BMDC, bone marrow-derived DC; DC, dendritic www.selleckchem.com/products/Rapamycin.html cells; HCV, hepatitis C virus; IFN-α, interferon alpha; IL-10, interleukin 10; MoDC, monocyte derived dendritic cells; PBMC, peripheral blood mononuclear cells. Phages from a library expressing 15-mer random peptides near the N-terminus of phage surface
protein pIII (a kind gift of GP Smith, University of Missouri-Columbia)19 were allowed to interact with biotinylated recombinant IL-10 (rIL-10) (Amersham Pharmacia Biotech, UK) as described.20 Three rounds of panning were carried out using 2.5, 0.02, and 0.002 μg/mL of rIL-10, respectively. After the third round, phages were eluted and their region coding for the 15-mer peptides was sequenced as described.20 Peptides identified using the phage library and used in initial screening assays as well as the human leukocyte antigen (HLA)-A2 restricted cytotoxic T lymphocyte (CTL) epitope from HCV NS3 1073-1081 and HCV NS3 peptide pools M2 and M4 were synthesized as described.21 Their purity was always above 80%. C-terminal amidated peptides p9 and p13, as well as control selleck kinase inhibitor peptide p301 (amino
acids 301-315 of HIV-1 gp120), were also purchased from NeoMPS (Strasbourg, France). MC/9 murine mast cell line (ATCC; Manassas, VA) was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 10% fetal bovine serum (complete medium; CM), and 5% rat T-STIM (BD-Biosciences, San Diego, CA). In IL-10-dependent proliferation assays, 2 × 104 cells were cultured in round-bottomed 96-well plates in CM containing
0.5 ng/mL of murine IL-4 (Peprotech, UK) and 1.25 ng/mL of human or murine rIL-10 (eBioscience, San Diego, CA), previously incubated for 2 hours with or without peptides. After 24 hours, 1 μCi of (methyl-3H)-thymidine (Amersham Life Science, Buckinghamshire, UK) was added per well and incubated for 12 additional hours before harvesting. Cells grown with or without IL-10 were used as positive (PC) and negative control (NC), respectively. Percentage of inhibition of IL-10 was calculated as: 100 × (cpmPC − cpmPT) / (cpmPC − cpmNC), where cpmPT corresponds to cpm obtained in the presence of IL-10 and peptides tested. Toxicity of the peptides was analyzed using similar bioassays with MC/9 MCE公司 cells but instead of rIL-10, murine GM-CSF (Peprotech, UK) at 0.01 ng/mL was used as stimulus. Peptide binding to IL-10 was analyzed by surface plasmon resonance using a BIAcore X Biosensor (BIAcore, AB, Uppsala, Sweden). IL-10 (R&D Systems) was covalently immobilized onto the surface of flow cell 2 (FC2) of a CM5 chip (BIAcore) as described.20 Flow cell 1 (FC1) without IL-10 was used as the reference flow cell. Peptide solutions (10 μM) were injected three times in 10 mM Hepes, 150 mM NaCl, 0.005% (v/v) Tween-20, 0.1 mg/mL BSA, pH 7.4 at a flow of 30 μL/min.