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Strategic deletion of genes in BCG leads to an enhancement in immunogenicity 2267476
Abstract Introduction Tuberculosis (TB) remains one of the most pressing public health crises, attributed to the bacterium Mycobacterium tuberculosis (Mtb) and resulting in a staggering 1.25 million deaths in 2023 alone. The only approved vaccine for TB is the Bacille Calmette-Guérin (BCG), which is typically administered at birth. While BCG is effective in preventing miliary and meningeal TB in children, it offers only limited protection against pulmonary TB in adults. This limitation highlights the urgent need for an improved BCG vaccine or alternative TB vaccines. Methods We hypothesized that the BCG vaccine could be enhanced by deleting genes that encode immune-evasive proteins. To test this hypothesis, we selected three specific genes: sapM, nuoG, and zmp1, whose products interfere with phagolysosomal (PL) fusion, autophagy, and apoptosis in macrophages. Results We created three genetically modified vaccine strains in the BCG Pasteur background: single knockout (SKO; ΔsapM), double knockout (DKO; ΔsapM and ΔnuoG), and triple knockout (TKO; ΔsapM, ΔnuoG, and Δzmp1). We then evaluated these genetically modified BCG strains for their potential to enhance PL fusion, autophagy, antigen presentation, and ex vivo immunogenicity. The results indicated that macrophages process SKO, DKO, and TKO strains more effectively via PL fusion and autophagy, thereby enhancing antigen presentation. The ex vivo immunogenicity results correlated with the in vitro results. Conclusion Our findings indicate that the immunogenicity of BCG can be improved by selectively deleting genes associated with immune evasion. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)
Antibody-mediated CAR T Platform for Targeting Tumor Cells and the Tumor Microenvironment 2301697
Abstract Introduction CAR T cell therapy has shown success in treating hematologic malignancies but faces challenges in solid tumors due to antigen heterogeneity and the complex tumor microenvironment (TME). CAFs expressing fibroblast activation protein (FAP) contribute to immune suppression by creating physical barriers and immune-suppressive environment that blocks T cell infiltration and persistence. Overcoming these barriers is crucial for enhancing CAR T cell therapy in solid tumors. Methods We engineered a modular CAR T cell platform using anti-FLAG CAR T cells conjugated with FLAG-tagged monoclonal antibodies, allowing flexible targeting of both tumor cells and CAFs. This platform was tested in B-cell lymphoma and pancreatic cancer models, both in vitro and in vivo. Cytotoxicity was evaluated through flow cytometry. TME remodeling and cytokine production were assessed using immunostaining, multiplex cytokine assays, and qPCR. In vivo efficacy was evaluated in subcutaneous and orthotopic models. Results In B-cell lymphoma, FLAG CAR T cells, combined with FLAG-tagged CD19 antibodies, exhibited strong anti-tumor activity, significantly increasing tumor cell lysis and achieving cytotoxicity comparable to traditional CD19 CAR T cell therapies. In solid tumor models, FLAG CAR T cells, in combination with FLAG-tagged antibodies targeting FAP+ CAFs and Claudin18.2+ tumor cells, enhanced immune cell infiltration and significantly suppressed tumor growth. This dual-targeting strategy disrupted the TME-induced immune suppression, reduced collagen deposition, and boosted T cell activity, resulting in notable tumor growth inhibition and prolonged survival in both subcutaneous and orthotopic pancreatic cancer models. Conclusion This CAR T cell platform enables dual targeting of tumor cells and CAFs, overcoming immune suppression in solid tumors. This approach enhances immune activation, reshapes the TME, and provides a promising strategy for improving CAR T cell therapy in solid tumors. Funding Source This study was funded by the National Natural Foundation of China (No. 81970632), Guangdong Science and Technology Department (Nos. 2020B1212060018 and 2020B1212030004), Guangdong Basic and Applied Basic Research Foundation 2025A1515011234. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Optogenetic inhibition of inflammasome in microglia to relieve neuropathic pain 2330993
Abstract Introduction The NLRP3 inflammasome has emerged as a central hub that converts diverse cellular stress signals into IL-1β—driven neuroinflammation in microglia under neuropathic pain. However, because NLRP3 functions as a multiprotein inflammasome complex, strategies that target NLRP3 alone face inherent limitations in controlling the spatial and temporal dynamics of inflammasome assembly and signaling. Methods To overcome these limitations, we developed NLRP3-TRAP (Temporally Restricted Assembled inflammasome Platform), an optogenetic system that enables spatiotemporally precise trapping of assembled NLRP3 inflammasomes using light. NLRP3 was tethered to a CIBN optogenetic module and co-expressed with a blue-light—responsive CRY2 variant (E281A, A9) in human microglial cells (HMC3). We evaluated multiple NLRP3—CIBN fusion designs to identify configurations compatible with efficient CRY2-mediated clustering. Using this optimized platform, we precisely analyzed NLRP3 clustering kinetics and light-dependent binding dynamics. Results Applying NLRP3-TRAP in LPS/ATP-stimulated HMC3 cells, blue-light illumination attenuated downstream inflammasome signaling, as evidenced by reduced levels of cleaved caspase-1 and mature IL-1β. For in vivo translation, NLRP3-TRAP were encapsulated into PLGA nanoparticles that are preferentially internalized by spinal microglia after intrathecal injection. In a spared nerve injury mouse model, repeated non-invasive LED stimulation produced reduction of pain behavior during post-injury days 4—12 compared with no-light controls, indicating a robust attenuation of neuropathic pain—like mechanical hypersensitivity. This behavioral improvement was accompanied by reduced microglial activation and decreased IL-1β expression in the spinal cord. Conclusion Together, these findings establish nano optogenetic modulation of microglial NLRP3 inflammasomes as a powerful strategy to dissect neuroimmune circuits and enable circuit-level anti-inflammatory interventions for neuropathic pain. Funding Source IBS-R001-Y4-2026-a00 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
IL-33-mediated Mast Cell Function is Female Dominant 2259527
Abstract Introduction Allergic asthma is more common and severe in women than in men. One component of allergic inflammation is IL-33, a cytokine released by the airway epithelium when damaged by allergens. Methods We used mouse bone marrow-derived mast cells for in vitro analysis of IL-33-induced cytokine production, inflammatory lipid secretion, and signal transduction. We also performed in vivo analysis of IL-33-induced peritonitis. Results Using both in vitro and in vivo models of IL-33-mediated inflammation, we found that IL-33 effects are more prominent in females. We show that mast cells cultured from female C57BL/6 or C3H/HeJ mice produce stronger IL-33-induced cytokines than matched male cultures. We also demonstrate that female-dominant responses include arachidonic acid metabolites. Importantly, we reveal that IL-33-mediated JNK activation is heightened in female mast cells. Surprisingly, ERK activation is greater in male mast cells. Conclusion Overall, these data support the hypothesis that specific cell signaling differences contribute to the female-dominant IL-33 response. Funding Source NIH 1R01AI164710 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)
Unique features of B. burgdorferi peptidoglycan drive persistence in discrete tissues and cause responses that mimic chronic illness 2257865
Abstract Introduction Each year there are roughly 500,000 new Lyme disease cases in the United States. Lyme disease, caused by Borrelia burgdorferi, presents with nonspecific flu-like symptoms, and can progress into more severe symptomology. Lyme arthritis is the most common late-stage symptom of Lyme disease affecting ∼10% of patients. Recent studies identified a specific bacterial component that persists within the synovial fluid of post treatment Lyme arthritis patients — B. burgdorferi peptidoglycan (BbPG). PG is a pathogen associated molecular pattern recognized by specific innate immune receptors and causes a proinflammatory response. Small changes in PG chemistry impact immune recognition. BbPG is unique compared to canonical PG chemistry, is released into the environment during normal bacterial growth, and is arthritogenic when injected into a murine model. Methods To investigate the relationship between BbPG and Lyme arthritis we developed a novel in vivo imaging system to track the biodistribution of fluorescent BbPG in real time in a murine model. This approach was coupled with multiomic studies to determine the impact of BbPG on specific tissues and cell types. Results These studies identified that BbPG persists within the murine liver for weeks compared to typical PG structures, is preferentially engulfed and retained by hepatocytes and Kupffer cells, and impacts pathways involving energy metabolism. Conclusion These findings highlight how the persistence of a unique pathogen associated molecular pattern can result in prolonged immune responses and chronic symptomology. Funding Source Steven and Alexandra Cohen Foundation, Global Lyme Alliance, Bay Area Lyme Foundation, NIAID Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
Evolution‐Driven Bio‐Composite Aerogels for Self‐Adaptive Thermal Regulation and Energy Storage
ABSTRACT Nature achieves remarkable multifunctionality by integrating chemically dissimilar phases into hierarchically organized architectures. Inspired by this principle, we develop a simple and universal strategy to construct bio‐composite aerogels by incorporating trivalent metal chlorides (MCl 3 ) into biopolymer chitosan (CTS) matrices. Coordination‐driven assembly in aqueous media enables the direct formation of metal ion‐coordinated chitosan (CTS–M) aerogels without external acids or additional crosslinkers. These aerogels exhibit reversible brittle‐to‐flexible transitions under humidity stimuli, together with exceptional mechanical resilience, enabling self‐adaptive thermal insulation under temperature extremes. Upon pyrolysis, the same precursor is converted into conductive carbon–metal oxide (C–M 2 O 3 ) aerogels, where metal oxide nanocrystals are embedded within an interconnected carbon framework. This structural integration couples a continuous electron‐transport network with redox‐active domains, thereby promoting charge‐transfer and increasing accessible storage sites. As a representative example, the C–V 2 O 3 cathode for aqueous zinc‐ion batteries (ZIBs) delivers excellent energy–power performance (656 Wh kg −1 at 200 W kg −1 , 178 Wh kg −1 at ∼20 000 W kg −1 ) with 85% capacity retention after 10 000 cycles, outperforming previously reported carbon–metal oxide systems. By linking adaptive thermal regulation and electrochemical energy storage through a single precursor‐to‐function pathway, this work establishes an evolution‐driven aerogel design paradigm for next‐generation multifunctional materials.
Harnessing Silicene‐to‐Silicic Acid Conversion for Organelle‐Specific Silica Deposition in Tumor Therapy
ABSTRACT Inducing localized mineralized lesions offers a promising drug‐free strategy for tumor suppression, yet calcium‐phosphate systems are limited by slow crystallization, high ion requirements, and poor organelle specificity. Here, we develop a silicene‐derived nanoplatform that serves as an “inorganic silicic acid reservoir”, enabling controlled, organelle‐specific biosilicification for cancer therapy. Silicene nanosheets are sequentially engineered with tannic acid and PEI‐anchored triphenylphosphonium (TPTS), conferring high colloidal stability, efficient endosomal escape, and selective mitochondrial targeting. Within the oxidative mitochondrial milieu, TPTS undergoes programmed hydrolysis to release Si(OH) 4 , which condenses in situ to form silica directly on mitochondrial membranes. The resulting confined mineral deposits disrupt membrane potential, impede metabolite trafficking, and precipitate a catastrophic energetic collapse that drives apoptosis. This platform delivers two major advances: (1) Intracellular mineralization redefinition—precursors shift from intrinsic labile physiological ions to exogenous bio‐orthogonal nano‐reservoir, enabling sustained, site‐specific silicic acid release; (2) High therapeutic potency – organelle‐level precise therapy surpasses conventional high‐dose‐dependent cellular‐scale mineralization, achieving 81.79% tumor inhibition in ectopic models and 65.81% even in the more challenging orthotopic TNBC models, without inducing systemic toxicity. Together, these results establish a generalizable paradigm for spatially programmed mineralization therapy and position silicene as a versatile foundation for next‐generation organelle‐targeted cancer interventions.
Structure-Guided Computational Engineering of pH-Selective B7-H3 CAR-T Cells to Mitigate On-Target/Off-Tumor Toxicity 2328499
Abstract Introduction Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in treating hematological malignancies. However, its application in solid tumors is severely limited by on-target/off-tumor (OTOT) toxicity due to the recognition of shared antigens on healthy tissues. While strictly tumor-specific antigens are ideal, they are scarce, and clinically relevant targets like B7-H3 often exhibit basal expression in normal organs. Consequently, strategies to restrict CAR-T activation strictly to the tumor site are urgently needed. The acidic tumor microenvironment (TME), a hallmark of solid tumors distinct from physiological pH, presents a unique opportunity to improve specificity. Methods In this study, we addressed OTOT toxicity by engineering pH-dependent CAR-T cells that selectively engage B7-H3 under acidic conditions. Utilizing a structure-based computational approach, we performed in silico dual-pH scanning mutagenesis–systematically substituting residues within the complementarity-determining regions (CDRs) and paratopic interfaces with either Histidine (H) or Aspartic acid (D). This combinatorial strategy was designed to identify optimal electrostatic networks that destabilize binding at neutral pH (7.4) while preserving or enhancing affinity at acidic pH (6.5), thereby creating a precise molecular switch for tumor-specific activation. Results We identified 16 scFv variants exhibiting significant pH-selective binding profiles. Upon incorporation into CAR constructs, two lead variants demonstrated pH-dependent functional activity. These engineered CAR-T cells showed abolished activation against B7-H3 cells at neutral pH but exhibited robust activation and cytotoxicity in acidic environments. Conclusion This study validates the feasibility of using computational design to harness the acidic TME for safety tuning. Our findings establish a generalizable paradigm for developing “smart” CAR-T therapies that decouple potent anti-tumor activity from systemic toxicity. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Single-Cell Multimodal Functional Glycan Profiling Identifies Novel T Cell Immunomodulatory Glycan Signatures 2309845
Abstract Introduction Glycans are key regulators of immune recognition, yet how endogenous human lectins interpret dynamic glycan changes at single-cell resolution remains poorly understood. Existing single-cell omics platforms do not measure functional glycan-lectin interactions, limiting the discovery of glycan-mediated immunoregulatory programs. To address this gap, we developed scGOAT-seq, a multimodal single-cell method that integrates DNA-barcoded recombinant human lectins with transcriptomic profiling to generate physiologically grounded readouts of functional glycan states across diverse immune contexts. Methods We curated and validated a panel of recombinant human lectins, including Siglec-7, Siglec-9, Siglec-15, Galectin-8/9, DC-SIGN, and MBL, that collectively span major mammalian glycan classes and applied them using scGOAT-seq on PBMCs under different immune perturbations. Results Applied to perturbed peripheral immunity, scGOAT-seq revealed stimulus-specific remodeling of Siglec-ligand landscapes that demarcate discrete immune activation states. In CD4 T cells, Siglec-9L and Siglec-15L distinguished immuno-metabolic, transitional, and effector-like activation programs, whereas Siglec-7L uniquely tracked IL-2-independent activation pathways associated with innate-like stimulation. To probe functional consequences of disrupting these pathways, we blocked Siglec-7/9 receptors during LPS stimulation. Blockade reduced Siglec-ligand display, rewired glycan landscapes across monocytes, NK cells, and T cells, and amplified inflammatory, metabolic, and cytotoxic transcriptional programs. These blockade-induced signatures showed prognostic relevance in TCGA kidney cancer datasets, highlighting their potential translational significance. Conclusion Together, scGOAT-seq defines a powerful framework for functional glycan profiling in single cells, revealing previously unrecognized glycan-defined T cell states and offering new avenues for diagnostic stratification and therapeutic glyco-engineering. Funding Source MIT Koch Institute Traditional Bridge Award, MIT School of Science Research Innovation Award, MIT Koch Institute Graduate Fellowship Topic Categories Technological Innovations in Immunology (TECH)
β-Catenin Stabilization Protects Against Alveolar Hemorrhage Through Amphiregulin and BATF-Mediated Regulatory T Cells 2300906
Abstract Introduction Alveolar hemorrhage (AH) is a life-threatening condition with a high mortality rate, yet the role of immune cells in its pathogenesis remains poorly defined. Here, we report the protective function of β-catenin stabilization under Lck promoter and its impact on AH. AH was induced using pristane, and CAT-Tg mice were significantly protected from lung damage, showing reduced proteinuria and decreased pulmonary proinflammatory cytokine production compared with wild-type (WT). This protection correlated with a marked increase in FOXP3+ regulatory T cells (Tregs) in CAT-Tg mice. We further identified a novel mechanism in which β-catenin stabilization enhances lung expression of Amphiregulin and BATF, two molecules essential for Treg function and tissue repair. Adoptive transfer of Tregs from CAT-Tg mice into WT mice with pristane-induced AH conferred superior protection, as evidenced by reduced lung inflammation and proteinuria. The systemic administration of a β-catenin agonist to mice with AH significantly attenuated disease severity. Our bioinformatic analysis confirmed that β-catenin stabilization upregulates pathways associated with tissue repair and immune homeostasis, including PI3K-Akt, angiogenesis, and STAT5 signaling. Methods Using transgenic mice with β-catenin stabilization (CAT-Tg), we demonstrate that CAT-Tg CD8+ T cells display increased central and effector memory subsets, elevated expression of CD44, CD122, Eomes, and T-bet, and reduced phosphorylation of STAT1, STAT3, and JAK1. Results These findings reveal that β-catenin stabilization protects against Alveolar hemorrhage by inducing a specialized T-cell phenotype and establishing a protective Amphiregulin—BATF—Treg axis. Conclusion This work not only uncovers a novel immunoregulatory pathway but also highlights β-catenin as a potential therapeutic target for AH and other cytokine storm mediated lung diseases Funding Source N/A Topic Categories Basic Autoimmunity (BA)
Messenger RNA vaccination produces functionally robust yet peripherally restricted tumor-specific cytolytic effector cells 2260948
Abstract Introduction Recent clinical trials have shown that messenger RNA vaccination against neoantigens can activate long-lasting CD8+ T cells against solid tumors. However, frequent doses appear to be needed to maintain this response. It is uncertain whether vaccination alone reliably recruits effector cells into tumors, particularly when antigen expression is low. We hypothesize that mRNA vaccination primes anti-tumor CD8+ cells, but this alone is not sufficient to recruit and sustain these cells within tumors. Methods We used a murine lung adenocarcinoma model; intratracheal lentiviral Cre recombinase was used to initiate KrasG12D/+; Trp53-/- tumors expressing two neoantigens. Mice were dosed intramuscularly with neoantigen-encoding mRNA. We analyzed CD8+ cell trafficking via flow cytometry, in vivo cytotoxicity assays, ex vivo transwell assays, and immunohistochemistry. Intravenous fluorescent labeling was used to track CD8+ T cell recruitment into tumors. Results In tumor-bearing mice, we observed a strong presence of neoantigen-specific CD8+ cells in the spleen one week post-vaccination. The majority of these cells were CX3CR1+ short-lived effectors. This was not seen among other antigen-experienced CD8+ cells in the spleen. However, these vaccine-primed effector cells mainly localized to the lung vasculature with limited tissue infiltration. In naïve mice, 99% of transferred neoantigen-pulsed splenocytes were cleared from the spleen within 20 hours. Conclusion Our data suggest that mRNA vaccination activates a strong effector population capable of clearing targets presenting neoantigens. However, these cells are ineffectively recruited from the vasculature, a potential barrier to strong anti-tumor immunity. We believe this is either due to a natural progression into short-lived effector cells after priming or inadequate signaling for tissue entry. Our current work aims to explore whether introducing a pro-migratory chemokine axis in the lung may improve recruitment of vaccine-primed cells into tumors. Funding Source NIH T32 Training Grant Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Regulatory T cells turn into multiple pro-inflammatory effector T cells in Mice with Atherosclerosis 2247491
Abstract Introduction Atherosclerosis is an immune-mediated disease in which regulatory T cells (Tregs) can lose lineage stability and convert into pro-inflammatory effector states (“exTregs”) that may accelerate plaque progression. However, the diversity, lineage relationships, and pathogenic roles of exTreg subsets in cardiovascular disease remain undefined. Methods Using Foxp3 lineage-tracker Apoe-/- mice, we uncovered two distinct Treg subsets (GFP+tdTomatolow and GFP+tdTomatohi), with only the tdTomatohi population differentiating into exTregs (GFP-tdTomatohi). To comprehensively characterize these populations, we performed large-scale single-cell CITE-seq and TCR-seq across spleen, draining (axillar and cervical), and non-draining lymph nodes from Apoe-/- mice on a Western diet. Results Density-based clustering and trajectory inference revealed at least seven exTreg subsets, including Tfh-like (the largest population), Th1-like, Th17-like, cytotoxic and proliferating exTreg-like populations. Pseudotime analyses supported a branched rather than linear trajectory, with tdTomatohi Tregs giving rise to multiple effector-like programs. TCR analyses showed clonal enrichment predominantly within Tfh- and cytotoxic-like exTregs, suggesting antigen-driven expansion. CITE-seq identified surface markers (e.g., CXCR5/CD185 for Tfh-like and CXCR6/CD186 for cytotoxic Th1-like exTregs). Spatially, exTregs accumulated in draining lymph nodes and spleen, and were detectable in atherosclerotic plaques by intravital microscopy. Conclusion Collectively, these data define a branched Treg-to-exTreg transition trajectory. Clonally expanded, antigen-experienced exTreg subsets that are expected to influence plaque biology. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Induction of Robust Immunity Against Hepatitis C Virus Using Rationally Adjuvanted Microneedle Patches Integrating A Secreted Form of E1E2 Heterodimer 2259568
Abstract Introduction Safe, effective, durable, and broadly deployable vaccines are needed for sustainable control of hepatitis C virus (HCV) that poses a significant global public health threat. We reported the preclinical development of a novel HCV vaccine candidate engineered by formulating a novel native-like secreted E1E2 immunogen and a well-established combinatorial adjuvant of QS-21 + 3D-(6-acyl) PHAD into our unique microneedle patches (MNPs). Methods In C57BL/6 mice, we evaluated (1) the local immunomodulation characteristics by RT-qPCR; (2) the local and systemic reactogenicity; (3) humoral responses by ELISA and pseudovirus neutralization; and (4) cellular responses by antigen-specific stimulation of isolated splenocytes, followed by intracellular cytokine staining and flow cytometry. Intramuscular vaccination (IM) was used as a benchmark group. In human skin explants, we studied the effect of MNP-delivered adjuvant on the subsets and phenotypes of skin-migratory dendritic cells by flow cytometry. Results Our HCV vaccine efficiently and safely (with no systemic and local reactogenicity) engineered the skin immune system in mice to induce proinflammatory milieu at the vaccine-targeted tissues with increased Nlrp3, Ifng, and Cxcl10 expression. In human skin explants, our HCV vaccine stimulated the migration of highly immunostimulatory antigen-presenting cells, supported by enhanced expression of co-stimulatory molecules, such as CD86 and CD83. Skin immunization of mice with our HCV vaccine elicited improved humoral (higher binding and neutralizing antibodies with enhanced Th1-skewing) and cellular polyfunctional T-cells responses compared to IM immunization. The formulation preserved potency for 3 months at 40 °C, indicating thermostability. Conclusion Unique safety, shelf-stability, innate and adaptive immunogenicity advantages of MNP-based skin immunization would enable the development of clinically translatable and globally accessible HCV vaccines with rationally designed antigens and adjuvants. Funding Source Department of Dermatology, University of Pittsburgh Topic Categories Vaccines and Immunotherapy (VAC)
Common and divergent dynamic changes of the human plasma metabolome across the first week of life in the Gambia & Papua New Guinea 2257987
Abstract Introduction The plasma metabolome changes markedly over the first week of life yet little is known regarding how these trajectories might differ across geographically distinct populations. Methods To gain insight into potential differences in ontogeny between geographic sites, newborn participants were enrolled in The Gambia (n = 45) and Papua New Guinea (n = 45). All participants had samples collected at birth and were randomized to have their second sample collected either on the first, third, or seventh day following birth. Global untargeted plasma metabolomics employed ultra-performance liquid chromatography mass spectrometry (Metabolon). Principal Component Analysis (PCA), regression methods, and enrichment analysis were used to identify significant differences in metabolite profiles between time points (p < 0.01, adjusted for multiple comparisons). Results PCA showed plasma metabolites clustering by day of sample collection and by site. The majority (62%) of metabolite features showed a significant difference based on time point: 46% of these were lipids and 17% were amino acids. Temporal dynamics revealed dramatic changes during the first week of life, with the most pronounced shifts occurring between D1 and D3. The most significant temporal changes were consistent across the two geographic sites, suggesting that the underlying biological processes are robust. Additionally, 24% of the metabolite features showed a significant difference between sites, with the most common being lipids (28.2%). Xenobiotics including caffeine and food metabolites were distinct in GAM vs. PNG, suggesting differences in maternal diet and environmental factors. Conclusion The plasma metabolome undergoes marked changes during the first week of human life, with distinct features across geographic locations, and warrants further exploration with respect to potential correlations to immune status and clinical outcomes, with particular emphasis on lipid pathways. Funding Source National Institute Of Allergy And Infectious Diseases of the U.S. National Institutes of Health under Awards Human Immunology Project Consortium (HIPC; U19AI118608) Topic Categories Computational and Systems Immunology (COMP)
In silico Analysis of the Anti-Pruritic Immunomodulatory Mechanisms of Chunwangbosim-dan Components Using Network Pharmacology and Molecular Docking 2333658
Abstract Introduction Chronic pruritus is associated with chronic inflammatory skin diseases and is often non-histamine-dependent, involving immune dysregulation and inflammatory signaling. Conventional antihistamine-based therapies often show limited efficacy, emphasizing the need for alternative approaches targeting upstream immune and inflammatory pathways. Chunwangbosim-dan (CWBSD) is a traditional East Asian herbal prescription. Among its components, Coptidis Rhizoma (CR) and Rehmanniae Radix (RR) were selected based on reproducible anti-inflammatory activity. We investigated their potential anti-pruritic effects using an in silico approach. Methods Compounds of CR and RR were identified from the TCMSP and TCMID, and their putative targets were predicted using SwissTargetPrediction. Pruritus-related genes were collected from GeneCards, OMIM, and TTD. Overlapping targets were used to construct PPI networks via STRING and h Cytoscape (Degree and MCC) to define hub genes. GO and KEGG enrichment analyses were performed using STRING. Molecular docking simulations were conducted using AutoDock Vina to evaluate binding affinities between representative compounds and key hub proteins. Results Network pharmacology analysis revealed that the selected components of CWBSD interacted with multiple pruritus-associated immune targets and were significantly enriched in inflammatory and immune signaling pathways, including MAPK, PI3K-Akt, JAK-STAT, IL-17, and chemokine signaling pathways. Molecular docking demonstrated strong and stable binding interactions between coptisine from CR and key signaling mediators such as AKT1, JAK2, and EGFR. In addition, isoacetoside from RR exhibited favorable binding affinities with MAPK family proteins, supporting their potential role in modulating inflammatory signal transduction. Conclusion CR and RR may modulate pruritus-relevant immune signaling through multi-target, multi-pathway actions, supporting experimental validation of CWBSD as a complementary strategy for chronic pruritus. Funding Source Portions of this work was carried out within the Korea Institute of Oriental Medicine, (grant number KSN18384113). Topic Categories Computational and Systems Immunology (COMP)
Immune resilience after pediatric cancer treatment 2309878
Abstract Introduction Long-term survivors of pediatric cancer remain at increased risk for chronic conditions and infection-related mortality, likely due to persistent immune dysfunction. However, the long-term immune landscape in adult survivors of childhood cancer remains poorly defined. Emerging hypothesis indicates immune health is a dynamic manifestation of the basal immune state and adaptive immune memory. Cancer and its treatment disrupt this balance, and it is unclear whether survivors’ immune systems return to their basal state or reconstitute to a new immune state post-treatment. Methods To address this, SOMASCAN serum sample measurement was done for cancer survivors who were diagnosed with Acute Lymphoblastic Leukemia (ALL) (n = 75) or Hodgkin’s Lymphoma (HL) (n = 102) from the St. Jude lifetime cohort (SJLIFE), selected based on the progression of clinical outcomes. Samples were profiled for immune and clinical markers and compared to age matched healthy control (HC) (n = 78). Additionally, single-cell sequencing was performed on a subset of individuals diagnosed with ALL (n = 6), HL (n = 6), and HC (n = 6), selected based on chronic disease burden (CDB) and frailty. Older HC samples (n = 3) was included to evaluate the similarity of survivor immune profiles to those of advanced age. Results Preliminary analyses suggest that pediatric survivors exhibit a significantly higher CDB compared to HC, based on regression models adjusted for demographic differences. Frailty independently predicted increased CDB, even after adjusting for age, sex, and race with prefrail/frail patients associated with higher CDB. Distinct protein markers showed associations with CDB in ALL and HL, based on regression-adjusted analyses. Markers included TNF-α, LEG9, Epo and IL-25 in ALL, and CXCL10, CCL3, CCL1 and IL-1β in HL. Conclusion These data highlight immune alterations in childhood cancer survivors. Ongoing analyses will aim to further characterize immune features following cancer treatment. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)
C-C Chemokine Receptor 7/Ligand Activation in T Cells Leads to Internalization and Recycling 2267578
Abstract Introduction CCR7 is a membrane-bound G protein-coupled receptor (GPCR), and trans-locates to membrane-bound compartments in T cells. We have found that in humans and in mice signaling through CCR7/CCL19 promotes rapid receptor internalization via arrestin-3, while CCR7/CCL21 is internalized more slowly and to a lesser extent, demonstrating distinct internalization pathways for each receptor/ligand combination. In this study we determined the kinetics, rab GTPases are small GTPase molecules that regulate vesicle and cell membrane trafficking of CCR7/ligand in human T cells. Methods To visualize CCR7 intracellular movement in T cells we used CEM leukemia cells stably expressing rab 5, 7 or 11 fused to mCherry. Cells were fixed and endogenous CCR7 was labeled, and single cells were imaged using a Zeiss LSM 900Airyscan 2 confocal microscope. Results Rab proteins recruit effector proteins that control lipid membrane organization. Phosphatidylinositol-3-kinase’s which phosphorylate various phosphatidylinositol lipids on the 3’ carbon of the inositol head group that serve as anchoring molecules for rabs. PI3K Class 2+ (PI3KC2+) works with rab 11 to recycle GPCRs post-internalization. On analysis of untreated cells we found that CCL19 promotes rapid trafficking of CCR7 to rab 5 early endosomes followed by trafficking to rab 11 recycling endosomes for up to 2 hr. Notably, a subset of CCR7 trafficked to Rab7 lysosomes, suggesting that the receptor is being degraded after prolonged stimulation. Using 500nM wortmannin, which blocks PI3KC2alpha, we found that recycling is dependent upon class 2 PI3Ks. In contrast, for CCR7/CCL21 we found that CCR7 remained at the surface of CEM cells associated with the membrane marker ras4. Conclusion These results indicate that CCL19 and CCL21 are differentially trafficked in T cells with most CCR7/CCL21 remaining at the cell surface, whereas CCR7/CCL19 is both recycled and degraded. CCR7/CCL19 trafficking is rab and class 2 PI3K dependent. Funding Source NIH SC1GM111172 Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Immunity to influenza B and cross-reactivity to Pandemrix/pH1N1 in narcolepsy 2261229
Abstract Introduction Narcolepsy type 1 (NT1) is caused by 85-95% hypocretin (HCRT) neuron loss due to a combination of genetic and environmental risk factors and associated with DQA1*01:02/DQB1*06:02 (DQ0602, 98%) and T cell receptor (TCR)gene AJ24/AJ28/BV4-2. Pandemic influenza A H1N1 (pH1N1) and Pandemrix vaccination in 2009-2010 induced NT1onset. Antigen specific T cell immune response in NT1 supports adaptive (auto)immunity against HCRT and Pandemrix/pH1N1. Despite this progress, the mechanism behind HCRT neuron loss and how the flu triggers a response against HCRT are unclear. Methods Binding affinity of B/Victoria candidate peptides to DQ0602 was next tested using a competition binding assay. Antigen-DQ0602 restricted/reactive T cells were further isolated using tetramer DQ0602 and single-cell RNA sequenced with 10X. Lastly, TCR clones were transfected into Jurkat 76 for TCR activation by a certain peptide presented by DQ0602. Results With thorough analysis of their sequences, we found a B/Victoria PB1 epitope that is homologous to Pandemrix/pH1N1 PB1 segment with only one amino acid difference. Using tetramer DQ0602, we detected reactive CD4+ T cells to this epitope in two cohort studies using different subjects. PB1 cells from the first cohort were sorted out for single-cell RNA sequencing and over 6,000 cells were recovered. Over 90% were effector memory T cells, over 86% were from multiple NT1 patients, and more than 35 clones were enriched at least 20 times, including 2 with over 500 times. These clones were activated by both peptides from B/Victoria and Pandemrix/pH1N1 under NFAT and NF-κB pathways but not AP-1, suggesting molecular mimicry between influenza A and B through PB1 epitope. In addition, we found TCR clones from baseline PBMCs were activated by Pandemrix/pH1N1. Conclusion Our research showed influenza B plays a role in NT1 pathogenesis and cross-reactivity to PB1 between B/Victoria and Pandemrix/pH1N1. Funding Source Wake Up Narcolepsy Topic Categories Basic Autoimmunity (BA)
PKN1 is a Novel Therapeutic Target for the Optimal Control of GvHD 2251611
Abstract Introduction We were the first to demonstrate that targeting JAK1/2 using baricitinib (BARI) and ruxolitinib (RUX) prevents and treats GvHD while enhancing multi-lineage hematopoietic reconstitution and graft-vs-leukemia (GvL) effect in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT). However, unlike BARI, RUX fails to completely prevent or treat GvHD. Methods To elucidate the mechanism underlying the superiority of BARI to RUX, we performed both mouse and human kinome analyses. After identifying PKN1 as the best candidate, we have generated T cell-specific PKN1 KO mice which are used as T cell donors in our mouse model of GvHD. Results We found that BARI, but not RUX, is a potent inhibitor of both murine and human PKN1. In addition, single-cell RNA sequencing of human PBMCs from patients in a phase 1 trial with BARI also showed downregulation of PKN1-associated genes. Further, mice transplanted with PKN1 KO T cells exhibited significantly reduced GvHD with higher frequencies of Tregs in the spleens, similarly to mice treated with BARI but not RUX. We next investigated if administration of RUX in addition to targeting of PKN1 could prevent GvHD as effectively as BARI. Briefly, we transplanted WT or PKN1 KO T cells along with WT T cell-depleted bone marrow cells (all from B6 mice) into lethally irradiated recipient mice (Balb/c), followed by subcutaneous injection of RUX (400 μg per mouse, 5 times/week). We found that RUX + PKN1 KO T cells demonstrated similar outcomes compared to BARI, showing significantly improved overall survival by reducing GvHD compared to either PKN1 KO T cells or RUX alone. Conclusion All of these findings suggest that targeting of PKN1 in addition to JAK1/2 is needed for optimal control of GvHD. Our work provides significant mechanistic insights into GvHD, its prevention and treatment with broad implications for the development and use of PKN1 inhibitors for solid organ transplant rejection and other chronic inflammatory diseases. Funding Source The American Cancer Society Topic Categories Transplantation Immunology (TRAN)
Targeting FOXP3 Isoforms disrupts Tregs suppression in Colorectal Cancer 2259576
Abstract Introduction MSI-H colorectal cancer (CRC) displays a highly immunogenic TME. FOXP3+ Tregs are key mediators of immunosuppression within the TME. Notably, human FOXP3 exists as two major isoforms: the full-length (FOXP3FL) and an exon 2-skipped (FOXP3dE2), but the impact of FOXP3 isoform switching in CRC remains unexplored. We hypothesized that reprogramming tumor-infiltrating Tregs to express the FOXP3dE2 isoform would attenuate their immunosuppressive activity and enhance anti-tumor immunity. Methods We utilized a novel mouse model selectively expressing only the FOXP3ΔE2 isoform, allowing isoform-specific functional analyses without inducing global Treg depletion or autoimmunity. This model was employed to investigate the role of the FOXP3ΔE2 isoform in MC38-derived CRC tumors through both in vitro supression assay and in vivo tumor development studies. Results FOXP3dE2 mice showed increased Ki67+ Tregs, indicating higher proliferation. In vitro suppression assay revealed ∼80% reduced suppression of CD4 T-cell proliferation by tumor-infiltrating dE2 Tregs as compared to WT Tregs. In vivo, FOXP3dE2 mice exhibited enhanced anti-tumor immunity, with reduced tumor volumes and weights in the CRC mouse model. Further analysis revealed that FOXP3dE2 tumors exhibit significantly enhanced cytotoxic CD8 T cells with an increased proportion of CD8 T cells expressing IFN-γ and granzyme B. Conclusion These findings highlight the potential of targeting Treg isoforms to promote anti-tumor responses and offer a new approach for MSI-H CRC immunotherapy, potentially overcoming current ICB limitations while avoiding systemic autoimmunity. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)