title: Human dendritic cells pulsed with specific lipopeptides stimulate autologous antigen-specific T cells without the addition of exogenous maturation factors.
abstract: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.
authors.affiliation: Department of Immunology, Monash University, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia.
authors.forename: K L
authors.initials: KL
authors.lastname: Jones
authors.forename: L E
authors.initials: LE
authors.lastname: Brown
authors.forename: E M Y
authors.initials: EM
authors.lastname: Eriksson
authors.forename: R A
authors.initials: RA
authors.lastname: Ffrench
authors.forename: P A
authors.initials: PA
authors.lastname: Latour
authors.forename: B E
authors.initials: BE
authors.lastname: Loveland
authors.forename: D M
authors.initials: DM
authors.lastname: Wall
authors.forename: S K
authors.initials: SK
authors.lastname: Roberts
authors.forename: D C
authors.initials: DC
authors.lastname: Jackson
authors.forename: E J
authors.initials: EJ
authors.lastname: Gowans
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D002454:Cell Differentiation; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry; D006801:Humans; D007156:Immunologic Memory; D008074:Lipoproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D016895:Culture Media, Serum-Free; D018984:Epitopes, T-Lymphocyte; D008074:Lipoproteins; D010455:Peptides