技术资料

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways,as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study,we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation,neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs,whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors,of which type I IFNs were one of the active components,played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types,MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore,signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs,with MyD88 playing a larger role than TRIF. In sum,in our experimental systems,TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation. View Publication

The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and,currently,it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells,and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly,our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells. View Publication

Human B cells can be cultured ex vivo for a few weeks,following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However,attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells,recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4,the main LPS receptor,prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells,accompanied by an increase in antigen-specific antibody secretion. This result suggested that some,but not all,B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6,a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo,the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria,until all pathogens have been cleared from the organism. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC,but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells,prerequisites for successful immunotherapy. Here,we investigated whether human tolDC,generated with dexamethasone and the active form of vitamin D3,maintained their tolerogenic function upon activation with LPS (LPS-tolDC),while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC,but not tolDC,expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore,LPS-tolDC were superior to tolDC in their ability to present type II collagen,a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic,naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype,although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential. View Publication

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally,copy number losses are identified using metaphase cytogenetics,whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such,is not often used clinically. Recently,introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study,we have applied 250K SNP array technology to detect previously cryptic chromosomal changes,particularly UPD,in a cohort of 301 patients with myelodysplastic syndromes (MDS),overlap MDS/myeloproliferative disorders (MPD),MPD,and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies,particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore,we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations,including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD,including CMML. View Publication

Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a procarcinogenic cytokine,IL-23,while inhibiting a central anticarcinogenic cytokine,IL-12,thereby shifting the balance of tumor immunity toward carcinogenesis. Stat3 induces expression of IL-23,which is mainly produced by tumor-associated macrophages,via direct transcriptional activation of the IL-23/p19 gene. Furthermore,Stat3 inhibits NF-kappaB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Tregs) express IL-23 receptor,which activates Stat3 in this cell type,leading to upregulation of the Treg-specific transcription factor Foxp3 and the immunosuppressive cytokine IL-10. These results demonstrate that Stat3 promotes IL-23-mediated procarcinogenic immune responses while inhibiting IL-12-dependent antitumor immunity. View Publication

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes,leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen,the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells,established B-leukemia cell lines,and animal models. In CLL cells,silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration,there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood,silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage,as evidenced by reactive oxygen species generation and membrane depolarization. In vivo,silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias. View Publication

AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4(+)CD7(-) Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1-mediated transformation,microarray analysis was performed to identify differentially expressed genes in AHI-1-suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4(+)CD7(-) Sezary cells from Sezary syndrome patients. HCK and BIN1 emerged as new candidate cooperative genes,with differential protein expression,which correlates with observed transcript changes. Interestingly,changes in HCK phosphorylation and biologic response to its inhibitor,dasatinib,were observed in AHI-1-suppressed or -overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells,which also exhibit differential MYC protein expression. In addition,aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells. View Publication

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are,in part,controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis,we have compared gene expression profiles of human erythroblasts,megakaryocytes,B cells,cytotoxic and helper T cells,natural killer cells,granulocytes,and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors,immunoglobulin superfamily members,and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude,ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition,we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg,GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data,which are freely accessible,will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. View Publication

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

Complete T cell development requires postthymic maturation,and we investigated the influence of this ontological period on the CD8 T cell response to infection by comparing responses of mature CD8 T cells with those of recent thymic emigrants (RTEs). When activated with a noninflammatory stimulus or a bacterial or viral pathogen,CD8 RTEs generated a lower proportion of cytokine-producing effector cells and long-lived memory precursors compared with their mature counterparts. Although peripheral T cell maturation is complete within several weeks after thymic egress,RTE-derived memory cells continued to express inappropriate levels of memory cell markers and display an altered pattern of cytokine production,even 8 weeks after infection. When rechallenged,RTE-derived memory cells generated secondary effector cells that were phenotypically and functionally equivalent to those generated by their mature counterparts. The defects at the effector and memory stages were not associated with differences in the expression of T cell receptor-,costimulation-,or activation-associated cell surface markers yet were associated with lower Ly6C expression levels at the effector stage. This work demonstrates that the stage of postthymic maturation influences cell fate decisions and cytokine profiles of stimulated CD8 T cells,with repercussions that are apparent long after cells have progressed from the RTE compartment. View Publication