搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

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

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation,in innate cell clearance of microbial pathogens,and in neural cell maintenance. However,the role of autophagy in T lymphocyte development and survival is not known. Here,we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5-/- chimeric mice,we found that Atg5-deficient T lymphocytes underwent full maturation. However,the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery,Atg5-/- CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore,Atg5-/- CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation. View Publication

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However,the factors that regulate the magnitude of ADCC are incompletely understood. In this study,we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M,and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting,a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs. View Publication

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

Mucosa-associated invariant T (MAIT) cells are a unique innate T cell subset that is necessary for rapid recruitment of activated CD4(+) T cells to the lungs after pulmonary F. tularensis LVS infection. Here,we investigated the mechanisms behind this effect. We provide evidence to show that MAIT cells promote early differentiation of CCR2-dependent monocytes into monocyte-derived DCs (Mo-DCs) in the lungs after F. tularensis LVS pulmonary infection. Adoptive transfer of Mo-DCs to MAIT cell-deficient mice (MR1(-/-) mice) rescued their defect in the recruitment of activated CD4(+) T cells to the lungs. We further demonstrate that MAIT cell-dependent GM-CSF production stimulated monocyte differentiation in vitro,and that in vivo production of GM-CSF was delayed in the lungs of MR1(-/-) mice. Finally,GM-CSF-deficient mice exhibited a defect in monocyte differentiation into Mo-DCs that was phenotypically similar to MR1(-/-) mice. Overall,our data demonstrate that MAIT cells promote early pulmonary GM-CSF production,which drives the differentiation of inflammatory monocytes into Mo-DCs. Further,this delayed differentiation of Mo-DCs in MR1(-/-) mice was responsible for the delayed recruitment of activated CD4(+) T cells to the lungs. These findings establish a novel mechanism by which MAIT cells function to promote both innate and adaptive immune responses. View Publication

CD4+ T cells recognize antigens through their T cell receptors (TCRs); however,additional signals involving costimulatory receptors,for example,CD28,are required for proper T cell activation. Alternative costimulatory receptors have been proposed,including members of the Toll-like receptor (TLR) family,such as TLR5 and TLR2. To understand the molecular mechanism underlying a potential costimulatory role for TLR5,we generated detailed molecular maps and logical models for the TCR and TLR5 signaling pathways and a merged model for cross-interactions between the two pathways. Furthermore,we validated the resulting model by analyzing how T cells responded to the activation of these pathways alone or in combination,in terms of the activation of the transcriptional regulators CREB,AP-1 (c-Jun),and NF-kappaB (p65). Our merged model accurately predicted the experimental results,showing that the activation of TLR5 can play a similar role to that of CD28 activation with respect to AP-1,CREB,and NF-kappaB activation,thereby providing insights regarding the cross-regulation of these pathways in CD4+ T cells. View Publication

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model,we investigated whether expression of CD1a and langerin,an LC-specific C-type lectin,imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin,placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. View Publication

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1,-2,and -3) signals through the same heterodimeric receptor complex,which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution,the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells,IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition,IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13,IFN-gamma,and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential. View Publication