Browse Articles

3D Assessment of Immunomodulatory Effects of Cytokine-treated Mesenchymal Stromal Cells Using AI-Based Organ and Tissue Detection Methods 2309067

Abstract Introduction Cell tracking and biodistribution studies are constrained by slow, labor-intensive workflows involving manual tissue segmentation and limited 3D visualization. BioInVision’s CryoViz™ creates high-resolution, microscopic color anatomical and molecular fluorescence volumes enabling 3D visualization and quantification of fluorescently labeled cells across a whole animal. Tissue Recovery and Molecular Analysis (TissueReM), an AI model for tissue segmentation, delivers repeatable biodistribution readouts that are faster than conventional harvesting-based assays. We applied our workflow consisting of CryoViz™ fee-for-service imaging and TissueReM AI-based segmentation to obtain 3D biodistribution analysis of cytokine treated Umbilical Cord Mesenchymal Stromal cells (ucMSCs) in whole mice. ucMSCs have immunomodulatory properties i.e. they modify T cell and NK cell behavior and play an important role in immunotherapy applications. Methods We intravenously injected mice with fluorescently labeled ucMSCs and imaged them with CryoViz™. From these image volumes, we segmented tissues by applying TissueReM’s convolutional neural network (CNN) AI model trained on paired CryoVizTM brightfield images and human expert segmented masks. We chose Dice Score, an accuracy metric that measures overlap with human expert ground truth, and organ-specific cell counts derived from AI to those obtained from human expert generated masks, to determine model performance. Results This resulted in Dice scores of 0.80 (lung), 0.91 (liver) and 0.83 (spleen) - major tissues analyzed in this study. TissueReM-derived cell counts were within 10% of the counts derived from human expert masks. This workflow preserves whole-body spatial context while reducing analysis time and improving consistency. Conclusion CryoViz™ Imaging combined with TissueReM is a highly efficient scalable alternative to traditional biodistribution analysis in cell therapy, supporting rapid optimization of dose and route while retaining anatomical context. Funding Source NIH/NIGMS Topic Categories Technological Innovations in Immunology (TECH)

Detection of secretory vesicles linked to Schistocephalus solidus-mediated immunomodulation 2260149

Abstract Introduction Parasite-induced immunosuppression increases the host’s susceptibility to secondary infection, thereby decreasing its fitness. Parasites primarily mediate host immunomodulation via the production of excretory secretory products (ESPs). The cestode, Schistocephalus solidus, modulates the immune response, behavior, and physiology of its host, the threespine stickleback fish, via ESPs. However, as previous work has almost exclusively focused on the host responses during late-stage S. solidus infection, the anatomical origins and timing of ESP production are unknown. Methods To address this gap, we used differential staining techniques to detect S. solidus secretory organs at multiple developmental stages: day 21 (immature, unsegmented) and 42 (mature, segmented) plerocercoids. These techniques involved Hematoxylin and Eosin (H&E) and immunohistochemical analysis through lectin staining. Results H&E staining revealed that d21 samples had a nucleated tegument, and did not contain secretory vesicles. Conversely, d42 samples showed an acellular tegument, and secretory vesicles. Lectin staining indicated the presence of abundant glycoprotein-containing secretory vesicles in the parenchyma of d42 samples, which were absent at d21. These results suggest that ESPs may be produced and stored in the secretory vesicles of the parenchyma, and that significant ESP production, and therefore immunomodulation, may not occur until later stages of plerocercoid development. Conclusion Supporting this finding, we observe significant, and wide-reaching immune suppression in d42-infected fish, with reductions in splenic melanomacrophages, IgM+ B cells, and CD4+ T cells. These preliminary findings and tools lay the foundation for future work, including confirmation of secretory vesicle contents and the characterization of ESP composition across cestode development. Funding Source University of Massachusetts Lowell Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Viral infection tunes B cell receptor copy number 2258730

Abstract Introduction The multivalent structures of membrane-bound and soluble immunoglobulins enable germline recognition of cognate antigen despite typically low-affinity binding. Avidity, the combined strength of multiple antigen:receptor interactions, enables an increase in apparent affinity by decreasing the antigen dissociation rate. B cell receptor (BCR) affinity for antigen is modulated by somatic hypermutation and affinity maturation; however, it is unclear how apparent affinity resulting from the avidity effect is regulated and what the consequences for humoral immunity are. Herein, we investigate how viral infection modulates B cell receptor copy number to regulate antigen recognition. Methods We engineered novel structure-based probes to count the number of antigen binding sites on mouse and human B lymphocytes. These probes consist of a kappa light chain specific nanobody (VHH) with a c-terminal avi-tag for site specific biotinylation. The biotinylated VHH is then conjugated to a monomeric streptavidin (mSA) labeled with one molecule of AlexaFluor488 (AF488) to produce VHH-mSA-AF488. In flow cytometry, we deploy commercial bead standards to interpolate molecular copy number from the MFI of B cell-bound VHH-mSA-AF488. Results Following influenza virus infection in mice, naïve BCR copy number increases on all B cells, both within the bone marrow and peripheral secondary lymphoid organs. Likewise, we show that the human naïve B cell repertoire increases antigen receptor density during COVID and then returns to baseline during convalescence. Kinetically, we find that higher BCR copy number provides a cellular avidity effect that lowers the threshold for receptor activation. Conclusion Collectively, our results define a process whereby the resting BCR is modulated at the germline repertoire level to maximize responsiveness to antigen. Funding Source F31AI186217 Topic Categories Viral Immunology (VIR)

Liver-targeted allergen immunotherapy rapidly and safely induces antigen-specific tolerance to treat allergic diseases 2256367

Abstract Introduction Allergic diseases are increasing worldwide, with current treatments managing symptoms rather than addressing the underlying immune response. Allergen immunotherapy holds the promise of long-term symptom relief through the development of allergen-specific tolerance; however, its use is hindered by lengthy protocols and debilitating side-effects, with ultimately inconsistent efficacy at preventing allergic disease. Methods To overcome these limitations, we developed LIT, a liver-targeted allergen immunotherapy, to rapidly and safely raise allergen-specific tolerance and provide long-term control of allergic disease without continuous intervention. LIT’s synthetic mannosylation of native allergens prevents antibody-mediated recognition and subsequent life-threatening anaphylaxis upon administration. LIT’s protein engineering prevents sensitization events that occur due to the proteolytic activity of native allergens which limits allergen immunotherapy effectiveness. Results In preclinical models of asthma and food allergies, LIT reduced clinical symptoms in as quick as two therapeutic interventions. LIT induces allergen-specific regulatory T cells in vivo, and these were necessary to mediate LIT’s protections. In allergic asthma, LIT reduced features of allergic disease, including airway eosinophilia, mucus hypersecretion, allergen-specific IgE and airway hyperresponsiveness. Furthermore, LIT provided a year-long control of disease symptoms in the absence of additional intervention. In food allergy, LIT protected mice from fatal food anaphylaxis and reduced clinical and serological features associated with the reaction. Conclusion Altogether, our data provides proof of concept that LIT provides durable control of allergic symptoms by inducing allergen-specific Tregs to control the immunological cause underlying the disease. As such, we propose LIT as a rapid, safe, and effective approach to allergen immunotherapy that holds promise for long-lasting control of allergic diseases and anaphylaxis. Funding Source Chicago Immunoengineering Innovation Center, Alper Fund Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Non-Competing Clones Overcome Therapeutic Antibody Interference in Flow Cytometry 2260752

Abstract Introduction The use of therapeutic antibodies in both basic research and clinical settings can interfere with flow cytometric analysis by masking target epitopes. This interference poses a significant challenge for identifying key cell populations and accurately assessing target expression levels. Methods To address this issue, we identified and validated antibody clones that do not compete with key therapeutic antibodies for binding, enabling reliable flow cytometry analysis even in the presence of these biologics. Results Specifically, we demonstrate non-competing clones for major therapeutic targets including CD38, PD-1, CD22, HER-2, and EGFR. These RUO antibodies retained strong binding despite pre-treatment with up to 40-fold excess of key therapeutic antibodies. Additionally, we provide a curated list of literature-reported non-competing clones for other targets. Recognizing the utility of both competing and non-competing clones in receptor occupancy assays, we also present clones that are blocked by their cognate therapeutic antibodies. Conclusion These data will support investigators in selecting appropriate antibody reagents tailored to their specific research needs when studying therapeutic biologics. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

CD1a-Mediated T cell responses abrogates Insulin Resistance in High Fat Diet T2D Mouse Model 2267311

Abstract Introduction Type 2 Diabetes (T2D) is characterized by adipose inflammation dominated by Th17 cells. Standard mouse models poorly recapitulate human T2D. We discovered CD1a, a lipid antigen-presenting molecule absent in mice, mediates protective T cell responses in T2D mouse models. We hypothesized that CD1a lipid antigen presentation drives inflammation in response to High Fat Diets (HFD). We generated transgenic mice expressing human CD1a (hCD1a) and fed them HFD 45% fat for up to 34 weeks. Adipose tissue immune infiltrates were characterized by flow cytometry. Methods We generated transgenic mice expressing human CD1a (hCD1a) and fed them HFD 45% fat for up to 34 weeks. Adipose tissue immune infiltrates were characterized by flow cytometry. Results hCD1a HFD had a significant increase of infiltrating CD8+ and CD4+ T Cells into adipose compared to WT after 34 weeks on diet. hCD1a HFD infiltrates were predominantly TCRβ+ and antigen-experienced when compared to WT HFD or hCD1a Low Fat diet (LFD) mice. hCD1a HFD mice had lower fasting insulin levels and HOMA-IR as early as week 6 on diet.They also had lower gonadal fat pad weight despite overall weight remaining comparable to WT HFD. Conclusion We demonstrate that CD1a mediates robust T cell infiltration into adipose tissue under HFD concurrent with protection from insulin resistance, suggesting a protective role for CD1a-mediated T cell inflammation. These data may explain limitations of traditional mouse models. This work identifies CD1a-lipid interactions as novel therapeutic targets and suggests immunomodulation strategies that enhance rather than suppress specific T cell responses in metabolic disease. Funding Source DP2 Grant NIH Topic Categories Immune Mechanisms of Human Disease (HUM)

Human NLRC4 and CASP4 inflammasomes activate GSDMD-mediated killing of S. flexneri independently of macrophage pyroptosis and bacterial cardiolipin 2254736

Abstract Introduction Elimination of the infecting pathogen is a core function of the immune system. Macrophages are a crucial component of innate immunity against bacterial infections as they internalize and kill intracellular bacteria. Although macrophage restriction of some intracellular bacteria depends on inflammasomes, the specific mechanisms of inflammasome-mediated killing of bacteria are incompletely understood. Methods The role of inflammasomes in macrophage killing of Shigella flexneri is assessed by quantification of intracellular bacteria during infection of wild-type human-derived THP-1 macrophages and macrophages deficient in inflammasome components. Chloroquine resistance assay is used to assess IFNγ impact on vacuolar escape. Pyroptosis and GSDMD pore formation are inhibited with glycine and disulfiram. To assess GSDMD-mediated killing, GSDMD-/- macrophages are used and GSDMD is expressed ectopically in HEK293T cells. Results I found that S. flexneri is restricted in human macrophages and that macrophage restriction is enhanced by priming with IFNγ. IFNγ does not impact bacterial internalization and vacuolar escape, indicating that the intracellular restriction takes place in the cytosol. IFNγ priming results in killing of S. flexneri independently of pyroptosis and macrophage lysis. I found that macrophage restriction depends on NLRC4-mediated activation of CASP1 and GSDMD in unprimed macrophages. In contrast, I found that CASP4 is required for efficient IFNγ-dependent killing of S. flexneri and that IFNγ upregulates CASP4 and enhances bacterial restriction independently of GBP1 and NLRP11. Expression of GSDMD in HEK293T cells leads to killing of intracellular S. flexneri. GSDMD kills S. flexneri lacking cardiolipin and when oxidative stress is inhibited. Conclusion GSDMD is the executioner of inflammasome-mediated killing of S. flexneri. A new mechanism of GSDMD antibacterial activity is identified as GSDMD kills S. flexneri independently of cardiolipin and oxidative stress. Funding Source NIH T32 AI007061, NIH F32 AI188955 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Real-time TIRF imaging reveals sequential recruitment of kindlin-3 and talin-1 during β2 integrin activation 2247812

Abstract Introduction Neutrophil recruitment involves a multi-step adhesion cascade crucial for innate immunity and depends on the activation of β2 integrins. The cytoplasmic adaptor proteins, kindlin-3 and talin-1 regulate the β2 integrin activation by binding to its β cytoplasmic tail, yet the sequence of their recruitment under physiological conditions remain poorly understood. Methods To address this, we generated double knock-in mice expressing fluorescently tagged mScarlet-kindlin-3 and eGFP-talin-1, which enabled real-time co-visualization of these adaptor proteins during β2 integrin activation. Results TIRF microscopy analysis of primary neutrophils revealed a novel two-stage recruitment sequence, wherein kindlin-3 was recruited first during rolling and within seconds of arrest, followed by talin-1 recruitment after firm adhesion. Gradually, the talin-1 signal intensity exceeded that of kindlin-3, suggesting a transition from kindlin-3 driven β2 integrin priming to talin-1 mediated cytoskeletal reinforcement and stabilization of adhesion sites. Conclusion In conclusion, this study refines current paradigms of β2 integrin activation by providing real-time evidence of unexpected sequential recruitment of kindlin-3 and talin-1 to the integrin cytoplasmic tail during the leukocyte adhesion cascade. Funding Source P01HL151433 Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Lipid Nanoparticle Co‐Delivery of mRNA and a Small Molecule Drug for Oral Cancer Chemoimmunotherapy

ABSTRACT Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5‐year survival rate is 50%. Strikingly, the overall incidence rate is projected to increase by 30% in the next 10 years, which may result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor genes (TSGs) and has a “cold” tumor microenvironment (TME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory “hot” tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co‐encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TME in part by repolarizing tumor‐associated macrophages (TAMs) to M1‐like phenotypes. When formulated together, our platform provides an all‐in‐one approach for OSCC, effective in both p53‐therapy‐susceptible and p53‐therapy‐resistant models. Additionally, this work offers a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.

Preserving a Kinetically‐Metastable Nanophase by Limited Calcination for High‐Performance Protonic Ceramic Cells

ABSTRACT Sluggish oxygen reduction/evolution reactions (ORR/OER) at the air electrode critically limit the efficiency of reversible protonic ceramic cells (r‐PCCs), yet conventional high‐temperature calcination of the air electrode leads to undesired surface passivation, while low‐temperature calcination leads to insufficient crystallization, both of which severely impair electrocatalytic activity. Here we employ a thermally‐limited calcination process to kinetically retain a nanophase in the air electrode that only forms at a selected calcination temperature (termed “metastable”), thereby forming rich heterointerfaces for active ORR/OER. Specifically, controlled calcination temperature induces selective Ce incorporation into the host lattice of Ba(Co,Fe,Y)O 3‐δ while preserving the metastable BaCeO 3 ‐related nanophase. This nanostructure enriches oxygen‐vacancy‐related defects, accelerates surface exchange and bulk diffusion, promotes proton incorporation, and improves thermomechanical compatibility with the electrolyte. The as‐developed electrode (BaCo 0.6 Fe 0.2 Y 0.1 Ce 0.1 O 3‐δ ‐BaCeO 3 ) exhibits a low resistance of 0.38 Ω cm 2 at 550°C. Single cells with this electrode deliver a high peak power density of 1.44 W cm −2 at 650°C and an electrolysis current density of −2.47 A cm −2 at 1.3 V. These findings establish a promising strategy of thermally‐limited calcination for designing high‐performance reversible protonic ceramic cells.

Laplace‐Pressure‐Stabilized Rutile Solid‐Solution Catalysts for Acidic Water Oxidation: Enabling DSA‐Inspired Compositions

ABSTRACT An early phase diagram study in 1967 revealed surprisingly wide immiscible gaps in the rutile‐type IrO 2 ─TiO 2 and IrO 2 ─SnO 2 systems, even though the three oxides share the same crystal structure and nearly identical ionic radii. This finding indicates that the formation of IrO 2 ‐based solid solutions, especially with oxides of different structures, is thermodynamically challenging. Using more than 20 foreign metal (M) cations, we demonstrate that single‐phase rutile (Ir, M)O 2 and (Sn, M)O 2 solid solutions containing up to 30 at.% M can be obtained when the crystal size is below 10 nm, whereas larger, submicron‐scale crystals exhibit immiscible behavior. In particular, complete solid solutions are achieved across the entire composition range in the nanoscale IrO 2 ─SnO 2 system. This enhancement in miscibility under high Laplace pressure is not restricted to specific foreign cations but represents a general phenomenon. These results are exploited to synthesize rutile‐phase nanocrystals incorporating DSA‐inspired quaternary cations (Sn, Nb, Ru, and Ir), which show high corrosion resistance and catalytic activity under anodic potentials. Quantitative electrochemical analyses further show that the catalytic durability of these quaternary nanocrystals, despite the significantly reduced Ir content, is comparable to that of IrO 2 during the oxygen evolution reaction in acidic water electrolysis.

The SOS response-induced cell division inhibitor YneA interacts with FtsW to block septal peptidoglycan synthesis in Bacillus subtilis

Computerized instrumentation system for assessing chargeability and deposition of charged spray droplets in agriculture

GLI2 Regulates T Cell Proliferation in Cooperation with IL-2 Signaling 2309246

Abstract Introduction GLI2, a transcription factor downstream of hedgehog (Hh) signaling, is essential in embryonic development. GLI2 is also known for regulating the proliferation of different cell types. Previous studies have shown that GLI2 plays a role in T helper 2 (Th2) polarization and asthma pathogenesis. However, its role in T cell homeostasis and proliferation remained poorly understood. Methods To investigate this, we used mice with conditional deletion of Gli2 in hematopoietic cells, Gli2fl/fl Vav_icre+/- (H-Gli2-/-) and Gli2fl/fl Vav_icre-/- (WT littermates). Results We observed a significantly increased frequency of CD4+ T cells and a corresponding reduction in CD8+ T cells in the spleen, lymph nodes, and peripheral blood of H-Gli2-/- mice compared to WT. However, this altered CD4/CD8 ratio persisted following immunization of mice with ovalbumin, indicating that immunization did not further modify the distribution of CD4+ and CD8+ cells. Additionally, T cells from H-Gli2-/- mice spleens exhibited markedly increased CD4+ and CD8+ T cell proliferation when stimulated in-vitro with anti-CD3 and anti-CD28. Importantly, supplementation with exogenous IL-2 enhanced the proliferation of T cells from WT, but not H-Gli2-/- mice, indicating that GLI2 restraints T cell proliferation through modulation of IL-2 or IL-2-dependent signaling or IL-2 enhances GLI2-mediated cell proliferation in WT cells. Ongoing studies using both H-Gli2-/- mice and mice expressing an active form of Gli2 (ΔN-Gli2+/-) models are focused on defining the molecular mechanism by which IL-2 modulates T cell proliferation in the presence of an intact GLI2, including its impact on IL-2 production, IL-2 receptor expression, and downstream STAT5 activation. Conclusion Together, these findings identify GLI2 as a critical regulator of T cell proliferation and may inform strategies to modulate immune responses in cancer and inflammatory diseases. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Is Toll-like Receptor 4 Required for Antibody Production?: Using Genetically Defined Mouse Models to Settle the Dispute 2260073

Abstract Introduction Toll-like receptors (TLRs) play an integral role in activating immune pathways in response to the presence of a pathogen. Although mechanisms of action in mediating innate immune responses are well-researched, it is still unclear whether TLR expression regulates adaptive immunity. Data from current literature lead to conflicting conclusions. Some studies show that TLRs, specifically TLR4, are critical to initiating adaptive immune responses, and therefore the production of antibodies, while others indicate that TLR signaling is not required. Methods This study uses a two week prime-boost schedule of intramuscular vaccination using the model antigen ovalbumin (Ova) in wild-type (WT) and TLR4-deficient (TLR4-/-) mice. Serum ELISAs indicate that TLR4-/- mice (strain B6(Cg)-Tlr4tm1.2Karp/J) have significantly less antibody production than WT mice (strain C57BL/6) after vaccination with Ova. To investigate whether the lack of TLR4 could be overcome by immunoadjuvants, TLR4-/- mice (strain B6(Cg)-Tlr4tm1.2Karp/J) were injected with adjuvanted Ova on days 1 and 14. The adjuvants CpG DNA (TLR9 ligand), PHAD (TLR4 ligand), and Alum (alhydrogel) were used. Serum was collected and antibody production was measured on days 14 and 28. Results Serum ELISAs indicate extremely low levels of antibody. To confirm the lack of antibody production is due to the TLR4 deficiency, the adjuvant study was repeated in a different strain of WT (C3HeB/FeJ) and TLR4-/- mice (C3H/HeJ). Serum ELISA data is inconclusive due to the lack of antibody production exhibited in the WT mice. Conclusion Further studies must be performed to determine whether insufficient antibody production is a phenotype exhibited by all TLR4-/- mice. Identifying pathways that are critical in stimulating adaptive immunity will potentially lead to the development of more effective vaccines, especially in immunosuppressed individuals. Funding Source Towson University Office of Undergraduate Research and Creative Inquiry 2025 Summer Project Grant and 2024 Fisher Endowed Chair of Biology Grant. Topic Categories Vaccines and Immunotherapy (VAC)

BTK Inhibition Resets Pathological B and T cell Subsets in Patients with IgG4-Related Disease 2258804

Abstract Introduction IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder marked by tissue infiltration of IgG4+ plasmablasts (PB) and elevated serum IgG4 levels. Bruton’s Tyrosine Kinase inhibition (BTKi) blocks key signaling pathways in B cells and showed promising efficacy in other autoimmune diseases. This is the first trial testing BTKi in IgG4-RD, and its effects were analyzed on a single-cell level. Methods 10 patients with IgG4-RD affecting the submandibular and/or lacrimal glands were enrolled in this single-site, open-label, investigator-initiated trial (NCT04602598). Patients received zanubrutinib orally (80mg BID) for up to 24 weeks. The primary endpoint was the change in glandular volume at week 24 compared to baseline. Single-cell transcriptomes and immune repertoires of PBMCs were analyzed at baseline, week 12, and week 24 and compared with matched healthy controls. Results Two patients terminated at week 4 due to adverse events. For the 8 remaining patients, treatment with zanubrutinib 80 mg PO BID resulted in significant reductions in lacrimal (—45.4% change) and submandibular gland volumes (—30.0% change, both p < 0.001) at week 24. Serum IgG4 levels decreased by a mean of 413 mg/dL and the IgG4‑RD Responder Index decreased by a mean of 4.6 points (p = 0.02). Single-cell sequencing analyses revealed diminished numbers of IgG4+ PB and atypical B cells (ABCs). Strikingly, BTKi most strongly affected IgG4+ and IgG2+ PB, restoring the composition of Ig subclasses to those seen in healthy donors. In ABCs, BTKi significantly downregulated genes associated with B cell receptor (BCR) signaling and antigen presentation. Numbers and activation levels of proliferating CD4+ T cells correlated with IgG4+ PB and ABCs, supporting the hypothesis that an extensive B cell / T cell cross-talk drives IgG4-RD pathology. Conclusion Zanubrutinib inhibits pathogenic B and T cell subsets in IgG4-RD in a targeted fashion and is a promising therapeutic for IgG4-RD. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Pannexin-1 promotes CD8+ T cell antitumor immunity against colorectal cancer in a gender-dependent way 2256739

Abstract Introduction Colorectal cancer (CRC) is the second leading cause of cancer death in the U.S., with stage IV patients showing poor five-year survival. Immune checkpoint blockade shows limited efficacy in CRC, highlighting the need to better understand how CD8+ T cells are regulated by the tumor microenvironment (TME). In the TME, CD8+ T cells often become exhausted and lose cytotoxic function. Terminally exhausted cells express high inhibitory receptors, PD-1, Tim-3, and low proliferative capacity; whereas stem-like exhausted cells can self-renew and respond well to checkpoint blockade. Metabolic regulation is critical for CD8+ T cells, including membrane channels that export metabolites. Our lab studies Pannexin-1 (PANX1), a channel that exports ATP and promotes memory CD8+ T cell survival via AMPK signaling. Methods We found that PANX1 is crucial for effective antitumor CD8+ T cell responses in CRC. High PANX1 expression correlates with increased CD8+ T cell infiltration and better prognosis in stage IV CRC patients. Using CRC mouse models, CD8-specific Panx1 knockout males, but not females, fail to control MC38 tumors, with tumor-infiltrating CD8+ T cells showing higher exhaustion, fewer stem-like cells in lymph nodes, and lower AMPK activity. Results Single-cell RNAseq and InfinityFlow confirm enhanced exhaustion signatures in PANX1-KO tumor-infiltrating stem-like exhausted cells, while wild-type tumors accumulate more effector-like (Zeb2+) CD8+T cells. Preliminary data suggests that AMPK overactivation (AICAR) significantly restores PANX1-KO exhausted CD8+ T cells. Finally, the gender-specific discrepancies may involve androgen receptor signaling, elevated in PANX1-KO exhausted cells. Ongoing studies examine whether PANX1-mediated AMPK activity regulates androgen receptor expression. Conclusion Together, these findings suggest PANX1 promotes CD8+T cell-driven anti-tumor immunity in CRC in a gender-dependent manner, potentially explaining sex-based differences. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Pharmacological Activation of STING attenuates the severity of schistosome immunopathology 2251618

Abstract Introduction Schistosomiasis, caused by parasitic worms, affects over 250 million people globally, with limited treatment options due to praziquantel’s inability to prevent reinfection or reduce immunopathology. In Schistosoma mansoni (S. mansoni) infection, egg-induced granulomatous inflammation in the liver and intestines is driven by CD4 T helper (Th) cell responses, with severe pathology in some individuals mediated by proinflammatory Th17 cells. We previously demonstrated that the stimulator of interferon genes (STING) mitigates schistosome egg-induced immunopathology by promoting type I interferon (IFN-I) production and suppressing Th17 responses. Here, we investigate the therapeutic potential of the STING agonist diABZI3 in a high-pathology CBA mouse model. Methods In vitro, diABZI3 pretreatment of bone marrow-derived dendritic cells (BMDCs) significantly enhanced IFNβ production while abolishing IL-1β and IL-17 expression in response to schistosome egg stimulation, an effect dependent on early administration. Results In vivo, a single dose of diABZI3 administered to S. mansoni-infected CBA mice reduced liver and intestine granuloma size, as well as IL-1β and CD209a levels, but induced the recruitment of Foxp3+ regulatory T cells. Furthermore, blocking STING degradation with bafilomycin A1 sustained STING signaling, leading to pronounced IL-1β suppression. Conclusion These findings highlight diABZI3 as a promising therapeutic agent for reducing schistosome-induced immunopathology and underscore the importance of early STING activation to modulate proinflammatory responses. Strategies to stabilize STING signaling may enhance therapeutic efficacy, though careful consideration of sustained activation is needed to avoid proinflammatory consequences. Funding Source NIAID grant R01 AI148656 to P.K. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Generation and Performance of a DNA Barcode-Based Nuclear Receptor Reporter Library 2267335

Abstract Introduction Nuclear receptors (NRs) are a large and distinctive class of ligand-regulated transcription factors that enable cellular sensing of hormone and metabolite gradients within and between tissues. They play essential roles in the regulation of immune cell development, function, and inflammation, making them among the most durable and efficacious drug targets for human diseases. However, roughly one-third of the 48 human NRs remain classified as ‘orphan’ receptors with no currently known physiological ligands. Conventional transcriptional reporter assays, though foundational in NR ligand discovery, are tedious, transient, and singleplex (one NR at a time), inherently favoring hypothesis- over discovery-driven research. Methods We develop a DNA barcode-based NR library that features 52 bi-component lentiviral vectors, including reporters for all 48 human NRs plus the Aryl hydrocarbon receptor (AhR), as well as one positive and two negative control constructs. Each reporter construct uses a dual anti-sense configuration to: (i) stably express one NR LBD-GAL4 fusion protein plus a barcoded mCherry reporter; and (ii) inducibly express a separately barcoded GAL4-responsive GFP reporter. Hence, NR activities can be quantified by either GFP/mCherry fluorescence ratios (singleplex; flow cytometry) or GFP/mCherry-linked barcode abundance ratios (multiplex; RNA-seq). Results We show that this barcode-based lentiviral reporter system performs similarly to a prior commercial multiplex NR reporter assay (trans-FACTORIAL; Attagene), but leverages workflows that are faster, more durable, more cost-effective, and more technically accessible. Conclusion We report the development and performance of a novel, stable, versatile, and multiplexable NR reporter library readable by either flow cytometry (singleplex) or RNA-seq (multiplex). Democratization of this multiplex reporter platform will support the expansion of NR ligand and drug discovery research. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Systemic CXCL13 as a possible sex-dependent biomarker to identify neuroinflammation in subclinical food allergies 2248073

Abstract Introduction Patient cohort studies have reported an association between subclinical food allergies and behavioral issues. Using an orally challenged subclinical cow’s milk allergy (CMA) mouse model, we previously demonstrated behavioral changes that were accompanied by neuroinflammation in male mice. While these observations highlighted the importance of acknowledging one’s hypersensitivity status, identifying individuals with subclinical food allergies is difficult, as they do not exhibit common allergic symptoms. In our CMA male mice, CXCR5-immunoreactive (CXCR5+) neurons, CD45+CXCR5+ leukocytes, and CXCL13, the chemokine for CXCR5, were increased in the frontal cortex. In addition, CXCR5+ cells were found along with the superior sagittal sinuses of the dura and decreased with CXCR5-blocking antibody in male, but not in female, CMA mice. These findings suggested that CXCL13/CXCR5-mediated central chemotaxis occurs in sensitized mice in a sex-specific manner. Methods Hypothesizing that the elevation of circulating CXCL13 could serve as a potential sex-dependent biomarker for detecting neuroinflammation in subclinically sensitized individuals, we measured plasma CXCL13 by ELISA. Results Plasma CXCL13 levels were strongly correlated with brain CD45+CXCR5+ cell number and moderately correlated with allergen-specific IgE levels in male CMA mice. Males showed 100% sensitivity (AUC = 0.9349) and females showed 88% sensitivity (AUC = 0.8151) for CXCL13 at 50% specificity; however, both sexes showed similar AUCs (0.9288, 0.9132) and sensitivities (92%, 95%) for BLG-specific IgE. Therefore, at a fixed specificity (50%), CXCL13 is a more sensitive biomarker for males than females. Conclusion Together, plasma CXCL13 could detect food-allergen-induced neuroinflammation as a systemic sex-dependent biomarker in subclinically sensitized individuals. Validating the diagnostic value of CXCL13 in different food allergies and elucidating the mechanism of allergen-triggered CXCL13 induction are warranted in future studies. Funding Source NIH RO1 Topic Categories Neuroimmunology (NEUR)