技术资料

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules,we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization,lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization,dependent on dynein motor function,occurred before MTOC polarization,and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion,one that may prepare,but not commit cells for precision secretory function. View Publication

PURPOSE: The class I phosphatidylinositol 3' kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. EXPERIMENTAL DESIGN: We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. RESULTS: We found that models harboring mutations in PIK3CA,amplification of human epidermal growth factor receptor 2,or dual alterations in two pathway components were exquisitely sensitive to the antitumor effects of GDC-0941. We found that several models that do not harbor these alterations also showed sensitivity,suggesting a need for additional diagnostic markers. Gene expression studies identified a collection of genes whose expression was associated with in vitro sensitivity to GDC-0941,and expression of a subset of these genes was found to be intimately linked to signaling through the pathway. CONCLUSION: Pathway focused biomarkers and the gene expression signature described in this study may have utility in the identification of patients likely to benefit from therapy with a selective PI3K inhibitor. View Publication

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg,PD-L1) on tumor cells; however,little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM),an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011,a novel anti-PD-1 antibody,enhances human NK-cell function against autologous,primary MM cells,seemingly through effects on NK-cell trafficking,immune complex formation with MM cells,and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered. View Publication

A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface,whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However,some individuals with the protective -35CC genotype exhibit high viral loads. Here,we investigated whether the ability of HIV-1 to replicate efficiently in the protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However� View Publication

The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene,a member of the homeobox gene family,is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system,in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development,Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells,or the earliest definitive hematopoietic progenitors. In addition,Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation,because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus,Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors. View Publication

During disease progression in myelodysplastic syndromes (MDS),clonal blasts gain a more aggressive nature,whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples,we showed that the expression of an immunoinhibitory molecule,B7-H1 (CD274),was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor,pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore,B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined,blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover,MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together,these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression,which may be associated with disease progression in MDS. View Publication

In mouse,a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However,translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here,we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs,human DNGR-1+ BDCA3hi DCs express Necl2,CD207,BATF3,IRF8,and TLR3,but not CD11b,IRF4,TLR7,or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8,but not of TLR7,and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably,DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. View Publication

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens,including live cells,without the need for chemi-selective stains. Using a microspectrometer,near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly,compared to proteins and lipids,the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus,we could identify intensity ratios of particular protein-related bands (e.g.,757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology,proliferation,or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid,noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs. View Publication

Compared with adults,pediatric mastocytosis has a relatively favorable prognosis. Interestingly,a difference was also observed in the status of c-kit mutations according to the age of onset. Although most adult patients have a D(816)V mutation in phosphotransferase domain (PTD),we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus KIT-PTD mutants were introduced into rodent Ba/F3,EML,Rat2,and human TF1 cells to investigate their biologic effect. Both ECD and PTD mutations induced constitutive receptor autophosphorylation and ligand-independent proliferation of the 3 hematopoietic cells. Unlike ECD mutants,PTD mutants enhanced cluster formation and up-regulated several mast cell-related antigens in Ba/F3 cells. PTD mutants failed to support colony formation and erythropoietin-mediated erythroid differentiation. ECD and PTD mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT,whereas AKT was only activated by ECD mutants. Consistently,AKT inhibitor suppressed ECD mutant-dependent proliferation,clonogenicity,and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EML-PTD cells,suggesting the differential role of AKT in those mutants. Overall,our study implied different pathogenesis of pediatric versus adult mastocytosis,which might explain their diverse phenotypes. View Publication

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups,making these studies difficult. Here,we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion,the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs,especially for their homing potential. View Publication

Defining the molecular mechanisms underpinning fetal (gamma) globin gene silencing may provide strategies for reactivation of gamma-gene expression,a major therapeutic objective in patients with beta-thalassemia and sickle cell disease (SCD). We have previously demonstrated that symmetric methylation of histone H4 Arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for recruitment of the DNA methyltransferase DNMT3A to the gamma-promoter,and subsequent DNA methylation and gene silencing. Here we show in an erythroid cell line,and in primary adult erythroid progenitors that PRMT5 induces additional repressive epigenetic marks at the gamma-promoter through the assembly of a multiprotein repressor complex containing the histone modifying enzymes SUV4-20h1,casein kinase 2alpha (CK2alpha),and components of the nucleosome remodeling and histone deacetylation complex. Expression of a mutant form of PRMT5 lacking methyltransferase activity or shRNA-mediated knockdown of SUV4-20h1 resulted in loss of complex binding to the gamma-promoter,reversal of both histone and DNA repressive epigenetic marks,and increased gamma-gene expression. The repressive H4K20me3 mark induced by SUV4-20h1 is enriched on the gamma-promoter in erythroid progenitors from adult bone marrow compared with cord blood,suggesting developmental specificity. These studies define coordinated epigenetic events linked to fetal globin gene silencing,and provide potential therapeutic targets for the treatment of beta-thalassemia and SCD. View Publication

Because video data are complex and are comprised of many images,mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article,we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments,hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies,recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth,three recipes were created. The first segmented the image into the colony and background,the second enhanced the image to define colonies throughout the video sequence accurately,and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes,the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared,results were virtually identical,indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition,other video bioinformatics recipes can be developed in the future for other cell processes such as migration,apoptosis,and cell adhesion. View Publication