技术资料

Mantle cell lymphoma (MCL) is a clinically aggressive and incurable form of non-Hodgkin lymphoma with very heterogeneous clinical and biological behaviors. Dysregulation of RNA splicing machinery is common in various types of cancer,including hematologic malignancies,and is associated with cancer progression. However,whether and how splicing factors,the spliceosome components responsible for pre-mRNA splicing,regulate MCL aggressive behaviors remain largely unknown. Bioinformatics analyses were employed to profile the mRNA expression of two key families of splicing factors,that are serine/arginine-rich (SR) and heterogenous nuclear ribonucleoprotein (hnRNP) families,in clinical specimens of MCL patients in comparison to normal B cells. Functional roles of splicing factors in MCL aggressive phenotypes defined as hallmarks of cancer were investigated in MCL cell lines. Kaplan–Meier survival analyses were performed to determine the clinical significance of splicing factors according to their gene expression level. Depletion of SRSF1,hnRNP F,and PTBP1,which are highly expressed in MCL clinical specimens,by CRISPR/Cas9 system significantly inhibit cell growth and proliferation,motility,and angiogenesis of MCL cells. SRSF1,hnRNP F (for Z-138 cells),and PTBP1 were found to mediate BTZ sensitivity in MCL cells,in agreement with an increase in caspase-3 activation and the occurrence of splicing events that favor the expression of pro-apoptotic isoforms of apoptosis regulatory genes. We also found that depletion of SRSF1,hnRNP F,and PTBP1 induces the expression of autophagy-related genes and the accumulation of autophagic vacuoles,which may act as one of the tumor-suppressive mechanisms. Survival analyses revealed that co-high expression of SRSF1,HNRNPF,or PTBP1 and oncogenic MYC predicts poor clinical outcomes in MCL patients. Our data describe the clinical significance of aberrant SRSF1,hnRNP F,and PTBP1 in MCL and their tumor-promoting roles via the regulation of cancer hallmarks,which could be important in understanding MCL pathogenesis and therapeutic development. View Publication

Over the past decade,significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation,with organoids emerging as key experimental models. These three‐dimensional in vitro cellular structures mimic the organization and functions of various gut regions,providing a powerful tool for research. By replicating critical stages of gut development,we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol,we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First,we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three‐dimensional spheroids form spontaneously,can be collected,embedded in an extracellular matrix,and cultured over time. During this phase,the organoid epithelium develops,supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture,HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development,model diseases,and test therapeutic agents. Human pluripotent stem cells can be guided through a stepwise differentiation process to produce self‐organizing intestinal and colonic organoids. The resulting organoids recapitulate fetal‐stage epithelial and mesenchymal organization,offering a powerful model to explore human gastrointestinal development and its disorders. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Older adults have decreased vaccine efficacy,but the adjuvanted recombinant VZV-gE zoster vaccine (RZV) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL),which is much less efficacious. RZV increased NK,monocyte,and DC activation in response to in vitro VZV-gE stimulation for up to 5 years post-vaccination,while ZVL increased only DC responses to VZV for up to 90 days. In purified monocyte and NK cell cocultures,RZV recipients showed increased responses to VZV-gE,HCMV and HSV antigenic stimulation post-vaccination. ATAC-seq analysis of purified monocytes revealed decreased accessibility in areas of the TGFβ1 gene. scRNA-seq and immunoproteomics confirmed decreased TGFβ1 transcription and translation,respectively. Exogenous supplementation and inhibition of TGFβ1 modulated in vitro monocyte responses to VZV-gE. In conclusion,RZV generated homologous (VZV-gE) and heterologous (HCMV,HSV) trained immunity in monocytes through genomic repression of the regulatory cytokine TGFβ-1. Cytokine modulation may represent a novel mechanism of generating trained immunity in myeloid cells. Author summaryOlder adults have decreased vaccine efficacy,but the adjuvanted recombinant varicella zoster virus (VZV)-gE zoster vaccine (RZV; Shingrix™) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL; Zostavax™),which is much less efficacious than RZV. We found that RZV increased the functionality of several innate immune cell subsets against VZV-gE other herpesviruses. The increase in functionality was associated with decreased production of the inhibitory cytokine TGFβ1,which may have resulted from decreased ability to use the TGFβ1 gene as a template for the synthesis of its product. We concluded that RZV generated homologous (VZV-gE) and heterologous (other herpesviruses) memory-like responses in innate immune cell subsets through genomic repression of the regulatory cytokine TGFβ-1. View Publication

Systemic sclerosis (SSc) is an autoimmune disease marked by fibrosis and extensive autoantibody production. Although B-cell depletion with rituximab (RTX) has shown clinical benefit,predictive biomarkers of response remain elusive. Here,we apply proteome-wide autoantibody screening using wet protein arrays covering 13,455 human antigens in serum samples from participants of the randomized trial of RTX. We identify a significant elevation in the total autoantibody levels in SSc compared to healthy controls,with greater reductions post-treatment observed in RTX high responders than in low responders. A stepwise selection highlights 88 clinically relevant autoantibodies,including those targeting G protein-coupled receptors. Among them,anti-C-C motif chemokine receptor 8 (CCR8) autoantibodies are functionally validated by cell-based assays using CCR8-overexpressing HEK293 cells. Furthermore,in a bleomycin-induced mouse model,anti-CCR8 antibody administration exacerbates dermal fibrosis and modifies immune cell infiltration. Although external validation with multiple comparison adjustment is further required,these findings reveal an autoantibody signature associated with therapeutic response and pathogenic potential in SSc,providing a foundation for precision immunotherapy and mechanistic insights into disease progression. B-cell depletion benefits systemic sclerosis,but predictive biomarkers remain limited. The authors here map autoantibody profiles using proteome-wide screening,identify C-C motif chemokine receptor 8-targeting autoantibodies with functional impact,suggesting novel pathophysiology and precision therapy targets. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs),only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients,the antitumor immune response is defective,conferring resistance and promoting tumor progression. Therefore,the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs),at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate,progression-free survival (PFS),overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy,higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs,identified patients with significantly worse survival. Furthermore,CD137⁺Tregs resulted also positively correlated with worse PS,while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab,with a significant increase in non-responsive patients during therapy. Interestingly,both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137−Tregs and LOX-1⁻PMN-MDSCs,respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis,as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy,improving patient selection and suggesting novel personalized therapies. View Publication

Endogenous long double-stranded RNAs (dsRNAs),which are not edited by the RNA editing enzyme ADAR1,may activate the antiviral dsRNA receptor MDA5 to trigger interferon-mediated immune responses. Among the large number of endogenous long dsRNAs,the key substrates that activate MDA5—termed as immunogenic dsRNAs—remain largely unidentified. Here we reveal that human immunogenic dsRNAs constitute a surprisingly small fraction of all cellular dsRNAs. We found that these immunogenic dsRNAs were highly enriched in mRNAs and depleted of introns,consistent with their role as cytosolic MDA5 substrates. We validated the MDA5-dependent immunogenicity of these dsRNAs,which was dampened following ADAR1-mediated RNA editing. Notably,immunogenic dsRNAs were enriched at genetic susceptibility loci associated with common inflammatory diseases,implying their functional importance. We anticipate that a focused analysis of immunogenic dsRNAs will enhance our understanding and treatment of cancer and inflammatory diseases,where the roles of dsRNA editing and sensing are increasingly recognized. The authors show that only a small subset of cytosolic double-stranded RNAs (dsRNAs) requires ADAR1-mediated RNA editing to evade an MDA5-dependent immune response. These immunogenic dsRNAs are enriched in mRNAs and overlap with GWAS signals for common inflammatory diseases. View Publication

Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However,it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here,we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors,ATG8-like proteins,or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy,while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening,protein engineering,and structural analysis,this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation. View Publication

Purpose: Noninfectious uveitis is a sight-threatening autoimmune eye disease lacking effective targeted therapies. Dipyridamole (DIP),a phosphodiesterase (PDE) inhibitor,has demonstrated anti-inflammatory properties in inflammatory diseases. However,its application in uveitis remains unexplored. Methods: We used single-cell RNA sequencing (scRNA-seq) data from experimental autoimmune uveitis (EAU) mice and uveitis patients to assess the potential association of PDE gene expression with disease development. Subsequently,EAU mice received oral DIP (300 mg/kg/day),starting at different time points (preventative,early-therapeutic,or late-therapeutic),and treatment efficacy was assessed. To explore immune components and signaling changes,we profiled cervical draining lymph nodes (CDLNs) from control,EAU,and DIP-treated mice by scRNA-seq and validated key findings with additional experiments. Mechanistically,pharmacologic interventions (an adenylyl cyclase inhibitor and the STAT3 agonist) were used in vitro. Results: Expression of several PDE genes correlated with uveitis severity in both human and mouse. Preventative DIP treatment most effectively reduced fundus inflammation in EAU and modulated the Teff/Treg ratio in the CDLNs and spleens. In vitro,DIP suppressed CD4+ T cell proliferation,and inhibited pathogenic Teff. scRNA-seq analysis revealed that DIP partially reversed EAU-induced transcriptional alterations,with notable changes in immune cell composition and pathway activity. Mechanistically,DIP downregulated STAT3 activity and PIM1 expression in Th17 cells via cAMP,suggesting the involvement of the cAMP-STAT3-PIM1 axis in modulating immune homeostasis. Conclusions: DIP ameliorated intraocular inflammation,modulated Th17/Treg balance,and reduced Th17 pathogenicity in EAU,potentially via cAMP-STAT3-PIM1 signaling. These findings highlight DIP as a promising therapeutic candidate for autoimmune uveitis. View Publication

Cutaneous tuberculosis (CTB) is an infectious disease highly associated with extracellular matrix remodeling and granuloma-driven fibrosis. Fibroblasts play crucial roles in this fibrotic process,but their specific roles in Mycobacterium tuberculosis (Mtb) skin infections remain unclear due to the lack of proper in vitro models. Here,we demonstrate that skin organoids (SKOs) derived from human induced pluripotent stem cells can model CTB infected by Mtb. Single-cell RNA analyses reveal an increase in fibroblasts,upregulation of genes involved in collagen synthesis,and enhanced collagen degradation induced by MMP2 and MMP14 in Mtb-infected SKOs. This is accompanied by the destruction of nerve cells and adipocytes. Importantly,the onset of fibrosis in Mtb-infected SKOs is dependent on the activation of the PI3K-AKT signaling pathway and transcription factor AP1 in fibroblasts. Pharmacological inhibition of PI3K-AKT and AP1 alleviates fibrosis and collagen deposition. Our findings have uncovered distinct alterations in cell populations during Mtb-induced skin fibrosis,highlighting the crucial roles of PI3K-AKT and AP1. The study demonstrates the utility of SKOs for investigating CTB pathogenesis and evaluating potential antifibrotic treatments. Cutaneous tuberculosis is an infectious disease associated with extracellular matrix remodeling and granuloma-driven fibrosis. Here,the authors present an in vitro model of this disease using skin organoids infected with Mycobacterium tuberculosis,and describe infection-induced alterations in specific pathways and cell populations. View Publication

Acute myeloid leukemia (AML) is primarily driven by leukemic stem cells (LSCs),the main cause of relapse and therapy resistance. Here,we discover that LSCs are predominantly small and mechanically soft. These mechanical properties enable their selective isolation using microfluidic chips. Single-cell RNA-sequencing of primary human AML bone marrow identifies enrichment of LSCs within the FSClow ALDH1A1+ subpopulation,which exhibits long-term stemness in functional assays. Notably,inhibiting ALDH1A1 in these cells promotes F-actin polymerization and increases cellular stiffness,reducing their stemness while enhancing their susceptibility to natural killer (NK) cell-mediated cytotoxicity. In AML patient-derived xenograft models,the combination of ALDH1A1 inhibition with NK cell therapy markedly suppresses leukemia progression. These findings suggest that targeting the mechanical properties of LSC offers a promising strategy to overcome AML treatment resistance,providing insights into stem cell mechanobiology and paving the way for combining targeted therapies with immunotherapy to improve clinical outcomes. Leukemic stem cells (LSCs) drive relapse and therapy resistance in acute myeloid leukemia (AML). Here,the authors show that increasing the stiffness of LSCs reduces their stemness and enhances their susceptibility to natural killer cell-mediated immunotherapy in AML. View Publication