This methodology enabled revealing of interference ofH
This methodology enabled revealing of interference ofH. to CD4+T-cells is a prerequisite for safety againstH. pylori[1, 2], and persistent colonization might result from deficient antigen presentation and CD4+T-cell activation. The induction of human being T-cell immunity by antigen-presenting cells (APCs) involvingH. pyloristimulation has been analyzed before [35]. Bacterial virulence factors, however , were shown to negatively affect human being CD4+T-cell activation by exerting direct anti-proliferative effects as well as by inhibiting T-cell cytokine production, such as interleukin-2 (IL-2) or interferon- (IFN-) [69]. In addition , the bacteria may directly modulate functions of human being APCs [10]. To study the functional consequences of a possible modulation of antigen-processing and demonstration in professional APCs byH. pylori, confounders such as direct bacterial effects on T-cell functions have to be eliminated. Here, we investigated antigen demonstration by human being monocyte-derived dendritic cells (DCs) to a murine CD4+T-cell hybridoma. Murine T-cell functions are not affected byH. pyloristimulation and CD4+T-cell-derived hybridoma cells respond to cognate JNJ-31020028 demonstration of T-cell epitopes relatively independent of additional costimulatory interactions by APCs. The use of T-cell hybridoma cells from human being leukocyte antigen (HLA)-transgenic mice as responder cells offers previously been shown to enable quantitative detection of Rabbit Polyclonal to GLCTK antigen digesting by human being APCs [11]. By applying this methodology to theH. pylorisystem, we show thatH. pyloriexposure hampers MHC-II-restricted antigen presentation by human DCs. This effect may rely on bacterial factors shared at least within additional Gramnegative bacteria as it can partly be mimicked when substitutingH. pyloriwith lipopolysaccharide (LPS) fromEscherichia coli (E. coli). == Components and methods == == Preparation of H. pylori == H. pyloriP12 crazy type strain was grown and prepared because described elsewhere [10]. For activation, H. pyloriwas adjusted to a multiplicity of infection (MOI) of 10. == T-cell hybridoma cell culturing == AG85Baa97-112-specific, HLA-DR1-restricted F9A6 cells [11] were grown in 75 JNJ-31020028 cm2flasks (TPP, Trasadingen, Switzerland) in DMEM medium (Gibco/Invitrogen, Life Technologies, Darmstadt, Germany), supplemented with 2 mM L-glutamine (Gibco), penicillin (100 U/ml), streptomycin (100 g/ml), gentamicin (30 g/ml), ciprofloxacin (10 g/ml), 0. 05 mM 2-mercaptoethanol (all from Sigma, Taufkirchen, Germany), and 10% fetal calf serum (Bio-chrom, Berlin, Germany) at 37 C and 5% CO2. Cells were passaged every second day time and harvested from day time 3 to day 10. == Generation of human being HLA-DR1+DCs == Healthy blood donors that volunteered were HLA-typed by the tissue typing laboratory from the Charit, Campus Virchow Klinikum (Berlin, Germany). DCs were generated because described previously [10]. JNJ-31020028 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated via Fi-coll density gradient centrifugation. CD14+monocytes were enriched by using magnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), and 3 106cells/well were cultured in six-well culture dishes (TPP) in complete RPMI medium, that contain 2 mM L-glutamine, 10 mM HEPES (Gibco), penicillin (100 U/ml), streptomycin (100 g/ml), 10% FCS, one thousand U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (sargramostim, Leukine, Berlex, Richmond, CA), and 100 U/ml recombinant IL-4 (R&D Systems, Wiesbaden-Nordenstadt, Germany). Fresh medium supplemented with cytokines was added every second day time to the wells, and cells were harvested between day time 6 and day 8. Immature DCs displayed a down-regulated CD14 expression and were further defined by the expression of high levels of CD11b and low levels of CD80 [10]. == Activation of cells with H. pylori == A total of 1 105F9A6 cells was stimulated for 1 h at 37 C withH. pyloriin RPMI medium supplemented with 10% FCS. The cells were washed twice and subsequently transferred to one well of a 96-well plate (TPP) precoated with 3 g/ml anti-CD3 mAbs (BD Pharmingen) for another 12 h of incubation. Intended for the activation of DCs withH. pylori, 2 106cells were incubated in 500 l RPMI medium that contain 10% FCS, 1000 U/ml of GM-CSF, and 100 U/ml recombinant IL-4 intended for 1 h at 37 C in the presence ofH. pylori. LPS derived fromE. coli(Invivogen, Toulouse, France) (100 ng/ml) was used as a control stimulus while cells managed in medium alone served as bad control. After 1 h of incubation,.