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T-Cell Activation Reflects the Intersection of Inflammation, Metabolic Dysfunction, and Vascular Risk in Prediabetic Obesity 2256824
Abstract Introduction Chronic low-grade inflammation is a hallmark of obesity and a key driver of metabolic dysfunction and cardiovascular disease. However, the immunological mechanisms linking inflammation to early metabolic and vascular risk in prediabetes remain unclear. Activated CD8+HLA-DR+ T cells represent a marker of sustained immune activation, yet their contribution to β-cell stress and cardiometabolic dysfunction has not been defined. Methods A cross-sectional study of 198 adults was conducted, stratified as lean healthy (n = 88), healthy obese (n = 69), and prediabetic obese (n = 41). Circulating CD8+HLA-DR+ T cells were quantified by flow cytometry and correlated with metabolic (glucose, insulin, C-peptide, HOMA-B%) and cardiovascular markers (lipids, blood pressure, MMP-9). Plasma cytokines were measured using a 38-plex Luminex assay. Findings were validated using public whole-blood transcriptomic data (GSE145412). Results Prediabetic obese individuals exhibited significantly higher frequencies of CD8+HLA-DR+ T cells compared with healthy obese and lean groups (p < 0.001). These activated T cells correlated positively with C-peptide, IL-6, TNF-α, IFN-γ, and IL-17A, and inversely with HOMA-B%, linking immune activation to β-cell stress and inflammation. CD8+HLA-DR+ frequency also associated with diastolic pressure, triglycerides, and MMP-9; multivariable analysis identified MMP-9 as an independent correlate (p = 0.04). Transcriptomic validation confirmed parallel immune-metabolic signatures in obese diabetic subjects. Conclusion Activated CD8+HLA-DR+ T cells emerge as key immune biomarkers linking inflammation, β-cell dysfunction, and cardiovascular risk in prediabetic obesity. Their association with MMP-9 highlights a potential immunometabolic axis driving vascular remodeling. These findings identify CD8+HLA-DR+ T-cell activation as a promising early indicator and therapeutic target for cardiometabolic risk prevention. Funding Source Kuwait Foundation for the Advancement of Science (KFAS)-RA MoH-2022-002 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Targeting NRAS-Derived Public Neoantigens in T Cell-Based Cancer Immunotherapy 2261145
Abstract Introduction T cells mediate anti-cancer immune responses through recognition of neoantigens - cancer-specific, mutation-derived peptides presented by MHC proteins. “Public” neoantigens are a subset of neoantigens derived from recurrently mutated driver genes and presented by common HLA alleles, making them ideal immunotherapy targets. NRAS is the second most frequently mutated RAS isoform, and its common hotspot mutations (Q61R, Q61K, and Q61L) give rise to a family of HLA-A*01:01 (A1)-restricted public neoantigens. Methods This study aims to elucidate how the NRAS Q61 neoantigens are structurally and biophysically distinct from their wild-type (WT) counterpart and to resolve the mechanisms that enable a panel of patient-derived TCRs to achieve neoantigen specificity. Results Differential scanning fluorimetry shows that each neoantigen and the WT peptide bind A1 with comparable affinity. X-ray crystal structures of the neoantigen/A1 and WT/A1 complexes reveal that the p7Q side chain of the WT peptide is “tethered” to the α2 helix of A1 via a network of hydrogen bonds. This network is disrupted with all neoantigens, increasing the solvent accessibility of the mutated p7 side chains and facilitating potential TCR interactions. In surface plasmon resonance (SPR) binding experiments, one TCR (TCR11L) binds all three neoantigens (Q61R, Q61K, Q61L) and the WT peptide with high affinity. Crystal structures reveal that TCR11L primarily contacts the penultimate p9E of the peptides, bypassing the p7 mutation site. However, T cell-based functional assays show that TCR11L responds only to the Q61K and Q61L neoantigens. Intriguingly, SPR kinetic analyses reveal significantly slower dissociation rates for TCR11L complexed with Q61K and Q61L compared to Q61R and WT, suggesting that the functional specificity of TCR11L is kinetically driven. Conclusion Collectively, these biophysical, structural, and functional studies reveal how TCR promiscuity can be exploited to enhance scalability in neoantigen-targeting therapies. Funding Source NIH R01CA286507 Topic Categories Vaccines and Immunotherapy (VAC)
Understanding the intracellular role of IL1α in regulation of immune suppressive myeloid cells in breast cancer 2260588
Abstract Introduction Breast cancer remains one of the most prevalent and deadly malignancies worldwide. The tumor microenvironment (TME) critically shapes tumor progression and immune regulation through cytokine signaling. Among these, interleukin-1 alpha (IL1α) is a key mediator of breast cancer—associated immunosuppression. We initially identified IL1α produced by CX3CR1+ macrophage subset within the TME. Deletion of IL1α reduced tumor growth and depleted this population, suggesting IL1α-driven myeloid reprogramming. We hypothesize that IL1α deficiency reprograms myeloid differentiation from monocyte-derived suppressive macrophages to inflammatory macrophage. Methods An in vitro bone marrow—derived macrophage (BMDM) model from wild-type (WT) and IL1α knockout (KO) mice was used to assess IL1α-dependent differentiation. Flow cytometry and qPCR characterized CD11b+CD11c+MHCII+ subsets and quantified inflammatory (iNOS, CD80) versus inhibitory (Arginase-1, CD206) markers. Functional assays, including T-cell proliferation and MDSC activity, evaluated immunoregulatory capacity. Seahorse analysis and mass spectrometry assessed mitochondrial metabolism and electron transport chain (ETC) integrity. Results IL1α KO BMDMs showed reduced F4/80 and CX3CR1 expression, a shift from CD11b+CD11c+MHCII^low to MHCII^high cells upon LPS activation, decreased inhibitory markers, and elevated inflammatory mediators. These macrophages exhibited reduced T-cell suppression and MDSC activity. Neutralization of extracellular IL1α in WT cells did not reproduce KO effects, supporting an intracellular role. IL1α loss impaired oxidative phosphorylation, and peptide inhibitor blocking IL1α mitochondrial localization reproduced this phenotype. Conclusion Our results show IL1α as a regulator of mitochondrial metabolism and myeloid differentiation. By stabilizing the ETC, IL1α sustains immunosuppressive macrophages, whereas its loss promotes inflammatory, anti-tumor myelopoiesis, identifying IL1α as a potential therapeutic target in breast cancer. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
TGF-β1 can convert hostile NK cells to friendly NK cells via downregulation of an activating receptor and upregulation of inhibitory receptors 2260153
Abstract Introduction In prenatal allotransplantation (Balb/c to C57BL/6), NK cells play a critical role in engraftment or rejection of graft in recipients. Adoptively transferred naïve hostile (Ly49D-AFG+) NK cells were converted into friendly phenotype (Ly49D+AFG+) in tolerant engrafter chimera mice. Therefore, we hypothesized that hostile NK cells can be converted into tolerant NK cells in tolerogenic (abundant TGF-b1) environment. Methods To test this hypothesis, isolated naïve hostile NK cells were cultured with allogeneic target cells in the presence e of high dose (1ng/ml) TGF-b1 or anti-CD48 antibody that stimulate NK cells to produce TGF-b. A week later, we analyzed the frequencies of activating NK cell receptor (Ly49D) and inhibiting receptor (Ly49A, F, and G) in hostile NK cell population. Results We found that hostile NK cells cultured with anti-CD48 and allogeneic target cells, expressed inhibitory receptor (Ly49AFG+) but decreased the frequency of activating receptor, Ly49D in hNK cell population. High dose TGF-b1 can convert hostile NK cells to friendly NK cells no matter with or without allo stimulation. Conclusion These results prove that NK cell tolerance can arise from down-regulation of activating receptor and/or expressing inhibitory receptors upon stimulation with allogeneic target cells in tolerogenic environment (presence of TGF-b1). It provides a possible tolerance therapy to protect graft in rejecting recipient following prenatal allo transplantaion. Funding Source Ann & Lurie Children’s Hospital research fund Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Dual Action of Phase Separation and Mechanical Locking Enabled Low‐Value Waste Wood Into High‐Performance Structural Phase‐Change‐Induced Self‐Healing Materials
ABSTRACT Wood is used as a high‐performance structural material in advanced buildings owing to its high mechanical strength, sustainability, and environmental friendliness. However, to overcome the intrinsic size limitation of natural wood, developing green adhesives with high strength, self‐healing, recyclability, and low cost to replace polluting petroleum‐based products has become the key objective for the next stage in wood adhesives research. Inspired by the multiscale structure of gecko toe pads, this work develops a biomimetic self‐healing adhesive featuring dual mechanisms of phase separation and mechanical interlocking. By introducing sodium acetate trihydrate (SAT) into a poly(vinyl alcohol) (PVA)/wood powder (W) adhesive, phase separation occurs to promote the formation of a dense hydrogen‐bonding network and a mechanically interlocked microstructure, which makes the green, sustainable PVA/W/SAT adhesive with high shear strength (5 MPa). Benefiting from SAT's excellent phase‐change capability, the material achieves phase‐change‐induced self‐healing at crack interfaces. The combination of superior mechanical performance, self‐healing functionality, and environmental friendliness makes the PVA/W/SAT adhesive a promising candidate for transforming waste wood into high‐performance planks, significantly reducing the carbon footprint of structural materials.
A CD19/BCMA Dual-targeting VHH format T-cell Engager with Novel CD3 Binder for Enhanced Potency and Safety Profile 2329221
Abstract Introduction B-cell depletion therapies targeting CD19 or BCMA have shown significant therapeutic potential in systemic lupus erythematosus (SLE) and other autoimmune diseases (AID). However, CD19-directed approaches alone may be insufficient, as long-lived plasma cells lacking CD19 but expressing BCMA can sustain disease activity. Therefore, a dual-targeting strategy addressing both CD19-positive B cells and BCMA-positive plasma cells may enable more complete elimination of pathogenic immune cells and improve treatment durability. Methods We developed FPE024, a CD19/BCMA dual-targeting T-cell engager (TCE) designed to enhance B-cell and plasma cell depletion. All components, including the CD3 nanobody, exhibit cross-species binding to both human and cynomolgus monkey antigens, enabling translational evaluation. In vitro cytotoxicity and cytokine release were assessed using tumor cells with varying CD19 and BCMA expression. B-cell killing was evaluated in PBMCs from healthy donors and SLE patients. In vivo efficacy was examined in NSG mouse xenograft models and cynomolgus monkeys. Results FPE024 induced potent cytotoxicity against CD19- and/or BCMA-expressing cells, with cytokine release comparable to approved TCEs, indicating a favorable safety profile. Similar B-cell depletion was observed in PBMCs from healthy donors and SLE patients. In NSG mice, FPE024 effectively eliminated target-expressing tumor cells at low doses. In cynomolgus monkeys, FPE024 achieved robust depletion of B cells and plasma cells at 0.1 mg/kg. Conclusion FPE024 is a potent CD19/BCMA dual-targeting T-cell engager that enables broad depletion of pathogenic B cells and plasma cells. Its strong preclinical efficacy and translational relevance support further development for autoimmune diseases requiring comprehensive B-cell depletion. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Uncovering the Elusive Nature of B Lymphocytes and Immunoglobulin in the Central Nervous System during Alzheimer’s Disease 2302553
Abstract Introduction Alzheimer’s Disease (AD) is a fatal, incurable neurodegenerative disease defined by amyloid-beta plaques, tau neurofibrillary tangles, chronic neuroinflammation, and elevated cognitive deficiency. Dysregulation of the Neuro-Immune axis, an immune cell network tasked with protecting the central nervous system (CNS), has been correlated to blood-brain-barrier dysfunction in early AD. This results in harmful inflammatory responses such as lymphocyte activation and autoantibody production; however, these CNS antibody-secreting cells are not properly defined. Genetic deletion of whole lymphocytes in AD murine models showed increased amyloid pathology and neuroinflammation, yet targeted B cell depletion resulted in less plaque burden and halting of memory defects. In addition, there is evidence of immunoglobulin deposition in the AD mice parenchyma. Thus, it is unclear if these lymphocytes are pathogenic or protective in AD, and what binding targets are driving antibody localization to the brain. Methods Using cerebrospinal fluid (CSF) samples from a clinical cohort of AD patients, we will characterize the CNS B lymphocyte and immunoglobulin compartment through three aims: (1) quantification of antibody titres and neuroinflammatory biomarkers (ELISA), (2) investigation of single-cell transcriptional differences and clonality (scRNAseq), and (3) identification of self-antigen targets via human proteomic assays (Microarray, PhIPseq). Results We predict AD CSF will have elevated levels of class-switched antibodies, inflammatory cytokines, and neuronal damage biomarkers. Single-cell sequencing will show that AD-derived B cells experience transcriptional reprogramming associated with increased activation, antibody production, and clonal expansion. Lastly, the proteomic assays will yield several antibody hits of targeted antigens consisting of human proteins present in the CNS niche. Conclusion Completing these specific aims will allow for a clearer understanding of the B cell and antibody response in AD. Funding Source NIH - Intramural Research Program (IRP) Topic Categories Neuroimmunology (NEUR)
Bhlhe40 promotes inflammatory property of ILC2s through differential regulation of IL-10 and GM-CSF expression 2308383
Abstract Introduction ILC2s (group 2 Innate lymphoid cells) are involved in type 2 immune responses during both phases of innate and adaptive response. The transcription factor Bhlhe40 has been reported to regulate the balance between inflammatory and anti-inflammatory reactions in CD4 T cells. However, although Bhlhe40 is also expressed by ILC2s, its functions in these cells are unknown. Methods By challenging Bhlhe40-germline knockout mice and ILC2-specific Bhlhe40 knockout mice with papain acutely, we found that eosinophils were reduced in these mice compared to their wild type controls, indicating that Bhlhe40 is required for ILC2 function in recruiting eosinophils in response to papain-induced lung damage. Results Interestingly, the expression of IL-5 known for recruiting eosinophils was not altered in Bhlhe40 deficient ILC2s. Instead, we found a reduction in GM-CSF expression but an increase in IL-10 production in the absence of Bhlhe40. To get a clearer picture of how Bhlhe40 regulates ILC2 function, we performed scRNA-seq and scATAC-seq of WT and Bhlhe40 knockout ILC2s, and found Bhlhe40 affected ILC2 subsets and regulated gene expression in distinct clusters of ILC2s. Further analysis of the Il10 locus identified several interesting elements at the Il10 promoter/enhancer regions that had differential gene accessibility between WT and knockout. Furthermore, we confirmed that Bhlhe40 can directly bind to Il10 locus in ILC2s by ChIP-Seq. On the other hand, no obvious changes in DNA accessibility were noted at the Csf2 (encoding GM-CSF) locus, suggesting Bhlhe40 may indirectly regulate GM-CSF expression. Conclusion Overall, our results demonstrate that just as its function in T cells, Bhlhe40 also regulates the balanced expression of inflammatory and anti-inflammatory cytokines in innate lymphoid cells. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Characterization of ENU-Induced Mutant Mice Expressing an Alternatively Spliced IL-15 Reveals Defective Antigen Presentation and CD8+ T Cell Immunity 2256931
Abstract Introduction Interleukin-15 (IL-15) is critical for the activation and expansion of CD8+ T cells and dendritic cells (DCs). We investigated the role of an alternatively spliced IL-15 variant, IL-15α”E7, using an ENU-mutagenized mouse line (P191) that exhibits elevated IL-15α”E7 expression. Following HSV-1 infection, CD8+ T cells in P191 mice showed impaired effector function and reduced proliferative capacity compared with wild-type controls. Methods To further define this defect, CTV-labeled gB-T1 transgenic CD8+ T cells, specific for the HSV-1 glycoprotein B (gB) epitope, were adoptively transferred into P191 and wild-type (WT) mice before HSV-1 footpad immunization. Results On day 3 post-immunization, proliferation of gB-T1 cells in the draining popliteal lymph node (LN) was comparable between groups. However, gB-T1 cell numbers in the peripheral LNs of P191 mice were reduced by 2-fold and 4.7-fold relative to wild-type (WT) mice on day 5 and 10, respectively. These findings indicate that IL-15α”E7 expression compromises sustained CD8+ T cell expansion despite normal early activation. Conclusion We hypothesize that IL-15α”E7 attenuates inflammatory responses and limits LN permeability, thereby restricting antigen dissemination and sustained antigen presentation. Alternatively, IL-15α”E7 may shorten DC lifespan, leading to insufficient support for prolonged CD8+ T cell responses. Future studies will employ fluorescent nanodiamonds as antigen mimetics to assess lymphoid permeability and DC expansion kinetics, aiming to elucidate how IL-15α”E7 expression modulates antigen distribution and DC-mediated antiviral immunity. Funding Source n/a Topic Categories Cytokines and Chemokines and their Receptors (CCR)
KDM5C-regulated anabolic metabolism is required for T cell expansion 2264712
Abstract Introduction Clonal expansion of T cells is a critical component of the adaptive immune response necessary for combating a diverse range of pathogens. Clonal expansion of T cells is a unique physiological process that enables the immune system to generate a large number and is highly controlled by metabolic reprogramming. This metabolic programming is orchestrated through signaling pathways such as PI3K, AKT/PKB and mTOR and key downstream including MYC, SREBPs and HIF1a that program anabolic metabolism. We have found that the KDM5C (JARID1C/SMCX) is a histone demethylase that removes methyl groups from lysine 4 on histone 3 (H3K4) is critical for CD8 T cell expansion during infection. We examined whether KDM5C links epigenetic modulation to the anabolic requirements of clonal expansion during immune responses. Methods We used adoptive T cell Tg models to establish that KDM5C is intrinsically required for T cell expansion. RNA-seq revealed extensive regulation of metabolic genes by KDM5C. We used metabolomics coupled with stable isotope tracing (SIL) to assess anabolism and immunoblot to validate the gene expression findings .CUT&TAG was used to define KDM5C genome occupancy. Results We found that KDM5C expression is rapidly upregulated following T cell activation and is necessary for the transcriptional activation of genes involved in anabolic metabolism. Loss of KDM5C results in reduced levels of metabolites central to nucleotide biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid synthesis. Correspondingly, we found that KDM5C promotes the expression of key transcription factors that drive anabolic programming via a histone-demethylase-independent mechanism. Conclusion This newly identified role positions KDM5C as a critical integrator of transcriptional and metabolic networks that sustain T cell clonal expansion during infection. Funding Source Van Andel Institute Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
The Ufmylation E3 Ligase, UFL1, is Essential for the Development and Functions of Invariant NKT Cells 2260769
Abstract Introduction Invariant natural killer T (iNKT) cells originate from a common lineage with conventional T cells but diverge to become distinct lymphocytes that specifically recognize glycolipid antigens and exert rapid effector functions upon activation. iNKT cells display superior tumor infiltration compared to conventional T cells and can reprogram suppressive immune cells into antitumor phenotypes, making them attractive candidates for immunotherapy. However, functional and numerical defects in iNKT cells have been reported in various human cancers and animal models. The mechanisms regulating iNKT cell development and function remain incompletely understood. UFL1, the E3 ligase of the novel ufmylation pathway, facilitates the transfer of ubiquitin-fold modifier 1 (UFM1) to target proteins, thereby modulating their functions. The role of UFL1 in iNKT cell development and function is currently unknown. Methods We used wild-type mice, mice lacking UFL1 in T cells, and Rag1-deficient mice in this study. Bone marrow chimeras, flow cytometry, and in vitro iNKT cell stimulation were used to assess iNKT cell development and proliferation. Cytokine assays and the B16F10 mouse melanoma model were employed to investigate iNKT cell functions. Results Mice lacking UFL1 in T cells exhibited decreased numbers of iNKT cells in the thymus, spleen, and liver. Furthermore, UFL1-deficient iNKT cells failed to attain full maturation. Bone marrow transplantation studies supported a cell-intrinsic role for UFL1 in regulating iNKT cell development. Functional analyses revealed that UFL1-sufficient iNKT cells produced significantly higher levels of IFN-γ and IL-4 than UFL1-deficient iNKT cells. In addition, UFL1 was required for iNKT cells to effectively inhibit melanoma metastasis to the lung. Conclusion Our findings identify a critical role for UFL1 in iNKT cell biology and provide new insights into how iNKT cells can be more effectively harnessed as frontline immune cells in combating cancer and infection. Funding Source NIH (NIAID) Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
Single-cell immunoprofiling of peripheral blood in non-infectious uveitis patients receiving TNF-inhibitors 2260690
Abstract Introduction Although TNF inhibitors (TNFi) are widely used in the treatment of non-infectious uveitis, the mechanisms underlying treatment response remain unclear. The purpose of this study was to characterize immune cell alterations and molecular responses to TNFi therapy in non-infectious uveitis. Methods Paired peripheral blood samples from 16 uveitis patients were collected before and after TNF inhibitor (TNFi) treatment was started. Single-cell RNA sequencing, surface-protein (CITE-seq), and T-cell receptor (TCR-seq) sequencing were performed on PBMCs using the 10x Genomics platform. Data processing, QC, batch correction, and clustering were performed using Seurat v5.3. Differential gene expression, surface protein analysis, and TCR clonotype assessment were analyzed between paired samples (obtained pre-treatment and while on treatment), as well as between clinical responders and non-responders. Results The most upregulated genes in pre-treatment samples compared to samples obtained on treatment were involved in antigen presentation and interferon signaling (HLA-DRB1, HLA-DRB5, CD74, AIF1, GNA15, ISG15, MX1). Treatment samples showed elevated expression of the S100A8, S100A12, FCN1, GZMB, PRF1, and GNLY genes, suggestive of enhanced innate immune regulation. The baseline samples from patients who were deemed responders showed reduced proportions of activated CD4+ and CD8+ T-cells, and increased levels of B cells, MAIT cells, and NK cells, in comparison to those who were non-responders. Additionally, at baseline, responders demonstrated higher regulatory and cytotoxic gene expression (GZMB, PRF1, GNLY, IL7R, KLRB1). Non-responders at baseline showed elevated expression of inflammatory genes (HLA-DRB1, CD74, ISG15, IFI44L). Overall, TCR analysis revealed decreased clonal expansion post-treatment. Conclusion Single-cell sequencing demonstrates that TNFi therapy reduces activated T-cell populations and downregulates genes associated with antigen presentation and interferon pathways. Funding Source NEI intramural research program, NEI career development award (K23EY036928) Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Side‐Chain Modulation in Diketopyrrolopyrrole‐Based Covalent Organic Frameworks for Fast and Durable Lithium‐Ion Batteries
ABSTRACT The development of high‐performance anode materials is essential for next‐generation rechargeable batteries. Herein, a series of pyrene‐linked diketopyrrolopyrrole (DKP)‐based covalent organic frameworks (COFs) is reported through systematic side‐chain engineering using methyl and ether functionalities. A stepwise synthetic strategy employing unsubstituted, methylated, and ether‐functionalized DKP monomers enabled precise tuning of pore environments, ion coordination, and electrochemical behavior. Among these materials, the ether‐modified Py‐DKPOMe COF exhibited outstanding lithium storage performance, delivering an initial reversible capacity of 265 mA h g − 1 and retaining over 100 mA h g − 1 at an ultrafast 20C rate, with 60 mA h g − 1 maintained at 25C. Remarkably, it achieved 80% state of charge within 61.7 s and exhibited excellent long‐term cycling stability, while the high lithium‐ion diffusion coefficient (7.12 × 10 −10 cm 2 s −1 ) confirmed the rapid ion transport facilitated by the ether‐functionalized chains. Interestingly, density functional theory (DFT) and nudged elastic band (NEB) calculations revealed enhanced Li + adsorption affinity and reduced migration barriers in Py‐DKPOMe COF. In addition, Py‐DKPOMe COF also demonstrated promising sodium‐ion storage (138 mA h g − 1 ). Full‐cell tests with a Li‐rich LNMO cathode verified its practical applicability, highlighting molecular‐level COF design as a powerful strategy for fast, durable organic electrodes.
A Glucocorticoid-Fas axis controls immune evasion from pan-cytotoxic lymphocytes during metastatic seeding 2334193
Abstract Introduction Metastasis is the major cause of death for patients with solid malignancies, such as Triple Negative Breast Cancer (TNBC). Metastatic lesions arise from cancer cells that disseminate from the original tumor mass and colonize new organs. While cancer cells in the tumor mass are protected by an immune-suppressive microenvironment, disseminated tumor cells (DTCs) in a new organ are vulnerable to immune attack. How some of these DTCs overcome immune surveillance remains unknown. Methods We used a visible antigen in a model of TNBC with cognate CD8+ T cells to interrogate mechanisms of immune evasion in early metastatic seeding. We focused on the lungs, as a major site of metastasis in TNBC and an organ with an active resident immune system. Results DTCs that escaped T cell killing showed a heightened activation of the Glucocorticoid Receptor (GR). Using a cancer cell specific GR knockdown, we showed that GR activity in DTCs was essential for resistance to both CD8+ T cells and Natural Killer (NK) cells. Notably, GR inhibition did not affect the primary tumor. To uncover how GR activity in DTCs dampened killing by NK and CD8+ T cells, we developed a labelling tool to tag immune cells in the DTC niche. Single cell analysis of these microscopic lesions uncovered Fas-FasL as a key pan-cytotoxic pathway against DTCs. Mechanistically, GR prevents NF-kB mediated expression of the cell death receptor Fas, inhibiting Fas-mediated DTC elimination. Pharmacologic inhibition of GR in combination with immune checkpoint blockade reduced metastatic burden and expanded lifespan in mice. Conclusion Thus, we discovered a mechanism of immune evasion that operates specifically during early metastatic seeding, illustrating the unique immune-cancer interactions at this stage in the metastatic cascade. Our findings suggest that there are therapeutic opportunities to eliminate DTCs, separately from treatments aimed at primary tumors, with GR inhibition as one promising target. Funding Source DOD/CDMRP/BCRP Era of Hope Scholar Expansion Award (W81XWH-14-1-0191), New York Stem Cell Foundation, Ludwig Cancer Center at Harvard and the Parker Institute for Cancer Immunotherapy (PICI) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
IL-1 signaling directs the prioritization of inflammatory sites in a model of post-operative pulmonary infection 2259548
Abstract Introduction Individuals recovering from surgery are at risk for nosocomial pneumonia. When this occurs, the innate immune system is faced with competing inflammatory sites in the injured skin and the infected lung, raising the question of how the immune response prioritizes its response. We hypothesized that a lung infection would disrupt the normal course of wound healing. Methods The impact of nosocomial pneumonia on laparotomy healing was assessed using data obtained from the ACS National Surgical Quality Improvement Program database. Mouse models were also developed to assess the impact of post-operative pulmonary infection on the innate immune response. Tail skin excision was followed by pulmonary K. oxytoca infection to measure the effect on rate of wound closure. Wounding by the dorsal subcutaneous implantation of polyvinyl alcohol sponges was followed by pulmonary K. oxytoca infection to assess cellular responses at the wound site. Results Laparotomy data showed that patients with pneumonia had higher rates of wound dehiscence. In mice, lung infection slowed skin wound closure and impaired wound innate immune responses. Wound fluid IL-1b and downstream chemokines were rapidly suppressed following lung infection. Local administration of rIL-1b and chemokines in mice with lung infection rescued wound inflammation, but at the expense of lung bacterial clearance. IL-1 is regulated by signaling inhibitors. IL-1RA, a non-signaling ligand of the IL-1 receptor, was upregulated in the bronchoalveolar lavage fluid and plasma of lung-infected mice. Lung infection also increased the expression of the IL-1 receptor decoy, IL-1R2, on blood and wound neutrophils. Administration of IL-1RA to wounded mice phenocopied the effects of lung infection. Conclusion These results show that the infected lung induces systemic IL-1 signaling inhibitors as a protective mechanism to tune distal inflammation, preventing leukocyte migration to the wound and allowing the innate immune system to prioritize inflammatory sites. Funding Source Defense Advanced Research Projects Agency (DARPA), Dean’s Areas of Emerging New Science Award (Brown U.), NIEHS T32-ES7272 (Training in Environmental Pathology), NIGMS COBRE Award P20GM10935, R01HL126887, R01HL165259-01A1, Carney Institute Innovation Awards Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Targeted immunosuppression with a tissue-selective PD-1 agonist prevents rejection of transplanted hearts 2308742
Abstract Introduction Systemic immunosuppression to prevent allograft rejection or treat autoimmune disease can risk infection and malignancy. To uncouple therapeutic immunosuppression from systemic toxicity, we engineered Location-Specific T-cell Inhibitory Molecules (LoSTIMs), Fc-silent bispecific antibodies that activate PD-1 and inhibit T-cells only when they engage a predefined tissue surface antigen. We hypothesized that tissue-selective PD-1 agonism could protect allografts from rejection while sparing systemic immunity. Methods We profiled PD-1 agonism, epitope specificity, and binding kinetics of anti-PD-1 antibodies, then engineered Fc-silent bispecific LoSTIMs. We evaluated antigen-dependent PD-1 agonism in Jurkat reporter assays and inhibition of T-cell activation in primary T cells. We tested a donor MHC class I (H-2Kb)-specific LoSTIM in fully mismatched heterotopic cardiac transplant models, assessing graft survival and immune cell phenotype by flow cytometry and spatial transcriptomics. Results LoSTIMs acted as conditional PD-1 agonists, inhibiting T cells only when engaging target antigen, and were FcγR independent, enabling Fc-silent designs that do not deplete PD-1+ T-cells and preserve physiologic immunity outside the target tissue. Systemic treatment with H-2Kb-specific LoSTIM durably prevented rejection of C57BL/6 allografts in BALB/c recipients, whereas monospecific anti-PD-1 or anti-H-2Kb did not. Flow cytometry and spatial transcriptomics showed reduced donor-specific priming and graft infiltration, loss of effector function in graft-infiltrating CD8+ T cells, and a shift toward less inflammatory, regulatory/progenitor-like T-cell populations. Third-party C3H cardiac allografts (H-2Kk) lacking the H-2Kb surface antigen were not protected, consistent with tissue-selective immunosuppression. Conclusion LoSTIMs provide systemically administered, tissue-restricted PD-1 agonism that prevents allograft rejection without global immune suppression and may be generalizable to other immune diseases. Funding Source ASCO Young Investigator Award (JT), Claudia Adams Barr Fellowship in Innovative Basic Cancer Research (JT) Topic Categories Transplantation Immunology (TRAN)
TLR4—Spike Interaction Mediates Metabolic Reprogramming in Human B Cells 2256897
Abstract Introduction Viral infections intricately modulate B cell immunity, yet the precise underlying mechanisms remain elusive. Methods We analysed B cells from COVID-19 patients, SARS-CoV-2 Spike protein treated human primary naive B cells and mice using flow Cytometry, immunohistochemistry staining and immunoprecipitation. Results This study identifies Toll-like receptor 4 (TLR4) on human B cells as a critical metabolic sensor that detects SARS-CoV-2 spike glycoprotein and triggers mitochondrial dysfunction characterized by reduced oxidative phosphorylation, structural alteration, and elevated reactive oxygen species, leading to bioenergetic deficits. Mechanistically, TLR4 activation enhances calcium signaling via upregulation of calcium transporters independently of B cell receptor (BCR) engagement, resulting in calcium overload that disrupts mitochondrial energy production. This cascade activates AMP-activated protein kinase (AMPK), suppresses mTORC1 signaling, and downregulates key transcription factors including c-Myc, thereby impairing IgG class switching and germinal center responses. Notably, TLR4-driven mitochondrial and metabolic dysfunctioncan be reversed by TLR4 blockade. Conclusion These findings underscore TLR4’s pivotal role in orchestrating calcium mobilization and metabolic regulation in B cells, unveiling a novel mechanism by which SARS-CoV-2 subverts innate immune sensing to evade protective immunity, offering new insights for vaccine design and therapeutic interventions. Funding Source C7156-20G; 17122915; 17114114; 24231222, COVID1903010 and COVID190123; EKPG22-01 Topic Categories Viral Immunology (VIR)
Multivariate Host-Pathogen Interactions Driving Heterogeneous Response to Influenza 2265319
Abstract Introduction Influenza infections differ greatly among individuals, with controlled human infection studies showing wide variation in viral load, immune activation, and symptom profiles. However, the biological and quantitative mechanisms underlying these differences remain unclear. Understanding how host and viral factors interact to drive this heterogeneity could provide insights relevant to both natural and experimental infections. Methods We used a data-driven, mechanistic modeling framework that integrates viral replication dynamics with immune responses in participants infected with influenza. The approach used digital twin and dimensionality reduction techniques to identify and simulate patterns of infection and immune control across individuals. Results The analysis identified distinct infection clusters arising from multivariate influences, and quantified the contribution of variability in inoculum size, virus infectivity, and baseline immunity. Important tradeoffs, such as between cell efficacy and expansion, were revealed, and host—pathogen interactions remained relatively consistent between primary infection and reinfection scenarios. Additional analyses of symptom data illustrated the model’s predictive value and revealed potential subjectivity that was independent of viral strain. Conclusion These findings illustrate the importance of mechanistic modeling in disentangling the complex determinants of influenza infection outcomes and suggest that individual-level variability may lead to shared patterns of disease resolution. Funding Source NIH NIAID Topic Categories Computational and Systems Immunology (COMP)
Immune Modulation of T Cell Dysfunction in Tibia Fracture Model of Complex Regional Pain Syndrome 2260770
Abstract Introduction Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder with no effective treatments. Increasingly, aberrant immune regulation has been recognized in the induction and maintenance of CRPS pathology. Less is known about the role of T cells in CRPS. More recently, resident memory T cells (Trm), which help define the response of local tissues, have been implicated in autoimmune diseases. We hypothesize that systemic T cell dysfunction in CRPS contributes to pathological Trm development leading to disease development and persistence. Targeting T cell dysfunction by repurposing FDA approved immune modulating therapies may provide a novel treatment strategy for CRPS. Methods Mouse tibia fracture model (TFM) of CRPS was generated and pain hypersensitivity was measured by von Frey and Hargreaves. Hind limb skin, lymph node, bone marrow, and PBMCs were collected at different time points. T cell populations were analyzed by flow cytometry. Drugs targeting T cell dysfunction were evaluated in fracture mice at 5 weeks post-fracture. Nur77-GFP reporter and TCR KO mice were used for measuring activation and pain behavior. RNA-seq and cytokine production assays were performed to assess drug efficacy and functional T cell phenotyping. Results TFM mice demonstrate increased presence of phenotypic Trm populations in the skin compared to control mice. Trm demonstrate increased CD103+ populations. Signaling markers associated with Trm development were upregulated in pathological Trm, which were autoreactive measured by activation in GFP reporter mice. Pharmacological blockade of JAK signaling reduced autoreactive Trm, pain and cytokine signaling. Experiments with TCR KO models and RNA-seq studies are ongoing. Conclusion CRPS currently has no approved therapies. Increases in pathological autoreactive skin Trm populations may provide a novel avenue for therapeutic intervention, where blockade of signaling for Trm development and maintenance could prove beneficial for attenuating pain in CRPS. Funding Source 4RF1NS130481-02 Topic Categories Basic Autoimmunity (BA)
Development of a novel preclinical immunocompetent model of glioblastoma that reflects tumor heterogeneity 2260765
Abstract Introduction Glioblastoma is an aggressive, lethal brain tumor with limited treatments and poor patient survival. Several murine tumor lines are used as models in preclinical studies, each with their own strengths and weaknesses, but little has been published about which most closely reflects human tumors in respect to cell composition, immune microenvironment and disease progression. Tumor heterogeneity is often given as a reason for high reoccurrence rate in GBM, but most preclinical work uses only one cell type at a time. A systematic, cross-species comparison of each model cell type and a mixture of all three to human tumor is critical to identify which model is best used preclinically for development of promising immunotherapies. Methods C57BL/6 mice were intracranially implanted with GL261, SB28, CT2A, and a combination of the three and monitored until humane endpoint. MRI, bulk RNAseq, and flow cytometry on brain and immune organs was performed. Additionally, tumor-bearing mouse brains and resected human GBM tissue were fixed and imaged by MRI. Results Differences in survival, tumor invasion, volume, gene expression profiles, and immune landscape were observed across both murine and human tumors. Bulk RNAseq results suggest model-specific patterns in highly expressed genes. Top genes for each murine tumor type were: immune evasion, including SERPINA3 (GL261), fibrosis programming, including collagen (CT2A), and cell adhesion and innate immunity, including CD44 and IFITm2 (SB28). Conclusion To our knowledge, we have made the first preclinical GBM model to truly capture tumor and immune heterogeneity. Differences in murine GBM tumor models and correlating aspects of human tumors offer promising insights for refining preclinical modeling strategies. Our approach serves as a more representative platform, improving translational relevance of available murine models for better immunotherapy development to improve outcomes for patients and their families affected by this devastating disease. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)