技术资料

Inadequate antigen-specific T cells activation hampers immunotherapy due to complex antigen presentation. In addition,therapeutic in vivo T cell expansion is constrained by slow expansion rates and limited functionality. Herein,we introduce a model fusion protein termed antigen-presenting cell-mimic fusion protein (APC-mimic),designed to greatly mimicking the natural antigen presentation pattern of antigen-presenting cells and directly expand T cells both in vitro and in vivo . The APC-mimic comprises the cognate peptide-human leukocyte antigen (pHLA) complex and the co-stimulatory marker CD80,which are natural ligands on APCs. Following a single stimulation,APC-mimic leads to an approximately 400-fold increase in the polyclonal expansion of antigen-specific T cells compared with the untreated group in vitro without the requirement for specialized antigen-presenting cells. Through the combination of single-cell TCR sequencing (scTCR-seq) and single-cell RNA sequencing (scRNA-seq),we identify an approximately 600-fold monoclonal expansion clonotype among these polyclonal clonotypes. It also exhibits suitability for in vivo applications confirmed in the OT-1 mouse model. Furthermore,T cells expanded by APC-mimic effectively inhibits tumor growth in adoptive cell transfer (ACT) murine models. These findings pave the way for the versatile APC-mimic platform for personalized therapeutics,enabling direct expansion of polyfunctional antigen-specific T cell subsets in vitro and in vivo . View Publication

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier,where the cells play essential roles in the digestion of food,absorption of nutrients and water,protection from microbial infections,and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling,which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells,indeed,is linked to gastrointestinal disorders such as irritable bowel syndrome,inflammatory bowel disease,and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and,therefore,homeostasis. Here,we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol,which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover,analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn’s Disease revealed that the dysregulation of peroxisome number,excessive cellular cholesterol,and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn’s Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis. Subject terms: Cell biology,Medical research View Publication

Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. In this study,we investigated the role of HES1,a member of HES family,in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry,histopathology analysis,and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers,decreases hematopoietic stem progenitor cell (HSPC) pool,results in defective multi-lineage differentiation. Functionally,fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1 fl/fl Flt3Cre embryos are resulted from decreased proliferation and elevated apoptosis,associated with de-repressed HES1 targets,p27 and PTEN in Hes1 -KO fetal HSPCs. Finally,pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. Together,our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis,and provide new insight into the differences between fetal and adult HSC maintenance. The online version contains supplementary material available at 10.1186/s13287-024-03836-8. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Mesenchymal stem cells (MSCs) have long been postulated as an important source cell in regenerative medicine. During subculture expansion,mesenchymal stem cell (MSC) senescence diminishes their multi-differentiation capabilities,leading to a loss of therapeutic potential. Up to date,the extrachromosomal circular DNAs (eccDNAs) have been demonstrated to be involved in senescence but the roles of eccDNAs during MSC. Here we explored eccDNA profiles in human bone marrow MSCs (BM-MSCs). EccDNA and mRNA was purified and sequenced,followed by quantification and functional annotation. Moreover,we mapped our datasets with the downloading enhancer and transcription factor-regulated genes to explore the potential role of eccDNAs. Sequentially,gene annotation analysis revealed that the majority of eccDNA were mapped in the intron regions with limited BM-MSC enhancer overlaps. We discovered that these eccDNA motifs in senescent BMSCs acted as motifs for binding transcription factors (TFs) of senescence-related genes. These findings are highly significant for identifying biomarkers of senescence and therapeutic targets in mesenchymal stem cells (MSCs) for future clinical applications. The potential of eccDNA as a stable therapeutic target for senescence-related disorders warrants further investigation,particularly exploring chemically synthesized eccDNAs as transcription factor regulatory elements to reverse cellular senescence. View Publication

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT,we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2),a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence,mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2,which could then serve as an early interventional target for HD. Subject terms: Huntington's disease,Mechanisms of disease,Stem cells View Publication

During embryogenesis,human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT),in which the HE cells undergo drastic functional and morphological changes from flat,anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development,molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study,by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures,we achieved real-time observation of the human EHT in vitro . By continuous observation and single-cell tracking in the culture,it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably,HSC precursor emergence was not linked to asymmetric cell division,whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT,and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development. View Publication

Clear cell renal cell carcinoma (ccRCC) demonstrates enhanced glycolysis,critically contributing to tumor development. Programmed death-ligand 1 (PD-L1) aids tumor cells in evading T-cell-mediated immune surveillance. Yet,the specific mechanism by which glycolysis influences PD-L1 expression in ccRCC is not fully understood. Our research identified that the glycolysis-related gene (GRG) HK3 has a unique correlation with PD-L1 expression. HK3 has been identified as a key regulator of O-GlcNAcylation in ccRCC. O-GlcNAcylation exists on the serine 900 (Ser900) site of EP300 and can enhance its stability and oncogenic activity by preventing ubiquitination. Stably expressed EP300 works together with TFAP2A as a co-transcription factor to promote PD-L1 transcription and as an acetyltransferase to stabilize PD-L1 protein. Furthermore,ccRCC exhibits interactive dynamics with tumor-associated macrophages (TAMs). The uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc),which serves as a critical substrate for the O-GlcNAcylation process,facilitates TAMs polarization. In ccRCC cells,HK3 expression is influenced by IL-10 secreted by M2 TAMs. Our study elucidates that HK3-mediated O-GlcNAcylation of EP300 is involved in tumor immune evasion. This finding suggests potential strategies to enhance the efficacy of immune checkpoint blockade therapy. Subject terms: Cancer metabolism,Renal cell carcinoma View Publication

As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line,and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically,mass culture to produce stock is no longer necessary; instead,a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small,closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study,atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment,expansion culture,and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion,atelocollagen beads enable 3D culture of iPSCs,and the quality of the obtained cells is at the same level as those derived from 2D culture. View Publication

Acute lymphoblastic leukemia (ALL) is the most common type of malignancy in children. ALL prognosis after initial diagnosis is generally good; however,patients suffering from relapse have a poor outcome. The tumor microenvironment is recognized as an important contributor to relapse,yet the cell-cell interactions involved are complex and difficult to study in traditional experimental models. In the present study,we established an innovative larval zebrafish xenotransplantation model,that allows the analysis of leukemic cells (LCs) within an orthotopic niche using time-lapse microscopic and flow cytometric approaches. LCs homed,engrafted and proliferated within the hematopoietic niche at the time of transplant,the caudal hematopoietic tissue (CHT). A specific dissemination pattern of LCs within the CHT was recorded,as they extravasated over time and formed clusters close to the dorsal aorta. Interactions of LCs with macrophages and endothelial cells could be quantitatively characterized. This zebrafish model will allow the quantitative analysis of LCs in a functional and complex microenvironment,to study mechanisms of niche mediated leukemogenesis,leukemia maintenance and relapse development. View Publication

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS,but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore,we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2,KIF5A,NEK1,TBK1,and TUBA4A) which highlighted many novel binding partners,and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system,and of ALS-associated C21ORF2 variants is largely unknown. Therefore,we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First,our data show C21ORF2 expression in ALS-relevant mouse and human neurons,such as spinal and cortical motor neurons. Further,the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients,but not isogenic controls,show increased apoptosis,and changes in DNA damage response,mitochondria and neuronal excitability. In addition,C21ORF2-V58L induced post-transcriptional downregulation of NEK1,an ALS-associated protein implicated in apoptosis and DDR. In all,our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. The online version contains supplementary material available at 10.1186/s40478-024-01852-6. View Publication

Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However,the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov),a known active constituent of Rhodiola,in IBD and the regulatory mechanisms. The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry,immunofluorescence,TUNEL,and EdU assays were applied to investigate the tight conjunction proteins expression,epithelial marker expression,number of apoptotic cells,and epithelial proliferation,respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally,RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably,Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly,Rov restored intestinal despair by increasing the number of Lgr5 + stem cells,Lyz1 + Paneth cells and Muc2 + goblet cells in intestines of colitis mice,displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids,Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5 +,Lyz1 + and Muc2 + cells) proliferation and defensin secretion,unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Rov holds potential as a natural prophylactic agent against IBD,with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy. View Publication