参考文献

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming,but systematic interrogation of transcription factors,and their interactions with each other,has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells,revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation,with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons,which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition. View Publication

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. View Publication

The developmental pathways of broadly neutralizing antibodies (bNAbs) against HIV are of great importance for the design of immunogens that can elicit protective responses. Here we show the maturation features of the HIV-neutralizing anti-V1V2 VRC26 lineage by simultaneously sequencing the exon together with the downstream intron of VRC26 members. Using the mutational landscapes of both segments and the selection-free nature of the intron region,we identify multiple events of amino acid mutational convergence in the complementarity-determining region 3 (CDR3) of VRC26 members,and determine potential intermediates with diverse CDR3s to a late stage bNAb from 2 years prior to its isolation. Moreover,we functionally characterize the earliest neutralizing intermediates with critical CDR3 mutations,with some emerging only 14 weeks after initial lineage detection and containing only {\~{}}6{\%} V gene mutations. Our results thus underscore the utility of analyzing exons and introns simultaneously for studying antibody maturation and repertoire selection. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that,in human,the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution,we observe a continuous but polarised organisation of the 49f+ compartment,where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics,whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state,distinct from lymphoid-primed multipotent progenitors,representing the earliest entry point into lymphoid commitment. View Publication

Proteasome inhibitors bortezomib,carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM),but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites,$\beta$5i,$\beta$1i,and $\beta$2i,which are different from their constitutive $\beta$5c,$\beta$1c,and $\beta$2c counterparts. Bortezomib and carfilzomib block $\beta$5c and $\beta$5i sites. We report here that pharmacologically relevant concentrations of $\beta$5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition,increasing immunoproteasome expression by interferon-$\gamma$ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102,an inhibitor of $\beta$2 sites,dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus,immunoproteasome inhibitors,currently in clinical trials for the treatment of autoimmune diseases,should also be considered for the treatment of MM. View Publication

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived,non-circulating memory T-cells,it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study,we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates-and thereby expected lifespans-of memory T-cells from BM,blood and lymph nodes (LN). While the fraction of Ki-67 positive cells,a snapshot marker for recent cell division,was higher in memory T-cells from blood compared to BM and LN,in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery,and are continuously recirculating between the blood,BM,and LN. View Publication

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse,we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding,at the molecular level,how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here,we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally,we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization,as well as T cell migration in vitro. Altogether,our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility. View Publication

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis,induced by injection of the TLR-2 ligand,zymosan,we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein,we show that only in low-dose injected mice,the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose,the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells. View Publication

Cellular senescence,which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2,can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing,including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however,a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis,hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition,and degeneration of cortical and hippocampal neurons,thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively,these results show that senescent cells have a role in the initiation and progression of tau-mediated disease,and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies. View Publication