Browse Articles

Macrophage—NK Cell Crosstalk via cGAS—STING Signaling Induces IFN-γ Responses Critical for Early Control of Borrelia burgdorferi Infection 2257978

Abstract Introduction Lyme disease, caused by Borrelia burgdorferi (B. burgdorferi), is shaped by complex interactions between innate & adaptive immunity. Previous studies show that B. burgdorferi activates cGAS—STING in macrophages by utilizing c-di-AMP as a PAMP, leading to type I interferon production. Building on this, we aimed to define the role of cGAS-STING signaling in coordinating crucial cell-to-cell crosstalk during the early stage of B. burgdorferi infection. Methods We used in vitro & ex vivo co-culture model to assess NK cell activation (IFN-γ production, CD69 expression & LDH release) following stimulation with B. burgdorferi-infected wild-type & cGAS-/- or STING-/- macrophages, neutrophils, or their supernatants. Phagocytosis & IFNAR blocking were performed to determine the mechanism of NK cell activation. Finally, we utilized cGAS-/-, STING-/-, IFNAR-/- & NK cell depletion mouse models to assess B. burgdorferi burden & dissemination. Results Macrophage-intrinsic cGAS—STING signaling, but not that of neutrophils, was essential for NK cell activation & subsequent IFN-γ production. Inhibition of cGAS—STING signaling in macrophages significantly reduced NK cell responses in co-cultures. This effect was largely dependent on macrophage phagocytic activity & mediated by macrophage-derived IFN-I. Blocking IFNAR on NK cells impaired their activation in response to the infected macrophage supernatant, confirming a required role for IFN-I signaling. Consistently, STING agonist treatment of macrophages enhanced NK cell activation, whereas direct stimulation of NK cells with STING agonist or B. burgdorferi failed to do so. In vivo, IFNAR deficiency or NK cell depletion significantly increased B. burgdorferi burden at the infection site, while loss of cGAS-STING signaling promoted bacterial dissemination. Conclusion These findings show that macrophage cGAS—STING signaling coordinates NK cell IFN-γ responses via type I IFN, establishing a critical, MyD88-independent axis for early immune control of B. burgdorferi. Funding Source R01 AI52235, R01 AI083640 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

PTHrP is a novel mediator of immune suppression in pancreatic cancer 2334262

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of common cancers. PTHrP (encoded by PTHLH) defines the most aggressive squamous PDAC subtype. We have shown PTHrP mediates tumor growth and metastasis. The impact of PTHrP on the tumor microenvironment (TME) remains unknown. Methods In vivo experiments used KrasG12D; p53R172H; Pdx1-Cre; YFP (KPCY) and KPCY-PthlhloxP/WT (KPCY-PthlhHet) genetically engineered mice or implantable cell lines. Orthotopic implantation experiments used Pthlh overexpressing KPCY tumor cells (herein PTHrPOE), PTHrPOE-Xcl1OE or empty vector control cells (Control). Tumor immunophenotyping by flow cytometry. IF microscopy performed on sample matched tumors. Immune mobilization studies quantified by flow cytometry of whole blood. Cytokine expression was performed using ELISA and cytokine array (Luminex). Transcriptomic analysis of bulk tumor RNA. NK cell killing assays used splenic NK cells co-cultured with target cells, function assessed with flow cytometry. Results Analysis of PTHrP-null tumors indicated that the tumor microenvironment (TME) was re-organized upon loss of PTHrP, suggesting PTHrP-mediated TME remodeling contributes to worse overall survival. Orthotopic implantation of (PTHrPOE) show Pthlh was sufficient to ablate dendritic cell (DC) and T cell recruitment to the TME. Transcriptomic and cytokine array analysis of bulk tumor revealed loss of a critical DC chemokine, Xcl1. Ex vivo analysis of NK cells show PTHrPOE cells inhibit NK cell activation and in vivo over expression of Xcl1 in PTHrPOE cells provides a survival benefit and imparts partial tumor control. Conclusion The work herein begins to elucidate a novel mechanisms of immune suppression in pancreatic cancer, driven by PTHrP. The PTHrP-dependent immunosuppressive TME excludes and inhibits effector immune cells, such as CD8+ T cells NK cells, and cDC1s. Intervention of PTHrP signaling in the TME may be a potent therapeutic strategy for the treatment of squamous PDAC. Funding Source NIH R00CA252153, NIH T32 GM135751, NIH T32AI095213 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Lattice‐Electron Synergistic Pinning Strategy for Intensified Regeneration of Spent Ternary Cathodes

ABSTRACT The hydrometallurgical recycling of spent lithium‐ion batteries (LIBs) confronts a fundamental scientific challenge in enhancing the structure while restoring the cathode chemistry, particularly in remedying the intrinsic Ni/Li antisite disorder and lattice distortions that dictate cycling stability. Herein, we propose a synergistic lattice‐electron pinning strategy induced by localized microenvironmental reconfiguration to achieve dual‐intensification of the regenerated structure and performance of retired LiNi 0.5 Co 0.2 Mn 0.3 O 2 (NCM523). The constructed localized strongly correlated Na–O–Ni ionic‐covalent‐bonding network enhances the strength of Ni–O bond, anchoring the lattice oxygen and ultimately effectively suppressing planar gliding in (003) facets. Simultaneously, the enlarged electronegativity difference between Na and O elevates the local electrostatic potential of Ni site, and the consequently increased Δ t drives the spin pinning of Ni 3+ from high‐spin ( t 2g 5 e g 2 ) to low‐spin ( t 2g 6 e g 1 ), significantly suppressing the superexchange interaction along Ni–O–TM bonds. The optimized regenerated cathode delivers a remarkable capacity retention of 69.6% after 400 cycles at 0.5 C, surpassing commercial NCM523 (42.3%) and unoptimized regenerated cathode (40.3%) by 164% and 172%, respectively, and ranks among the top‐performing regenerated NCM cathodes to date. This work highlights the significance of pinning effect in hydrometallurgical and establishes a sustainable‐route for converting spent LIBs into high‐quality regenerated cathodes.

Confinement‐Engineered Ir‐IrO ₂ Interfaces Activate Hydrogen‐Bond‐Mediated Oxide Pathway Mechanism for Durable Acidic Water Oxidation

ABSTRACT Achieving durable acidic oxygen evolution at low iridium loadings remains the bottleneck for proton exchange membrane water electrolysis (PEMWE). Conventional Ir catalysts are limited by adsorbate evolution mechanism scaling and lattice‐oxygen‐induced degradation, while the oxide pathway mechanism (OPM) has remained inaccessible due to the instability of sub‐2.9 Å Ir─Ir dual sites under high anodic potentials. Here, we report a confinement‐engineered strategy that localizes Ir precursors within the nanochannels and interlayer galleries of a layered covalent organic framework, yielding ultrathin twinned Ir‐IrO 2 interfaces with a contracted 2.68 Å Ir─Ir dual‐site geometry. Operando spectroscopic analyses establish that these interfaces activate the oxide pathway during OER. Kinetic analysis reveals a hydrogen‐bond‐mediated OPM (HB‐OPM), in which hydrogen bonding between surface intermediates ( * OH··· * OH/ * O···HO * ) and electric‐field‐induced reorganization of interfacial water cooperatively lowers the O─O coupling barrier and suppresses Ir over‐oxidation. This catalyst achieves low‑iridium (0.28 mg Ir   cm −2 ) PEMWE performance with high activity (2 A cm −2 at 1.71 V) and exceptional durability, operating for over 5500 h at 1 A cm −2 with a decay rate of 3.5 mV kh −1 . These results demonstrate that confinement‐engineered Ir‐IrO 2 interfaces provide an effective strategy for activating OPM and support HB‐OPM as a key mechanism for designing durable, low‐Ir acidic OER catalysts.

Withdrawal: Human calpain-3 and its structural plasticity: Dissociation of a homohexamer into dimers on binding titin

Synergistic approach to enhancing interlayer bonding in 3D printed concrete via water to binder ratio and water retention addition

Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology 2309432

Abstract Introduction Microthrombus formation is a determinant of COVID-19 severity; however, the in vivo spatiotemporal mechanisms of thrombogenesis remain unclear. Here, we used an in vivo imaging system to elucidate the dynamics of neutrophils and platelets in the pulmonary microcirculation and to understand how SARS-CoV-2 infection compromises lung perfusion. Methods We used in vivo two-photon excitation microscopy in a Biosafety Level 3 facility to visualize cellular pathophysiology in Ob/Ob (obesity model) mice infected with a fluorescent-reporter SARS-CoV-2 virus. We quantified intravascular neutrophil motility, platelet aggregate size, and pulmonary capillary perfusion. Adhesion molecule expression on lung neutrophils was assessed with flow cytometry. Clinical relevance was validated by re-analyzing scRNA-seq datasets of peripheral neutrophils from COVID-19 patients. Results In vivo imaging revealed that SARS-CoV-2 infection significantly prolonged neutrophil adhesion to pulmonary vessel walls in Ob/Ob mice. This stagnation was driven by upregulated CD44, CD62L, and E-selectin on neutrophils. Adherent neutrophils frequently captured platelets, forming complexes that evolved into microthrombi and impaired pulmonary perfusion. Neutrophil depletion reduced thrombus formation and extended survival. Human scRNA-seq data analysis confirmed elevated CD44 and SELL (CD62L) expression in neutrophils from severe COVID-19 cases. Conclusion We found that upregulated adhesion molecules on intravascular neutrophils trigger vessel wall adhesion, acting as scaffolds for platelet aggregation. This cascade drives microthrombus formation and pulmonary perfusion defects, highlighting neutrophil adhesion as a therapeutic target for severe COVID-19. Funding Source This research was supported by a Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP19fk0108113, JP20fk0108412, JP21fk0108552, JP243fa627001) by a Japan Program for Infecti Topic Categories Viral Immunology (VIR)

Homogenously-sized chlamydial nanovaccine efficiently activates dendritic cells by caveolin endocytosis to boost innate immune responses 2260757

Abstract Introduction Previously, we employed a heterogeneously-sized PLGA (poly D, L-lactide-co-glycolide) encapsulating chlamydial major outer membrane protein (MOMP) nanovaccine (CnV), which induced Th1 immune responses in bone marrow-derived dendritic cells (BMDCs). Herein, we employed a microfluidics-assisted fabrication technique to obtain homogenously-sized CnV-H to assess the effects of size and homogeneity on innate immune responses. We hypothesized that due to homogeneity, CnV-H will bolster Th1 immune responses in BMDCs as compared to CnV. Methods The characterization data revealed that CnV-H had an average size of 166 nm, a surface charge of -2 mV and exhibited homogeneity as evidenced by a low polydispersity index (PDI > 0.06) and as confirmed by scanning electron microscopy. Contrastingly, CnV had an average size of 180 nm, a surface charge of -12 mV and a PDI (> 0.1), indicating heterogeneity. To assess in-nate immune responses, BMDCs were stimulated for 24 hours with CnV-H and or CnV (0.1, 1, and 5 µg/mL of encapsulated-rMOMP). Supernatants were collected for cytokine-specific ELISAs, and cells were processed for gene-expression analysis using TaqMan qPCR or were stained with fluorochrome-conjugated antibodies specific for co-stimulatory and antigen-presentation molecules for flow cytometry assessment. Results The data showed that higher concentrations (1 and 5 µg/ml) led to activation of the caveolin en-docytosis pathway, enhanced expression of pathogen pattern recognition receptors (TLR-2), co-stimulatory molecules (CD40, CD80, CD86), antigen-presentation molecules (MHC-II), and significantly increased Th1-cytokines (IL-6, IL-12p40) production, particularly TNF-α, compared to the CnV-stimulated BMDCs. The negative control CnV or CnV-H did not induce any cytokine production. Conclusion In summary, homogeneously-sized CnV-H is more efficient at activating dendritic cells, potentially for robust antigen-presentation, likely due to the uniform activation of the caveolin endocytosis pathway. Funding Source This research was supported by the National Science Foundation (NSF)-HBCU-UP (HRD-1911660) and the NSF-EiR (2200529) grants to Dr. Vida A. Dennis at Alabama State University. Topic Categories Vaccines and Immunotherapy (VAC)

Expanding Antibody Labeling Options: TrailBlazer™ Tag and StarBright™ Dye Label Kits 2259389

Abstract Introduction Multiplexing experiments are often constrained by the lack of ideal fluorophores for certain antibodies or experimental needs, limiting experimental design in flow cytometry, western blotting, and microscopy applications. While custom antibody-dye conjugates can be generated with labeling kits, choices are usually limited to standard fluorophores (such as FITC), which often restricts panel expansion and improvement. Methods   Results The new TrailBlazer Tag and StarBright Dye Label Kits are a significant advancement in overcoming this issue. These innovative kits directly label antibodies with premium StarBright Dyes, with options spanning the five main laser lines. Our data shows the one-year stability of TrailBlazer Kit—conjugated antibodies, which is due to the novel SpyTag-SpyCatcher technology used, making them ideal for longer-term studies without the need to perform multiple labeling reactions. Data demonstrates the robust and reproducible performance of kit-labeled antibodies across different applications is comparable to catalog StarBright Dye—conjugated antibodies. Additionally, flow cytometry data show that kit-labeled StarBright Dye antibodies are compatible with multiple staining buffers and fixation/permeabilization buffers and can be easily included in multiplexing panels. Conclusion In summary, the novel TrailBlazer Labeling Kits are a valuable tool for scientists, offering the flexibility of self-conjugation to generate high-quality, multidimensional data in complex biological analyses. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Dynamic Gene Expression and Immune Cell Remodeling in DN T Cell-Mediated Antitumor Response to Breast Cancer 2257888

Abstract Introduction In the Ja281 KO mouse model, we found that transferring TCRαβ+CD4⁻CD8⁻ double-negative (DN) T cells into these mice inhibited breast cancer (BC) growth. This DN T cell transfer-mediated antitumor response depends on host NK cells and IFNγ, but is independent of IFNγ and perforin produced by the donor DN T cells. The underlying mechanism is unknown. Since implanted tumor cells fail to grow in the transferred mice, it is challenging to study the associated cellular and molecular processes. Methods Herein, we conducted a transcriptomic analysis to identify gene signatures related to cell signaling and functional changes. RNA sequencing was combined with antibody-mediated cell depletion to investigate the dynamic cellular and molecular changes. Results Results showed that 42 hours after tumor inoculation, the major biological processes and signaling pathways significantly upregulated in DN T-transferred mice compared to wild-type controls included glycolysis, fatty acid metabolism, enhanced NK cell activation and IFNγ production, and elevated CCL8 expression. By day 3 post-inoculation, IFNγ-induced signaling pathways were significantly elevated in the tumors of DN T-transferred mice. Additionally, myeloid cells were observed infiltrating the tumor site. On days 3 and 5 after tumor inoculation, genes related to antigen presentation, T cell differentiation and activation were consistently upregulated in tumors from DN T-transferred mice. Interestingly, antibody-mediated depletion of CD8+ but not CD4+ T cells partially restored BC growth in some the transferred Ja281 KO mice, although tumor development remained significantly delayed compared to non-transferred controls. Conclusion In summary, DN T cell transfer promotes glycolysis and fatty acid oxidation shortly after tumor inoculation, induces NK cell activation and type 1 myeloid cell infiltration within 2—3 days, and is followed by enhanced T cell activation. CD8+ T cells appear to play a role in the sustained control of BC growth in this model. Funding Source NIH/NCI grant R01CA276284 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Thymocyte development and transcription factor expression are disrupted by Runx2 deficiency 2259108

Abstract Introduction Transcriptional regulation during T cell development in the thymus is critical for the maturation of naive T cells that will seed the periphery. Several key signals, including T cell receptor signaling and co-stimulation, impact expression of transcription factors which guide thymocyte fate via processes such as survival and lineage commitment. Runt-related transcription factors (Runx) have been described as key mediators of development processes for several lymphocyte subsets. While Runx1 and Runx3 have been demonstrated to impact CD8 T cell development, studies on Runx2 are limited. Findings from experiments using Runx2 overexpressing mice suggest a role of Runx2 in β-selection during the double-negative stage of T cell development. Whether deficiency in Runx2 impacts thymocyte development at stages other than β-selection is currently unknown. Methods To explore whether Runx2 has a key role in T cell maturation, we evaluated its expression in thymocytes by flow cytometry and analyzed double-negative (DN), double-positive (DP), and single-positive (SP) T cell development in 5-week-old C57BL/6J wild-type and T cell-conditional Runx2-deficient (runx2-/-) mice. Results We found that while Runx2 expression varied by T cell maturation stage, Runx2 was highly expressed in DN thymocytes. Accordingly, the frequency of DN thymocyte subsets was altered in the thymus of runx2-/- mice when compared to the frequency present in wild-type mice. SP CD4 and CD8 T cell maturation, as measured by CD24 expression, was also impaired in T cell conditional runx2-/- deficient mice. Furthermore, expression of the transcription factors Runx3, ID2, and RORγT was altered in select runx2-/- thymocyte subsets. Conclusion Together, our results suggest that the transcription factor Runx2 is involved in the transcriptional coordination of thymocyte development and maturation. These and future studies aim to help further understand the role of Runx2 in the formation of mature T cells. Funding Source UCF Foundation Topic Categories Hematopoiesis and Immune System Development (HEM)

Iron metabolism In Dendritic Cells is important for Induction of Follicular Helper T Cell Subsets in Allergy 2258031

Abstract Introduction Allergic asthma affects 25 million of people in the United States and 300 million people worldwide. There are treatments to reduce symptomology, but currently there is no cure. Thus, additional research is warranted. Dendritic cells (DCs) are crucial bridges between the innate and adaptive immune systems, capturing antigens, processing them, and presenting them to T cells. They are crucial to initiating and shaping adaptive immune responses. Follicular T helper cells (Tfh) are a specialized subset of CD4+ helper T cells that are important in the development of antibody-producing B cells. CD71, the transferrin receptor, is a cell surface glycoprotein that plays a crucial role in iron uptake. Tfh13 cells are the specialized subset of Tfh that induce production of high affinity IgE. Here, we hypothesized that iron uptake was important for Tfh polarization, especially Tfh13 polarization. Methods C57/BL6 mice were treated with Ferristatin II intranasally to inhibit iron uptake prior to the allergen extract, Alternaria alternata administered intranasal. The mice were also treated with Deferiprone, a cell permeable iron chelator, intraperitonially prior to the intranasal instillation of allergen extract, Alternaria alternata. On day 8, lymphadenectomy was performed, cells were processed for Flow cytometry, and data analysis was done using Flowjo. Statistical analyses were performed using Ordinary One-way ANOVA. Results With Ferristatin II treatment, we saw significant decrease in differentiation to Tfh13, while Tfh2 and Th2 cells were not significantly affected. with Deferiprone, a cell permeable iron chelator, we observed significant differences in Tfh2 and Tfh13 while Th2 cells were unchanged. We also observed a novel subset of allergen-carrying CD71hi cDC2 in the draining lymph node after allergen sensitization by flow cytometry. Conclusion CD71hi cDC2 population may be important for the polarization of the T follicular helper cells, thereby influencing antibody production of B cells. Funding Source NHLBI-NIH (R01HL162991) to RKM Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Immunogenicity of Double-Stranded RNA in mRNA Vaccines and its Impact on Immune Cell Function 2335037

Abstract Introduction Double-stranded RNA (dsRNA) is a natural activator of innate immune responses in mammalian cells and can form as a limited byproduct during in vitro transcription of mRNA vaccines Methods We sought to further characterize humoral and cell-mediated immune responses to varying levels of dsRNA using a prototype nucleoside-modified mRNA (modRNA) based respiratory syncytial virus (RSV) vaccine. The modRNA lipid nanoparticle formulations contained defined quantities of a full-length dsRNA generated from complementary sense and antisense strands of RSV modRNA. Results In vivo analysis in BALB/c mice revealed that increasing levels of dsRNA impurities in a modRNA LNP vaccine delivered intramuscularly led to a modest induction of inflammatory cytokines, including Type I interferons, but had no effect on neutralizing antibody titers or the functionality of CD4+ and CD8+ T cells. In parallel, in vitro analysis of human PBMCs exposed to the same formulations showed no significant changes in inflammatory cytokine production or cell-mediated immunity; Increasing levels of dsRNA impurities did not alter population composition, antigen-presenting cell (APC) activation, or CD4+ and CD8+ T cell exhaustion, indicating limited immunostimulatory potential of N1-Methylpseudouridine (m1ψ) modified dsRNA in immune cells. Conclusion Together, these results improve our understanding of potential dsRNA impurities and immune responses in mRNA vaccines. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

<i>E. coli</i> Extracellular Matrix: A Tunable Composite With Hierarchical Structure

ABSTRACT Escherichia coli ( E. coli ) biofilms consist of bacteria, an extracellular matrix (ECM) mainly made of curli amyloid fibers, phosphoethanolamine‐modified cellulose (pEtN‐cellulose), and water. While curli amyloid fibers contribute to biofilm rigidity, pEtN‐cellulose contributes to their cohesion. This work explores the interplay between these fibers, and how their interactions influence biofilm structure and mechanical properties. We performed a multiscale analysis on E. coli biofilms grown using strains producing curli and pEtN‐cellulose, and only curli and only pEtN‐cellulose in co‐seeded ratios. Micro‐indentation experiments, confocal microscopy, and cryo‐FIBSEM 3D imaging revealed a composite‐like behavior of the biofilm, where its mechanical properties depend on ECM composition and organization. Spectroscopic analysis of the extracted fibers showed that their biophysical properties are influenced by their pEtN‐cellulose to curli ratio and assembly. We propose that pEtN‐cellulose swelling is constrained by its interactions with rigid curli fibers. The reference E. coli strain maximizes this effect by assembling a curli/pEtN‐cellulose hybrid material at the sub‐micron scale, where its composition, interactions, and architecture can explain biofilm emergent properties. This knowledge on microbial ECM assembly opens new avenues for engineering living materials, especially for the use of bacterial biofilms as a source of bio‐sourced materials.

Stable Protein‐Based G‐Quadruplex‐Derived Supramolecular Bioinks as Tunable ECM‐Mimetic Constructs Assembled by Combining Non‐Covalent and Covalent Strategies

ABSTRACT G‐quadruplex hydrogels hold great promise for biofabrication owing to their dynamic supramolecular nature, provided their inherent instability under physiological conditions is overcome. Here, a bioinspired strategy that synergistically combines supramolecular self‐assembly, under macromolecular crowding conditions, with in‐bath enzymatic covalent crosslinking was employed to create stable, protein‐based G‐quadruplex‐derived hydrogels. Mimicking the crowded intracellular milieu, the addition of Ficoll enhances G‐quadruplex stability and tunes the rheological behavior, while transglutaminase‐mediated crosslinking reinforces the network, preserving its structural integrity over extended periods. This combined approach yields printable bioinks with optimal viscosity, yield stress, and shear‐thinning properties, enabling the fabrication of complex, multilayered 3D constructs that support enhanced cell viability and proliferation within an extracellular matrix (ECM)‐mimetic fibrillar environment. Moreover, the modulation of the crosslinking density allows controlling cellular responses, offering a versatile platform for tailoring the biomechanical microenvironment. This study establishes a new class of hybrid G‐quadruplex hydrogel bioinks, exhibiting unprecedented stability under physiological conditions, biofunctionality, and off‐the‐shelf availability, unlocking their potential for advanced tissue engineering and regenerative medicine strategies.

EGFR endocytosis down-regulates binding to EphA2 at the plasma membrane

A synergistic coordinate and attention module for pipeline weld surface defect detection

Programmable Panels: A Web-Based Design Studio to Accelerate Custom Cytometry Panel Development for Clinical Immune Profiling 2309669

Abstract Introduction Each year, 800+ Phase I oncology trials launch, and ∼75% require custom cytometry panels to quantify immune cell frequencies. Multiparameter cytometry panel development can require evaluation of &amp;gt; 700 parameters (40 markers × 6 titrations × 3 metrics) to optimize staining in multiparameter assays. Traditional workflows take 3—12 months, delaying sample collection and PK/PD, exploratory, MOA, and biomarker data generation. To address this, we developed Programmable Panels, a web-based, self-service platform enabling rapid cytometry panel design. Methods A web-based platform was developed to enable rapid design of custom immune profiling panels. Users define disease-relevant cell types, stimulation conditions, and target markers using an integrated marker library that is updated in real time as antibody clone—conjugate combinations are verified across sample types and fixation methods. Antibody clones are recommended based on live versus fixed samples, assay application, and historical performance. An interactive gating hierarchy is generated during panel assembly to define population structure and downstream analysis. Optional expert cytometrist support is available for troubleshooting and optimization. Results Using this platform, custom cytometry panel design and build time was reduced significantly. Automated clone selection and real-time gating visualization minimized manual review of &amp;gt; 700 parameters per panel and reduced design errors. Both self-guided and expert-assisted workflows supported rapid iteration, enabling earlier study initiation and consistent panel configuration across clinical and translational use cases. Conclusion By reducing cytometry panel development timelines, this approach accelerates study start-up and data generation for PK/PD, exploratory, MOA, and biomarker analyses. The platform enables scalable, reproducible immune monitoring by reducing manual panel build effort and improving the quality of cytometry panel design. Funding Source Teiko Topic Categories Translational and Interventional Immunology (TI)

Characterizing spatial functional microniches with SpaceTravLR 2260916

Abstract Introduction The advent of spatial omics has revolutionized our understanding of tissue biology. While in-silico perturbation methods and foundation models aim to model the impact of genetic perturbations, these methods are limited to single-cell approaches lacking spatial resolution. We address this major unmet need by developing SpaceTravLR, a novel interpretable machine learning approach that generalizes across tissues and species, uncovering spatial microniches linked to functional outcomes. Methods SpaceTravLR predicts how single or combinatorial genetic perturbations rewire signals across the tissue neighborhood, defining novel spatial niches across a range of tissues at different scales of organization, disease, and developmental contexts. All predictions are made solely based on context-specific spatial omic data with no prior knowledge, yet they align closely with findings from mechanistic experiments. Critically, interpretable machine learning enables the generation of mechanistic hypotheses underlying identified niches. Results SpaceTravLR discovered a novel mechanism for CCR4 that could not be captured by existing approaches that drives the compartmentalization and spatial location of a pathogenic population of Th2 cells in allergic asthma, which was validated in a murine model. Conclusion Overall, SpaceTravLR provides a novel interpretable and experimentally validated framework for uncovering how genes act individually and combinatorially through cell-intrinsic and cell-extrinsic circuits to shape spatial tissue organization and function. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

Necroptosis in SJS/TEN: RIPK1 and RIPK3 Expression and Implications for Disease Pathogenesis 2259694

Abstract Introduction Stevens—Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) are rare, potentially life-threatening skin conditions, often triggered by medications. They inflict damage on various epithelial and mucosal surfaces, including but not limited to the eyes, lungs, and GI tract. The pathogenesis of SJS/TEN is still unknown, however, several studies have concluded that necroptosis, a type of cell death pathway, is likely involved. Targeting the molecular players of necroptosis, such as RIPK1 and RIPK3, could offer therapeutic benefits in the treatment of these severe disorders. This study expands on previous work from our lab, which demonstrated through immunohistochemistry that there is significant expression of RIPK3 in SJS/TEN biopsy samples. Methods The study examined the expression levels of RIPK1 and RIPK3 in skin biopsies from patients with biopsy- confirmed SJS/TEN, using lichen planus as a positive control and normal skin as a baseline control. Immunohistochemistry was employed for this analysis. Additionally, the circulating levels of RIPK1 and RIPK3 in the plasma of SJS/TEN patients, mixed pathology patients for positive controls, and normal human plasma for baseline controls was measured using quantitative ELISA kits. Results Increased expression of RIPK1 and RIPK3 was noted in SJS/TEN skin biopsy and plasma samples when compared to both positive and baseline controls. Conclusion These findings strengthen the evidence that necroptosis plays a role in the pathogenesis of SJS/TEN and highlights the involvement of RIPK1 and RIPK3 as key inflammatory mediators, as well as, targets for therapeutic intervention. Funding Source Illinois Society for the Prevention of Blindness (ISPB) Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)