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Venoms comprise highly sophisticated bioactive molecules modulating ion channels,receptors,coagulation factors,and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research,we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis,pore formation,and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements,we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic,ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms,we found that the pore-forming latrotoxin ?-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes,while the lytic bee peptide melittin is not selective. Furthermore,evaluation of snake venoms showed that Elapid species induced rapid membrane lysis,while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits,aligning with clinical observations at venom level,beyond discriminating among ion pore-forming,membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic,cardiotoxic and neurotoxic effects,and assist in identifying venom components that hold the potential to benefit humankind. Graphical abstractImage 1 Highlights•Optimization of bioassays to study venoms strengthens the discovery of novel drugs and envenomation treatments•We developed a high-content bioassay measuring DNA and [Ca2+]i that investigates multiple mechanisms in venom biology•This bioassay monitored membrane integrity,ion channels and ion pore formation to unravel venom's mechanism of action•We found the latrotoxin ?-Lt1a strikingly prefers neuron-like cells while the ?-helical melittin is non-selective•Evaluation of Elapid and Viper snake venoms demonstrates that this bioassay predicts the phylogeny and clinical findings View Publication

ABSTRACTGenetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study looked at people from 12 unrelated families with variants in the gene SMPD4,a neutral sphingomyelinase that metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These individuals have severe developmental brain malformations,including microcephaly and cerebellar hypoplasia. The disease mechanism of SMPD4 was not known and so we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia,a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells lacking SMPD4 exhibit neural progenitor cell death and have shortened primary cilia,which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development. Summary: Mouse and human stem cell models of SMPD4 loss of function demonstrate that SMPD4 promotes cilia function and neural development. View Publication

IntroductionBAP1 is a deubiquitinase (DUB) of the Ubiquitin C-terminal Hydrolase (UCH) family that regulates gene expression and other cellular processes,through its direct catalytic activity on the repressive epigenetic mark histone H2AK119ub,as well as on several other substrates. BAP1 is also a highly important tumor suppressor,expressed and functional across many cell types and tissues. In recent work,we demonstrated a cell intrinsic role of BAP1 in the B cell lineage development in murine bone marrow,however the role of BAP1 in the regulation of B cell mediated humoral immune response has not been previously explored. Methods and resultsIn the current study,we demonstrate that a B-cell intrinsic loss of BAP1 in activated B cells in the Bap1 fl/fl Cγ1-cre murine model results in a severe defect in antibody production,with altered dynamics of germinal centre B cell,memory B cell,and plasma cell numbers. At the cellular and molecular level,BAP1 was dispensable for B cell immunoglobulin class switching but resulted in an impaired proliferation of activated B cells,with genome-wide dysregulation in histone H2AK119ub levels and gene expression. Conclusion and discussionIn summary,our study establishes the B-cell intrinsic role of BAP1 in antibody mediated immune response and indicates its central role in the regulation of the genome-wide landscapes of histone H2AK119ub and downstream transcriptional programs of B cell activation and humoral immunity. View Publication

Humanized mice are limited in terms of modeling human immunity,particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated,genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells,followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system,including marginal zone B cells,germinal center B cells,follicular helper T cells and neutrophils,and develop well-formed lymph nodes and intestinal lymphoid tissue,including Peyer’s patches,and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses,entailing somatic hypermutation,class-switch recombination,and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination,THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain,with blood incretion of human cytokines,including APRIL,BAFF,TGF-β,IL-4 and IFN-γ,all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses,THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics. Humanized mice have been a valuable tool for modeling human immunology but are limited in their ability to model human antibody responses. Here the authors present their THX humanized mouse that does model human antibody responses and test its suitability for vaccination and autoimmunity studies. View Publication

AbstractThe landscape of cancer treatment has been transformed by immune checkpoint inhibitors; however,the failure to benefit a large number of patients with cancer has underlined the need to identify promising targets for more effective interventions. In this study,we leverage 23andMe,Inc.’s large-scale human germline genetic and health database to uncover the previously unknown role of UL16-binding protein 6 (ULBP6),a high-affinity NK group 2D (NKG2D) ligand,in cancer and its promise as an immuno-oncology therapeutic target. We confirm ULBP6 expression in human tumors and demonstrate that soluble ULBP6 shed from tumors circumvents NKG2D activation provided by membrane-anchored NKG2D ligands to inhibit immune cell activation and tumor cell killing. Based on these findings,we developed 23ME-01473,a humanized Fc effector–enhanced antibody that binds to ULBP6 and its closely related family members,ULBP2 and ULBP5. 23ME-01473 effectively blocks soluble ULBP6-mediated immunosuppression to restore the NKG2D axis on NK and T cells to elicit tumor growth control. Moreover,the Fc effector–enhanced design of 23ME-01473 increases its binding affinity to fragment crystallizable gamma receptor IIIa,which,together with 23ME-01473’s binding to membrane-anchored ULBP6/2/5 on cancer cells,allows for augmented antibody-dependent cellular cytotoxicity induction,providing a second activation node for NK cells. Our studies demonstrate the therapeutic potential of an Fc effector–enhanced anti-ULBP6/2/5 antibody to reinvigorate NK cell and T-cell activation and cytotoxicity for the treatment of cancer.Significance:This study emphasizes the utility of population-based genome-wide assessments for discovering naturally occurring genetic variants associated with lifetime risks for cancer or immune diseases as novel drug targets. We identify ULBP6 as a potential keystone member of the NKG2D pathway,which is important for antitumor immunity. Targeting ULBP6 may hold therapeutic promise for patients with cancer. View Publication

Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile arthritis,accompanied by cytokine storm and hemophagocytosis. In addition,COVID-19–related hyperinflammation shares clinical features of MAS. Mechanisms that activate macrophages in MAS remain unclear. Here,we identify the role of miRNA in increased phagocytosis and interleukin-12 (IL-12) production by macrophages in a murine model of MAS. MAS significantly increased F4/80+ macrophages and phagocytosis in the mouse liver. Gene expression profile revealed the induction of Fcγ receptor–mediated phagocytosis (FGRP) and IL-12 production in the liver. Phagocytosis pathways such as High-affinity IgE receptor is known as Fc epsilon RI -signaling and pattern recognition receptors involved in the recognition of bacteria and viruses and phagosome formation were also significantly upregulated. In MAS,miR-136-5p and miR-501-3p targeted and caused increased expression of Fcgr3,Fcgr4,and Fcgr1 genes in FGRP pathway and consequent increase in phagocytosis by macrophages,whereas miR-129-1-3p and miR-150-3p targeted and induced Il-12. Transcriptome analysis of patients with MAS revealed the upregulation of FGRP and FCGR gene expression. A target analysis of gene expression data from a patient with MAS discovered that miR-136-5p targets FCGR2A and FCGR3A/3B,the human orthologs of mouse Fcgr3 and Fcgr4,and miR-501-3p targets FCGR1A,the human ortholog of mouse Fcgr1. Together,we demonstrate the novel role of miRNAs during MAS pathogenesis,thereby suggesting miRNA mimic–based therapy to control the hyperactivation of macrophages in patients with MAS as well as use overexpression of FCGR genes as a marker for MAS classification. View Publication

IntroductionThe effector function of T cells is regulated via immune checkpoints,activating or inhibiting the immune response. The BTLA-HVEM complex,the inhibitory immune checkpoint,may act as one of the tumor immune escape mechanisms. Therefore,interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA,thus disrupting the BTLA-HVEM interaction. These peptides’ structure and amino acid sequences are based on the gD protein,the ligand of HVEM. Here,we investigated their immunomodulatory potential in melanoma patients.MethodsFlow cytometry analyses of activation,proliferation,and apoptosis of T cells from patients were performed. Additionally,we evaluated changes within the T cell memory compartment.ResultsThe most promising compound – Pep(2),increased the percentages of activated T cells and promoted their proliferation. Additionally,this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells.DiscussionWe conclude that the examined peptide may act as a booster for the immune system. Moreover,the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds. View Publication

Given the marginal penetration of most drugs across the blood-brain barrier,the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication,respectively. Similarly,LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab,respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice,which rely on myeloid cells and lymphocytes for their efficacy. Overall,this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM. Ultrasound-mediated blood-brain barrier opening has been exploited to improve drug delivery in the brain. Here the authors show that low-intensity pulsed ultrasound in combination with intravenous injection of microbubbles enhances the delivery of doxorubicin and anti-PD1 in gliomas,improving anti-tumor immune responses. View Publication

BackgroundGraft-versus-host disease (GVHD) and relapse are major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Metabolites play crucial roles in immune regulation,but their causal relationships with GVHD and relapse remain unclear.MethodsWe utilized genetic variants from genome-wide association studies (GWAS) of 309 known metabolites as instrumental variables to evaluate their causal effects on acute GVHD (aGVHD),gut GVHD,chronic GVHD (cGVHD),and relapse in different populations. Multiple causal inference methods,heterogeneity assessments,and pleiotropy tests were conducted to ensure result robustness. Multivariable MR analysis was performed to adjust for potential confounders,and validation MR analysis further confirmed key findings. Mediation MR analysis was employed to explore indirect causal pathways.ResultsAfter correction for multiple testing,we identified elevated pyridoxate and proline levels as protective factors against grade 3–4 aGVHD (aGVHD3) and relapse,respectively. Conversely,glycochenodeoxycholate increased the risk of aGVHD3,whereas 1-stearoylglycerophosphoethanolamine had a protective effect. The robustness and stability of these findings were confirmed by multiple causal inference approaches,heterogeneity,and horizontal pleiotropy analyses. Multivariable MR analysis further excluded potential confounding pleiotropic effects. Validation MR analyses supported the causal roles of pyridoxate and 1-stearoylglycerophosphoethanolamine,while mediation MR revealed that pyridoxate influences GVHD directly and indirectly via CD39 + Tregs. Pathway analyses highlighted critical biochemical alterations,including disruptions in bile acid metabolism and the regulatory roles of vitamin B6 derivatives. Finally,clinical metabolic analyses,including direct fecal metabolite measurements,confirmed the protective role of pyridoxate against aGVHD.ConclusionsOur findings provide novel insights into the metabolic mechanisms underlying GVHD and relapse after allo-HSCT. Identified metabolites,particularly pyridoxate,may serve as potential therapeutic targets for GVHD prevention and management.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. Key points1. Strong evidence has found the protective effect of pyridoxate against aGVHD and has been validated in patients.2. Mediated MR analysis suggests that pyridoxate may reduce aGVHD risk by increasing CD39+ Tregs level.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. View Publication

Systemic inflammatory conditions are classically characterized by an acute hyperinflammatory phase,followed by a late immunosuppressive phase that elevates the susceptibility to secondary infections. Comprehensive mechanistic understanding of these phases is largely lacking. To address this gap,we leveraged a controlled,human in vivo model of lipopolysaccharide (LPS)-induced systemic inflammation encompassing both phases. Single-cell RNA sequencing during the acute hyperinflammatory phase identified an inflammatory CD163+SLC39A8+CALR+ monocyte-like subset (infMono) at 4 h post-LPS administration. The late immunosuppressive phase was characterized by diminished expression of type I interferon (IFN)-responsive genes in monocytes,impaired myelopoiesis and a pronounced attenuation of the immune response on a secondary LPS challenge 1 week after the first. The infMono gene program and impaired myelopoiesis were also detected in patient cohorts with bacterial sepsis and coronavirus disease. IFNβ treatment restored type-I IFN responses and proinflammatory cytokine production and induced monocyte maturation,suggesting a potential treatment option for immunosuppression. Stunnenberg et al. use a model of lipopolysaccharide injection in humans to characterize the transcriptomic landscape of bone marrow and blood immune cells during the hyperinflammatory and immunosuppressed phases of systemic inflammation. View Publication

BackgroundChimeric antigen receptor T (CAR-T) cell therapy holds promise for cancer treatment,but its efficacy is often hindered by metabolic constraints in the tumor microenvironment. This study investigates the role of glutamine in enhancing CAR-T cell function against ovarian cancer.MethodsMetabolomic profiling of blood samples from ovarian cancer patients treated with MSLN-CAR-T cells was conducted to identify metabolic changes. In vitro,glutamine pretreatment was applied to CAR-T cells,and their proliferation,CAR expression,tumor lysis,and cytokine production (TNF-α,IFN-γ) were assessed. Mechanistic studies focused on the mTOR-SREBP2 pathway and its effect on HMGCS1 expression,membrane stability and immune synapse formation. In vivo,the antitumor effects and memory phenotype of glutamine-pretreated CAR-T cells were evaluated.ResultsElevated glutamine levels were observed in the blood of ovarian cancer patients who responded to MSLN-CAR-T cell treatment. Glutamine pretreatment enhanced CAR-T cell proliferation,CAR expression,tumor lysis,and cytokine production. Mechanistically,glutamine activated the mTOR-SREBP2 pathway,upregulating HMGCS1 and promoting membrane stability and immune synapse formation. In vivo,glutamine-pretreated CAR-T cells exhibited superior tumor infiltration,sustained antitumor activity,and preserved memory subsets.ConclusionsOur findings highlight glutamine-driven metabolic rewiring via the mTOR-SREBP2-HMGCS1 axis as a strategy to augment CAR-T cell efficacy in ovarian cancer.Trial registrationNCT05372692Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06853-0. View Publication

Pathogenic mutations in Bcl2-associated athanogene 3 (BAG3) cause genetic dilated cardiomyopathy (DCM),a disease characterized by ventricular dilation,systolic dysfunction,and fibrosis. Previous studies have demonstrated that BAG3 mediates sarcomeric protein turnover through chaperone-assisted selective autophagy to maintain sarcomere integrity in the human heart. Although mouse models provide valuable insights into whole-organism effects of BAG3 knockout or pathogenic variants,their utility is limited by species-specific differences in pathophysiology,which often do not translate to humans and contribute to the failure of clinical trials. As a result,the development of induced pluripotent stem cell-derived cardiomyocytes (iCM) and engineered heart tissues presents a promising alternative for studying adult-onset cardiac diseases. Here,we used genome engineering to generate an isogenic pseudo-wild-type control cell line from a patient-derived iPSC line carrying a pathogenic BAG3 variant,clinically presenting with DCM. While monolayer iCMs recapitulated some features of BAG3-mediated DCM,such as reduced autophagy,mitochondrial membrane potential,and decreased HSPB8 stability,they failed to develop the age-associated impairment in sarcomere integrity. Therefore,we developed a mature,patient-specific,human engineered heart tissue model of BAG3-mediated DCM and compared it to its isogenic healthy control. We demonstrated successful recapitulation of adult-like features of the clinically observed disorganized sarcomeres and impaired tissue contractility,thereby providing a platform to investigate BAG3-related pathophysiology and therapeutic interventions. Graphical abstract View Publication