Cell Reports Medicine

Preterm birth (PTB) is a leading cause of perinatal and infant mortality worldwide. PTB can be induced by systemic maternal or intra-uterine infection or by sterile intra-amniotic inflammation driven by alarmins such as interleukin-1 (IL-1). We reported earlier that Broad-Spectrum Chemokine Inhibitor (BSCI) prevented PTB in murine and non-human primate models of infection-mediated preterm labor. Here, we investigated whether BSCI can prevent PTB in pregnant C57BL/6 mice following ultrasound-guided intra-amniotic injection of IL-1 (400 ng per sac) on gestational day (GD)16.5. Half the mice received BSCI (10 mg/kg, intravenous daily) beginning GD15.5 and through to term. The impact of IL-1 alone or IL-1 plus BSCI was assessed on (i) injection-to-delivery interval, fetal survival, placental and neonatal weight; (ii) cytokine and chemokine levels in maternal plasma and amniotic fluid (by Luminex assay) and inflammatory gene expression in maternal and fetal tissues (by Real-Time RT-PCR); (iii) global multi-omic profiling of myometrial tissues, including gene expression (RNA sequencing), chromatin accessibility and regulatory landscape (ATAC-seq), and protein abundance profiling by tandem mass spectrometry (TMT-MS proteomics); (iv) uterine leukocyte infiltration (by immunofluorescence with automated quantification). Pre-treatment with BSCI i) prevented IL-1-induced PTB; (ii) significantly attenuated cytokine and chemokine signals in maternal plasma, myometrium, decidua, and placenta, and amniotic fluid; (iii) suppressed myometrial contraction-associated genes, including Nfkb1, Ptgs2, Akr1c18, and Gja1; (iv) prevented broad IL-1-induced changes in myometrial gene expression, chromatin accessibility, and proteomic extracellular matrix (ECM) structural remodeling; (v) reduced uterine F4/80+ macrophage counts and changed M1-M2 balance. BSCI-treated dams that delivered at term had live pups with normal placental and fetal weight. Taken together, BSCI reduced the incidence of IL-1-mediated PTB and maintained uterine quiescence by suppressing uterine inflammation and global changes in labor gene expression and chromatin accessibility. BSCI represents a promising therapeutic approach for PTB prevention in high-risk pregnant women.

Immune checkpoint inhibitor-induced myotoxicity (ICI-M) comprises myocarditis, myositis, and myasthenia gravis-like syndrome, demanding rapid recognition and therapy. Using immunophenotyping and transcriptomics analysis from blood and muscle, we identified distinct CD38hi and KIR+ CD8 T cells in ICI-M. Abatacept rescued patients and altered the composition and clonality of these cells. Dynamics of CD38hi and KIR+ CD8 T cells effectively supported therapeutic monitoring, offering personalized treatment in life-threatening irAEs.

mRNA-lipid nanoparticle (LNP) vaccines are detectable in human blood after vaccination, but platform-specific differences in systemic persistence and transcript integrity remain poorly defined. We analyzed serial blood samples from 73 participants receiving Moderna mRNA-1273 (three formulations), Pfizer/BioNTech BNT162b2, or an investigational receptor-binding domain (RBD) mRNA vaccine (three different doses). Using droplet digital polymerase chain reaction (ddPCR) assays, we quantified total and long-range linked ("intact") vaccine mRNA, and we measured vaccine-specific ionizable lipids by liquid chromatography-mass spectrometry (LC-MS). Across platforms, mRNA decay was fastest for mRNA-1273, intermediate for BNT162b2, and slowest for the RBD vaccine, with ionizable lipid decay following the same rank order. Notably, intact spike mRNA declined two-fold faster after mRNA-1273 than BNT162b2 vaccination. Kinetics modelling revealed platform-dependent coupling of mRNA and lipid kinetics: intact mRNA tracked closely with SM-102 for mRNA-1273, whereas ALC-0315 persisted longer than intact mRNA for BNT162b2. A ten-fragment linkage ddPCR panel spanning the spike transcript showed lower linkage toward 3'-proximal regions that mirrored the administered mRNA-1273 formulation. Together, these data establish a quantitative framework for benchmarking mRNA-LNP platform kinetics and transcript integrity in humans.

We studied the effect of stimulating innate immune function in tumor-associated myeloid cells (TAMs) in medulloblastoma (MB) and diffuse midline glioma (DMG), using a polyoxazoline nanoparticle formulation of the TLR7/8 agonist resiquimod (ResiPOx). Children with MB and DMG need novel therapeutic strategies to improve outcomes and reduce recurrence. We investigated the effect of systemically administered ResiPOx on TAMs in MB and DMG using endogenous MB and DMG models in immune-competent mice and identified multiple mechanisms of anti-tumor effect. We packaged resiquimod into polyoxazoline micelles to generate ResiPOx. We studied ResiPOx efficacy as a single agent or paired with radiation therapy (RT). We determined ResiPOx pharmacokinetics (PK) using tritium-labeled resiquimod and mass spectroscopy imaging (MSI). We determined ResiPOx pharmacodynamics (PD) using flow cytometry immunohistochemistry, bulk and single-cell RNA-seq and immunoblotting. We then studied ResiPOx safety and PD in a non-human primate model using rhesus macaques. ResiPOx formulation improved the blood-brain barrier penetration and anti-tumor efficacy of resiquimod. ResiPOx treatment extended progression-free survival (PFS) in mice with MB and DMG. In both tumor types, ResiPOx expanded TAM populations and reprogrammed TAMs toward anti-tumoral states, characterized by activation of IFN{beta} and extrinsic apoptosis pathway signaling, antigen presentation, and T cell activation signatures. In rhesus macaques, systemic ResiPOx administration was well tolerated and induced brain transcriptional responses that resembled ResiPOx responses in DMG and MB mouse models, indicating common effects across species from mice to non-human primates, and highlighting potential for similar effects in patients. ResiPOx is a brain-penetrant immunomodulatory therapeutic that reshapes the immune-privileged brain tumor microenvironment. Systemic administration activates myeloid-driven anti-tumoral immunity mediated by microglial and macrophage TAMs, and improves survival in preclinical models of DMG and MB.

Despite recent advances in immunotherapy for advanced malignancies, Pancreatic ductal adenocarcinoma (PDAC) remains largely refractory to current immunotherapy due to dense fibrosis, limited antigen presentation, and myeloid-driven immune suppression. Here we report the tumor-targeting, immune remodeling, and safety profiles of the attenuated Salmonella enterica serovar Typhimurium strain CRC2631, and of iSTORM, a next-generation derivative engineered for tumor-localized CMTM6 silencing. CRC2631 preferentially colonizes orthotopic and genetically engineered PDAC tumors, with enrichment in primary lesions and metastases. Tumor-localized CRC2631 induces chemokine and adhesion programs consistent with leukocyte recruitment, increases intratumoral activated T-cell fractions, and triggers transcriptional signatures aligned with innate sensing, interferon signaling, antigen-processing and presentation, and apoptosis programs. iSTORM extends this platform by delivering CMTM6-targeting shRNA to modulate a PD-L1-stabilizing, myeloid-associated immune-evasion programs within tumor-colonized tissue. Compared with CRC2631, iSTORM increases intratumoral CD8+ T cells, shifts T-cell state toward activation with reduced exhaustion-prone features, strengthens antigen-presentation programs, and achieves deeper tumor control. A lyophilized formulation preserves immune remodeling while improving deployability. Mechanistically, glycan arrays and functional studies support mannose-rich glycan-guided tumor engagement. iSTORM toxicity studies, including systemic cytokine, hematologic, blood chemistry, and lethality demonstrate a favorable safety profile. Collectively, these findings establish iSTORM as a safe, programmable, CMTM6-silencing microbial immunotherapy platform that selectively targets and penetrate PDAC tumors to unleash anti-tumor immune activities. What is already known on this topicPDAC is highly resistant to immune checkpoint blockade because dense stroma and myeloid-dominated suppression prevent effective T-cell infiltration; attenuated Salmonella strains can selectively colonize tumors but first-generation agents showed limited efficacy and safety concerns. What this study addsThis study defines CRC2631/iSTORM as a tumor-selective microbial immunotherapy that exploits surface-exposed, mannose-rich N-glycans to colonize PDAC, delivers CMTM6 silencing, and restores CD8+ T-cell activation and tumor control in models resistant to PD-1 blockade immunotherapy. How this study might affect research, practice or policyThese findings provide a mechanistic blueprint for glycan-guided, CMTM6-targeted bacterial "living drugs," support rational combination strategies for deepening therapeutic effect, and establish a lyophilized, biocontained platform that could be developed into scalable microbial immunotherapies for PDAC and other immunologically cold solid tumors.

PurposeBispecific T cell engagers (BiTEs) have recently been approved as a locoregional immunotherapy for malignant ascites. Although ascites is recognised as a lipid-rich, immunosuppressive environment, the mechanisms by which ascites, particularly its lipid components, suppress antitumour immunity remain poorly understood. Here, we investigated the impact of ascites-associated lipids on T cell immunosuppression and assessed whether lipid modulation could enhance the efficacy of BiTE therapy. Experimental DesignTranscriptomic profiling was performed on T cells treated with acellular ascites fluid to identify gene expression signatures associated with ascites exposure. Functional assays were conducted to evaluate the effects of ascites-associated lipids on T cell activation and cytotoxicity. In parallel, T cells were cocultured with ovarian cancer cells and EpCAM-targeting BiTEs in the presence or absence of a lipid-removal agent to assess how lipid depletion affected BiTE efficacy. ResultsT cells exposed to acellular ascites fluid exhibited an enriched transcriptomic signature associated with cholesterol efflux and incomplete fatty acid oxidation, which are metabolic features often found in exhausted T cells. These alterations converged on a metabolically imbalanced state linked to impaired plasma membrane signalling. Lipid removal from ascites selectively rescued CD137 expression but not CD25, and restored BiTE-mediated cytotoxicity, suggesting a differential impact of lipid metabolism on TCR complex-dependent versus cytokine-driven activation pathways. ConclusionsThese findings identified lipid as a driver for T cell dysfunction in ovarian cancer ascites. Removal of ascites lipids restored T cell activation and augmented BiTE-mediated cytotoxicity, supporting a combination approach to potentiate BiTE therapy in malignant ascites. Translational RelevanceMalignant ascites represents a lipid-rich, immunosuppressive tumour microenvironment that is increasingly targeted by emerging T cell-based therapies. Although EpCAM-targeting bispecific T cell engagers (BiTEs) have recently been approved for malignant ascites and multiple similar BiTEs are in clinical development, the mechanisms by which ascites impairs T cell function and potentially limits therapeutic efficacy remain poorly understood. Using patient-derived ascites throughout, this study demonstrated that lipid metabolic reprogramming, rather than immune checkpoint upregulation, was a driver of T cell dysfunction. Importantly, we demonstrated that lipid removal from ascites rescued T cell function and restored BiTE efficacy, identifying a targetable metabolic barrier to immunotherapy. While EpCAM was used as a proof-of-concept target, we anticipate the metabolic insights and therapeutic strategies identified here will be equally applicable to other BiTE and CAR-T platforms, supporting a new combination approach for the treatment of malignant ascites.

Head and neck squamous cell carcinoma (HNSCC) remains associated with substantial morbidity and a 5-year overall survival rate of approximately 60%, reflecting persistent radio- and chemo-resistance and the lack of effective precision medicine strategies. Patient-Derived Tumor Organoids (PDTO) constitute promising functional models that may predict individual treatment response. In this study, we generated PDTO from surgically resected HNSCC of the oral cavity, oropharynx, larynx, and hypopharynx. A total of 20 long-term PDTO lines were established, maintaining growth over seven passages and successfully cryopreserved, capturing the molecular and clinical diversity of the patient cohort. These PDTO faithfully recapitulated histological features, major tumor marker expression, and the genomic and transcriptomic landscapes of their tumors of origin, with stability over time. Functional assays revealed heterogeneous responses to cisplatin and X-rays. Importantly, in vitro sensitivity of PDTO was associated with clinical outcome of patients at 24 months. Cisplatin response of PDTO predicted prognosis with 66.7% sensitivity and 100% specificity, while X-ray response showed 91.7% sensitivity and 75% specificity. Notably, all patients whose PDTO were classified as resistant to both cisplatin and X-rays experienced relapse and/or death within 24 months. Collectively, the successful long-term expansion and cryopreservation of HNSCC PDTO establish a stable and scalable preclinical resource that captures the molecular and clinical heterogeneity of the disease. This biobank provides a valuable platform for mechanistic studies and for the evaluation of innovative therapeutic strategies. This cohort represents one of the largest clinically annotated HNSCC PDTO collections to date, demonstrating a robust association between PDTO response to cisplatin and X-rays and patient prognosis. These findings support the predictive potential of PDTO-based functional assays and argue for their integration into standardized, rapid, and miniaturized precision oncology workflows for HNSCC.

GLP-1 receptor agonists have reshaped obesity therapeutics, but their impact on neuropsychiatric outcomes remains poorly characterized. From 29 million patients in a large federated data platform across the USA, including 489,785 semaglutide treated patients, we conducted an observational study integrating longitudinal neuropsychiatric outcomes. From this population, we assembled a cohort of 63,215 patients with baseline neuropsychiatric conditions before treatment initiation and evaluated 24 incident neuropsychiatric outcomes. In propensity-matched comparator analyses, during the 2 year time-period from treatment initiation, semaglutide was associated with broadly lower neuropsychiatric event risk than metformin, SGLT2 inhibitors, and DPP-4 inhibitors. Within the semaglutide-treated cohort, higher attained dose during the first two years after the first prescription ("pre-landmark period") was associated with significantly lower incidence during the following two years ("post-landmark period") of diagnostic codes associated with substance-related disorders (P<0.001), mood disorders (P<0.001), anxiety- and stress-related disorders (P<0.001), CNS atrophies (P<0.001), neuromuscular disorders (P=0.013), eating/sleep/behavioral disorders (P=0.022), and personality/impulse-control disorders (P=0.028). Consistent with previous clinical trials, the post-landmark incidence of dementia or CNS degenerative diseases was similar between the high-dose and low-dose semaglutide cohorts (P=0.15). For most neuropsychiatric diagnoses, post-landmark incidence was strongly associated with the maximum attained semaglutide dose during the pre-landmark period, but incident cognitive symptoms and speech/language symptoms were more closely linked to the pre-landmark weight-loss magnitude (p<0.001 and p<0.003, respectively). Bulk and single-cell transcriptomic analyses demonstrated GLP1R expression in CNS tissues (hypothalamus, caudate, putamen, nucleus accumbens, cerebellum) and peripheral nerves. Age-associated heterogeneity in GLP1R expression was evident in several of these compartments including the caudate nucleus, suggesting dynamic changes in the availability of the neurobiological substrate for semaglutide response. Together, these data support a model in which semaglutide confers a sustained, dose-dependent, weight loss-independent benefit across multiple neuropsychiatric conditions via direct CNS target engagement. This observational study motivates prospective clinical studies and mechanistic analyses to clarify the impact of GLP-1 receptor agonists on human neuropsychiatric pathways and disease processes.

The intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

For mRNA-based cancer gene therapy, we engineered a membrane-bound fusion protein combining interferon-{gamma} (IFN{gamma}) with the Fas intracellular domain (FasICD) to couple local IFN{gamma} signaling with Fas-driven apoptotic tumor cell death. IFN{gamma}-FasICD was robustly expressed on the plasma membrane after mRNA transfection. In murine cancer cell lines, IFN{gamma}-FasICD mRNA reduced viability within 24 h, resulting in [~]50% cell death in MC38 cells and [~]75% in B16OVA cells, exceeding the cytotoxicity of the FasICD-deleted control (IFN{gamma}-Fas{Delta}). Mechanistically, IFN{gamma}-FasICD induced predominantly apoptotic rather than necrotic cell death. IFN{gamma}-FasICD also activated IFN{gamma} receptor signaling in both cancer and the immune cells, inducing IFN{gamma}-responsive genes in IFN{gamma}R-high B16OVA cells and triggering STAT1 phosphorylation in co-cultured splenocytes. For in vivo delivery, IFN{gamma}-FasICD mRNA was formulated in lipid nanoparticles (LNPs), enabling strong intratumoral expression that peaked at [~]3 h and persisted for more than 48 h. Repeated intratumoral injections of LNP-formulated IFN{gamma}-FasICD mRNA suppressed the growth of established B16OVA and MC38 tumors and improved survival, with [~]40% and [~]20% of mice surviving beyond 30 days, respectively. IFN{gamma}-FasICD treatment remodeled the tumor microenvironment by increasing tumor-infiltrating CD45+ cells and CD8+ T cells, while further reducing FOXP3+ regulatory T cells. Moreover, NK/NKT cells and cDC1/cDC2 populations were increased, and their activation was enhanced. In tumor-draining lymph nodes, IFN{gamma}-FasICD mRNA promoted dendritic cell migration and increased priming and differentiation of CD8+ T cells toward effector and memory phenotypes, accompanied by enhanced functional activation of IFN{gamma}-producing CD8+ T cells and highly cytotoxic NK cells in peripheral blood. Overall, our findings provide a mechanistic foundation for cytokine-death receptor fusion proteins as an in vivo antitumor strategy that can reprogram tumor cells into localized sources of both apoptotic signals and immune-activating cues.

BackgroundCoronavirus outbreaks remain a persistent threat to global health, and vaccines based primarily on spike-specific immune responses are susceptible to antigenic variation. T-cell immunity directed against conserved internal viral proteins may provide a complementary and more variant-tolerant strategy for next-generation coronavirus vaccines. MethodsWe combined machine learning-guided antigen prioritization with ex vivo functional immunological validation to identify conserved non-spike T-cell targets across betacoronaviruses. Candidate sequences were screened for immunogenicity using primary human peripheral blood mononuclear cells from healthy donors using intracellular cytokine staining and activation-induced marker assays. Top-ranked conserved regions were incorporated into multiepitope mRNA constructs, and their intracellular expression and HLA class I presentation were confirmed by immunopeptidomics. Immunogenicity was further evaluated ex vivo and in vivo using mRNA immunization of mice and T-cell FluoroSpot assays. FindingsAcross a panel of 97 peptides derived from 19 viral proteins, evolutionary conservation across distinct betacoronavirus taxa was strongly associated with functional T-cell immunogenicity in human donors. Highly conserved peptides elicited significantly stronger and more frequent CD4 and CD8 T-cell responses than taxon-restricted peptides. Multiepitope mRNA constructs encoding conserved regions were efficiently expressed and presented on HLA class I molecules and induced T-cell responses in human PBMCs. In mice, mRNA immunization with conserved multiepitope constructs generated robust interferon-{gamma}- and interleukin-2-producing T-cell responses that exceeded those induced by unconserved control constructs. InterpretationThese results link evolutionary conservation to functional cellular immunogenicity and demonstrate the feasibility of multiepitope mRNA delivery for inducing conserved coronavirus-directed T-cell responses. Although protective efficacy remains to be established, conservation-guided antigen selection represents a scalable strategy for developing T-cell-focused vaccines with broad lineage coverage, supporting pandemic preparedness beyond spike-centered immunity. FundingThe research was supported by CEPI, NEC, University of Oslo and Oslo university hospital. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrior coronavirus vaccine development has focused predominantly on spike protein-directed neutralizing antibodies. While highly effective against matched strains, spike-centered immunity is vulnerable to antigenic drift and lineage-specific escape. Multiple observational and experimental studies have shown that T-cell responses, particularly against internal viral proteins, are more conserved and correlate with reduced disease severity and cross-variant recognition. Epitope prediction algorithms and immunoinformatics approaches have been widely used to nominate candidate T-cell targets; however, systematic functional validation of conserved non-spike antigens across betacoronaviruses in primary human immune systems, combined with antigen presentation data and in vivo vaccine testing, has remained limited. Searches of PubMed and bioRxiv up to December 2025 using terms including "coronavirus T-cell vaccine," "conserved coronavirus epitopes," "betacoronavirus cross-reactive T cells," and "mRNA T-cell vaccine" identified studies demonstrating cross-reactive T-cell immunity and computational epitope selection, but few integrated machine-learning-guided antigen prioritization with ex vivo human functional screening, immunopeptidomics, and in vivo mRNA immunization in a unified workflow. Added value of this studyThis study provides an integrated experimental and computational framework for identifying and validating conserved non-spike T-cell antigens across betacoronaviruses. We functionally screened a panel of candidate peptides derived from multiple viral proteins and demonstrated that evolutionary conservation across species is strongly associated with T-cell immunogenicity. We further demonstrate that multiepitope mRNA constructs encoding these top-ranked conserved regions can be intracellularly expressed, presented on HLA class I molecules to induce polyfunctional T-cell responses in primary human PBMCs. Finally, in vivo mRNA immunization in mice induces robust interferon-{gamma} and interleukin-2 T-cell responses exceeding those induced by unconserved control constructs. Together, these findings link evolutionary conservation to functional cellular immunogenicity and extend beyond in silico prediction by demonstrating antigen processing, presentation, and immunogenicity across human and murine systems. Implications of all the available evidenceCollectively, the available evidence indicates that T-cell immunity directed toward conserved internal coronavirus proteins represents a complementary and potentially more variant-tolerant axis of vaccine design than spike-only strategies. Our findings suggest that evolutionary conservation can serve as a practical selection principle for prioritizing T-cell antigens with broad lineage coverage and that multiepitope mRNA delivery is a feasible platform for inducing such responses. While direct protection and heterologous challenge studies will be required to establish clinical efficacy, the integration of computational prioritization with functional validation supports a scalable approach to pandemic preparedness that may be applicable to other rapidly evolving viral families.

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we demonstrate that subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across four immunologically and hormonally distinct murine BC models. Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4+ and CD8+ T cells. When layered onto this immunogenic milieu, CD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 33% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Efficacy required both CD4+ and CD8+ T cells, and complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T cell activation. Together, these findings establish T-FUS as an immune-potentiating partner for CD40 agonism, capable of driving durable, robust BC regression and immunological memory. This work positions T-FUS+CD40 agonism as a clinically scalable, in situ vaccination-like strategy with potential to benefit breast cancers, including luminal subtypes, that remain largely refractory to immune checkpoint blockade.

Rare cancers are individually uncommon but collectively represent a substantial share of cancer burden, with limited systemic treatment options for many entities. Molecular profiling identifies targetable alterations, but actionable findings are limited and responses can vary despite a matched target. This motivates complementary approaches that directly assess tumor drug response. Here, we establish a biopsy-compatible ex vivo drug sensitivity testing platform optimized for low input and reproducibility. Patient-derived material was tested either directly or following ex vivo expansion. Functional profiling was performed within clinically relevant timelines across models from 126 patients with rare advanced solid tumors. Drug responses were consistent between model types. In most samples, we identified at least one potentially active compound, supporting feasibility at biopsy-scale. High in vitro sensitivity was associated with clinical benefit and progression-free survival. These findings support functional drug sensitivity testing as a complementary component in precision oncology for adults with rare cancers. Statement of SignificanceThis study presents a biopsy-compatible drug sensitivity testing platform for phenotype-based therapy stratification in rare cancers. It identifies actionable ex vivo drug responses and shows associations with clinical outcome in patients treated with screened therapies. These findings support functional testing as a complementary additional layer of stratification for therapeutic prioritization.

Age-defined type 1 diabetes (T1D) endotypes, T1DE1 and T1DE2, are characterized by reproducible differences in pancreatic immunopathology and clinical course. In particular, these endotypes differ in the extent and composition of lymphocytic insulitis and in the extent of loss of insulin-producing {beta} cell mass, at diagnosis. However, blood-based biomarkers that may distinguish these endotypes and inform the underlying immune-islet biology axis at diagnosis remain limited. Here, we characterized the clinical features and profiled circulating microRNAs (miRNAs) in plasma from two independent INNODIA cohorts of individuals with newly diagnosed stage 3 T1D (discovery, n=115; replication, n=147), stratified into age-defined endotypes (T1DE1, <7 years; T1DE2, [&ge;]13 years; and intermediate T1DInt, 7-12 years). Differential-expression and age-adjusted models were coupled to orthogonal ddPCR validation. Putative miRNAs cellular sources were inferred using reference miRNA expression atlases. Biological context was explored via correlations of miRNAs with whole-blood transcriptomics. Clinically, T1DE1 was associated with lower {beta}-cell function and higher first-year C-peptide decline, alongside distinct islet autoantibody patterns, consistent with an immunologically aggressive endotype. Small RNA-seq analysis and ddPCR validation identified a reproducible signature in which miR-150-5p, a B-and T-lymphocyte related miRNA, and miR-375-3p, a {beta} cell enriched molecule, were consistently increased in T1DE1 compared with T1DE2 across both cohorts. MiR-150-5p retained robust association with T1DE1 even after age adjustment, and neither miRNA was associated with age in non-T1D pediatric datasets, supporting T1D endotype specificity. The increased circulating miR-150-5p signal was not explained by differences in peripheral blood B-or T-cell frequencies in high-parameter flow-cytometry subsets, and its levels correlated inversely with whole-blood expression of the immune-associated miR-150-5p target genes MPPE1 and RABGAP1L. Finally, applying a rule-based combined classifier (miR-150-5p and miR-375-3p "high") achieved re-stratification of T1D individuals, including those in the intermediate age group, into two miRNA-defined groups with distinct {beta} cell functional trajectories. Collectively, these data suggest circulating miR-150-5p and miR-375-3p as non-invasive biomarkers linked to endotype-associated biology at T1D diagnosis, with potential utility for endotype-centered stratification and trial enrichment.

Atopic dermatitis (AD) flares are frequently accompanied by Staphylococcus aureus overgrowth and activation of quorum sensing-regulated virulence pathways that amplify inflammation and barrier dysfunction. Because commensal members of the skin microbiome can inhibit S. aureus colonization and virulence, we hypothesized that a consortium sourced from healthy human skin could therapeutically target S. aureus and ameliorate AD. We curated 180 skin-derived bacterial strains and used kChip, an ultrahigh-throughput coculture platform, to profile S. aureus physiological response to over four million combinations consisting of two, three, or seven cocultured strains. This screening identified Ensemble No.2 (ENS-002), a three-strain consortium that strongly suppressed S. aureus growth and virulence in follow-up microtiter assays and a Reconstructed Human Epidermis S. aureus Activity (RHESA) model. ENS-002 is now undergoing development as a topical live biotherapeutic product (LBP) treatment for AD.

Triple negative breast cancer (TNBC) patients with comorbid Type 2 diabetes (T2D) show worse survival compared to nondiabetic TNBC patients. Immune checkpoint blockade (ICB) has unclear benefit in TNBC. Immune suppression in T2D, and use of metformin, an activator of 5 Adenosine Monophosphate-activated Protein Kinase (AMPK), in such patients, prompted us to examine AMPK regulation of immune checkpoint expression. Improved ICB efficacy may optimize outcomes for certain TNBC patients. We have also been exploring the role of Bromodomain and ExtraTerminal domain (BET) proteins (BRD2, BRD3, BRD4) in regulation of checkpoint molecules in immune cell subsets, including CD4+, CD8+ T cells, and NK cells. BET proteins are important transcriptional co-regulators, critical for proliferation and metastasis in many cancer types, including TNBC. We observed differential BET regulation of immune checkpoint proteins, specifically TIM-3, TIGIT, PD-1 and CTLA-4, on CD3/CD28-stimulated peripheral blood mononuclear cells by flow cytometry. Chemical inhibition of AMPK with Compound C, and with the pan-BET inhibitor JQ1 or the BRD4-selective PROTAC inhibitor MZ-1, revealed that BET proteins regulate PD-1 and CTLA-4 through an AMPK-dependent pathway and TIM-3 and TIGIT through an AMPK-independent pathway. Personalized approaches to ICB treatment of TNBC patients with comorbid T2D should improve outcomes.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma. Although many patients respond well to R-CHOP immunochemotherapy, those with the activated B-cell (ABC) subtype are often refractory or relapse. Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have improved outcomes, but acquired resistance limits their long-term efficacy. Here, we modeled the development of ibrutinib resistance in ABC-DLBCL and investigated whether the BCR-signaling regulator microRNA-28 (miR-28) can block this process. Using flow cytometry-based competition assays, multicolor clonal barcoding, transcriptomic profiling, and xenograft models, we found that miR-28 expression impairs the emergence of ibrutinib-resistant ABC-DLBCL cells. Mechanistically, miR-28 interferes with the clonal selection process triggered by ibrutinib treatment and rewires transcriptional programs by downregulating mitochondrial and mTOR signaling pathways critical for resistance development. Furthermore, the miR-28-repressed gene signature associated with ibrutinib resistance correlates with improved survival in ibrutinib-treated patients from the PHOENIX trial cohort with the MCD genetic subtype, which is associated with ABC-DLBCL. Finally, the targeted therapeutic delivery of miR-28 via aptamer-guided nanoparticles suppresses ibrutinib-resistant tumor growth in vivo. These findings identify miR-28 as an effective inhibitor of ibrutinib resistance, underscoring its translational potential as an adjunct strategy in ABC-DLBCL therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/687947v2_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@8e5913org.highwire.dtl.DTLVardef@17a19c3org.highwire.dtl.DTLVardef@13178f2org.highwire.dtl.DTLVardef@fb5d9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biomarkers for the early identification of acute coronary syndrome (ACS) risk remain inadequately investigated, particularly in patients with type 2 diabetes mellitus (T2DM), for whom timely clinical intervention may substantially enhance prognostic outcomes. The gut microbiota and serum metabolites may serve as pivotal mediators in the occurrence and progression of ACS among patients with T2DM, and whether these factors can be used for the precise discrimination of patients with T2DM complicated by ACS remains to be explored. Overall, 76 participants were enrolled (38 patients diagnosed with T2DM complicated by ACS and another 38 with T2DM without ACS). 16S rRNA sequencing combined with untargeted LC-MS metabolomics revealed a dysregulated gut-serum axis in patients with T2DM complicated by ACS: enrichment of proinflammatory microorganisms (Enterococcus spp.), reduction of butyrate producers (Butyricimonas spp.) and concomitant dysregulation of circulating lipid metabolites-upregulation of PC(16:0/9:0 (CHO)) and arachidonic acid alongside downregulation of cholesterol sulfate. By integrating multiomics data and applying various feature selection methods, we subsequently identified six key biomarkers. The final constructed combined model robustly distinguished patients with T2DM complicated by ACS from those with T2DM alone (AUC = 0.983), outperforming the other single omics models. Our study revealed that the gut microbiota and related serum metabolites serve as key mediators in the onset and progression of ACS among patients with T2DM, and demonstrated their potential value as noninvasive biomarkers for the early diagnosis of T2DM complicated by ACS. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSWe combined 16S rRNA sequencing with untargeted LC-MS metabolomics to dissect the gut-serum axis in patients with T2DM complicated by newly diagnosed ACS. We identified distinct gut microbial and serum metabolic signatures that distinguish ACS progression within the T2DM population. A multiomics classifier integrating clinical, microbial, and metabolic variables achieved robust diagnostic performance (AUC = 0.983) and outperformed single omics models. What Are the Clinical Implications?Integrated multiomics biomarkers facilitate the early identification of ACS progression in patients with T2DM, offering novel avenues for precision prevention, dynamic clinical surveillance, and individualized therapeutic strategies. Dysregulations of the gut microbiota and serum metabolome may play an important role in the development and progression of ACS in patients with T2DM.

Diffuse large B-cell lymphoma (DLBCL) is a common aggressive form of Non-Hodgkin lymphoma. Tetraspanin CD37 is highly expressed on mature B cells and being studied as a therapeutic target for NHL, including DLBCL. DuoHexaBody-CD37 is a biparatopic antibody with an E430G hexamerization-enhancing mutation targeting two non-overlapping CD37 epitopes shown to promote complement-dependent cytotoxicity. However, the impact of DuoHexaBody-CD37 on direct cytotoxic signaling has not yet been studied. Here we demonstrate that DuoHexaBody-CD37 induces direct cytotoxicity in DLBCL-derived tumor cell lines independent of the subtype. DuoHexaBody-CD37 induced significant CD37 clustering and was retained at the cell surface in contrast to rituximab, which was internalized. Unbiased screening identified the modulation of 26 (phospho)proteins upon DuoHexaBodyCD37 treatment of primary B cells or DLBCL cells. Whereas DLBCL cells predominantly upregulated p-SHP1(Y564) upon DuoHexaBody-CD37 treatment, primary B cells showed significantly increased p-AKT(S473) and MAPK signaling which is linked to cell survival. Studies using CD37-mutants identified the N-terminus to be involved in DuoHexaBody-CD37-induced signaling. Finally, DuoHexaBody-CD37 treatment inhibited cytokine pro-survival signaling in DLBCL cells. These findings provide novel insights into the signaling functions of CD37 upon DuoHexaBody-CD37 treatment, and open up opportunities for developing CD37-targeted immunotherapy in combination with small molecule inhibitors to maximize tumor cell death.

Semaglutide is often optimized for weight loss, but whether longer-term cardiovascular benefit tracks achieved weight loss or therapeutic exposure levels remains unclear. Using a federated deidentified U.S. electronic health record network of 29 million patients, including 505,874 semaglutide-treated individuals, we leveraged multimodal AI technologies to analyze 47,199 patients with baseline cardiovascular disease. We quantified dose escalation and weight change during the 0-2-year period after semaglutide initiation (landmark period) and assessed cardiovascular outcomes during the 2-4-year period (post-landmark). In propensity-matched comparisons during the landmark period, semaglutide was associated with lower cardiovascular events than metformin, DPP-4 and SGLT2 inhibitors. Higher maximum semaglutide dose was associated with greater weight loss during the landmark period (3.15% additional weight loss per 1 mg increase; r=0.97, P<0.001), and lower post-landmark risk of all-cause mortality (RR 0.42, p<0.001), composite cardiovascular events (death, myocardial infarction, or stroke; RR 0.51, p<0.001), cerebrovascular disease (RR 0.50, p<0.001), heart failure (RR 0.55, p<0.001), and valvular/rheumatic heart disease (RR 0.71, p=0.025). In contrast, greater achieved weight loss during the landmark period did not show a consistent monotonic association with lower post-landmark cardiovascular risk (All-cause mortality p-value=0.14, composite cardiovascular endpoint p-value=0.55). Integrating insights from a single cell GLP1R expression atlas was used to infer how semaglutide pharmacology may tie into heart-specific signaling, beyond what is reflected by body-weight reduction alone. The strongest prevalence-weighted GLP1R signal was observed in the pancreas, followed by the heart, where GLP1R engagement potential (GEP) was considerable across cardiomyocyte, cardiac endothelial, and rarer immune cell populations. Together, semaglutide cardiovascular benefit appears organized more by maximum dose attained than by achieved weight-loss magnitude, setting the stage for beyond-obesity trial designs that integrate whole-body spatial intelligence.