技术资料

Improving effector T-cell functions is highly desirable for preventive or therapeutic interventions of diverse diseases. Signal transducer and activator of transcription 3 (Stat3) in the myeloid compartment constrains Th1-type immunity,dampening natural and induced antitumor immune responses. We have recently developed an in vivo small interfering RNA (siRNA) delivery platform by conjugating a Toll-like receptor 9 agonist with siRNA that efficiently targets myeloid and B cells. Here,we show that either CpG triggering combined with the genetic Stat3 ablation in myeloid/B cell compartments or administration of the CpG-Stat3siRNA drastically augments effector functions of adoptively transferred CD8+ T cells. Specifically,we show that both approaches are capable of increasing dendritic cell and CD8(+) T-cell engagement in tumor-draining lymph nodes. Furthermore,both approaches can significantly activate the transferred CD8(+) T cells in vivo,upregulating effector molecules such as perforin,granzyme B,and IFN-γ. Intravital multiphoton microscopy reveals that Stat3 silencing combined with CpG triggering greatly increases killing activity and tumor infiltration of transferred T cells. These results suggest the use of CpG-Stat3siRNA,and possibly other Stat3 inhibitors,as a potent adjuvant to improve T-cell therapies. View Publication

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known,but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG,OCT4,and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression,and increases the number of stem cells and their capacity for invasion,properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors. View Publication

The mTOR protein is a master regulator of cell growth and proliferation,and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1,which was identified in a biochemical mTOR assay,we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1),which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM,respectively. Moreover,Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC(50) = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

The active vitamin A metabolite retinoic acid (RA) imprints gut-homing specificity on lymphocytes upon activation by inducing the expression of α4β7 integrin and CCR9. RA receptor (RAR) activation is essential for their expression,whereas retinoid X receptor (RXR) activation is not essential for α4β7 expression. However,it remains unclear whether RXR activation affects the RA-dependent CCR9 expression on T cells and their gut homing. The major physiological RA,all-trans-RA,binds to RAR but not to RXR at physiological concentrations. Cell-surface CCR9 expression was often induced on a limited population of murine naive CD4(+) T cells by all-trans-RA or the RAR agonist Am80 alone upon CD3/CD28-mediated activation in vitro,but it was markedly enhanced by adding the RXR agonist PA024 or the RXR-binding environmental chemicals tributyltin and triphenyltin. Accordingly,CD4(+) T cells treated with the combination of all-trans-RA and tributyltin migrated into the small intestine upon adoptive transfer much more efficiently than did those treated with all-trans-RA alone. Furthermore,naive TCR transgenic CD4(+) T cells transferred into wild-type recipients migrated into the small intestinal lamina propria following i.p. injection of Ag,and the migration was enhanced by i.p. injection of PA024. We also show that PA024 markedly enhanced the all-trans-RA-induced CCR9 expression on naturally occurring naive-like regulatory T cells upon activation,resulting in the expression of high levels of α4β7,CCR9,and Foxp3. These results suggest that RXR activation enhances the RAR-dependent expression of CCR9 on T cells and their homing capacity to the small intestine. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study,we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover,they contributed directly to neovasculogenesis in ischemic mouse hind limbs,thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells. View Publication

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover,safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple,nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe,efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research,disease modeling,and regenerative medicine. ?? 2010 Elsevier Inc. View Publication

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However,despite the activity of this agent,PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus,we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal,we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment,exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together,our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells. View Publication

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells,we show,using single-cell biochemical analyses,that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction,as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance,and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation. View Publication

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here,endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo,reduced mammosphere formation,and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely,lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2,Nanog,and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog,KLF4,and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo. View Publication

Pulmonary bacterial infections are a leading cause of death. Since the introduction of antibiotics,multidrug-resistant Klebsiella pneumoniae became an escalating threat. Therefore,development of methods to augment antibacterial defense is warranted. Neutrophil recruitment is critical to clear bacteria,and neutrophil migration in the lung requires the production of ELR(+) CXC chemokines. Although lung-specific CXCL1/keratinocyte cell-derived chemokine (KC) transgene expression causes neutrophil-mediated clearance of K. pneumoniae,the mechanisms underlying KC-mediated host defense against K. pneumoniae have not been explored. In this study,we delineated the host defense functions of KC during pulmonary K. pneumoniae infection using KC(-/-) mice. Our findings demonstrate that KC is important for expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine,and activation of NF-κB and MAPKs in the lung. Furthermore,KC derived from both hematopoietic and resident cells contributes to host defense against K. pneumoniae. Neutrophil depletion in mice before K. pneumoniae infection reveals no differences in the production of MIP-2 and LPS-induced CXC chemokine or activation of NF-κB and MAPKs in the lung. Using murine bone marrow-derived and alveolar macrophages,we confirmed KC-mediated upregulation of MIP-2 and activation of NF-κB and MAPKs on K. pneumoniae infection. Moreover,neutralizing KC in bone marrow-derived macrophages before K. pneumoniae challenge decreases bacteria-induced production of KC and MIP-2,and activation of NF-κB and MAPKs. These findings reveal the importance of KC produced by hematopoietic and resident cells in regulating pulmonary host defense against a bacterial pathogen via the activation of transcription factors and MAPKs,as well as the expression of cell adhesion molecules and other neutrophil chemoattractants. View Publication

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However,the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2,whereas eosinophils and neutrophils expressed its cell-surface receptor,Robo1. Compared to neutrophils,eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1),leading to activation of Cdc42,recruitment of PI3K to Robo1,enhancment of eotaxin-induced eosinophil chemotaxis,and exaggeration of allergic airway inflammation. Notably,OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis,with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration,whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast,Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation,which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation. View Publication