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CMPK2 controls metabolic adaptation and GDF15 induction in inflammatory macrophages 2300489
Abstract Introduction CMPK2 is a mitochondrial monophosphate nucleotide kinase robustly induced during inflammatory macrophage activation, increasing the production of both mtDNA and the nucleotide analog ddhCTP, which activate NLRP3 and cGAS/STING pathways and act as anti-viral molecules, respectively. However, its role in macrophage immunometabolism is not well defined. Here, we studied how CMPK2 influences macrophage metabolic rewiring and inflammatory programs. Methods Bone marrow-derived and peritoneal macrophages from WT and myeloid-specific CMPK2 knockout (KO) mice were treated with LPS, LPS+IFNg, and IL-4 for 24 hours. GDF15 expression and release were assessed by RNA sequencing, qPCR, WB, and ELISA. ATP production, OCR, and ECAR were measured by Cell-Titer Glow and Seahorse bioanalyzer, and the ETC complex abundances by WB. Glycolytic and TCA cycle metabolites were measured by 1H-NMR and GC/MS. Human osteoarthritis (OA) synovium from total knee arthroplasty patients was analysed for CMPK2, GDF15, and macrophage markers (CD68, TREM2, CD206) by IHC. Results CMPK2 KO macrophages showed impaired bioenergetics, including reduced total ATP, diminished glycolytic- and OxPhos-derived ATP, and marked lower basal, maximal, and spare respiratory capacity. CMPK2 deficiency also reduced ETC Complex I and III and decreased pyruvate, lactate, and multiple TCA cycle intermediates, including succinate, fumarate, and itaconate. This was accompanied by reduced GDF15 mRNA, protein, and release in inflammatory macrophages and LPS-challenged mice. Synovial CD68+TREM2+ macrophages located in the inflamed synovial lining in human OA synovium highly expressed CMPK2 and GDF15. Conclusion CMPK2 is required for the metabolic and bioenergetic programs that sustain inflammatory macrophage activation and GDF15 induction, and emerges as a potential therapeutic target for inflammatory diseases, modulating inflammatory metabolism and GDF15-driven pathways. Funding Source Health Science Research Grant, UC San Diego Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
Comprehensive Mapping of Immune Nanobody Repertoires with NanoMAP 2267451
Abstract Introduction Nanobodies have recently emerged as an alternative to classical antibodies in therapeutic and diagnostic contexts, promising improved stability and simpler manufacturing. However, many labs still rely on low throughput conventional screening methods for nanobody discovery. Here we report streamlined experimental and computational tools for discovery of nanobodies, permitting deep characterization of the binding properties of immune repertoires. Methods To improve nanobody discovery, we developed NanoMAP, an integrated experimental and computational pipeline for nanobody discovery. We immunized alpacas with a pool of antigens, and created a phage display library from circulating B-cells. We then panned this phage display library on each antigen separately, and used competitors or antigen variants to assess complex binding phenotypes of the immune repertoire. Finally, we sequenced the panned libraries and developed a clustering method that allows data to be aggregated within B-cell clonal families, improving signal-to-noise ratios and reducing the complexity of the repertoire. Results We tested NanoMAP on three distinct pools of targets, collecting data on close to 1M unique nanobody sequences. We found that our specialized clustering method outperformed standard sequence clustering, producing clonal families that are coherent in sequence and function. Conclusion By aggregating sequencing data within clonal families, NanoMAP produced reliable and rich data on binding phenotypes for each antigen. Using this information, we discovered nanobodies recognizing functionally relevant, and evolutionarily conserved sites on each antigen, demonstrating the broad utility of our methods. Funding Source NIAID R01AI25704, NIGMS 5K12GM133314-07 Topic Categories Computational and Systems Immunology (COMP)
Regulatory T cell migration and accumulation in tumors and secondary lymphoid tissue during murine 4T1 breast cancer progression 2305484
Abstract Introduction Infiltrating regulatory T cells (Tregs) within tumor microenvironments suppress tumor immune responses and are correlated with increased tumor growth. However, the mechanisms regulating distribution of Tregs within tumors or nearby tumor-draining lymph nodes (dLN) is not fully understood. During lymphocyte trafficking, L-selectin functions to regulate migration of lymphocytes to lymph nodes through high endothelial venules (HEV). L-selectin can also mediate lymphocyte trafficking to sites of inflammation by binding to ligands present on inflamed endothelium. Methods Using a murine 4T1 breast cancer model in L-selectin deficient mice (Foxp3EGFP/L-selectin-/- ), Treg cell migration to tumors, draining and non-draining lymphoid tissue during tumor progression was tracked and quantified by flow cytometry. Results Here we demonstrated L-selectin facilitates Treg cell distribution and migration within secondary lymphoid and 4T1 tumor tissue. Treg populations preferentially accumulated in tumor dLNs and increased at higher rates than conventional CD4+ T cell populations during tumor progression. L-selectin deficient mice (Foxp3EGFP/L-selectin-/- ) had significantly reduced numbers of Treg populations within the dLN, ndLN and within tumor tissue. Migration of Tregs into dLNs occurred primarily from the blood through HEV and was L-selectin-dependent. However, Treg cells were also able to enter dLNs from the tumor through afferent lymphatics. In L-selectin deficient mice, Treg cell migration into solid tumor tissue was also reduced during late stage tumors. Conclusion Therefore, these studies demonstrate Treg influx into tumor and dLNs occurs by specific homing of Tregs to these tissues and may be dependent on Treg expression of L-selectin. Funding Source UWM Chancellor’s Office and the UWM Graduate School Research Growth Initiative (RGI) program; UWM Graduate School through an Advanced Opportunity Program (AOP) Fellowship Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)
Ptpn22 Restricts Antiviral B cell Response by Modulating BCR Signaling 2256188
Abstract Introduction B cell receptor (BCR) signal strength governs effector functions that promote viral clearance. Defining how BCR signaling is regulated during infection can uncover mechanisms shaping antiviral B cell responses. Ptpn22, commonly associated with autoimmunity, encodes the immune-specific tyrosine phosphatase PEP, a proposed regulator of BCR signaling whose role remains controversial and unexplored in viral infection. PEP-deficient (PEP-null) mice infected with the chronic virus LCMV-Cl13 clear infection, whereas wild-type (PEP-WT) mice do not, suggesting PEP suppresses antiviral immunity and promotes viral persistence. However, if and how PEP may be restricting antiviral B cell function remains unknown. We hypothesize that PEP negatively regulates BCR signaling, thereby constraining B cell activation and effector function during chronic infection. Methods To investigate this, we first examined BCR signaling kinetics in PEP-WT and PEP-null B cells following anti-IgM stimulation by western blot. To assess downstream effects, we next used flow cytometry to profile PEP-null B cell subsets in spleen, lymph nodes, and bone marrow after various days post LCMV-cl13 infection. Finally, we evaluated PEP-null B cell function by quantifying antiviral antibody responses across infection timepoints. Results Key findings confirm that PEP-null B cells exhibit accelerated signaling, with earlier phosphorylation of SYK (3 min) and ERK (15 min) relative to PEP-WT. Correspondingly, PEP-null mice displayed an altered B cell landscape with increased plasmablasts and plasma cells, as well as higher anti-LCMV-Cl13 antibody titers detected up to three days earlier than PEP-WT. Conclusion Together, these findings demonstrate that PEP-null B cells have enhanced and accelerated BCR signaling, promoting rapid differentiation of antibody-secreting cells and elevated antibody production during chronic viral infection. This work defines a novel role for Ptpn22 and PEP in tuning BCR signaling and regulating humoral immunity. Funding Source CBID CoBRE Research Grant (KU)-P20GM113117* Topic Categories Viral Immunology (VIR)
Antigen dependency defines memory and exhausted CD8 T cell fates 2257447
Abstract Introduction Antigen experienced T cell subsets are heterogenous, and can be described based on their phenotype, function, and migration properties. We wished to better understand how antigen stimulation regulates the trafficking, survival, differentiation and developmental plasticity of T cell subsets. Methods We developed a new mouse model that allows for the in vivo excision of the P14 TCR on a defined fraction of antigen-specific CD8 T cells. Specifically, we engineered a novel tamoxifen-inducible TCR-knockout transgenic mouse using a rAAV6 vector encoding for the floxed P14 TCR gene. This model allows for permanent cessation of antigen stimulation on select T cells at time points of our choosing. Results After acute infection with LCMV Armstrong, elimination of the TCR on established memory CD8 T cells maintained stable subset composition, but resulted in detectable downregulation of exhaustion markers, indicating that TCR sensing occurs among steady-state memory T cells. TCR deletion during chronic infections (LCMV Minnesota and LCMV Cl13 ± aCD4 depletion) affected both the population structure and phenotype. Terminal (Tex/TXt), as well as progenitor exhausted (Tpex/TXp) T cell subsets substantially declined after TCR deletion in persistently viremic infection with LCMV Cl13 + aCD4. However, the loss of Tpex/TXp after TCR deletion was moderated in settings of chronic infections without CD4 T cell depletion, in which most host viral load was more tightly controlled. Conclusion These results demonstrate that the antigen dependence of Tpex/TXp depends on context, and a subset may survive in the absence of further antigen stimulation, which has important implications for maintaining immunity to chronic infections and cancer. Funding Source Walter Benjamin Program, DFG Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Gestational diabetes fetal programming activates the PI3K-Akt and NETosis inflammatory axis in neonatal endothelial colony-forming cells 2259927
Abstract Introduction Gestational diabetes mellitus (GDM) is a critical model for studying the developmental programming of cardiovascular risk (CR), where intrauterine inflammation primes the offspring for future diseases. To understand this early-life predisposition, we investigated the functional phenotype of neonatal ECFCs isolated from cord blood of infants born to women with GDM versus lean controls. Methods We performed a transcriptomic analysis utilizing publicly available bulk RNA sequencing data for 60 individuals (SRA Project PRJNA952510). Quality control and filtering was first applied to the sequencing data, derived from well-characterized neonatal ECFCs (CD31+/vWF+), where all samples met the RNA Integrity Number > 7.0 threshold. Results Our analysis reveals a shift toward a pro-inflammatory and pro-thrombotic phenotype already present in GDM-exposed neonatal ECFCs, indicating the advanced programming of fetal endothelium by the GDM environment. Specifically, the transcriptome displayed robust enrichment for the PI3K-Akt signaling pathway (AKT3, HSP90AA1), acting as a central hub for chronic cellular stress and inflammatory survival. Crucially, pathway analysis demonstrated significant upregulation of genes promoting extracellular matrix remodeling and, subsequently, the highly destructive process of neutrophil extracellular trap (NET) formation (SELE, SPP1). This suggests that neonatal ECFCs are actively upregulating molecules that initiate ECFC-neutrophil crosstalk. Conclusion Our data unveils a novel mechanism where GDM exposure activates an underlying inflammatory nexus (PI3K-Akt) in neonatal ECFCs, directly promoting vascular immunopathology via NETosis at birth. These findings position neonatal ECFCs as dysregulated early drivers of sterile inflammation in GDM-exposed offspring compared to lean-exposed controls. Mechanistically, this effect is mediated by the hyperactivation of the PI3K-Akt/NET axis, identifying this pathway as promising target for early-life intervention to mitigate lifetime CR. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)
T6B-mediated inhibition of the miRISC decreases myeloid-derived suppressor cell abundance and enhances survival in a murine ovarian cancer model 2259723
Abstract Introduction MicroRNAs play a critical role in regulating post-transcriptional gene expression. Dysregulation of microRNA concentrations has been associated with tumor initiation, progression, and metastasis in various cancer models. Previous studies demonstrated that global inhibition of the microRNA-induced silencing complex (miRISC) assembly can be accomplished through peptide-mediated interreference. Here, we implement a similar mechanism by overexpressing the peptide (T6B) in ovarian cancer (OvCa) cells to evaluate the effect of T6B-mediated tumor miRISC inhibition on the immune tumor microenvironment (TME). Methods In vivo murine studies using mFTCβ +/- T6B OvCa cells were conducted to assess the impact of T6B-mediated tumor miRISC inhibition on the immune TME. Harvested tumors and ascites were used to evaluate immune cell phenotypes and cytokine distribution via flow cytometry, immunohistochemistry (IHC), and Western blotting. RNA sequencing of mFTCβ cells and NanoString analyses of harvested tumors were used to identify T6B-mediated gene expression levels compared to controls. Results We observed an increased probability of survival in T6B-overexpressing mFTCβ tumor-bearing mice compared to control tumors. Flow cytometric and IHC analyses of ascites and tumors, respectively, revealed an increase in dendritic cell and macrophage populations, as well as a decrease in Ly6G+ granulocytic cells in T6B-overexpressing tumors. Gene expression profiling of myeloid cells revealed that tumor T6B downregulated the expression levels of factors associated with immunosuppression, such as Cxcl2, Arg1, and Marco. Moreover, the TME of T6B-overexpressing tumors exhibited decreased inflammatory factors — such as IL-6, CXCL1, CCL5, CCL11, and G-CSF — compared to control. Conclusion The results of this study offer significant insight into the immune TME of T6B-overexpressing OvCa tumors, providing the groundwork for further exploration into utilizing tumor-specific T6B-mediated inhibition of miRISC formation. Funding Source MWRI Startup Fund (Awarded to Sandra Cascio) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Bioinspired Rheological Sensing for Robotic Liquid Identification in Sealed Containers via Ultrafast Incipient Slip Detection
ABSTRACT The rapid, non‐invasive identification of sealed liquids is crucial for ensuring counter‐terrorism security and improving operational efficiency in various scenarios such as airports and large‐scale events. Existing technologies are limited by invasive procedures or reliance on vision. In this research, we developed a bioinspired iontronic tactile sensor with an interlocked “protrusion‐groove” structure. Its unique biomimetic structure allows for the detection of static pressure and incipient slip within a single device, with a peak sensitivity of up to 1873.83 kPa − 1 in the low‐pressure regime and a slip response time as fast as 46 ms. A robotic fingertip integrated with this sensor can effectively capture the transient signals generated by shaking liquids. Furthermore, by extracting physical features from the signals and applying a Random Forest classifier, we developed a non‐visual liquid identification system that achieves an accuracy of 99.04% ± 0.47% for different liquids. This work provides a practical solution for non‐visual liquid identification and demonstrates a robust approach for enabling robots to achieve multimodal tactile perception.
Antioxidant Proline Carbon Dot‐Composited Poly(L‐methionine) Hydrogel Restores Mitophagy to Alleviate Cellular Senescence in Intervertebral Disc Degeneration
ABSTRACT The senescence of nucleus pulposus cells (NPCs) is a hallmark pathological feature of intervertebral disc degeneration. Senescent NPCs exhibit mitochondrial dysfunction and impaired mitophagy, thus compromising mitochondrial turnover and quality control. Current therapeutic strategies predominantly target external risk factors but do not restore mitophagy. In this study, a proline carbon dot‐composited poly(L‐methionine) (EG 45 M 25 /Pro‐CD) hydrogel is developed and employed to re‐establish the impaired mitophagy pathway. Expression levels of Parkin and phosphorylated Parkin increase, indicating successful activation of the mitophagy pathway. Restoration of mitochondrial function markedly reduces oxidative stress and the senescence‐associated secretory phenotype in NPCs. The levels of type II collagen and aggrecan recover to 84.63% and 77.31% of their normal ones, respectively. In an animal model, the administration of Pro‐CD in a thermo‐sensitive and injectable hydrogel effectively preserves the water content and height of intervertebral discs while maintaining the structural integrity and functional activity of NPCs. The mean Pfirrmann grade improves from 5.00 in the Control group to 3.33 after treatment, whereas the disc height index recovers to 82.06% of the physiological value. Collectively, our findings demonstrate that the EG 45 M 25 /Pro‐CD composite hydrogel has therapeutic potential to promote endogenous disc regeneration by enhancing mitophagy, re‐establishing redox homeostasis, and alleviating NPC senescence.
Integrated multi-omics analysis identifies microbial and metabolic signatures and drivers of CNS autoimmunity 2305382
Abstract Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system driven by genetics and the environment. People with MS exhibit distinct gut microbiomes and altered systemic bacterial produced metabolites, including tryptophan-derived products. However, defining microbial metabolic drivers of MS remains challenging. We previously showed that colonization with Limosilactobacillus reuteri (L. reuteri) worsens disease in a model of MS, experimental autoimmune encephalomyelitis (EAE), in a tryptophan-dependent manner. Methods We integrated microbiomic and metabolomic datasets from a longitudinal EAE study using high and low tryptophan diets in mice colonized or not with L. reuteri to identify coordinated microbe—metabolite modules, and used Random Forest modeling to identify predictors of EAE severity. Candidate metabolites were tested in vivo for their effects on EAE and immune response. Results During short-term dietary intervention, L. reuteri colonization had a greater effect on the gut microbiome than did tryptophan bioavailability. With longer dietary exposure and EAE progression, high dietary tryptophan and L. reuteri colonization synergized to elicit profound changes in the microbiota, including altered abundance of distinct Lachnospiraceae, Blautia coccoides, and Akkermansia muciniphila. Multiomic integration revealed distinct clusters of metabolites and microbiota enriched for functional pathways, including bile acid and tryptophan metabolism. Metabolites outperformed microbiota in predicting EAE severity, identifying p-cresols and indoles as associated with disease worsening. Treatment with p-cresol or 3-indoleglyoxylic acid exacerbated EAE, enhanced proinflammatory T cell responses and increased cerebellar pathology. Conclusion These data indicate that dietary response is modulated by gut microbiome composition and that integrated microbiomic-metabolomic analyses can predict drivers of disease worsening in MS. Funding Source R01 NS097596 from NIH/NINDS and RG-2407-43682 from the NMSS to DNK and F31NS120381-01A1 from NIH/NINDS to TLM, training grant T32AI055402-16A1 to Dr. Gary Ward Topic Categories Mucosal and Regional Immunology (MUC)
Structural characterization of broadly neutralizing and potent V1/V2 apex antibodies elicited in rhesus macaques through immunization 2267435
Abstract Introduction Eliciting broadly neutralizing antibodies (bnAbs) that target the HIV-1 envelope glycoprotein (Env) remains a central goal of HIV vaccine design. Among these, antibodies directed against the V1/V2 apex epitope represent an important class of bnAbs with potent and broad neutralizing activity. As HIV-1 bnAbs often require extensive somatic hypermutation, a complete understanding of bnAb maturation pathways is essential to guide bnAb development during immunization. Methods Here, we combine negative-stain electron microscopy polyclonal epitope mapping (nsEMPEM) and cryo-electron microscopy (cryo-EM) to characterize potent and broad V1/V2 apex antibodies elicited in rhesus macaques immunized with the germline targeting immunogen CAP256.GT1. Results nsEMPEM analyses identified apex-targeting antibodies present in sera twelve weeks after the priming immunization, indicating successful engagement of desired precursor lineages. Polyclonal epitope mapping and biophysical characterization of antibody responses to candidate immunogens guided subsequent boosting strategies and successfully facilitated bnAb development. High-resolution cryo-EM structures of bnAbs isolated over a year after the priming immunization revealed that these antibodies recapitulate the binding modes and structural features observed in human-derived V1/V2 apex bnAbs isolated from chronically infected donors, further supporting the utility of nonhuman primate models for evaluating germline-targeting immunogens. Conclusion Together, these findings demonstrate the applicability of structural and polyclonal epitope mapping approaches in guiding rational vaccine design. Funding Source Howard Hughes Medical Institute Emerging Pathogens Initiative; Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD); National Institutes of Health National Institute of Allergy and Infectious Diseases (NIH NIAID) Grants R01 AI 167716, R61 AI 161818, P01 AI 177683 Topic Categories Vaccines and Immunotherapy (VAC)
Adenosine Suppresses Microglial Immunity in Breast Cancer Brain Metastasis 2306834
Abstract Introduction Breast cancer brain metastasis (BCBM) is a fatal and increasingly common complication in breast cancer patients. The blood—brain barrier (BBB) limits the efficacy of many conventional therapies, contributing to poor prognosis. Immune cells, however, can cross the BBB, making immunotherapy a promising treatment strategy. Microglia, the brain’s resident macrophages, are first responders in central nervous system diseases and can eliminate cancer cells via phagocytosis. Microglia are part of a larger immune microenvironment and interact with various other immune cells, including regulatory T cells (Tregs). Tregs normally maintain self-tolerance, but can also suppress anti-tumor immunity. However, the interaction between Tregs and microglia during BCBM remains poorly understood. Preliminary analyses highlight an adenosine-mediated signaling axis, suggesting a mechanism by which Tregs may inhibit microglial function. We hypothesize that Treg-derived adenosine, produced via CD73, suppresses microglial anti-tumor activity by signaling through the adenosine A3 receptor (A3AR) on microglia. Methods Here, we employ a combination of in vitro and in vivo studies. In vitro, microglia were treated with lipopolysaccharide and adenosine, and activation was assessed by flow cytometry. In vivo, we modeled BCBM using intracranial injection of E0771 breast cancer cells in mice, treated animals with a CD73-blocking antibody, and analyzed immune responses by flow cytometry. Results In vitro, we demonstrated that treatment of microglia with soluble adenosine reduced microglial activation. Treatment of mice bearing BCBM with a CD73 blocking antibody lead to a moderate decrease in tumor burden, expansion of T cells, and increased microglial antigen presentation. Conclusion These findings support that Treg CD73-derived adenosine may contribute to BCBM immunosuppression through A3AR signaling in microglia. Funding Source NIH T34GM136498 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Therapeutic Efficacy of a Novel Oral NRF2 Activator in a Murine Ulcerative Colitis Model 2256177
Abstract Introduction Ulcerative colitis (UC) arises from three interconnected pathological processes: impaired epithelial barrier function, persistent mucosal inflammation, and elevated oxidative stress. NRF2 (NF-E2—related factor 2), a transcription factor that serves as a central coordinator of cellular defense programs, orchestrates antioxidant defenses, inhibits inflammatory pathways, and facilitates epithelial regeneration. Beyond its cytoprotective functions, NRF2 activation regulates intestinal immunity and influences UC development. To address the multifactorial drivers of UC, we have developed a novel, potent, and selective oral NRF2 agonist that shows efficacy in a preclinical model of UC. Methods MTAI-1025 was evaluated for in vivo efficacy in a 14-day DSS-induced mouse colitis model. Mice received compound (30, 60, or 100 mg/kg, PO, daily from days -1 to 12), vehicle, or a reference anti-IL-12p40 monoclonal antibody (10 mg/kg, IP on days 6 and 9). Inhibition of immune-related signaling was determined in LPS/IFN-g-stimulated monocyte-derived DCs. MoA studies were conducted using HEK293 cells stably transfected with FLAG-NRF2 and HA-KEAP1 (wt or C151) or HEK293 cells stably transfected with split-luciferase tagged Cul3 or NRF2 and KEAP1. Results In the DSS model, MTAI-1025 dose-dependently reduced DAI and body weight loss. Treatment also improved mucosal architecture, decreased inflammatory cell infiltration, and lowered pro-inflammatory cytokine levels (IL-1β, IL-6, CXCL1) compared with controls. Dose-dependent activation of the NRF2 transcriptional pathway was confirmed by induction of NQO1 mRNA. MTAI-1025 reduced cytokine secretion in MoDC cells and specifically regulated the dynamics of the NRF2/KEAP1/Cul3 complex at KEAP1 C151. Conclusion These results demonstrate that MTAI-1025 effectively attenuates inflammation and exhibits efficacy in an in vivo model of UC, supporting its potential as a first-in-class oral NRF2-targeted therapy for UC. Funding Source Montai Therapeutics Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Exploring the origins of adaptive immunity 2257946
Abstract Introduction Jawless vertebrates (Agnathans), such as the sea lamprey, persist today with minimal evolutionary changes compared to their first appearance on Earth nearly 500 million years ago. Unlike jawed vertebrates (Gnathostomes), lampreys evolved a distinct adaptive immune system that uses variable lymphocyte receptors (VLRs) instead of immunoglobulins as their antigen receptors. Their B- and T-like lymphocytes proliferate in response to antigenic stimulation, yet it remains unknown if these cell types coordinate together. Furthermore, lampreys lack canonical lymphoid organs such as spleen, bone marrow, and lymph nodes. Therefore, where do immune niches exist in the sea lamprey, and does coordinated lymphocyte response contribute to adaptive immunity? Methods We have transcriptionally characterized VLRA+ T-like cells and VLRB+ B-like cells in circulation. Furthermore, we have developed methods for flow cytometry and IHC in the blood, gills, liver, and intestine of the sea lamprey. We are assessing the sea lamprey for immune niches and the necessity of T-like cells in the adaptive immune response. Results We have transcriptionally characterized VLRA+ T-like cells and VLRB+ B-like cells in circulation. Furthermore, we have developed methods for flow cytometry and IHC in the blood, gills, liver, and intestine of the sea lamprey. Currently, we are assessing the sea lamprey for immune niches and the necessity of T-like cells in the adaptive immune response. Conclusion This work will provide insights into how a convergently evolved adaptive immune system functions and lay the foundation for future investigation into lymphocytes of the sea lamprey. Funding Source The Hypothesis Fund Topic Categories Veterinary and Comparative Immunology (VET)
Peroxisome proliferator-activated receptor delta loss reduces alloreactive CD4 T cell proliferation and lessens graft-versus-host-disease severity 2260348
Abstract Introduction Our lab previously demonstrated that murine alloreactive T cells increase fatty acid oxidation (FAO) during graft-versus-host disease (GVHD), and more recently, we observed higher levels of peroxisome proliferator-activated receptor delta (PPARγ) in donor T cells after transplantation. Therefore, we hypothesize that PPARγ-driven FAO in T cells plays a role in GVHD development. Methods We first conducted in vitro assays on wild-type (WT) T cells using a PPARγ-specific agonist. Then, we created a T cell conditional PPARγ knockout (KO) mouse strain to supply donor T cells for our GVHD model. Results We found that GW501516-treated T cells stimulated by MLR had increased transcript levels of FAO genes: a 2-fold increase in acetyl-CoA acyltransferase 2 (Acaa2), a 4-fold increase in perilipin-2 (Plin2) in CD8 T cells as well as a 6-fold (Acaa2) and an 8-fold (Plin2) increase in CD4 T cells (multiple t-test, p = 0.01). PPARγ agonism of MLR-stimulated T cells also increased FAO in CD8 and CD4 T cells in radiolabeled palmitate oxidation assays (unpaired t-test, p = 0.01). In our GVHD model, deleting PPARγ in donor T cells improved survival (median: WT = 20; KO = 33) compared to WT donor T cell recipients (one-way ANOVA, p = 0.01). Notably, on Day 7 after transplantation, we saw a significant decrease in proliferating donor CD4 T cells, but not CD8 T cells, from PPARγ KO donors. We also noted reduced proliferation of CD4 PPARγ KO T cells on Day 5 post-transplant and during in vitro stimulation assays (Walsh t-test, p = 0.01). In the context of a CD4-only GVHD model, survival in PPARγ KO recipients was even more improved compared to WT recipients (median: WT = 51; KO=undef). Flow cytometry and histology analysis on Day 21 post-transplant showed lower donor lymphocyte frequencies and fewer total donor CD4+ T cells in the liver and small intestines of PPARγ KO recipients. Conclusion These data overall indicate that the loss of PPARγ impairs the ability of donor T cells to infiltrate target organs during GVHD progression. Funding Source T32- AI074490 from the NIAID (Dr. Angus Thomson, PI) Topic Categories Transplantation Immunology (TRAN)
The Impact of Environmental Respiratory Hazards on Pulmonary Macrophages 2259898
Abstract Introduction Environmental respiratory hazards (ERH) are associated with pulmonary disease and lung cancer. Yet, the mechanisms by which ERH disrupt lung immunity, and pulmonary macrophages (pMac) in particular, are largely unknown. Methods FVB mice were exposed to aerosolized ERHs for 1wk or 4wks and pMacs were assessed by ATACseq and RNAseq. Primary and Raw264.7 macrophage function was determined after co-culture with ERHs. Spatial transcriptomics (GeoMx) for pMac and epithelial gene expression was performed on paired non-malignant lung tissue and lung tumors from patients (N = 28). Patient ERH exposure levels were constructed from 1-year prior to surgery using census tract-level daily PM2.5, ozone, and NO2 estimates from the EPA and confirmed with polarized light microscopy. Results Increasing duration of ERH exposures in mice resulted in a durable epigenetic shift in pMacs from pro-resolution to tissue remodeling. Functionally, ERHs inhibited wound healing and phagocytosis which could be reverted by enhancing mitochondria with the AMPK agonist AICAR. In ERHHigh patients, pMacs in lung tissue expressed gene profiles of complement and coagulation driven by KLF4, PPARγ, and RUNX1. By contrast, pMacs in ERHLow lung drove chemokine signaling via RELA/RELB, STAT4, and IRF8. Notably, epithelial cells surrounding pMacs expressed divergent transcriptional profiles between ERHHigh and ERHLow patients. Tumor-associated macrophages (TAMs) from ERHHigh patients were transcriptionally poised for ECM remodeling and expressed KLF4 and RUNX2, whereas TAMs in ERHLow tumors were enriched for lipoprotein metabolism with RELA, IRF6, and SMAD4 expression. Conclusion ERH exposures result in epigenetic dysregulation in pMacs, inhibiting function, in part, through decreased bioenergetics. Clinically, ERHs were associated with distinct molecular pathways in pMacs which altered surrounding epithelium and may inform precision drug development and risk modeling in lung disease. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)
MOCVD‐Grown MoS <sub>2</sub> Wafers as a Transfer‐Free Platform for Top‐Gate Devices via Dry Interface Engineering
ABSTRACT We uncover the electronic origin of hidden interfacial doping in monolayer MoS 2 single‐crystal wafers grown on sapphire by metal–organic chemical vapor deposition (MOCVD) and establish a transfer‐free top‐gate device platform. Despite structural perfection, as‐fabricated devices exhibit degenerate electron doping and lack a clear off state. Hall measurements quantify an interfacial electron density of 2.7 × 10 12 cm −2 , evidencing substantial charge transfer across the nominal van der Waals interface. Interface‐sensitive spectroscopy, lateral force microscopy, and thermal desorption analysis reveal a buried sulfate‐derived layer accompanied by a water‐like interfacial structure that acts as an intrinsic electron donor. A purely dry H 2 /Ar annealing process selectively removes these species, suppressing charge transfer and restoring intrinsic FET characteristics without transfer or wet processing. Through this dry interface engineering approach, we demonstrate MOCVD‐grown single‐crystal MoS 2 wafers as a robust, transfer‐free platform for the reliable evaluation of intrinsic gate stacks and device performance.
Integrin αV-Mediated Spatially Restricted Activation of Autocrine TGF-β1 Programs Cytotoxic Tissue-Resident NK Cell Differentiation 2310290
Abstract Introduction Tissue-resident natural killer (trNK) cells differentiation requires TGF-β1 for tissue residency. As TGF-β1 often suppresses NK function, the mechanism by which human NK cells acquire residency without compromising cytotoxicity remains unclear. We hypothesized that Integrin αV-mediated spatially restricted activation of autocrine TGF-β1 programs cytotoxic trNK cells in the presence of IL-15, thereby preserving effector function. Methods Conventional PBNK cells were cultured on integrin αVβ8- coated surfaces supplemented with IL-15. Phenotype, signaling, cytotoxicity (against SCC cells ), and transcriptional profiles were characterized using flow cytometry, functional assays, SMAD2/3 phosphorylation analysis, and bulk RNA-seq. Human NSCLC CosMx SMI datasets were used for spatial transcriptomics to map trNK cells and integrin αV expressing populations. Results Integrin αVβ8 exposure elicited a trNK-like phenotype in IL-15-stimulated PBNK cells while retaining strong tumor cell killing. Mechanistically, IL-15 induced latent TGF-β1 secretion, which accumulated on the NK cell surface and was locally activated by integrin αVβ8. This resulted in spatially confined, autocrine TGF-β1 signaling, triggered low-level SMAD2/3 phosporylation. Transcriptomic analysis revealed enrichment of cytotoxic, metabolic, and integrin-associated pathways, distinct from NK cells differentiated with high-dosage soluble TGF-β1 treatment. Spatial transcriptomics of a human NSCLC showed that CD49a+CD103+ trNK cells preferentially localized within stromal regions, in close proximity to epithelial cells expressing integrin αV. Conclusion We uncover a novel integrin αV-dependent mechanism for the programming of highly cytotoxic CD49a+CD103+ tissue-resident NK cells. This involves PBNK cell-secreted latent TGF-β1, followed by its localized activation via epithelial integrin αV (αVβ6/αVβ8). This spatially confined signaling drives low-level SMAD2/3 phosphorylation to establish tissue residency while preserving effector function. Funding Source NIH (1R35DE030054-01) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Immunomodulatory Tr1 CD4 T cells induced by engineered V1-deleted HIV vaccine candidate decrease the risk of SIV acquisition in macaques 2267572
Abstract Introduction The development of an effective anti-HIV vaccine remains a critical tool to halt the HIV epidemic, particularly due to the limits of the PrEP strategies. CD4+ T lymphocytes play a crucial role in vaccine efficacy; however, they are also the primary target cells for HIV infection. Given these opposing roles, we hypothesized that an in-depth characterization of CD4+ T cell responses to vaccination will elucidate mechanisms of protection. Methods To test this hypothesis, we immunized rhesus macaques using a prime-boost strategy that included a DV1-DNA prime and boosts with ALVAC alone or in combination with DV1-gp120 protein, followed by intravaginal exposures to SIVmac251. We then integrated data obtained by flow cytometry and plasma proteome analyses, as well as transcriptome and chromatin accessibility analyses of CD3+ cells, to investigate how the vaccine shapes the CD4+ T cell immunity and how these responses cooperate in reducing the acquisition. Results We found that DV1 DNA/ALVAC/gp120 vaccine elicited envelope-specific immunomodulatory Tr1 CD4+ T cells, and that total Tr1 responses were associated with a reduced risk of viral acquisition. Transcriptome analyses of CD3+ cells identified vaccine-induced Tr1, as well as IL-27, gene signatures that were associated with a reduced risk, confirming the protective role of these cells. Furthermore, the study of the epigenetic landscape of CD3+ cells revealed that the epigenetic reprogramming of enhancer region upstream of the transcription factors BATF and IRF-1, which are involved in the development of Tr1 cells, correlated with lower viral acquisition. Conclusion These data suggest that immunoinhibitory Tr1 cells contribute to vaccine efficacy by decreasing inflammation and potentially counteracting the development and recruitment of HIV target cells. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)
A systematic evaluation of T lymphocytes and metabolic parameters in childhood obesity: new therapeutic targets for the disease? 2308210
Abstract Introduction According to the World Obesity Federation, it is estimated that 206 million children and adolescents aged 5-19 are obese, and this number will rise by 2030. Due to its high incidence, obesity has become a public health concern, which contributes to alterations in metabolic parameters such as insulin resistance, hyperglycemia, and dyslipidemia. Studies have also reported changes in the number and function of immune cells in obese children. This systematic review aims to determine whether there is a correlation between the number and functions of T lymphocytes and metabolic parameters. Methods A systematic literature search of PubMed, SciElo, and Scopus databases identified studies relevant to our aim, which were included for analysis and discussion. Results Results from the articles showed an increase in Th17, CD8+T effector memory cells, and B cells, but a decrease in T regulatory and natural killer cells in obese children. These results also suggested a correlation between circulating Th17 frequency and fasting insulin, erythrocyte count, and glucose. A relationship was also identified between the Th17/Treg ratio and both total plasma cholesterol and systolic blood pressure. CD8+T effector memory cell counts showed an association with total and visceral fat mass. Similarly, the number of circulating CD19+ B cells was linked to the percentual fat mass, the android/gynoid fat mass ratio, inflammatory markers, and leptin levels. Additionally, both the frequency and absolute number of CD3+CD4+ T cells showed an association with fat mass and circulating leptin. Conclusion Our findings highlight that T cell alterations in obese children are closely related with metabolic parameters, including glucose, insulin, and lipid profile. A better understanding of this association in further research may reveal whether these T cells may be considered new markers for childhood obesity and its clinical outcomes, as well as whether they may represent a promising therapeutic target in obesity. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)