Browse Articles

Deoxyguanosine Kinase Deficiency Couples Purine Metabolism to Innate Immune Activation and Lipid Accumulation in Hepatocytes 2309176

Abstract Introduction Mitochondrial DNA depletion syndromes (MDS) are fatal pediatric disorders with few treatment options. Deoxyguanosine kinase (DGUOK), a mitochondrial nucleoside salvage enzyme, is classically linked to impaired mtDNA replication. However, patients with DGUOK deficiency can develop hepatic inflammation and steatosis despite intact mtDNA, suggesting additional pathogenic mechanisms. As DGUOK regulates intracellular deoxyadenosine (dAdo) levels, we hypothesized that DGUOK deficiency initiates a purine-driven immunometabolic program contributing to liver pathology. Methods DGUOK was acutely depleted in human hepatocytes using siRNA to model early disease states prior to mtDNA loss. Innate immune activation and metabolic remodeling were assessed by RNA sequencing, hERV analysis, and RT-qPCR. Lipid accumulation was quantified by BODIPY and Oil Red O staining, flow cytometry, and electron microscopy. Global DNA methylation was measured by 5-mC ELISA, and purine imbalance was modeled by exogenous dAdo supplementation. Results Early DGUOK depletion preserved mtDNA copy number but induced robust type I interferon signaling, ISG expression, and hERV derepression. Transcriptomic analysis revealed suppression of lipid metabolic pathways alongside enrichment of purine and methylation-associated networks. DGUOK-deficient hepatocytes accumulated neutral lipid droplets prior to mitochondrial dysfunction, a phenotype recapitulated by dAdo treatment in wild-type cells and associated with global DNA hypomethylation. These immunometabolic signatures parallel interferon activation and steatosis in DGUOK deficient mice. Conclusion These data support a model in which DGUOK deficiency first triggers a purine-epigenetic axis that drives innate immune activation and lipid dysregulation, preceding mtDNA depletion. This framework links early hepatocyte dysfunction to inflammatory liver pathology in DGUOK-deficient mice, identifying purine metabolism and downstream interferon signaling as tractable preclinical targets in MDS. Funding Source This work was supported by philanthropic and institutional funding from the Kyowa Kirin Interactive Fund (award no. 18040-01-403), the National Institutes of Health grant R01 CA256133 (award no. 20071-04-403), and the University of California San Diego gr Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Tyrosine Phosphorylation of Themis Induced by TCR and Cytokine Signaling Is Regulated by Multiple Src Family and Janus Family Kinases 2253039

Abstract Introduction TCR and cytokine signaling play critical roles in thymocyte development and peripheral T cell homeostasis. Themis is a key regulator of signaling pathways in both developing thymocytes and mature T cells. Mice deficient in Themis fail to produce mature thymocytes due to defective positive selection. Previous studies have shown that tyrosine phosphorylation of Themis at Y34, mediated by the kinase Lck and regulating interaction with the phosphatase Shp1, is essential for its role in modulating TCR signaling during CD4 SP thymocyte development. However, the mechanism by which Themis regulates cytokine signaling, particularly in CD8 SP thymocytes and peripheral CD8+ T cells, remain poorly understood. Methods To identify kinases that regulate Themis phosphorylation, we cloned potential kinases expressed in T cells and co-expressed them with Themis in 293T. To determine which tyrosine residues are targeted by these kinases, we individually mutated all 18 tyrosine sites of Themis (Y-F) and assessed the impact of each mutation on kinase-dependent phosphorylation. Furthermore, to examine the intracellular localization and trafficking of Themis following TCR and/or cytokine stimulation, we generated a series of Themis—EGFP fusion proteins. Results In addition to Lck, Themis was phosphorylated by several other Src family kinases and members of the Janus kinase family. We have successfully generated a set of Themis Y-to-F mutants and are currently mapping which tyrosine residues are regulated by these kinases. Moreover, we constructed seven Themis—EGFP fusion variants, inserting EGFP at different positions within Themis. Most of these fusion proteins retained functional activity, as evidenced by their constitutive binding to Grb2, which is a characteristic feature of Themis. Conclusion Themis is regulated by multiple kinases, including members of the Src and Janus kinase families, suggesting that Themis integrates signals from both TCR and cytokine pathways to coordinate T cell development and homeostasis. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Epithelial antigen compartmentalization dictates CD4+ T cell fate during C. rodentium infection 2267571

Abstract Introduction While intestinal epithelial cells (IECs) upregulate MHCII during inflammation and can serve as non-professional antigen-presenting cells, their specific contribution to pathogen-specific CD4+ T cell immunity and immunological memory formation remains poorly defined. Methods We utilized Citrobacter rodentium infection (murine model of EPEC) with single-cell RNA-seq, engineered bacteria expressing model antigens in distinct compartments, and proximity-labeling approaches. Conditional IEC-specific MHCII deletion and inducible post-infection deletion strategies assessed functional requirements for CD4+ T cell responses. Results C. rodentium triggered sustained MHCII expression across all IEC lineages persisting >2 months post-clearance. IEC MHCII proved essential for pathogen-specific CD4+ TRM formation–conditional knockout shifted CD4+ T cell fate from TRM to TCM populations. scRNA-seq revealed MHCII deletion induced aberrant expression of central memory genes (Tcf7, Lef1, Bcl6) in residual TRM cells, while WT TRM showed robust TCR activation signatures (Fos, Jun, Jund) and Runx3 expression. Type III secretion-delivered cytosolic antigens drove TRM via autophagy-dependent IEC processing, whereas surface antigens promoted TFH differentiation in lymphoid tissues. Proximity labeling confirmed direct, MHCII-dependent IEC-CD4+ TRM synapses, particularly with CD103+ subsets. Conclusion We establish IECs as orchestrators of mucosal memory, demonstrating that IEC-MHCII actively suppresses TCM programming while promoting TRM differentiation through sustained TCR signaling. This antigen compartmentalization paradigm redefines CD4+ fate decisions–cytoplasmic antigen targeting via epithelial presentation could selectively generate protective TRM. These mechanisms illuminate IBD pathogenesis where dysregulated epithelial antigen presentation may disrupt TRM/TCM balance, perpetuating chronic inflammation. Funding Source F30DK135345 Topic Categories Mucosal and Regional Immunology (MUC)

KLF2 regulates CD8 T cell differentiation and migration in infection and cancer 2254075

Abstract Introduction Besides acute infection, CX3CR1+ effector-like CD8 T cells also appear in chronic infection and cancer. Whether there is a central transcriptional regulator of the effector fate is unclear. Methods Here, using joint single-cell profiling of the transcriptome and epigenome of chimeric antigen receptor (CAR) T cells, we identified KLF2 as a hub transcription factor in the gene regulatory network of effector-like CAR T cells. Results KLF2 deficiency abolished effector-like differentiation in both CAR T cells and antiviral CD8 T cells. KLF2 deficiency also impaired the differentiation of the stem-like subset. In both chronic viral infection and cancer, KLF2 deficiency impaired CD8 T cell infiltration to non-lymphoid tissues. However, unlike CAR T cells, KLF2-deficient antiviral CD8 T cells showed a greater expansion in the lymphoid tissue. Single-cell transcriptomics analysis revealed that KLF2-deficient CD8 T cells upregulated the transcriptional signature of terminally exhausted T cells and downregulated the signature of effector-like T cells, suggesting a central role of KLF2 in regulating the bifurcation of effector versus terminal exhaustion cell fates. In addition, the TOX-controlled transcriptional program was upregulated in KLF2-deficient CD8 T cells. KLF2 deficiency downregulated multiple genes associated with T cell migration including S1pr1. S1PR1 deficiency impaired the effector-like subset, whereas deficiency of CD69, a negative regulator of S1PR1, increased the effector-like subset. Notably, aging downregulated KLF2 in antigen-specific CD8 T cells, impaired the differentiation of the CX3CR1+ subset, and upregulated the TOX-mediated transcriptional program. Conclusion Thus, our study demonstrates KLF2 as a central regulator of the effector fate and migration of CD8 T cells. Funding Source NIH, CRI, AFAR, V Foundation Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Leveraging extrafollicular B cells for the treatment of solid tumors 2260703

Abstract Introduction Intratumoral memory B cells (MBCs) correlate with immunotherapeutic response in cancer patients. MBCs are often associated with germinal center reactions but may also be cued by the extrafollicular (EF) response, which predominates in chronic infections and autoimmunity. In autoimmunity, EF responses are driven by toll-like receptor (TLR) 7, but in the cancer setting we show TLR9 stimulation results in more functional subsets, suggesting the initial activation signal is crucial for effective B cell responses. Our lab has demonstrated that EF MBC subsets are dysfunctional in the tumor microenvironment, defined by hyporesponsiveness to BCR stimulation, impaired antibody secreting cell differentiation, and poor antibody production. We showed that EF MBCs accumulate in the peripheral blood and tumors of head and neck cancer patients, correlating with advanced stage disease. Importantly, EF populations predominately generate IgG, poising this MBC subset as an antigen specific population that may contribute to antitumor immunity through the production of tumor reactive antibodies. Thus, we hypothesize that reinvigoration of EF MBCs may contribute to tumor-specific immunity through antibody dependent cell cytotoxicity Methods This study utilizes ex vivo profiling of patients tumors and in vitro co culture systems. Results Here, we investigate discrete phenotypes of EF MBC subsets within head and neck cancer patients, finding that EF MBCs are phenotypically diverse in co-expression of integrin-adhesion molecules (CD11c, CD18), and inhibitory receptors (PD-1, FcRL5). We show that expression of FcRL5 is associated with blunted proliferation in tumor infiltrating B cells and advanced stage tumors. Blocking FcRL5 in vitro restores of proliferative capacity and increases differentiation to antibody secreting cell subsets. Conclusion Together, these data suggest that FcRL5 is a negative regulator of MBC function, highlighting a potential new axis for B-cell specific therapies in the treatment of solid tumors. Funding Source NIH F31 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Resolving the Strength–Modulus–Elasticity Tradeoff in Elastomers Using Dual Phase‐Separated Nanodomains

ABSTRACT Achieving elastomers that simultaneously combine ultrahigh strength, high modulus, and excellent elasticity remains a longstanding challenge because these properties are intrinsically conflicting. Here, we report a dual phase‐separated nanodomain strategy that resolves this trade‐off by transforming reversible cross‐links into spatially confined reinforcing nanodomains. Elastomers are fabricated via copolymerization of rigid aromatic polyurea segments with flexible poly(urethane‐urea) chains containing acylsemicarbazide moieties. The resulting elastomers exhibit an exceptional combination of mechanical properties, including tensile strength of 104.6 MPa, Young's modulus of 43.1 MPa, toughness of 350 MJ m −3 , and full recovery after 600% strain. Small‐angle x‐ray scattering and electron microscopy reveal two distinct nanodomains originating from self‐assembled aromatic polyurea segments and acylsemicarbazide‐stacked hydrogen‐bond arrays, respectively. Their synergistic reinforcement increases matrix rigidity while preserving entropy elasticity, enabling the simultaneous realization of ultrahigh mechanical robustness and excellent elastic recovery. The elastomers further demonstrate outstanding puncture resistance, environmental stability, healability, and reprocessability. When used as binders for carbon‐fiber fabrics, the composites achieve record‐high fracture energies of up to 2059 kJ m −2 , owing to the exceptional mechanical robustness and energy dissipation of the elastomer, together with strong elastomer‐fiber interfacial adhesion. This dual nanodomain design provides a novel route to high‐performance elastomers that transcend conventional strength‐modulus‐elasticity trade‐offs.

PAX8 controls proximal tubule epithelial identity and stress response through epigenetic modification of distal regulatory elements

Proteomic signatures reveal stage-specific progression across gastrointestinal–neuropsychiatric comorbidity: a deep learning and multi-state trajectory analysis

Deep functional interpretation of influenza-induced ciliary gene dysregulation through stepwise Large Language Model profiling 2301536

Abstract Introduction The interpretation of large-scale transcriptional data remains a significant challenge in functional genomics, particularly in complex biological contexts such as host-pathogen interactions. Methods We present a systematic approach combining air-liquid interface (ALI) cultures with stepwise Large Language Model (LLM) analysis to achieve deep functional interpretation of ciliary gene regulation during influenza infection. From 2,828 differentially expressed genes, initial high-throughput LLM screening identified 29 genes with high confidence scores specifically associated with ciliated cell biology. We conducted detailed functional profiling of these candidates through human-in-the-loop validation. Results Analysis revealed a coordinated program of ciliary gene dysregulation. Key genes, including DNAH5, DYNC2H1, and DNAAF4-CCPG1, showed consistent downregulation by 24-48 hours post-infection. This pattern was validated across independent datasets from Influenza, Rhinovirus, and SARS-CoV-2 infections, suggesting a conserved mechanism of mucociliary clearance impairment across respiratory viral infections. Conclusion By focusing our analysis on ciliated cell-associated genes, we uncovered specific mechanisms of viral pathogenesis while establishing a generalizable framework for context-aware interpretation of complex biological datasets. Funding Source National Institute of Allergy and Infectious Diseases (NIAID) Topic Categories Viral Immunology (VIR)

UVRAG and the VPS34 complex II regulate macropinocytosis via the synthesis of PI(3)P on the plasma membrane 2259463

Abstract Introduction Macropinocytosis is central to critical macrophage immune functions including wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Methods We performed whole-genome screens to identify key regulators of macropinocytosis. Phospho-proteomic analysis was conducted after depletion of the key negative regulator PAK2. Lattice lightsheet microscopy (LLSM), utilizing macrophages expressing 2xFYVE-mScarlet reporter, tracked PI(3)P dynamics in macropinocytic cups and nascent macropinosomes. Results Uvrag and other members of VPS34 complex II, which catalyzes PI(3)P synthesis, was identified as a top positive regulator. Conversely, the loss of PAK2 increased macropinosome formation, confirming it as a key negative regulator. PAK2 depletion reduced phosphorylation of GAPex-5 (Rab5 GEF) and Rabankyrin-5 (Rab5 effector), both of which were required for efficient macropinocytosis. LLSM confirmed PI(3)P localization in macropinocytic cups prior to closure, as well as on large (0.5-3 μm) nascent macropinosomes and smaller (< 0.5 μm) vesicles moving toward the plasma membrane. This data suggests PI(3)P supports macropinocytosis by recruiting GAPex-5, which activates the Rab5/Rabankyrin5/Rabenosyn5 pathway to facilitate SNARE-mediated fusion preceding macropinocytic cup closure. Conclusion This work elucidates the mechanisms underpinning PI(3)P regulation of macropinocytosis and elucidates the mechanisms of PAK2-mediated negative regulation of Rab5 activity and macropinocytosis. Unlocking mechanisms of macropinocytosis may reveal new therapeutic targets for in vivo study of this process and better understanding of the role of macropinocytosis plays in macrophage cell biology. Funding Source NIH COBRE Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Investigating the Role of Mast Cell Condensates in Regulating Macrophage Phenotype and Dynamics in Tissue Repair 2309191

Abstract Introduction The peritoneal cavity contains diverse immune cells that coordinate complex processes in response to injury and disease. During tissue repair, these cells form large repair aggregates within the cavity, however, the exact mechanism coordinating this function remains unclear. Large peritoneal macrophages (LPMs) appear to be the dominant cells that form the aggregates at the site of injury, however mast cells were seen to also be prevalent participants. Methods In-vitro assays were performed using peritoneal fluid to induce LPM aggregation via stimulation with ATP and adding back calcium. Confocal microscopy was used to visualize aggregation formation and identify extracellular granules released by mast cells during aggregation. Rheometry and fluorescence recovery after photobleaching (FRAP) were used to characterize these extracellular condensates after isolation and assess their material properties. Lattice-light sheet microscopy was used to visualize the release of these extracellular condensates by mast cells. Results Stimulation of the peritoneal fluid revealed that mast cells were responsible for the release of large (∼1 μm) avidin-positive extracellular condensates that localized between aggregating LPMs. Confocal microscopy demonstrated that the extracellular condensates were also positive for macrophage receptor with collagenous structure (MARCO), a scavenger receptor known to be involved with macrophage aggregation. These granules were further defined to be membraneless condensates using rheometry and FRAP imaging where both MARCO and avidin demonstrated recovery. Lattice light sheet microscopy revealed thin, actin-positive filapodia extending hundreds of microns in length coming from the mast cell, which grab condensates and deposit them on neighboring cells at the injury site, creating a limited sphere of influence. Conclusion These findings suggest that mast cell-released condensates play a part in macrophage aggregation and drive the tissue-repair mechanism in the peritoneal cavity. Funding Source Canadian Research Excellence Chair (CERC), Infrastructure Operating Fund (IOF), Canadian Foundation for Innovation (CFI), and Bruce Mitchell Research Program Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

T Cell-Specific Deletion of Glutaminase (Gls1) Lowers AutoAb Production in Lupus Mice by Altering Metabolism and Function of Follicular Helper T Cells 2253101

Abstract Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease marked by high-affinity, class-switched autoantibodies (autoAbs) produced by germinal center (GC) B cells with follicular helper T (Tfh) cell assistance. We previously used pharmacological inhibition with 6-Diazo-5-oxo-L-norleucine (DON) and T-cell-specific deletion of Gls1 (Gls1 KO) in Sle1b autoimmune mice and B6 controls, showing altered glutamine metabolism in SLE Tfh cells. Here, we investigate the mechanisms by which T-cell-intrinsic glutamine metabolism supports Tfh function and autoAb production. Methods B6 and Sle1b KO mice with controls were assessed ex vivo for autoAbs, class switching, GC responses, and Tfh markers by ELISA and flow cytometry. Seahorse metabolic assays, in vitro Tfh—B co-cultures, and RNA-seq assessed metabolic, functional, and transcriptional changes. Post-transcriptional proteasomal regulation was tested with MG132. Results Gls1 KO reduced Tfh cell function, autoAb production, and GC expansion in SLE-prone mice, underscoring the critical, T cell-intrinsic role of glutamine in pathogenic Tfh function. Seahorse assays showed reduced mitochondrial respiration, glycolysis, and spare respiratory capacity uniquely in Sle1b KO relative to Sle1b CD4+ T cells. In vitro Sle1b KO Tfh cells induced lower Ab production and class switching from co-cultured B6 B cells. ICOS expression was downregulated in DON-treated and Gls1 KO Tfh cells, linking glutaminolysis to its regulation and GC function. Tfh RNA-seq revealed broad transcriptional changes on the Sle1b background, indicating autoimmune-specific reliance on glutamine. Yet, Icos transcripts were unchanged, suggesting post-transcriptional regulation. Proteasome inhibition did not restore ICOS expression. Conclusion These findings identify glutaminolysis as a key metabolic pathway driving SLE Tfh function and autoAb production. Glutaminolysis regulates ICOS post-transcriptionally, revealing a novel proteasome-independent glutamine-dependent regulation of ICOS. Funding Source R37AI128901 Topic Categories Basic Autoimmunity (BA)

HuR—SerpinB9 Axis Regulates Tumor T-Cell Fitness and Persistence via Post-Transcriptional Control 2267776

Abstract Introduction Post-transcriptional regulation (PTR) plays a crucial role in shaping T-cell gene expression programs that adapt to their microenvironment. Among RNA-binding proteins (RBPs) involved in PTR, Human antigen R (HuR; ELAVL1) binds to AU-rich elements within mRNAs encoding regulators of proliferation, inflammation, and immune responses. While HuR controls cytokine expression and T-cell development, its role in effector function and persistence remains unclear. Methods We utilized a T cell—specific HuR knockout (HuRfl/fl CD4-Cre) mouse model and employed flow cytometry, RIP-seq, and transcriptomic profiling to identify HuR-bound targets and their downstream effects. Functional analyses included chronic stimulation followed by characterization, ELISA, HuR and SerpinB9 overexpression, adoptive transfer into Rag ko mice (colitis), and the B16-F10 tumor model to assess T-cell function and persistence. Results Using this approach, we found that HuR-deficient T cells exhibit increased effector cytokine secretion, decreased CD27 expression, elevated KLRG1 levels, and enhanced SA-β-Gal activity, characteristics of replicative senescence. Adoptive transfer of HuR-deficient T cells caused less severe colitis in Rag KO mice despite their effector phenotype, suggesting impaired persistence. Corroborating these findings, HuR expression was significantly reduced in tumor-infiltrating and aged T cells. Conversely, HuR overexpression decreased senescence and restored memory marker expression in both mouse and human aged T cells and TILs. Mechanistically, HuR bound transcripts involved in immune regulation, including SerpinB9, a granzyme B inhibitor vital for cytotoxic T-cell survival. Overexpressing SerpinB9 in wild-type and HuR-deficient T cells enhanced persistence under chronic stimulation and improved tumor control. Conclusion These findings highlight a HuR-SerpinB9 axis that regulates T-cell senescence and persistence, offering a potential therapeutic target in cancer and autoimmune diseases. Funding Source This work was supported by NIH grants R01CA300284, R01CA290201, R01CA282408, R42CA239952, R01DE030013, R01CA250458. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Single-cell profiling of CSF immune dynamics following intracranial B7-H3 CAR T cell therapy for pediatric brain tumors 2254484

Abstract Introduction CAR T cell therapy is a promising approach for pediatric patients with aggressive brain tumors. Understanding immune responses within the central nervous system (CNS) is critical for optimizing CAR T cell therapy against pediatric brain tumors. In the Loc3CAR clinical trial (NTC05835687), B7-H3- CAR T cells were administered intracranially via an Ommaya reservoir. To investigate therapy-induced immune changes in the cerebrospinal fluid (CSF), we performed longitudinal single-cell RNA sequencing to profile immune populations before and after CAR T cell infusions. Methods CSF samples were collected immediately before and 24 hours after serial intracranial infusions from pediatric patients enrolled in Loc3CAR. Samples were processed for single-cell transcriptomic and TCR profiling using 5’ chemistry from 10x Genomics platform. Cell types were annotated by canonical markers and automated classification. Results Despite low baseline CSF cellularity, most samples yielded high-quality single-cell data. Cell counts increased markedly 24 hours post-infusion, revealing robust immune recruitment. Across patients, CAR+ T cells became detectable and increased over successive infusions, suggesting persistence and expansion in the CNS. Notably, regulatory T cells (Tregs) increased post-infusion, paralleling the expansion of CAR+ T cells. In parallel, nonclassical monocytes showed strong cyclical increases after each infusion, coinciding with reduced macrophage and dendritic cell frequencies. Cell-cell interaction analysis indicated strong incoming signaling toward CAR+ T cells and extensive outgoing signaling from various myeloid subsets. Conclusion Our study shows that single-cell profiling of CSF immune cell components is feasible and revealed dynamic immune remodeling following intracranial B7-H3 CAR T therapy. Repeated infusions promoted CAR+ T cell accumulation but also expanded potentially suppressive Tregs and signaling-active myeloid subsets, highlighting targets for future therapeutic refinement. Funding Source SJCRH/ALSAC CeTII / CePIO, NIH NCI Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Helios promotes CD8 T cell effector differentiation by modulating IL-2 signaling 2260989

Abstract Introduction Helios (Ikzf2) belongs to a family of transcription factors that regulate lymphoid differentiation and function. In thymic-derived regulatory T cells, Helios promotes suppressive function. Previously, we observed that Helios is induced in CD8 T cells under tolerance-inducing contexts. The aim of this work was to analyze the signals that drive Helios expression and determine the roles it plays in CD8 T cell function. Methods We established an in vitro assay to identify factors that modulate Helios expression. Productive CD8 activation, achieved by high affinity ligands, inhibited Helios expression. Results In contrast, activation with low-affinity ligands led to robust Helios upregulation. Pharmacological dissection of signaling pathways indicated that activation of the IL-2-STAT5 pathway inhibited Helios induction. We analyzed Helios expression kinetics in CD8 T cells during an acute infection, where clonal expansion relies on IL-2. We observed a transitory Helios expression that peaked at 72 hours post infection. Ikzf2-deficient CD8 T cells exhibited impaired proliferation, expansion, and effector responses when exposed to cognate antigen in the context of infections (i.e. Listeria and LCMV-Arm). Transcriptomic analysis (scRNA-seq) showed that absence of Helios was associated with an expansion of cells with memory/progenitor features, suggesting that effector differentiation is altered in Ikzf2-deficient CD8 T cells during acute infections. Furthermore, transcripts encoding phosphatases implicated in IL-2 signaling were more abundant in Helios-deficient cells. Conclusion As IL-2 signaling plays a key role in the acquisition of effector functions in T cells, Helios could act as a repressor of CD8 T cell effector functions through the modulation of IL-2 signaling. In conclusion, Helios is essential for CD8 T cell effector differentiation. This regulatory mechanism could represent a novel target able to improve or restrain CD8-mediated immune responses in the context of autoimmunity or cancer. Funding Source CONAHCYT 303067 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Structural architecture of a hybrid β-barrel assembly machinery BAM-TAM complex in Borrelia burgdorferi

Brachiocephalic vein distensibility during passive leg raising in healthy adults

Abstract Passive leg raising (PLR) is a validated test of fluid responsiveness, but the value of brachiocephalic vein (BCV) distensibility as an ultrasound marker remains uncertain. In this prospective study, 129 volunteers were assessed for eligibility and 122 were analyzed (45 nonfasting controls, 77 fasting). Cardiac output (CO) was measured before and after PLR by echocardiography, and BCV distensibility was calculated from respiratory diameter variation. PLR increased CO in both groups ( P  < 0.001). Median ΔCO was lower in fasting participants than in controls (21.05 [12.93–37.32] vs. 29.88 [22.13–39.86], P  = 0.038), with lower responder rates at ΔCO ≥ 10% (79.2% vs. 95.6%, P  = 0.016) and ΔCO ≥ 15% (74.0% vs. 91.1%, P  = 0.032). ΔBCV distensibility did not differ between groups and was not associated with ΔCO overall (Spearman r  = 0.148, P  = 0.105). ROC analysis showed poor discrimination (AUC 0.38 overall, 0.51 in controls, 0.31 in fasting participants for ΔCO ≥ 15%), and exploratory adjusted analyses did not support an independent association between fasting status and responder status. BCV distensibility showed limited value and poor discriminatory performance as a marker of PLR-induced cardiac output change in spontaneously breathing healthy adults.

FXR-Driven Macrophage Reprogramming Enhances Tumor Immunity and Limits Colorectal Cancer Growth 2301033

Abstract Introduction FXR is best known for regulating bile acid metabolism but also has emerging immune functions. How FXR influences tumor-associated macrophages (TAMs) in colorectal cancer is still unclear. Because TAM-driven immune programs strongly affect tumor progression, defining whether FXR shapes macrophage-mediated tumor immunity is biologically and therapeutically important. We examined whether pharmacologic FXR activation enhances anti-tumor macrophage activity and whether these effects require macrophage-intrinsic FXR. Methods To evaluate FXR gain-of-function, mice in CT26 and AOM-DSS Colitis-associated CRC models were treated with the FXR agonist obeticholic acid (OCA). To test loss-of-function, macrophage-specific FXR-deficient mice (Fxrfl/fl;Cx3cr1-Cre) were generated. Tumor burden, proliferation, and gene expression were examined by caliper measurement, histology, and qPCR. TAM states were analyzed by flow cytometry to quantify M1-like versus M2-like markers, co-stimulatory molecules, and antigen-presentation capacity. Results FXR activation significantly reduced tumor size and weight in both CT26 and AOM—DSS models. OCA treatment downregulated key oncogenes, decreased proliferative markers, and hifted TAMs toward an immunostimulatory profile, with increased M1-associated markers, enhanced co-stimulatory signaling, and elevated antigen-presentation pathways. In contrast, macrophage-specific FXR loss markedly accelerated tumor growth, increased spleen weight, heightened oncogene expression, and drove TAMs toward an immunosuppressive, M2-like phenotype with reduced antigen-presentation ability. Conclusion These findings identify macrophage FXR as a critical regulator of tumor immunity in CRC. FXR activation enhances anti-tumor macrophage programming and suppresses tumor progression, while macrophage-intrinsic loss of FXR impairs immune activation and promotes tumor growth. FXR represents a promising therapeutic target for reconditioning the tumor microenvironment in CRC. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Effects of type 2 diabetes mellitus on fibrosis formation in post-tuberculosis lung disease 2259589

Abstract Introduction Tuberculosis disease (TB) is a significant global health challenge, affecting 10 million people and causing 1.3 million deaths annually. While most Mycobacterium tuberculosis (Mtb) infections resolve without causing disease, some cause residual lung damage that can lead to fibrosis. People with type 2 diabetes mellitus (T2DM) are more susceptible to Mtb infection and have worse TB outcomes, including exacerbated post-tuberculosis lung disease (PTLD). T2DM-mediated mechanisms for fibrosis formation in PTLD have not yet been fully characterized. Methods C3HeB/FeJ mice were used to induce T2DM using a combination of a high fat diet and streptozocin. Both T2DM and non-T2DM mice were infected with 102 CFU Mtb for 8 weeks prior to receiving TB chemotherapy. Samples were obtained at 8 weeks post-infection (p.i.) and 16 weeks p.i. (at end of treatment) for histological, immunological, and western blot analysis. Whole body plethysmography was performed at 4 week intervals throughout the study to determine changes in lung function over time. Results C3HeB/FeJ mice developed T2DM as determined by blood glucose level monitoring. During TB treatment, mice with T2DM showed higher levels of pro-fibrotic cytokines (e.g. IL-17A) and altered lung function in comparison to non-T2DM mice. Additionally, expression of latent TGFβ and proteins involved in TGFβ-mediated pathways affecting fibroblasts differed between groups. Histology revealed differences in granuloma formation and collagen accumulation between T2DM and non-T2DM mice. Conclusion These results indicate that T2DM affects pro-inflammatory drivers of fibrosis formation, resulting in significant physiological differences. The C3HeB/FeJ mouse shows potential as a translational model to examine the underlying mechanisms of fibrosis formation in PTLD, including as exacerbated by T2DM. Funding Source Institute for Human Infections and Immunity and Center on Lung Disease, Inflammation, and Remodeling Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Harnessing the Potential of mRNA Vaccines Against Hepatitis C Virus 2309530

Abstract Introduction Chronic hepatitis C virus (HCV) infection affects 58 million people globally and remains a major cause of cirrhosis and hepatocellular carcinoma. Aproximately 1 million new infections 250,000-related deaths occur annually, yet no vaccine exists. WHO has identified HCV as a top-priority virus requiring a vaccine to achieve viral hepatitis elimination. Increasing evidence implicates liver-resident memory CD8+ T (TRM) cells as key mediators of protection against hepatotropic pathogens. We therefore investigated whether mRNA vaccines can be engineered to establish robust intrahepatic TRM responses against HCV. Methods Nucleoside-modified mRNA encoding HCV genotype 3a NS5B polymerase was formulated in ALC-0315 or MC3 lipid nanoparticles (LNPs) or in DOTAP/DOPE liposomes to form lipid-complexed RNA (LPX). To enhance immunogenicity, LPX was co-formulated with the invariant NKT agonist α-galactosyl-ceramide analogue (α-GCB). BALB/c mice (n = 5/group) received 5 µg mRNA per dose: via intramuscular, intradermal or intravenous routes. HCV-specific CD8+ T cells were quantified using flow cytometry, IFN-γ ELISPOT and intracellular cytokine staining in liver and the spleen. Results All formulations induced systemic and intrahepatic NS5B-specific CD8+ T cells, but only LPX-mRNA induced bona fide liver TRM cells. Co-delivery with α-GCB significantly increased TRM magnitude and numbers while homologous boosting further expanded durable intrahepatic TRM cells persisting to day 60 post-vaccination. Conclusion This study provides the first evidence that a HCV vaccine can elicit liver-resident memory CD8+ T cells. LPX’s hepatic tropism, combined with α-GCB driven NKT activation, efficiently imprints robust, durable liver TRM responses, supporting LPX-mRNA/α-GCB as a promising, platform for next-generation HCV vaccines Funding Source ACH4 (Australia) Topic Categories Vaccines and Immunotherapy (VAC)