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The development of hematopoietic cell lineages is a highly complex process governed by a delicate interplay of various transcription factors. The expression of these factors is influenced,in part,by epigenetic signatures that define each stage of cell differentiation. In particular,the formation of B lymphocytes depends on the sequential silencing of stemness genes and the balanced expression of interdependent transcription factors,along with DNA rearrangement. We have investigated the impact of Dido3 deficiency,a protein involved in chromatin status readout,on B cell differentiation within the hematopoietic compartment of mice. Our findings revealed significant impairments in the successive stages of B cell development. The absence of Dido3 resulted in remarkable alterations in the expression of essential transcription factors and differentiation markers,which are crucial for orchestrating the differentiation process. Additionally,the somatic recombination process,responsible for generation of antigen receptor diversity,was also adversely affected. These observations highlight the vital role of epigenetic regulation,particularly the involvement of Dido3,in ensuring proper B cell differentiation. This study reveals new mechanisms underlying disruptive alterations,deepening our understanding of hematopoiesis and may potentially lead to insights that aid in the development of therapeutic interventions for disorders involving aberrant B cell development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-025-01394-x. 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

IntroductionImmune resilience is the capacity of the immune system to recover from sepsis-induced organ injury and reestablish host defense. While sepsis survivors are often viewed as immunocompromised,recent studies suggest that some may acquire adaptive immune traits that enhance resistance to secondary infections.MethodsWe employed a murine cecal ligation and puncture (CLP) model to study polymicrobial sepsis and subsequent immune responses. Listeria monocytogenes was used as a model intracellular pathogen to assess immune protection. We analyzed myeloid cell phenotypes using single-cell RNA sequencing and evaluated lipid metabolic changes through quantitative RT-PCR,immunohistochemistry,serum cytokine assays,and plasma lipidomics.ResultsSepsis-surviving mice showed enhanced resistance to Listeria infection. Single-cell RNA sequencing revealed transcriptional reprogramming in splenic CD11b+Ly6Chigh myeloid cells,including downregulation of lipoprotein lipase and lipid efflux genes. CD11b+ myeloid cells exhibited increased lipid droplet accumulation,accompanied by elevated serum interferon-gamma (IFN-γ) levels. Plasma lipidomics identified systemic lipid remodeling,with increased phosphatidylserine and decreased phosphatidylinositol and phosphatidylglycerol levels.DiscussionOur findings suggest that sepsis survival induces lipid metabolic reprogramming in myeloid cells,contributing to enhanced immunity against intracellular pathogens. These insights reveal potential therapeutic targets within lipid metabolic pathways to improve host defense in sepsis survivors. View Publication

BackgroundIn persons living with HIV,antiretroviral therapy (ART) reduces HIV RNA in their plasma and increases CD4 + T lymphocytes,thus restoring their immune function and reducing mortality rates.MethodsThe heavy and light chains of B cell receptor (BCR) were amplified,sequenced,analyzed,and determined to be anti-CD4 mAb. The cytotoxicity of NK cells mediated by the anti-CD4 mAb was assessed using CCK-8,flow cytometry,ELISA,and western blotting. Detecting the viability/regulation of CD4 cells involved inhibiting the attachment of autoantibodies against CD4 to crucial receptors and detecting the inhibition of key molecules in B cells to produce anti-CD4 mAb in patients with immune non-responders (INR). Furthermore,through Phage Random Peptide Library Screening,we discovered that the AAPMFHSSVQLP-CD4 peptide has an affinity for the anti-CD4 mAb.ResultsAdministering anti-CD4 mAb enhanced NK cytotoxicity. The simultaneous administration of anti-CD4 mAb alongside GST-CD4 alleviated the harmful impacts of anti-CD4 mAb on the CD3 + population in humanized mice,and HIV virus (p24). Individuals diagnosed with INR displayed abnormal B cell activity,particularly with elevated BAFFR expression and increased levels of anti-CD4 mAb. Nevertheless,suppression of BAFFR hindered B cell function and decreased the production of anti-CD4 mAb. In HIV-infected individuals,the dysregulation of B-cells led to the production of anti-CD4 mAb,which in turn facilitated NK cell cytotoxicity and the CD4 + T effect by upregulating the expression of BAFFR.ConclusionThe dysregulation of B-cells in person living with HIV increased the production of anti-CD4 mAb,which in turn promoted NK cell cytotoxicity and the CD4 + T effect.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01286-3. Highlights1) B-cell dysregulation increased anti-CD4 mAb levels.2) B cells are abnormally active in patients with INR.3) Knockdown of BAFFR obviously reduced the secretion of anti-CD4 mAb.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01286-3. View Publication

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms,where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans,most commonly p.R525H. However,the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood,partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report,conducted to pursue this objective,we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants,leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells,accompanied by R-loop accumulation,which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations,gene expression profiles were largely similar between p.R525H-positive and -negative cells. However,ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile,highlighting an intricate interplay shaping the patient’s MDS phenotype. View Publication

The human bone marrow (BM) microenvironment involves hematopoietic and non-hematopoietic cell subsets organized in a complex architecture. Tremendous efforts have been made to model it in order to analyze normal or pathological hematopoiesis and its stromal counterpart. Herein,we report an original,fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the hematopoietic differentiation. This fully-human Artificial Marrow Organoid (AMO) model is highly efficient to recapitulate MSC support to myeloid differentiation and NK cell development from the immature CD34 + HSPCs to the most terminally differentiated CD15 + polymorphonuclear neutrophils,CD64 + monocytes or NKG2A-KIR2D + CD57 + NK subset. Lastly,our model is suitable for evaluating anti-leukemic NK cell function in presence of therapeutic agents. Overall,the AMO is a versatile,low cost and simple model able to recapitulate normal hematopoiesis and allowing more physiological drug testing by taking into account both immune and non-immune BM microenvironment interactions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-07717-9. View Publication

ObjectiveBifidobacterium is known to be depleted in patients with atopic dermatitis (AD). This study aims to investigate the potential prophylactic effects of Bifidobacterium animalis subsp. lactis BX-BC08 (B. lactis BX-BC08) in a murine model of AD.DesignThe immunosuppressive and anti-inflammatory effects of BX-BC08 were evaluated in a MC903-induced AD mouse model. Gut microbiota composition was analyzed by metagenomic sequencing,while high-performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to identify anti-inflammatory molecules produced by B. lactis BX-BC08.ResultsBX-BC08 significantly attenuated pro-inflammatory responses,scaling and swelling in the MC903-induced AD like murine model compared to controls. Fecal microbial profiling revealed an enrichment of probiotics and a reduction of pro-inflammatory bacteria in BX-BC08 treated mice. Metabolic analysis of BX-BC08 bacteria culture supernatant and treated mice identified a significant enrichment of alpha-Ketoglutaric acid (AKG). Functional validation in the murine AD model demonstrated that AKG strongly suppressed T helper 2 (Th2)-driven pro-inflammatory responses.ConclusionBX-BC08 mitigates AD-like inflammation by producing the anti-inflammatory metabolite AKG. BX-BC08 could serve as a novel prophylactic agent for AD prevention.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06769-9. View Publication

Aging is associated with immune dysfunction,but long-term endurance training may confer protective effects on immune cell function. This study investigates how natural killer (NK) cell phenotypes,functional markers,and metabolism differ between endurance-trained and untrained older adults. Ex vivo expanded NK cells from endurance-trained (63.6 ± 2.1 years) and untrained (64.3 ± 3.3 years) males were exposed to adrenergic blockade (propranolol; 0–200 ng/mL) or mTOR inhibition (rapamycin; 10–100 ng/mL),both with or without PMA-induced inflammatory stimulation. Flow cytometry assessed NK subsets,activation (CD38,CD57,CD107a,NKG2D),senescence (KLRG1),and inhibitory markers (PD-1,LAG-3,TIM-3,NKG2A). Seahorse analysis measured metabolic parameters. Trained participants displayed healthier immune profiles (lower NLR,SII) and higher effector NK cells with lower cytotoxic subsets. Propranolol at 100 ng/mL blunted PMA-driven increases in CD57,CD107a,and NKG2D,while potentiating regulatory markers KLRG1,LAG-3,and PD-1 in the trained group,indicating stronger immunoregulation. With rapamycin,trained NK cells preserved NKG2D and CD107a at 10 ng/mL,maintaining cytotoxicity and degranulation. In contrast,at 100 ng/mL rapamycin plus PMA,trained NK cells shifted toward an effector phenotype with higher CD57 and CD107a,yet a blunted PMA-increased LAG-3 and TIM-3,suggesting resistance to exhaustion. PD-1 and KLRG1 remained elevated,reflecting balanced immune control. Mitochondrial analysis revealed that trained NK cells exhibited higher basal and maximal OCR,greater spare respiratory capacity,and OCR/ECAR ratio,reflecting superior metabolic fitness. These findings indicate that endurance-trained older adults have NK cells with greater functional adaptability,reduced senescence,and enhanced metabolism under inflammatory and pharmacological stress.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06057-y. 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

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success,but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion,hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet,the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable,multiplexed modulation of gene expression with greater safety. Here,we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes,PDCD1 and LAG3,both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion,limiting functional assessment,gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore,transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features. Graphical abstract Epigenome editing is used to engineer CAR T cells targeting prostate cancer by stably silencing the PDCD1 and LAG3 genes,which encode key inhibitory checkpoint receptors. This DNA break-free approach enhances safety by avoiding genomic damage and holds promise as a next-generation strategy for safer,more durable cancer immunotherapy. View Publication

BackgroundMacrophages play a crucial role in the development of early-onset preeclampsia (EOPE),which may be closely associated with an imbalance in macrophage M1/M2 polarization. Mesenchymal stem cell (MSC)-derived apoptotic vesicles (apoVs) have anti-inflammatory,tissue repair,and immunomodulatory functions. MSC-apoVs may ameliorate EOPE by regulating macrophage polarization,but the underlying mechanisms remain to be clarified.MethodsMacrophage infiltration and M1/M2 polarization were first analyzed in the placentas of PE patients and normal pregancies to identify macrophage alterations in EOPE placentas. MSC-apoVs were extracted and characterized. The effects of MSC-apoVs on macrophage polarization and trophoblasts invasion were validated in vivo and in vitro. miRNA transcriptomic sequencing of MSC-apoVs was conducted to identify key miRNAs involved in macrophage M2 polarization and to investigate upstream and downstream regulation factors,which were further validated in vivo and in vitro.ResultsThe proportion of M2 macrophages was significantly reduced in EOPE placentas. MSC-apoVs carrying high levels of miR-191-5p recruited macrophages,downregulated CDK6 protein expression,stabilized mitochondrial membrane potential (MMP),and promoted M2 polarization of macrophages. This enhanced the invasion of trophoblasts and improved EOPE pregnancy outcomes in mice,including reduced blood pressure,decreased urine protein,and improved embryo quality. Overexpression of miR-191-5p mimics in MSC-apoVs further alleviated EOPE-related symptoms,whereas inhibition of miR-191-5p reduced the therapeutic effect of MSC-apoVs. Further experiments confirmed that M2 macrophages polarized by MSC-apoVs promote trophoblasts invasion by secreting platelet-derived growth factor-AB (PDGF-AB),which binds to platelet-derived growth factor receptor-beta (PDGFR-β) on trophoblasts,directly activating the downstream PI3K-AKT-mTOR signaling pathway,thereby improving EOPE.ConclusionOur findings reveal the crucial role of M2 macrophages in the pathogenesis of EOPE. MSC-apoVs with high miR-191-5p recruit macrophages,downregulate CDK6,stabilize MMP,and promote M2 polarization,increasing PDGF-AB secretion,which enhances trophoblasts invasion and thereby treat EOPE. Therefore,MSC-apoVs therapy may serve as a promising strategy to improve the prognosis of EOPE.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04546-5. View Publication

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however,its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here,we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids,revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation,expressed SOX2,and lacked CRX,consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas,particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities,demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1,Flavopiridol) and the cell-cycle inhibitor Volasertib,indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively,our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma,establishes a robust and clinically relevant human retinal organoid platform,and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype. View Publication