MC/9 cells were cultured for 1 hour with rIL-10 (0 25 ng/mL) or r

MC/9 cells were cultured for 1 hour with rIL-10 (0.25 ng/mL) or rIL-9 (Sigma; 12.5 U/mL) previously incubated for 1 hour with or without peptides (100 μg/mL) or anti-IL-10 neutralizing antibody (eBioscience) (1 μg/mL). Then cells were harvested and phospho-STAT-3 (signal transducer and activator of transcription 3) and actin content was determined by immunoblot as described.22 Blood was obtained from the Blood Bank of Navarra after informed consent. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) (2 × 105 cells/well) were stimulated in RPMI 1640 CM in 96-well plates with the Toll-like receptor 9 (TLR9) oligodeoxinucleotide

ligand CpG 221623 (Sigma-Genosys) (5 μg/mL) for pDC analysis, or with a monolayer of 2 × 104 irradiated control or CD40L-expressing BHK cells24 (kindly provided by H. Engelmann; Munich, Germany) Ensartinib purchase for mDC analysis, with or without recombinant HCV core protein (2.5 μg/mL) (BioDesign; Memphis, TN). IL-10-binding peptides (100 μg/mL) or anti-IL-10 antibody were added simultaneously to some wells in triplicate. Two days later, supernatants were harvested and enzyme-linked immunosorbent assay (ELISA) was used to measure content of IFN-α (Mabtech, CT99021 mw Sweden), IL-12 and IL-10 (BD-Biosciences,

San Diego, CA). Human monocyte-derived DC (MoDC) (105 cells/well), prepared as described,25 were stimulated with lipopolysaccharide (LPS) (1 μg/mL). Murine bone marrow-derived DC (BMDC) were prepared as described21 from C57BL/6 or HHD mice (transgenic for human HLA-A2.1 and beta-2 microglobulin molecules; a gift of Dr. F. Lemonnier, Institute Pasteur, Paris, France). Animals received humane care, maintained in pathogen-free conditions, and treated according to guidelines of our Institution PDK4 Review Board. Murine BMDC were cultured at 106 cells/mL with LPS (0.5 μg/mL) and peptide

1073-1081 (10 μg/mL) or were infected with a recombinant adenovirus encoding HCV NS3 (AdNS3) as described.21 In all cases, cells were cultured with or without IL-10 peptide inhibitors (100 μg/mL) for 24 hours, harvested, washed, and used for in vitro stimulation experiments or immunization, in the case of murine DC. To measure IL-12 production by human mDC, PBMC were stimulated with CD40L as described above, and monensin (BD-Biosciences) was added for the last 4 hours of culture. Then cells were stained with a cocktail of FITC-labeled anti-CD3, -CD14, -CD16, -CD19, -CD20, and -CD56, PerCP-labeled anti-HLA-DR and APC-labeled anti-CD11c antibodies (all from BD-Biosciences). After fixation and permeabilization, PE-labeled anti-IL-12 antibodies (Miltenyi, Germany) were added and IL-12 production was analyzed in the mDC population, gated as Lin-HLA-DR+CD11c+. Cells were acquired in a FACSCalibur flow cytometer (BD-Biosciences) and analyzed with Flowjo software (Tree Star, Ashland, OR).

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