title: Generation and characterization of an immunogenic dendritic cell population.
abstract: Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.
authors.affiliation: The Liver Research Center, Providence, Rhode Island 02903, USA; The Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, USA.
authors.forename: Stephan
authors.initials: S
authors.lastname: Gehring
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Gregory
authors.forename: Philip
authors.initials: P
authors.lastname: Wintermeyer
authors.forename: Maryann
authors.initials: M
authors.lastname: San Martin
authors.forename: Costica
authors.initials: C
authors.lastname: Aloman
authors.forename: Jack R
authors.initials: JR
authors.lastname: Wands
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D019013:CD40 Antigens / Q000737:chemistry / Q000276:immunology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007114:Immunization / Q000379:methods; D018189:Immunomagnetic Separation; D016130:Immunophenotyping; D008024:Ligands; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000276:immunology*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D019013:CD40 Antigens; D016207:Cytokines; D008024:Ligands; D008070:Lipopolysaccharides; D017361:Viral Nonstructural Proteins; D051941:fms-Like Tyrosine Kinase 3; C497970:FLT3 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase