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Metabolic Regulation during Stress Erythropoiesis 2257677
Abstract Introduction During Anemia of Inflammation (AI), pro-inflammatory cytokines skew hematopoiesis towards myelopoiesis and impair homeostatic steady-state erythropoiesis, thus decreasing overall erythroid output. These same signals promote stress erythropoiesis (SE), a compensatory pathway that produces a bolus of erythrocytes through rapid proliferation of stress erythroid progenitors (SEPs) and differentiation to mature erythrocytes. We identified nitric oxide (NO) as a signal that rewires metabolism to promote SEP proliferation while also inhibiting differentiation. NO drives the formation of multi-enzyme condensates called purinosomes that allow for efficient metabolic channeling of intermediates of de novo purine biosynthesis. Methods Murine (C57BL/6) bone marrow cells were isolated and grown in specialized media that mimic the proliferation stage of SE. Cultures were treated with 1400W, a nitric oxide synthase (NOS2) inhibitor, or vehicle control. To investigate purinosome formation and activity, we utilized a flow cytometry-based proximity ligation assay and isotope tracing. Results Treatment with 1400W decreased SEP proliferation, purinosome formation, and purine levels compared to wild-type (WT) control. Moreover, glutamine, a key metabolite in NO production, is utilized in WT SEPs to form purines and pyrimidines. We predict incorporation of glutamine in nucleotides will decrease with 1400W treatment, and ongoing studies will investigate such changes. Results will then be compared to Nos2 KO SEPs. Conclusion The role of NO is implicated in sickle cell disease, where patients exhibit SE but limited NO availability, which could be the cause of anemia. Additionally, SE is effective in responding to acute inflammatory insults, but the SE response to chronic inflammation is impaired leading to AI. Understanding how SEPs are metabolically regulated to support their proliferation and maturation into erythrocytes during inflammatory stress would allow us to identify new targets for therapies for AI. Funding Source NIH HL146528, NIH DK138865, USDA-NIFA Hatch Project PEN04960 accession #7006577, T32DK120509, Penn State College of Agricultural Sciences Graduate Student Grant Program (2024) Topic Categories Hematopoiesis and Immune System Development (HEM)
The mTOR pathway in pristane -induced lupus models 2218025
Abstract Introduction mTOR signaling is overactive in systemic lupus erythematosus (SLE) and plays a key role in the disease pathogenesis by disrupting the function of immune cells. Among them, mTOR altered macrophage function through helping polarize anti-inflammatory M2 macrophage. Rapamycin (an mTOR inhibitor) can induce a shift toward the pro-inflammatory M1 macrophage. Pristane ( a hydrocarbon) induces a lupus-like autoimmune syndrome, and it induces serious life-threatening lung Diffuse Alveolar Hemorrhage (DAH) in C57BL/6 mice, but not in Balb/c mice. M1 macrophage are central to the pathogenesis of DAH, contributing to the ongoing inflammation and damage in the lung tissue. mTOR pathway overactivation contributes to inflammation and autoimmunity, making it a promising therapeutic target in SLE. Methods flow cytoemtry and real time PCR Results We found rapamycin can induce DAH development in 60% of pristane treated Balb/c mice and aggravate DAH development in C57BL/6 mice from 60% to 80%. Circulating CD138+ proinflammatory monocytes significantly increased in Rapamycin treated pristane mice. Further study found that mTOR activity is higher in pristane treated Balb/c mice than C57BL/6 mice and pristane could increase mTOR activity in RAW cells in vitro . Conclusion These findings indicate the activity of mTOR, the central regulator of metabolic reprogramming, in myeloid cells influences the clinical phenotype of SLE. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Delineating the role of CXCR6 for the formation and maintenance of CD8+ tissue-resident memory T cells in the gut 2260742
Abstract Introduction The CXCR6-CXCL16 axis has emerged as a critical regulator of T cell migration across various barrier tissues, thereby shaping localized immune memory. CXCR6 may help maintain the retention and function of CD8+ T cells in multiple tissues after infection, influencing not only immediate effector responses but also the long-term memory landscape of T cell populations. However, definitive in vivo data on the effect of CXCR6 on CD8+ T intraepithelial lymphocytes in the intestine (siIELs) are lacking. Methods Using the lymphocytic choriomeningitis virus (LCMV) Armstrong mouse model, we aim to examine the differential effects of CXCR6 deletion in the gut and identify the tissue-specific producers of the CXCR6 ligand, CXCL16. For this, we employ spectral flow cytometry and plan to use bulk and single-cell RNA sequencing, mass spectrometry imaging, and spatial transcriptomics. Results Preliminary adoptive transfer experiments showed that, when assessed in bulk, the ratio of CXCR6-deficient to wild-type P14 CD8+ T cells was lower among siIELs at day 7 after acute infection. Notably, within this population at the same time point, CXCR6 deficiency led to reduced CD69 expression and a reciprocal increase in CD103. Additionally, CD103+CD69+ KO CD8+ T cells were increased in the gut compared to adoptively transferred WT cells. Meanwhile, the expression of CD127 and KLRG1 remained unchanged. Conclusion Together, these findings suggest that CXCR6 plays a role in maintaining immune homeostasis and supporting the adaptive immune response in the gut during the early stages of acute infection. Funding Source In-institute funding. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Sonoenzymatically Triggered Cascading Degradation of Bioresorbable Materials for On‐Demand Transient Triboelectric Implants
ABSTRACT Bioresorbable materials provide temporary biomedical support but often generate wear debris during degradation, leading to immune responses and long‐term complications. Here, we present a sonoenzymatic activation (SEA) materials strategy for on‐demand, cascading in vivo degradation of bioresorbable systems. By coupling ultrasound‐triggered mechanical disintegration with enzyme‐catalyzed molecular degradation, we design a bioresorbable composite embedding enzyme‐encapsulated, biocompatible metal‐organic frameworks (BCEM). Ultrasound‐induced cavitation enables temporally controlled enzyme release, which cleaves ester bonds in the polycaprolactone matrix. In vivo, BCEM undergoes rapid mechanical fragmentation within 15 min under medically relevant ultrasound (20 kHz, 1.0 W cm − 2 ), followed by enzyme‐assisted boosted degradation that reduces the persistence of residues over the 14‐day observation period. This SEA strategy establishes a general materials platform for bioresorbable implants with programmable lifetimes and non‐invasive elimination.
Imprinted antibody lineages can persist for half a century and dominate cross-neutralizing recall responses to poliovirus 2310306
Abstract Introduction Humoral immunity depends on a dynamic interplay between durable, circulating polyclonal IgG antibodies and long-lived memory B cells (MBC). However, the clonal relationship between these long-lived compartments remains poorly defined, particularly decades after childhood vaccination. Methods To investigate the durability of immunological memory to poliovirus (PV), we analyzed the molecular features of the recall response to inactivated poliovirus vaccine (IPV) boosting in healthy adults who had received their primary immunizations 24—55 years earlier during childhood in the United States. Using IgG immunoproteomics (Ig-Seq) coupled with ex vivo activation of MBCs via the Modular Immune In vitro Construct (MIMIC) system, we distinguished the respective contributions of LLPCs (as proxied by secreted plasma IgG proteomics) and MBCs to the anti-PV antibody response. Results IPV immunization elicited highly skewed plasma IgG recall repertoires, dominated by a small subset (∼8%) of total antibody lineages that were detectable prior to boosting (i.e., “pre-existing”). These IgG lineages spiked logarithmically in abundance (∼102-104 fold-increase) and potently cross-neutralized multiple PV serotypes in vitro (titers ≥ 1:105). Notably, these dominant plasma IgG lineages were clonally linked to class-switched MBCs that circulated in blood 21 days post-boost and bore heavily mutated VH and VL variable region genes. Importantly, IPV-primed ex vivo B-cell cultures yielded clonal expansions that mirrored the dominant post-boost plasma IgG lineages in vivo, providing an in vitro mechanistic correlate of recall immunity. Conclusion Together these findings demonstrate a decades-long interconnectivity between persistent MBC clones and the serological IgG repertoire. They further highlight the central role of immunological imprinting, whereby cross-reactive MBC clones–originally primed in childhood–can be reactivated to dominate the circulating IgG recall response more than 50 years after initial exposure. Funding Source DARPA Topic Categories Vaccines and Immunotherapy (VAC)
Mitochondrial DNA oxidation propagates autoimmunity by enabling plasmacytoid dendritic cells to induce TFH differentiation 2253320
Abstract Introduction The border between chronic inflammation and autoimmunity is obscure. It is unclear whether persistent innate immune activation breaches self-tolerance and initiates long-lasting autoimmune responses. A key danger signal that triggers sterile inflammation through NLRP3 inflammasome-mediated caspase-1 activation is Ox-mtDNA, an alarmin that enters the cytoplasm in response to mitochondrial stress. In addition to generating 8-oxo-deoxyguanosine (8-Oxo-dG)-containing DNA, which binds NLRP3, mtDNA oxidation promotes its cleavage into small fragments that pass through mitochondrial pores. Along with pro-IL-1β processing, activated Casp1 cleaves gasdermin-D to generate plasma membrane pores through which Ox-mtDNA escapes to the extracellular space and reaches the circulatory system. However, whether circulating Ox-mtDNA is simply a disease sign or an immunopathogenic driver, is unknown. It is also not clear whether and how mtDNA oxidation dictates its immunostimulatory activity. Methods pDC and CD4+ T cell co-culture Flow cytometry Histological evaluation and immunohistochemistry Immunofluorescence and confocal microscopy scRNA sequencing Results Sustained Ox-mtDNA release, triggered by a prototypical NLRP3 inflammasome activator, induces autoantibody production and glomerulonephritis in mice. Similar autoimmune responses, dependent on plasmacytoid dendritic cells (pDCs) and follicular helper T (TFH) cells, are elicited by in vitro-generated Ox-mtDNA, but not by non-oxidized mtDNA. Although both mtDNA forms are internalized by pDCs and induce interferon-α, only Ox-mtDNA stimulates autocrine interleukin (IL)-1β signaling that induces co-stimulatory molecules and IL-21, which enable mouse and human pDCs to induce functional TFH differentiation, supportive of autoantibody production. Conclusion These findings underscore the role of pDC-generated IL-1β in autoantibody production and highlight Ox-mtDNA as an important autoimmune trigger, suggesting potential therapeutic opportunities. Funding Source n/a Topic Categories Basic Autoimmunity (BA)
Rapid Identification of Aerobic Growth in Sepsis Pathogens by Flow Cytometry 2254627
Abstract Introduction Sepsis is a life-threatening syndrome with 30% mortality in severe cases. Early therapy within 6 hours improves prognosis by 50%, but conventional identification by blood culture and subsequent analyses requires ≥24 h. During this delay, broad-spectrum antibiotics are used, and prolonged administration promotes antimicrobial resistance, emphasizing the need for faster growth characterization methods. We aimed to establish flow cytometry as a tool for rapid assessment of aerobic proliferation. Specifically, we examined whether scatter-based analysis could discriminate aerobic from non-aerobic bacterial strains within 6 hours, across inoculum sizes. We also assessed detection sensitivity, reproducibility of scatter metrics, and temporal changes in bacterial distributions to provide experimental evidence supporting early pathogen discrimination. Methods Two bacterial strains known to proliferate aerobically and three that do not were adjusted to 1, 10, 10², and 10³ CFU/mL. Samples were incubated aerobically and analyzed with the UF-5000 system at 0, 1, 2, 3, and 6 hours. Growth kinetics were quantified, and forward scatter and side scatter distributions were evaluated. The area encompassing 95% of bacterial events was calculated. Results Enterococcus faecalis and Escherichia coli became detectable within 6 hours at ≥ 10 CFU/mL. Their bacterial counts rose progressively after 3 hours, with clear proliferation by 6 hours. Scatter plots showed expansion of the 95% event area to > 50 times of baseline at 6 hours. Non-aerobic strains (Bacteroides fragilis, Prevotella melaninogenica and Clostridium perfringens) remained undetectable even after 6 hours at 10³ CFU/mL and showed no significant change in scatter distribution. Conclusion Flow cytometry enables fundamental assessment of aerobic growth capacity within 3 hours. Differences in bacterial proliferation under aerobic conditions can be detected at an early stage, offering a basis for experimental discrimination of sepsis pathogens. Funding Source JSPS KAKENHI, JP 24K11096 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
The Ikaros zinc finger transcription factor Aiolos promotes the formation of lung-resident memory CD8+ T cells 2257320
Abstract Introduction Influenza A virus (IAV) is responsible for half of all pandemics since the early 1900s, and since 2024, influenza infection has caused over 27,000 deaths in the U.S. alone. The economic burden of influenza is estimated to be $11.2 billion yearly in the U.S, with direct medical costs accounting for $3.2 billion. As IAV’s rapid mutation heightens the risk for future pandemics, effective and durable vaccines are urgently needed. Current IAV vaccines generate neutralizing antibodies against mutation-prone external IAV proteins, limiting their longevity. Resident memory CD8+T cells (TRM) offer an alternative as they, like other T cells, can recognize conserved internal IAV proteins. Uniquely, TRM reside within peripheral tissues like the lungs, enabling rapid response against respiratory viruses like IAV, and making them ideal vaccine targets. However, the molecular pathways governing TRM formation remain incompletely understood. Methods Here, we identify the transcription factor Aiolos as a novel inducer of TRM formation. TCR-transgenic OT-I CD8+ T cells were adoptively transferred into CD8 knockout mice, which were then infected with PR8-OVA (murine-adapted IAV). Lungs were collected at the effector and memory timepoints for flow cytometric analysis. Results We found that Aiolos-deficient murine CD8+ T cells expressed less CD103–a canonical TRM marker that facilitates lung retention–compared to wild-type (WT) controls. There were indeed significantly fewer Aiolos-deficient CD8+ T cells residing in the lungs after IAV infection at the memory timepoint. Mechanistically, we observed that Aiolos-deficient CD8+ T cells express less TGFβR1 (a TRM inducer) and more Eomes (a TRM repressor) than WT cells. Conclusion Collectively, our preliminary data suggest that Aiolos enhances lung-resident TRM formation and is a strong candidate target for improving durable immunity against IAV infection. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Long-term maintenance of CD8+ tissue resident memory T cells in the upper respiratory tract 2257745
Abstract Introduction CD8+ tissue-resident memory T cells (TRM) in the upper respiratory tract (URT) limit viral replication and prevent spread to the lower respiratory tract (LRT), where infection often causes significant morbidity and mortality. While long-term maintenance of URT TRM is essential for this protection, data demonstrating this feature and exploring mechanisms of maintenance is limited. Methods Using mouse models of intranasal and intraperitoneal vaccination, we show that URT CD8+ TRM can develop without local respiratory antigen, a feature distinguishing them from their LRT counterparts. Further, we demonstrate that URT TRM are maintained up to 8 months post-vaccination in both the presence and absence of local antigen, whereas LRT TRM experience gradual decline over time. Results To further elucidate the transcriptional cues governing long-term maintenance of URT TRM after parenteral vaccination, we performed bulk RNA sequencing 6 months post-intranasal and intraperitoneal priming. We are currently working to assess transcriptional patterns of URT TRM under these distinct antigen conditions. In addition, we are utilizing CD103-Cre reporter mice to track the contribution of effector memory cell recruitment or homeostatic proliferation in maintenance of the URT CD8+ TRM pool. Conclusion Our work aims to identify the mechanisms that regulate CD8+ TRM maintenance in the upper respiratory tract, offering valuable insights for T-cell-based vaccine strategies targeting respiratory viruses. Funding Source NIH R35 HL150803; NIH F31 HL176090-02 Topic Categories Vaccines and Immunotherapy (VAC)
A neonatal bias towards CX3CR1 ++ memory CD8+ T cells is associated with impaired control of Cytomegalovirus in early life 2232221
Abstract Introduction Congenital cytomegalovirus (CMV) is a leading cause of hearing loss and neurological deficits. A risk factor for congenital CMV is primary exposure of pregnant individuals to the virus in bodily fluids. CMV infected adults stopped shedding virus in bodily fluids within ∼6 months of seroconversion compared to children who continued shedding virus in bodily fluids for ∼4 years after seroconversion. Methods Here we sought to replicate this phenomenon in a mouse model with the Murine strain of CMV by infecting neonatal and adult mice. We then examined the cellular intrinsic differences between neonatal and adult CD8+ T cells by performing adoptive co-transfers into congenic hosts. Our group utilized spectral flow cytometry, bulk RNA-sequencing and in vivo animal imaging to better characterize this phenomenon. Results Infected neonatal mice continued to shed virus for >12 weeks post-infection, while infected adult mice stopped shedding at 4 weeks post-infection. Both adult and neonatal CD8+ T cells upregulated CX3CR1 in response to CMV infection, however, neonatal CD8+ T cells terminally differentiated into a unique CX3CR1 ++ population that was functionally impaired in the ability to make IFNγ. This ‘dysfunctional’ CX3CR1 ++ population in neonates was specific to CMV, as neonatal CD8+ T cells in response to vaccinia made robust IFNγ. Higher expression of CX3CR1 on neonatal CD8+ T cells had a physiological role by promoting the T cells to stay vascularized, while adult CD8+ T cells were readily able to disseminate into the extravascular space of tissues. Conclusion This work provides the first identified mechanism that explains why the host immune response of neonates fails to control CMV viral replication and shedding. Funding Source American Association of Immunologist (AAI) — Postdoctoral Intersect Fellowship for Computational Scientists and Immunologist Topic Categories Viral Immunology (VIR)
Targeting the Idiotype: Nanobody-Based PET Imaging of Antigen-Specific T Cells in Experimental Autoimmune Encephalomyelitis 2260726
Abstract Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by demyelination and neuroinflammation. Autoreactive CD4+ T cells that recognize myelin-derived antigens, such as myelin oligodendrocyte glycoprotein (MOG), drive infiltration of immune cells into the brain and spinal cord. Experimental autoimmune encephalomyelitis (EAE), a well-established murine model of MS, recapitulates these features, including activation of MOG-specific T cells (2D2 T cells). Yet, the direct in vivo visualization of these antigen-specific T cells remains challenging. Methods An alpaca was immunized with recombinant 2D2 TCR, and phage display subsequently yielded nanobodies recognizing distinct 2D2 epitopes. We performed adoptive transfers (CD45.1 with CD45.2 2D2 CD4+ T cells) for binding and functional readouts. For imaging, D3 was site-specifically sortase-conjugated to DFO-PEG and labeled with 89Zr. Immune-PET was performed in 2D2-TCR transgenics, Rag⁻/⁻ recipients of 2D2 T cells, and C57BL/6 mice given 2D2 T cells and induced with MOG35—55 EAE. Results Six nanobodies (2A7, 2C1, 2C3, D3, E10, H4) bound distinct 2D2 epitopes with nanomolar affinity. In flow cytometry, D3 enabled single-reagent detection of 2D2 T cells with low background, obviating αTCR-Vα3.2/β11 panels and congenic gating. Functionally, 2C3 and D3 blocked antigen-dependent activation in vitro. 89Zr-DFO-PEG-D3 produced high-contrast PET: it specifically detected 2D2 T cells in 2D2-TCR mice; visualized homing to secondary lymphoid organs in Rag⁻/⁻ recipients; and revealed a distinct spinal-cord signal in EAE mice consistent with infiltrating 2D2 T cells. Conclusion Idiotype-specific nanobodies enable non-invasive, longitudinal imaging of defined antigen-specific T cells in vivo. The approach enables precise, longitudinal monitoring and functional modulation of defined T-cell subsets and can be generalized to additional TCR—pMHC pairs, providing immunologists a practical alternative to conventional antibody panels. Funding Source National Multiple Sclerosis Foundation Topic Categories Technological Innovations in Immunology (TECH)
Phase‐Engineered Ru‐MoC Heterointerfaces for Efficient Alkaline Hydrogen Evolution by Boosting Interfacial Water Dissociation
ABSTRACT Alkaline hydrogen evolution (HER) performance of Ru catalysts is limited by the high activation energy barriers of water dissociation and the poisoning of active sites by OH * intermediates. Herein, the Mo x C nanoclusters with distinct crystalline phases (MoC and Mo 2 C) are supported on hierarchical N‐doped carbon nanocages (hNCNC). Long‐range disordered Ru nanoclusters are induced to grow on the MoC nanoclusters due to the presence of Mo vacancies, while crystalline Ru nanoclusters grow on both Mo 2 C nanoclusters and hNCNC due to fewer defects. The Ru‐MoC/hNCNC achieves an ultralow overpotential (@10 mA cm −2 ) of 20 mV, a high turnover frequency of 21.8 H 2 s −1 and robust durability in 1 M KOH, much superior to the counterpart of Ru‐Mo 2 C/hNCNC. The anion‐exchange membrane water electrolysis device with Ru‐MoC/hNCNC cathode delivers an industrial‐scale current of 1 A cm −2 at ≈1.65 V in 1.0 M KOH. The theoretical calculation, together with in situ Raman spectra, reveals that the Ru‐MoC heterointerfaces promote the water dissociation kinetics on Mo sites, leading to the detached formation of OH * on MoC and H * on Ru. This spatial decoupling of OH * and H * can mitigate the OH * poisoning of Ru and promote subsequent H 2 formation, thereby demonstrating excellent alkaline HER performance.
Quartz‐Like Supramolecular Glass Enabled by Host–Guest Size Mismatch
ABSTRACT Supramolecular glasses are conventionally restricted by a fundamental trade‐off between mechanical robustness and deep‐ultraviolet (UV) transparency, due to the ubiquitous reliance on aromatic building blocks. Herein, we overcome this limitation by reporting a “quartz‐like” supramolecular glass assembled from fully saturated aliphatic macrocycles and lithium salts, enabled by a host–guest size mismatch strategy. Crystallographic elucidation reveals that the undersized Li + guest induces severe conformational frustration within the macrocycle, which, in concert with weak anion coordination (PF 6 − ), kinetically suppresses crystallization. In contrast, size‐matched analogues (Na + /K + ) or strongly coordinating anions (Cl − /NO 3 − ) assemble as ordered crystalline solids. The resulting material resolves the traditional property conflict, exhibiting fused‐silica‐like transparency (>95%) spanning the deep‐UV to near‐infrared regions, while maintaining a high Young's modulus of ∼4.85 GPa, dynamic regenerative repair capability, and robust interfacial adhesion. Notably, the glass demonstrates exceptional stability against high‐intensity UV and γ ‐ray radiation. This study establishes mismatch‐induced frustration as a versatile conceptual framework for engineering optically silent supramolecular materials that decouple mechanical strength from optical bandgap.
LatY136F mice: a preclinical model for IgG4-related disease and autoimmune disorders 2328676
Abstract Introduction Experimental preclinical models are powerful research tools to explore autoimmune diseases as well as the efficacy and mechanism of action for novel therapeutics. Mice with a loss-of-function mutation in the LAT adaptor (LatY136F) develop an autoimmune and type 2 inflammatory disorder called defective LAT signalosome pathology (DLSP). DLSP manifestations involve accumulation of Th2 effector cells, polyclonal B cell activation, IgG1/IgE hypergammaglobulinemia and systemic autoimmunity with nephritis and severe proteinuria. The pathogenic CD4+ T cells developing in LatY136F mice have been solely analyzed in bulk, precluding to determine whether the LatY136F mouse constitutes at single-cell resolution an authentic preclinical model of human IgG4-Related Disease (IgG4-RD). This study aims to validate the LatY136F mouse as a preclinical tool for investigating the pathogenesis and treatment of IgG4-RD and type 2 autoimmune diseases at the single-cell level. Methods We used single-cell omics approaches to analyze the cellular trajectory leading to pathogenic CD4 T cells responsible for the LATY136F DLSP, a mimic of IgG4-RD. Results Single cell omics results showed that CD4+ Tfh and CTL, activated B cells, and plasma cells were found in LATY136F spleen and lung. Such cell constellation entailed all the cell types causative of human IgG4-RD. Importantly, targeted therapies using monoclonal antibodies and CAR T-cells effectively reversed LATY136F disease manifestations. Conclusion Our findings elucidate the etiology of the LatY136F DLSP and qualify it as a model of IgG4-RD and of autoimmune and inflammatory disorder. Our model is a useful tool to facilitate therapeutic target discovery and the preclinical evaluation of drug candidates intending to treat IgG4-RD, type 2 immune disorders and inflammatory fibrotic conditions. Funding Source - Centre National de la Recherche Scientifique (CNRS) - Institut National de la Santé et de la Recherche Médicale (INSERM) - The European Research Council under the European Union’s Horizon 2020 research and innovation program (Grant agreement 787300 t Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Immunomodulatory effects of non-psychoactive cannabinoids in a model of metabolic dysfunction-associated steatotic liver disease (MASLD) 2232983
Abstract Introduction Emerging evidence suggests that activation of different immune cell populations contributes to the progression of MASLD to advanced MASH. Thus, with lipogenic and fibrotic markers, exploring liver innate and adaptive immune cell activation and recruitment can enhance understanding of MASLD progression and therapeutic targets. Previous studies showed CBG, a non-psychotropic cannabinoid, reduces inflammation in MCD diet-induced MASH, while CBD alleviates alcohol-induced liver steatosis. Considering this, the study explores the efficacy of CBG alone or with CBD in MASLD-associated inflammation and metabolic disorders. Methods In vivo, blood glucose levels, serum insulin, and adiponectin are analyzed. Inflammation by innate immune cells is assessed by IHC for CD45, CK19, F4/80 (Kupffer), and infiltrated macrophages—M1 and M2, and LPS-induced IL-6 production from isolated hepatocytes. Adaptive immunomodulation is assessed by T cell numbers (CD4+ and CD8+) +/- the treatments in peripheral blood, spleen, and liver. In vitro, steatosis is induced in hepatocytes by free fatty acid, followed by CBG or CBD to evaluate effects on fat accumulation. Results In vivo, CBG decreases Kupffer cells, M1 macrophages, and ductular proliferation, but increases M2 macrophages in both sexes. It also improves steatosis, increases adiponectin, and lowers insulin. In vitro, CBG decreases fat accumulation, while the combo reduces IL-6. Conclusion Together these data suggest a promising therapeutic potential of CBG in MASLD. Funding Source National Institute of Health (NIH), [Grant number: 1R15DK131507-01A1] Topic Categories Immune Mechanisms of Human Disease (HUM)
BCG vaccine induced CD4 T cell activation is dependent on processing by cDC1 and cDC2 subsets of human dendritic cells 2259597
Abstract Introduction Tuberculosis due to Mycobacterium tuberculosis (Mtb) kills a million people each year and the widely used BCG vaccine is partially effective. We examined the mechanism of its antigen processing in human PBMC derived DCs since they are critical for T cell activation preceding TH1 immunity. Methods Human cDC1 and cDC2 were bead purified from culture enriched PBMCs, infected with BCG Copenhagen vaccine, followed by gene expression analysis using a RT2-PCR array and in vitro assays to measure BCG infected DCs how well they present Antigen85B-derived peptide to CD4 T cells. Results The gene expression array showed that BCG infected cDC1 strongly express autophagy related ATG5 gene along with Cathepsins and HDAC1, which drives DC differentiation. These biomarkers were greatly reduced in cDC2. BCG infected cDC1 overlaid using specific CD4-T cells secreted higher levels of IL-2 than BCG Infected cDC2. Blockade of HDAC1 using vorinostat reduced antigen presentation in cDC1 but not cDC2. Likewise, 3-methyladenine blockade of autophagy, reduced antigen presentation only in cDC1. We propose that cDC1 has autophagy-dependent MHC-II antigen presentation to CD4 T cells unlike cDC2, and hypothesize that targeting BCG during vaccination to cDC1 subset may enhance its efficacy. Conclusion We propose that cDC1 has autophagy-dependent MHC-II antigen presentation to CD4 T cells unlike cDC2, and hypothesize that targeting BCG during vaccination to cDC1 subset may enhance its efficacy. Funding Source 1R01AI-187440 NIAID, NIH Topic Categories Vaccines and Immunotherapy (VAC)
Enhanced CD25 expression and IL-2/STAT5 signaling characterize β-galactosidase+ senescent and aged CD4+ T cells 2257543
Abstract Introduction Alterations to the IL-2 signaling axis contribute to age-related immune decline, with increased IL-2RA (CD25) expression linked to impaired T cell function. However, the contribution of age-associated T cell senescence to this phenomenon is unknown. Using a biochemical senescence-associated β-galactosidase (SA β-Gal) assay, we examined IL-2-STAT5 signaling in total and senescent CD4+ T cells during aging. Methods PBMCs from 65 older (≥60 years) and 54 young (18-40 years) adults from an ongoing R01 study (NCT04487041) were analyzed by flow cytometry for T cell phenotype and SA β-Gal expression. IL-2-induced STAT5 phosphorylation was assessed by phosphoflow in FoxP3- CD4+ T cells and quantified as the fold-change (FC) in median fluorescence intensity (MFI) after IL-2 stimulation. Bulk RNA-seq was performed on anti-CD3/anti-CD28-stimulated SA β-Gal+ and SA β-Gal- CD4+ T cells FACS-sorted from 3 older donors. Flow data were analyzed in OMIQ with Welch’s or paired t-tests. RNA-seq analysis was performed with DESeq2 and EnrichR in R. Results Older adults had higher frequencies of CD25 + (7.1% vs. 6.1%, p = 0.03) and SA β-Gal + (8.1% vs. 5.6%, p = 0.005) CD4+ T cells and elevated IL-2-induced STAT5 (2.9 vs. 1.4, p < 0.0001). CD25 expression was ∼3-fold higher in SA β-Gal+ vs. SA β-Gal- T cells (p < 0.0001). We identified 2,130 DEGs in SA β-Gal+ vs. SA β-Gal- T cells, with upregulation of senescence genes CDKN1A (p21) and CDKN2A (p16), and Th1 transcription factor TBX21 (T-bet), and downregulation of follicular helper (Tfh) differentiation genes TCF7, LEF1, SELL, and CCR7. FoxP3 expression was not increased in SA β-Gal+ cells. IL-2-STAT5 signaling was the second most enriched MSigDB term in SA β-Gal+ T cells. Conclusion CD25+ and senescent CD4+ T cells accumulate with age and exhibit enhanced IL-2-STAT-5 signaling and Th1 polarization, and reduced Tfh-associated gene expression, providing a potential mechanism through which CD4+ T cell senescence contributes to age-related immune dysfunction. Funding Source National Institute on Aging (NIA) - National Institutes of Health (NIH) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Differential effect of Recombinant Zoster Vaccine (RZV) and Zoster Vaccine Live (ZVL) in a controlled human infection model (CHIM) of VZV reactivation 2307815
Abstract Introduction HZ results from uncontrolled VZV reactivation. Both ZVL and RZV protect against HZ, with RZV (AS01B-adjuvanted gE glycoprotein) showing higher efficacy. To investigate mechanisms underlying this difference, we developed a CHIM mimicking VZV reactivation. Methods A live VZV challenge (intradermal ZVL) was given to 105 adults ≥50 years immunized with ZVL or RZV 6-12 months (Recent) or ≥ 5 years (Remote) prior. We profiled virologic, transcriptional and immunologic responses in skin and blood on Days 0, 1, 3 and 7 post-challenge. Results Presence of VZV mRNA in all D1 skin biopsies and of VZV DNA in 74% D1 blood samples indicated ongoing viral replication that tapered through D7. D3 DNAemia was more common in Remote than Recent vaccinees, consistent with waning immunity. Plasma cytokines increased on D1 in all groups with higher D3-D7 levels in the Remote ZVL. Blood immune cell profiling showed higher D1 CD8+CTL and D3 CD4+CTL in Recent RZV, and higher D7 T cell proliferation in Remote ZVL. Higher CD4+CTL on D1 correlated with lower incidence of DNAemia on D3 indicating control of viral replication. Local responses included transcriptome changes from D1 in both groups; broader and more persistent cytokine induction in ZVL than RZV from D1; and D1 CD8+CTL and Teff expansion in RZV and D3-D7 innate and T cell expansion in ZVL. Overall, ZVL had delayed T cell and higher inflammatory responses than RZV. TRB sequence matching of VZV- and gE-specific cells derived from blood with D3 skin and D0 total blood showed higher total VZV-specific T cell clonotypes in skin and higher CD4+ and CD8+ gE-specific clonotypes in skin and blood in RZV than ZVL. Conclusion The CHIM effectively reproduced VZV reactivation. Early robust CD4+CTL responses in blood correlated with protection against VZV replication. Higher abundance of gE-specific T cells in blood, faster mobilization to the site of VZV infection, and lower local inflammation conferred by RZV than ZVL vaccination may explain its superior protective effect. Funding Source 1U01AI141919-01; 2U01AI141919-06 (NIAID) Topic Categories Vaccines and Immunotherapy (VAC)
Characterization of Non-Neutralizing Antibody Responses in a Dengue Virus-4 Human Challenge Model 2243013
Abstract Introduction Primary infection with one of the four dengue viruses (DENV-1—4) induces serotype-specific antibodies (Abs) that confer durable protection and cross-reactive Abs that offer transient immunity. While neutralizing Ab titers are commonly used to assess protection, they do not reliably predict disease outcome, and the role of Abs that mediate non-neutralizing effector functions remains poorly understood. We present an approach to evaluate the non-neutralizing Ab (nNAb) response to experimental DENV infection in humans. Methods In a phase 1 open-label study, 14 dengue—naïve subjects were subcutaneously inoculated with a slightly attenuated, live DENV-4 strain, H-241. Plasma was collected from pre- and 6 months post-infection to measure Ab-dependent complement deposition, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity (ADCC). All assays used target cells infected with DENV or transfected to express DENV-derived surface-bound NS1 protein. Results Infected subjects generated DENV-4-specific complement-fixing Abs and anti-NS1 Abs that facilitated monocyte-mediated phagocytosis of target cells. DENV-4-specific Abs also mediated NK cell activation in a subset of subjects. Conclusion Future work will focus on extending this functional study to recipients of a tetravalent vaccine followed by DENV-1 challenge to investigate how nNAbs correlate with disease protection/risk, and broaden our understanding of immune defense against dengue beyond neutralizing Abs. Funding Source DOD Topic Categories Viral Immunology (VIR)
Carbonic anhydrase 1 is a potent therapeutic target for mucosal and connective tissue mast cells 2261031
Abstract Introduction Mast cells (MCs) are innate immune cells that play an important role in mediating allergic and inflammatory responses. MCs are derived from hematopoietic progenitor cells (HPCs) which migrate from the bone marrow to the periphery where they mature and reside. Mature mast cells are further classified as connective tissue mast cells (CTMC) and mucosal mast cells (MMC) based on their location, granule content and function. Upon activation, mature mast cells play important roles in promoting inflammatory diseases like allergic inflammation, mastocytosis, and autoimmune diseases via their release of diverse effector molecules. Despite the therapeutic need, a gap in understanding the factors that regulate mast cell development has limited our ability to design novel therapeutics to target mast cells. Our previous studies have identified a population of HPCs that possess mast cell potential and are defined by their expression of the metabolic enzyme carbonic anhydrase (Car)1. Here we show that genetically and pharmacologically targeting Car1 is sufficient to prevent the development of both CTMC and MMC in models of mastocytosis and atopic dermatitis-like diseases. Collectively, these studies demonstrate that Car1 is a viable therapeutic target for the treatment of pathologic CTMC as well as MMC and identify Car1 as a critical regulator of these distinct mast cell populations. Methods Genetically and pharmacologically targeting Car1 in both CTMC and MMC in models of mastocytosis and atopic dermatitis-like diseases. Results Here we show that genetically and pharmacologically targeting Car1 is sufficient to prevent the development of both CTMC and MMC in models of mastocytosis and atopic dermatitis-like diseases. Conclusion Collectively, these studies demonstrate that Car1 is a viable therapeutic target for the treatment of pathologic CTMC as well as MMC and identify Car1 as a critical regulator of these distinct mast cell populations. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)