技术资料

Graft‐versus‐host disease is mediated by donor‐derived T cells reactive against the recipient's broadly expressed minor histocompatibility antigens (mHA). Regulatory T cells (Treg) have been explored as a therapeutic approach for chronic GVHD (cGVHD). The promising results from polyclonal Treg trials in this setting have led us to develop a Treg product specific for mismatched minor antigens between patient and donor (mTreg),circumventing broad immune suppression risks. HLA‐matched siblings of opposite sexes were used to obtain the sister's CD4+CD25hiCD127low Treg for co‐culture with the respective brother's dendritic cells as a source of mismatched mHA. We have established the optimal culture conditions resulting in the highest mTreg proliferation and viability. Comprehensive phenotyping during the ex vivo selection shows PD‐1,CTLA‐4,CD39 and HLA‐DR expression. Transcriptomic analysis revealed a switch in metabolic process,and up‐regulation of functional Treg genes. Furthermore,mTreg possess specific and potent suppressive activity,in which there is a dependency on cell‐to‐cell contact and a role for HLA class II expression on mTreg. This protocol would allow the generation of Treg specific to an array of mHA from the recipient's healthy tissues,likely providing a directed and strong suppression of cGVHD. We optimised a protocol for mHA‐specific Treg (mTreg) selection in an HLA‐matched context while defining its phenotype,transcriptional state and function. mTreg were highly activated and exerted specific,HLA class II‐,contact‐dependent suppression. This protocol can be explored as a highly personalised antigen‐specific Treg‐based therapy in future clinical trials for cGVHD. View Publication

CD8+ T cells are an important weapon in the therapeutic armamentarium against cancer. While CD8+CD103+ T cells with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognoses,the tumor microenvironment also contains dysfunctional exhausted T (TEX) cells that exhibit a variety of TRM-like features. Here we deconvolute TRM and TEX cells across human cancers,ascribing markers and gene signatures that distinguish these populations and enable their functional distinction. Although TRM cells have superior functionality and are associated with long-term survival post-tumor resection,they are not associated with responsiveness to immune checkpoint blockade. Tumor-associated TEX and TRM cells are clonally distinct,with the latter comprising tumor-independent bystanders and tumor-specific cells segregated from cognate antigen. Intratumoral TRM cells can be forced toward an exhausted fate when chronic antigen stimulation occurs,indicating that the presence or absence of continuous antigen exposure within the microenvironment is the key distinction between tumor-associated TEX and TRM populations. These results highlight unique functions for TRM and TEX cells in tumor control,underscoring the need for distinct strategies to harness these populations for cancer therapies. Here the authors show that tissue-resident memory and exhausted T cells in tumors are distinct populations that are shaped by relative presence or absence of TCR signals,suggesting that a tailored therapeutic strategy is needed to target each subset. View Publication

Background: The pathology of Alzheimer’s Disease (AD) is characterized by aggregates of amyloid beta (Aβ) peptides and neurofibrillary tau tangles. Increased blood-brain barrier (BBB) permeability and reduced Aβ clearance,which signal neurovascular dysfunction,have also been proposed as early markers of AD. Despite intense scrutiny,the mechanisms of AD remain elusive and novel treatments that address core symptoms of dementia are limited. New alternative methods (NAMs) aim to develop in-vitro translational models that recapitulate human pathology more accurately than previous models and could contribute to the development of new therapies. Methods: Here,we developed a NAM model of the cortical neurovascular unit (NVU) using brain cells derived from human induced pluripotent stem cells (hiPSCs) from a patient with AD and a healthy individual. Differentiated neurons,astrocytes,pericytes,microglia,and brain-like microvascular endothelial cells were cultured in a microphysiological system to create a brain-chip model to evaluate NVU-related endpoints. Results: Compared to control,AD brain-chips had reduced claudin-5 and ZO-1 expression and increased paracellular permeability. AD brain-chips also had decreased activity of the efflux transporter P-glycoprotein (P-gp),but its expression was unchanged. In AD brain-chips,levels of Aβ42,total tau,and p-tau 181 were decreased in protein lysates from the brain channel,while levels of total tau and p-tau 181 were increased in protein lysates from the vascular channel. Finally,AD brain-chips had increased levels of the proinflammatory markers IL-6 and MCP-1 in effluent from both brain and vascular channels. Conclusion: In this brain-chip model,we showed Aβ-independent NVU dysfunction that was related to neuroinflammation and vascular tau accumulation. This study demonstrates the utility of the brain-chip model to evaluate changes in NVU functions induced by AD-like pathology and highlights donor-specific responses associated with the use of hiPSC-derived models. View Publication

Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation,driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response,leading to hepatocyte damage,senescence,and impaired liver regeneration. While the underlying mechanisms remain incompletely understood,increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here,we identify iRhom2 as a key regulator of immune-mediated liver injury,orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages,yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts,particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress,preserved mitochondrial function,and attenuated apoptosis,indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators,with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably,HMGB1 release occurs independently of TACE catalytic activity,suggesting an alternative unexplored regulatory mechanism. Furthermore,co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition,driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly,HMGB1 neutralization enhances hepatocyte viability and mitigates senescence,underscoring its pathogenic role. Additionally,HMGB1 knockdown in macrophages protects hepatocytes,though p21 expression remains unaffected,hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival. View Publication

The immunoregulatory effects of probiotics have been widely studied,particularly in maintaining immune balance. Conventional in vitro functional screening of probiotics relies on fresh donor-derived primary immune cells,which often exhibit significant inter-individual and temporal variability,limiting reproducibility and interpretation. As an alternative,human-induced pluripotent stem cell (iPSC)-derived dendritic cells were co-cultured with five probiotic strains in the current study to evaluate their immunomodulatory interactions. To assess whether cytokines produced by probiotic-stimulated dendritic cells can influence T cell differentiation,human CD4+ T cells were exposed to the conditioned medium derived from co-cultures. Enzyme-linked immunosorbent assay results demonstrated that iPSC-derived dendritic cells secreted cytokines at distinct concentrations in response to different probiotic strains,suggesting that these cells can distinguish between different microbial stimuli,and supporting their use in functional probiotic screening. Among the five strains tested,Lactiplantibacillus plantarum LPA-56,Limosilactobacillus reuteri RU-23,and Lactobacillus fermentum Fem-99 induced cytokine production levels that promoted the differentiation of the human CD4+ T cells into regulatory T cells. These findings demonstrate that iPSC-derived dendritic cells have immunomodulatory potential,are reliable for in vitro screening of probiotics,and offer a promising strategy for selecting potent immunoregulatory probiotic candidates. View Publication

Interleukin-17 (IL-17) plays a central role in the pathogenesis of various autoimmune diseases. Soluble CD14 (sCD14),a marker of innate immune activation,is elevated in several inflammatory conditions. However,its influence on IL-17 production and the differentiation of Th17 cells remains poorly understood. We found that sCD14 enhances Th17-associated cytokine production and up-regulates critical transcription factors such as STAT3 and RORC. Notably,sCD14's effect on Th17 polarization was mediated indirectly through autologous sCD14-treated peripheral blood mononuclear cell (PBMC) supernatant (sCD14-PBMC-Sup). Additionally,we identified a distinct cytokine profile enriched for pro-inflammatory cytokines and chemokines in sCD14-treated T cells,further reinforcing the Th17-promoting role of sCD14. Interestingly,gamma-aminobutyric acid (GABA),a metabolite elevated in sCD14-treated monocytes,was identified as a potential contributor to Th17 polarization. GABA supplementation in T-cell cultures enhanced IL-17A secretion,indicating its role as a signaling molecule in T-cell differentiation. Our findings also revealed the expansion of innate lymphoid cell (ILC)2/3-like cells in T-cell cultures exposed to sCD14-PBMC-Sup and GABA,highlighting the potential role of monocytes in Th17-mediated immunity. Furthermore,while sCD14 promoted Th17 polarization,it simultaneously impaired T-cell activation and proliferation,suggesting an immunosuppressive effect mediated by soluble factors released from monocytes. These results underscore the dual role of sCD14 in modulating T-cell responses,promoting Th17 differentiation while suppressing T-cell effector functions. This study identifies a previously unrecognized role for sCD14 in promoting Th17 induction,highlighting its contribution to immune regulation and its potential as a therapeutic target in Th17-driven autoimmune conditions. View Publication

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central roles in cellular signaling,yet their promise remains largely untapped due to technical challenges inherent to profiling them. Here,we employ N-glycoproteomics to analyze 85 patient-derived xenografts (PDXs),constructing Glyco PDXplorer—an in vivo pan-cancer atlas of cancer-derived surface proteins. We develop a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validate FAT2 as a squamous-cancer-enriched surface protein minimally detected in normal tissue. Functional studies reveal that FAT2 is essential for head and neck squamous cancer (HNSC) cell growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR)-T cells targeting FAT2 demonstrate anti-tumor activity. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal platform to inform therapeutic target discovery across cancers. Graphical abstract Highlights•Pan-cancer landscape of cancer-derived cell surface proteins detected in vivo•Discovery pipeline to prioritize proteins as immunotherapy target candidates•Validation of FAT2 as an SCC surface protein with minimal normal tissue expression•FAT2 CAR-T cells demonstrate anti-tumor activity in pre-clinical models Govindarajan et al. leverage N-glycoproteomics and PDX models to decode the in vivo cancer cell surfaceome and establish Glyco PDXplorer—a target discovery platform. The identification and validation of FAT2 as a previously undescribed,actionable antigen demonstrates the utility of Glyco PDXplorer for uncovering therapeutic vulnerabilities. View Publication

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior,drug response,and cell–matrix interactions. Here,we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates,facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation,GelMA preparation,bioprinting,and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research,including studies of metastasis,drug resistance,cancer cell lipid metabolism,and TME-associated hypoxia. View Publication

Neural crest cells contribute to craniofacial formation by differentiating into skeletogenic mesenchyme and neuro-glial lineages. Using Smart-seq2 single-cell transcriptomics,we show that mesenchymal fate commitment correlates specifically with the expression of rRNA-modifying and ribosome assembly factors,rather than structural ribosomal proteins. Notably,EMG1 and NHP2 introduce key post-transcriptional modifications into 18S rRNA,including m¹acp³ψ at U1248,which requires TSR3 for final maturation. Disrupting NHP2 or TSR3 in vitro and in vivo perturbs cranial neural crest differentiation; post-migratory temporal knockout of Polr1a or Polr1c also causes craniofacial malformations. These findings align with cell type-specific m¹acp³ψ levels during neural crest differentiation. Given the neural crest contribution to neuroblastoma,we analyze patient data to find that elevated ribosomal control and rRNA-modifying proteins predict poorer outcomes. Complementary experiments in neuroblastoma cell lines reveal functional roles for TSR3 and WDR74 in mesenchymal-like tumor states. Together,our results link rRNA modifications and ribosome assembly to fate decisions,suggesting ribosomal heterogeneity shapes both normal development and tumor progression. Neural crest cells differentiate into skeletogenic mesenchyme and neuro-glial lineages,thereby contributing to craniofacial formation. Here,single-cell analysis of cranial neural crest shows that specific rRNA modification and ribosome assembly factors contribute to skeletogenic fate. Their disruption causes craniofacial defects,while high levels in neuroblastoma predict poor survival. View Publication

BackgroundDiabetic peripheral neuropathy (DPN) is one of the most prevalent and debilitating complications of diabetes,marked by chronic neuroinflammation,immune dysregulation,and progressive neuronal degeneration. Current treatments offer limited efficacy,largely focusing on symptomatic relief rather than addressing the underlying disease mechanisms. There is a critical need for disease-modifying therapies that target the molecular basis of DPN.ResultsIn this study,we developed a novel targeted nanotherapeutic system—ZH-1c-EVs@SIN—composed of neural stem cell-derived extracellular vesicles (NSC-EVs) modified with the ZH-1c aptamer and loaded with the anti-inflammatory compound sinomenine (SIN). This system was specifically designed to target microglia and inhibit the WNT5a/TRPV1 signaling pathway. Transcriptomic profiling of microglia revealed key gene networks implicated in DPN pathology and responsive to SIN treatment. Functional assays demonstrated that ZH-1c-EVs@SIN facilitated a shift in microglial phenotype from pro-inflammatory M1 to anti-inflammatory M2,significantly reduced inflammatory cytokine expression,and restored levels of neuronal regulatory proteins. Nanoparticle tracking analysis and transmission electron microscopy confirmed optimal vesicle size and morphology,while fluorescence imaging showed efficient uptake by microglia. In vivo studies in a murine model of DPN revealed marked improvements in pain-related behavior and histopathological signs of nerve damage.ConclusionZH-1c-EVs@SIN represents a promising therapeutic strategy for DPN,offering targeted immunomodulation and enhanced neural repair via regulation of the WNT5a/TRPV1 signaling axis. This nano-delivery platform introduces a novel and precise approach to intervening in diabetic neuropathy and may be applicable to other neuroinflammatory conditions.Graphical abstractMechanism of ZH-1c-EVs@SIN Mediating the WNT5a/TRPV1 Pathway to Improve Immune-Inflammatory Homeostasis in the Treatment of DPN in Mice. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication