参考文献

Background and Objectives: Serum myelin oligodendrocyte glycoprotein (MOG) antibodies are a hallmark of the newly defined neuroinflammatory disease entity MOG antibody–associated disease (MOGAD). Yet,the lack of patient-derived recombinant human MOG (hMOG)–reactive autoantibodies limits investigations into the molecular mechanisms by which these autoantibodies mediate CNS pathology,thereby hindering rational therapeutic approaches. To understand the origins and disease-relevant mechanisms of autoantibodies in MOGAD,we generated and characterized monoclonal anti-hMOG antibodies (MOG-mAbs) from circulating B cells of patients with MOGAD.Methods: We isolated MOG-specific B-cell receptor (BCR) sequences from unique circulating B-cell clones of 6 patients with MOGAD using an antigen selection approach. BCR sequences were expressed as immunoglobulin (Ig)G1 antibodies,and their molecular features,epitope specificity,and binding to MOG isoforms were investigated. The MOG-mAbs’ ability to mediate antibody-dependent cellular phagocytosis (ADCP),natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC) toward MOG-expressing cells was assessed by live cell-based assays.Results: Of the 15 MOG-mAbs generated,4 revealed evidence of affinity maturation,whereas the remaining 11 were germline encoded. Binding capacities to hMOG varied considerably,with the most frequent putative epitope mapping to a region that includes residue P42. The efficacy of these antibodies in mediating ADCP,ADCC,and CDC of MOG-expressing cells was heterogeneous and associated with their binding characteristics to MOG and its isoforms.Discussion: Taken together,the molecular characteristics and binding patterns of these patient-derived MOG-mAbs reveal a diverse repertoire of MOG-binding autoantibodies with pathogenic capacity in vitro. Consequently,these well-characterized patient autoantibodies offer a foundation for developing in vivo models of MOGAD,serve as tools to standardize diagnostic assays,and guide development of therapeutic strategies targeting either B cells or autoantibodies and their effector functions. View Publication

Francisella tularensis (Ft),a Gram-negative bacterium that causes tularemia,possesses a non-inflammatory atypical LPS (LPSFt) that is highly immunogenic through unknown mechanism. We previously showed that immunization with LPSFt,a type 2 T-independent (TI) antigen,elicits protective LPSFt-specific IgM (IgMFt) and IgG3Ft by B1 cells in a mechanism dependent on the IL-5 produced by innate lymphoid cells type 2 (ILC2). Here,we examined the role of complement in the B1 cells’ response against LPSFt. C3-/-,C1q-/- and C4-/- mice immunized with LPSFt failed to produce IgMFt and IgG3Ft. In contrast,the response of Cfb-/- and Mbl1/Mbl2-/- mice was comparable to that of WT mice. Thus,activation of the classical complement cascade,but not the alternative or the Mannose Binding Lectin pathway,is required for activation of B1 cells and production of LPSFt-specific antibodies. Complement activation generates the C3d fragment,which opsonizes antigens for recognition by complement receptor-2 (CR2),and the C3a and C5a anaphylatoxins. Our results show that C3d opsonized LPSFt and that the response to immunization was dependent on CR2 expression by B1 cells. Importantly,the response to LPSFt immunization was also drastically decreased in C3ar1-/-,but not in C5ar1-/- mice. C3a induced IL-5 in ILC2,which supported B1 cells activation. Decreased antibody production in C3ar1-/- and Cr2-/- mice correlated with increased susceptibility to tularemia. Together,these results demonstrate that the high immunogenicity of LPSFt depends on two effector mechanisms triggered by activation of the classical complement pathway: 1) tagging of LPSFt with C3d fragment,leading to its interaction with CR2 expressed by B1 cells; 2) production of the anaphylatoxin C3a that stimulated IL-5 secretion by ILC2. Our study increases our understanding of the B1 cells’ response to TI-2 antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions. Author summaryThe lipopolysaccharide (LPS) of the bacterium Francisella tularensis strongly stimulates B cells for antibody production independently of T cell help through unknown mechanism. In the present study we examined the role of the complement cascade in this process. We found that production of antibodies against this LPS depends on activation of the classical complement pathway but not the MBL-dependent lectin or the alternative pathways. Following complement activation,LPS became tagged with the C3d complement fragment leading to its interaction with the complement receptor CR2 expressed by B cells. Complement activation also resulted in production of the anaphylatoxin C3a that was required for B cells activation,possibly through induction of IL-5 by innate lymphoid cells 2. Our study increases our understanding of the B cells’ response to T-independent antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions. View Publication

SF3B1,a component of the U2 snRNP pre-mRNA splicing factor,plays a critical role in splicing and is frequently mutated in cancer,particularly hematologic malignancies. We investigated the effects of the most common SF3B1 mutation,heterozygous substitution of Lysine 700 to Glutamate (K700E),in human embryonic stem cells (hESC),using CRISPR-Cas9 to generate heterozygous SF3B1K700E clones. Interestingly,we observed the upregulation of several key transcription regulators associated with hematopoiesis and a broad range of immune genes in SF3B1K700E hESCs. Despite differences in the transcriptional and splicing profiles between hESC and myelodysplastic syndrome (MDS) cells harboring the SF3B1K700E mutation,several common immune gene programs were identified in both cell types. To elucidate the molecular mechanisms underlying dysregulated gene expression in SF3B1K700E hESCs,we mapped actively engaged RNA polymerase II (RNA Pol II) using Precision Run-On sequencing (PRO-seq). These analyses revealed that the SF3B1K700E mutation alters RNA Pol II elongation properties. Specifically,we observed a general increase in pause release in SF3B1K700E hESCs,consistent with recent work in leukemia cells suggesting that the SF3B1K700E mutation affects early transcription elongation. Taken together,our study identifies several candidate genes that could contribute to the SF3B1 mutated phenotype and clarifies the role for the U2 snRNP and pre-spliceosome assembly on transcription by RNA Pol II. Further,our data suggest that mutations of SF3B1 impact immune gene expression independent of cell type,providing new insights into the role of SF3B1K700E in hematologic malignancies. View Publication

Lens transparency relies on proper intercellular communication. Exosomes are crucial mediators of intercellular communication and play a key role in organ homeostasis and development. However,their presence and dynamics in the lens remain unclear. Here,we report endogenous exosomes in the zebrafish lens using cryaa-driven Cd63-AcGFP labeling. Live imaging revealed dynamic exosome movement within lens cells and their potential transfer to adjacent tissues. Additionally,we found that the biogenesis of Cd63+ exosomes in the lens is regulated by the Syntenin-a pathway. And Syntenin-a knockdown delayed lens development by impairing lens cell differentiation,highlighting the potential role of lens cell–derived exosomes. Furthermore,ROR1+ lens progenitor cell-derived extracellular vesicles promoted lentoid differentiation in vitro,with proteomic analysis suggesting underlying mechanisms. Overall,our study addresses the gap in direct observation of endogenous lens exosomes,providing foundational insights into lens pathophysiology and a potential strategy for modulating the lens microenvironment. Visualization of endogenous exosomes in the zebrafish lens reveals their dynamic roles in intercellular communication and development,with Syntenin-a–regulated exosome biogenesis influencing lens cell differentiation. View Publication

The T-cell receptor (TCR) initiates T-lymphocyte activation,but the mechanism of TCR activation remains uncertain. Here,we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent,the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast,the HLA-bound complex adopts the open and extended conformation,and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together,these results establish allosteric conformational change during TCR activation,reveal avenues for immunotherapeutic engineering,and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here,the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs,revealing large conformational changes during activation. View Publication

MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening,we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover,we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program,as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets,reduces ribosome biogenesis,and impairs protein synthesis. Mechanistically,CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci,facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore,combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo,leading to more pronounced tumor growth suppression. Overall,our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation,suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB. MYC-driven medulloblastoma is an aggressive pediatric tumor with limited treatment options. Here,the authors show that CDK8 regulates ribosome biogenesis and that combined inhibition of CDK8 and mTOR demonstrates therapeutic efficacy in mouse models of this cancer. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. 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

Neural crest induction begins early during neural plate formation,requiring precise transcriptional control to activate lineage-specific enhancers. Here,we demonstrate that SALL4,a transcription factor associated with syndromes featuring craniofacial anomalies,plays a crucial role in early cranial neural crest (CNCC) specification. Using SALL4-het-KO human iPSCs to model clinical haploinsufficiency,we show that SALL4 directly recruits BAF to CNCC-lineage specific enhancers at the neuroectodermal stage,specifically when neural crest gene expression is induced at the neural plate border. Without functional SALL4,BAF is not loaded at chromatin,leaving CNCC enhancers inaccessible. Consequently,the cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression,despite normal neuroectodermal and neural plate progression. Moreover,by performing SALL4 isoform-specific depletion,we demonstrate that SALL4A is the isoform essential for CNCC induction and specification,and that SALL4B cannot compensate for SALL4A loss in this developmental process. In summary,our findings reveal SALL4 as essential regulator of BAF-dependent enhancer activation during early stages of neural crest development,providing molecular insights into SALL4-associated craniofacial anomalies. View Publication

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