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Selective Electrochemical NO <sub>x</sub> Reduction to N <sub>2</sub> for Sustainable Ammonia Energy Systems
ABSTRACT Ammonia‐based energy systems offer a pathway toward carbon‐free energy, yet NO x emissions from ammonia combustion hinder the realization of a sustainable closed‐loop. Electrochemical NO x reduction reaction (eNO x RR) provides a promising route to convert NO x to benign N 2 under mild conditions. However, under the low NO x concentrations typical of practical exhaust streams, N 2 selectivity is limited by competing hydrogenation, hydrogen evolution, and inefficient intermediate utilization. Here, we present a mechanistic perspective on selective NO x ‐to‐N 2 conversion, highlighting that eNO x RR proceeds through *NO‐mediated pathways with N 2 O as a key intermediate, while low surface coverage restricts N–N coupling. We propose that integrating tandem catalysis with interfacial microenvironment regulation offers a unified strategy to enhance N 2 selectivity. Finally, we outline key challenges for practical implementation under gas‐phase and dilute conditions, providing design principles for efficient electrochemical denitrification. This framework has the potential to enable scalable NO x removal and support a closed‐loop ammonia energy system.
Atomic‐to‐Nanoscale Engineering of Piezocatalytic Heterojunctions for Ultrasound‐Triggered Osteomyelitis Therapy
ABSTRACT Osteomyelitis, primarily caused by methicillin‐resistant Staphylococcus aureus (MRSA), poses critical clinical challenges, including drug‐resistant infections and insufficient bone regeneration. Herein, we propose a mechano‐electro‐chemical energy conversion paradigm based on an atomic‐to‐nanoscale synergistic strategy, realized by constructing a heterojunction between sulfur‐doped zinc oxide and bismuth ferrite (S‐ZnO/BiFeO 3 ). At the atomic level, sulfur doping induces lattice distortion and oxygen vacancies, narrowing the bandgap of ZnO. At the nanoscale level, sulfur doping modulates crystal growth, transforming ZnO nanoblocks into thinner nanosheets that establish intimate interfacial coupling with BiFeO 3 . This dual‐scale synergy amplifies the piezoelectric coefficient (d 33 ) tenfold, significantly enhancing the ultrasound (US)‐responsive piezocatalytic output. Under US, the enhanced piezocatalytic field facilitates directional charge migration and continuous reactive oxygen species (ROS) generation, achieving a 99.45% antibacterial rate against MRSA while effectively inhibiting biofilm growth. Transcriptomic profiling reveals that this energy conversion process eliminates MRSA via a multi‐target collapse of membrane defense, ion homeostasis, and energy metabolism. Concurrently, S‐ZnO/BiFeO 3 promotes osteogenic differentiation through US‐triggered microcurrents and Zn 2+ release. In vivo results validated robust bactericidal potency, osseointegrative kinetics, and excellent biocompatibility. This study establishes a mechano‐electro‐chemical energy conversion paradigm via atomic‐to‐nanoscale engineering, offering a high‐performance platform for precise piezocatalytic therapy.
Engineered Nanobodies for Durable Tolerance in Allergic Asthma 2260092
Abstract Introduction Allergic asthma is caused by hypersensitivity to airborne allergens such as dust mites and pollen. Current treatment includes allergen avoidance, systemic desensitization and symptom relief, none of which provide an effective cure. Thus, there is unmet need for transformative therapies that induce durable, antigen-specific tolerance in asthma. Methods Our nanobody-engineering approach co-delivers the allergen and glucocorticoids (dexamethasone) to all antigen-presenting cells (APCs) by targeting major histocompatibility complex class II (MHCII). This co-delivery ensures presentation of the allergen in a tolerogenic context and leads to anergy and/or deletion of allergen-specific B and T lymphocytes. Results We have established this approach in an ovalbumin model of allergic asthma. We found that intranasal delivery outperforms retro-orbital injection in a prophylactic setup. In pre-sensitized mice, a single dose of nanobody-adduct was sufficient to intercept the immunoglobulin (Ig) response and reduce allergic airway inflammation. We even see great response in mice treated in between ovalbumin challenges as model for intermittent asthma episodes. We have analyzed systemic and intranasal delivery by positron emission tomography and probed the engaged APC subsets by multiparametric cytofluorometry. We found that intranasal instillation persists in the lung for over 72 hours (PET) and directly engages B cells (flow), particularly pronounced in pre-sensitized mice. To assess the translational potential, we developed nanobody-adducts of house dust mite (HDM) allergen Der P 2 and tested this in patient samples of known HDM allergy. Conclusion Allergic asthma is a common disorder affecting &gt;150 million people worldwide, though no curative treatment is available. We established a potent approach to intercept allergic airway inflammation in an ovalbumin mouse model. We will now analyze B cell and other APC subsets by single cell RNA sequencing to delineate the molecular mechanisms of tolerance induction. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)
Human gut microbe, Parabacteroides distasonis, accelerates diabetes onset in mice without causing gut dysbiosis 2258225
Abstract Introduction Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic, insulin-producing beta cells by autoreactive T-cells. T1D incidence is increasing worldwide but it is poorly understood what activates autoreactive T-cells. Over 90% of anti-insulin mouse CD4 T-cells target amino acids 9-23 of the insulin B chain (insB:9-23). The gut microbiome is thought to contribute to T1D development. We previously showed Parabacteroides distasonis (Pd) peptide HPRT:4-18 cross-reacted with insulin B:9-23-specific mice and patient T-cells. We hypothesize Pd accelerates diabetes onset via molecular mimicry. Methods Specific pathogen-free non-obese diabetic (NOD) mice were oral gavaged with saline, live Pd or heat-inactivated Pd. Germ-free NOD mice were orally gavaged with Pd. Insulitis, gut microbiome diversity and the serum metabolome were assessed. Results Live but not-heat inactivated Pd increased severe insulitis. Gut permeability and metabolites were unaltered with minimal changes in gut microbiome diversity. Germ-free Pd-colonized mice had more severe insulitis but gut permeability and metabolite composition did not change compared to the saline-treated mice. Conclusion The findings further support our hypothesis that Pd accelerates T1D onset via insulin molecular mimicry. To further test the molecular mimicry hypothesis, NOD mice are orally gavaged with engineered Escherichia coli which secretes wild-type or mutant HPRT, containing the insB:9-23 mimic. Funding Source G. Harold and Leila Y. Mathers Charitable Foundation Research Grant No. MF-1905-00311, Juvenile Diabetes Research Foundation Grant No. 1-INO-2022-1108-A-N the Beatson Foundation(2023-003), and a Diabetes Research Connect grant. Topic Categories Basic Autoimmunity (BA)
Succinate Fuels CCR4+ Treg Expansion and Immune Escape in Aging Ovarian Tumors 2256159
Abstract Introduction Ovarian cancer (OvCa) remains the most lethal gynecologic malignancy, with older women experiencing the poorest outcomes. Aging profoundly remodels the ovarian tumor immune microenvironment (TME), promoting regulatory T cell (Treg) mediated immune evasion; however, the underlying metabolic mechanisms remain unclear. Methods Using young (3-month) and aged (22-month) female mice injected intraperitoneally with ID8p53⁻/⁻ OvCa cells, we investigated how age associated metabolic reprogramming influences tumor immunity Results Aged OvCa bearing mice exhibited accelerated tumor progression, reduced survival (median 54 vs. 72 days), decreased CD4+ and CD8+ T cells, and an expanded Treg population. Metabolomic profiling revealed increased succinate accumulation in ascites, tumor tissue, and serum of aged mice and elderly OvCa patients. Exogenous succinate enhanced CCR4+FOXP3+ Treg frequency and upregulated IL-10 and TGF-β secretion. Elevated levels of CCL22, a CCR4 ligand, were also detected in ascites from aged mice and patients, suggesting a chemokine-driven reinforcement of Treg recruitment. PBMCs from aged patients displayed an OXPHOS-reprogrammed CCR4+FOXP3+ Treg subset with heightened suppressive capacity, markedly inhibiting CD4+ and CD8+ effector cytotoxicity. Treatment with AA6, an α-ketoglutarate dehydrogenase inhibitor that blocks succinate synthesis, reduced CCR4+FOXP3+ Treg accumulation, restored effector T-cell function, and significantly prolonged survival in aged OvCa-bearing mice. Conclusion Together, these findings reveal a novel succinate—CCR4 metabolic—chemokine axis that drives Treg mediated immunosuppression in aged OvCa. Targeting this pathway offers a promising strategy to restore anti-tumor immunity and overcome age-related immunotherapy resistance in ovarian cancer. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Early establishment of Virtual memory T cells in the neonatal lung 2259596
Abstract Introduction CD8+ T virtual memory (TVM) cells are antigen-inexperienced, memory-like cells that arise early in life. To date, most TVM studies have focused on adult, splenic TVM populations and failed to consider TVM function at their origin and in mucosal sites. Here, we sought to determine how TVM populate the lung during early life and may impact subsequent immunity. Methods TVM in the lung tissue versus the circulation were studied using a novel intravascular staining protocol via the retro-orbital sinus that we optimized for use in neonates. Lungs were subsequently phenotyped and analyzed by flow cytometry for CD8+CD44+CD122+CD49d⁻ TVM across postnatal (P) days P3—P14. To explore the contribution of cytokine signaling in neonatal lung TVM establishment and localization, parallel analyses were performed in IL-15 knockout neonates. Results In naïve neonatal mice, TVM were detected in the lung parenchyma as early as P3. While total numbers of overall lung TVM increased with age, this expansion was primarily driven by circulating TVM. While lung-resident TVM remained numerically stable, expression of Ki-67 suggested local proliferation. In P5 naïve IL-15 knockout neonates, lung-resident TVM were completely absent, accompanied by approximately 70% reduction in circulating lung TVM and ∼50% reduction in splenic TVM numbers compared to wild-type controls. Conclusion Our findings demonstrate that T virtual memory cells emerge in the neonatal lungs shortly after birth, establishing a stable resident pool capable independent of infection. Moreover, IL-15 may specifically contribute to the early establishment and maintenance of the TVM niche in the lung parenchyma, with circulating and splenic niches only partially dependent on IL-15. Together, these data suggest that neonatal TVM are regulated distinctly from adults, with IL-15 being requisite for maintaining TVM at the respiratory barrier early in life. Funding Source NIH/NAID Grant #P01AI139449 Topic Categories Mucosal and Regional Immunology (MUC)
AHR-activated Stab1+Ly6G+ macrophages elicit resolution of pulmonary fibrosis-associated pathology 2328474
Abstract Introduction In response to various inflammatory stimuli, bone marrow-derived macrophages replace tissue-resident macrophages and mediate tissue inflammation to reestablish tissues homeosis. When inflammation becomes persistent and uncontrolled, however, inflammatory macrophages differentiate into pro-fibrotic macrophages, causing dysregulated fibrotic reactions. It remains largely unknown if macrophages with fibrosis-resolving capacity emerge during fibrogenesis. Methods Using the hyperlipidemia drug fenofibrate that was identified here as a novel ligand for the aryl hydrocarbon receptor (AHR), we showed that its activation promoted Ly6G+ lung macrophage differentiation and activation, resulting in resolution of pulmonary fibrosis. Results Fenofibrate-activated AHR in macrophages created a complex with c-MAF and induced expression of unique sets of genes that were critical in multiple resolving processes of lung homeostasis. Ly6G+ macrophages produced three categories of effector molecules produced by Ly6G+ macrophages to divide labor for resolution of pulmonary fibrosis-associated pathology: 1) GM-CSF drove monocyte-derived macrophage differentiation toward alveolar macrophages and Ly6G+ macrophages themselves; 2) spermidine promoted alveolar regeneration; and 3) metalloproteases (in particular, MMP14) recovered collagen deposition. Lungs of pulmonary fibrosis patients contained reparative, anti-fibrotic Stabilin-1 + (Stab1+) macrophages corresponding to mouse Ly6G+ macrophages and these cells could be differentiated from inflammatory monocytes after AHR stimulation with fenofibrate. Conclusion Thus, our study suggests that AHR-mediated activation of reparative, anti-fibrotic lung macrophages may provide a therapeutic strategy for the treatment of pulmonary fibrosis. Funding Source BK-21 FOUR program through National Research Foundation of Korea (NRF) under Ministry of Education, Republic of Korea Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
MEK/ERK Mediated Diffuse Alveolar Hemorrhage in Murine Lupus 2261092
Abstract Introduction About 3% of patients with lupus develop severe diffuse alveolar hemorrhage (DAH) with pulmonary vasculitis. C57BL/6 (B6) mice with pristane-induced lupus also develop DAH, but BALB/c mice are resistant. DAH is independent of Toll-like receptor signaling and other inflammatory pathways. This study examined the role of the MEK1/2 pathway Methods B6 and BALB/c mice were treated with pristane with or without inhibitors of MEK1/2 (trametinib/GSK1120212 [GSK]), ERK1/2 (SCH772984 [SCH]), JNK, or p38. Effects on lung hemorrhage and hemostasis were determined Results GSK and SCH abolished DAH, whereas JNK and p38 inhibitors were ineffective. Apoptotic cells were present in lung samples from pristane-treated mice but not in mice receiving pristane and GSK, and endothelial dysfunction was normalized. Expression of the ERK1/2-regulated transcription factor early growth response 1 increased in pristane-treated B6, but not BALB/c, mice and was normalized by GSK. Pristane also increased expression of the anticoagulant genes Tfpi and Thbd in B6 mice. The ratio of Tfpi to tissue factor (F3) to Tfpi increased in B6 (but not BALB/c) mice and was normalized by GSK. Circulating thrombomodulin protein levels increased in B6 mice and returned to normal after GSK treatment. Consistent with augmented endothelial anticoagulant activity, pristane treatment increased tail bleeding in B6 mice. Conclusion Pristane treatment promotes lung endothelial injury and DAH in B6 mice by activating the MEK1/2-ERK1/2 pathway and impairing hemostasis. The hereditary factors determining susceptibility to lung injury and bleeding in pristane-induced lupus are relevant to the pathophysiology of life-threatening DAH in systemic lupus erythematosus and may help to optimize therapy. Funding Source NIH, University of Florida Gatorade fund Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Myeloid—Vascular Crosstalk as a Regulator of Secondary Brain Injury After Stroke 2310349
Abstract Introduction Secondary brain injury after stroke is a major factor in neurological outcomes, yet the mechanisms behind it remain poorly understood. Evidence suggests interactions between myeloid cells (both circulating and resident) and the blood—brain barrier (BBB) may explain inflammatory patterns and region-specific vulnerability. We explored this “myeloid-vascular crosstalk” as a framework to understand secondary injury. Understanding these interactions could help identify high-risk patients and guide early treatment. Methods We conducted a systematic review and meta-analysis (PRISMA) of studies from 1990—2025 examining inflammatory biomarkers in ischemic or hemorrhagic stroke. Biomarkers included serum, CSF, and tissue-level measures reflecting myeloid activation, endothelial signaling, or immune trafficking. Using a Bayesian hierarchical model, we assessed links between biomarker trajectories and secondary injury outcomes such as edema, hemorrhagic transformation, and neurological decline. Results Across heterogeneous stroke cohorts, indicators of myeloid activation (specifically circulating monocytes, IL-1β, TNF-α) demonstrated stronger and consistent associations with secondary brain injury than endothelial markers alone. Experimental data suggests post-stroke endothelial activation triggers myeloid recruitment, thereby amplifying vascular permeability. Despite differences in biomarker timing, cumulative evidence establishes immune activation as a key mediator of secondary injury progression. Conclusion Our findings support that myeloid—vascular crosstalk plays a role in secondary brain injury after stroke. Posterior circulation and deep subcortical regions appear especially vulnerable, suggesting that early modulation of immune—vascular interactions could reduce severe complications. This study supports a coordinated myeloid-vascular framework, rather than single biomarkers, to help improve risk stratification and guide future targeted immunomodulatory therapies. Funding Source n/a Topic Categories Neuroimmunology (NEUR)
An Ion‐Based Strategy Toward Synergistic Surface Functionalization Combining the Osteogenic Properties and NIR‐Mediated Antibacterial Activity of PEEK
ABSTRACT Persistent implant‐associated infections critically compromise the longevity and success of orthopedic prostheses. Herein, we present a facile surface‐engineering strategy to construct a multifunctional PEEK‐based implant by introducing manganese‐chelated polydopamine (Mn@p) and mesoporous bioactive glass nanoparticles (MBGNs) onto sulfonated PEEK (SPEEK). The resulting Mn@p/MBG‐SPEEK exhibits a hierarchically porous three‐dimensional architecture with markedly enhanced hydrophilicity and surface roughness. This tailored interface shows in vitro bioactivity and cytocompatibility, significantly promoting the osteogenic differentiation of MC3T3‐E1 cells through the activation of PI3K/Akt/mTOR and AP‐1 signaling pathways, as evidenced by the upregulation of ALP, OCN, OPN, and Runx2 expression. Meanwhile, the pro‐angiogenic potential of Mn@p/MBG‐SPEEK is supported by the upregulation of VEGF and CD31 in HUVECs and enhanced capillary‐like network formation. Notably, Mn@p/MBG‐SPEEK demonstrates efficient light‐to‐heat conversion and potent antibacterial efficacy against Staphylococcus aureus under near‐infrared (NIR) irradiation. Density functional theory (DFT) calculations further reveal that Mn chelation narrows the HOMO‐LUMO energy gap and facilitates charge separation, thereby amplifying photothermal and ROS‐mediated antibacterial effects. Collectively, this study establishes a versatile and scalable route to enhance the biological performance of PEEK implants, offering a conceptual framework for integrating ion‐assisted therapy with phototherapy toward next‐generation bioactive and infection‐resistant orthopedic materials.
Advanced Machine Learning Across Multiple Vaccines to Predict Durable Immunogenicity using the Immune Signatures Data Resource 2260430
Abstract Introduction Understanding vaccine durability is key to designing immunizations with long-term efficacy. Leveraging the Immune Signatures Data Resource, a compendium of transcriptomic and immunological responses from 1405 healthy adults (18+ years) across 24 vaccines, we investigated shared immune mechanisms underlying durable antibody responses. The dataset spans live (yellow fever, smallpox), recombinant viral-vector (Ebola), inactivated (influenza), and glycoconjugate (pneumococcal) vaccines. Methods Data preprocessing included imputation, normalization, and alignment across post-vaccination time points. We applied advanced machine learning (ML) frameworks to predict antibody immunogenicity and durability. Feature selection for high-dimensional, low-sample-size multi-omics datasets was performed using HSIC Lasso to identify predictors of antibody responses. Selected features served as input to ensemble, regularized regression, and gradient-boosting models (e.g. DT, RF, LASSO, XGB, CatBoost). We compared single-target and multi-output approaches, evaluating stacked, chained, and wrapper-based strategies, and implemented multi-layer neural networks to capture complex relationships among immune features. Results Post-vaccination time points explained ∼15% of the total variance, indicating shared immune kinetics across vaccine types, while age and sex contributed minimally. Gradient-boosting and multi-output modeling approaches achieved the highest predictive accuracy across vaccines, highlighting the value of integrating correlated outcomes. Neural network models similarly captured complex, nonlinear immune signatures, albeit with reduced explainability. Conclusion Conserved transcriptional modules, particularly interferon-signaling and plasmablast-related pathways, emerged as strong predictors of antibody durability. This integrative ML framework enables identification of key immune signatures critical for developing vaccines with durable responses, advancing data-driven strategies for systems vaccinology. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)
Targeting IL-22 activity to the gut epithelium confers disease benefit without provoking skin inflammation 2258200
Abstract Introduction IL-22 is a cytokine acting on surface-lining epithelial cells to induce antimicrobial defense and barrier regeneration. It has gained clinical interest for the treatment of inflammatory bowel disease due to its non-immunosuppressive mechanism. Prior clinical efforts have delivered IL-22 systemically, but development was constrained by dose-limiting skin toxicity. This presents an opportunity to widen the therapeutic index by targeting IL-22 to gut epithelium while avoiding keratinocytes and other IL-22-responsive cells. Methods A cell-surface protein specifically expressed on gut epithelium was identified using Ampersand’s proprietary Address Map. Candidate AND-Body molecules were engineered by pairing an Address binder with a modified IL-22 which signals only when the Address binder is engaged. Activity was evaluated in vitro and in vivo in rodents and in non-human primates (NHPs) for potency on gut vs. skin tissue. Results Studies on human cell lines showed that the AND-Body AMP-220 has &gt;500-fold increased potency on enterocytes compared to a non-targeted IL-22 control, with no activity on keratinocytes. Mouse studies showed enrichment of AMP-220 in small intestine and colon relative to non-targeted tissues. This was accompanied by highly selective activity in small intestine and colon when compared to a non-targeted IL-22 Fc control. Moreover, AMP-220 delivered significant benefit in both therapeutic and prophylactic models of DSS-induced colitis. In NHPs the AND-Body demonstrated robust induction of IL-22 target genes in colon with minimal evidence of skin activity or systemic inflammation. Conclusion By identifying a gut-specific address and tuning of gut-targeting and cytokine effector moieties, our IL-22 AND-Body shows potent and specific activity on human enterocytes, gut-specific PK/PD and efficacy in rodents, and localized activity in NHPs. These results support continued advancement of AMP-220 through IND-enabling studies and highlight the potential of precision-targeted cytokines. Funding Source n/a Topic Categories Cytokines and Chemokines and their Receptors (CCR)
A Hybrid Grading Model for Immunology Education: Balancing Rigor and Equity 2256290
Abstract Introduction Immunology, a field known for its complexity and clinical significance, often imposes high cognitive and emotional demands on students. Traditional grading systems can increase stress and impede teamwork, while purely pass/fail systems might reduce accountability and recognition of achievement. Methods We at Chicago State University used appropriate surveillance methods, such as direct observation, digital monitoring, biometric sensors, and self-reporting tools. Ensured ethical practices by obtaining informed consent, protecting privacy, and maintaining transparency. Used tools such as LMS analytics, screen-monitoring software, and video analysis platforms to collect data. Analyzed the data using qualitative or quantitative methods. Results This study, conducted at Chicago State University’s College of Health Sciences and Pharmacy, introduced a hybrid grading model for undergraduate immunology courses that combines traditional letter grades with pass/fail assessments to improve both academic rigor and student well-being. The proposed model assigns letter grades to core knowledge assessments–such as exams, quizzes, and final evaluations–which count for 50% of the overall grade. The other 50% is based on pass/fail assessments, including lab practical, group projects, participation, and reflective writing. This approach fostered skill development, collaboration, and engagement without increasing grade pressure, creating a more inclusive and supportive learning environment. Conclusion This flexible approach encourages intrinsic motivation, reduces anxiety, and promotes equity while maintaining academic standards. By aligning assessment methods with the diverse learning objectives of immunology–such as knowledge retention, practical skills, and collaborative inquiry–the hybrid grading system provides a versatile framework suitable for various science curricula. Future research should assess its impact on student performance, engagement, and long-term retention of immunological concepts. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)
Mapping the chromatin landscape of the mouse immune system with low-input automated CUT&RUN 2260000
Abstract Introduction Understanding how immune cells develop and function requires insight into the epigenomic mechanisms that regulate gene expression. While many genomic studies focus on transcriptional outputs, changes in the chromatin landscape play a central role in shaping lineage commitment. The mammalian immune system is composed of highly diverse and dynamic cell types, but detailed epigenomic studies have been severely limited by technical challenges in profiling rare cell populations. Methods We developed and validated a low-input, automated CUT&RUN workflow that incorporates standardized sample preparation to ensure reliable generation of data at the consortium scale. This method minimizes sample handling and applies internal controls to monitor assay performance during experimental and sequencing stages. Results Extensive optimization of assay conditions and antibody reagents enabled robust mapping of histone post-translational modifications (PTMs) from as few as 10,000 cells per reaction. Applying this approach, we profiled &gt;170 immune subpopulations collected from 11 ImmGen consortium labs over two years. Conclusion These innovations establish a scalable, high-resolution platform for profiling chromatin landscapes from minimal cell inputs. Our automated CUT&RUN pipeline enables standardized, reproducible analysis across diverse immune cell types and can distinguish technical issues from true biological insights. Together, these advances lay the foundation for a companion study presenting the first comprehensive epigenomic atlas of immune lineages and provide a framework for studying chromatin regulation in rare or limited samples across the life sciences. Funding Source NIH R44 AI167215 Topic Categories Technological Innovations in Immunology (TECH)
Evaluating immunotherapy response in bladder cancer patients by spatially interrogating PD-1/PD-L1 interaction 2258170
Abstract Introduction Protein-protein interaction is one of the many mechanisms where individual cells communicate with nearby cells or extracellular matrix to modulate the environment. Visualizing these interactions using a spatial platform can validate known interactions implicated in disease pathology, especially cancer. We developed the ProximityScope™ assay that can be used with the RNAscopeTM Multiomic LS assay to visualize protein interactions, proteins, and mRNA on a single tissue section. Methods Antibodies were conjugated to oligonucleotides designed to bind RNAscope signal amplification complexes. The entire workflow was performed using the fully automated Leica BOND RX system. The assay detects 1 interaction with 5 RNA/protein targets and was deployed on samples from patients with localized, muscle-invasive bladder cancer who received neoadjuvant anti-PD-L1 checkpoint inhibitor therapy prior to surgery on a clinical trial (NCT02451423). To characterize tumor-immune microenvironment of these patients, PD-1/PD-L1 interactions were assessed with protein markers for immune and tumor cells (CD3, CD4, CD8, FoxP3, and PanCK) and activation markers (IFNG, GZMB, GZMK). Results We visualized PD-1/PD-L1 interactions, proteins and RNA targets within the tumor before and after immunotherapy treatment. The PD-1/PD-L1 interaction between CD4+ or CD8+ T cells and CK+ tumor cells, showed significant reduction in responder samples than non-responder samples. Additionally, responder samples showed a decrease in percent of tumor-infiltrating regulatory and cytotoxic T cells. These results demonstrate that while anti-PD-L1 may not induce an increase in CD8+ T cell recruitment overall, this treatment modulates PD-1/PD-L1 interaction between specific cell populations. Conclusion Our findings underscore the importance of spatial evaluation of PD-1/PD-L1 interaction alongside cell phenotyping protein and effector molecule encoding RNA in assessing anti-PD-L1 treatment responses. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
De novo generation of innate memory-like T cells occur early during human immune development 2254150
Abstract Introduction Infants rely heavily on innate immunity while adaptive memory develops. A subset of T cells, termed “virtual memory” or innate-like memory T cells, bridges innate and adaptive immunity, but their emergence in early human life is unclear. Infants receiving gene therapy for X-linked severe combined immunodeficiency (SCID-X1), whose immune systems reconstitute de novo, offer a unique model to study this process. We investigated whether innate-like memory CD8+ T cells arise during immune reconstitution and defined their epigenetic, transcriptional, and functional features. Methods Longitudinal blood samples from SCID-X1 infants post—gene therapy were analyzed. Flow cytometry identified CD8+ subsets expressing NKG2A. TCR sequencing assessed clonal diversity, while DNA methylation and RNA profiling defined molecular signatures. Functional assays measured cytokine-induced, antigen-independent IFN-γ production after IL-12 and IL-18 stimulation. Results Early after gene therapy—induced immune reconstitution, infants’ peripheral blood showed a relative enrichment of innate-like memory CD8+ T cells marked by NKG2A expression. These NKG2A+ CD8 T cells displayed innate-associated transcriptional signatures and a TCR repertoire distinct from conventional naïve/memory subsets. DNA methylation profiling confirmed a subset-specific epigenetic signature, including open (poised) regions at effector genes, consistent with readiness for rapid response. Functionally, when stimulated ex vivo with IL-12+IL-18, NKG2A+ T cells produced IFN-γ in an antigen-independent fashion, indicative of innate-like responsiveness. Together, these features distinguish them from conventional antigen-driven memory CD8 T cells. Conclusion Innate-like memory CD8+ T cells (NKG2A+ “virtual memory” T cells) arise early in human life during immune reconstitution and are epigenetically and transcriptionally primed for rapid, antigen-independent effector responses. Funding Source NIH (R01AI114442; R01CA237311; P01-HL053749; U54AI082973;P01AI138962), The ASSISI Foundation of Memphis, AlexLemonade Stand, SU2C, Key for a Cure Foundation, NRSA(F32-CA250155), CIRM (CLIN2-10830; CLIN2-09504), and theAmerican Lebanese Syrian Associated Topic Categories Hematopoiesis and Immune System Development (HEM)
Monocyte-Derived ADA2 Regulates Myeloid—T Cell Crosstalk and Drives Pro-Inflammatory Responses Linked with Intestinal Inflammation in Crohn’s Disease 2307789
Abstract Introduction Crohn’s disease (CD) is a chronic inflammatory disorder marked by dysregulated immune responses. Purinergic signaling contributes to mucosal injury, yet the role of adenosine-metabolizing enzymes remains unclear. Adenosine deaminase 2 (ADA2), a secreted myeloid-derived enzyme that converts adenosine to inosine, has been insufficiently studied in CD. We investigated whether monocyte-derived ADA2 is linked to intestinal inflammation. Methods Twenty-nine patients with CD undergoing endoscopy at the University Hospital Heidelberg were enrolled. Paired inflamed and non-inflamed intestinal tissue samples were collected. ADA2 activity and cytokine/chemokine levels were measured in tissue digestion medium. Tissue-derived cells were analyzed by flow cytometry to characterize monocyte populations and their intracellular ADA2 expression. Results ADA2 activity was increased in inflamed versus non-inflamed tissue, along with pro-inflammatory mediators IL-6, IL-1β and IFN-γ. Intracellular ADA2 was reduced in CD14+ monocytes from inflamed regions, suggesting that the protein was actively secreted into the extracellular space. Notably, ADA2 activity was significantly associated with CXCL10 levels, a chemokine for CXCR3-dependent cell recruitment. Accordingly, the CXCR3+ cells were increased specifically within ADA2+CD14+ monocytes in inflamed tissues, while no difference was noted in ADA2-CD14+ population. In general, ADA2+CD14+ monocytes exhibited a pro-inflammatory signature enriched in TNFa, IL-23, and IFN-γ, compared to ADA2-CD14+ monocytes. Subsequent analysis of bulk RNA-seq data of ADA2-stimulated macrophages confirmed upregulation of IL23, IL1B, and IL6 -key drivers of pathogenic Th17 cell differentiation. Consistently, ADA2 stimulation of peripheral blood mononuclear cells increased the frequency of CCR6+ CD4+ Th17-like cells. Conclusion These findings suggest ADA2 as a previously unrecognized regulator of myeloid—T cell crosstalk in CD, highlighting a potential pathogenic axis and therapeutic target. Funding Source German Research Foundation (DFG), Heidelberg University Faculty of Medicine Topic Categories Immune Mechanisms of Human Disease (HUM)
Combination of 3D and 2D Small and Wide Angle X‐Ray Scattering Imaging Reveals Diminished Bone Quality in the Superior Human Femoral Neck Cortex
ABSTRACT The human femoral neck is particularly vulnerable to fracture, with failure most often initiating in the superior region. While age‐related microstructural changes such as cortical thinning and increased porosity are well established, the contribution of material properties at the lamellar and mineralised collagen fibril (MCF) levels remains poorly understood. Here, regional differences in nanostructural properties of cortical bone from 78 femoral necks obtained from 44 donors aged 54–96 are investigated using a combined 2D and 3D X‐ray scattering imaging approach. We identified misalignment between the scattering signals arising from the MCF bundles – specifically those associated with mineral inclusions in the collagen fibril gap regions, the mineral nanostructure, and the mineral crystal lattice – suggesting the presence of distinct mineral phases within and around the collagen fibers. Despite substantial intra‐sample variability, the superior region displays on average more oblique MCF orientations, larger and thicker mineral platelets arranged in a less‐ordered structure, greater misalignment between mineral and collagen at the MCF level, and possibly stiffer collagen fibers, with no significant trends observed with donor age or sex. The cumulative effect of these material property differences may contribute to the increased susceptibility of the superior cortex to compressive failure.
Unbiased Multiparameter Analyses Reveal Mechanisms of Variable Restimulation-Induced Cell Death Sensitivity across Human T cell Populations 2251586
Abstract Introduction Restimulation-Induced Cell Death (RICD) is a crucial self-regulatory apoptosis pathway that constrains effector T cell expansion during an adaptive immune response. Our lab and others have implicated multiple genes involved in regulating RICD sensitivity, but molecular mechanisms that explain highly hetergeneous RICD susceptibility across healthy human donors remain nebulous. We hypothesized that relative RICD sensitivity could be explained by specific gene expression programs that correspond to functional differentiation states of multiple, distinct effector T cell subsets. Methods We took an unbiased approach to this question by amassing extensive phenotypic data on T cells collected from &gt;50 healthy human donors, employing spectral flow cytometry and scRNA-seq on effector T cells from the most highly resistant vs. sensitive individuals. Results Our results unveiled enrichment of CD8+ T cells expressing senescence (CD57, NKG2D) and exhaustion (TIGIT) markers in highly sensitive donors; indeed, FACS-based purification confirmed CD8+CD57+ T cells were markedly more susceptible to RICD compared to CD57- counterparts. Moreover, scRNA-seq revealed upregulation of PI-3K pathway genes in RICD sensitive donors, consistent with enhanced PI-3K signaling in senescent T cells. RICD sensitive donors also harbored more “aged” CD4+ T cells expressing ZEB2 and cytotoxicity genes (PRF1, GZMB/K, GNLY, NKG7). Conversely, RICD-resistant CD4+ and CD8+ effector T cells were enriched for genes/markers corresponding to more stem-like properties (IL7R, SELL, CD27, LRRN3, LEF1, BACH2). Indeed, more naïve/central memory T cells were observed in initial PBMC collected from RICD-resistant donors. Conclusion Collectively, our work provides the first “atlas” of human RICD variability that can be used to elucidate and predict relative RICD susceptibility across humans donors, informing new approaches for optimizing adoptive T cell immunotherapies and understanding capricious T cell responses to infection/immunization. Funding Source NIH R35GM139619 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
IgA is necessary and sufficient to protect against norovirus infection 2258197
Abstract Introduction Human norovirus (HNV) causes up to 700 million infections and 200,000 deaths each year, primarily affecting young children in low-resource settings. Despite this significant public health and economic impact, no licensed vaccines or anti-viral therapies exist for HNV. Several vaccine candidates are currently in advanced clinical trials, including virus-like particle (VLP), adenoviral (Ad5), and mRNA lipid nanoparticle (LNP) based vaccines. While these candidates show promise, none have demonstrated complete or long-term protection. The mechanisms underlying protective immunity to HNV remain poorly defined, posing a major barrier to rational vaccine design. Technical limitations make it challenging to directly interrogate mechanisms of HNV immunity, as there are no scalable cell culture, reverse genetics, or small animal models. Murine norovirus (MNV) is a genetically tractable model for HNV that shares many fundamental properties including minimal intestinal pathology, rare epithelial cell tropism, and fecal-oral transmission. Methods We used a panel of genetically and pharmacologically modified mice to investigate the correlates and cellular determinants of protection to MNV infection. Results T cell—dependent IgA is necessary to control MNV replication in the small intestine, whereas CD8+ T cells are dispensable. IgA plasma cells mediate protection from re-infection and dimeric IgA, but not monomeric, is sufficient to confer complete protection against MNV infection. In contrast, despite generating a robust systemic IgG response, systemic vaccination with the viral capsid protein (VP1) fails to protect mice, mirroring the recent failure of a systemic VLP vaccine in infants. Conclusion Our work shows that mucosal IgA is central to controlling norovirus infection in a murine model and provides important insights for rational vaccine design and HNV immunotherapeutics. Funding Source n/a Topic Categories Viral Immunology (VIR)