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Identification of a FoxP3-specific small molecule degrader with potent antitumor immunotherapeutic efficacy 2309510
Abstract Introduction T regulatory cell (Treg) infiltration and accumulation within solid tumors leads to tumor immune evasion and is a major barrier to immunotherapy efficacy. Thus, immunotherapeutic strategies aimed at reducing Treg abundance and/or suppressive function hold significant promise for cancer treatment. Efforts to modulate Tregs have largely relied on non-specific molecular or cellular approaches; however, targeting Forkhead box protein 3 (FoxP3), the master transcription factor for Tregs holds significant promise. Here, we report the first FoxP3-specific small molecule degrader which promotes proteasomal degradation of FoxP3 and partially reduces Treg suppressive function. Methods To identify potential hits, we used high content imaging to screen a 640 small, electrophilic compound library. The top hits were resynthesized and validated by flow cytometry in human Treg-like MT-2 cells, leading to identification of our lead compound, termed FD03. To explore the mechanism by which FD03 mediated FoxP3 reduction, we employed co-immunoprecipitation and immunoblotting techniques. We then evaluated therapeutic efficacy of our compound in ex vivo and in vivo settings using flow cytometry as a readout. Results After identifying FD03 as the lead compound, we further characterized its efficacy finding that FD03 had a EC50 of ∼5-10 uM in primary murine and human Tregs. Mechanistically, FD03 degrades FoxP3 in part through facilitating FoxP3 interaction with its E3 ligase, STUB1, to promote FoxP3 ubiquitination-mediated proteasomal degradation. Finally, we characterized the therapeutic potential of FD03: we illustrated that FD03 can reduce Treg suppressive function. Importantly, we demonstrated that FD03 treatment in tumor-bearing mice can decrease tumor burden and skew the immune cell landscape towards an inflammatory phenotype. Conclusion Overall, we demonstrated proof-of-concept and feasibility of targeting FoxP3, which has been known to be “undruggable”, in a chemical manner by small molecules. Funding Source 1F31CA287701-01, T32GM15538-10, 1T32GM149439-01 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Membrane‐Confined Proton Management Orchestrates Proton‐Electron Transfer for Efficient Photocatalytic Hydrogen Peroxide Synthesis
ABSTRACT Coordinating proton and electron transport remains a central challenge in photocatalysis, particularly for H 2 O 2 synthesis, where two‐electron oxygen reduction requires synchronized proton delivery and charge transfer. Here, we report hydroxyl‐functionalized three‐dimensional covalent organic framework (COF) membranes that regulate proton‐coupled electron transfer within ordered nanochannels. Phenolic hydroxyl groups are precisely embedded in the membrane pores as internal proton reservoirs, while the continuous COF framework provides pathways for photogenerated electron transport. By tuning hydroxyl density, the dihydroxylated COF‐2OH membrane establishes an optimal proton‐management microenvironment, promoting O 2 adsorption, charge separation, superoxide/*OOH intermediate formation, and dynamic proton recycling. Under one‐sun irradiation in pure water and ambient air, COF‐2OH achieves an H 2 O 2 production rate of 21.79 mmol g – 1 h – 1 without sacrificial agents or cocatalysts, greatly outperforming the corresponding powder catalyst. Mechanistic experiments and simulations reveal that membrane confinement and hydroxyl‐mediated proton buffering jointly synchronize proton and electron fluxes during continuous photocatalysis. The free‐standing membrane can also be integrated into a tubular Al 2 O 3 ‐supported reactor for in situ H 2 O 2 ‐driven photo‐Fenton degradation of organic pollutants. This work establishes proton‐managing COF membranes as programmable reaction interfaces for efficient solar H 2 O 2 production and integrated water treatment.
Mapping immune landscapes and immunopathomechanisms of inflammatory skin diseases through single-cell multi-omic analysis 2334083
Abstract Introduction Inflammatory skin diseases are a heterogeneous group of chronic disorders characterized by persistent or recurrent inflammation with complex and multifactorial immunopathomechanisms. These diseases share overlapping clinical features and some overlapping molecular mechanisms, despite heterogeneity and complex pathophysiology, although their pathophysiology remains only partially understood. To guide treatment strategies and predict therapeutic response, characterization of shared or disease-specific pathomechanism is needed. In addition, how the clinical and immunological phenotypes of these diseases differ by ancestry remains underexplored. Methods Here, we newly generated a single-cell atlas of inflammatory skin diseases, atopic dermatitis (AD), generalized pustular psoriasis (GPP), plaque psoriasis (PSO), hidradenitis suppurativa (HS) and vitiligo (VIT). Our dataset includes single-cell RNA sequencing of 77 tissue biopsies and 85 peripheral blood mononuclear cell (PBMC) samples from Korean patients, and single-cell resolution spatial transcriptomics on 35 tissue biopsies, with a custom add-on panel to refine immune signatures. Results The tissue dataset enabled cross-disease stratification at the sample level, where heterogeneity in keratinocytes shaped by disease-specific cytokine milieus is a major determining factor in disease stratification. In addition, we identified some disease-specific features, especially in immune cell subsets including resident memory T cells and macrophage subsets, which we show can inform potential druggable targets. In parallel, we have profiled PBMCs using mass cytometry for protein level validation of immune phenotypes. Conclusion These integrated approaches will enable discovery of unique molecular signatures of inflammatory skin diseases, offering insights for the development of treatment strategies and predictive biomarkers for inflammatory skin diseases with overlapping phenotypes. Funding Source Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : RS-2024-00403375) Topic Categories Computational and Systems Immunology (COMP)
Subunit Vaccination Using Atomic Layering Thermostable Antigen and Adjuvant (ALTA®) Platform Elicits Enhanced Humoral and Cellular Immune Responses 2258445
Abstract Introduction Creating effective and thermostable vaccines is of significant relevance for global health. The Atomic Layering Thermostable Antigen and Adjuvant (ALTA®) platform combines spray drying to encapsulate antigens in a sugar matrix for improved thermostability followed by alumina layer coating for temporal control over in vivo release. Given these advantages, understanding the immune response to ALTA® formulated antigens is important. Here, the immune response to ALTA® formulated antigen was described and compared to a set of adjuvanted liquid formulations. In addition, critical attributes of the ALTA® platform driving its immunogenicity were determined. Methods B6 mice were administered OVA antigen formulated with ALTA® platform or liquid adjuvants, including Alhydrogel®, AddaVax™, Alhydrogel®+CpG. The humoral and cell-mediated responses were measured by ELISA and flow cytometry. Fluorescent OVA was utilized to characterize antigen-presenting cells (APCs) at the site of injection and in lymph nodes. Results Increased and lasting antigen-specific antibody titers post administration of ALTA® OVA demonstrated robust and durable humoral response. In contrast to adjuvants, ALTA® injected mice produced both IgG2c and IgG1 indicating a more balanced Th1/Th2 response. Importantly, ALTA® OVA elicited robust humoral response at lower doses of aluminum than Alhydrogel®. At all antigen doses, the magnitude of OVA-specific CD8+ T cell response was significantly higher after ALTA® administration. The adaptive immune response and numbers of OVA+ APCs were lower after injection of placebo ALTA® mixed with OVA in diluent than ALTA® formulated OVA. This result demonstrated that antigen encapsulation contributes to the immunogenicity of the platform. Conclusion ALTA® formulated protein antigen elicits strong humoral and cell-mediated immunity suggesting a potential broad applicability of the platform to vaccines against various diseases, including intracellular infections and cancer. Funding Source The Gates Foundation INV-064824, The Gates Foundation INV-076862 Topic Categories Vaccines and Immunotherapy (VAC)
Defining viral-host mechanisms of neuroinflammation after human metapneumovirus infection 2257071
Abstract Introduction Human metapneumovirus (hMPV) is a leading cause of severe respiratory illness in infants, and can lead to the rare development of acute neurological complications such as encephalitis and seizures. Severe early-life viral infections with hMPV, RSV and other respiratory viruses have been linked to long-term neurodevelopmental impairments, including cognitive, motor, and behavioral deficits. However, the viral-host mechanisms driving neurotropism and neuroinflammation associated with these clinical outcomes remain poorly understood. Methods Here, we have modeled HMPV-infection of the central nervous system (CNS) using human primary neuronal culture, human brain organoid models and in vivo mouse models of intranasal HMPV infection. In these models we used proteomics, single-cell genomics and functional studies to determine the mechanisms of neuroinflammation driven by HMPV infection. We then utilized a high-throughput small-molecule drug screen to identify novel inhibitors of HMPV infection. Results We demonstrated that hMPV can directly infect and replicate within neuronal and nonneuronal cells in the CNS leading to activation of proinflammatory innate immune responses and markers of neuronal damage using human cell and organoid models. Moreover, we identified different signatures of HMPV innate immune activation compared to a related respiratory virus, RSV. In a mouse model, we found that after Intranasal hMPV infection, virus was detected in both lung and brain tissues with inflammatory signatures. Finally, we performed a drug repurposing screen to identify novel antiviral drugs that could be used to block HMPV infection of both respiratory and CNS tissues. Conclusion These findings establish HMPV as a virus capable of CNS infection and inflammation, identify me mechanisms of host-virus immune interactions and identified novel antiviral drugs that could be used as therapies to mitigate both respiratory and neurological complications associated with HMPV infection. Funding Source Paul Henson Endowment, Children’s Mercy Kansas City Topic Categories Viral Immunology (VIR)
The Intestinal Microbiome Differentially Modulates SARS-CoV-2-Induced Pathology and Host Immune Response in Male and Female Mice 2261020
Abstract Introduction Several reports have shown disparity in male and female responses to SARS-CoV-2 infection. Males often develop move severe acute infection, while the incidence of females developing long-COVID out-numbers males. Commensal microbes play a vital role in shaping both the mucosal and systemic immune response to pathogens. Using germ-free mice, we investigated whether commensal microbes play a role in male and female host susceptibility to SARS-Cov-2 infection. Methods Age-matched male and female germ-free (GF) and control specific pathogen free (SPF) C57BL/6 mice were intranasally infected with the mouse-adapted SARS-CoV-2 MA10. Body weight, temperature, and behavior scores were monitored to assess the severity of disease. Innate responses in the lungs and peripheral blood were determined by flow cytometry analysis of innate subsets, viral load in lung tissues, and the profile of host mRNA responses. Results Male and female GF mice differentially responded to SARS-CoV-2 infection. Thus, female GF mice also showed no change in body temperature, while the infected male GF mice displayed the characteristic drop in body temperature seen in SPF C57BL/6 mice. However, neither male nor female GF mice lost weight after infection. Blood collected three days post infection, showed higher numbers of CD11b+CD11c+ and CD11b+Ly6G+ cells in male GF compared to female GF mice. Further, the lungs of male GF mice contained higher titers of live SARS-CoV-2 MA10 virus than female GF mice four days post infection. Ongoing scRNAseq analysis of male and female GF mice lungs are expected to identify mechanisms underlying these differences. Conclusion Our results suggest that commensal microbes-dependent and -independent mechanisms contribute to the difference of outcome after SARS-CoV-2 infection of males and females. Cellular and molecular mechanisms underlying these differences are being investigated. Funding Source NIH P01AI175399 Topic Categories Mucosal and Regional Immunology (MUC)
Targeting the KRAS G12D mutant suppresses tumor growth in a syngeneic mouse cholangiocarcinoma model through activation of immune cells 2258895
Abstract Introduction Cholangiocarcinoma (CCA), is an aggressive malignancy often diagnosed at an advanced, inoperable stage, with 5-year survival rate of 3%. KRAS mutations are frequent driver alterations linked to poorer prognosis in this cancer. The KRAS G12C mutation inhibitor Adagrasib has been approved by the FDA for the treatment of advanced non-small-cell lung cancer, indicating that the KRAS mutation is a druggable target. However, KRAS mutant inhibitors have yet to be employed in CCA treatment. Methods A mouse CCA model, designated AKP, was generated by crossing genetically engineered mouse strains harboring Alb-Cre, LSL-KrasG12D, and p53L/L. Subsequently, the AKP-M4 cell line was derived by serially passaging tumor cells from AKP mice across three generations. Sanger sequencing and immunohistochemical (IHC) staining were used to determine the KRAS G12D mutation and cytokeratin 19, respectively. MTT assay was used to assess the cell proliferation inhibition by MRTX-1133. Immune cell profiles in AKP-M4-bearing mice treated with MRTX-1133 were determined by flow cytometry. Results Sanger sequencing confirmed G12D mutation in the KRAS gene in AKP-M4 cells. IHC analysis demonstrated that AKP-M4 tumors expressed cytokeratin 19, a key marker for CCA. The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133. We further assessed the anti-cancer efficacy of MRTX-1133 in AKP-M4-bearing C57BL/6 mice, observing a significant dose-dependent reduction in tumor volume and weight. Flow cytometry analysis showed that MRTX-1133 elevated CD4+ T cells, CD8+ T cells, and M1 macrophages, while decreasing MDSCs and M2 macrophages. Notably, PD-L1 expression on tumor cells was diminished. Conclusion KRAS G12D mutant inhibition reduces cancer cell proliferation and stimulates immune cells. This supports the development of KRAS mutation inhibitor for CCA treatment. Funding Source National Science and Technology Council, Taiwan Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Distinct Inflammatory Programs Underlie the Intramuscular Lipid Nanoparticle Response 2267321
Abstract Introduction Developments in mRNA Lipid nanoparticle (mRNA/LNP) technologies have heralded a new era of both vaccines and therapeutics, raising questions about their immunogenicity. While robust immune responses are required for effective vaccination, aberrant inflammation can hinder efficacy of RNA therapeutics. Understanding mechanisms underlying these disparate immunological responses is critical for development of safe and effective delivery systems for vaccines and gene therapies. Methods We sought to understand if ionizable lipid chemistry determines an LNP’s immunogenic potential in vivo as well as the underlying cellular mechanisms. To this end we employed a combination of flow cytometric, transcriptomic, and serological techniques to understand the muscle tissue restricted immunomodulatory mechanisms of LNPs harboring different lipid chemistries. To determine upstream regulators we integrated transcriptomic, cell reporter and genomic screening studies. Results We find that ionizable lipid chemistry determines magnitude of inflammatory gene expression locally within injected muscle. Additionally, LNPs induce rapid and robust neutrophil infiltration into injected muscle and ionizable lipid chemistry determines the magnitude of infiltration. We find that systemically ionizable lipid chemistry and RNA immunogenicity act synergistically within critical antigen presenting cells. Ionizable lipid chemistry determines the efficiency that cDC2 and B cells are transfected as well as their subsequent activation. Conclusion Based on these data we propose a classification of ionizable lipids based on their acute immunomodulatory capacity: Class A inflammatory and Class B non-inflammatory. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)
CD4-Targeted CAR-Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia 2224687
Abstract Introduction Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Current Immunotherapies, including chimeric antigen receptor (CAR) T cell strategies, are limited by the scarcity of antigens that are present on AML cells but absent from hematopoietic stem and progenitor cells (HSPCs). To identify a potential therapeutic target, we analyzed two publicly available single-cell RNA sequencing datasets and found that CD4 is frequently expressed on AML blasts but absent from HSPCs, supporting its potential as a novel CAR target. We developed a CD4-targeted CAR using CD3+CD4⁻CD8⁻ double-negative T cells (CAR4-DNTs). DNTs offer a unique therapeutic platform: they naturally lack CD4, preventing fratricide, and elicit endogenous anti-leukemic activity. Their feasibility and safety as an allogeneic cell therapy platform was demonstrated in a Phase I trial. This work aims to establish CAR4-DNTs as a next-generation, off-the-shelf CAR-T cell therapy for AML. Methods Donor-derived DNTs were engineered to express a CD4-CAR. Cytotoxicity, persistence, and cytokine secretion were evaluated against AML cell lines, CD4+ and CD4-knockout AML variants, and primary AML samples. NSG-SGM3 mice engrafted with luciferase+ MV4-11 cells received CAR4-DNTs, untransduced (UT) DNTs, or PBS. Disease progression was monitored by bioluminescence imaging and survival analysis. Results CAR4-DNTs were successfully manufactured without fratricide. They exhibited significantly enhanced and more durable cytotoxicity than UT-DNTs against AML cell lines and patient samples. Importantly, CAR4-DNTs retained endogenous cytotoxicity towards CD4-negative AML cells. In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse. Conclusion These findings establish CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development. Funding Source Distinguished Doctoral Recruitment Scholarship, The C17 Council, Alberta Cancer Foundation, Alberta Children’s Hospital Research Institute Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Synergistic Interfacial Dangling‐Bond Passivation for Enhanced Moisture Resistance in Perovskite Photovoltaics
ABSTRACT Control of the crystallization process and post‐treatment are key to the fabrication of high‐performance perovskite photovoltaics. During the natural crystallization of perovskite thin films, uncapped dangling bonds readily form at interfaces, induce severe nonradiative recombination losses, and act as preferential sites for the ingress of moisture and oxygen, accelerating a continual phase transitions and structural decomposition. If this kind of continual degradation is not adequately addressed, it will be difficult to achieve good stability in perovskite solar cell devices. Here, a multifunctional small molecule, tetrahydrothiophene‐2‐carboxylic acid (TTA), is introduced as a single interfacial modifier for post‐treatment to simultaneously achieve effective defect passivation and enhance environmental stability. The carboxyl group of TTA strongly coordinates with dangling bonds, suppressing deep‐level defects, while sulfur in the tetrahydrothiophene ring interacts with free PbI 2 to restrain further perovskite decomposition. TTA can form a dense, hydrophobic interfacial layer that blocks water penetration. As a result, TTA‐modified perovskite solar cells achieve a high‐power conversion efficiency of 26.7%, with a fill factor of 85.5% and an open‐circuit voltage of 1.20 V. Notably, unencapsulated devices retain 92.1% of their initial efficiency after 200 h at 70% relative humidity, and 92% after 800 h of continuous illumination under AM 1.5G.
Early-Life Overnutrition Reprograms Metabolism and Immunity to Promote Sex-Biased CNS Autoimmunity 2333917
Abstract Introduction Multiple sclerosis (MS) is a chronic autoimmune disease with a strong female bias and no curative therapy. MS incidence has increased too rapidly to be explained by genetics alone, implicating environmental factors. Childhood and adolescent obesity are established risk factors for MS, particularly in females. However, how early-life overnutrition programs metabolism and immunity to influence CNS autoimmunity remains poorly defined. Methods To model early-life overnutrition without dietary confounders, we employed postnatal overfeeding (PNO) via litter size reduction in C57BL/6 mice. Long-term metabolic phenotyping was performed using indirect calorimetry and glucose tolerance testing. Baseline immune phenotypes were assessed by flow cytometry, cytokine production following T cell stimulation, and genetic disruption of type I interferon signaling. CNS autoimmunity was evaluated using experimental autoimmune encephalomyelitis (EAE), including active disease induction and adoptive transfer approaches. Results PNO resulted in durable increases in body weight, visceral adiposity, and induced long-term metabolic reprogramming, including increased oxygen consumption and mid-life glucose intolerance. Female, but not male, PNO mice exhibited altered immune homeostasis, characterized by increased thymus and spleen size and enhanced IFN-γ production by CD4+ T cells. This Th1 bias persisted in T cell-specific IFNAR1-deficient mice, indicating independence from type I interferon signaling. Consistent with these changes, PNO selectively exacerbated EAE severity in females, while male PNO mice showed no disease enhancement despite increased adiposity. Conclusion Together, these findings demonstrate that early-life overnutrition durably reprograms metabolic and immune states in a sex-dependent manner and selectively enhances susceptibility to CNS autoimmunity in females. Ongoing studies aim to define the mechanisms by which metabolic programming affects immune development to drive sex-biased autoimmune disease. Funding Source Canadian Institutes of Health Research (CIHR) Topic Categories Basic Autoimmunity (BA)
IL-10 suppresses adaptive immune responses during Borrelia burgdorferi infection 2258748
Abstract Introduction Borrelia burgdorferi (Bb), the causative agent of Lyme disease, causes persistent infections in their reservoir hosts, including mice. B cell responses to Bb can control disease but cannot clear infection. Prior work in the lab identified a rapid collapse of infection-induced germinal centers (GC) and impaired long-term humoral immunity to Bb and unrelated vaccine antigens. IL-10, a potent immunoregulatory cytokine, is strongly induced during Bb infection, limiting Bb tissue clearance. However, the mechanisms by which IL10 acts to limit effective immunity remains unknown. Here we investigated whether blockade of IL10 can improve adaptive immunity to Bb. Methods Groups of C57BL/6 mice were treated with anti—IL-10 receptor antibody (αIL-10R) or control rat IgG prior to infection with Bb. Some mice were immunized in addition with influenza virions in alum. Serum antibody responses were measured by ELISA. Flow cytometric analysis assessed GC B and T follicular helper (Tfh) cell populations, and PCR for Bb flaB on various tissues quantified bacterial loads. Results On day 30 after infection, αIL-10R—treated mice had increased total and Bb-specific serum IgG, as well as enhanced responses to influenza vaccination. Flow cytometry demonstrated increased GC B and Tfh populations, and qPCR revealed reduced Bb loads compared to control-treated mice. Using IL-10—GFP reporter mice, we identified CD4+ T and B cells as predominant sources of IL-10. Conclusion Ongoing studies using CD4-Cre and mb1-Cre IL-10flx/flx mice will define the impact of each IL10 cell source on humoral immune responses to Bb and on GC maintenance. These results demonstrate the critical role of Bb-induced IL10 in suppression of effective adaptive immunity to Bb, identifying IL-10 as a potential therapeutic target for improving immunity and Bb clearance in Lyme disease patients. Funding Source NIH/NIAID R01 AI157007 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Neutrophil Reprogramming Fuels Immunosuppression in Staphylococcus aureus Biofilm Infection 2256977
Abstract Introduction Neurosurgical procedures such as craniotomies carry up to a 15% infection risk, predominantly due to Staphylococcus aureus (S. aureus) biofilms. These biofilms resist antibiotics and promote immune suppression, enabling chronic infection. Granulocytic myeloid-derived suppressor cells (G-MDSCs) emerge as key mediators of this immunosuppression, but the mechanisms driving their development, presumably from neutrophils, remain poorly defined. Methods We exposed bone marrow-derived neutrophils to S. aureus biofilm and analyzed phenotypic and functional changes via flow cytometry, RNA- sequencing, and T cell suppression assays. Genetic knockout models lacking Toll-like receptor 2 (TLR2) or MyD88 were used to delineate host signaling pathways. Additionally, we screened the S. aureus Nebraska Transposon Mutant Library (NTML) to identify bacterial factors responsible for neutrophil reprogramming. Results Exposure to S. aureus biofilm transformed neutrophils into G-MDSC-like cells, exhibiting elevated CD11b, CD14, and PD-L1 expression, reduced bactericidal capacity, and suppression of T cell proliferation. TLR2-deficient neutrophils showed markedly reduced acquisition of these traits, while MyD88 deletion completely prevented the shift, implicating MyD88-dependent pathways. NTML screening identified specific bacterial TLR2 ligands as essential for driving this reprogramming. Conclusion Our findings reveal that S. aureus biofilms exploit TLR2-MyD88 signaling to reprogram neutrophils into immunosuppressive G-MDSC-like cells. This host-pathogen interaction underlies biofilm persistence and highlights both bacterial-derived TLR2 ligands and host TLR2 signaling as promising therapeutic targets to restore antimicrobial immunity in neurosurgical infections. Funding Source CHRI Mini grant Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
Inhibition of Fatty Acid Metabolism in Alloreactive T Cells Minimizes Acute Graft-versus-host Disease 2303327
Abstract Introduction Acute Graft-Versus-Host Disease (GVHD) is a complication of allogeneic blood and marrow transplantation in which alloreactive T cells destroy tissues in the liver, skin, and gastrointestinal tract. The dual energetic demands of activation and proliferation push alloreactive T cells into adopting fatty acid oxidation (FAO), a pathway that then becomes a therapeutic target for mitigating GVHD. Here we demonstrate the results of genetic deletion of FAO machinery in donor T cells, and administration of a novel FAO-targeting pharmaceutical as a GVHD treatment. Methods We utilized a C57BL/6 donor into B6D2F1/J recipient model of GVHD. Post-transplant, recipient clinical scores were calculated weekly and weight assessed twice weekly. Genetic FAO inhibition was achieved through a T cell-specific knockout (KO) of carnitine palmitoyltransferase 1a in C57BL/6 mice; recipient mice received either KO (n = 16) or wild-type (n = 16) splenic T cells in transplant. For compound testing, recipient mice with wild-type T cell transplants received either the drug (n = 12) or vehicle control (n = 13) intraperitoneally at days 3, 7, and 10 post-transplant. Results KO T cell recipients exhibited increased survival (Mantel-Cox p = 0.008). While the ongoing drug treatment experiment has not found a significant survival improvement, the drug-treated group exhibits a trend towards greater weights at day 21 post-transplant (Student’s t-test p = 0.06). Conclusion Our results find that genetic inhibition of FAO reduces GVHD; as such, pharmacological inhibition of mitochondrial FAO could theoretically produce the same effect. Although preliminary results do not reveal a survival benefit to drug treatment, optimization of dosing strategy and formulation is underway to clarify the drug’s potential therapeutic benefit. Further investigations will characterize its role in post-transplant weight recovery and impact on specific T cell subpopulations early after transplantation, and determine whether similar effects manifest in human T cells. Funding Source Department of Pediatrics, University of Minnesota Topic Categories Transplantation Immunology (TRAN)
From Mouse to Clinic: Non-Invasive Swab-Based Profiling of Inflammatory and Barrier Markers in Atopic Dermatitis 2258968
Abstract Introduction Atopic dermatitis (AD) is a chronic inflammatory skin disease driven by immune dysregulation and impaired barrier function, including altered ceramide composition. In clinical practice–especially in children–non-invasive tools such as epidermal curettage, microneedle patches, and tape stripping are widely used to monitor skin biomarkers and predict therapeutic efficacy. However, these techniques remain underused in preclinical models, where invasive biopsies are still standard, limiting longitudinal monitoring and translational relevance. Methods This study assessed the feasibility, safety, and translational value of a non-invasive skin surface sampling strategy to monitor immune dysregulation and barrier alterations in two murine AD models. AD-like lesions were induced by chronic oxazolone application on the back or repeated MC903 treatment on the ear. Weekly samples were collected using cotton-tipped swabs for multiplex cytokine assays and ceramide profiling, while glass beads were rolled over the skin for lipid extraction and LC-MS/MS quantification. Data from non-invasive samples were compared with full-thickness biopsies analyzed at endpoint by immunoassays and lipidomics. Results Repeated sampling over 20 days caused no visible damage, distress, or interference with disease progression. Key cytokines (IL-4, IL-6, TNF, IL-1β) and ceramides were consistently detectable on the skin surface and correlated with tissue levels. Ceramide profiles reflected barrier dysfunction and inflammation, supporting their value as non-invasive biomarkers. Conclusion This dual-parameter, minimally invasive approach enables longitudinal monitoring of both immune and barrier markers in preclinical AD models. It aligns with clinical strategies, supports the 3Rs principles and enhances the translational value of murine studies. Funding Source n/a Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)
Rapid Assembly of Oligo Pools into Medium-Scale T Cell Receptor Lentiviral Libraries 2267508
Abstract Introduction Synthesis and construction of T cell receptor cDNA is one of the main bottlenecks to large-scale T cell receptor (TCR) antigen screening. New technologies allow for massive screening of TCRs against hypothesized antigens[1,2,4], including the multiplexed barcoded pMHC tetramer screening technology[2] developed in our lab. However, validation of discovered TCRs requires synthesis and cloning that costs upwards of $100 per TCR and is unsustainable for antigen screening labs. Methods have been published that seek to overcome this bottleneck[5,6] but have significant start-up costs and take multiple days of benchwork. Herein we describe a workflow that rapidly generates small pools of TCRs at a cost of ∼ $10 per TCR. Methods In this method, we synthesized pools of alpha and beta CDR3s linked in single oligos to ensure pairing fidelity of the two chains. A proprietary set of CDR3-TRV overlap sequences were computationally generated with codon substitution to maximize overlap diversity while maintaining homogenous melting temperature and GC content. Our method only requires 6 steps which can be completed in one day. In our preliminary investigation, we successfully synthesized and assembled a pool of 13 TCRs through a series of assembly PCRs. Results Sequencing of the plasmid pools confirmed that all TCRs were present in the final plasmid pool. Transduced J76 cells were stained with anti-human TCR antibody and tetramer confirming expression of the antigen specific T-cell clones in the pool. Conclusion This novel method overcomes a major bottleneck in antigen screening pipelines by providing rapid synthesis and assembly of TCRs in a pool. Future work will identify the upper limit of TCR assembly for this method and demonstrate its screening utility using tetTCR-seqHD. (1) Dezfulian M, +, Cell 2023; (2) Zhang SQ, +, Nat. Biotech. 2018; (4) Birnbaum M, +, Cell 2014; (5) Moravec Z, +, Nat. Biotech. 2025; (6) Ganglione S, +, BioRxiv 2025 Funding Source NIH R33 IMAT 2021 Topic Categories Technological Innovations in Immunology (TECH)
Functionally divergent resident memory T cells localize to lung and lymph node following influenza infection 2220996
Abstract Introduction Resident memory T cells (TRM) mediate localized immunity in barrier tissues while central memory T cells (TCM) recirculate through lymphoid organs to surveil for reinfection. Although TRM are classically associated with peripheral non-lymphid tissues, they have also been identified within lymph nodes (LNRM) where mechanisms guiding their formation and their role during secondary pathogen encounters remain poorly understood. Moreover, functional distinctions between TCM, LNRM, and lung TRM (LungRM) have not been clearly defined. Methods Flow cytometry, immunofluorescent imaging, RNA+ATAC sequencing, cytotoxic assays Results Using longitudinal antibody labeling, we tracked the migration of T cells after influenza infection and demonstrate that CD69+CD103+ T cells in lymph nodes are bona fide resident. LNRM accumulate throughout lung-draining lymph nodes and are present within all LN compartments including, sub capsular sinus, T cell zone and germinal centers. LNRM formation is conserved across viral strains, antigen specificities, and precursor populations. Epigenetic and transcriptional profiling reveal that LNRM are uniquely poised for cytotoxicity whereas LungRM resemble exhausted cells with elevated expression of inhibitory receptors and increased chromatin accessibility at the Pdcd1 locus. Regulatory network analysis of transcription factors, combined with target gene expression and chromatin accessibility identified key gene networks differentiating TCM, LNRM and LungRM. Upon antigen re-encounter, LNRM produce more IFNγ, are more proliferative, and are more cytotoxic than LungRM. Conclusion These findings establish LNRM as a distinct and durable memory population bridging features of circulating and tissue-resident cells. Our results suggest that LNRM may play a previously unappreciated role in orchestrating local recall responses within secondary lymphoid organs and provide superior effector functions than LungRM. Funding Source Ludwig Center Postdoctoral Fellowship Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Mechanically Robust and Depolymerizable Polyesters With Near‐Complete Monomer Recovery for Circular Additive Manufacturing
ABSTRACT Designing polymers that combine tunable macromolecular architecture with complete chemical recyclability remains a fundamental challenge in sustainable polymer chemistry. Here, we report a δ‐lactone copolymer platform synthesized via controlled ring‐opening polymerization of δ‐dodecalactone (δ7) and δ‐valerolactone (δ0), enabling regulation of molecular architecture, mechanical response, and depolymerization pathways within a single polyester chemistry suitable for additive manufacturing (AM; or 3D printing). Segmental programming of δ7 and δ0 domains yields a synergistic combination of chain mobility and reversible crystalline reinforcement, producing recyclable, high‐stiffness 3D‐printing precursors with elastic modulus elevated by 2–3 orders of magnitude relative to prior δ‐lactone materials. End‐group functionalization generates methacrylate‐terminated copolymers and acrylate‐terminated macromonomers that undergo efficient photopolymerization without disrupting the depolymerizable backbone. Blending these components produces photocurable formulations with rheology governed by intrinsic polymer design, enabling compatibility with direct ink writing and digital light processing via temperature‐mediated control of segmental crystallinity. This strategy eliminates external monomers or permanent crosslinkers, preserving chemical integrity and recyclability. Increasing molecular weight shifts the system into an entanglement‐dominated thermoplastic regime for fused filament fabrication. Sequential thermal–catalytic unzipping exploits ceiling‐temperature differences to recover δ7 and δ0 monomers with ∼95% efficiency even from crosslinked networks, with repolymerization yielding materials indistinguishable from virgin polymers.
Characterization of γδ TCR ligand expression using a novel Vγ4Vδ7 TCR tetramer 2335176
Abstract Introduction γδ T cells are evolutionarily conserved innate-like T cells that have been implicated in infectious disease, autoimmunity, and cancer. While there is growing appreciation of the roles these cells play in shaping the immune response, our understanding of γδ T cell biology has lagged behind that of their iNKT and MAIT cell counterparts. This gap in understanding can largely be attributed to our poor understanding what γδ T cell receptors recognize. We recently reported that a significant fraction of IFN-γ-producing Vγ4 γδ T cells are characterized by the preferential usage of a Vγ4Vδ7 TCR. Here, we set out to identify and characterize the binding partners of the Vγ4Vδ7 TCR under normal homeostatic conditions. Methods A recombinant soluble Vγ4Vδ7 TCR tetramer was produced and used to screen and identify Vγ4Vδ7-specific populations from normal mouse tissues using flow cytometry under different binding conditions. Results When tetramer staining was conducted at 4°C, we found that Vγ4Vδ7 TCR bound to a rare population of B cells in both the spleen and lungs of B6 mice. Interestingly, however, when tetramer staining was conducted at 23°C or 37°C, we found that Vγ4Vδ7 TCR bound to a wide variety of cells in the same tissues, while an irrelevant human TCR exhibited no binding under similar conditions. Vγ4Vδ7 tetramer binding to cells was inhibited at both temperatures through addition of unlabeled Vγ4Vδ7 TCR monomer, and permeabilization of cells prior to staining resulted in a significant increase in Vγ4Vδ7 TCR staining. Conclusion Taken together, the data suggest the presence of an intracellular Vγ4Vδ7 TCR ligand that is broadly distributed among a wide variety of cell types, and that a small subset of B cells expresses this ligand on the cell surface. These data are consistent with a model in which the Vγ4Vδ7 TCR recognizes a conserved intracellular ligand that may be translocated to the cell surface under specific conditions. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Glucocorticoids redirect naïve T cells to the bone marrow for preservation in malnourished mice 2253552
Abstract Introduction Malnutrition impairs immunity and contributes significantly to child mortality. This depressed immunity involves the impairment of T cells. During malnutrition, effector T cells experience a decline in number, proliferative capacity, and cytokine secretion. As little is known about changes involving naïve T cells, we conducted a study to assess the impact of malnutrition on the naïve T cell population. Methods B6, B6 Thy1.1, and Nr3clfl/fl CD4.cre + (conditional glucocorticoid receptor knockout/cGRKO) mice were randomly assigned to an ad libitum (AL) or a malnutrition (MAL) diet. After one week of MAL, cells were isolated from the femoral bone marrow (BM) and axillary, brachial, and inguinal lymph nodes (LN). Flow cytometry was used to identify naïve (CD44lo) T cells and assess apoptosis. In an adoptive transfer experiment, cells from AL Thy1.1+ and MAL Thy1.1- donor mice were labeled and retro-orbitally injected into AL Thy1.1- and MAL Thy1.1- recipients. Recipients were euthanized two hours after injection, and percent change in the AL: MAL naïve T cell ratio was calculated. Cells from the spleen and BM of MAL and AL cGRKO mice were stained with anti- CD44, CD62L, CD4, CD8, CXCR4, and CCR7 and acquired by flow cytometry. Results The number of naïve T cells decreased in LN and increased in BM during MAL, and MAL naïve T cells in BM were less apoptotic than controls. MAL naïve CD4+ T cells preferentially migrated to BM while AL control cells preferentially migrated to LN in a T cell-intrinsic fashion. T cell-specific lack of the glucocorticoid receptor greatly reduced the number of naïve T cells in BM of MAL mice. T cell sensitivity to glucocorticoids was also required for naïve CD4+ T cells’ elevated expression of CXCR4 in the spleen and CCR7 in BM during MAL. Conclusion These findings suggest that naïve T cell migration to BM during MAL is intrinsic and requires sensitivity to glucocorticoid. This phenomenon is likely adaptive and intended to preserve naïve T cells during malnutrition. Funding Source National Science Foundation grant numbers (1951881) and (1920116) awarded to M.R. Gubbels Bupp Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)