Browse Articles

Dietary consumption of psyllium modifies the microbiota to support the development of small intestinal CD4+ CD8αα+ intraepithelial T cells 2253124

Abstract Introduction Dietary fiber has a role in changing the composition of the gut microbiota, which impacts how the immune system functions. Dietary fiber is made of complex polysaccharides with many different structures. While these fibers are mostly indigestible by the host, they are a major nutrient source for members of the microbiota that encode large enzymatic machinery to break down these complex carbohydrates. Our lab has previously shown that a low fermentable fiber diet causes impaired development of small intestinal T cell populations such as CD4+ CD8αα±, also called double positive intraepithelial lymphocytes (DP IELs), which have a local regulatory function in the small intestine; and Th17 cells, which help regulate commensal bacteria colonization. This study aims to identify fiber structures that can support the development of the DP IELs when supplemented in the diet. Methods To do this, we compared the immune cell populations in the intestines of mice fed a standard chow diet and low fiber diet to low fiber diets supplemented with specific fibers, such as inulin and psyllium. Inulin is known to improve gut health by supporting the colonization of commensal bacteria such as Bifidobacteria and Lactobacilli. Psyllium can decrease gut inflammation and improve gut health by supporting the colonization of bacteria that produce butyrate. Results Our analyses show that a diet supplemented with psyllium was able to modify the microbiota and support the development of DP IELs. Conclusion In further studies we will identify the bacteria metabolizing psyllium and the mechanism by which it restores the development of DP IELs. This knowledge will allow us to identify fibers that are beneficial for the microbiota and support our immune system. Funding Source R011DK129950 Topic Categories Mucosal and Regional Immunology (MUC)

Optimal NAD+ level is a prerequisite for small intestinal secretory cell differentiation 2299891

Abstract Introduction NAD+ is key to cellular metabolism. NAMPT is the rate limiting enzyme in NAD+ synthesis, accounting for 90% of NAD+ in the body. Children harboring loss-of-function mutations of NAMPT had reduced secretory cell (SecC; goblet, Paneth) densities in the small intestine. SecCs play key roles in gut innate immunity. How NAD+ regulates SecC homeostasis is unclear. We hypothesized that NAMPT-dependent NAD+ production supports sirtuin (SIRT)-dependent Notch degradation, a necessary step in ATOH1 activation and gut SecC differentiation. Methods Intestinal epithelial cells (IECs) from wild type (WT) mice were used for snRNAseq. Various Nampt conditional knockout mice were generated: IECs (NamptΔIEC, via VilCre), SecCs (NamptΔAtoh1, via Atoh1CrePR), and Paneth cells (NamptΔPC, via Defa4Cre). Poly(I:C) was used to induce gut injury. NAD+ levels were defined by mass spectrometry. Immunoblot was used to define Notch intracellular domain (NICD), HES1, ATOH1 and acetyl-NFκB (SIRT activity). Results snRNAseq showed Nampt expression in all IEC cell types. NamptΔIEC IECs and organoids showed reduced SecC densities, without SecC apoptosis, suggesting a differentiation defect. WT organoids contained higher NAD+ levels compared to stem cells. NamptΔAtoh1 and NamptΔPC mice showed no reduction in SecCs, suggesting NAD+ is critical in SecC differentiation before ATOH1 activation. There was increased NICD and HES1 and repressed ATOH1 and SIRT activity in NamptΔIEC IEC and organoids. This was phenocopied in mice and organoids with inhibition of SIRT members. NamptΔIEC mice had more severe Poly(I:C) injury. NAD+ precursor nicotinamide ribonucleoside or overexpression of the tryptophan metabolism enzyme IDO1 rescued SecC density in NamptΔIEC mice and organoids and ameliorated Poly(I:C) injury. Conclusion Appropriate levels of NAD+ during the early phase of IEC differentiation is necessary to generate gut SecCs. This study provides a mechanistic link between cellular metabolic state and SecC proportions in the gut. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)

CX3CR1+ myeloid cell-intrinsic NOD2 signaling limits Crohn’s disease-associated intestinal fibrosis 2254570

Abstract Introduction Crohn’s disease (CD) is a chronic inflammatory bowel disease that often progresses to fibrostenotic disease, requiring surgical intervention. Intestinal fibrosis (IF) is a debilitating outcome of chronic inflammation, excessive collagen deposition, abnormal cellular functions and defective tissue remodeling. CD stems from a complex interplay of host genetics, immune dysregulation, microbial imbalance, and environmental cues. Notably, loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) — a cytosolic innate-immune receptor and the strongest genetic risk factor for CD — increase susceptibility to IF. Yet, the cell-intrinsic mechanisms by which NOD2 deficiency promotes IF remain unclear. Methods We employed single-cell RNA sequencing (scRNA-seq) in a chronic chemical-induced colitis model using littermate wild-type and Nod2-/- mice. Complementary approaches, including qRT-PCR, immunohistochemistry, RNA in situ hybridization, flow cytometry, and conditional knockout mice, were used to validate cell populations and molecular signatures associated with IF. Results scRNA-seq revealed a distinct Pi16+ progenitor population, termed FAET (Fibroblast and Endothelial Transition) cells, emerging in the fibrotic gut. Trajectory analysis indicated that FAET cells in Nod2-/- mice aberrantly differentiate into inflammation-associated fibroblasts (IAFs) rather than endothelial cells (EndoCs), leading to crypt microvasculature loss. Cellular interactome and genetic studies identified CX3CR1+ myeloid cell-intrinsic NOD2 signaling maintains CX3CR1+CD206+ gut resident macrophages that favor a niche conducive for FAET-to-EndoC transition over IAF differentiation, promoting wound healing. Consistently, ileal biopsies of CD patients carrying NOD2 variants showed reduced CD206+ macrophages and disrupted stromal-immune communication networks. Conclusion CX3CR1+ myeloid cell-intrinsic NOD2 signaling establishes a pro-restitutive stromal-immune niche that constrains IF in CD. Funding Source The American Association of Immunologists (AAI) Intersect Fellowship Program for Computational Scientists and Immunologists, Canadian Institute of Health Research (CIHR) Fellowship Program Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

TyCHE enables time-resolved lineage tracing of B cells in primary and recall germinal center reactions 2261047

Abstract Introduction Phylogenetic methods for cell lineage tracing have driven significant insights into B cell development, immune responses, and viral evolution. They have been used to identify the ignition date of the HIV-1 pandemic, and the spread of SARS-CoV-2 globally. While most methods estimate mutation trees, time-resolved lineage trees are more interpretable and could relate cellular migration and differentiation to perturbations like vaccines and drug treatments. However, somatic mutation rates vary dramatically by cell type, significantly biasing existing methods. For example, B cells undergo periods of rapid somatic hypermutation during immune responses before becoming quiescent memory cells. Methods We introduce TyCHE (Type-linked Clocks for Heterogenous Evolution), a Bayesian phylogenetics package that infers time-resolved phylogenetic trees of populations with distinct evolutionary rates. To validate our model, we developed an agent-based simulation package, SimBLE. Results Using SimBLE simulations, we show TyCHE estimates more accurate tree topologies, node dates, and ancestral cell types than existing methods. Further, we show how TyCHE can use BCR sequences to accurately reconstruct both primary GC reactions in HIV infection and recall GC reactions following influenza vaccination. We also use TyCHE to infer clinically realistic temporal evolution of a glioma tumor lineage and progression of a bacterial lung infection. Conclusion TyCHE is tailored to the unique challenges of B cell phylogenetic inference and generates highly accurate trees for B cell and non-B cell heterogeneously evolving populations. These advances in time-resolved phylogenetic inference open new avenues for understanding how cell proliferation, differentiation, and migration shape responses to vaccines, drug treatments, and other stimuli. TyCHE and SimBLE are available as open-source software packages compatible with the BEAST2 and Immcantation ecosystems. Funding Source National Institute for Allergy and Infectious Diseases grant R00AI159302 Topic Categories Computational and Systems Immunology (COMP)

Soret Effect‐Based Ionic Thermoelectric Converters: Principles, Device Physics, and Applications

ABSTRACT Ionic thermoelectric (iTE) materials have been attracting more and more attention, because they can be used to harvest heat into electricity through the Soret effect. In comparison with electronic thermoelectric materials, they have distinctive advantages including high thermopower, high mechanical flexibility, and low intrinsic thermal conductivity. But they cannot be directly adopted as the active materials of the conventional thermoelectric generators (TEGs) because the ions cannot transport across the electrodes into the external circuit. Several types of devices were reported to use iTE materials for heat harvesting. Their principles and device physics are notably different from the conventional TEGs. This article provides a review on the recent advancements in the understanding of ionic thermopower and the development of iTE‐based energy converters. To have continuous power generation even under steady temperature gradient, the iTE materials should be integrated with electronic materials. Apart from waste heat harvesting, they can have important applications in other areas, such as self‐powered sensors, human‐machine interfaces, fire protection, smart buildings, wound monitoring/repair and iTE‐enhanced catalysis. At last, perspectives including key challenges and opportunities toward scalable and robust iTE devices are provided.

Atg23 interacts with both the N- and C-termini of Atg9 via a hydrophobic binding pocket

Increased renal PCSK9 expression may be partly responsible for the elevated serum PCSK9 level and hypercholesterolemia in primary nephrotic syndrome

Akkermansia genetic variation in vitamin K production as a potential modulator of MS disease progression 2300114

Abstract Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) with heterogenous clinical presentation and disease course. MS is driven by genetic and environmental risk factors, including altered gut microbiome composition and associated bacterial metabolites. A key feature of the MS gut microbiome is increased abundance of Akkermansia, a genus recently reclassified into several distinct species. How Akkermansia species- or strain-level variation influences MS initiation, severity, or progression is unclear. Methods In a longitudinal study of people with MS, we profiled the baseline gut microbiome, correlating microbial features to disease progression; with key findings replicated in additional patient cohorts. Inferred metagenomic analysis, in silico pathway reconstruction, and bacterial comparative genomics identified metabolic drivers of disease worsening, which were validated using in vitro bacterial culture and in vivo metabolite treatment in a mouse model of MS. Results 41 bacterial species were associated with disease worsening, including depletion in Akkermansia muciniphila, while A. timonensis and A. massiliensis showed minimal or opposite associations. These findings were replicated in the International MS Microbiome Study, where A. muciniphila abundance correlated with MS severity. Metagenomics revealed reduced microbial vitamin K2 (VitK) biosynthesis linked to Akkermansia. Genomic analysis of Akkermansia isolates identified species and strains with deletions or variants in enzymes central to VitK biosynthesis, mirrored by differential in vitro VitK production. In mouse models of MS, VitK supplementation reduced CNS autoimmunity in females, while VitK-deficient diets increased coagulopathy in males. Conclusion Akkermansia species-level genetic variation, particularly within the VitK pathway, may modify MS disease progression in a sex-dependent manner. Akkermansia and VitK may be indicated as a tailored therapeutic approach to modulate MS disease progression. Funding Source National MS Society Postdoctoral Fellowship Topic Categories Mucosal and Regional Immunology (MUC)

Natural variation in the activity of RUNX transcription factors pattern the development of effector and memory CD8 T cells 2309563

Abstract Introduction CD8 T cells respond to viral infection and generate memory populations that protect against subsequent encounters with the same antigen. The transcription factor (TF) RUNX3 is required for the formation of circulating and tissue-resident memory CD8 T cells; however, the roles of the co-expressed paralogs RUNX1 and RUNX2 in this process remain poorly defined. Methods We used P14 TCR-transgenic CD8 T cells which recognize LCMV-Armstrong, a virus that produces an acutely resolving infection in mice. Chromatin accessibility at RUNT motifs was assessed by ATAC-seq in RUNX3-depleted P14 cells at day eight post-infection. The three RUNT TFs, or their obligate co-factor CBFβ, were individually depleted by retrovirally delivered shRNAmirs in Tcf7-reporter P14 cells. These experiments were integrated with previously generated scRNA-seq data from day 8 P14 cells. De novo transcriptome assembly of bulk RNA-sequencing data was used to identify expressed RUNX TF splice isoforms. Results Depletion of RUNX3 resulted in an incomplete loss of chromatin accessibility at RUNT motifs, suggesting compensation by RUNX1 or RUNX2. RUNX1 directed formation of CX3CR1hiKLRG1hi terminal effector cells, whereas RUNX3 counteracted this program to generate a stable CX3CR1intKLRG1int precursor population that gives rise to tissue-resident and central memory CD8 T cells. Depletion of RUNX2 had minimal effects on most effector populations but was required for the formation of Tcf7hiCX3CR1loKLRG1lo central memory precursors. These findings were corroborated by single-cell RNA-sequencing, which revealed variation in RUNX TF activity correlated to distinct transcriptional states. De novo transcriptome assembly further identified differential usage of Runx1 and Runx2 splice isoforms that differed by inclusion of an ETS interaction motif. Conclusion The three RUNX TFs, and their distinct splice isoforms, likely compete at RUNT motifs, resulting in variation of RUNX activity that directs formation of specific CD8 T cell states. Funding Source NIH U19AI109976, R01AI095634 , P01AI145815 (M.E.P.) AHA 24PRE1191318 (D.S.A.) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Genetic linkage analysis identifies SNX3 as a regulator of antigen cross-presentation during anti-tumor antibody therapy 2309751

Abstract Introduction Trastuzumab, an anti-HER2 targeted immunotherapy (ITx), has changed the landscape of HER2+ breast cancer survival, but drug resistance and disease recurrence still present a problem. Patients who develop anti-tumor immunity after targeted ITx show improved long-term benefit, but host-intrinsic mechanisms regulating immune priming remain unclear. Methods (Diversity OutbredxBALB/c)F1 (DOF1) mice bearing HER2/neu+ TUBO mammary tumors were treated with anti-HER2/neu mAb 7.16.4. Genetic linkage analysis was performed in R/qtl2 to identify loci associated with response and select Collaborative Cross (CC) mouse models with driver genetics. scRNAseq was performed on CD45+ TUBO infiltrates from CCF1 mice. To measure cross-presentation, DC2.4 dendritic cells were transduced with shSNX3 or control lentiviruses and cocultured with TUBO cells expressing ovalbumin (OVA) prior to the addition of OVA-specific OT-I CD8 T cells. scRNAseq was performed on 46 trastuzumab-treated HER2+ breast cancer samples (∼60% responders) collected prior to therapy. Results We find that host genetic background influences endogenous immune priming after targeted ITx in mice. DOF1 TUBO-bearing mice exhibited differential response to 7.16.4 treatment; ∼45% of mice eliminated primary tumor with protection against contralateral rechallenge. Genetic linkage analysis and tumor immune infiltrate scRNAseq identified SNX3 as a potential regulator of response to targeted ITx. We observed increased OT-I activation in the presence of DC2.4/shSNX3, suggesting SNX3 negatively regulates antigen cross-presentation. Supporting this hypothesis, our human breast cancer scRNAseq dataset showed an inverse correlation of trastuzumab response with dendritic cell SNX3 expression. Conclusion We demonstrate that genetic background influences response to targeted ITx and identify SNX3 as a regulator of response through antigen presentation to CD8 T cells. Future studies aim to leverage SNX3 as a novel actionable gene to improve targeted ITx outcomes. Funding Source R37CA220482, P20CA290450, P30CA022453, Pardee Hospital Foundation, DMC Foundation Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

CD15+ Myeloid Cells from Patients with Disseminated Coccidioidomycosis Inhibit CD4+ T Cell Proliferation 2260247

Abstract Introduction Valley fever (VF) is a respiratory disease caused by Coccidioides and is estimated to cost over 3 billion in medical costs annually. In VF, the infiltration of Ly6G+ cells into the lungs has been reported in mice and humans, but the role of these cells has not been fully uncovered. Ly6G+ cells share a phenotype with myeloid-derived suppressor cells (MDSCs), which suppress protective immunity. We previously showed, Ly6G+ cells enriched from the lungs of non-protected mice suppressed CD4+ T cell proliferation and exacerbated disease in vaccinated mice. Thus, a portion of the infiltrated Ly6G+ cells comprises MDSCs, which contribute to the pathogenesis. Parallelly, we have analyzed PBMCs from VF patients, to investigate whether these myeloid cells are immunosuppressive. We hypothesize CD15+ cells isolated from the PBMCs of disseminated patients suppress CD4+ T cell function to a greater extent than those isolated from recovered patients. Methods PBMCs from recovered (n = 18), pulmonary (n = 5), and disseminated (n = 11) VF patients were evaluated for CD15 + (HLA-DR-CD11b+CD15+). A functional assay was performed in recovered (n = 10), pulmonary (n = 4), and disseminated (n = 6) patients. CD15+ cells were enriched and combined with autologous CD4+ T cells at a 1:1 ratio with anti-CD3/CD28 beads for 72 hours. Results Flow cytometric analysis revealed that the composition of CD15+ myeloid cells in PBMCs from three patient cohorts was comparable, consistent with other reports of microbial infection. Indeed the CD15+ cells from disseminated patients suppressed a significantly higher percentage of CD4+ T cell proliferation than those from recovered and pulmonary patients. Conclusion These data demonstrate that CD15+ myeloid cells from disseminated patients suppress CD4+ T cells, a function shared with MDSCs. This discovery is parallel to our previous report that MDSCs are recruited into the lungs and exacerbate disease in Coccidioides-infected mice. Our data provide an insight on the role of CD15+ myeloid cells in VF. Funding Source The National Institute of Allergy and Infectious Diseases, National Institutes of Health, provided funding for this research under grants R01 AI135005 (CYH), U19AI166761 (CYH), and training grant T32AI184340 (RN and MMB). Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Human Treg Deep Phenotyping by Flow Cytometry: Advancing Immunosafety Assays in Autoimmune and Healthy Populations 2305384

Abstract Introduction The FDA 2025 roadmap for reducing animal testing in preclinical safety studies has accelerated the need for robust human in vitro models in Immunosafety. Regulatory T cells (Tregs) are central to immune tolerance and play a critical role in autoimmune disease pathogenesis and therapeutic response. This study aimed to develop a comprehensive flow cytometry panel for deep phenotyping of human Tregs, facilitating their characterization in both healthy and autoimmune populations. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from consented donors. A 13-color flow cytometry panel was designed for compatibility with Treg isolation kits. The panel was tested with PBMCs isolated from healthy donors and autoimmune donors with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), or type I diabetes (T1D). Additionally, Tregs were purified and expanded using CD3/CD28 stimulation and IL-2 supplementation and expanded Tregs were tested side-by-side with PBMCs from matched donors. High-dimensional analysis was performed using FlowJo plugins (UMAP, FlowSOM) to validate gating strategies and marker expression. Results The panel reliably identified Treg subsets, with Helios and FoxP3 distinguishing thymic-derived Tregs. High-dimensional analysis confirmed equivalence between conventional gating and unsupervised clustering. Compared to healthy donors, Helios was significantly upregulated in SLE donors. ICOS was significantly downregulated in medicated SLE and RA donors, and CD95 was significantly downregulated in SLE and RA donors. LAG3 staining showed no difference between healthy and autoimmune donors. Conclusion The deep phenotyping Treg flow cytometry panel and Treg expansion protocols enhance the Immunosafety toolbox for human in vitro and in vivo immune toxicity testing. These methods support the transition to complex in vitro human models and provide valuable tools for future studies and regulatory assessments. Funding Source n/a Topic Categories Basic Autoimmunity (BA)

Liver sinusoid constraints make random search of CD8 T cells for Plasmodium parasites efficient 2254515

Abstract Introduction A relatively small number of liver-localized CD8 T cells can provide sterilizing protection against exposure to Plasmodium sporozoites. Previously published mathematical model-based simulations of randomly searching for an infection T cells, assuming that T cells move in open space or on a regular lattice, suggested that some degree of attraction is needed for cells to find the infection. Yet, in our previous experiments we failed to find evidence of attraction towards the infection in most T cells. Because the movement of liver-localized T cells is confined to sinusoids, the blood vessels of the liver, T cells may in fact be attracted to the infection site via the sinusoids. Methods In new experiments we imaged Plasmodium-specific CD8 T cells, sporozoites, and liver sinusoids simultaneously. We also developed a new method to model imaged sinusoids as graphs and to simulate movement of T cells on graphs, and new tests to detect biased cell movement on graphs. Results As before, our previously published open space/3D-based metric suggested that few T cells are attracted to the infection site in presence of moderate cluster of T cells. However, with our new graph-based metric we could not detect attraction when constraining T cell movement to sinusoids. Surprisingly, simulations of T cells searching for an infection via liver sinusoids showed that a relatively small number of T cells (1 cell/imaging volume or < 10^5 cells/liver), moving without attraction at experimentally observed speeds, is sufficient to find the infection within 24 hours, well below the expected lifespan of the Plasmodium liver stage in mice. Conclusion Our results suggest that the constrained environment of the tissue structure makes a random search more effective, and that detecting T cell attraction towards specific areas may arise as an artifact of metrics designed for open space/3D. Our results thus call for a re-evaluation of the implicit assumption that attraction is necessary for T cells to find infections in tissues. Funding Source R01GM118553, R01AI158963 Topic Categories Vaccines and Immunotherapy (VAC)

Brain Fog in Infection-Associated Chronic Illnesses (IACI): Novel Insights from Neurocognitive and Molecular Profiling of the MAESTRO Clinical Study 2261026

Abstract Introduction There is a complex interplay between the host immune system and pathogens in Infection-Associated Chronic Illnesses (IACI), including those following Lyme disease and COVID-19, which pose a growing public health concern with limited diagnostic and therapeutic options. Among their most debilitating and least understood symptoms is “brain fog.” Objective measures of neurocognitive dysfunction remain insufficient. The MIT MAESTRO study addresses this gap through a comprehensive, multi-dimensional approach to characterize neurocognitive and physiological changes in IACI. Methods The MAESTRO Study profiles individuals with IACI from Lyme and COVID through neurological assessments and commercially available neurocognitive tests, including eye movement tracking, EEG, and cognitive test battery. Additional assessments include autonomic dysfunction, proxy cerebral blood flow, capillaroscopy, hypermobility testing, integrated with extensive surveys and deep biological profiling (urine, sweat, saliva, blood, vaginal, and rectal swabs) for pathogen loads, dysbiosis, genomics, metabolomics, and proteomics, aiming to uncover molecular and neuroimmune signatures underlying these complex conditions. Results We found significant neurocognitive differences unique to individuals with IACI. Greater symptom burden correlated with objective outcomes. Reaction times across modalities were consistently slower in IACI participants than controls. Preliminary results suggest that barrier permeability may relate to cognitive dysfunction. Protemotic findings suggest associations between neuroinflammatory features and reaction time, indicating potential physiological links to neurocognitive function. Conclusion Using commercially available tools to objectively assess neurocognitive impairments may improve clinical care for individuals with IACI. Integrating neurocognitive, physiological, and molecular data could help identify early changes and enable longitudinal follow up and tracking. Funding Source NIH RO1 AI 178713, Emily and Malcolm Fairbairn, Mass Life Sciences or Mass Life Science Center (MLSC), The Manton Foundation Topic Categories Neuroimmunology (NEUR)

Kinetics study reveals interconnected relationships of component activities in trifunctional RNA capping enzymes

Severe pollution with toxic elements in road dust from Baia Mare city, Romania

pH-Dependent Innate Immune Circuitry Confers Tolerance to Systemic Inflammation 2303150

Abstract Introduction PH homeostasis is essential for physiological function, and disruptions in pH are a common feature of inflammation and disease. Immune cells monitor pH changes through specialized receptors, including the proton-sensing G-protein-coupled receptor GPR65. It is widely expressed on tissue-resident immune cells and detects subtle pH variations within the physiological range (pH 7.0-7.4). Methods We compared WT and Gpr65 whole-body knockout mice as well as cell-type specific Gpr65 knockouts, to study the role of GPR65-mediated pH sensing in regulating tolerance to systemic inflammation. Results We identify GPR65 as a critical regulator of tolerance to systemic inflammation. GPR65-deficient mice exhibit markedly increased lethality following systemic bacterial infection, despite normal pathogen burden and clearance. This heightened sensitivity is recapitulated during LPS-induced sepsis. We found that late-phase IL-17A production from peritoneal γδ T cells is both necessary and sufficient to drive the enhanced lethality in Gpr65⁻/⁻ mice. Strikingly, IL-17A levels are controlled by a “dual-safe” myeloid- γδ T cell circuit: GPR65 limits myeloid-derived IL-1β and IL-23 that are required for activating γδ T-cell IL-17A production, while simultaneously constraining the intrinsic responsiveness of γδ T cells to inflammatory cues. Thus, GPR65 signaling in either compartment of the myeloid—γδ T-cell axis is sufficient to maintain tolerance to systemic inflammation. Mechanistically, GPR65-mediated pH sensing regulates both the magnitude and kinetics of IL-10 expression in myeloid cells, which is crucial for restraining IL-1β and IL-23. In γδ T cells, GPR65 dampens cytokine sensitivity through intrinsic rewiring of transcriptional programs. Conclusion Together, this work reveals that GPR65 enforces a “dual-safe” innate immune circuit that calibrates the balance between protective immunity and inflammatory pathology, establishing pH sensing as a fundamental environmental checkpoint in immune-mediated disease. Funding Source NIH NIDDK, Cystic fibrosis foundation, NIH NIGMS Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

MR1-driven activation of MAIT cells orchestrates anti-tumor immunity in melanoma 2309830

Abstract Introduction Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that recognize riboflavin-derived metabolites presented by the monomorphic antigen-presenting molecule MR1. MAIT cells are enriched in barrier tissues and tumors, yet their role in cancer remains controversial. Recent evidence suggests that appropriate activation can reprogram MAIT cells toward cytotoxic, Th1/NK-like effector functions. Here, we examined whether MR1-dependent activation of MAIT cells promotes anti-tumor immunity in cutaneous melanoma. Methods MAIT cells were activated in vivo using the MR1 agonist 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) combined with the TLR9 agonist CpG. Tumor growth was monitored longitudinally. Immune infiltrates in skin and tumors were analyzed by multiparametric flow cytometry using MR1 tetramers. MR1 dependency and host antigen presentation were assessed using MR1 knockout and conditional MR1 deletion models. Antibody-mediated depletion was used to define the contribution of downstream immune subsets. Results MAIT cell activation significantly delayed melanoma growth compared to control-treated mice. Tumor suppression was strictly MR1 dependent, as anti-tumor effects were lost in MR1-deficient hosts. MAIT activation induced robust expansion and accumulation of MAIT cells in skin and tumor tissue and was associated with increased infiltration of NK cells and CD8+ T cells. Depletion studies demonstrated that MAIT cells and NK cells were required for tumor control. Conditional deletion of MR1 in host myeloid cells impaired MAIT-mediated anti-tumor immunity, indicating a critical role for MR1-dependent antigen presentation within the tumor microenvironment. Conclusion These data demonstrate that MR1-dependent activation licenses MAIT cells as potent anti-tumor effectors that coordinate broader immune responses in melanoma, supporting MR1-MAIT targeting as a potential immunotherapeutic strategy. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Indoleamine 2,3-dioxygenases-isoforms in the host anti-tuberculous immunity and vaccine response 2260176

Abstract Introduction Tuberculosis (TB) is a major public health burden. One microbial immune-regulatory mechanism is pathogen-driven modulation of host immunometabolism via the tryptophan catabolizing indoleamine 2,3-dioxygenases (IDO1 and IDO2). We used gene knockout (KO) mice and IDO inhibition to study how these enzymes impact anti-TB immunity and vaccine efficacy. Methods We used ido1-/-, ido2-/-, and ido1/ido2-/- mice, in conjunction with targeted pharmacological IDO inhibition to define the roles of IDO isoforms during Mtb infection and BCG immunization. Results Biochemical analysis revealed that the tryptophan catabolic pathway was active in Mtb infected tissue in mice. Flow cytometric analysis of lungs and lymph node (LN) cells from mice infected with Mtb Erdman showed that IDO1 expression is in general, higher than that of IDO2 in dendritic cells (DCs), plasmacytoid DCs, and macrophages. Results derived from IDO-deficient mice and inhibitor studies showed that i) the two isoforms differentially modulate Mtb susceptibility, as assessed by tissue bacterial loads and mortality rates, in a dose- and infection phase-dependent manner; impacting ii) the Th1, Th17, neutrophilic, Treg and the TNF+ CD4+ T cell response; iii) IDO1 inhibition during BCG vaccination resulted in higher levels of central memory CD4+ T cells (TCMs) in the lungs and LNs, compared to that observed in immunized mice without inhibitor treatment; iv) Mtb challenge in BCG vaccinated, IDO inhibitor-treated mice exhibited diminished tissue bacillary loads that was associated with reduced levels of granulomatous inflammation. Conclusion Our findings suggest that IDO1 and IDO2 serve as immunometabolic checkpoints in TB infection and vaccine response. We propose that targeting IDO1 during immunization could enhance vaccine-elicited immunity, possibly via the augmentation of lung and LN TCM response. This metabolic modulation may be applicable to other microbes to augment vaccine immunogenicity. Funding Source NIH NIAID R01 AI137344 Topic Categories Vaccines and Immunotherapy (VAC)

MTHFR Allele and One-Carbon Metabolic Profile Predict Severity of COVID-19 Disease 2260350

Abstract Introduction Alterations in the methionine cycle, a key component of the one-carbon metabolism pathway that is critical for viral propagation and disease progression, may serve as early indicators of severe SARS-CoV-2 infection. Methods As part of the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study, we performed a longitudinal analysis of untargeted plasma metabolic profiles of hospitalized adult COVID-19 patients followed from admission to 1-year post-discharge. Using a 7-point ordinal scale, patients were clustered into 5 disease trajectory groups based on respiratory illness during hospitalization and targeted metabolomics was assessed using liquid chromatography-mass spectrometry. Results Metabolic profiling revealed that patients in more severe disease trajectory groups (TG4-TG5) showed alterations in the methionine cycle. Specifically, S-adenosylmethionine (SAM), a methyl group donor important for gene expression and methylation of DNA, RNA and proteins, was highly abundant among the more severe trajectory groups (TG4-TG5) compared to the less severe trajectory groups (TG1-TG3). In addition, we identified the C766T allele mutation of the MTHFR gene, a common polymorphism, as a genetic contributor to the methionine pathway and predictor of disease trajectory. Conclusion These results highlight the potential of metabolic characterization of the methionine pathway in combination with screening for the common genetic MTHFR variant as a practical approach for precision COVID-19 management. Funding Source NIH (3U01AI167892-03S2, 3U01AI167892-01S2, 5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI057229-18, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, and 5T32DA018926-18, 3U19AI1289130, U19AI128913-04S1, and R01AI122220, UM1TR004528); NSF DMS2310836 Topic Categories Computational and Systems Immunology (COMP)