Browse Articles

Adapting the sequential infection mouse model to improve the translation of preclinical cancer research and immunotherapy 2257903

Abstract Introduction Specific Pathogen Free (SPF) mice have been critical in understanding cancer-immune interactions and immunotherapies. However, they often fail to recapitulate the complex immune environment of human tumors. A main reason is their immature immune system, which lacks microbiota adaptations, infections, and vaccinations. The use of “dirty” mice through co-housing has started to address this issue, but practical issues preclude their widespread use. To overcome this, we utilized a sequential infection (SI) model to establish a more complex immunity in SPF mice for cancer research. Methods Starting from 4 weeks old, SPF mice were sequentially exposed to MHV68, MCMV, influenza A (H1N1), and Citrobacter rodentium at 2-week intervals. SI mice were subjected to experiments at 18-22 weeks old. Results SI mice exhibited a more experienced immune system enriched in effector memory and tissue-resident memory T cells, changes in bone marrow progenitors, and elevated circulating antibody, compared to mock-infected (mi)SPF mice. When compared to miSPF mice, SI mice treated with an anti-CD28 super-agonist reacted similarly to adult humans, including limited Treg expansion and elevated serum inflammatory cytokines. In tumor models, SI mice exhibited accelerated growth of orthotopic breast cancer and melanoma models. Further, SI mice showed decreased responsiveness to anti-PD1 and anti-CTLA4 blockade, better recapitulating the response rates observed in humans. Compared to miSPF mice, tumors in SI mice were infiltrated with less tumor-specific CD8 T cells and increased memory “bystander” CD8 T cells, with additional alterations in the myeloid cells. Conclusion These observations suggest that prior pathogen encounter significantly influences the tumor and response to immunotherapy, which better resembles adult humans. We propose that SI mice can enhance the translatability of preclinical cancer studies and reveal novel cancer-mediated immune regulation. Funding Source NIH R21, 2025-2027 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

An HIV V1V2 mRNA-LNP vaccine induces strong, cross-clade functional antibody responses in mice and rhesus macaques 2307530

Abstract Introduction Antibodies against the V1V2 domain of the HIV envelope glycoprotein have been identified as a potential correlate of reduced infection risk in three large HIV vaccine efficacy trials. Although vaccine-elicited anti-V1V2 antibodies do not mediate potent virus-neutralizing activities, they mediate Fc-dependent effector functions. Strategies that enhance the magnitude and quality of these responses may improve HIV vaccine efficacy. Methods We examined a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccine encoding the V1V2 domain of CRF_01.AE244 on the 2J9C scaffold. Immunogenicity was assessed in three animal models: mice, rabbits, and rhesus macaques, following two or more immunizations. Antibody binding, cross-reactivity, and Fc-mediated functions were quantified using Luminex antigen-binding assays, virion- and cell-surface Env recognition assays, antibody-dependent cellular phagocytosis (ADCP), and Fc receptor binding. Results In mice two doses of the V1V2 mRNA-LNP vaccine elicited robust, cross-reactive serum IgG responses that recognized soluble Env and native Env on cells and virions. Importantly, murine IgG2a antibodies were generated that displayed Fc-mediated activities. However, in rabbits the elicited responses were directed mainly against the scaffold, with minimal V1V2-specific antibodies even after four vaccinations. In contrast, rhesus macaques developed high-level V1V2-specific antibodies responses after two vaccinations, comparable to those observed in mice. Conclusion The V1V2 mRNA-LNP vaccine is highly immunogenic in mice and rhesus macaques, eliciting cross-clade functional antibodies capable of recognizing native Env. The marked species-dependent differences, most notably the weak V1V2 targeting in rabbits, underscore the importance of selecting appropriate animal models for preclinical HIV vaccine evaluation and may provide insights relevant to vaccine testing against other pathogens. Funding Source VA Merit Review I01BX005616, NIH R01 AI148327 Topic Categories Vaccines and Immunotherapy (VAC)

Longitudinal single-cell lineage mapping reveals TCR motifs shape CD8+ T cell fate and expansion 2310264

Abstract Introduction When a naïve CD8+ T cell encounters a viral antigen from an acute infection, it undergoes cellular proliferation produce descendants with identical T cell receptors (TCRs), termed clones. These clones expand during an effector phase to clear the virus, then contract before forming a stable memory population. While cellular differentiation toward effector or memory fates occurs within clones, it remains unclear whether this process is stochastic or TCR-dependent. Due to the vast diversity of TCRs and the difficulty of tracing individual clones within a naturally arising repertoire, how clones dynamically progress throughout an acute infection remains unknown. Methods Using single-cell RNA/TCR sequencing in an LCMV Armstrong model of acute viral infection, we showed that clonal distributions in blood reflect those in spleen and lymph nodes. This enables non-lethal blood sampling for longitudinal tracking of clonal evolution throughout infection. We then applied the TCRdist algorithm, which groups complementarity-determining region 3 (CDR3) into TCR motifs based on amino acid sequence similarity. Results We traced hundreds of clones and found that memory T cell clonal distribution is surprisingly not congruent with their early clonal dominance. Instead, we find that early central memory precursor frequency predicts memory clonal dominance. These analyses revealed that TCR sequence not only determines antigen specificity, but also influences proliferation and fate decisions during the immune response. Conclusion Overall, our findings provide novel insights into the evolution of a full polyclonal repertoire during acute viral infection, and uncovers a previously under-appreciated role for the TCR itself in shaping clonal expansion and differentiation. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Modelling of early-life immunity shaped by contrasting environments 2256917

Abstract Introduction The neonatal period represents a critical window of immune development, during which environmental exposures influence health long term. Prior data capturing immune maturation in human newborns are limited to date. This multicohort study involves 1,000 infants across contrasting environmental contexts including rural African and urban European regions to map early-life immune trajectories and identify exposure-dependent variation. Methods Peripheral whole blood samples are collected longitudinally during the first years of life. Spectral flow cytometry and mass cytometry is used to study immune cell composition and functional maturation, while targeted plasma proteomics quantifies key soluble proteins. Metadata on key clinical and biometric features as well as health outcomes complement the analysis. Integrated computational modelling combines trajectory inference and modeling to reconstruct immune development and link molecular signatures to health endpoints. Results High-dimensional profiling reveals substantial location-dependent heterogeneity in early immune trajectories. Conserved maturation patterns are identified across cohorts, alongside exposure-dependent deviations. Early-life factors–such as perinatal antibiotic exposure–are associated with persistent shifts in cellular profiles. Multimodal models predict exposure status and early health outcomes, highlighting coordinated maturation of different immune modules shaped by the environment. Conclusion This large-scale longitudinal study provides a multidimensional view of human neonatal immune development. By integrating cellular, proteomic, and contextual data, it delineates conserved and modifiable features of immune ontogeny. The findings demonstrate how early exposures can reshape developmental trajectories, offering a foundation for predictive and preventive strategies that promote healthy immune maturation in early life. Funding Source German research council postdoc stipend Topic Categories Immune Mechanisms of Human Disease (HUM)

Contribution of T Cell Receptor Signaling Pathways to Ribosomal Biosynthesis 2260618

Abstract Introduction In the early phase of T cell activation the cells increase in size about five times before they divide about three times per day. These processes require the rapid upregulation of macromolecular biosynthesis. We have asked how TCR signaling pathways upregulate ribosomal biosynthesis. Methods We visualized ribosomal biosynthesis by cytometric quantification: The cellular RNA content was detected by dye stainings, rRNA precursors by FISH probes and translational activity by puromycilation. Results We have seen previously that T cells lacking mTORC1 express just half the amount of ribosomes and translational capacity. Cells lacking the mTORC1 targets S6 kinases 1 and 2 display a high sensitivity to Raf, MEK and Rsk inhibition, indicating a partial compensation by the MAP kinase pathway. Cells deleted of the Malt1 or Bcl10 components of the CBM complex or an inhibited Malt1 paracaspase activity display a reduction of rRNA synthesis of about one third, while cells with an inhibited Conclusion These data show that the MAP kinase and NF-kB signaling pathways contribute to the upregulation of ribosomal biosynthesis in activated T cells and thus to their increased biomass production. Funding Source German Research Council Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

HLA-E-restricted KIR+ CD8+ regulatory T cells are enriched during allograft rejection and suppress activated CD4+ T cells in vitro and ex vivo 2308005

Abstract Introduction CD8+T cells expressing NK cell receptors have regulatory function. We have shown that alloreactive CD4+T cells upregulate Qa-1 (human HLA-E) stress-peptide complex in mice, enabling elimination by Qa-1—restricted Ly49+CD8+Tregs and promoting graft survival. We hypothesized that analogous human CD8+Tregs exist, express killer immunoglobulin-like receptor (KIR), are enriched in graft rejection, can be expanded, and suppress HLA-E—upregulated alloimmune CD4+ T cells. Methods Cryopreserved PBMCs from kidney transplant recipients (KTRs) with no rejection (NR), antibody-mediated rejection (AMR), acute cell-mediated rejection (ACR), lupus nephritis, and healthy controls (HC) were phenotyped by flow cytometry. KIR+CD8+T cells from healthy apheresis leukoreduction samples were used to study in vitro expansion and CD4+T-cell suppression. In human kidney organoid (HKO) humanized murine (NSG-BLT) model, KIR+CD8+T cells were isolated to assess ex vivo expansion and suppression of alloreactive CD4+T cells. Results Inhibitory KIR (iKIR) are homologous to murine Ly49 in CD8+ Tregs. In KTRs, frequency of iKIR+ CD8+Tregs were significantly higher in AMR than HC. KIR2DL2/3 and KIR3DL1 subsets of iKIR+CD8+Tregs were most enriched. KIR2DL2/3+KIR3DL1+CD8+Tregs(CD8+Tregs) were enriched in alloimmunity compared to HC and demonstrated effector memory phenotype. IL-15-dependent in vitro expansion of FACS-sorted CD8+Tregs >50-fold in 3weeks while maintaining perforin and granzyme-B expression. In vitro expanded CD8+Tregs suppressed activated HLA-E—upregulated CD4+T cells significantly more in the presence of peptide. In a humanized murine transplant model (HKO-NSG-BLT), we showed that ex vivo—expanded CD8+Tregs eliminated alloreactive CD4+T cells in an antigen-dependent manner. Conclusion In alloimmunity, KIR+CD8+Tregs form a distinct subset of T cells enriched during rejection and suppress activated CD4+T cells in vivo and ex vivo, supporting their development as cell therapy against allograft rejection. Funding Source n/a Topic Categories Transplantation Immunology (TRAN)

Uncovering Synergistic Pathways for T Cell Stemness and Improved Adoptive Cell Therapy Outcomes 2257598

Abstract Introduction Adoptive T cell therapy (ACT) has revolutionized cancer therapy with remarkable success in liquid cancers, but efficacy in solid tumors remains limited due to poor T cell infiltration and persistence. Stem cell memory T cells (Tscm) are a potent subset with enhanced self-renewal, persistence, and anti-tumor responsiveness. Skewing T cells towards a Tscm phenotype prior to transfer represents a promising strategy to improve ACT outcomes. While pathways, such as PI3K-AKT-mTOR and Wnt/β-catenin, are implicated in Tscm differentiation, the interactions between these complex pathways in regulating stemness and anti-tumor function remain poorly understood, representing a potential therapeutic target to improve efficacy. Methods This study evaluated whether combining small molecules targeting distinct pathways could synergistically enhance Tscm differentiation and anti-tumor efficacy. We used small molecule PI3K, AKT, and GSK3 inhibitors, known regulators of Tscm differentiation, either as single agents or in combination, to condition CD8+ OT-1 T cells prior to ACT. Cells were characterized by flow for stemness markers (e.g., TCF1, FOXO1, Slamf6) and exhaustion markers (e.g., PD-1, Tim-3, Lag-3). Conditioned T cells were adoptively transferred into tumor-bearing mice, and tumor size and survival were monitored. Results In vitro, dual treatments enhanced expression of Tscm-associated markers (TCF1, FOXO1, Slamf6) and reduced expression of PD-1, Tim-3, and Lag-3. In vivo, the combination of a PI3K inhibitor and a GSK3 inhibitor significantly reduced tumor size. Conclusion These findings suggest that the PI3K-AKT-mTOR and Wnt/β-catenin pathways interact to regulate Tscm differentiation, enabling enhanced anti-tumor activity. These findings provide valuable insights into the mechanisms driving stemness and persistence, offering a promising framework for advancing ACT strategies in melanoma and other cancers, with strong potential to improve clinical outcomes. Funding Source NIH grants: AI155015 and CA257954 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Determining the impact of microbial dysbiosis on immune and barrier function in long COVID 2304111

Abstract Introduction Over 11 million people in the US are affected by long-COVID syndrome, or post-acute sequelae of SARS-CoV-2 infection (PASC), occurring in 10% of those infected. Despite advances, PASC studies remain largely observational due to the lack of a good animal model and the disease’s heterogeneity. It is therefore imperative to discover the underlying causes of PASC and develop effective treatments. Recent studies showed that SARS-CoV-2 Spike RNA can persist in the colon for>2 years after infection. Given the frequency of microbial dysbiosis and T cell hyperactivation in PASC, we reasoned that a gut viral reservoir may cause T cell dysfunction by altering the microbial metabolome. Methods To investigate viral persistence, we acquired colon biopsy patients from the University of Chicago colonoscopy clinic and conducted RNAscope targeting SARS-CoV-2 Orf1ab RNA. On the same patient’s stool, we did metabolomic and metagenomic analysis to investigate differences in metabolites from PASC patients. Subsequently, we probed how exposure to PASC metabolites affected T cell activation and metabolism by conducting flow cytometry experiments on T cells exposed to autologous or long COVID metabolites. Results Using colon biopsy samples from colonoscopy patients, we found evidence of SARS-CoV-2 Orf1ab RNA persistence in patients with persistent symptoms after COVID-19. We also found PASC patient stool was enriched in pro-inflammatory tryptophan metabolites. After TCR stimulation, T cells exposed to long COVID metabolites upregulated the inhibitory marker PD1 while increasing fatty acid synthesis/OXPHOS and reducing fatty acid oxidation, demonstrating that long COVID metabolites reduced the ability of T cells to activate metabolic programs leading to functional impairment. Conclusion Taken together, our data show that viral persistence may be a frequent occurrence linked to long COVID symptoms and may play a role in promoting a dysregulated inflammatory state by altering the metabolite microenvironment of the gut. Funding Source Center for Intestinal Inflammatory Diseases (C-IID) P30 DK042086 Topic Categories Mucosal and Regional Immunology (MUC)

Nanomaterials Drive In Vivo CAR Immune Cells Engineering

ABSTRACT Chimeric antigen receptor (CAR) immune cell therapy has emerged as a cornerstone of modern cell‐based medicine, demonstrating potent clinical efficacy against a range of malignant tumors and autoimmune diseases. Nevertheless, conventional ex vivo CAR immune cell manufacturing is hindered by complexity, high costs, and significant inter‐individual variability, which have limited its broad clinical application. These bottlenecks have prompted a paradigm shift toward in vivo CAR engineering, wherein nanocarriers directly deliver genetic material to circulating or tissue‐resident immune cells, substantially simplifying the production process. This review first outlines the evolution of CAR immune cell therapy and key limitations of ex vivo approaches and then examines major delivery platforms for in vivo approaches. Comparative analyses are presented across delivery efficiency, cellular tropism, and expression kinetics, with particular emphasis on delineating the mechanistic distinctions and application boundaries between transient and durable expression strategies. We further dissect the critical challenges, including receptor‐mediated non‐specific immune activation, nanocarrier‐associated immunogenicity constraining repeated administration, and delivery barriers imposed by the solid tumor microenvironment. Finally, the intrinsic trade‐off between therapeutic durability and biosafety across distinct delivery platforms is discussed, along with the necessity that their clinical translation hinges on systematic optimization of delivery precision, immune compatibility, and expression controllability.

Asialoglycoprotein receptor subunit Asgr1a loss results in attenuated cholesterol absorption in zebrafish fed a western diet

Parents’ awareness of respiratory syncytial virus (RSV) and its prevention: a cross-sectional study in Riyadh, Saudi Arabia

Abstract Respiratory Syncytial Virus (RSV) represents a major public health challenge as a primary cause of lower respiratory tract infections in infants and young children globally, with an estimated 33 million cases and 100,000 deaths annually in children under five. Despite recent advances in RSV immunoprophylaxis, parental understanding of RSV epidemiology, transmission dynamics, vulnerable populations, and available preventive strategies remains inadequately characterized in Middle Eastern populations. This study aimed to assess the prevalence and determinants of parental awareness regarding RSV and its prevention among parents in Riyadh, Saudi Arabia. We conducted a community-based cross-sectional survey among parents residing in Riyadh, Saudi Arabia, between February and March 2026. Using convenience sampling through online platforms, we recruited parents with at least one child. A structured, self-administered Arabic questionnaire assessed demographic characteristics, prior RSV awareness, knowledge of transmission routes and clinical manifestations, recognition of high-risk groups, and awareness of preventive immunization. Data were analyzed using descriptive statistics and chi-square tests to examine associations between demographic variables and RSV awareness. Among 214 eligible parents, the majority were female and held bachelor’s degrees. Only 35.0% (95% CI: 28.7–41.8%) had previously heard of RSV. Knowledge deficits were substantial across multiple domains, with considerable proportions unable to identify transmission mechanisms, symptoms, or high-risk populations. The majority were unaware that preventive injections exist. Among those aware of RSV prevention, social media was the predominant information source, while healthcare professionals accounted for a small minority. Educational level showed a positive but non-significant association with RSV awareness. This study reveals critically low prevalence of RSV awareness among parents in Riyadh, with substantial knowledge gaps across all domains of RSV epidemiology and prevention. These findings underscore an urgent public health need for multi-level interventions targeting both community education and healthcare system strengthening. Integrating RSV education into maternal-child health services and developing culturally appropriate health communication strategies are essential to reduce the burden of RSV-related morbidity and mortality in Saudi Arabia.

Skin Gamma Delta T Cell Gene Expression Highlights Stress Response in Alopecia Areata 2258104

Abstract Introduction Alopecia areata is an autoimmune skin disease characterized by nonscarring, patchy hair loss, and affects roughly 2% of the global population. The onset of alopecia areata involves both genetic and environmental factors, leading to the loss of immune privilege. γδ T cells help regulate skin homeostasis and are elevated during alopecia areata; however, their role in disease progression remains unclear. Methods By reanalyzing publicly available single-cell RNA sequencing data from mouse and human skin, we identified genes upregulated in γδ T cells during alopecia areata. In humans, V•1 T cells respond to stress in alopecia areata by upregulating heat shock response genes (HSP70, HSP90AA1, HSPA1A, HSPA1B, DNAJB1). We validated HSP70 upregulation by skin γδ T cells in the C3H/HeJ mouse model of alopecia areata using immunofluorescent microscopy. Results Further, murine skin gd T cells also up-regulate chaperones, including CD74 and receptors that regulate T cell activation, such as Ly6A/E, in alopecia areata. We quantified CD74 and Ly6A/E expression in the skin of mice with alopecia areata and found upregulation at the hair follicle, upper dermis, and epidermis. To identify which cells are regulating gd T cell stress and activation, we further examined single-cell RNA sequencing data and have identified the genes expressed by helper CD4+ T cells to regulate gd T cell stress and activation responses during alopecia areata. Conclusion Our findings show that T cells are undergoing a prominent stress response in response to alopecia areata, which leads to their activation, making them interesting targets for therapeutics. Funding Source National Institute of Health Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Reliable Antibodies for Immunology Research: TREM1 as a Case Study of Abcam’s Enhanced Validation Framework 2307284

Abstract Introduction Reliable antibody performance is essential for translational research, particularly when investigating disease-associated targets such as, Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), a key modulator of innate immune responses implicated in inflammatory conditions. Conventional antibody validation methods often fall short in addressing the complexity of human tissue expression and disease heterogeneity. Methods To overcome these limitations, an enhanced validation framework was developed that integrates disease-relevant tissue profiling. This approach combines multiple validation steps, including specificity testing and generating detailed IHC expression profiles in selected formalin- fixed paraffin-embedded (FFPE) human tissue microarrays (TMA) using automated immunohistochemistry (IHC) platforms. As a case study, a recombinant rabbit monoclonal antibody (RabMab®) targeting TREM1, clone EPR22060-229 (ab225861), was validated across multiple IHC automated platforms. Results In both normal and cancer tissues, the antibody showed positive staining in interstitial regions and infiltrating lymphocytes. Its high specificity was further validated through co-localization assays with CD14, a well-established myeloid marker, across COVID-19, glioblastoma, and inflammatory bowel disease (IBD) samples, confirming accurate detection of TREM1-expressing cells. Conclusion This study highlights the value of enhanced antibody validation in de-risking reagent selection and ensuring robust, reproducible results in translational research. The TREM1 case exemplifies how rigorous Abcam’s antibody validation can accelerate discovery and translational applications. Funding Source n/a Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Diverse epitopes in anti-PF4/heparin antibodies predicted by in silico docking 2310336

Abstract Introduction Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by antibodies that recognize platelet factor 4 (PF4) when it forms complexes with heparin (PF4/H). These antibodies engage the Fc receptor on platelets, resulting in aberrant platelet activation and thrombus formation. The epitope on PF4 recognized by HIT antibodies has been partially characterized using polyclonal IgG as well as KKO, a mouse-derived “HIT-like” antibody. However, HIT is a polyspecific disorder, and the epitopes involved may not be fully elucidated. Methods Anti-PF4/H antibodies were cloned from HIT patients previously. The structures of these antibodies were modeled from amino acid sequence using AlphaFold3. These models were then docked to PF4 using the ClusPro webserver, and the top clusters were loaded into PyMol and PDBePISA for further analysis of interacting residues. Fab versions of cloned antibodies were used to assess competitive binding, using inhibition of platelet activation measured by flow cytometry as a readout. Results Residues on PF4 within 4Å of the antibody were recorded, and the percentage overlap of these residues with those involved in KKO binding was calculated. Percentage overlap of residues ranged from 0% to 48%. However, KKO fab was able to completely inhibit P-Selectin expression for all tested clones, including the clone with 0% overlapping residues. Conclusion In silico docking methods suggest that anti-PF4/H antibodies in HIT patients bind to diverse epitopes on PF4. Many of the tested clones bound to overlapping regions on the PF4 surface, but one clone was predicted to bind to a completely distinct epitope from that bound by KKO. However, validation of this finding using an in vitro platelet activation assay found that KKO still effectively inhibited the ability of all antibody clones to activate platelets, suggesting that despite the diversity in predicted epitopes, the tested HIT clones share a method of platelet activation susceptible to inhibition by KKO fab. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

Metformin modulates macrophage response to conditioned medium collected from triple-negative breast cancer cells 2257225

Abstract Introduction Triple-negative breast cancer (TNBC), a subtype of breast cancer (BrCa) lacking both hormone and HER2 receptors, accounts for 15-20% of all BrCa cases, has a high recurrence rate and is likely to become resistant to traditional chemotherapies. Metformin, a widely used T2D drug, has shown promising anti-cancer effects against some cancers. Its primary mode of action involves the activation of AMP-activated protein kinase, leading to reduced cancer cell proliferation and growth, and alterations in the tumor microenvironment (TME). Interestingly, while evidence suggests a protective effect against ER-positive BrCa, there is less evidence of a benefit for ER-negative or TNBC. We speculate that this could be due to differences in the TME. Macrophages play a critical role in the TME of BrCa, where they can influence disease progression. Methods In these studies, we investigated the capability of metformin to block the effect of tumor factors from a mouse TNBC cell line (4T1) on mouse macrophages (RAW 264.7). Macrophages were treated with tumor supernatants in the presence or absence of different concentrations of metformin and then, production of nitrites and expression of costimulatory molecules was evaluated. In addition, direct effect of metformin on macrophage inflammatory activation and migration was evaluated. Results We determined that tumor conditioned medium induces the generation of nitric oxide by macrophages and an increase in CD80 expression. Metformin treatment was able to counteract this effect. Further, metformin had a dose dependent effect on macrophage migration and production of nitric oxide in response to LPS. Conclusion The response of BC patients to metformin has been contradictory, with no benefit observed for TNBC. We consider that the differences could be associated with the nature of the macrophage population in different BrCa types, with metformin decreasing macrophage responses independently of their M1 or M2-like phenotype. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Unique immunization strategies elicited antibodies in mice binding to the specific GPCR target on live cell surface across a panel of Class A GPCRs 2260717

Abstract Introduction G protein-coupled receptors (GPCRs) regulate nearly every aspect of human physiology and diseases, and form one of the largest drug target families. GPCRs share a conserved structure with 7 transmembrane α-helices. Class A GPCRs, Rhodopsin-like receptors, are the biggest and most distinct class of the GPCR subfamily in humans. Generation of antibodies binding to a GPCR protein on the cell surface can be a very useful tool to detect the target expression and drug discovery potential. The structure complexity and dynamic conformations make it very challenging to develop antibodies binding GPCRs on the cell surfaces. To advance the GPCR antibody discovery, we have developed unique fit-for-target immunization approaches to generate GPCR-specific antibodies in mice. Methods GPCR protein sequence and structure for each target was analyzed using multiple parameters. Based on each target’s unique sequence and structure features, we design fit-for-target immunogens, formulations and immunization regimens and conduct immunizations in mice. The antibody responses immune mouse serum samples were evaluated against each specific GPCR target antigen by ELISA and target expressing cells by flow cytometry.. Results For this study, we selected a panel of 10 Class A GPCR targets, including GPR19, GPR56, LGR4, NK3R, NTR1, GIPR, LPHN3, CRFR2, CALCRL and P2RY1. The immunization with each target generated target-specific and high titers of antibody responses in mice for all 10 targets. The immune mouse serum samples could bind to the relevant specific target antigen by ELISA and target-expressing cells on live cell surfaces by flow cytometry. Conclusion The innovative fit-for-target immunization strategies can be advanced platforms to generate antibodies against GPCRs for reagent antibody development and drug discovery. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

Endogenous neoantigen specific antibodies mediate anti-tumor responses and determine vaccine efficacy 2308188

Abstract Introduction The role of B cells and antibodies in cancer immunity remains unresolved, with studies reporting both protective and deleterious effects across tumor types. A mechanistic explanation for this variability has been lacking. We hypothesized that neoantigen membrane localization governs whether endogenous antibodies drive tumor control. Methods We analyzed The Cancer Genome Atlas (TCGA) data to assess the prognostic significance of intratumoral IgG. Syngeneic tumors were engineered to express membrane-localized or cytoplasmic neoantigens, then evaluated for growth, antibody class switching, and immune composition. CXCL13 and IL-21-expressing cells were used to enhance IgG responses. Adenoviral vaccines encoding wild-type Trop2 or the T256R variant were employed to test how antigen localization influences vaccines. Results TCGA analyses showed that the prognostic significance of IgG varies markedly across cancer types. In vivo, we found that membrane-localized nantigens, unlike cytoplasmic antigens, stimulated robust class-switched IgG production, enhanced myeloid cell activation, and limited tumor growth, independent of CD8 T cells. Alteration of antigen location to the membrane was sufficient to convert colorectal cancer models (where IgG predicts poor outcomes) into settings in which antibodies contributed to tumor control. These effects required CD4 T cell help and intact Fc receptor signaling. Consistent with this effect, expression of CXCL13 and IL-21 amplified antibody-mediated tumor control, with the combination providing the greatest benefit. Finally, the Trop2 T256R neoantigen, which disrupts membrane trafficking and reduces antibody accessibility, led to markedly diminished vaccine-induced tumor control, despite generating comparable T cell responses to wild-type Trop2. Conclusion This work defines antigen membrane localization as a key determinant of productive humoral anti-tumor immunity, providing a mechanistic basis for incorporating humoral immunity into cancer immunotherapy. Funding Source This work was supported by the National Institutes of Health (NIH) (R01CA238217-01A1/02S1 to ZCH), the Department of Defense (DOD) (W81XWH-20-1-0346 to ZCH and W81XWH-21-2-0031 to HKL/ZCH), and institutional support from the Office of Physician Scientist Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Systemic Proteomic Remodeling Following Nephrectomy in Renal Cell Carcinoma 2257687

Abstract Introduction Renal cell carcinoma (RCC) exerts widespread systemic effects that extend beyond the tumor microenvironment. However, how the removal of the primary tumor reshapes the circulating proteome remains largely undefined. Characterizing these changes may reveal biomarkers of immune recovery and mechanisms by which tumors disrupt systemic immune function. Methods We profiled plasma from 16 RCC patients collected before and after nephrectomy using the SomaLogic SOMAscan 11k assay v5.0, which quantifies 11,000 proteins. Data were z-score normalized and scaled in R. Differential abundance was analyzed using paired t-tests with false discovery rate (FDR) correction. Pathway analysis was performed using the GSEA package. Protein-protein correlation networks were constructed using the igraph package, and global topological parameters (node degree, edge strength, and network density) were compared before and after nephrectomy. Results Nephrectomy induced broad proteomic remodeling consistent with systemic homeostatic restoration. Differential abundance and pathway enrichment analysis identified 1,737 significantly altered proteins (FDR < 0.05), with decreases in pathways and mediators of immune suppression following tumor removal. Network analysis demonstrated a reduction in extremes in protein-protein correlations, node degree, and interaction strength, indicating a loss of tumor-driven network polarization. Together, these results suggest that the presence of RCC disrupts protein co-regulation, which normalizes following tumor removal. Conclusion Our findings demonstrate that RCC drives coordinated systemic proteomic dysregulation that is partially reversed after nephrectomy. The observed reduction in network extremes reflects a global rebalancing of circulating protein interactions, providing insight into how tumors perturb systemic physiology. Plasma proteomics and network metrics may offer sensitive indicators of systemic immune recovery and potential biomarkers of post-surgical immune restoration. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

15-Lipoxygenase expressing neutrophils promote Acute Lung Injury resolution 2306687

Abstract Introduction The acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure among critical ill patients with limited available therapeutic options. Dysregulated unresolving inflammation and extensive lung injury are main features of ARDS. There is a urgent need for a better understanding of the biochemical and immunological events in ARDS that inform novel treatment strategies. Methods We used a mouse model of self-limited acid-induced ALI by selectively instilling hydrochloric acid to the left lung of adult mice. The time-course of lung disease was evaluated by consolidation by micro-computed tomography (mCT) and total protein in bronchoalveolar lavage (BAL), while inflammation was assessed by histology and flow cytometry. Results Neutrophils, that were rapidly recruited into the lungs, decreased over time during resolution while macrophage numbers, significantly reduced during initial stages, increased. Immunophenotyping of leukocytes recruited to the lungs during the resolution of injury uncovered a neutrophils’ subset that was expressing sialic acid—binding, Ig-like lectin F (SiglecF) and Alox15 (15-lipoxygenase). These cells were distinct from eosinophils and transcriptionally, phenotypically, and functionally different from the SiglecF-negative neutrophils. The roles for SiglecF-positive neutrophils were further assessed by in vitro functional studies and adoptive transfer experiments. Depletion of neutrophils impaired epithelial repair, while the adoptive transfer of SiglecF-positive neutrophils accelerated epithelial cell restitution. Among the mechanisms associated with the SiglecF-positive neutrophils in lung repair was the production of Alox15-derived specialized pro-resolving mediators and CSF1. In patients with respiratory failure, ALOX15+ neutrophils were present in the BAL and their frequency correlated with improved oxygenation. Conclusion In summary, we identified protective roles for a distinct subset of tissue neutrophils during the resolution of lung injury. Funding Source Pew Charitable Trusts, grant Latin American Fellowship 00034105 ; and American Lung Association, Catalyst Award CA- 1420662. Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Phosphonate‐Substituted Chitosan With Complete Non‐Conjugation Facilitates Efficient Hole Injection in Perovskite Light‐Emitting Diodes

ABSTRACT Perovskite light‐emitting diodes (PeLEDs) promise efficient, pure‐color next‐generation displays but suffer from charge injection imbalance due to energy barriers at the indium tin oxide (ITO)/perovskite interface. To address this, we introduce non‐conjugated phosphorylated chitosan (NCPC) as an efficient hole injection layer (HIL) for the first time. NCPC enhances interfacial interactions with ITO and the hole‐transport layer (HTL), effectively reducing their energy barrier and achieving favorable energy level alignment. This significantly lowers both charge injection barriers and interfacial nonradiative recombination. Incorporating NCPC markedly improves device performance. Red, green, and blue PeLEDs show maximum external quantum efficiency (EQE max ) enhancements of 25.35% (670 nm), 25.45% (512 nm), and 14.22% (489 nm), respectively, compared to control devices using PEDOT:PSS HIL. Notably, the red PeLED EQE max at 670 nm ranks among the top two reported values. The facilitated hole injection also reduces turn‐on voltages. This work advances the design of efficient HILs for PeLEDs and demonstrates the potential of environmentally friendly natural materials for high‐performance displays.