Browse Articles

Disparate immunity proteins independently inactivate an antibacterial nuclease toxin

Obstructive Hypertrophic Cardiomyopathy

Uncovering reliability blind spots in transportation AI: an edge-case-based validation approach for transportation systems

Abstract With artificial intelligence (AI) technology expanding into high-risk domains, criteria for evaluating its reliability and safety are required, as AI deployment may otherwise be constrained by future government regulation or societal backlash. In particular, verifying reliability in unexpected scenarios, such as edge cases, has emerged as a key challenge for implementing safe AI systems. This study introduces a supplementary validation approach to support the reliability testing of AI systems in high-risk domains. Considering the case of AI-based vehicle license plate recognition (VLPR), this study proposes edge-case-based validation approaches specifically designed for certification bodies. These approaches aim to address reliability validation needs that fall outside the well-defined operating regions covered by conventional testing. For VLPR systems, edge cases were identified as adverse-condition samples misrecognized by a baseline model and were then evaluated using a resolution-enhanced model. Under rainy conditions, the conventional recall metric reported 99.2%, whereas evaluating the same system on the extracted edge cases yielded 93.3%—a gap the aggregate metric did not make explicit. These findings highlight the potential for certification bodies to utilize this approach as a qualitative validation tool to uncover reliability blind spots in such systems. While the study focuses on VLPR systems, the proposed methodology is, in principle, transferable to other AI-based applications, although this remains to be tested. Future studies will aim to quantify edge case extraction techniques and examine their adaptability to diverse AI systems across various domains. This study provides preliminary, case-study insights that may contribute to improving the safety and reliability of AI systems in high-risk environments.

CD19 targets in the brain parenchyma attracts murine CD19-directed CAR T cells to infiltrate 2259766

Abstract Introduction Neurotoxicity is a common complication of CD19-directed CAR T cell therapy for leukemia and lymphoma patients. We have previously shown that murine CD19-directed CAR T cells can induce neurotoxicity in immunocompetent non-tumor bearing mice. There is concern that CAR T cells have targets other than B cells in the brain so we are investigating whether CAR T cells infiltrate the normal brain in a targeted fashion, or if they are simply surveilling. Methods Wild type BALB/c mice received 10x10^6 murine CD19-directed CAR T cells and 10x10^6 mock-GFP T cells I.V.. We characterized T and B cells distribution in brain, dura, skull, and spleen using flow cytometry. Then longitudinally tracked the behavior of T cells in the brain and meninges using in vivo two photon imaging. Finally, we assessed CAR T cell distribution in an immunocompetent CD19+ RCAS-TVA glioma model. Results CAR to mock T cell ratios in the brain and leptomeninges are 6 fold higher than in spleen (n = 6 mice, p = 0.0001) on day 6, suggesting they are preferentially infiltrating and not a reflection of relative abundance. In vivo two photon imaging showed many extravasated and actively migrating CAR T cells are in the dura and leptomeninges, with far fewer in the parenchyma. No mock T cells were seen in brain parenchyma (n = 3 mice). When an infiltrative CD19+ brain tumor target is present, CAR T cells successfully infiltrate the brain parenchyma more than both non-target brain tumor and non-tumor bearing mice. Conclusion CAR T cells are rarely seen in the brain parenchyma in non-tumor bearing mice. However, massive CAR T cell infiltration in the brain parenchyma is seen with CD19+ infiltrative brain tumor targets. The ability of CD19 directed CAR T cells infiltrating CNS after target stimulation has therapeutic implications for treating CNS autoimmunity and malignancy. Funding Source NIH R37CA275954 (PI: Juliane Gust), NIH R03NS135446 (PI: Juliane Gust), and NIH K08NS118138 (PI: Juliane Gust) Topic Categories Neuroimmunology (NEUR)

Cytokine changes following methotrexate treatment in rat adjuvant-induced arthritis 2257725

Abstract Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in joint inflammation leading to systemic bone and cartilage destruction. The most common first-line treatment for RA is the disease-modifying antirheumatic drug methotrexate (MTX). MTX is though to work in RA by suppressing the autoimmune response by regulating leukocyte activity and protecting joint tissues. Rat adjuvant-induced arthritis (AIA) is a rodent model that mimics the pathogenesis of human RA. In this study, we will investigate the effects of MTX on cytokine expression in rat AIA joint and spleen tissues. Methods Rat AIA studies were performed, where MTX was administered preventatively 3 times before the onset of arthritis symptoms (day 9). Rat ankle joints and spleens were harvested from AIA rats. Tissue samples were homogenized, and protein extracts from MTX-treated animals and control AIA rats were assessed for 36 different cytokines using a cytokine array. Cytokines with altered expression in MTX-treated animals were quantified using specific ELISAs. Results Animals that received MTX treatment did not develop arthritis as determined by articular index scoring and joint circumference measurements, compared to control animals. MTX-treated AIA animals showed significant decreases in ankle MCP-1, ICAM-1, and CINC-1 levels and spleens from these animals had significantly reduced CINC-1 levels. Conclusion The reduced levels of CINC-1 and MCP-1 in joint tissues of MTX-treated animals could result in reduced neutrophil and monocyte recruitment, resulting in the reduced joint inflammation. Additionally, the reduction of ICAM-1 could be responsible for the reduced migration of immune cells into the MTX treated joints. Finally, the reduction of acute phase reactants, including CINC-1, in the spleen may be responsible for the reduction in systemic inflammation seen with MTX treatment. Further studies could focus on other cytokines and signaling pathways to develop a better understanding of the mechanism of action of MTX. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Bile acid sequestrants modulate microbiome diversity and host immunity during enteric microbial infection 2254635

Abstract Introduction Approximately 90 million American adults are hypercholesterolemic, with an estimated 43 million either using or eligible for cholesterol-lowering therapies such as bile acid sequestrants. Beyond their role as end-products of cholesterol catabolism, bile acids regulate diverse biological processes in both intestinal and extra-intestinal organs. Increasing evidence highlights their crucial role in maintaining intestinal homeostasis under both physiological and pathological conditions. While bile acid sequestrants effectively reduce serum cholesterol, their impacts on intestinal homeostasis in healthy individuals and during enteric infections remain unclear. Methods In this study, we investigated how bile acid sequestration influences mucosal immune responses and the ability of enteric microbial pathogens to colonize the small intestine in vivo. We further examined the effects of sequestration on intestinal microbial communities before and during infection. Results Our results provide clear evidence that bile acids act as limiting factors in restricting microbiome diversity and richness. Mice administered a bile acid sequestrant exhibited significantly enhanced microbial diversity and richness, which correlated with increased resistance to enteric infection. This protective effect suggesting that enhanced microbial ecosystem diversity is a key driver of resistance. As expected, the antibiotic-driven microbiome depletion caused increased susceptibility to Giardia infection. Together, these findings reveal a previously underappreciated role of bile acid sequestrants in shaping the intestinal microbiome. Conclusion By altering bile acid signaling and downstream pathways, sequestration not only lowers cholesterol but also modulates host immunity during enteric infections. These insights may have important implications for patients taking bile acid sequestrants as part of cholesterol-lowering therapy. Funding Source This study was financially supported by a startup fund (20344-8015) from the Department of Biomedical Sciences, School of Medicine and Health Sciences, the University of North Dakota; a Dean’s Meritorious Pilot Grant, School of Medicine and Health Science Topic Categories Mucosal and Regional Immunology (MUC)

Epitope-Specific Neutralizing IgG4 Antibodies Define Natural and Acquired Tolerance in Peanut Allergy 2244882

Abstract Introduction While early introduction has clinically transformed our care of food allergy patients by inducing long-lived natural tolerance in children, the underlying mechanism of natural tolerance remains unclear, though serum IgG4 levels have been shown to increase in tolerant and peanut consuming adolescents. In oral immunotherapy (OIT), neutralizing IgG4 antibodies have been associated with sustained tolerance. We hypothesize that neutralizing IgG4 antibodies (nAbs) are also induced in peanut-allergic individuals with high thresholds of reactivity. Methods To study this, we developed three biomarker assays: an indirect ELISA which was previous benchmarked in tolerance after OIT, an indirect bead-based assay (iBBNA), and a direct bead-based assay (dBBNA). The ELISA and iBBNA using Ara h 2 specific epitope-specific nAbs to epitopes 1.2 and 3 that block neutralizing epitopes on Ara h 2. The dBBNA uses a mutant Ara h 2 that preserves epitopes 1.2 and 3 and therefore measures nAb levels. Protein G purified serum from peanut-allergic individuals with a low (< 144 mg, n = 10) or high reactivity (>144mg, n = 10) peanut were used for profiling. Results Naturally tolerant individuals exhibited robust neutralizing IgG4 responses, mirrored by OIT subjects achieving sustained unresponsiveness, but absent in transiently desensitized patients. The iBBNA, validated against inhibitory ELISA (R² = 0.9, CV = 2.5%), reliably distinguished durable from transient OIT responses. Conclusion These findings establish neutralizing IgG4 as a shared feature of natural and therapeutic tolerance, supporting their use as predictive biomarkers and guiding hypo-allergen design. Funding Source R01 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Phosphatase DUSP23 induces Th2-mediated allergic diseases by preventing GATA3 degradation 2253381

Abstract Introduction Dual-specificity phosphatase 23 (DUSP23) is involved in the regulation of placental development and cell division. The role of DUSP23 in immune regulation remains unknown. Methods We studied the role of DUSP23 in T-cell function using DUSP23 knockout (KO) mice, T-cell-specific DUSP23 conditional knockout (T-DUSP23 cKO, DUSP23f/f;CD4-Cre) mice. We performed in vitro T-cell differentiation, KLH immunization mouse model, ovalbumin (OVA)-induced allergic asthma mouse model, and dinitrofluorobenzene (DNFB)-induced atopic dermatitis mouse model. The functional effects of these DUSP23 variants are being studied by mutagenesis and immunoblotting analysis. Results We found that IL-4 production of the KLH-restimulated T cells was significantly decreased by DUSP23 knockout. In vitro Th2 differentiation was reduced using splenic T cells from DUSP23 KO mice. The lung inflammation and the skin inflammation were attenuated by T-cell-specific DUSP23 conditional knockout. The Th2 cytokines such as IL-4, IL-5, and IL-13 levels in the bronchoalveolar-lavage fluid (BALF) and serum were significantly decreased in OVA-challenged DUSP23 cKO mice. Interestingly, DUSP23 overexpression induced GATA3 protein levels, whereas GATA3 protein levels were reduced in T-DUSP23 cKO T cells. Furthermore, DUSP23 prevented FBXW7-induced K48-linked ubiquitination and proteasomal degradation of GATA3 protein, leading to induction of GATA3 protein levels and subsequent Th2 cytokine production. Consistently, DUSP23 and GATA3 protein levels were increased in T cells from patients with Th2-biased asthma and atopic dermatitis (AD). Data mining of whole exome sequencing (WES) datasets derived from human AD patients showed that multiple DUSP23 gene variants occurred in AD patients. Conclusion Taken together, DUSP23 induces Th2-mediated inflammation by preventing FBXW7-mediated GATA3 degradation, contributing to Th2-mediated allergic diseases. Funding Source National Health Research Institutes, Taiwan Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

A Complete Multiomic Solution: WTA NEXT Powers Deep Immune Profiling Across mRNA, Surface Proteins, and VDJ in One Assay 2259618

Abstract Introduction Single-cell profiling has evolved to simultaneously capture mRNA, surface proteins, and immune receptor repertoires. However, challenges such as low-abundance transcript detection, sequencing cost, and background noise from unbound antibodies continue to limit assay performance. Methods We present an updated plate-based workflow featuring WTA NEXT, a high-sensitivity whole transcriptome analysis (WTA) assay that significantly improves gene and molecule recovery per cell. Benchmarking against the previous WTA version demonstrated a 27% increase in median genes per cell and a 30% gain in median molecules per cell. These improvements enhance cell classification resolution, rare transcript detection, and overall assay efficiency. Results Combined with modular lyophilized antibody panels–available for T, B, NK, tumor, and antigen-presenting cells –WTA NEXT supports flexible multiomic profiling. Researchers can tailor experiments using different panel combinations, and the format allows drop-ins without affecting performance when additional markers are needed. Conclusion The plate-based format enables post-capture washes to reduce ambient antibody background, while signal-dampening strategies improve detection of low-abundance proteins. The updated full-length TCR/BCR (VDJ) assay offers increased sensitivity for low abundance clonotypes, enabling robust repertoire profiling. When integrated with transcript and protein data, this assay reveals deeper insight into clonal expansion, cell state, and functional diversity. Together, these innovations deliver a sensitive, modular, and cost-efficient workflow for high-content single-cell immune profiling. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Enhancing Flow Cytometry Multiplexing Panels with New StarBright™ Spectral Dyes 2259336

Abstract Introduction Full spectrum cytometry is increasingly preferred over conventional cytometry for its ability to generate data from larger, more complex panels. However, progress is hampered by the limited availability of suitable dyes. Many existing fluorophores are dim, have poor stability, lack a unique emission wavelength, or have high spectral overlap, limiting panel optimization and affecting data quality. Results Bio-Rad’s StarBright Dyes for flow cytometry have expanded to 36 fluorophores with the addition of four new Spectral StarBright Dyes, purpose-built for spectral cytometry, where no fluorophores with similar peak emissions exist. They address key challenges by providing bright, narrowly defined emission spectra, allowing larger panels to be acquired. Using data obtained from a Cytek Aurora (Cytek Biosciences), we show that these new dyes also enhance existing panels by replacing suboptimal traditional fluorophores, minimizing spectral overlap and spreading without compromising data quality Conclusion New Spectral StarBright Dyes provide the same benefits as current StarBright Dyes and are compatible with the existing dyes and other fluorophores, ensuring seamless integration into established workflows. The wide choice available simplifies panel design and offers alternatives for conventional dyes, offering improved performance while maintaining compatibility with standard protocols. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Discovery and engineering of novel IgM proteases for the treatment of autoimmune and inflammatory diseases 2253144

Abstract Introduction IgM is a multifunctional immunoglobin that acts as the first line of defense against systemic pathogens. IgM’s multimeric structure, together with its complement binding ability, makes IgM a highly potent inflammation driver. The presence of autoreactive IgM antibodies causing complement dependent damage has been described in inflammatory and autoimmune conditions such as Cold Agglutinin Disease (CAD) and Multifocal Motor Neuropathy (MMN) . For this reason, the development of IgM autoantibodies degrading therapy represents an attractive approach. Methods Employing Seismic’s IMPACT platform and evolutionary screening, we identified several novel and selective IgM proteases. Discovered proteases cleave soluble IgM in plasma and the IgM BCR in B cells from healthy individuals. Results CAD is an autoimmune hemolytic anemia, caused by IgM autoantibodies that bind to red blood cells (RBC), causing agglutination and activate complement cascade resulting in RBC death. Seismic’s IgM protease cleaves RBC bound IgM, preventing agglutination in whole blood from CAD donors, an unmet need not targeted by current approved treatment. In addition, IgM cleavage reduced cold-induced complement deposition on RBC from CAD patients. Conclusion Further engineering and optimization with our IMPACT Platform for the novel proteases is currently under way. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Gram‐to‐Ton Synthesis of Single‐Atom Materials via Low‐Hydroxyl‐Coverage Surface Collision

ABSTRACT Single‐atom materials (SAMs) possess promising properties for chemical engineering, energy, environmental protection, and ecological remediation applications, yet their practical application remains constrained by the scarcity of scalable and sustainable synthesis methods. Here, we report a low‐hydroxyl‐coverage surface collision (LSC) strategy to prepare universal single‐atom materials (Fe, Co, Ni, Mn, Mg, Al) on (hydr)oxide supports from gram to ton scales. Amino acids are utilized to precisely control surface hydroxyl coverage, resulting in activating metal sites for cation exchange while preventing metal aggregation. The self‐designed production facility achieves a daily capacity of 1 ton (annual capacity > 300 tons) and reduces carbon emissions by 67.8‐72.0% compared to conventional synthesis methods, which breaks the traditional limitations of mass/heat transfer and solvent use. The representative single‐atom material with asymmetric Fe 1 –O–La sites facilitates ligand exchange between hydroxyl and phosphorus by tailoring the coordination structure of hydroxyl. Impressively, the composite of Fe 1 –OLa with bentonite achieves the phosphorus removal efficiencies of 98.7% in the eutrophic lake water (Xingyun Lake, China), controlling the concentration below the eutrophication threshold (0.02 mg L − 1 ). Our work establishes a general and industrial platform for manufacturing high‐performance single‐atom materials, bridging a critical gap between laboratory innovation and large‐scale engineering applications.

Myeloid-specific interferon regulatory factor 5 promotes bone formation via orchestration of osteoclast lineage-osteoblast coupling

A customisable environmental sensor proof-of-concept to enable prescription medicine reuse

Abstract The main barriers preventing medicine reuse are concerns of costs and safety risks. To address this, we demonstrate a proof-of-concept Smart Packaging and Returns Assessment System (SPaRAS): A customisable smart sensor that validates the storage conditions of medicines while in patient care and streamlines the examination of returned medicines using Near Field Communication (NFC) technology. Here we evaluated the lifespan of the system over various sample rates; calculating the power consumption, memory usage, and the time taken to download data on return. When running at the same sample-rate to sensors already used in the UK National Health Service (NHS), SPaRAS can collect data for 17 days. This is long enough to be used within a larger trial for prescription medicine reuse. If a custom circuit board was designed to reduce power consumption, the device could run for 337 days with its current memory capacity. With further in-situ testing, this would be able to verify the conditions of most medicines.

TcBuster-M™ Enables Efficient Non-Viral Delivery of Multicistronic CD19-CAR Constructs in T and NK Cells 2259709

Abstract Introduction Viral vectors are widely used for immune cell engineering but pose challenges including limited cargo capacity and high production costs. TcBuster-M™, a transposase-based editing platform, offers a non-viral cell engineering alternative with broader cargo capacity and commercial availability. Methods Peripheral blood-derived T and NK cells were edited via electroporation with the TcBuster-M transposase and a multicistronic CD19-CAR transposon. Cells were expanded and assessed for cell growth and viability in addition to CAR expression and cell phenotype by flow cytometry. Genomic integration of the CD19-CAR was assessed by dPCR. Cytotoxicity was evaluated using a luciferase-based CD19+ target cell assay and cytokine secretion profile by Simple Plex (Ella). Results TcBuster-M-mediated cell engineering achieved high CD19-CAR expression in both T and NK cells while preserving cell viability and growth. Edited cells demonstrated potent, target-specific cytotoxicity and favorable cytokine secretion. Genomic analysis revealed stable integration with copy number variations below eight. Conclusion The TcBuster transposon system supports rapid, cost-effective cell manufacturing and enables delivery of complex therapeutic cargos, positioning it as a robust alternative to virus-mediated editing systems for immunotherapy development. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Loss of LDH-A accelerates CD8+ T memory cell differentiation 2252161

Abstract Introduction Lactate Dehydrogenase A (LDH-A) catalyzes the final step of glycolysis and is critical for hematopoietic stem cell survival and pluripotency. While activated T cells depend on glycolysis for effector functions, the role of LDH-A in CD8+ T cell differentiation remains unclear. Methods We investigated this using conditional LDH-A deletion in OT-I TCR transgenic mouse T cells. Results Following Listeria monocytogenes—OVA infection, OTI/LDH-A-/- CD8+ T cells showed reduced expansion at day 8 but higher frequency of central memory precursors. Transcriptomic analysis revealed enrichment of Bcl6 and Tcf7, markers of TMEM identity. Upon rechallenge at day 60, OTI/LDH-A-/- TMEM cells mediated superior pathogen clearance capacity compared to control OTI TMEM, leading to significantly reduced CFUs. To test anti-tumor immunity, we transferred OTI or OTI/LDH-A-/- cells into syngeneic hosts bearing B16-F10-OVA melanoma. Surprisingly, hosts receiving OTI/LDH-A-/- cells developed larger tumors with fewer CD8+ T cell infiltrates, which exhibited features of exhaustion, including elevated TIM-3 and reduced SLAMF6 and CD62L expression. Conclusion Our findings reveal that LDH-A deficiency skews CD8+ T cells toward a memory-like program, enhancing recall responses to infection but impairing effector differentiation and tumor control. Thus, LDH-A emerges as a key regulator balancing memory and effector differentiation and function across distinct immune contexts. Funding Source R01CA238263, R01CA257672 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

IL-15—Driven NK-like Activation Is Augmented in Senescent CD57+CD8+ Memory T Cells 2255278

Abstract Introduction IL-15 can stimulate CD8+ memory T (Tm) cells to exert NKG2D-mediated NK-like cytotoxicity in a TCR-independent manner, which can contribute to immunopathological host injury during viral infections. However, it has not been fully elucidated if senescent status of CD8+ Tm cells determines the degree of IL-15—induced NK-like activation. Here, we examined IL-15 responsiveness of replicative-senescent CD57+CD8+ Tm cells compared to CD57-CD8+ Tm cells and investigated the underlying molecular mechanism. Methods We obtained PBMCs from healthy donors (20-74 years) and analyzed CD57+CD8+ and CD57-CD8+ Tm cells by flow cytometry and bulk RNA-seq following ex vivo IL-15 stimulation. In addition, we performed single nucleus-multiome analysis (snRNA/ATAC-seq) of CD57+CD8+ and CD57-CD8+ Tm cells. We further analyzed single-cell transcriptomes of bystander CD8+ T cells identified with dCODE MHC-I multimers from PBMCs of patients with acute HAV infection. Results We demonstrated that senescent CD57+CD8+ Tm cells exhibit augmented IL-15—induced NK-like activation compared to non-senescent CD57-CD8+ Tm cells. The degree of IL-15—induced NK-like cytotoxicity was correlated with age and the proportion of CD57+CD8+ Tm cells. Furthermore, CD57+CD8+ Tm cells exhibited stronger NK-like activation than CD57- counterparts in patients with acute HAV infection, where the NK-like bystander activation of memory CD8+ T cells is well-exemplified. Notably, CD57+CD8+ Tm cells exhibited upregulation of Aiolos, known as a key regulator of AP-1 that is critical for IL-15—induced NK-like activation of CD8+ Tm cells. Conclusion Our findings reveal a mechanism by which augmented IL-15—induced NK-like activation of senescent CD57+CD8+ Tm cells is associated with enhanced bystander activation of CD8+ T cells in the elderly, providing insights for novel molecular targets for the treatment of aging-associated immunopathology. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Unraveling genomic regulatory complexity by adding chromatin accessibility to the long-read sequencing toolkit 2247245

Abstract Introduction Chromatin structure regulates gene expression, shaping immune cell responses and differentiation. Profiling accessibility, transcription factor binding, and DNA methylation is key to understanding immune regulation. Short-read sequencing (SRS) methods like ATAC-seq and ChIP-seq provide valuable insights but lack resolution and cannot capture multiple chromatin features together, limiting our ability to define their coordinated roles in immune function. Methods We optimized Fiber-seq, a multiomic long-read sequencing (LRS) method to simultaneously profile chromatin accessibility and endogenous methylation using LRS. Fiber-seq uses a non-specific DNA methyltransferase to label accessible DNA by creating N6-methyladenosine, a mark rarely found in eukaryotic genomes. This rapid enzymatic incubation also preserves endogenous DNA methylation so that both methylation marks can be detected by direct LRS. Results We validated this new method using human lymphoblast cells (K562). Aggregate Fiber-seq accessibility profiles were highly concordant with published ATAC-seq datasets. We confirmed that 6mA-labeling preserved sequencing accuracy and detection of CpG methylation. We also found nucleosome and protein footprints could be inferred from Fiber-seq data. We have validated multiple transcription factor inferred footprints are highly concordant with published CUT&RUN or ChIP-seq datasets. Further, these footprints are captured at a per-molecule and near-base pair resolution, uncovering multiple binding events in close proximity, which is usually obscured by lower resolution SRS-based methods. Conclusion Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variation on single molecules at near—base-pair resolution. This integrated view reveals how genetic and epigenetic features interact to regulate gene expression and provides a powerful new framework for dissecting immune cell function and disease mechanisms. Funding Source NIH R44 GM148145 Topic Categories Technological Innovations in Immunology (TECH)

Microbially Induced CD8+ Tissue-Resident Memory T Cells Rewire Microglial Function and Suppress Neurogenesis in the Brain 2255642

Abstract Introduction T cells are increasingly recognized as long-term residents of the adult brain, displaying regional diversity and accumulating with age; however, functional consequences of T cell residency on brain microenvironment are not well-understood, especially in non-aged populations. Conventionally used young adult Specific Pathogen Free (SPF) mice exhibit low numbers of T cells in the brain compared to young adult humans, limiting translational relevance. In this work we utilize our Specific Pathogen Experienced (SPExp) mouse model, in which prior peripheral infections lead to significant enrichment of tissue-resident memory (Trm) T cells in the brain, generating a neuroimmune state more closely resembling humans. Using this model we recently demonstrated that the presence of T cells in the brain influences seizure outcomes in young mice. Methods Here we combined spectral flow cytometry, single-cell RNA sequencing, and intravital imaging to interrogate the impact of CD8 Trm on brain microenvironment. Results At memory timepoints post peripheral pathogen exposures we identified robust T cell-dependent transcriptional changes in microglia. Differential expression revealed distinct primed and senescence-associated microglial phenotypes enriched for cytokine signaling, antigen presentation and neuroimmune crosstalk; with reversal upon CD8 T cell depletion. Intravital imaging demonstrated that CD8 Trm are motile and form sustained contacts with microglia in vivo, corresponding to the changes in activation state. Additionally, KEGG/GO analyses suggest induced microglial states downregulate support for neuronal development. Consistently, SPExp mice exhibited CD8 Trm localization at neurogenic borders and decreased neural stem cell proliferation, which was restored after CD8 depletion. Conclusion Together, these findings identify CD8 Trm as key regulators of microglial function and neurogenesis, establishing a new paradigm for adaptive immune regulation of brain function at homeostasis. Funding Source Howard Hughes Medical Institute (HHMI) Hanna H. Gray Fellowship Topic Categories Neuroimmunology (NEUR)

A humanized mouse model to study immune complex processing and clearance 2259807

Abstract Introduction The complement system performs diverse functions spanning immune surveillance, pathogen defense, and developmental regulation. Upon antigen recognition, antigen—antibody immune complexes (ICs) activate complement and are opsonized with C3b. In humans, C3b-opsonized ICs bind to complement receptor 1 (CR1) on erythrocytes (E), a process termed immune adherence (IA), which directs IC trafficking to the liver and spleen for clearance. In contrast, in mice, ICs do not bind erythrocyte CR1 but associate with complement factor H (FH) on platelets (Plts), limiting the translational relevance of murine models for human IA biology. Another nuance increasing the complexity of these interactions is that aside from FH on platelets, in mice and humans, the liver generates FH and releases it into the plasma. Plasma FH deficiency causes diseases such as dense deposit disease. We hypothesized that expression of human CR1 on erythrocytes, combined with platelet-specific FH deletion while preserving plasma FH, would replicate the human IA pathway. Methods To achieve this goal, we generated FHfl/fl mice and crossed them with Pf4-Cre mice to selectively deplete FH in platelets, maintaining normal plasma FH. These were then bred with CR1hu mice to yield FH fl/fl Pf4-Cre CR1huTg offspring (CR1hu on erythrocytes, no FH on platelets, and intact plasma FH). Results Characterization confirmed platelet-specific FH deletion, preserved systemic complement regulation, and physiological CR1 expression on erythrocytes. Ongoing studies are assessing erythrocyte CR1hu as the IA receptor and tracking IC trafficking in vivo. Conclusion This novel humanized immune-adherence mouse model replicates the human CR1-dependent pathway for immune complex clearance, providing an essential tool to investigate complement-mediated mechanisms and develop targeted therapies for immune complex-driven diseases. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)