Browse Articles

Molecular characterization of the IgE serological repertoire in pediatric peanut allergy 2267488

Abstract Introduction Though recent strides in peanut allergy prevention have changed the clinical thinking about pediatric treatment, the antibodies responsible for severe allergic reactions, IgE, remain poorly understood, especially at the molecular level. The advent of BCR-seq–deep sequencing of the B cell receptor (BCR) repertoire–has illuminated molecular features of pathogenic IgE producing B cells causing food allergies. However, the degree to which the transcribed BCR repertoire represents the circulating secreted immunoglobulin (Ig) repertoire is not known. Methods Here, we use parallel BCR- and Ig-seq methods to describe the circulating IgE, IgG, and IgA repertoires in pediatric donors with peanut allergy. Results Tandem liquid chromatography mass spectrometry allows for relative quantitation of serological lineages, identification of CDRH3 motifs underlying peanut allergy, description of antibody clone convergence, and analysis of lineage overlap between circulating isotypes. Conclusion Because the correlation between peanut-specific IgE and peanut allergy is weak, comprehensive characterization of the IgE serological repertoire will inform diagnostic opportunities as well as elucidate mechanisms underlying B cell and Ig trafficking in food-allergic individuals. To our knowledge, this is the first molecular-level proteomic IgE repertoire analysis completed. Funding Source Texas Biologics Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

CD43 regulates T-cell function and survival in a context-dependent manner 2299796

Abstract Introduction CD43 (sialophorin) is a sialomucin widely expressed on hematopoietic cells that governs T-cell adhesion, trafficking, and activation. We tested whether CD43 sets proliferation and survival thresholds in T-cell subsets. Methods Lymph-node T cells from WT and CD43KO C57BL/6 mice were stimulated with anti-CD3/CD28 or PMA/ionomycin; proliferation (CFSE), viability, Bax/Bcl-xL, and GLUT1, CD25, and CD69 were quantified by flow cytometry. For antigen-specific responses, CFSE-labeled OT-I/OT-II WT or CD43KO T cells were adoptively transferred into OVA-immunized recipients. Results Under anti-CD3/CD28, CD43KO CD4 and CD8 proliferated less than WT, indicating that CD43 supports TCR/CD28-driven activation. With PMA/ionomycin, CD43KO CD4 proliferated less, whereas CD43KO CD8 proliferated more. Loss of CD43 pushed CD4 toward a pro-apoptotic, hypoproliferative state characterized by an increased Bax/Bcl-xL ratio, while CD43KO CD8 retained a balanced Bax/Bcl-xL and showed more GLUT1+ and CD69+ cells, consistent with elevated metabolic/activation thresholds. CD25 was unchanged. In vivo, OT-I CD43KO cells expanded 5-fold over WT, whereas OT-II CD43KO cells expanded 4-fold less than WT. Conclusion CD43 has a context-dependent, subset-specific role in T-cell responses differentially tuning CD4+ and CD8+ activation and expansion. CD4+ cells are more prone to activation-induced death and require CD43 for proper expansion, whereas CD8+ cells are restrained by CD43, limiting excessive activation. Thus, CD43 shapes the magnitude and quality of T-cell immunity across contexts. Funding Source Funded by DGAPA/UNAM IN222523, Postdoctoral fellow by SECIHTI, 6002103. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

GPR25 as a novel target to boost anti-tumor TRM responses 2300820

Abstract Introduction Tissue-resident memory CD8 T (TRM) cells are central to durable antiviral and antitumor immunity, yet the molecular cues that program their formation remain incompletely defined. We identified the orphan G protein—coupled receptor GPR25 as enriched in TRM cells and induced by TGF-β, suggesting a role in TRM differentiation and maintenance. Methods Using adoptive-transfer models of viral infection and tumor challenge, we compared trafficking, differentiation and recall responses of wild-type and Gpr25-deficient CD8 T cells. Single-cell RNA sequencing dissected transcriptional programs altered by Gpr25 deficiency. We validated TGF-β downstream signaling by modulating GPR25 expression and tested reported ligands (CXCL17 and cleaved 4CysCXCL17) in orthogonal β-arrestin and calcium-mobilization assays. Finally, we implemented a high-throughput screening (HTS) pipeline–based on inducible GPR25 reporter cell lines and calcium/β-arrestin readouts–to discover small-molecule GPR25 agonists. Results Gpr25-deficient CD8 T cells trafficked normally but failed to develop and sustain TRM populations in lung and liver, with reduced stem-like features (including TCF1), and exhibited impaired secondary TRM expansion after rechallenge. Transcriptomes of Gpr25-deficient cells exhibited reduced TGF-β—responsive signatures, and SMAD2/3 phosphorylation in vitro. Besides, we were unable to replicate reported CXCL17—GPR25 agonism across multiple assay platforms. Leveraging inducible GPR25 reporter lines, our HTS workflow reliably detects ligand-dependent and ligand-enriched activities and is poised to identify small-molecule agonists. Conclusion GPR25 is a key regulator of TRM differentiation and maintenance through enhancement of TGF-β signaling. Failure to validate CXCL17 as the endogenous ligand underscores the need for systematic deorphanization. Our HTS platform positions us to discover GPR25 agonists with the potential to augment TRM-mediated antitumor immunity and translate into novel immunotherapeutics. Funding Source William K. Bowes Jr Foundation (to P.V.), Whittaker Foundation (to c.h.o.), curebound Discovery grant 24Dg05 (to P.V. and m.R.J.) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Recent Advances in Interfacial Chemistry for Solid‐State Lithium–Sulfur Batteries

ABSTRACT All‐solid‐state Lithium–sulfur batteries (ASSLSBs) have emerged as a highly promising candidate for next‐generation energy storage systems, featuring a unique combination of earth‐abundant sulfur cathodes, high‐capacity lithium metal anodes, and nonflammable solid‐state electrolytes (SSEs). These components collectively circumvent the energy density limitations (<300 Wh kg −1 ) and safety concerns for the conventional liquid electrolyte‐based lithium‐ion batteries. However, the high impedances at the SSEs/electrode interfaces, at both Li anodes and sulfur cathodes, impede efficient charge transfer and Li stripping/plating kinetics, indicating a critical bottleneck. This review focuses on the mechanistic dynamics governing these solid‐state interfaces. We provide an in‐depth analysis of the origin and evolution of SSEs/electrode interfaces and their impact on the electrochemical performance. Furthermore, we systematically evaluate state‐of‐the‐art strategies for deciphering solid‐state sulfur conversion reactions and Li plating/stripping processes, as well as for enhancing the interfacial stability and reaction kinetics. Finally, we examine the gap between current achievements in laboratories and the industrial requirements for practical ASSLSBs, followed by actionable perspectives. This review is expected to provide valuable insights for solid‐state battery community and facilitate the realization of high‐performance ASSLSBs.

Comparative genomic and gene expression study of Dirigent genes in genus Dendrobium: a bioinformatics analysis

CD8 T cells require IFNg to induce disease in a murine model of Alopecia Areata 2310132

Abstract Introduction Alopecia Areata (AA) is a prevalent autoimmune disease affecting 2% of the population and presents as nonscarring hair loss. In AA, CD8 T Cells have been found infiltrating around and within the hair follicle and are identified as main drivers of disease. Our transcriptional analysis has revealed that CD8 T cells in the skin of AA patients are poised to make effector molecules such as interferon gamma (IFNg), perforin, and granzymes. Similarly, in the C3H/HeJ murine model of AA, pathogenic NKG2D+ CD8 T cells expressed more IFNg, perforin, and Granzyme B than non-pathogenic T cells. Given the close spatial relationship between CD8 T cells and the hair follicle end target, our objective was to assess the contributions of CD8 T cell-derived molecules during disease. Methods We assessed the role of CD8 T cell molecules in a C3H/HeJ murine model of AA, wherein pathogenic CD8 T cells are sorted from the skin draining lymph nodes of AA mice, expanded in vitro, and intradermally injected into naïve recipients. We used RosaERTcreIFNgKO or RosaERTcrePrfKO mice, capable of tamoxifen-inducible global knockdown of IFNg or Perforin, as donors. Mice globally deficient in the IFNg-receptor(IFNgrKO), bone marrow chimeras with an IFNgrKO hematopoietic compartment, or WT mice were used as recipients. Results We observed perforin deficient CD8 T cells were capable of transferring disease in a manner comparable to WT cells. Conversely, IFNg deficient CD8 T cells failed to transfer disease. Furthermore, global IFNgrKO mice were incapable of developing AA following CD8 T cell induction and bone marrow chimeras containing an IFNgrKO hematopoietic compartment were also unable to develop disease. Conclusion Overall, our findings suggest that perforin is dispensable, while IFNg is a crucial pathogenic effector molecule employed by CD8 T cells in AA. Sensing of IFNg by the immune compartment was a vital step of disease pathogenesis, and future studies may decipher how IFNg sensing by the hair follicle impacts disease. Funding Source n/a Topic Categories Basic Autoimmunity (BA)

Simplifying CD19 CAR Detection: A Bright Single-Step Solution for Flow Cytometry 2259753

Abstract Introduction Accurate flow cytometric detection of CD19 CAR-expressing cells is critical across CAR-T cell discovery and translational research workflows. However, variability in surface CD19 CAR protein expression presents challenges in reliably identifying CAR+ cells. Additionally, detection using recombinant CD19 protein is hindered by its instability in soluble form. Methods To address these limitations, BD® has developed a novel, bright, and stable one-step CD19 CAR Detection Reagent, available in multiple fluorochrome formats to support diverse panel designs and instrument configurations. Results We demonstrate that this reagent specifically stains CD19 CAR-expressing CHO cells, Jurkat CAR-T cells, and primary human CD19 CAR-T cells. Furthermore, it performs effectively in lysed whole blood, multi-color panels, and workflows involving intracellular staining. Notably, CAR-T cells spiked into donor PBMCs were detected at frequencies as low as < 0.04% of live cells, highlighting its potential utility in studies of CAR-T cell persistence. Minimal background staining was observed in CAR-negative and mismatched CAR populations, and low-expressing CAR cells (CARlo) were distinguishable from negative cells Conclusion Overall, these findings highlight the BD® CD19 CAR Detection Reagent as a robust solution for overcoming key challenges in CD19 CAR+ cell detection, enabling progressive CAR-T cell research. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

IL2 signaling and Foxp1 expression maintain Treg identity in the absence of Foxp3 2260415

Abstract Introduction Regulatory T cells (Treg) have a pivotal role in tolerance and immune homeostasis. In autoimmunity, Treg are destabilized and degenerate into exTregs, loosing Foxp3 expression and compromising their regulatory function, while gaining a T effector cell (Teff)-like phenotype. Methods By utilizing a Foxp3 loss-of-function mutation model, we show that Treg development gives rise to a heterogeneous population of Treg cells (ΔTreg) in periphery. ΔTregs that retain the core Treg transcriptome and epigenetic imprint have residual suppressive activity and can be classified based on the expression of CD25. On the other hand, ΔTregs that acquire Teff transcriptome promote autoimmunity, are characterized by loss of CD25 and Foxp1 expression and impaired suppressive activity. Further, depletion of ΔTregs in DSS induced colitis model reduced the severity of disease confirming the teff like function of these cells. We herein propose a stepwise degeneration of ΔTregs from CD25+ Treg like population to CD25— Teff like population and finally exTregs with a loss of Foxp3 locus activity, driven by reduced IL2/CD25 signaling and Foxp1 expression while acquiring Teff programs. Results Treatment with IL2/αIL2, CDK8/19 inhibitor and STAT5b gain of function prevent the degeneration of ΔTreg cells in vivo resulting in increased frequency of CD25+ ΔTregs and improvement in the scurfy phenotype, supporting the role of IL2 signaling in maintaining Treg cell attributes of ΔTreg cells in absence of Foxp3 expression. ΔTreg-specific Foxp1 deletion led to a steep decrease in the frequency of ΔTreg, lower CD25 expression, increased T-bet expression, and a reduction in the Foxp3 locus activity, suggesting that Foxp1 loss accelerate the degeneration process of ΔTreg cells. Conclusion Our results indicate that IL2 signaling is sufficient to maintain ΔTreg regulatory capacity, while Foxp1 expression is necessary to stabilize their identity and prevent terminal degeneration. Funding Source NIH NIAD Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Proinflammatory Genes are Transcriptionally Suppressed by Type I Interferon Through Undescribed Non-canonical Mechanisms 2259728

Abstract Introduction Type I interferons (IFN) are crucial for effective antiviral immune responses. Paradoxically, they exhibit poorly defined immunosuppressive properties in conditions such as bacterial infections or multiple sclerosis. Previous work has shown that IFNβ suppresses transcription of key proinflammatory genes leading to immunosuppression; however, the total repertoire of genes impacted is unknown. Methods To determine the immune response genes transcriptionally suppressed by IFNβ, RNA sequencing was performed on bone marrow—derived macrophages (BMDMs) pretreated with IFNβ prior to stimulation with ligands for two distinct Toll-like receptors (TLRs). Results Differential expression analysis revealed that IFNβ pretreatment reprograms TLR-induced transcriptional responses, uncovering a unique coordinately regulated cluster of TLR-induced genes suppressed by IFNβ that lack shared promoter-proximal sequence motifs. Pathway enrichment analysis indicated preferential suppression of immune-related pathways, including cytokine—cytokine receptor interactions, IL-17 signaling, and neutrophil chemotaxis. Although IFNs are known to positively regulate gene expression via the ISGF3 complex, IFNβ-mediated transcriptional suppression persisted in STAT1- and STAT2-deficient BMDMs, suggesting an alternative regulatory complex. Conclusion This study defines a novel population of type I interferon—inhibited genes (TIIGs) regulated independently of ISGF3, further defining IFN’s role in non-viral inflammation. Funding Source R21 AI180181-01 T32 AI095190 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Critical role of 4-1BB:4-1BBL axis in humoral immune responses against Plasmodium 2242516

Abstract Introduction Several costimulatory and coinhibitory signaling axis have been reported to modulate anti-Plasmodium immune response, albeit the precise role of 4-1BB:4-1BBL axis has yet to be investigated. Methods We used 4-1BB KO and 4-1BBL KO mice to investigate the effects of each receptor during Plasmodium infection. To temporally modulate 4-1BBL signaling, we treated WT mice with a 4-1BBL blocking antibody (TKS-1) at different timepoints. Considering the scenario of 4-1BB binding to Galectin-9 (GAL9) as previously reported, certain experiments involve treating WT mice with GAL9 blocking antibody. To understand the functional significance of 4-1BB-4-1BBL axis, we also performed single cell RNA-, V(D)J-, and CITE-seq on sort-purified WT, 4-1BB and 4-1BBL KO T and B cells sourced from three-way bone marrow chimeric mice infected with a rodent-specific Plasmodium strain. Results Here, we show that complete loss of 4-1BB resulted in delayed parasite control, impaired GC responses and memory B cell recall. The absence of 4-1BBL, however, resulted in enhanced parasite control without influencing the GC response. Early blockade of 4-1BB:4-1BBL signaling by using TKS-1 recapitulated 4-1BB KO data, an effect that was absent when 4-1BBL was blocked at effector time points. While exploring the possibility of other 4-1BB binding partners such as GAL9, we observed that combined blockade of GAL9 and 4-1BBL resulted in loss of parasite control as well as loss of protection following lethal challenge, similar to 4-1BB KO mice. Conclusion While these data show a preferential binding of 4-1BB to 4-1BBL mediating an inhibitory effect, our data suggest that 4-1BB can bind to GAL9 in the absence of 4-1BBL and potentiate stimulatory signals. Our future experiments are directed towards understanding the precise and differential role of ligands of 4-1BB and their temporal effects in governing anti-Plasmodium humoral immunity. Funding Source This work was supported by 1R01GM125907 to S.E.L., the RFUMS startup funds, Schweppe Scholar Award, 1R01AI182217 to R.V., and the AAI 2024 Careers in Immunology Fellowship to C.C. and R.V. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Antibodies cloned from a patient with Idiopathic CD4 Lymphopenia induce potent anti-T and B cell complement-dependent lysis 2304145

Abstract Introduction Idiopathic CD4 Lymphopenia (ICL) is a rare immunodeficiency disorder characterized by an unexplained CD4+ T-cell lymphopenia. Often, these patients harbor additional deficiencies in CD8, B, and/or NK cells and, therefore, may suffer from opportunistic infections. We previously found that half of patients with ICL possess auto-antibodies-(Ab) against a broad spectrum of molecules, including those expressed on the membranes of B and T-cells, suggesting a potential role of these anti-lymphocyte Ab (ALAb) in their lymphopenia/s. We focused here on a patient (ICL-9) whose IgM ALAb induced complement dependent cytotoxicity (CDC) against healthy donor’s (HD) lymphocytes and against B-cell lymphoma cell lines. Methods We performed single-cell B-cell receptor (BCR) sequencing of sorted ICL-9’s B cells, cloned 21 IgM BCR sequences, and screened them for specificity against B/T lymphocytes and Raji B-cell line. The positive reactive clones were further tested for CDC of their cellular targets. Results The cloned ALAb belonged to the previously described autoreactive VH4-34 family. Ten of the auto-Ab recognized B and/or T cells, with three of them inducing lysis of their targets by CDC. A particularly potent clone, named Aliron, induced CDC of 50% of B lymphocytes at 0.01 nM concentration, showing a 100-fold higher CDC potency than anti-CD20 Ab Rituximab. At higher concentrations, Aliron was also able to kill T-cells by CDC. Further, Aliron induced CDC of all seven B-cell lymphoma lines tested. We are currently analyzing the in vivo activity of these IgM monoclonal Abs in humanized mouse models. Conclusion From a single patient with ICL we cloned and characterized IgM Abs that recognized membrane targets on T and B lymphocytes and induce CDC. The discovery of these ALAb support their possible pathogenic role on ICL and they might develop into potential future clinical applications. Funding Source NIAID, NIH Topic Categories Immune Mechanisms of Human Disease (HUM)

Creation of the first gluten allergenicity map using diploid, tetraploid, and hexaploid wheats in a mouse model 2300763

Abstract Introduction Gluten allergy is a growing global disease affecting individuals with high risk of potentially deadly anaphylactic reactions. The relative intrinsic allergenicity of glutens from the three commercially grown wheat species (Diploid Triticum monococcum, tetraploid Triticum durum, hexaploid Triticum aestivum) is unknown. Here, using a recently described adjuvant-free mouse model, we tested the hypothesis that the three different wheat species will exhibit natural variation in their gluten allergenicity. Methods Groups of Balb/c mice (n = 10/group) were transdermally sensitized to alcohol-soluble or acid-soluble gluten extracts followed by elicitation of systemic anaphylaxis. Initial studies were performed to validate the model for glutens from the three wheat species. Sensitization was measured by specific IgE levels. Anaphylaxis was measured by rectal thermometry. Mucosal mast cell response was quantified by measuring MMCP-1 elevation in the blood. Results Both glutens from all three wheat species elicited robust sIgE responses, as well as systemic anaphylaxis. However, comparative mapping analysis revealed differences in capacity to elicit sIgE among the three wheat species with T. aestivum being the most potent in both gluten extracts. Hypothermic shock response (HSR) analysis revealed that the three species elicited similar kinetics and intensity of anaphylaxis. Nevertheless, when analyzing Mucosal Mast Cell Response (MMCR), it was revealed that the glutens from T. aestivum (hexaploid) emerged as the most potent elicitor. Conclusion Collectively, these results yield a comparative map of the natural variation in intrinsic allergenicity potencies of the three consumed wheat glutens for the first time. This map may be utilized for: i) quantitative comparison the allergenic potential of glutens from genetically engineered wheats and processed wheat products against existing wheat glutens; and ii) the identification of potentially hyper/hypo/iso-allergenic species/varieties of wheats. Funding Source USDA/NIFA: MICL02486, 02796, 01699; AFRI: 2018-67017-27876 (MICL05103) Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Inhibition of Innate Immune Allorecognition Using Nanobiologics Effectively Overturns Ongoing T Cell Mediated Allograft Rejection 2305425

Abstract Introduction Acute T cell mediated rejection (TCMR) episodes are inadequately controlled by rejection treatment and impede long-term survival. Given that TCMR is driven by innate allorecognition memory (Allo-IMem), we investigated whether specifically inhibiting Allo-IMem using nanobiologics would overturn TCMR. Methods WT B6 recipients of Balb/c islet allografts were left untreated and then treated at the peak of TCMR (days 7-12) with innate immune cell specific high-density lipoprotein nanobiologics. Nanobiologics were either unloaded (control; U-NB) or loaded with the mTOR inhibitor rapamycin (mTORi-NB; 5mg/kg) and compared to free rapamycin (Rapa; 5mg/kg). Results We uncovered that Allo-IMem signals through mTOR, and unlike Rapa treatment, mTORi-NB inhibits Allo-IMem compared to U-NB. Remarkably, mTORi-NB treatment at the peak of TCMR overturned rejection in 90% of recipients and was therapeutically advantageous over Rapa treatment. scRNA-seq analysis of graft-infiltrating innate cells (day 10) demonstrated that mTORi-NB inhibited cell cycle, glycolysis, and type I/II interferon signaling, and promoted TGF-β signaling pathways, compared to U-NB. Multiphoton intravital microscopy of islet allografts on day 10 revealed that mTORi-NB significantly reduced both the dynamics (speed and distance traveled) and intracellular Ca+ signaling in effector T cells, compared to U-NB. Mechanistically, mTORi-NB significantly reduced the antigenic presentation capacity of graft-infiltrating innate cells compared to U-NB. In addition, both graft infiltrating effector T cell proliferation and IFNγ+ numbers were significantly decreased with mTORi-NB compared to U-NB. Conclusion Taken together, we demonstrate that inhibiting Allo-IMem using innate cell specific mTORi-NB effectively overturns TCMR, thus providing a novel targeted therapeutic treatment for acute rejection in transplant patients. Funding Source NIH U01 U01AI132758 (GC)↑, Thomas E. Starzl Postdoctoral Fellowship in Transplantation Biology (HB)↑ Topic Categories Transplantation Immunology (TRAN)

Escaping the Scaling Relationships in Oxygen Reduction Catalysis: Implications for PEM Fuel Cells

ABSTRACT The growing interest in the hydrogen economy has sparked renewed interest in proton exchange membrane fuel cells (PEMFCs); however, the high cost and limited supply of noble metals like platinum remain a major obstacle to their widespread use. A primary bottleneck is the electrochemical oxygen reduction reaction (ORR) owing to its complex, multi‐intermediate pathway constrained by fundamental scaling relationships that limit the performance of even the most effective catalysts. Herein, we argue that interfacial heterogeneity, integrating symmetry‐breaking perturbations that span the subsurface lattice, the chemisorption layer, and the near‐interface solvation volume, provides the mechanistic basis for decoupling the adsorption energetics of ORR intermediates and overcoming these linear scaling relationships. This outlook critically examines recent strategies pursued to this end, including strain engineering, atomically dispersed sites, doping, interfacial field effects, and confinement, that modulate intermediate energetics to varying degrees, yet full thermodynamic decoupling remains elusive in practice. We, therefore, contend that the most promising path forward lies in engineering the interface holistically, integrating the lattice, chemisorption, and solvation contributions rather than tuning any one in isolation, while co‐optimizing for durability and transport dynamics in order to translate these mechanistic insights into practical PEMFCs.

Model-free predictive control strategy for three-level dual active bridge converter based on novel sliding mode observer

Exploring the antiviral potency of synthetic Cationic lipo-oligopeptides (CLOPs) against Herpes simplex virus type 1 2310162

Abstract Introduction Herpes simplex virus type 1 (HSV-1) is a common human pathogen that can infect sensory neurons and establish latency, where it remains dormant and shielded from the host’s immune defenses and the effects of antiviral drugs. To worsen this situation, prolonged use of current antiviral drugs may induce resistance. Hence, there is a need for alternative treatments to manage HSV-1 infections. In our study, we evaluated the antiviral potency of proprietary synthetic Cationic lipo-oligopeptides (CLOPs) against HSV-1. We hypothesized that CLOPs would inhibit the activity of the HSV-1 strain (F) either by disrupting its membrane envelope or by inducing endogenous mediators. Methods We utilized the TaqMan quantitative polymerase chain reaction and screened a range of non-toxic concentrations (12.5-100 µg/mL) of CLOPs for their antiviral effects on HSV-1. We also evaluated the impact of CLOPs on HSV-1-induced inflammatory responses using the Enzyme-Linked Immunosorbent Assay. Additionally, we evaluated the CLOPs’ effects on HSV-1 plaque-forming efficiency and the expression of glycoproteins D & B using the plaque reduction assay and immunofluorescence assay techniques, respectively. Results Our results showed that CLOPs (OB1111 and TP359) substantially reduced the viral load of HSV-1 in HaCaT cells. CLOPs exhibited anti-inflammatory effects by successfully reducing the production of IL-6 in HaCaT cells exposed to HSV-1. The plaque reduction assay demonstrated that CLOPs inhibited HSV-1 plaque-forming ability and altered the morphology of the plaques in Vero cells, with OB1111 and TP359 exhibiting the maximum inhibition observed. We also observed reduced expression of HSV-1 glycoproteins D & B, as well as cytopathic effects, in Vero cells following OB1111 treatment. Conclusion These findings support the potential of select CLOPs as promising therapeutic candidates for managing HSV-1 infection. Research is underway to investigate the mechanisms of the observed antiviral activity of CLOPs in HaCaT cells. Funding Source This research was supported by the National Science Foundation (NSF) HBCU-UP (HRD-1911660) grant and the Ph.D. Program in Microbiology at Alabama State University Topic Categories Vaccines and Immunotherapy (VAC)

Extracellular vesicles and cytokines released from Pseudomonas aeruginosa infected macrophages activate cardiac fibroblasts 2259917

Abstract Introduction Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative, opportunistic pathogen and a major cause of severe pneumonia. Infections caused by P. aeruginosa carry a high mortality rate, ranging from 32% to 42.8%. Notably, the risk of pneumonia is strongly associated with cardiovascular diseases (CVD), particularly heart failure. Methods In our previous studies, we demonstrated that P. aeruginosa lung infection leads to significant cardiac electrical disturbances and left ventricular (LV) dysfunction, despite minimal bacterial dissemination to the heart. To further investigate the underlying mechanisms, we identified that extracellular vesicles (EVs) released from both host cells, used LC-MS/MS to identify the EV content, and systemically administered outer membrane vesicles (OMVs) via tail vein injection to study the effects in vivo. Cardiac fibroblasts, key components of heart tissues, play a critical role in cardiac inflammation and damage upon activation. Using human cardiac fibroblasts (HCFs), we examined the effects of conditioned media (C-media), which contains both cytokines and EVs, on fibroblast activation. Results Our data revealed that exposure to C-media significantly elevated inflammatory markers CXCL10, IRF1, CXCL2, TNFα, and IL6 at both 6 and 24 hours, as detected by next-generation mRNA sequencing and confirmed by ELISA and qPCR. The EVs carried bacterial products, including toxins (as shown by LC-MS/MS), and tail vein injection of the OMVs resulted in pronounced LV dysfunction and increased mortality. Conclusion Mechanistically, these findings suggest that EVs upregulate the TNF signaling pathway, leading to increased cytokine production, leukocyte recruitment, and severe cardiac inflammation and dysfunction. In summary, our data indicate that P. aeruginosa infection induces the release of EVs that activate cardiac fibroblasts and impair cardiomyocyte contractility, contributing to cardiac dysfunction. Funding Source AI145262 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

B cells facilitate immune evasion of metastasizing cancer cells in tumor-draining lymph nodes 2260657

Abstract Introduction Lymphatic colonization correlates with worse prognosis in several cancers, as lymph nodes act as a reservoir of malignant cells, allowing their dissemination to distant organs. More recently, lymph node metastasis was found to promote systemic immunosuppression, further highlighting the need for a better understanding of the mechanisms supporting its formation. How tumor cells can enter the draining lymph node where anti-tumor immunity is primed and overcome immune recognition remains unknown. Methods To address this outstanding question, we developed a mouse model of trackable pancreatic cancer highly metastatic to the draining lymph node in a well-defined timely manner. We used this model to characterize tumor-draining lymph nodes at the time of tumor cell seeding compared to unperturbed lymph nodes by both single cell RNA-sequencing and spatial multiplexed immunofluorescence. Results Unexpectedly, this unbiased comprehensive analysis of pre-metastatic tumor-draining lymph nodes revealed that the most striking changes corresponded to B cells. We thus investigated the contribution of B cells to the metastatic cascade in genetic models of several B cell deficiencies and antibody-mediated depletion. We observed that metastatic pancreatic cancer cells fail to successfully colonize tumor-draining lymph nodes in the absence of B cells. Interestingly, this phenotype was not specific to our pancreatic cancer model as similar findings replicated in metastatic melanoma. We found that immunosurveillance by T cells is impaired in tumor-draining lymph nodes in the presence of B cells. Additionally, B cells support metastasis through antigen presentation on MHCII. Conclusion Collectively, these data revealed that B cells are required for metastasis formation as they facilitate immune evasion of disseminated cancer cells in the tumor-draining lymph node where anti-tumor T cell immunity is orchestrated. Funding Source Landry Cancer Biology Consortium (Harvard Medical School) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

mRNA Nanotherapy targeting Kv1.3 channels enables in vivo reprogramming of T cells for cancer therapy 2260072

Abstract Introduction Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a suboptimal immunotherapy response. T cell-mediated cytotoxicity is crucial for antitumor immunity; however, in HNSCC, tumor-infiltrating T cells are functionally impaired, partly due to downregulation of Kv1.3 potassium channels that support Ca2+-dependent effector functions. We developed a targeted mRNA nanotherapy to restore Kv1.3 expression in T cells. Methods Kv1.3 mRNA was synthesized via in vitro transcription and encapsulated within lipid nanoparticles functionalized with anti-CD5 antibodies (CD5-NPs) to ensure T cell specificity. Functional assays and flow cytometry were performed in vitro, and efficacy was tested in vivo using humanized NSG mice bearing Cal27 tumor xenografts. Results CD5-NPs selectively attached to and were internalized by T cells, delivering Kv1.3 mRNA into the cytosol. This resulted in upregulated Kv1.3 protein levels, enhanced Kv1.3 activity, and Ca²+ influx, leading to increased effector cytokine and cytotoxic molecule secretion in T cells. Local in vivo delivery of CD5-NPs reduced tumor growth and increased CD8+ T cell infiltration and granzyme B expression, demonstrating the restoration of antitumor activity. Conclusion Restoring Kv1.3 expression via CD5-NPs enhances Ca²+-dependent T cell effector functions, thereby overcoming the immune dysfunction in HNSCC. This study introduces a mechanistically informed mRNA nanotherapy platform with broad potential for T cell—based cancer immunotherapy. Funding Source NIH (grant 5R21 CA277341-02) to Dr. Laura Conforti Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

A New Therapeutic Strategy for Multiple Sclerosis: DRAK2 Inhibition Promotes Induction of Regulatory T Cells via Metabolic Reprogramming 2256515

Abstract Introduction Multiple Sclerosis (MS) is an autoimmune disease where autoreactive T cells cause damage in the central nervous system. Current therapies make patients vulnerable to infection and cancer during treatment, emphasizing the need for safer approaches. Previous study shows that DAP kinase-related apoptosis-inducing protein kinase 2 (DRAK2)-deficient mice are resistant to experimental autoimmune encephalomyelitis (EAE) without any defect in T cell recruitment to CNS upon infection. This suggests that DRAK2 inhibition suppresses autoreactive T cells in mice. Methods T cell activation, PBMC isolation from whole blood, flow cytometry, cell staining for confocal imaging, LC-MS for metabolomics, western blotting. Results Drak2-/- T cells induced higher Treg numbers upon activation, and mouse in vivo data suggests that EAE resistance in Drak2-/- mice is due to higher number of Tregs suppressing inflammation that can lead to autoimmunity. DRAK2 deficiency also resulted in higher oxidation of cellular lipids, which is connected to its higher autophagic flux. Our studies have shown that Drak2-/- T cells undergo faster beta oxidation, which affects TCR signaling that eventually favors AMPK signaling over mTOR signaling for T cell expansion. Newly synthesized DRAK2 inhibitors exhibited promising results with Treg induction accompanied by a decrease in cellular lipid contents in both mouse and human T cells upon T cell activation. Remarkably, T cells from MS patients also showed Treg induction followed by DRAK2 inhibitor treatment, with altered metabolic signatures. Conclusion Altogether, the results of DRAK2 studies demonstrate that DRAK2 regulates metabolic signals to determine T cell fate. Further studies on how DRAK2 regulates T cell metabolism may offer new insights to understand T cell differentiation and homeostasis. If DRAK2 inhibitors can induce a significant number of suppressive Tregs that can successfully target autoreactive T cells, it will offer a new avenue on a future MS therapeutics development. Funding Source NIND/NIH - T32 Training Program in Stem Cell Translational Medicine for Neurological Disorders Topic Categories Therapeutic Approaches to Autoimmunity (THER)