技术资料

Thrombopoietin (Tpo),which binds to its specific receptor,the Mpl protein,is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor,as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565,Mpl-Y599 and Mpl-Y604. Here,we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599,mutation of which resulted in thrombocytopenia,deficient megakaryopoiesis,low hematopoietic stem cell (HSC) number and function,and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN),where Mpl is required for pathogenesis,thrombocytosis was dependent on intact Mpl-Y599. In contrast,Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression,and exacerbated thrombocytosis associated with MPN. View Publication

SummarySevere COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However,our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here,we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation,via toll-like receptor 2 signaling,and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli,releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung,indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance,contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections,and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection. Graphical abstract Highlights•SARS-CoV-2 envelope (E) protein activated innate immune cells through TLR2•E protein promoted a long-term tolerant immune state after initial activation•Monocytes in this tolerant state had reduced responsiveness to secondary stimuli•E protein priming reduced lung inflammation markers to LPS in neonatal mice Molecular biology; Immunity; Components of the immune system; Virology; Transcriptomics. View Publication

Alternative splicing events are a major causal mechanism for complex traits,but they have been understudied due to the limitation of short-read sequencing. Here,we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer’s disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms,providing insight into the human genome evolution. In addition,some of the isoforms are expressed in a cell-type specific manner,whose alternative 3’-UTRs usage contributes to their specificity. Further,we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing. This paper unveils the complexity of human immune cell splicing,highlighting cell-specific isoforms and establishing connections between alternative splicing and complex traits. These findings have implications for understanding diseases and the evolution of the genome. View Publication

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication

IntroductionImmune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason,we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature.MethodsBM samples from four SLE patients,six healthy controls,and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples,and single-cell RNA-sequencing was performed.ResultsA total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers,which were characteristic of each cell type,were identified. We identified three HSPC subpopulations,one of which consisted of proliferating cells (MKI67 expressing cells),one T-like,one B-like,and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures,in healthy BM of HSPC clusters 3 and 4 when compared with CB,and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations.DiscussionThis study revealed both quantitative—as evidenced by decreased numbers of non-proliferating early progenitors—and qualitative differences—characterized by an IFN signature in SLE,which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE,which may account for the immune aberrancies and the cytopenias in SLE. View Publication

Background:Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore,administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way.Methods:Primary human NK cells were treated with PKC modulators (bryostatin-1,prostratin,or the designed,synthetic bryostatin-1 analog SUW133),and evaluated by examining expression of activation markers by flow cytometry,analyzing transcriptomic profiles by RNA sequencing,measuring cytotoxicity by co-culturing with K562 cells,assessing cytokine production by Luminex assay,and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells.Results:PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines.Conclusions:Although PKC modulators have some significant effects on NK cells,their contribution in “kick and kill” strategies is likely due to upregulating HIV expression in CD4+ T cells,not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the “kick” rather than the “kill” arm of this HIV cure approach. View Publication

IntroductionSepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness.MethodsCellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression,RNA velocities,and biologic process clustering.ResultsWe identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally,we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome.DiscussionThe origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however,these cells exhibit a dynamic phenotype with considerable plasticity. View Publication

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here,we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore,gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks),and it was accompanied by epigenetic remodeling events. Finally,we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion,we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing. Graphical abstract David Fenard and colleagues developed a lentiviral backbone for the multiplexing of up to three ZF-R sequences,allowing an efficient,stable,and specific epigenetic control of multiple genes in T cells or Tregs after a single lentiviral transduction event. View Publication

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2–6,suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes,promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function,memory potential and metabolic fitness in settings of chronic stimulation,and exhibited enhanced persistence and tumour control in vivo. By contrast,overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably,FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes,underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells,and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory,and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity. View Publication

IntroductionB-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important.MethodsWe identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family,TRPV5,as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses,we generated a murine TRPV5 knockout (KO) by CRISPR–Cas9. ResultsWe found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K–RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly,calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however,differential expression of other calcium-regulating proteins,such as ORAI1,may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this,TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly,we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. DiscussionThus,our findings identify a role for TRPV5 in BCR signaling and B-cell activation. View Publication

AbstractLong single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology,yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli ‘helper’ strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability,scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA. Graphical Abstract Graphical Abstract View Publication

AbstractPolysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous,immune,and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases,including cancers and mental health disorders. However,the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase,endoneuraminidase NF (EndoN),and the catalytically inactive polySia lectin EndoNDM,have been extensively used for studying polySia. However,EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species,the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples,while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM,which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein,QSOX2,which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology. View Publication