Thus, immunization with IC-loaded BMDCs induces both Th1 and CD8 effector immunity. Sciences University, Portland, Oregon, USA) and Alan Frey (New York University School of Medicine, New York, New York, USA). All mice entered the study at 6 to 8 8 weeks old. Cell lines. The MO-4 cell lines are stable OVA cDNA transfectants of B16F10 (provided by Alan Houghton, Memorial Sloan-Kettering Cancer Center, New York, New York, USA), and B16 cell lines (a C57BL/6 melanoma cell line) were obtained from the American Type Culture Collection (Manassas, Virginia, USA). Cell culture media consisted of RPMI-1640 (Cellgro, Herndon, Virginia, USA) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS). Geneticin (1 mg/ml) was added every week for maintenance of selective pressure on stable transformants. Antigens and other reagents. The H-2KbCrestricted peptide SIINFEKL, corresponding to amino acid residues 257C264, and the H-2DbCrestricted 323C339 peptide (ISQAVHAAHAEINEAGR) of chicken SN 38 OVA were synthesized Rabbit Polyclonal to ZNF695 by American Peptide Co. (Sunnyvale, California, USA) and by New England Peptide (Fitchburg, Massachusetts, USA) and highly purified ( 95%) as assessed by HPLC. Crystallized and lyophilized OVA was obtained from Worthington Biochemical Corp. (Lakewood, New Jersey, USA). The lyophilized rabbit IgG fraction to chicken albumin (anti-OVA) was purchased from Cappel ICN (Aurora, Ohio, USA). Peroxidase/antiperoxidase ICs were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). All reagents were SN 38 tested for measurable endotoxin activity with the Limulus Amebocyte Lysate kit (Biowhittaker, Walkersville, Maryland, USA) and were assessed directly for DC maturation activity. No reagent induced any change in the immunophenotype of DCs following 48 hours of incubation. Generation of bone marrow DCs. The procedure used for the generation of DCs was that described by Lutz et al. (24). After removing all muscle tissues from the femurs and tibiae, the bones were placed in a 100-mm dish with RPMI-1640. Both ends of the bones were cut with scissors in the dish, and then the marrow was flushed out using 2 ml of RPMI-1640 with a syringe and 25-gauge needle. The cell suspension was passed through nylon mesh to remove small pieces of bone and debris, and red cells were lysed with ammonium chloride. After washing, 2 105 cells were placed in a non-tissue-culture-coated Petri dish per ml of medium (RPMI, 10% FCS, penicillin, streptomycin, and L-glutamine) supplemented with either 20 ng/ml rGM-CSF (PeproTech Inc., Rocky Hill, New Jersey, USA) or 10% GM-CSF containing supernatant obtained from the GM-CSF transfectant J558L B cell hybridoma cell line (provided by Alan Houghton). At day 3, equal volume of fresh medium and SN 38 GM-CSF was added to the plate. At day 6, 50% of the medium was aspirated and replaced with equal volume of fresh medium containing GM-CSF. DCs were harvested between days 7 and 10 of culture based on the morphological accumulation of medium-sized 10- to 50-cell DC aggregates, which appear at this time loosely attached to adherent cells. Immaturity of cells was confirmed by flow cytometric determination of low-level expression of both MHC class II and CD86. DCs were collected from plates by gentle aspiration and collection of nonadherent cells, which uniformly bore the DC immunophenotypic signature CD11c+CD14C. Endocytosis and phagocytosis assays Cellular immunofluorescence assessment of OVA uptake. Immature wild-type (WT) or SN 38 bone marrowCderived dendritic cells (BMDCs) were plated on lysine-coated coverslips and incubated with 10 g/ml OVA alone, 50 g/ml anti-OVA, or OVA-ICs (50 g/ml anti-OVA and 10 g/ml OVA) for 30 minutes. Cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100. Cells were blocked in PBS/1% BSA, then stained with rabbit anti-OVA IgG (1:100 in PBS/1% BSA), washed, and visualized with FITCCanti-rabbit IgG (1:100 in PBS/1% BSA) using an E500 fluorescent microscope (Nikon Inc., Melville, New York, USA) and the RT.