Browse Articles

Identifying the Mechanism of CAR T Cell Metabolic Improvements Associated with Compound 991 Treatment 2310041

Abstract Introduction Chimeric Antigen Receptor (CAR) T cells are a revolutionary treatment option for aggressive relapsed and refractory blood cancers. Unfortunately, CAR T cell cure rates remain low (∼50%), in part due to issues with T cell persistence and in vivo function. Treating CAR T cells with Compound 991, an AMPK agonist, has shown promise by increasing the in vivo efficacy of CAR T cells. However, the mechanism behind how AMPK activation improves in vivo CAR T cell performance is unknown. Methods To identify candidates downstream of AMPK in human T cells, we performed phosphoproteomic analysis on cell lysates following 991 treatment. Phosphorylation results were then confirmed by immunoprecipitation of candidate samples and immunoblotting using an anti-phosphoserine antibody. CRISPR technologies were used in parallel to eliminate the target protein of interest. Results Phosphoproteomic analysis of cell lysates from 991-treated T cells revealed increased phosphorylation of several proteins. Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2), known to be a direct target of AMPKs when phosphorylated on site (Ser151) stood out with an observed log2 fold change in phosphorylation of 8.081 following 991 treatment (p-value< 0.001). ARHGEF2 protein could be immunoprecipitated from 991-treated cells, with detection by phosphoserine antibodies ongoing. Further, we have identified a guide RNA which convincingly targets ARHGEF2 expression, with initial decreases of > 34% at the protein level following CRISPR treatment. Conclusion Phosphoproteomic analysis following 991 treatment detects increased phosphorylation of ARHGEF2, a protein that serves a diverse array of functions in human cells. These data suggest that AMPK, working through ARHGEF2 phosphorylation, could be the mechanistic link to improved T cell metabolism following AMPK activation. We now have the tools to both define phosphorylation of ARHGEF2, as well as delete ARHGEF2 in T cells, to determine whether our suggested, novel mechanism is true. Funding Source University of Minnesota Department of Pediatrics Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Therapeutic potential of bovine type III interferon (IFN-γ3) as anti-bovine viral diarrhea virus agent 2305378

Abstract Introduction Recombinant bovine interferon lambda 3 (bovIFN-γ3) can effectively clear bovine viral diarrhea virus (BVDV) in cell cultures. Based on this finding, we evaluated whether a replication-defective human adenovirus type 5 (Ad5) vector expressing bovIFN-γ3 can reduce the severity of BVDV infection and protect against thymus-associated lymphoid follicle depletion in calves. Methods Twenty-four colostrum-deprived calves were divided into six groups as follows: group 1: uninoculated control (n = 3); group 2: BVDV challenged control (n = 3); group 3: Ad5-blue (empty vector) subcutaneous [SQ] and BVDV (n = 3); group 4: Ad5-blue intranasal [IN] and BVDV (n = 3); group 5: Ad5-bovIFN-γ3 -SQ and BVDV (n = 6); group 6: Ad5-bovIFN-γ3 IN and BVDV (n = 6). Calves in groups 3-6 received the respective Ad5 vectors (∼7 × 10e10 plaque forming units (PFU) per animal) either IN or SQ one day before and one day after BVDV IN challenge (∼6 × 10e7 TCID50 per animal). Nasal swabs and blood samples were collected throughout the study, and calves were euthanized 14 days after BVDV challenge. Results Calves inoculated with BVDV experienced a transient decline in lymphocytes and monocytes during the study period. Real-time PCR analysis showed no significant changes in BVDV shedding or viremia amongst the groups. A lack of BVDV-induced thymic weight reduction was observed in calves treated with Ad5-bovIFN-γ3 (groups 5 and 6) at necropsy. A greater protection of thymus-associated lymphoid follicles was observed in calves receiving Ad5-bovIFN-γ3 SQ (group 5). Conclusion These findings indicate that two doses of SQ administration of Ad5-bovIFN-γ3 (∼7 × 10e10 PFU/animal) can protect the thymus and prevent BVDV-induced thymic lymphoid depletion. However, higher doses or multiple days of administration (Ad5-bovIFN-γ3) may be required to fully prevent BVDV shedding and viremia. Funding Source USDA CRIS 5030-32000-229-000-D Topic Categories Vaccines and Immunotherapy (VAC)

Oral STAT6 Inhibitor EPS-3903 Demonstrates Good Preclinical In Vivo Tolerability without Reactive Metabolites or Metabolic/Safety Liabilities 2257506

Abstract Introduction Reactive metabolites can lead to idiosyncratic toxicity in humans, and thus their assessments are critical for drug development. EPS-3903 is a potent, selective, potentially best-in-class STAT6 inhibitor for the treatment of type 2 inflammatory diseases including asthma and atopic dermatitis. Here we present the in vitro and in vivo reactive metabolite evaluations and in vivo tolerability in preclinical species. Methods Reactive metabolite assessments were conducted in liver S9, hepatocytes, HepatoPac, and animal plasma across multiple species. Preclinical in vivo tolerability was determined in mice. Results No reactive metabolites or glutathione (GSH) adducts were detected in vitro (including mice, rats, dogs, monkeys, and humans) or in vivo across preclinical species. EPS-3903 exhibits drug-like properties with excellent in vitro-in vivo correlation for the evaluation of reactive metabolites. EPS-3903 was well tolerated in mice at doses up to 250 mg/kg for 8 days with steady-state plasma exposure reaching 918 µg-hr/mL. The steady-state plasma exposure provides a safety margin up to 160-fold over the projected exposure at human efficacious doses. Conclusion EPS-3903 generated no reactive metabolites in vitro or in vivo, minimizing metabolic/safety liabilities in humans. The highly favorable preclinical in vivo tolerability of EPS-3903 supports its development as a potential oral therapeutic for the treatment of allergic diseases including asthma and atopic dermatitis. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Investigation of the Impact of BECC-Derived Adjuvants in Allergen-Specific Immunotherapy 2260252

Abstract Introduction Allergen specific immunotherapy (AIT) is a treatment for allergies that requires a gradual increase in exposure to an allergen to eventually allow patient tolerance through shifting memory B cells from producing allergic antibody isotype IgE towards production of non-allergic isotypes like IgG2a. Immunoadjuvants can be added to AIT to improve the efficacy of the treatment as these adjuvants create a more robust immune response when combined with an antigen. Methods A murine model of ovalbumin allergic asthma in BALB/c mice was used to investigate the capacity of lipid A mimetic BECC adjuvants 438 and 470 in combination with AIT to decrease measures of allergy. Blood serum was collected at different time points throughout this study and antibody production was measured using antigen-specific indirect serum enzyme-linked immunosorbent assays (ELISAs). Results The antibodies evaluated were IgE, IgG1, and IgG2a. The abundant production of antibody isotype IgE is a defining characteristic of allergic response while antibody isotypes IgG1 and IgG2a allowed for visualization of whether the adjuvanted treatment led to a shift toward tolerance. Lung histopathology was also analyzed to determine airway inflammation and lung epithelial thickness. Conclusion Ongoing experiments measure antibody production at intermediate time points as well as investigate proteins of interest. This potential improvement of AIT has the potential to greatly help allergy sufferers worldwide. Funding Source OURCI Summer Project Grant, Fisher College Endowed Chair, Bridges to the Doctorate, FSCM Student Research Grant Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Tailoring NINJ1 for the Control of Plasma Membrane Rupture in Regulated Cell Death 2260243

Abstract Introduction Cell death enables host cells to eliminate pathogens and maintain tissue homeostasis. Consequently, dysregulation of cell death pathways contributes to a wide range of pathologies, including cancer, autoimmune diseases, and aging. Most of the cell death pathways culminate in plasma membrane rupture (PMR), a process actively executed by NINJ1. In resting cells, inactive NINJ1 exists as homodimers, but upon activation of upstream cell death triggers, NINJ1 oligomerize to induce PMR. Previous studies have identified key functional residues within NINJ1 but the mechanism underlying PMR require further elucidation. Methods To better understand the functional importance of NINJ1 in lytic cell death, we engineered NINJ1 knockout (KO) macrophages by CRISPR-Cas9. To gain insight into the mechanisms regulating NINJ1 activity, we evaluated the ability of specific ectopically expressed NINJ1 point mutants to mediate lytic death. Results Upon LPS and Nigericin treatment, NINJ1 was shown to be essential for LDH release, but dispensable for NLRP3 expression, Gasdermin D (GSDMD) cleavage, and propidium iodide (PI) uptake. These findings corroborate that NINJ1 is not required for initial PM permeabilization but is crucial for the subsequent PMR during lytic cell death. When overexpressed in HEK 293T cells K45Q, D53A, Q91A and G95L mutations resulted in a pronounced loss-of-function effect. When co-expressed in an overexpression model of auto-activation of wild-type (WT) NINJ1, these mutants markedly attenuated the PMR activity of WT NINJ1, suggesting a true dominant-negative effect as opposed to just loss-of-function. A dose-response analysis revealed that these mutants inhibit WT NINJ1 activity at ratios below 1:1, likely pointing to additional mechanisms for NINJ1 regulation beyond stabilization of homodimerization. Conclusion Our results confirm the central role of NINJ1 in PMR and highlight the functional significance of specific residues, advancing our mechanistic understanding of NINJ1 inhibition. Funding Source G.G.R supporteed by the Ramón Areces Foundation Research Fellowship. C.L.E. supported by the Ragon Early Independence Fellowship, Karin Grunebaum Cancer Research Foundation Faculty Research Fellowship, and NIH grant R35GM159938. Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Beyond IFNs: How IRFs suppress pathogenic inflammation during viral infection 2305581

Abstract Introduction Type I interferon (IFN) responses are central to antiviral defense and are driven by IFN regulatory factors (IRFs), which are classically known as transcription factors that induce IFNs and antiviral genes. Our work identifies non-transcriptional activities of IRF3 and IRF7, including IRF3-dependent apoptosis of virus-infected cells and a distinct mechanism that suppresses NF-κB—driven inflammatory gene induction. These pathways shape the host response to viral infection and modulate immunopathology. Methods We used primary bone marrow-derived macrophages, IRF3/IRF7 transcriptionally inactive mutants, and IRF3 single-action (transcriptionally inactive) knock-in mouse models to dissect non-canonical IRF functions. NF-κB activity was assessed by cytokine expression by qRT-PCR, and protein interactions by immunoprecipitation and proximity ligation assay. In vivo relevance was evaluated in parainfluenza virus infection models using physiological and inflammatory readouts. Viral antagonism was examined through interaction and functional inhibition assays. Results IRF3 displayed two transcription-independent effector functions: induction of apoptosis in infected cells and direct repression of NF-κB-dependent inflammatory gene expression (RIKA). This anti-inflammatory IRF3 activity in macrophages was essential for protection against viral lung inflammation in mice. IRF7, which contains a similar NF-κB—binding motif, also suppressed inflammatory gene induction, revealing a broader non-canonical regulatory module within the IRF family. Several viral proteins selectively blocked this IRF:NF-κB interaction, demonstrating an evolved viral strategy to overcome IRF-mediated inflammation control. Conclusion IRF3 and IRF7 function beyond transcriptional activities to restrain inflammatory signaling and limit viral immunopathology. The newly defined non-canonical IRF pathways represent critical determinants of antiviral inflammation, targets of viral antagonism, and offer new avenues for therapeutic modulation. Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

A multimodal atlas of COVID-19 severity identifies hallmarks of dysregulated immunity 2258066

Abstract Introduction The alpha-variant wave of the COVID-19 pandemic provided a unique opportunity to study, at single-cell resolution, how near-universal exposure to the same pathogen can lead to either effective or dysfunctional immune responses in humans. Methods We analyzed 2.5 million circulating immune cells from 428 patients across time points (840 PBMC samples), encompassing three contemporaneous SARS-CoV-2 cohorts: acutely infected patients at five WHO disease severity levels and three time points, patients from the first randomized control trial to study efficacy of tocilizumab in management of COVID-19, and convalescent patients three months after infection. We used linear modeling to integrate multiple data types – single-cell RNA-seq, CITE-seq, TCR and BCR sequencing, viral load measurements, viral neutralization assays, detection of 75 autoantibodies, HLA genotype data, and serum proteomics covering 1,463 targets – to derive the most comprehensive view to-date of the biological features of COVID-19 disease severity. Results We show that myeloid-derived suppressor cells (MDSCs) act as a key immunologic pivot point in severe COVID-19. Myeloid dysfunction is marked by impaired antigen presentation and drives a non-productive adaptive immune response. Severe disease is also linked to autoantibodies targeting type I interferons, specific HLA-DQB1 allelic variants, and serum IL-6 levels. Tocilizumab treatment eliminates CLU-expressing MDSCs and ISG-positive myeloid subsets, restores antigen presentation, and reactivates productive adaptive immunity. In convalescence 3-months post-infection, we found persistently high ICOS expression in regulatory T cells. Conclusion Overall, we define distinct innate and adaptive host immune responses associated with acute, IL-6—responsive, and convalescent SARS-CoV-2 infection. Our multimodal and high-dimensional dataset with curated clinical metadata provides a foundational and clinically relevant resource for modeling host immune response biology in health and disease. Funding Source We acknowledge the following funding sources: this work was supported by several training grants, including a NIAID grant T32AR007258 (to KS), three NHLBI grants 5T32HL116275-13 (to CC), 5T32HL129970-09 (to APN), and the K08HL157725 (to PS), as well as the American Heart Association Career Development Award (to PS). PS was also supported by the Brigham and Women’s Hospital Innovation Evergreen Fund. EY was supported by funding from the Stanford Medical Scholars program. RJX acknowledges supports from NIH DK43351 and U19AI142784. RJX and AR were supported by the Manton Foundation and the Klarman Cell Observatory. PJU was supported by Third Rock Ventures; Henry Gustav Floren Trust; Stanford Department of Medicine Team Science Program; Stanford Medicine Office of the Dean; and National Institutes of Health R01 grants AI175771 and AI182319-02. RPB acknowledges funding support from the Massachusetts General Hospital Executive Committee on Research, the American Lung Association, and the Broad Institute’s Next Generation Scholar award. MBG, MRF, and NH were supported by an American Lung Association COVID-19 Action Initiative grant. MBG and MRF were supported by a grant from the Executive Committee on Research at MGH. NH acknowledges was supported by NIH/NIAID U19 AI082630, a Chair and gift from Sandra, Sarah, and Arthur Irving. ACV acknowledges funding support from the COVID-19 Clinical Trials Pilot grant from the Executive Committee on Research at MGH; a COVID-19 Chan Zuckerberg Initiative grant (2020-216954); the funds from the Manton Foundation and the Klarman Family Foundation; the Broad Institute’s Next Generation Scholar award; the MGH Howard M. Goodman Fellowship; the National Institutes of Health (DP2CA247831); work at the Broad Institute was supported by a gift from an anonymous donor. Topic Categories Computational and Systems Immunology (COMP)

A new TLR-7 agonist stimulates anticancer immunity through the activation of epithelial cell cytokine response 2257774

Abstract Introduction Lung cancer remains one of the deadliest types of cancer, supporting the development of new therapeutic approaches. While various immunotherapies were developed in the past decade, their efficacy remains limited. Recently, we demonstrated that the neoadjuvant treatment with nanoparticle FB-631, a TLR-7 agonist which is well tolerated in humans, significantly prevented tumor cell implantation in the lung and the development of metastases in the CMT-167 mouse model of lung adenocarcinoma. This was achieved in part by improving the natural killer cell and CD8 T cell responses in the lung. To further address the mechanisms by which FB-631 mitigates cancer development, we verified whether this drug also favors an anticancer immune response in lung epithelial cells. Methods We first verified whether mouse and human lung epithelial cells (CMT-167 and H226) incorporate FB-631 using star red-coupled nanoparticle and fluorescence microscopy. Then, the production of anticancer cytokines by epithelial cells was evaluated using flow cytometry. Results We demonstrated that FB-631 enters mouse (CMT-167) and human (H226) epithelial cells, allowing its interaction with and stimulation of intracellular TLR-7. Furthermore, we observed that CMT-167 cell stimulation with FB-631 increases the percentage of cells expressing IFN-g, IL-1a and IL-6. Finally, we also report higher per cell-expression of TNF-a, IL-1a and IL-6 in CMT-167 epithelial cells. Conclusion Those results suggest that FB-631activates TLR-7 by entering epithelial cells, triggering an anticancer cytokine production in this cell line. These results will be complemented by assays in human epithelial cells, to support the transferability of this technology to human lung cancer. Funding Source Fondation de l’IUCPQ Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Particulate Photocatalytic Water Splitting for Solar Energy Conversion: From Material Design to Scalable Demonstration

ABSTRACT Photocatalytic water splitting using particulate semiconductors represents a promising strategy for sustainable hydrogen production driven by solar energy. However, realizing practical solar photocatalytic hydrogen generation requires not only the development of highly efficient photocatalyst materials but also the design of integrated systems that are scalable, stable, and economically viable. This review presents a comprehensive overview of recent advances in photocatalytic material design, photogenerated charges regulation, surface catalysis, and scalable system‐level integration for overall water splitting. Emphases are placed on emerging light‐absorbing materials with tunable band structures, strategies to enhance photogenerated charge separation, innovations in dual‐cocatalyst engineering, and reverse reaction suppression techniques. Recent progresses in Z‐scheme photocatalytic systems are also highlighted, which offer alternative paradigms for achieving solar water splitting. Furthermore, advancements toward scalable applications are further presented, including the development of photocatalytic panel reactors and the hydrogen farm project (HFP) strategy, which offers a practical solution for solar energy storage and conversion without the need for H 2 /O 2 separation. Looking ahead, despite significant progress, key challenges such as limited solar‐to‐hydrogen (STH) efficiencies, suboptimal visible‐light harvesting, and scale‐up constraints persist. Addressing these issues through interdisciplinary research will be pivotal in realizing practical solar hydrogen production and advancing a sustainable energy future.

An siENPP1‐Delivering Bimetallic MOF Nanocomplex Enables Triple Activation of the cGAS‐STING Pathway for Synergistic Triple‐Negative Breast Cancer Therapy

ABSTRACT Triple‐negative breast cancer (TNBC) is associated with poor prognosis due to its highly heterogeneous tumor immune microenvironment and the lack of actionable molecular targets, which collectively limit the efficacy of current therapies. To address these challenges, PEI/siENPP1@Mn‐ZIF‐8 (PEMZ) is reported, an integrated gene–chemodynamic–immune nanocomplex that activates the cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) pathway through a previously unattainable triple‐activation mechanism. PEMZ efficiently encapsulates siENPP1, protects it from degradation, and enables acid‐responsive cytosolic release, resulting in sustained ectonucleotide pyrophosphatase/phosphodiesterase 1 ( ENPP1 ) silencing and accumulation of tumor‐derived cyclic 2′,3′‐GMP‐AMP (cGAMP). Simultaneously, the Mn 2+ /Zn 2+ ions released from the trienzyme‐mimetic nanocomplex, which exhibits peroxidase‐, catalase‐, and glutathione peroxidase‐like activities, amplify intracellular reactive oxygen species, deplete glutathione, and trigger mitochondrial dysfunction. This coordinated redox disruption induces immunogenic cell death and further enhances STING signaling. In vitro and in vivo studies demonstrate that PEMZ markedly suppresses TNBC proliferation and metastasis, promotes dendritic cell maturation, and increases intratumoral CD8 + T‐cell infiltration, achieving potent tumor inhibition with minimal systemic toxicity. This work establishes PEMZ as a mechanistically integrated nano‐immunotherapy platform, offering a promising and broadly applicable strategy for the precision treatment of TNBC and other ENPP1‐overexpressing cancers.

Intragenomic riboregulatory interaction modulates the conformation of the hepatitis C virus 3′X RNA

Emotion regulation difficulty mediates the relationship between right dorsolateral prefrontal cortex spontaneous activity and problematic smartphone use

SARS-CoV-2 endoribonucleaseU and ORF6 work synergistically to antagonize innate immune signaling 2327626

Abstract Introduction Coronaviruses (CoVs) produce dsRNA during genome replication and mRNA synthesis. Upon sensing dsRNA, host cells activate numerous antiviral pathways. CoVs encode multiple proteins that antagonize antiviral responses. The conserved CoV nsp15 contains an endoribonuclease (EndoU) that cleaves viral ssRNA to limit dsRNA accumulation. Similarly, the viral protein, NS6, encoded by open reading frame (ORF)6 is expressed by severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 and has been shown to block STAT translocation, but its absence has minimal impact on IFN-stimulated genes (ISGs). A viral genome has limited coding capacity, and ORFs encoding proteins without a significant function are typically mutated or deleted over time. Therefore, we infer that ORF6 plays an essential role in immune evasion, perhaps dependent upon other viral proteins. Methods A recombinant SARS-CoV-2 encoding an inactive EndoU (nsp15mut) and an interrupted ORF6 (ORF6stop) was constructed. Viral kinetics and innate immune induction were assessed in both Calu-3 cells and primary nasal air-liquid interface (ALI) cultures using RT-qPCR and Western blot analysis. Results Replication of SARS-CoV-2 nsp15mut/ORF6stop in nasal ALI cultures was attenuated at both 33 °C and 37 °C compared to WT SARS-CoV-2. A significant increase in type I and III IFN transcripts was also observed in nasal ALI cultures infected with nsp15mut/ORF6stop compared to WT and each single mutant. Robust induction of various ISGs were also observed via Western blot analysis, with a faster induction observed at 37 °C. Conclusion The synergistic relationship between nsp15 and ORF6 is essential for efficient SARS-CoV-2 replication. We hypothesize that since nsp15 functions by reducing dsRNA upstream of ORF6, nsp15 acts as a more potent antagonist leaving little activity for ORF6 to account for. However, when both EndoU activity and NS6 are absent, a robust induction of IFN and ISGs are induced to levels higher than that of the nsp15mut alone. Funding Source This work was supported by National Institutes of Health grants R01 AI140442 (SRW), R01AI169537 (SRW&NAC), RO1A1AI161175 (LM-S); Department of Veterans Affairs Merit Review 1-I01-BX005432-01 (NAC&SRW); the Penn Center for Research on Emerging Viruses (SRW Topic Categories Viral Immunology (VIR)

A phenotypic switch confers resistance to CAR T cell immunotherapy in neuroblastoma 2305524

Abstract Introduction CAR T cell immunotherapy has had clinical success for liquid tumors but has faced variable potency for solid tumors, encompassing complete tumor clearance to no response. CAR-T responses are known to depend on many biological and clinical factors, but we lack a mechanistic framework that moves these qualitative observations towards quantitative predictors of CAR-T efficacy. Our goal is to determine the mechanisms by which CAR T cells clear tumors, quantitatively model the predictive features, and develop clinical interventions to increase potency. Methods We developed an experimental model in neuroblastoma, a pediatric cancer of the neural crest, by screening for gene knockouts that confer resistance to CAR T cells. We co-cultured the resistant lines with CAR T cells and tracked tumor growth, prepared cells for flow cytometry, and collected supernatants for cytokine quantification. We integrated the multimodal data into a machine-learning model to determine predictors of tumor clearance. Results The model performed remarkably well, so we identified the features most important for model predictions. The top features included cytokines (TNF, IFNγ), death receptors (Fas), and tumor antigen levels. We asked how the neuroblastoma lines would respond to cytokines alone, and we found similar patterns of resistance as with the CAR T cell co-cultures, indicating resistance independent of ligand availability. We then explored the transcriptional differences between knockout and wildtype cells by RNA sequencing. Interestingly, the genetic knockouts that conferred CAR-T resistance caused neuroblastomas to adopt a distinct resistance phenotype with key ubiquitous pathways downstream. Conclusion The data indicate that neuroblastoma may escape CAR T cells by undergoing a phenotypic switch that confers resistance to cytokine death signals. We are excited by the clinical prospect of knowing how to supplement or block cytokines to increase CAR T efficacy by anticipating and countering the phenotypic switch. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

GIBLE: Inferring time-resolved B cell lineage trees from spatial BCR sequencing 2257851

Abstract Introduction The spatial dynamics of B cell affinity maturation during germinal center reactions are critical for effective immune responses. Using B cell receptor (BCR) sequences, computational models can infer the history of mutations within a B cell lineage by building phylogenetic lineage trees. These lineage trees are a powerful way to infer past B cell dynamics during infection, vaccination, and autoimmune diseases. Recently developed spatial sequencing techniques generate BCR sequences across 2D slices of tissue, but no methods exist that use this data to infer the spatial evolution of B cell lineages. Methods Here, we introduce GIBLE (Geographic Inference of B Cell Lineage Evolution), a novel Bayesian phylogenetic model that reconstructs spatial locations of unobserved, ancestral B cells, explicitly links mutation rates with physical locations, and quantifies the direction of B cell migration across tissue sections. We applied GIBLE to characterize B cell maturation in the human tonsil. Results We find that clonal lineages can seed new germinal centers, leading to parallel evolution of distinct, localized evolutionary clades. Conclusion GIBLE provides a robust and broadly applicable framework for integrating spatial and phylogenetic data to trace B cell evolution in space and time during immune responses. Funding Source National Cancer Institute grant T32CA134286 Topic Categories Computational and Systems Immunology (COMP)

Mechanisms regulating ferritinophagy during infection with Mycobacterium tuberculosis induces ferroptosis and orchestrates TB pathogenesis 2255488

Abstract Introduction Ferroptosis, an iron-dependent mode of cell death, has emerged as a critical nexus during infection with Mycobacterium tuberculosis (Mtb), with studies reporting its inextricable link to Mtb-induced necrosis, inflammation, tissue damage and dissemination. Amidst the plethora of reports reporting the role of an impaired antioxidant system as a driver of Mtb-induced ferroptosis, we provide evidence of dysregulated iron metabolism and identify a novel post-translational modification regulating the process. Mtb infection induces ferritinophagy, the autophagic turnover of the intracellular iron chelator- ferritin, due to the accumulation of the cargo receptor, nuclear receptor co-activator 4 (NCOA4). We identify a crucial role for the methylation of NCOA4 in regulating its interaction with ferritin within the iron-replete conditions of Mtb-infected cells. Methods Using loss-of-function studies, we delineate the role of NCOA4-dependent ferritinophagy in regulating the three hallmarks of ferroptosis, availability of iron, reactive oxygen species and lipid peroxidation, during Mtb infection. Following the identification of a host arginine methyltransferase, PRMT5, in methylating NCOA4 and enhancing its cytoplasmic retention during Mtb infection, we extended our studies to BALB/c mice infected with Mtb that were treated with a PRMT5 inhibitor and assessed for mycobacterial dissemination and survival. Results We observe that the increased flux in ferritinophagy contributes to the availability of ferrous iron and is a prerequisite for Mtb-induced ferroptosis. Specifically, we found that pharmacological inhibition or knockdown of PRMT5 rescues iron homeostasis, concomitantly decreasing lipid peroxidation and ferroptosis in vitro and in vivo, thereby restricting Mtb burden. This aided in rescuing the infected mice from severe TB-like pathology. Conclusion Thus, our findings shed light on the novel roles of PRMT5 and NCOA4 during Mtb infection, and their possible implication in potentiating anti-TB responses. Funding Source Department of Biotechnology, Government of India (DBT number BT/PR47843/MED/29/1631/2023, BT/PR41341/MED/29/1535/2020, BT/PR27352/BRB/10/1639/2017 and BT/PR13522/COE/34/27/2015) Department of Science and Technology, Government of India (DST) (EMR/2014/000 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Steady-state immune surveillance of the nasal mucosa by neutrophils 2260397

Abstract Introduction The nasal cavity serves as a critical barrier, filtering airborne pathogens to prevent them from reaching the lungs. Immune cells in the nasal mucosa neutralize these pathogens, protecting against local infection. However, the composition, origins, and functions of these immune populations remain largely unexplored. Our research identified a prominent population of extravascular neutrophils (EVNs) in the nasal mucosa during homeostasis. Methods The nasal mucosa was accessed through a burr hole in the skull and imaged by two-photon excitation to visualize EVN dynamics under steady-state conditions. Neutrophil subsets were identified by fluorescent surface labeling. Parabiosis determined tissue residency, and single-cell and bulk RNA sequencing identified transcriptional differences between subsets. Results EVNs were categorized into three subsets: nN1, nN2, and nN3. We found that nN1 neutrophils reside in bone marrow adjacent to the nasal cavity and likely migrate directly into the surrounding mucosa via bone conduits. By contrast, most nN2 neutrophils access the nasal mucosa from the blood stream and then differentiate into nN3 neutrophils within 5—6 days. Functionally, nN2 neutrophils express genes characteristic of conventional neutrophils and are the primary subset responsible for phagocytosis of bacterial and fungal pathogens. Meanwhile, nN3 neutrophils progressively downregulate neutrophil specific genes and upregulate genes associated with antigen-presenting cells. Indeed, nN3 neutrophils constantly acquire material from surrounding cells under steady-state conditions, and effectively cross-present exogenous antigens to CD8+ T cells in vitro. Conclusion The nasal mucosa harbors a diverse population of EVNs with distinct origins, phenotypes, and specialized functions. These findings highlight the complexity of the nasal immune landscape and shed light on its essential and unique role in host defense. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Inflammatory monocyte recruitment depends on adventitial fibroblast-derived CCL2 following influenza A virus infection 2260912

Abstract Introduction Immune-mediated tissue damage is the major driver of severe disease after respiratory viral infections. Lung fibroblasts are emerging as central regulators of immune responses in the lungs after respiratory viral infection and have been shown to contribute to immunopathology. Recent studies have identified diverse fibroblast lineages residing in distinct lung compartments. Adventitial and peribronchial fibroblasts are located near blood vessels and larger airways in the proximal lung, and alveolar fibroblasts reside in the distal alveoli. The functional responses of these lineages and their crosstalk with immune cells during influenza A virus infections are still unclear. Methods We performed in-vitro cytokine and BAL fluid stimulations in primary fibroblast lineages, and in-vivo influenza A virus infections and determined fibroblast activation using flow cytometry. Results Our in-vitro and in-vivo experiments showed that inflammatory pathways, including IFNα and NFkêžµ signaling were preferentially activated in adventitial fibroblasts compared to alveolar or peribronchial fibroblasts. Our in-vitro experiments further demonstrated that this inflammatory activation was in part intrinsically programmed in these cells and were independent of culture conditions. Using reporter mice, we found that C-C motif chemokine ligand 2 (CCL2), a chemokine required for monocyte recruitment, was primarily expressed by adventitial fibroblasts at 6 days post-infection. Fibroblast-specific knockout of CCL2 in mice significantly reduced CCL2 levels in the blood and respiratory tract and resulted in a profound reduction in the number of monocytes recruited to the lung during infection compared to the wild type controls. Conclusion These findings identified adventitial fibroblasts as a key cell type involved in regulating early monocyte recruitment to the lung through chemokine production and highlight lung fibroblasts as a potential therapeutic target to modulate immunopathology following respiratory viral infection. Funding Source Supported by National Heart, Lung, and Blood Institute (NHLBI) - R01HL170121 , California Institute for Regenerative Medicine (CIRM) Predoctoral Fellowship - EDUC4-12759 Topic Categories Viral Immunology (VIR)

Circulating Monocytes are Associated with Centralized Pain in Patients with Rheumatoid Arthritis 2253199

Abstract Introduction Over half of patients with RA report clinically meaningful pain, despite treatment with disease-modifying antirheumatic drugs (DMARDs). While joint inflammation is a known cause of pain in patients with rheumatic diseases, emerging data indicate that many patients also suffer from centralized or nociplastic pain. There is a critical unmet need to characterize the altered cellular state that distinguish patients with centralized pain. Methods In this study, we recruited RA patients with minimal joint inflammation but varying levels of pain. All patients consented under the IRB protocol underwent quantitative sensory testing (QST) to assess centralized pain and provided blood for immune profiling using state-of-the-art multi-parameter flow cytometry with a custom panel designed to identify key immune cell types and activation states. Samples from patients in the study with the highest and lowest levels of nociplastic pain were additionally used to generate single-cell RNA-sequencing (scRNA-seq) libraries. The resulting sequencing data was processed with cellranger pipeline and analyzed using the Seurat package in R. Results We were able to collect 44 usable samples from patients who successfully consented and completed all study requirements. Supervised and unsupervised analysis of the multi-parameter flow data demonstrated that the proportions of monocyte subpopulations were positively correlated with pain levels. Moreover, preliminary analyses of scRNA-seq from 6 patients confirmed that the ratio of monocytes to T cells was increased in the high pain compared to the low pain group. Conclusion Our preliminary results indicate that monocytes are associated with centralized/nociplastic pain in RA patients. With further analysis, we aim to validate these results in the larger dataset and determine whether there are robust transcriptional changes in specific cell types. These studies provide novel insights into the role of circulating immune cells in altered CNS pain regulation in RA patients. Funding Source RRF, NIH Topic Categories Translational and Interventional Immunology (TI)

IL-5 Receptor Alpha Positive Lungs Cells Protect Against Acute Lung Injury 2257777

Abstract Introduction Acute Lung Injury (ALI) has a variety of etiologies that ultimately result in barrier dysfunction, which can lead to edema and hypoxemic respiratory failure. Previously, we demonstrated that IL-5 signaling has a protective effect on mice in the bleomycin mouse model of ALI. Mice lacking IL- 5Rα succumb to ALI and have more edema in the lung while treatment with IL-5 protects mice. Interestingly, these effects where independent of eosinophils suggesting a role for other IL-5 responding cells. We discovered that epithelial cells increased their expression of IL-5Rα seven days post bleomycin and that these IL-5Rα+ epithelial cells were highly proliferative. Additionally, IL-5Rα-positive B cells were increased in the lung tissue seven days post bleomycin administration, whereas eosinophil levels were comparable to uninjured mice. Methods To investigate the requirement of IL-5Rα in epithelial and B cells during bleomycin, a Cre-lox mouse model was utilized to specifically of delete IL-5Rα in these populations. These conditional knockouts were then subjected to bleomycin-induced ALI. Results While IL-5Rα+ epithelial cells are more proliferative, conditional depletion of IL-5Rα in lung epithelium did not impact survival during ALI. Depletion of IL-5Rα in B cells also did not have a difference in survival compared to controls however, B cell subsets were skewed towards B-2 cells. This suggests that multiple cell types are responding to IL-5 and their effect alone may not affect protection during bleomycin induced acute lung injury. Conclusion During bleomycin-induced ALI, exogenous IL-5 is protective, and we demonstrate that both lung epithelial cells and B cells respond to IL-5 through proliferation and subset differentiation, respectively. It is possible multiple IL-5 responding cells are together involved in protective mechanisms during ALI. As IL-5 signaling is a target in the clinic for reducing eosinophils, this novel IL-5 protective pathway during ALI should be considered in these patients. Funding Source T32 AI007046 Topic Categories Mucosal and Regional Immunology (MUC)