技术资料

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents,yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers,it faces major limitations in solid tumors due to immune evasion,antigen loss,and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells,including multiple killing mechanisms and lower risks of graft-versus-host disease,neurotoxicity,and cytokine release syndrome,making them promising candidates for off-the-shelf cell therapies. However,unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment,persistence,and tumor-mediated suppression. To overcome these barriers,we developed a cost-effective method to engineer CAR NK cells targeting CD70,a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor,creating “armored” CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression,secrete IL-15,and demonstrate improved cytotoxicity,persistence,and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA. Graphical abstract Engineered “armored” CAR NK cells targeting CD70 overcome immune suppression in osteosarcoma,enhancing persistence,tumor homing,and cytotoxicity. This study presents a promising off-the-shelf immunotherapy approach for relapsed and metastatic OSA,offering a potential advance where current treatments have stagnated for decades. View Publication

Background: Chronic myocarditis (CMYO) progresses to fibrosis and heart failure,yet no therapies effectively target fibrosis. Fibroblast activation protein (FAP) marks pathogenic myofibroblasts,but its therapeutic potential remains unexplored in inflammatory settings.Methods: Using bulk/scRNA-seq of human myocarditis samples,we identified FAP as a fibrosis-specific marker. We engineered FAP-targeted CAR-T (FAP.CAR-T) cells and tested their efficacy in autoimmune (EAM) and viral (CVB3) myocarditis models. Human cardiac organoids (hCOs) treated with IL-17A modeled inflammatory fibrosis.Results: FAP expression correlated with fibrosis severity in patients (r = 0.96,P = 0.0028). In EAM and CVB3 models,FAP.CAR-T cells reduced fibrosis by 65% and 55%,respectively (P < 0.001),restored ejection fraction to higher than 65%. hCOs treated with FAP.CAR-T cells showed 55% less fibrosis (P < 0.05). No toxicity was observed in healthy mice.Conclusions: FAP.CAR-T cells eliminate fibrosis-driving myofibroblasts,reversing cardiac dysfunction in chronic myocarditis. This strategy,validated in human organoids,offers translatable immunotherapy for fibrosis-driven heart disease. View Publication

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

INTRODUCTION: Alzheimer's disease (AD) pathology is complex and involves mitochondrial dysfunction. There are emerging therapies targeting mitochondrial function in clinical trials for AD. This highlights the need for biomarkers that measure mitochondrial function. METHODS: We determined the utility of a novel blood‐based mitochondrial biomarker,the mitochondrial functional index (MFI),in the context of AD in a pilot study.RESULTS: In vitro and in vivo models of AD had a reduced MFI. MFI was lower in human AD subjects and APOE ????4 carriers. Receiver operating characteristic analysis showed MFI had a higher area under the curve than other plasma biomarkers. The MFI biomarker correlated with the Mini‐Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR) scale. DISCUSSION: This study highlights the potential utility of MFI as a functional blood‐based mitochondrial biomarker to interrogate energy metabolism. Ongoing studies are examining the relationship of MFI with brain energy metabolism outcomes. Highlights: The MFI biomarker is reduced in cell and animal models of AD. The MFI biomarker is reduced in human AD subjects and APOE ε4 carriers. The MFI biomarker can discriminate between subjects with normal cognition and AD with better performance than other plasma biomarkers. The MFI biomarker correlates with cognitive scores. 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 discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM: To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1,iMSC3),an iMatrix-free method (iMSC2),growth factor supplementation (iMSC4),and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria,requiring expression of surface markers (CD105,CD73,CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration. RESULTS: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001,respectively),while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01,respectively). Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001),reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION: While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications. 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