Browse Articles

Lymphocyte Inhibition by Cell Wall Components of Batrachochytrium dendrobatidis (Bd) 2333163

Abstract Introduction The chytrid fungus, Batrachochytrium dendrobatidis (Bd), is linked to continuing global amphibian population declines. Bd causes the lethal disease chytridiomycosis when swimming zoospores attach, encyst, form a germ tube, and invade epidermal skin cells. Disruption of the host skin results in death. Although amphibians have robust immune defenses, this fungus has evolved mechanisms to evade immunity. Previous studies have revealed three small metabolites released by Bd that inhibit lymphocyte responses. We hypothesized that another molecule or molecules associated with the fungal cell wall is also inhibitory for lymphocytes. Methods To investigate the role of cell wall components, we fragmented mature zoosporangia, isolated the cell wall components lacking other soluble factors, and cultured them with proliferating frog spleen cells (Xenopus laevis) or Jurkat T cells. The cell wall fractions were boiled or treated with a mixture of carbohydrase enzymes to investigate the nature of potential inhibitory factors. Results Enriched cell wall fragments alone inhibited proliferation of both frog spleen cells and Jurkat T cells in a dose-dependent manner. Boiling of the cell wall fragments or treatment with carbohydrase enzymes reduced their capacity to inhibit lymphocyte proliferation. Conclusion These results suggest that the active inhibitory factor in the Bd cell wall is a glycosylated protein or a protein-carbohydrate complex. Ongoing studies aim to further understand the nature of the lymphocyte inhibitory factors associated with the chytrid cell wall. Support: Vanderbilt SYBBURE Searle Undergraduate Research Program and NSF IOS-2147467. Funding Source NSF IOS-2147467 Topic Categories Veterinary and Comparative Immunology (VET)

Aim2 inflammasome dependent IL1β/IL-18/Gasdermin D signaling is required for initiation of stress erythropoiesis in anemia of inflammation 2258281

Abstract Introduction Anemia of inflammation (AI) accompanies chronic inflammatory diseases. While steady-state erythropoiesis makes ∼2.5 × 106 RBCs/s, inflammatory cytokines shift hematopoiesis toward myeloid effectors suppressing erythroid differentiation and causing AI. To sustain red-cell output stress erythropoiesis expands self-renewing stress erythroid progenitors (SEPs) that rapidly proliferate and differentiate into RBCs. We investigated Aim2 inflammasome control of early SEPs and caspase-1—dependent maturation of IL-1β/IL-18 and the pore-forming protein gasdermin D (GSDMD). Methods Primary mouse marrow was cultured in a two-phase stress-erythropoiesis system: an Epo-free hypoxic expansion phase (BMP4, Hedgehog, GDF15, SCF on macrophage stroma) yielding immature SEPs, followed by Epo-driven differentiation. Perturbations included recombinant IL-1β/IL-18, IL-18 neutralization, and marrow from Aim2⁻/⁻, Gsdmd⁻/⁻, or Il1r1⁻/⁻ mice. Proliferation (viable counts), phenotype (flow cytometry), and output (BFU-E/CFU-E) were measured. In vivo, heat-killed Brucella abortus (HKBA) induced sterile inflammatory anemia; hematocrit, spleen, and serum were profiled for AIM2/caspase-1/IL-1β/IL-18 and SEP abundance. Results Loss of AIM2 and GSDMD impaired proliferation of immature SEPs and reduced BFU-E. Deletion of Il1r1(Receptor for IL1β) or neutralization of IL-18 similarly decreased early SEP expansion, whereas exogenous IL-1β/IL-18 enhanced it. In-vitro,IL-1β increased IL-1R1 on SEPs and upregulated PU.1, enriched in the most immature subsets. In HKBA anemia, Aim2⁻/⁻ mice showed reduced hematocrits and contracted immature SEPs. Il1r1⁻/⁻ and Gsdmd-/- mice failed to mount an effective response due to defects in the splenic monocyte/macrophage population due to impaired IL-1β signaling. Conclusion The AIM2/GSDMD/IL-1β/IL-18 axis is a primary driver of early SEP expansion, mechanistically linking dsDNA sensing to cytokine maturation, IL-1R upregulation, and PU.1 modulation. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)

The inflammatory response and immune cell status following radiation exposure influence the host’s susceptibility to new infections 2306220

Abstract Introduction Tissue-targeted and whole-body irradiation (WBI) are widely used to treat patients with solid tumors and hematologic malignancies, respectively. Although medical countermeasures (MCMs) are in place to reduce the side effects of radiation exposure (RE), understanding how RE affects host immune status is critical to improve the protection against infection and sepsis. Methods We conducted a retrospective cohort study using the SlicerDicer™ tool to identify patients admitted to the University of Iowa Hospitals and Clinics (UIHC) from 2015 to 2025. Patients with a documented history of ionizing RE who later developed infection or sepsis were included; non-RE patients served as controls. For mouse studies, wild-type C57BL/6 mice were subjected to 0, 2, or 5Gy doses of WBI. Ten or more days after RE, mice received either lipopolysaccharide (LPS) or underwent cecal ligation and puncture (CLP) to induce sepsis. For infection studies, mice were challenged with Streptococcus pneumoniae or attenuated Listeria monocyogenes following RE. Results Patients with a history of RE exhibited a higher risk of developing infection, sepsis, and septic shock compared with non-RE controls. Although the incidence of infection and sepsis was highest shortly after RE, it declined over time. Interestingly, RE conferred WBI dose-dependent protection against two different LPS doses in mice, as reflected by improved control of pro-inflammatory cytokines compared with 0Gy mice. Notably, RE mice remained susceptible to polymicrobial CLP sepsis despite displaying inflammatory responses comparable to 0Gy mice. RE, also impaired cell-mediated immunity, thus failing to clear bacterial pathogens. While treatment with ampicillin at 12 hours post-infection improved survival, RE mice still exhibited defective pathogen clearance. Conclusion Thus, immune cellularity and inflammation control after RE determine the host’s ability to clear infection and improve survival. These findings help guide future work to improve MCMs for RE patients. Funding Source n/a Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Immunophenotyping of T-cell Responses from Immunocompromised Hosts in Response to Vaccination 2255680

Abstract Introduction Immunocompromised hosts, including those with lymphoid or hematological malignancies and solid organ transplant recipients, are more susceptible to contracting infections and thus require protection from various pathogens but often have reduced responses to vaccination. Repeat immunizations against various pathogens including SARS-CoV-2, influenza, and Respiratory Syncytial Virus (RSV) have been offered, and we have previously demonstrated that the cellular immune response to such immunizations was sub-optimal, but more preserved than the humoral immune response, in terms of frequencies of post-immunization antigen-specific T cells. Objective To evaluate functional and phenotypic fingerprints of vaccine-induced T-cell responses of several cohorts of immunocompromised patients. Methods Peripheral blood draws from immunocompromised hosts were collected pre-vaccination and post-vaccination (SARS-CoV-2 mRNA vaccine, SARS-CoV-2 adjuvanted protein vaccine, and RSV adjuvanted or non-adjuvanted protein vaccine). Antigen-specific cellular immune responses were captured using the Activation-Induced Marker (AIM) assay, enabling flow cytometry-based quantification of antigen-specific T cells and additional immunophenotyping. Antigen-specific T cells assessed include SARS-Cov-2, influenza, and RSV. Results Human in vitro modeling was successfully utilized to complete deep immunophenotyping of T cells responses both pre- and post-vaccination. An increase in antigen-specific T cells could be observed in a subset of each cohort. Ongoing analyses will reveal whether specific T-cell functionalities or phenotypes are associated with successful increase in T-cell frequencies post-immunization. Conclusion T cell responses from immunocompromised hosts demonstrated heterologous responses to vaccination. These results could be contributed to varying medication types utilized by patients and additional investigation is required to understand the continuation of immunosuppressive drugs on vaccine responses. Funding Source NIH, Barber and Amos Hostetter, and Chleck Foundation Topic Categories Vaccines and Immunotherapy (VAC)

Time‐Encoded Geometric Encryption Enabled by Shape‐Memory Hydrogel with Photoisomerization‐Gated Autonomous Recovery

ABSTRACT Leveraging spatiotemporal shape‐changing of information‐bearing morphologies, materials with predetermined autonomous‐shape‐morphing potentially offer a unique form of time‐encoded geometric communication in contrast to existing optical encryption methods. However, fundamental mechanisms and control strategies underlying the spatiotemporal programmability remain insufficiently explored, thus the information capacity and security levels are still restricted. Here we report a non‐thermal and spatiotemporally controllable strategy to gate the morphing kinetics of shape‐memory hydrogel via photoisomerization. Upon functionalization of azobenzene moieties, multiple amide‐amide hydrogen bonds are incorporated into the poly(acrylamide) hydrogel. These hydrogen bonds, with strong time‐temperature dependence, act as stress‐damping units that decelerate the network's inherent elastic recovery, providing a mechanism for time‐encoded autonomous‐shape‐morphing. Photoisomerization of the pendant azobenzene moieties modulate the thermodynamic state of the hydrogen bonds, thus can control the morphing kinetics. Through orthogonal time and photo‐spatial programming of the dynamic interactions, the hydrogel can autonomously execute sophisticated shape transformations along predetermined pathways, thereby serving as a carrier for storing geometric information. This work demonstrates the feasibility of time‐encoded shape morphing and establishes an alternative strategy to conventional optics‐based encryption, providing enhanced data capacity and security through time‐dependent geometric encoding.

Upregulation of Fatty Acid Metabolism in Activated Synovial CD4 T Cell Populations in Rheumatoid Arthritis 2259803

Abstract Introduction Rheumatoid arthritis (RA) is an autoimmune disease involving joint damage driven by infiltrating immune populations. The RA synovium accumulates T peripheral helper cells (Tph), a T cell population that expresses high levels of PD-1, ICOS, and CXCL13 and lacks CXCR5. Circulating T cells from RA patients show a range of metabolic abnormalities. However, the metabolic features of synovial Tph cells and how these features regulate function remain unclear. Methods We utilized SCENITH to interrogate bioenergetic profiles of T cells from seropositive RA synovial fluid. Single cell RNA-seq data of T cells from RA synovial tissue was analyzed to identify metabolic genes upregulated in synovial Tph cells. We confirmed protein expression of an identified target, FATP2, in RA synovial fluid T cells. BODIPY 493/503 and BODIPY FL-C16 were used to assess neutral lipid content and fatty acid uptake capacity in RA synovial fluid T cells. BODIPY FL-C16 was used to assess the impact of FATP2 on fatty acid uptake capacity in CRISPR-modified human memory CD4 T cells. Results SCENITH revealed that Tph cells showed the highest baseline puromycin binding, reflecting high metabolic activity, and reduced oligomycin sensitivity among RA synovial fluid T cells. Single cell RNA-seq and flow cytometric analysis identified the selective upregulation of FATP2 within RA synovial Tph cells. Consistent with this, RA synovial fluid Tph cells have heightened neutral lipid content and fatty acid uptake capacity. CRISPR deletion of FATP2 in human memory CD4 T cells led to decreased fatty acid uptake. Conclusion Tph cells show heightened global metabolic activity compared to other T cells in RA synovial fluid. Synovial fluid Tph cells display increased lipid accumulation, fatty acid uptake capacity, and selective upregulation of FATP2 suggesting an altered lipid metabolism. FATP2 serves a non-redundant role in exogenous fatty acid uptake in human CD4 T cells. Funding Source This work is funded in part by a Burroughs Wellcome Fund Career Award in Medical Science and R01 AR078769 from NIAMS (to DAR). RTB is supported by T32 AR007530 from NIAMS. Topic Categories Basic Autoimmunity (BA)

Hybrid Cytometry: automated live-cell tracking with a final snapshot of fixed-cell phenotyping to study immune-synapse dynamics 2263890

Abstract Introduction Even advanced flow cytometry and fluorescence microscopy fall short in linking dynamic immune cell functions and phenotypes at high plexity. We aimed to develop an automated workflow that bridges this gap, capturing mixed culture dynamics with deep phenotyping and optional integration with high-resolution imaging or single-cell RNA sequencing, tracking individual cells throughout the experiment. Methods We implemented an imaging-based workflow integrating automated liquid handling, fluorescence whole-plate scanning, and tracking of sequential activation cycles in live cells, together with corresponding cytokine or surface marker expression in fixed cells. Datasets were computationally aligned to correlate up to 3-plex dynamic functional readouts with phenotypic features. A plexity of 12—20 markers can be achieved through sequential immunostaining. Results This centrifugation-free pericellular liquid exchange workflow enables us to: • Track individual T cells through repeated peptide stimulations of human PBMCs; • Validate activation by downstream cytokine or surface marker staining; • Increase per sample data yield up to 20—100-fold versus ELISpot or flow cytometry; • Visualize transient immune synapses and interactions including antigen presentation, cross-presentation, T-cell—B-cell cooperation, dynamic killing, and Teff—Treg proximity. Conclusion This method provides a scalable, high-content approach for low-volume, single-cell immunology supporting target discovery, vaccine design, and mechanistic research. Funding Source 100XBIO Inc. (Private Investment Funding) Topic Categories Technological Innovations in Immunology (TECH)

Distinct Immune Signatures are Associated with Exposure History in Human Malaria 2259590

Abstract Introduction As a surrogate to interrogating the diverse human immune response to natural malaria, we examined responses to four sequential controlled human malaria infection (CHMI) in malaria-naïve adults. Methods We used a multi-modal approach, integrating: 1) single-cell RNA-sequencing and Over-Representation Analysis (ORA) of naive peripheral blood mononuclear cells (PBMC) stimulated with Pf schizonts; 2) longitudinal plasma cytokine analysis; and 3) comparative cytokine profiling and Principal Component Analysis (PCA) of CHMI participants and Ghanaian children presenting with malaria illness. Results The naive monocyte response to Pf in vitro involves the simultaneous upregulation of pro-inflammatory cytokines and key regulatory molecules. ORA confirmed this paradox, showing co-activation of pro-inflammatory (NF-κB, TNF) and anti-inflammatory (IL-10) signaling pathways, alongside a decrease in host phagocytosis, cell metabolism, pathogen recognition and presentation. In vivo, both CHMI and natural Pf exposures trigger a similar paradoxical response. The maturation of the CHMI response is characterized by a progressively IL-10-dominant signature over four infections and a consistent post-infection increase in IL-18. PCA revealed a specific cytokine balance that differentiates the CHMI participants and Ghanaian children. Conclusion A paradoxical immune response was noted in both malaria-naïve and malaria-endemic individuals with PBMC expression of pro and anti-inflammatory cytokines. When compared to the CHMI participants response, the higher IL-10 and IL-18 and lower IL-21 and IL-4 response in Ghanaian children is consistent with high exposure in endemic areas modulating monocyte activation. Defining these different developmental and endpoint signatures is key for evaluating vaccine strategies and understanding naturally-acquired immunity. Funding Source 5R01AI177698-03 - National Institutes of Health (NIH) Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

UCD200/206 scleroderma-prone chickens show aberrant innate immune function 2335106

Abstract Introduction Non-communicable diseases (e.g., autoimmunity) typically are multifactorial diseases, requiring genetic, immune system, and environmental factors for expression. The UCD200/206 chickens spontaneously develop scleroderma/systemic sclerosis (SSc). The hallmarks of SSc are fibrosis (e.g., dermis of the skin), vasculopathy, and vascular occlusion leading to tissue pathology in highly vascularized organs (lungs and kidneys). Initial observable abnormalities manifest one to two weeks post-hatch and include severe comb swelling and erythema, leading to necrosis and partial or complete loss of the comb. Dermal lesions include swelling, induration, and loss of feathers, and become evident at 3 weeks of age, in the dorsal neck region. In human SSc, there is growing evidence for the role of innate immunity and aberrant innate immune system function in the development of SSc. Methods To determine innate immune response capabilities of UCD200/206 and healthy Leghorn controls, we examined inflammatory responses initiated by innate immunity activators using the minimally invasive growing feather (GF) cutaneous bioassay. Each test-material was administered by intradermal injection multiple GF-pulps of a bird, and GFs collected before-and at various times post-injection for ex vivo analyses. The test-materials were lipopolysaccharide, peptidoglycan, Mycobacterium spp., and Salmonella spp. bacterins. Collected GFs were used for immunofluorescent staining and leukocyte population analysis by flow cytometry, and cytokine mRNA expression analyses by qRT-PCR. Results Aberrant innate inflammatory responses to test-materials, especially to components of gram+ bacteria and particulate antigens were observed, contributing to new knowledge of the temporal, qualitative, and quantitative aspects of inflammatory responses in the SSc-chicken model. Conclusion The spontaneous development of SSc in UCD200/206 chickens provides opportunity to dissect the interactions involved in non-communicable diseases with immunopathology. Funding Source Tyson Professorship in Avian Immunology Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Exogenous IFNα Impairs Neural Communication by Disrupting Endothelial Mitochondrial Function at the Blood-Brain Barrier 2258446

Abstract Introduction Type I interferon alpha (IFNα) is essential to maintain immune surveillance, but, when abnormally elevated, drives neuroinflammation, blood-brain barrier (BBB) breakdown, and neuropsychiatric dysfunction. However, the mechanisms underlying IFNα-induced brain homeostatic imbalance remain poorly understood. Methods We investigated the impact of IFNα on BBB integrity by intravenously administering IFNα to young adult mice. Mice received daily treatment for 4 days, followed by the assessment of vascular leakage using FITC—dextran and tight junction Claudin-5 (CLDN5) continuity. The consequence of vascular leakage is analyzed by astrocytic morphology, and interferon-stimulated gene (ISG) expression in the hippocampus. Endothelial mitochondria were examined for number and NADPH oxidase 2 (Nox2) levels, with IFNAR1 blockade used to test signaling involvement. Neuronal activity in hippocampal CA3 was quantified by c-fos immunostaining, and ongoing studies include flow cytometry of macrophage accumulation and in vivo two-photon imaging of vascular—immune—neural interactions. Results IFNα-treated mice exhibited multiple vascular leakage sites, indicating compromised BBB stability. Capillary analysis showed disrupted CLDN5 junctions, astrocytic end-feet retraction, and significant hippocampal ISG upregulation. Endothelial mitochondria displayed reduced abundance and elevated Nox2 expression that is reversed by IFNAR1 blockade, implicating endothelial IFNα—IFNAR1 signaling in mitochondrial stress. Consequently, neuronal activation was elevated in hippocampal CA3 neurons, suggesting enhanced hippocampal input relevant to memory processing. Conclusion These findings demonstrate that IFNα induces endothelial mitochondrial stress to disrupt BBB integrity, establishing a mechanistic link between neuroinflammation, vascular dysfunction, and altered brain immune—neuronal dynamics. Funding Source NIH P50MH112491 Topic Categories Neuroimmunology (NEUR)

Investigation of the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in SARS-CoV-2 pathogenesis 2308341

Abstract Introduction Acute lung injury and persistent inflammation caused by SARS-CoV-2 may lead to further complications after the virus gets cleared. People with Cystic fibrosis (pwCF) are highly vulnerable to pulmonary diseases due to mutations in the CFTR gene. However, there is limited research about SARS-CoV-2 severity in pwCF and subjects carrying a single variant of this gene. Methods Middle-aged (4-5 months old) and elderly (>18 months) mice expressing various levels of CFTR (WT, CFTR Knock out (KO), and heterozygous for CFTR (HET)) were infected with the mouse-adapted SARS-CoV-2 virus, MA10. Body weight and body temperature were measured in addition to spike specific antibodies detected by ELISA, flow cytometry on immune cells from blood samples, and lung histology. Results Middle-aged KO mice significantly lost less weight than WT and HET mice for both male and female mice. However, HET males lost significantly more weight compared to WT and KO male mice (p = 0.0058 and 0.0004, respectively). HET mice body weight recovery took much longer especially in males for both middle-aged and aged mice (∼28 days for middle-aged mice vs 21 days for KO mice, and 51 days for aged mice vs 25 days for KO mice). Conclusion This study highlights the significance of carrying CFTR mutation in susceptibility and lung pathology to COVID-19. Our data show that the severity of SARS-CoV-2 infection in different mice CFTR genotypes is highly dependent on the age and gender. Funding Source Internal OSU funding Topic Categories Viral Immunology (VIR)

Whole Body Irradiation (WBI) impairs antigen-independent functions of memory CD8 T cells 2255916

Abstract Introduction Radiation exposure is increasing due to its expanding use in the medical, energy production, and space travel industries. It has adverse effects on the memory CD8 T cell compartment, including permanent numerical loss, reduced cytokine production following antigen (Ag) stimulation, and diminished proliferative capacity upon in vivo challenge. However, it remains unclear how WBI affects bystander function of memory CD8 T cells (non-Ag-driven IFNγ production) and their trafficking to sites of inflammation. Here we define how radiation exposure impacts memory CD8 T cell sensing and responsiveness to inflammatory cues in the environment. Methods 1M P14 CD8 T cells from control (0 Gy) or irradiated (5 Gy) mice were subjected to scRNA-seq to analyze expression of cytokine/chemokine receptors and signaling-related genes. To evaluate how this transcriptional profile translated to functional bystander activation, mice containing 1M and quaternary (4M) P14 CD8 T cells were exposed to either 5 Gy or 0 Gy WBI, then challenged with virulent L. monocytogenes (LM) lacking gp33 expression at different days post-WBI. Spleens were harvested 20 hrs post-infection and incubated with brefeldin A (BFA) for one hour prior to intracellular staining. Results Pathway analysis of scRNA-seq data revealed differential expression of genes involved in cytokine sensing/signaling in irradiated 1M P14 CD8 T cells. Consistent with this, both 1M and 4M CD8 T cells from irradiated mice produced significantly less IFNγ and exhibited reduced expression of activation markers CD25 and CD69 following LM infection, despite higher bacterial burdens in vivo. Conclusion Collectively, data demonstrate that irradiation impairs the bystander functions of both 1M and 4M CD8 T cells. Together with previous observations showing radiation-induced inhibition of Ag-specific responses, our results highlight a multifaceted disruption of memory CD8 T cell immunity–insights that are critical for the development of effective post-radiation therapies. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Correction to “Multi‐State Probabilistic Computing Using Floating‐Body MOSFETs Based on the Potts Model for Solving Complex Combinatorial Optimization Problems”

Investigating Cytotoxic Function in CD4+ T-cells for Cancer Immunotherapy 2260082

Abstract Introduction T-cell-based immunotherapies like ACT have mostly focused on CD8 T cells, but cytotoxic CD4 T cells (CD4 CTLs) are increasingly recognized as contributors to anti-tumor immunity. As such, we created a CD4 T-cell activating nanoparticle platform, termed the artificial antigen presenting cell (aAPC). Under Th1-skewing conditions, aAPC-stimulated CD4 T cells upregulate granzyme B and mediate cell lysis. We hypothesize that aAPCs can provide a tunable system to probe how environmental cues influence CD4 T-cell cytotoxicity. Methods aAPCs are formed by covalently conjugating signal 1 to amine-coated iron-oxide nanoparticles. CD4 T-cells from transgenic OT-II mice were isolated from organs and cultured in vitro with aAPCs and soluble signals 2 and 3 for 7 days. Cells were expanded in the presence of CD4 subset-polarizing mixes (Th1, Th2, or Treg), using aAPCs of varying diameters (70 to 4000 nm), or with aAPCs presenting different signal 1 molecules (I-ab OVA or anti-CD3). CD4 CTL and CD4 phenotypic markers (CD44, CD62L, and granzyme B) were assessed with flow cytometry. Results aAPCs were used to activate OT-II CD4 T cells in vitro. Th1-skewing conditions promoted expression of cytotoxic-associated transcription factors. Th2- and Treg-skewing conditions failed to induce granzyme B. Smaller aAPCs (70 and 250 nm) supported greater cytotoxic function than larger particles (4 um). Antigen-specific stimulation elicited stronger cytotoxicity than non-specific stimulation. Higher IL-2 concentrations increased both frequency and expression of granzyme B, and favored an effector over memory phenotype. Conclusion We have shown that aAPCs can drive differentiation of CD4 CTL in vitro. Furthermore, we show that CD4 CTL phenotype and function are tunable by cytokine concentration and milieu, particle size, and antigen specificity. These findings lay the groundwork for further investigation of this non-canonical T-cell subset, with the ultimate goal of improving the efficacy of T-cell-based cancer immunotherapies. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

miR-10a induces IL-33/ST2 signaling and Tregs to promote adipose browning and attenuate adipose tissue inflammation 2253749

Abstract Introduction Obesity is a pandemic, characterized by infiltrating macrophages and T cells in adipose tissue (AT) which triggers chronic inflammation. microRNA (miR) specifically downregulation of miR-10a in AT contributes to inflammation and alters adipocyte biology in obesity. In this study, we explored whether administering miR-10a mimic in the high-fat diet (HFD) mice alters AT inflammation, adipogenesis, and browning during obesity. Methods C57BL/6J mice were fed HFD for eight weeks and administered eight doses of miR-10a or scrambled RNA control (Sc-Control) via intravenous injection for next four weeks under HFD. Body weight, metabolic parameters were measured, and AT was collected to evaluate the metabolic and immune signatures by histology, flow cytometry, and RT-PCR analysis at the experimental endpoint. We also evaluated browning gene induction by IL-33 in 3T3-L1 adipocytes using in vitro analysis. Results Body weight change, fat depots, blood glucose, adipocyte size, and crown-like structures were reduced prominently in the miR-10a group as compared to Sc-Control. miR-10a administration in mice exhibited better glucose tolerance by IPGTT. Activated T cells, macrophages, and dendritic cells in AT were decreased in the miR-10a group as compared to control mice. Furthermore, regulatory T cells (Tregs) and IL-33-expressing macrophages and immune cells in AT were elevated, whereas inflammatory genes (TNF-α, NF-kB, STAT3) expression was suppressed in the miR-10a-treated group. We also noticed elevated expression of IL-25 in CD4 T cells and ST2 receptor on immune cells in AT of miR-10a group. Intriguingly, AT browning genes (PGC1-α, CIDEA, FNDC5) were notably increased in the miR-10a group. We also found that IL-33 stimulated similar thermogenic gene expression and reduced lipid deposition in 3T3-L1 adipocytes. Conclusion The results suggest that miR-10a stimulates the AT browning program and attenuates adipocyte hypertrophy and inflammation by inducing IL-33/ST2 signaling and Tregs in AT. Funding Source NIAID R01 AI140405 Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Bridging the Gap in Flow Cytometry Training: A Practical Workflow for Teaching Autofluorescence Management in Immunology 2261098

Abstract Introduction Cellular autofluorescence, caused by endogenous biomolecules, can obscure low-intensity fluorophore emissions, leading to compromised data interpretation in flow cytometry. This issue is particularly challenging for trainees and early-career researchers, who may lack experience in spectral unmixing and tissue-specific background management. Methods We, therefore, developed a practical workflow that introduces researchers to autofluorescence as a consistent, tissue-dependent variable and provides structured guidance for improving panel design and data analysis. This workflow integrates unstained controls and autofluorescence extraction during unmixing to refine gating and improve dim-marker resolution (Cytek Aurora (16V—14B—10YG—8R), SpectroFlo software). Results Integrating this training into Core workflows improved researchers’ ability to distinguish true fluorescence from background signals and enhance their understanding of tissue-specific autofluorescence. The approach reduces troubleshooting time, increases reproducibility, and empowers users to apply autofluorescence management strategies to their own experiments independently. Conclusion This workflow addresses a critical blind spot in flow cytometry education by transforming autofluorescence from a confounding variable into a teachable and manageable feature of experimental design. It enhances data quality, supports reproducibility, and builds core competencies in flow cytometry for immunology research. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)

Design and evaluation of engineered MyD88 fusion proteins for enhancing CAR-T therapy 2333869

Abstract Introduction MyD88 is a scaffold protein that plays a key role in propagating innate immune signals. Recent work has shown that a chemically induced dimerization (CID) platform comprising MyD88 fused to the intracellular domain of CD40 can enhance CAR-T cell fitness via multiple mechanisms. However, this system was associated with toxicity in the clinic due to excessive pro-inflammatory signaling. We sought to better understand the mechanistic levers by which engineered MyD88 assembles and propagates pro-inflammatory signals, with the goal of developing next-generation variants with improved safety profiles. Methods Using structural analysis and bioinformatics, a series of engineered MyD88 fusion proteins were designed, cloned, and expressed in Jurkat cells and primary human T cells in combination with the Tmod™ NOT logic-gated CAR-T platform. Effects on NF-kB induction, cytokine production and cell expansion were evaluated. Results Consistent with previous reports, we observe that Myd88-based CID systems synergize with TCR signaling pathways in human T cells. We demonstrate that engineered MyD88 assembly can be modulated by either raising or lowering the stability of an initial dimeric “seed.” We then characterize how the integration of either natural or synthetic TRAF recruitment motif(s) impart distinct cytokine profiles, enabling the construction of variants with reduced pro-inflammatory signaling. Conclusion Our results shed light on the mechanisms underlying inducible Myddosome assembly and offer insight into why MyD88 is particularly amenable to CID. We show that functional outputs can be modulated via altering assembly thresholds as well as tuning signaling motifs, two parameters which help to define design goals for next-generation immunotherapies integrating innate and adaptive signals. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

Mitochondrial ATP production links chromatin accessibility to T cell differentiation and function 2258461

Abstract Introduction Immune elimination of chronic infection or cancer requires cytotoxic CD8 T cells that adopt and maintain an effector phenotype. Cytotoxic T cell function is a bioenergetically demanding process and T cells subjected to chronic antigen exposure have compromised effector function despite high rates of glycolysis. Here we report the ability of the short chain fatty acid, D-α-hydroxybutyrate, to act as a signaling molecule that increases mitochondrial ATP production and drives the conversion of proliferating T cells into cytotoxic effector cells. DAHB signaling switches ATP production from glycolysis to fatty acid oxidation, even in glucose-replete media. This conversion suppresses both AMPK phosphorylation and the integrated stress response (ISR) in activated T cells while significantly elevating the level of the phosphagen, phosphocreatine (PCr). Both the PCr bioenergetic reserve and oxidative phosphorylation were required for T cell effector differentiation. DAHB-induction of CD8-effector gene transcription was coupled to bioenergetics by enhanced ATP-dependent remodeling of chromatin accessibility at effector gene loci. DAHB enhanced CD8 T cell antitumor activity both in vitro and in vivo, and DAHB treatment of transferred T cells led to persistent in vivo antitumor effects. Together, these findings link cellular bioenergetics to the regulation of chromatin accessibility and gene expression required to support effector function. Methods Murine T cells were tested in response to D-α-hydroxybutyrate by flow cytometry, RNAseq, ATACseq and metabolomics. Results DAHB promotes OXPHOS by inducing fatty acid oxidation which leads to an elevated creatine-phosphate buffer. Creatine-phosphate regenerates ATP to support chromatin remodeling and expression of effector genes. DAHB enhances immune checkpoint blockade and T cell adoptive transfer in tumor settings. Conclusion Mitochondrial ATP and phosphocreatine supports chromatin remodeling for cytotoxic T cell gene expression. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Interrogating T-Cell Receptor Interactions with Peptide-MHC Through Structure-Guided Mutational Analysis 2219106

Abstract Introduction Studies have shown that germline contacts may underlie MHC restriction, supporting the idea that TCR—MHC specificity is partially encoded in the germline (Garcia et al., 1996; Marrack & Scott-Browne, 2010). In this study, we aimed to investigate how germline-encoded TCR contacts influence TCR—pMHC interactions and T cell activation. Methods Nine MART-1 peptide variants (a melanoma-associated antigen), generated by single alanine substitutions, were pulsed onto T2 cells (1 × 10⁵/well) and co-cultured with TCR-transduced Jurkat T cells at a 1:1 ratio for 14 hours. Activation was assessed by CD69 staining and NFAT-GFP reporter signal via flow cytometry. Four α and two β chain TCR variants were generated by single alanine substitution at predicted polar contacts within germline-encoded CD1 and CD2 regions. Jurkat T cells were co-transduced with the α and β chain lentiviruses, then co-cultured for 14 hours with T2 cells pulsed with peptides that had retained activity in the initial screen. Activation was assessed by CD69 and NFAT-GFP expression via flow cytometry. Results Two MART-1 variants, MART1-1A (E1A, 15.6%) and MART1-9A (T9A, 11.5%), induced dose-dependent activation. Another peptide, MART-91, derived from Mycobacterium and previously reported to share a similar motif with MART-1, induced the strongest activation (28.6%). Based on these results, MART1-1A and MART-91 were selected for testing TCR variants. While substitutions in the β chain yielded inconclusive results, disrupting polar contacts in the α chain abolished activation in response to both peptides. In contrast, the germline TCR (Germline-TCR156) maintained robust activation, particularly in response to MART-91. Conclusion These findings support a role for germline-encoded TCR residues in modulating TCR—MHC recognition and specificity, enabling tolerance of variation at peptide contact sites. Future studies will aim to repeat β chain experiments to clarify the ambiguous results, which may have been due to a failed transfection. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Macrophage migration inhibitory factor as a microbial modulator of colitis-associated colorectal cancer 2256450

Abstract Introduction The gut microbiome is altered in inflammation and colorectal cancer (CRC), yet how proinflammatory factors induce dysbiosis, which promotes CRC development, remains unclear. Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule produced by various cell types that protects against multiple pathogens but is also linked to inflammatory diseases and cancer progression. The present study examined the role of MIF in shaping the microbiota configuration and its effect on the development of colitis associated with CRC (CAC). Methods We identified the microbiota through 16S rRNA sequencing of fecal samples from BALB/c MIF-knockout (MIF-KO) and wild-type (WT) mice with azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CAC and compared them with healthy MIF-KO and WT mice (CTL). Additionally, WT mice were purchased from Harlan vivarium and received fecal transplants from MIF-KO and WT mice and subsequently subjected to AOM/DSS-induced CAC (WT-Harlan/MIF-KOv CAC and WT-Harlan/WTv CAC, respectively). Microbial dysbiosis was analyzed via linear discriminant analysis effect size (LEfSe). Results The MIF-KO CAC mice developed a greater number of tumors and exhibited pronounced dysbiosis, with an increased abundance of inflammation- and CRC-associated bacteria such as Muribaculaceae, compared to WT CAC mice. Fecal transplantation from MIF-KO donors to WT-Harlan increased the tumor number and size, and an increase in Muribaculaceae levels relative to those in WT-Harlan/WTv CAC mice was also observed. Conclusion The absence of MIF promotes a dysbiotic microbial environment that enhances tumor development, with Muribaculaceae, an inflammation- and CRC- associated bacteria, emerging as a potential microbial signature of MIF deficiency, even in fecal transplant conditions. This is the first demonstration of the role of MIF in the development of gut microbiota in the pathogenesis of CRC. Funding Source State Council of Science and Technology of the State of Mexico (COMECyT), CIKAS-FICDTEM25-019 and the Support Program for Research Projects and Technological Innovation (PAPIIT)-UNAM, IV200425 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)