技术资料

It has been recently shown that increased oxidative phosphorylation,as reflected by increased mitochondrial activity,together with impairment of the mitochondrial stress response,can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD+-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance,leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors,without concurrent HSC exhaustion,improves survival by 80{\%},and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice,NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+-boosting strategies on the most primitive blood stem cells,establishing a link between HSC mitochondrial stress,mitophagy,and stem-cell fate decision,and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy. View Publication

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A,POLR3B,and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report,we describe three individuals with biallelic variants in POLR3GL,a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual,we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis,oligodontia,short stature,and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A,POLR3B,POLR1C,and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected. View Publication

Bacteriophage are abundant at sites of bacterial infection,but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf,and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-beta (TRIF)-dependent type I interferon production,inhibition of tumor necrosis factor (TNF),and the suppression of phagocytosis. Conversely,immunization of mice against Pf prevents Pa wound infection. Thus,Pf triggers maladaptive innate viral pattern-recognition responses,which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection. View Publication

Microbial infections are most often countered by inflammatory responses that are initiated through the recognition of conserved microbial products by innate immune receptors and result in pathogen expulsion1-6. However,inflammation can also lead to pathology. Tissues such as the intestinal epithelium,which are exposed to microbial products,are therefore subject to stringent negative regulatory mechanisms to prevent signalling through innate immune receptors6-11. This presents a challenge to the enteric pathogen Salmonella Typhimurium,which requires intestinal inflammation to compete against the resident microbiota and to acquire the nutrients and electron acceptors that sustain its replication12,13. We show here that S. Typhimurium stimulates pro-inflammatory signalling by a unique mechanism initiated by effector proteins that are delivered by its type III protein secretion system. These effectors activate Cdc42 and the p21-activated kinase 1 (PAK1) leading to the recruitment of TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase kinase kinase 7 (TAK1),and the stimulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) inflammatory signalling. The removal of Cdc42,PAK1,TRAF6 or TAK1 prevented S. Typhimurium from stimulating NF-kappaB signalling in cultured cells. In addition,oral administration of a highly specific PAK inhibitor blocked Salmonella-induced intestinal inflammation and bacterial replication in the mouse intestine,although it resulted in a significant increase in the bacterial loads in systemic tissues. Thus,S. Typhimurium stimulates inflammatory signalling in the intestinal tract by engaging critical downstream signalling components of innate immune receptors. These findings illustrate the unique balance that emerges from host-pathogen co-evolution,in that pathogen-initiated responses that help pathogen replication are also important to prevent pathogen spread to deeper tissues. View Publication

Lupus glomerulonephritis (GN) is an autoimmune disease characterized by immune complex-deposition,complement activation and glomerular inflammation. In lupus-prone NZM2328 mice,the occurrence of lupus GN was accompanied by a decrease in Treg cells and an increase in proinflammatory cytokine-producing T cells. Because IL-33 in addition to IL-2 has been shown to be important for Treg cell proliferation and ST2 (IL-33 receptor) positive Treg cells are more potent in suppressor activity,a hybrid cytokine with active domains of IL-2 and IL-33 was generated to target the ST2+ Treg cells as a therapeutic agent to treat lupus GN. Three mouse models were used: spontaneous and Ad-IFNalpha- accelerated lupus GN in NZM2328 and the lymphoproliferative autoimmune GN in MRL/lpr mice. Daily injections of IL233 for 5 days prevented Ad-IFNalpha-induced lupus GN and induced remission of spontaneous lupus GN. The remission was permanent in that no relapses were detected. The remission was accompanied by persistent elevation of Treg cells in the renal lymph nodes. IL233 is more potent than IL-2 and IL-33 either singly or in combination in the treatment of lupus GN. The results of this study support the thesis that IL233 should be considered as a novel agent for treating lupus GN. View Publication

Intratumoral (IT) STING activation results in tumor regression in preclinical models,yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here,clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution,low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation,and in the context of optimized T cell responses,TNFalpha is dispensable for tumor control. In a poorly immunogenic model,S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity. View Publication

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation,sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci,cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells,with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors. View Publication

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) are a promising class of immunotherapeutics that activate innate immunity to increase tumour immunogenicity. However,the efficacy of CDNs is limited by drug delivery barriers,including poor cellular targeting,rapid clearance and inefficient transport to the cytosol where STING is localized. Here,we describe STING-activating nanoparticles (STING-NPs)-rationally designed polymersomes for enhanced cytosolic delivery of the endogenous CDN ligand for STING,2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). STING-NPs increase the biological potency of cGAMP,enhance STING signalling in the tumour microenvironment and sentinel lymph node,and convert immunosuppressive tumours to immunogenic,tumoricidal microenvironments. This leads to enhanced therapeutic efficacy of cGAMP,inhibition of tumour growth,increased rates of long-term survival,improved response to immune checkpoint blockade and induction of immunological memory that protects against tumour rechallenge. We validate STING-NPs in freshly isolated human melanoma tissue,highlighting their potential to improve clinical outcomes of immunotherapy. View Publication

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However,functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells,pointing out the need of mandatory BCR co-factors in this process. Here,we investigated benefits of several BCR co-stimulatory molecules (IL-2,IL-4,IL-15,IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand,IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition,we established a proliferative advantage for ZAP70 positive CLL cells,associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover,the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies. View Publication

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However,responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here,we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction,cell-cycle progression,and transcriptional responses in HSPC subpopulations,with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation,constraining proliferation,yield,and engraftment of edited HSPCs. However,functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs. View Publication

Epstein-Barr Virus latent membrane protein-1 (LMP1) interacts with the SUMO-conjugating enzyme Ubc9,which induces protein sumoylation and may contribute to LMP1-mediated oncogenesis. After analyzing human lymphoma tissues and EBV-positive cell lines,we now document a strong correlation between LMP1 and sumo-1/2/3 or SUMO-1/2/3 levels,and show that LMP1-induced sumo expression requires the activation of NF-kappaB signaling through CTAR1 and CTAR2. Together,these results point to a second mechanism by which LMP1 dysregulates sumoylation processes and adds EBV-associated lymphomas to the list of malignancies associated with increased SUMO expression. View Publication