Browse Articles

Single-Cell Multi-Omics Reveals T Cell Dysregulation in Sarcoidosis 2310194

Abstract Introduction Sarcoidosis is a systemic inflammatory disease characterized by granulomatous inflammation that affects multiple organs. Prior studies suggest that infection may trigger an antigen-mediated T cell response. However, the underlying immunological mechanisms remain largely unclear. Understanding these mechanisms is needed to identify biomarkers and new treatment targets. Methods We established a systems immunology framework to integrate single-cell RNA sequencing and T cell receptor profiling to unravel the landscape of sarcoidosis. We analyzed immune cell composition, cell—cell communication, and signaling pathways in patients with sarcoidosis and healthy controls. We also used an independent public dataset to test changes in immune cell abundance. In addition, we performed pseudo bulk analysis to study signaling changes in regulatory T cells. Results We identified major immune alterations in sarcoidosis. γδT cells were enriched, while Treg cells were reduced, in patients compared to controls. Cell-cell communication analysis revealed increased signaling pathways through immune checkpoint and innate-like pathways, including HLA-E—CD94/NKG2A and ANXA1-FPR1. In contrast, signaling linked to leukocyte recruitment were reduced. Treg cells exhibited diminished outgoing signals in antigen presentation and immune regulation pathways. In an independent dataset, Treg abundance was significantly reduced in patients. Further pseudobulk analysis also showed a significant downregulation of the KLRB1/CD161 signaling, which strengthens our predicted changes in cell-cell communication. Conclusion This study defines a distinct immune dysregulation pattern in sarcoidosis marked by loss of regulatory T cell function and altered T cell communication. These findings provide new insight into disease mechanisms and point to specific immune pathways that may serve as biomarkers or therapeutic targets. Ongoing analysis of T cell receptor data will help identify disease-related antigens and clonal T cell features. Funding Source The authors declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by startup funds from the Immunology Center of Georgia (IMMCG), Medical College of Georgia, Augusta Universi Topic Categories Computational and Systems Immunology (COMP)

Negative charges in the surface of the SARS-CoV-2 spike protein promote autoantibody generation in hospitalized COVID-19 patients 2300244

Abstract Introduction Autoreactive antibodies are pathological in many infectious diseases and can lead to chronic autoimmune disease. Severe COVID-19 has been shown to elicit strong autoantibodies with broad reactivity towards both viral and self-antigens, and recent work has shown mRNA vaccinees develop similar autoantibodies but to a lesser extent. Determining the drivers of these antibodies is critical for understanding how to circumvent infection-driven autoimmunity. Methods To do this, we generated recombinant antibodies from plasmablasts and expanded B-cells of severe COVID-19 patients using single-cell RNA sequencing and tested their reactivities. Results We found a subset of cloned antibodies exhibited polyreactivity towards both viral and self-antigens. Notably, despite overlapping antigen specificities, analysis using a protein language model revealed no shared paratope convergence across these clones. Instead, structural analysis revealed that autoreactivity was highly correlated with cationic residues in the heavy chain (H)CDR3, and docking predictions showed charge-based dependency rather than epitope similarity as the most likely mode of interaction. Germline reversion, which reduced positive charge in HCDR3s, led to diminished viral binding and reduced affinity for nuclear antigens, implicating charge as a driver of reactivity. Conclusion These findings, together with independent evidence that the SARS-CoV-2 spike protein has evolved to lose negative surface charges over time, suggest a model in which electrostatic surface charge of the virus may favor the expansion of highly charged antiviral antibodies that are inadvertently autoreactive. Our study identifies charge-based interactions as a unifying mechanism connecting antiviral immunity to autoantibody emergence in both infection, and potentially vaccination, providing a conceptual framework for understanding and mitigating infection-related autoimmunity. Funding Source R01HL161127 Topic Categories Basic Autoimmunity (BA)

Mechanistic models coupled with sequencing data for the prediction of variant/cell type-specific IL-10 responses 2332592

Abstract Introduction IL-10 plays a central role in limiting inflammation by suppressing pro-inflammatory cytokine production, reducing antigen presentation, and supporting regulatory T cell survival, but is also known to produce immune stimulatory functions on immune cell types like CD8+ T cells. This pleiotropy could be the cause behind the limited success of IL-10-based therapies, as therapeutic effects depend strongly on cell type and dose. Methods In this work, we built mathematical models of the IL-10 pathway that capture the events from IL-10 dimerization to the binding to surface receptors until STAT1/STAT3 phosphorylation. Using these models, we want to give a mechanistic reasoning on how different, opposite responses can emerge in IL-10 signaling, centering ourselves in the IL-10 response at the levels of pSTAT phosphorylation and downstream transcriptional programs. Results Using Bayesian-based model selection, we provide a mechanistic explanation for disentangling pro- and anti-inflammatory differential responses in pSTAT1/pSTAT3 dose-response curves. On top of that, through correlations of cell-type-specific pSTAT model predictions with changes in gene expression, we identified a set of IL-10-responsive genes that reflect a cell-type-independent regulatory program. Repeating the same analysis with a curated list of IL-10 DEGs, we suggest IL-10 could be aggregating distinct transcriptional programs engaged at different signaling strengths, explaining dose-dependent IL-10 effects like CD8+ T cell proliferation at high IL-10 concentrations. Conclusion Our study highlights the potential of computational modeling as a powerful framework to refine our understanding of the IL-10 pathway and to begin disentangling the pleiotropic effects of IL-10 from pathway mechanisms to downstream transcriptional responses, with the future goal of supporting the rational development and clinical translation of more precise and effective IL-10-based therapies. Funding Source Spanish Ministry of Science, Innovation and Universities Topic Categories Computational and Systems Immunology (COMP)

Comparative analysis of baseline and bovine viral diarrhea virus-induced interferon stimulated gene expression in bovine turbinate and kidney cells 2305626

Abstract Introduction Bovine viral diarrhea virus (BVDV) is a significant economic concern for the American cattle industry. Transcriptome analyses of BVDV-infected animals have shown that the virus alters host gene expression, particularly within immune-related pathways. Differences in BVDV-mediated interferon stimulated gene (ISG) expression have not been studied in a controlled cell culture system. This study aims to investigate selected early ISG transcriptome profiles in two bovine cell lines infected with BVDV at different multiplicities of infection (MOI). Methods Madin-Darby bovine kidney epithelial cells (MDBK), and bovine turbinate primary neonatal-derived cells (BTu) were infected with a noncytopathic BVDV1a strain (PI28) at two different MOIs (0.5 and 50) and transcriptomic analysis was completed at 8-, 24-, and 72-hours post-infection. Thirty ISGs were analyzed to compare their baseline and BVDV-induced gene expression across the three timepoints. Results Of the 30 ISGs, 11 have higher baseline transcription in MDBKs: ISG20, IFITM3, RIG-I, USP18, CASP4, CASP8, TRIM21, TRIM14, LGALS3BP, RNASEL, and IFI6. Only 4 ISGs, SOCS3, ALCAM, ICAM1, and CD47 have higher baseline transcription levels in BTus than MDBKs. BVDV infection elicited prolonged 8—24-hour gene expression and increased transcription of RIG-I, Mx1, MDA5, CASP4, CASP8, LGALS3BP, TRIM21, and TRIM14 in MDBK cells in the 50 MOI condition and reduced transcription in the 0.5 MOI condition at 24 hours. In BTus, SOCS3, ALCAM, ICAM1, and CD47 demonstrated BVDV-induced increases with 50 MOI infection and decreases with 0.5 MOI infection at 24 hours. Conclusion BVDV modulates the host innate immune response, as illustrated by the modulation in ISG transcription in vitro. These effects are viral load-dependent, with the low titer infections (0.5 MOI) reducing ISG gene expression below cellular control levels, whereas high titer infections (50 MOI) increase ISG gene expression. Funding Source USDA/Agricultural Research Service, Project 5030-32000-229-000D Topic Categories Veterinary and Comparative Immunology (VET)

Exploring the role of immune-related microRNAs in newborn calves’ circulation 2259711

Abstract Introduction Bovine colostrum provides nutrients and bioactive factors including antibodies, microRNAs (miRNAs), among others that are essential for newborn calves. We aimed to assess immune miRNAs in bovine colostrum and to evaluate the role of circulatory miRNAs in neonatal calf health. Methods Beef-on-dairy calves were randomly assigned to a colostrum-fed (CF) or colostrum-deprived (CD) group after birth (n = 10/group). Calves in the CF were tube-fed pooled colostrum (10% BW/calf) ∼4 hours after birth, while calves in the CD were fed 2.8L of a commercial milk replacer (MR) as their first feeding. Whole blood was collected from all calves prior to and after their first feeding. Colostrum, MR, and blood samples were subjected to RNA extraction, small RNA sequencing using Illumina platform, and differential expressed miRNAs were analyzed using DESeq2 in R. Significance was declared at P-adj ≤ 0.05. Results MicroRNAs miR-22-3p and miR-2285 were upregulated in bovine colostrum and in blood of calves that were CF vs CD post-first feeding. In contrast, circulatory miR-6775 and miR-21-5p were downregulated post-feeding in CF relative to CD. miR-22-3p is involved in T cell differentiation while miR-2285 has been linked to milk fat metabolism in mammary epithelial cells. Conclusion Our findings highlight functional miRNAs that may modulate immune pathways in calves. However, the ability to intestinal absorption of microRNAs from colostrum remains unclear. Funding Source Agriculture and Food Research Initiative Competitive Grant no. 2023-67015-39081 from the United States Department of Agriculture (USDA) — National Institute of Food and Agriculture (NIFA) Topic Categories Veterinary and Comparative Immunology (VET)

Evaluating the impact of the CD47 on RBC morphology following sublethal irradiation 2261226

Abstract Introduction Acute radiation syndrome (ARS) results from exposure to ionizing radiation during medical treatment, nuclear accidents, & space travel. A common side effect of ARS is bone marrow damage & anemia. Healthy red blood cells (RBCs) highly express CD47. A previous study has shown progressive decrease in CD47 expression by RBCs following radiation exposure, suggesting RBC damage & death. Preliminary data from our lab shows Cd47-/- mice are highly susceptible to radiation. Clearance of damaged RBCs is typically mediated by SIRPa expressing phagocytes following decrease in CD47 expression by RBCs. Recent work has revealed RBCs can also undergo programmed inflammatory cell death known as spectosis. To this end, we evaluated RBC death & morphology, as well as anemia following radiation in the absence of CD47. Methods We non-lethally irradiated C57BL/6, Cd47-/-, & Cd47-/-Sirpa-/- (DKO) mice. Mice were bled retroorbitally at multiple time points during the recovery period. We evaluated RBC morphology via blood smears, total white blood cells, RBCs, and complete blood counts. Results We observed slight anemia in Cd47-/- compared to controls, and even more exaggerated anemia in DKO. We also observed more morphological abnormalities in the absence of CD47 than in controls. Conclusion This data suggests that CD47 is required for efficient recovery of the hematopoietic compartment following radiation. This is a potential mechanism mediating susceptibility of Cd47-/- to ionizing radiation. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)

Remote Modification‐Induced Butterfly Effect on Nonfullerene Acceptor Aggregation for Efficient Organic Solar Cells

ABSTRACT Organic solar cells (OSCs) possess unique advantages for next‐generation photovoltaic applications, yet their power conversion efficiencies (PCE) are still constrained by substantial voltage losses. Simultaneously achieving high luminescence and charge transport properties of nonfullerene acceptors (NFAs) remains a major challenge due to the complex, nonlinear relationship between molecular structure and solid‐state packing. In this study, we demonstrate a molecular design concept based on the “butterfly effect” that enables precise control over intermolecular packing in A–DA′D–A‐type NFAs through subtle substituent modifications on the remote phenyl ring. Systematic modulation with methoxy, methyl, hydrogen, and fluorine groups reveals distinct packing configurations: methoxy‐substituted AQxPO enhances photoluminescence quantum yield (PLQY to 9.96%) via depressed end–bridge (E–B) stacking but disrupts vital charge transport pathways, while fluorine‐substituted AQxPF promotes E–B/end–end (E–E) stacking, enhancing carrier transport at the cost of reduced PLQY (4.78%). Notably, the hydrogen‐substituted AQxPH optimally suppressing detrimental E–B stacking while maintaining efficient E–E interactions, yielding both high PLQY and superior charge transport. Consequently, the D18:AQxPH‐based binary OSC achieves a remarkable PCE of 20.9% with a high V OC of 0.925 V at an ultralow non‐radiative energy loss of 0.176 eV. This work provides a design principle to overcome OSC efficiency bottlenecks through precise stacking control.

Anode Compatibility of Halide Solid‐State Electrolytes

ABSTRACT Recently, lithium‐metal‐halide (Li‐M‐X) superionic conductors have emerged as promising solid‐state electrolyte (SSEs) for all‐solid‐state lithium batteries (ASSLBs), owing to their high Li + conductivity, excellent compatibility with cathodes, and good mechanical deformability. However, many of these conductors exhibit chemical and electrochemical instability when in contact with the reductive anodes, which hinders the direct use of lithium metal as anodes with extraordinarily high specific capacities and limits the overall energy density of halide‐based ASSLBs. Therefore, enhancing the anode compatibility of halide SSEs has become a critical task in ASSLB development and has attracted broad research interest. Herein, the underlying mechanisms responsible for the instability of halide SSEs against the lithium anode are elucidated based on both experimental observations and theoretical calculations. Recent strategies and progress aimed at improving the compatibility of halide SSEs with lithium anodes are summarized. Moreover, the effects of pressure and volume changes on the interfacial compatibility between halide SSE and lithium metal are discussed. In addition, the current challenges and future research directions are analyzed, aiming to provide theoretical insights and guidance to support further advancements in Li‐M‐X type solid‐state electrolytes and all‐solid‐state lithium batteries.

Green synthesis of CeO2 nanoparticles using strawberry and mulberry extracts: effect of calcination temperature on photocatalytic performance and cellular toxicity

Dual Role of IgA in Gut Integrity and Pro-Inflammatory Activation During Atherogenesis and Sleep Fragmentation 2261038

Abstract Introduction Atherosclerosis is a chronic disease involving immune dysregulation and the formation of dangerous plaque in vessels. IgA maintains gut homeostasis but may also contribute to inflammation in the circulation under pathological conditions. Sleep fragmentation (SF), common in modern life, exacerbates systemic inflammation and atherosclerosis. To date, the role of IgA in atherogenesis and in SF-accelerated atherosclerosis is unclear. Methods To test the role of SF on mucosal immunity in atherosclerosis, Apolipoprotein E-deficient (Apoe-/-) mice were fed a high-fat diet (HFD) and placed on an SF or activity control regimen for 12 weeks. We tested the role of IgA on macrophage phenotype and foam cell formation, in vitro. Finally, to test the role of IgA in atherogenesis, we generated atherosclerosis-prone IgA-deficient low-density lipoprotein receptor-deficient Iga-/-Ldlr-/- mice and placed them on HFD feeding for 12 weeks. Results SF promoted atherosclerosis in HFD-fed Apoe-/- mice. SF also elevated intestinal reactive oxygen species (ROS) and IFNγ, and significantly reduced IgA+ B cell frequency and tight junction protein expression in the gut of HFD-fed SF vs AC Apoe-/- mice.12 wks HFD-fed Iga-/-Ldlr-/- mice showed attenuated plaque burden and reduced leukocyte infiltration into the aorta vs HFD-fed Ldlr-/- controls. IgA binds to pathogenic oxidized LDL (oxLDL) and thus may potentially reduce foam cell formation. In vitro, mouse and human IgA, when co-incubated with oxLDL, reduced oxLDL uptake by macrophages but enhanced their activation and ROS, suggesting a pro-inflammatory role of IgA in the circulation.Thus, IgA plays a paradoxical role in SF-accelerated atherosclerosis, preserving gut barrier integrity while promoting pro-inflammatory myeloid activation. Conclusion These findings suggest IgA as a key immunomodulator at the interface of mucosal immunity and vascular inflammation. Funding Source American Heart Association Topic Categories Mucosal and Regional Immunology (MUC)

Complex intra-host SARS-CoV-2 evolution following monoclonal antibody pre-exposure prophylaxis 2309595

Abstract Introduction Monoclonal antibodies have emerged as a prophylactic strategy to prevent symptomatic SARS-CoV-2 infection in immunocompromised individuals. However, the evolutionary and clinical implications of breakthrough infections remain unclear. Methods An 80-year-old male with a haematological/oncological diagnosis received a 2000 mg intravenous infusion of sotrovimab in March 2023 and was diagnosed with COVID-19 by RT-qPCR from a nasopharyngeal swab in August 2023. Weekly samples (n = 24) were collected through February 2024 (171 days). All samples underwent whole-genome sequencing, with select mutations subjected to functional assessment. Results Sequencing identified the GE.1 lineage at all timepoints. An intra-host recombination event in ORF1ab (positions 8980-11203) was detected 100-107 days post-infection, followed by a 14-fold increase in viral load (7.42E+06 to 1.00E+08 RNA copies/mL) and a marked shift in the mutation landscape. E340D, a sotrovimab-resistance mutation, fluctuated over time and was selected 107 days post-infection. We assessed five spike mutations — V36M, S98F, and V213G in the N-terminal domain, Y505P in the receptor-binding domain, and P681Q near the S1/S2 cleavage site–and additionally evaluated the impact of E340D. V36M conferred the highest infectivity across all cell lines, with the most significant effect in low-TMPRSS2 cells. While all mutations showed enhanced infectivity with the addition of E340D, the effect was most pronounced in mutations with lower baseline infectivity. The addition of E340D significantly decreased relative neutralizing titres for V36M, S98F, and V213G, enabling escape from neutralizing antibodies in XBB-responsive individuals. Patient neutralizing activity was absent pre-sotrovimab, and sotrovimab-induced neutralization was further compromised by selection of E340D. Conclusion Fixation of E340D abolished the surrogate defence provided by sotrovimab, eliminating neutralizing activity and exerting a positive epistatic effect on emerging mutations. Funding Source GSK; Gates-Cambridge scholarship Topic Categories Vaccines and Immunotherapy (VAC)

Centrosome-associated assembly and trafficking coordinate spatial activation of the NLRP3 inflammasome in primary human monocytes 2310149

Abstract Introduction The spatial organization of NLRP3 inflammasome activation in human monocytes is poorly understood. By examining Toxoplasma gondii infection of primary human monocytes, we identified centrosome-linked and PIKfyve-dependent endosomal pathways that coordinate NLRP3 inflammasome assembly. Methods Inflammasome assembly and activation were analyzed in T. gondii-infected human monocytes using a combination of super-resolution and live-cell imaging along with biochemical and immunological assays. Pharmacological inhibitor experiments and inducible gene knock-out systems were used to gain mechanistic insights into pathways of inflammasome assembly during infection. Results Super-resolution imaging revealed that infection drives NLRP3 redistribution from diffuse cytosolic puncta to discrete pericentrosomal clusters coinciding with active caspase-1 in primary human monocytes. Dynamic live-cell imaging of NLRP3—mNeonGreen-expressing cells during infection captured directed movement of NLRP3 puncta toward the γ-tubulin-defined microtubule-organizing center and coalescence into a radial, spoke-like structure, indicating the centrosome as a principal scaffold for inflammasome organization. This configuration required the centrosomal kinase NEK7, and pharmacological disruption of the CPAP-β-tubulin scaffold abolished ASC assembly and downstream inflammasome activation under both T. gondii infection and canonical LPS + ATP stimulation. Consistent with the vesicle-like dynamics of NLRP3 assembly, inhibition or degradation of PIKfyve, a key regulator of early endosomal homeostasis, caused aberrant retention of NLRP3 within EEA1+ early endosomes and markedly reduced inflammasome activation and IL-1β cleavage while leaving NF-κB-dependent priming intact. Conclusion Collectively, these results delineate a coupled centrosomal-endosomal system that spatially governs NLRP3 inflammasome activation in primary human monocytes and reveals assembly checkpoints that determine inflammasome competence. Funding Source NIH Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Aryl Hydrocarbon Receptor Inhibition Activates Mito-calcium/mtROS/IL-12 Axis in Dendritic Cells to Promote Tumor Suppression 2299915

Abstract Introduction IL-12 generated by dendritic cells (DCs) plays a pivotal role in activating an effective anti-tumor response. Specific genetic manipulation of DCs to enhance tumor infiltration and localized IL-12 secretion is an attractive therapeutic strategy for cancer immunotherapy. Methods A genome-wide CRISPR/Cas9 knockout screening identified negative regulators of IL-12 production in bone marrow-derived dendritic cells (BMDCs). Adoptive transfer studies established the role of the Aryl hydrocarbon receptor (AhR) in tumor growth. The immune cell neutralization and two-photon live imaging were employed to determine the effector cells involved in AhR inhibition-induced tumor suppression, and the molecular pathways were explored using bulk RNA sequencing, TEM, and flow cytometry. AhR inhibition in human DCs validated their role in activating tumor-killing effector cells. Results CRISPR/Cas9 screening in BMDCs identified aryl hydrocarbon receptor (AhR) as an important negative regulator of IL-12 production. DC-specific knockout of AhR suppresses tumor growth by promoting the infiltration of IL-12+ DCs and CD107a+ NK1.1+ cells. Antibody blockade studies in MC38-implanted AhRΔDC mice reveal NK cells as the predominant effector cells mediating tumor suppression. AhR inhibition promotes NK cell migration and interaction with TIDC within the TME. AhR-deficient TIDCs enhanced the mtROS production while preserving structure and membrane potential. AhR-deficient DCs sensitized TLR4 signaling and enriched mitochondria-ER contacts (MERCs), which leads to IP3R/VDAC1/MCU-mediated mitochondrial calcium influx, mtROS, and IL-12 production. AhR inhibition in human DCs enhanced tumor-killing function in NK cells in an mtROS/IL-12-dependent manner. Conclusion AhR ablation in DCs releases the inhibitory brake on the IP3R-gated mito-calcium/mtROS/IL-12 axis. This facilitates DC-NK infiltration and crosstalk within the TME. AhR and MERCs could be potential therapeutic targets in DCs for future cancer immunotherapy. Funding Source n/a Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)

Cytokine training primes NK cells for enhanced antiviral activity against JC polyomavirus 2259891

Abstract Introduction JC polyomavirus (JCPyV) is a neurotropic DNA virus that causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease in immunocompromised individuals. There is currently no standard treatment option, highlighting the need for new immune-based approaches. Natural killer (NK) cells contribute to antiviral defense, and our lab previously showed NK-mediated control of JCPyV infection. Cytokine-induced memory-like (CIML) NK cells generated with IL-12, IL-15, and IL-18 display enhanced effector function in cancer settings, but their role in JCPyV infection is undefined. Methods NK cells from healthy donor PBMCs were stimulated for 16 h with IL-12, IL-15, and IL-18, followed by a 6—7-day rest. Fresh and IL-15—only NKs served as controls. CIML NKs were either unstimulated or restimulated with the same cytokine cocktail during co-culture with JCPyV-Turbo—infected SVGA cells. Viral control was measured by VP1 and T-antigen expression, and NK function by IFN-γ production and CD107a degranulation via flow cytometry. Exhaustion markers (PD-1, TIGIT, TIM-3) were also assessed. Results CIML NK cells showed enhanced IFN-γ and CD107a expression compared to fresh and IL-15—treated NKs, with modestly improved control of JCPyV infection. Restimulated CIML NKs exhibited the strongest IFN-γ and CD107a responses, indicating that these cells are primed for rapid recall and heightened activation upon secondary stimulation. CIML NKs also upregulated PD-1, TIGIT, and TIM-3, consistent with activation-linked exhaustion. Conclusion CIML NK cells enhance antiviral responses against JCPyV, and restimulation further amplifies their effector capacity, supporting a memory-like phenotype. These findings highlight the potential of trained NK cells as immunotherapy for persistent viral infections such as PML. Ongoing work uses transcript-level flow cytometry to determine whether checkpoint receptor upregulation reflects transient activation or functional exhaustion. Funding Source R01NS116278 Topic Categories Viral Immunology (VIR)

Wild-Derived Microbiota Protect Against Diet-Induced Obesity via an Immune-Mediated Effect on Brown Adipocytes 2305928

Abstract Introduction The prevalence of obesity and metabolic diseases increased in industrialized nations in parallel to reduced diversity of gut microbiota. We recently demonstrated that the reconstitution of conventional laboratory mice with complex microbiota from wild mice protects against diet-induced obesity. Methods To determine the mechanism for this protection, we studied energy expenditure, adipocyte transcriptome and immune response of a colony mice with complex natural microbiota (Wildlings) that was generated by rederiving C57BL/6 embryos in wild mice. Results Wildlings were protected against diet-induced obesity throughout life and irrespective of sex, diet, genetics. Wildlings had increased energy expenditure, which decreased significantly in thermoneutral conditions but remained higher than that of conventional laboratory mice on the same genetic background. Single-nuclei RNA sequencing revealed that the wild-derived microbiota induced changes in the brown adipocyte transcriptome, that indicated increased thermogenic activity. While conventional laboratory mice acquired the protective phenotype only when cohoused with Wildlings in the first two weeks of life, germ-free mice acquired it even after week-10 of age. This suggests that the timing of exposure to the natural microbiota relative to the induction of immune responses is relevant. Increased type 2 responses in brown adipose tissue did not contribute to the obesity protection phenotype, because IL-33 KO and STAT6 KO Wildlings were protected from diet-induced obesity. Rather, recruitment of bone marrow-derived monocytes was essential because CCR2 KO Wildlings showed reduced energy expenditure and no protection from diet-induced obesity. Transfer of Wildling bone marrow cells into CCR2 KO Wildling pups reconstituted the phenotype. Conclusion Collectively, these results demonstrate that wild-derived microbiota protect against diet-induced obesity via immune-mediated induction of brown adipose tissue thermogenesis. Funding Source Intramural Research Program of NIDDK, NIH Topic Categories Cytokines and Chemokines and their Receptors (CCR)

B Cells Break the Structure Rule: Recognition of Intrinsically Disordered Epitopes in the SARS-CoV-2 Spike Model 2259769

Abstract Introduction Antibody recognition has long been defined by order–a stable, well-folded epitope that fits its antibody like a key in a lock. Yet many viral proteins contain intrinsically disordered regions (IDRs): dynamic, shape-shifting segments that defy this rule. Whether antibodies can recognize such flexible regions–and by what mechanism–remains largely untested. The SARS-CoV-2 Spike furin-cleavage loop (PRRAR↓S) is one such IDR, essential for viral activation yet conformationally fluid. We asked whether murine B cells can mount specific antibody responses to structural disorder itself. Methods A panel of eight peptide probes was designed–five structured Spike epitopes and three disordered furin-loop peptides preserving the PRRAR motif. Using the 10x Genomics BEAM-Ab platform, we profiled splenic B cells from mice immunized with full-length Omicron BA.1 Spike and recovered over one hundred paired heavy- and light-chain sequences binding the barcoded probes. Antibody—peptide interactions were modeled with IgFold, docked using ClusPro, and evaluated by ΔG analyses; molecular dynamics assessed complex stability. Results BEAM-Ab identified productive clonotypes recognizing both structured and disordered Spike epitopes at comparable frequencies (∼46% vs 54%). Several antibodies bound directly to the PRRAR motif–a region previously considered too mobile for precise recognition. Docking and ΔG analyses supported formation of energetically favorable antibody—peptide complexes localized to the furin-loop region, with predicted affinities comparable to structured epitopes. Structural modeling further indicated that antibody engagement can stabilize the disordered loop through induced ordering upon binding to paratopes of matched flexibility Conclusion These findings show that B cells can generate specific antibodies against intrinsically disordered viral epitopes, redefining the structural limits of immune recognition and highlighting flexibility as an antigenic feature exploitable for antibody and vaccine design. Funding Source NIAID/NIH Topic Categories Computational and Systems Immunology (COMP)

Methionine metabolism shapes epigenetic and functional programs of developing CD8+ T cells 2259369

Abstract Introduction Host defense evolves across the lifespan, with newborns and infants exhibiting distinct susceptibilities to infection compared to older children and adults. Neonatal CD8+ T cells exhibit distinct transcriptional and functional states, favoring effector differentiation over memory formation. These developmental differences arise from epigenetic regulation of chromatin accessibility and transcription factor networks governing effector, memory, and exhaustion programs. Our preliminary data identify methionine metabolism as a key regulator of these programs. Methionine-derived S-adenosylmethionine (SAM) fuels histone methylation and other transmethylation reactions; increased methionine utilization in neonatal cells may support these epigenetic circuits, whereas its restriction impairs activation, proliferation, and effector differentiation. Methods To dissect developmental regulation of T cell function, we integrated RNA sequencing, CUT&RUN profiling, and cellular immunology of activated cells to define age-specific transcriptional and chromatin landscapes. Targeted metabolomics quantified nutrient utilization, and CRISPR-Cas9 knockouts identified transcriptional regulators driving developmental divergence. Results Methionine emerged as one of the most differentially regulated pathways, with neonatal cells expressing higher levels of histone methyltransferases and demethylases. They also deposit activating H3K4 methylation marks at Ikzf2 (Helios) and Eomes loci. Helios expression was higher in neonatal cells and was rescued by SAM under methionine restriction. Functional assays demonstrated neonatal dependence on methionine for survival, proliferation and memory formation. Isotope tracing further showed faster methionine turnover in neonatal cells. Conclusion Together, these findings suggest a methionine-dependent epigenetic regulation as a mechanism driving early-life T cell responses and propose metabolic interventions to enhance immunity and optimize T cell—based therapies in infancy. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)

Hydrogel‐Woven COF Membranes With Orthogonal, Opposite Thermoresponsive Nanochannels: Overcoming the Permeance‐Selectivity Trade‐off

ABSTRACT Smart membranes, characterized by unique stimuli‐responsive channels to dynamically alter their chemistry and structure for orchestrating mass transport, are highly promising to gain extraordinary sieving performance over conventional membranes. However, smart membranes are locked by formidable trade‐off between permeance and selectivity during stimuli‐responsive process, owing to their synchronous, homogeneous stimuli‐responsive changes in channels. Here, we discover a new kind of smart membranes, hydrogel‐woven covalent organic framework (HW‐COF) membranes with orthogonal, opposite thermoresponsive nanochannels, overcoming the permeance‐selectivity trade‐off for achieving extraordinary aqueous separation. The key of HW‐COF membranes lies in the photothermal‐driven in‐situ confined weaving of poly( N ‐isopropylacrylamide) hydrogel networks within orthogonal in‐plane pores and interlayer channels of two‐dimensional COF membranes for yielding mechanical interlocking architecture. Distinct from the non‐interlocked counterpart, HW‐COF membranes showcase a positive response in in‐plane pores yet a negative response in interlayer channels. This unique thermoresponsive behavior can be tailored by the synergy of in‐plane pores and interlayer channels, allowing HW‐COF membranes to obtain markedly positive response in both permeance and selectivity, almost not achievable with reported smart membranes. As a concept of demonstration, HW‐COF membranes can be applied for sieving water isotopologue with ultrahigh selectivity (90.9%) and permeance (16.6 L m −2  h −1 bar −1 ) using low‐grade heat as stimuli.

Dual-network synergy for inventory forecasting: integrating adaptive feature dynamics and recursive perception correction

Leveraging modular TriKE-PACC molecules capable of dual-antigen targeting to enhance NK-cell immunotherapy in AML 2257266

Abstract Introduction Natural Killer (NK) cells are potent effectors of the innate immune system that kill tumor cells via natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). In acute myeloid leukemia (AML), tumor heterogeneity and antigen escape challenge current therapies, resulting in a 5-year survival rate of < 40%. To overcome these barriers and build on our ongoing clinical trial of a CD33-targeted Tri-Specific Killer Engager (TriKE), we engineered TriKEs with Poly Antigen Cytokine Complexes (TriKE-PACCs), which combine dual AML-specific targeting with NK activation and persistence. Methods TriKE and TriKE-PACC constructs were expressed in Expi293 cells and purified by affinity chromatography. TriKE-PACCs consist of an anti-CD16 sdAb, an IL-15 moiety, and an anti-AML scFv or sdAb with a PACC arm containing IL-15Rα linked to an additional anti-AML scFv or sdAb. AML antigens were evaluated on cell lines and 20 primary samples by flow cytometry. Antigen escape was modeled using CRISPR knockout AML lines or primary patient samples. NK activation (CD107a, IFNγ, TNFα) and cytotoxicity were measured using flow cytometry and Incucyte assays. Results Our examination of AML patient samples revealed diverse antigen profiles, highlighting the need for modular TriKE-PACC designs. The TriKE-PACCs enhanced CD107a, IFNγ, and TNFα production and exhibited improved cytotoxicity compared to single-antigen TriKEs, demonstrating resistance to antigen escape. Conclusion TriKE-PACCs offer a customizable, modular approach to treating AML heterogeneity and antigen escape with NK cell-based immunotherapies. By combining IL-15 and CD16 signaling with dual tumor-antigen targeting, we enhance NK cell proliferation and antitumor activity compared to TriKEs or IL-15 therapies alone. These data support further preclinical development and testing of modular and personalized AML immunotherapies to improve upon our current CD33 TriKE clinical trial. Funding Source NIH/NCATS: 1T32TR004376 and 1UM1TR004405 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)