Health Grant R21AI147187 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Characterising humoral and airway immunity following Respiratory Syncytial Virus infection challenge in older adults 2309278

Abstract Introduction Respiratory syncytial virus (RSV) is a major cause of respiratory viral illness in older adults, yet the effect of ageing on systemic and mucosal immunity against RSV infection remain unclear. Using a RSV controlled human infection model (CHIM) in young and older adults, we examined early humoral and mucosal immunity, identified age-related immune differences, and explored immune correlates of protection at both systemic and airway sites. Methods Twenty-eight older (60—75 years) and 8 young (18—55 years) adults were inoculated intranasally with RSV A Memphis-37. Viral loads were measured by qPCR. Symptoms and paired serum, nasal, and bronchoalveolar lavages (BAL) were collected longitudinally. Neutralising antibodies and F and G protein—specific responses were quantified. Samples from a previous young adult CHIM using the same virus were also included. Results Older adults showed higher infection rates (68% vs 31%) and greater viral shedding. Pre-existing serum neutralising titres correlated with protection in older, but not young adults. Serum IgG responses were comparable across age groups and targeted pre-F epitopes. Nasal IgA responses while intact overall, showed limited boosting of pre-F-specific antibodies, that have the most potent neutralising activity. Multiplex analysis confirmed these patterns and demonstrated increased G-specific nasal IgA in older infected adults, which was correlated with protection. A similar pattern was also seen in BAL samples, but these did not correlate with protection from infection. Conclusion RSV CHIM in older adults is safe and reveals preserved systemic neutralising and IgG responses. Healthy older adults, however, were more susceptible to infection and exhibited distinct mucosal antibody profiles, with G-specific nasal IgA emerging as a potentially strong correlate of protection. These findings have implications for vaccine design in older adults, particularly as current approved vaccines only target RSV pre-F protein. Funding Source National Institute of Health Research; United Kingdom Research and Innovation Topic Categories Viral Immunology (VIR)

The membrane distal domain of CD16a shapes the NK cell ADCC immune synapse 2309381

Abstract Introduction Natural Killer (NK) cells are a powerful and versatile part of the human immune system. The molecular mechanisms of their antibody reliant response, antibody dependent cellular cytotoxicity (ADCC), are not fully understood. The cell surface receptor CD16a is known to bind to IgG at the membrane proximal domain (D2), but the role of the membrane distal domain (D1) is unknown. Methods Using two nanobodies (C21 and C28) that bind to CD16a D1 at distinct epitopes without blocking IgG binding, we were able to probe the role of the CD16a D1 in shaping the NK cell immune synapse during ADCC. Using real-time ADCC analysis, we examined how binding of these nanobodies altered NK cell ADCC activity toward Her2+ SKOV3 cells treated with Trastuzumab. We subsequently used confocal and stimulated emission-depletion (STED) imaging of NK cell immune synapses on supported lipid bilayers to determine the role of CD16a D1 in synapse structure and CD3-zeta (CD3z) phosphorylation. Results We found that ADCC activity is disrupted in a dose-dependent manner in the presence of C28 but not C21. Our STED imaging and analysis revealed that ADCC ablation was due to disruption of CD3z phosphorylation, disruption of normal NK cell attachment, and alteration of actin synaptic structure. Conclusion Our data support the hypothesis that CD16a D1 may be involved in signaling with an unknown co-activating receptor/protein, explaining why blocking it has negative effects on ADCC and synapse formation. Our data could point to a universal function of membrane distal domains, which are structurally conserved across all Ig receptors. Further, our findings have implications for antibody and cellular therapeutic engineering efforts aimed at enhancing in vivo NK cell ADCC activity during treatment for broad types of cancer and viral infections. Funding Source NIH/NIAID R01 AI167646 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Correction to “Highly Specific Cytokine Receptor‐Targeting Chimeras for Targeted Membrane Protein Degradation and Sensitization of Osimertinib in EGFR‐Mutated Non‐Small‐Cell Lung Cancer”