参考文献

Enhancers and transcription factors (TFs) are crucial in regulating cellular processes. Current multiomic technologies to study these elements in gene regulatory mechanisms lack multiplexing capability and scalability. Here we present single-cell ultra-high-throughput multiplexed sequencing (SUM-seq) for co-assaying chromatin accessibility and gene expression in single nuclei. SUM-seq enables profiling hundreds of samples at the million cell scale and outperforms current high-throughput single-cell methods. We demonstrate the capability of SUM-seq to (1) resolve temporal gene regulation of macrophage M1 and M2 polarization to bridge TF regulatory networks and immune disease genetic variants,(2) define the regulatory landscape of primary T helper cell subsets and (3) dissect the effect of perturbing lineage TFs via arrayed CRISPR screens in spontaneously differentiating human induced pluripotent stem cells. SUM-seq offers a cost-effective,scalable solution for ultra-high-throughput single-cell multiomic sequencing,accelerating the unraveling of complex gene regulatory networks in cell differentiation,responses to perturbations and disease studies. This work presents SUM-seq,an ultra-high-throughput method for co-profiling chromatin accessibility and gene expression in single nuclei across multiplexed samples,advancing the study of gene regulation in diverse biological systems. 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

AbstractObjectiveNatural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However,clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells,resulting in the generation of supercharged NK (sNK) cells.Methods and analysisWe explored the proteomic,transcriptomic and functional characterisation of sNK cells using cell imaging,flow cytometric analysis,51-chromium release cytotoxicity assay,ELISA,ELIspot,IsoPLexis single-cell secretome analysis,proteomic analysis,RNA analysis,western blot and enzyme kinetics.ResultsWe found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1,JUN,BHLHE40,ELF1,MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation,respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.ConclusionsThese results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process. View Publication

BackgroundMonocytes have been confirmed to increase in persistently food-allergic children. A phenomenon of innate immune memory,called trained immunity,has also been observed in monocytes from allergic children. However,the underlying mechanism remains poorly understood.MethodsWe enrolled a cohort of HDM-allergic children alongside age-matched healthy controls and established an HDM-sensitized allergic mouse model. Flow cytometric analyses were conducted to quantify monocyte frequencies in clinical cohorts and experimental animals. We performed integrated transcriptomic profiling via RNA-seq combined with chromatin occupancy analysis using CUT&Tag technology in parallel human and murine samples to elucidate the molecular mechanisms.ResultsIn our study,we demonstrated a reduced H3K27me3 methylation level accompanied by an increased proportion and a proinflammatory transcriptional memory in monocytes from house dust mite (HDM)-allergic human subjects. The same transcriptional and epigenetic phenotype was also confirmed in HDM-sensitized mice. Finally,the administration of GSK-J4,which upregulates H3K27me3 level in murine monocytes,attenuated the inflammatory response in vitro and in vivo.ConclusionsOur study confirms that H3K27me3 methylation modulates the trained immunity in monocytes and regulates HDM-allergic diseases through an inflammatory-dependent mechanism. View Publication

SummaryStudying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression,such as experimental autoimmune encephalomyelitis (EAE),is often limited by the availability of gene-edited mice. Here,we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction,preparation of retroviruses,viral delivery,and Th17 differentiation. We then detail procedures for in vivo functionality analysis.For complete details on the use and execution of this protocol,please refer to Zhong et al.1,2 Graphical abstract Highlights•Steps for designing and cloning dual U6gRNA cassettes to delete a specific CRE•Instructions for optimized retrovirus production and transduction into CD4+ T cells•Guidance on Th17 differentiation and confirmation of CRE deletion in cultured T cells•Procedures for adoptive transfer of CRISPR-edited Th17 cells to assess in vivo function Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression,such as experimental autoimmune encephalomyelitis (EAE),is often limited by the availability of gene-edited mice. Here,we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction,preparation of retroviruses,viral delivery,and Th17 differentiation. We then detail procedures for in vivo functionality analysis. View Publication

AbstractUnsupervised clustering is a powerful machine-learning technique widely used to analyze high-dimensional biological data. It plays a crucial role in uncovering patterns,structures,and inherent relationships within complex datasets without relying on predefined labels. In the context of biology,high-dimensional data may include transcriptomics,proteomics,and a variety of single-cell omics data. Most existing clustering algorithms operate directly in the high-dimensional space,and their performance may be negatively affected by the phenomenon known as the curse of dimensionality. Here,we show an alternative clustering approach that alleviates the curse by sequentially projecting high-dimensional data into a low-dimensional representation. We validated the effectiveness of our approach,named automated projection pursuit (APP),across various biological data modalities,including flow and mass cytometry data,scRNA-seq,multiplex imaging data,and T-cell receptor repertoire data. APP efficiently recapitulated experimentally validated cell-type definitions and revealed new biologically meaningful patterns. View Publication

BackgroundLevels of the human cationic antimicrobial host defence peptide LL-37 are enhanced in the lungs during neutrophilic airway inflammation. LL-37 drives Th17 differentiation,and Th17 cells produce IL-17A and IL-17F which form the biologically active heterodimer IL-17A/F. While IL-17 is a critical mediator of neutrophilic airway inflammation,LL-37 exhibits contradictory functions; LL-37 can both promote and mitigate neutrophil recruitment depending on the inflammatory milieu. The impact of LL-37 on IL-17-induced responses in the context of airway inflammation remains largely unknown. Therefore,we examined signaling intermediates and downstream responses mediated by the interplay of IL-17A/F and LL-37 in human bronchial epithelial cells (HBEC). As LL-37 can become citrullinated during airway inflammation,we also examined LL-37-mediated downstream responses compared to that with citrullinated LL-37 (citLL-37) in HBEC.ResultsUsing an aptamer-based proteomics approach,we identified proteins that are altered in response to IL-17A/F in HBEC. Proteins enhanced in response to IL-17A/F were primarily neutrophil chemoattractants,including chemokines and proteins associated with neutrophil migration such as lipocalin-2 (LCN-2). We showed that selective depletion of LCN-2 mitigates neutrophil migration,functionally demonstrating LCN-2 as a critical neutrophil chemoattractant. We further demonstrated that LL-37 and citLL-37 selectively suppress IL-17A/F-induced LCN-2 abundance in HBEC. Mechanistic studies revealed that LL-37 and citLL-37 suppresses IL-17 A/F-mediated enhancement of C/EBPβ,a transcription factor required for LCN-2 production. In contrast,LL-37 and citLL-37 enhance the abundance of ribonuclease Regnase-1,which is a negative regulator of IL-17 and LCN-2 in HBEC. In an animal model of allergen-challenged airway inflammation with elevated IL-17A/F and neutrophil elastase in the lungs,we demonstrated that CRAMP (mouse orthologue of LL-37) negatively correlates with LCN-2.ConclusionsOverall,our findings showed that LL-37 and citLL-37 can selectively suppress the abundance of IL-17A/F-mediated LCN-2,a protein that is critical for neutrophil migration in HBEC. These results suggest that LL-37,and its modified citrullinated form,have the potential to negatively regulate IL-17-mediated neutrophil migration during airway inflammation. To our knowledge,this is the first study to report that the immunomodulatory function of LL-37 enhances the RNA binding protein Regnase-1,suggesting that a post-transcriptional mechanism of action is mediated by the peptide.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12950-025-00446-w. View Publication

Zhang et al. demonstrate that the expression of a mutated CARMIL2 protein in CD28-deficient mice induces most of the developmental and functional consequences known to result from CD28 costimulation and in turn triggers potent tumor-specific T cell responses resistant to PD-1 and CTLA-4 blockade. Naive T cell activation requires both TCR and CD28 signals. The CARMIL2 cytosolic protein enables CD28-dependent activation of the NF-κB transcription factor via its ability to link CD28 to the CARD11 adaptor protein. Here,we developed mice expressing a mutation named Carmil2QE and mimicking a mutation found in human T cell malignancies. Naive T cells from Carmil2QE mice contained preformed CARMIL2QE-CARD11 complexes in numbers comparable to those assembling in wild-type T cells after CD28 engagement. Such ready-made CARMIL2QE-CARD11 complexes also formed in CD28-deficient mice where they unexpectedly induced most of the functions that normally result from CD28 engagement in a manner that remains antigen-dependent. In turn,tumor-specific T cells expressing Carmil2QE do not require CD28 engagement and thereby escape to both PD-1 and CTLA-4 inhibition. In conclusion,we uncovered the overarching role played by CARMIL2-CARD11 signals among those triggered by CD28 and exploited them to induce potent solid tumor–specific T cell responses in the absence of CD28 ligands and immune checkpoint inhibitors. View Publication

Metabolite nutrients within the tumor microenvironment shape both tumor progression and immune cell functionality. It remains elusive how the metabolic interaction between T cells and tumor cells results in different anti-cancer immunotherapeutic responses. Here,we use untargeted metabolomics to investigate the metabolic heterogeneity in patients with colorectal cancer (CRC). Our analysis reveals enhanced S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) metabolism in microsatellite stable (MSS) CRC,a subtype known for its resistance to immunotherapy. Functional studies reveal that SAM and SAH enhance the initial activation and effector functions of CD8+ T cells. Instead,cancer cells outcompete CD8+ T cells for SAM and SAH availability to impair T cell survival. In vivo,SAM supplementation promotes T cell proliferation and reduces exhaustion of the tumor-infiltrating CD8+ T cells,thus suppressing tumor growth in tumor-bearing mice. This study uncovers the metabolic crosstalk between T cells and tumor cells,which drives the development of tumors resistant to immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40170-025-00394-2. View Publication

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR),with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here,we introduce enGager,an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types,showing up to 6-fold higher efficiency compared to unfused Cas9,especially for large transgenes in primary cells. Notably,enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells,enhancing anti-tumor functionality. This ‘tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer,more efficient alternative to viral vectors for therapeutic gene modification. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9. View Publication