参考文献

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here,we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF,a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30{\%} engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF,as evidenced by up to 18{\%} HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells,defined by the markers CD34+CD90+CD45RA-,allowing for a 10-fold reduction in the number of transplanted target cells,thus considerably reducing the need for editing reagents. The frequency of engrafted,gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases. View Publication

Hypertrophic cardiomyopathy (HCM) is an inherited cardiomyopathy characterized by left ventricular hypertrophy ≥15 mm in the absence of loading conditions. HCM has a prevalence of up to one in 200,and can result in significant adverse outcomes including heart failure and sudden cardiac death. An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells obtained from the whole blood of a 38-year-old female patient with HCM in which genetic testing identified the well-known pathogenic p.Arg403Gln mutation in myosin heavy chain 7. iPSCs express pluripotency markers,demonstrate trilineage differentiation capacity,and display a normal 46,XX female karyotype. This resource will allow further assessment of the pathophysiological development of HCM. View Publication

Phospho flow is a powerful approach to detect cell signaling aberrations,identify biomarkers and assess pharmacodynamics,and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here,B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions,fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes. View Publication

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast,human T cells,a major anticancer effector cell,only rarely express Siglecs. However,here,we report that the majority of intratumoral,but not peripheral blood,cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity,cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement,which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1,but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted,glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset,while confining T-cell activation to the tumor microenvironment. View Publication

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15,a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients,significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues,and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors,we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore,STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM,53BP1,and MDC1. Furthermore,protein levels of these DNA damage response molecules are reduced by IL-15,as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally,pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. View Publication

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However,how DCs display functional plasticity and control host immune responses have not been fully understood. In this study,we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR),a C-type lectin receptor (CLR) expressed on human DCs,resulted in rapid activation of Syk,followed by PLCgamma2 and PKCdelta engagements. However,different from other Syk-coupled CLRs,including Dectin-1,signaling cascade through DC-ASGPR did not trigger NF-kappaB activation. Instead,it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB,which consequently bound to IL10 promoter and initiated cytokine expression. In addition,DC-ASGPR ligation activated Akt,which differentially regulated the activities of GSK-3alpha/beta and beta-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway,which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses. View Publication

The cancer stem cell (CSC) model suggests that a subpopulation of cells within the tumor,the CSCs,is responsible for cancer relapse and metastasis formation. CSCs hold unique characteristics,such as self-renewal,differentiation abilities,and resistance to chemotherapy,raising the need for discovering drugs that target CSCs. Previously we have found that the antihypertensive drug spironolactone impairs DNA damage response in cancer cells. Here we show that spironolactone,apart from inhibiting cancerous cell growth,is also highly toxic to CSCs. Notably,we demonstrate that CSCs have high basal levels of DNA double-strand breaks (DSBs). Mechanistically,we reveal that spironolactone does not damage the DNA but impairs DSB repair and induces apoptosis in cancer cells and CSCs while sparing healthy cells. In vivo,spironolactone treatment reduced the size and CSC content of tumors. Overall,we suggest spironolactone as an anticancer reagent,toxic to both cancer cells and,particularly to,CSCs. View Publication

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However,the efficiency with which they transduce target cells varies significantly,in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning,production,and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene. View Publication

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized,especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here,we show,using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy,that urethral macrophages contain integrated HIV-1 DNA,RNA,proteins and intact virions in virus-containing compartment-like structures,whereas viral components remain undetectable in urethral T cells. Moreover,urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide,while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor,CD206 and interleukin-4 receptor,but not CD163. To our knowledge,these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication. View Publication

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules,including PD-1,are preferentially expressed at the surface of persistently infected cells. However,whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals,we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely,PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication