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

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

The intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naive T(reg) cells with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of T(reg) impairments in multiple sclerosis (MS) is still contradictory,we analyzed naive PD1- and PD1+ T(reg) cells in peripheral blood and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS) patients and of healthy control subjects. Results showed that 1) CSF PD1- T(reg) cells were significantly augmented in MS patients; 2) PD1- T(reg) cells were significantly increased in the peripheral blood of patients with stable disease (SMS) compared to those with acute (AMS) disease,and in patients responding to glatiramer acetate (COPA) compared to AMS- and COPA-unresponsive patients; and 3) PD1+ T(reg) cells were similar in CSF and peripheral blood of all groups analyzed. PD1- T(reg) cells were not increased in the peripheral blood of interferon-beta (IFNbeta) -responsive patients,but the suppressive ability of T(reg) cells was significantly higher in SMS and in COPA- or IFNbeta-responsive compared to AMS- and COPA-unresponsive individuals. The data herein suggest that PD1- T(reg) cells play a pivotal role in MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNbeta. View Publication

Bacillus anthracis secretes two bipartite toxins,edema toxin (ET) and lethal toxin (LT),which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor,the catalytic subunit of ET,is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here,we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN,a marker of immature DCs,and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta,like LPS-matured DCs. Interestingly,cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET. View Publication

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or,when activated with IFN-gamma,an APC phenotype. Herein,TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta,IL-6,IL-8/CXCL8,and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB,inducible NO synthase (iNOS),and TRAIL upon TLR activation in MSC and macrophages,but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless,TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells,as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence,TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition,IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context,a mechanism that could be applied in a cell-based vaccine. View Publication

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine,but not human cells,and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models,NAb binding to porcine endothelial cells will likely induce complement activation,lysis,and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts,either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis,direct xenogeneic NK lysis,NAb-dependent ADCC,and adhesion of human NK cells under shear stress. Homologous recombination,panning,and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line,PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However,direct xenogeneic lysis of PED2*3.51,mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92,was not reduced. Furthermore,adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion,removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC,but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity,indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells. View Publication

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors,but the functional capacity of DC must be assessed in detail,especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant,providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol,resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors,or TLRs,do not show these functional features. Therefore,SeV-infected DC have the potential for DC-directed immunotherapy. View Publication

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here,we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen,while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner,while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells,a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack. View Publication

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa,which,through their ability to synthesize retinoic acid (RA),appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC,CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs,but not CX3CR1-expressing cells,migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover,CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs,whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens. View Publication

Wild-type (WT) NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) when given 0.05% NaI in their drinking water,whereas B cell-deficient NOD.H-2h4 mice are SAT resistant. To test the hypothesis that resistance of B cell-deficient mice to SAT was due to the activity of regulatory CD4+CD25+ T (T reg) cells activated if autoantigen was initially presented on non-B cells,CD25+ T reg cells were transiently depleted in vivo using anti-CD25. B cell-deficient NOD.H-2h4 mice given three weekly injections of anti-CD25 developed SAT 8 wk after NaI water. Thyroid lesions were similar to those in WT mice except there were no B cells in thyroid infiltrates. WT and B cell-deficient mice had similar numbers of CD4+CD25+Foxp3+ cells. Mice with transgenic nitrophenyl-specific B cells unable to secrete immunoglobulin were also resistant to SAT,and transient depletion of T reg cells resulted in severe SAT with both T and B cells in thyroid infiltrates. T reg cells that inhibit SAT were eliminated by day 3 thymectomy,indicating they belong to the subset of naturally occurring T reg cells. However,T reg cell depletion did not increase SAT severity in WT mice,suggesting that T reg cells may be nonfunctional when effector T cells are activated; i.e.,by autoantigen-presenting B cells. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. 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

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication