title: Sequential delivery of maturation stimuli increases human dendritic cell IL-12 production and enhances tumor antigen-specific immunogenicity.
abstract: BACKGROUND
  Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model.
  
  
  METHODS
  Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-gamma release Elispot assay using a CD8(+) MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-gamma release Elispot assay.
  
  
  RESULTS
  DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009).
  
  
  CONCLUSIONS
  Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.
authors.affiliation: Department of Surgery, Duke University Medical Center and Durham VA Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Kalady
authors.forename: Mark W
authors.initials: MW
authors.lastname: Onaitis
authors.forename: Sirisha
authors.initials: S
authors.lastname: Emani
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Abdel-Wahab
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Tyler
authors.forename: Scott K
authors.initials: SK
authors.lastname: Pruitt
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000276:immunology*; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen; D019013:CD40 Antigens / Q000378:metabolism; D023201:CD40 Ligand / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D016922:Cellular Senescence; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D000939:Epitopes; D006098:Granulocytes; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D007376:Interleukin-2 / Q000096:biosynthesis*; D007519:Isoantigens / Q000378:metabolism / Q000494:pharmacology; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000494:pharmacology; D010641:Phenotype; D015398:Signal Transduction; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000473:pathology; D014763:Viral Matrix Proteins / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D019013:CD40 Antigens; C497964:CD86 protein, human; D000939:Epitopes; C531098:HNA-4A antigen; D007376:Interleukin-2; D007519:Isoantigens; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D014763:Viral Matrix Proteins; D023201:CD40 Ligand; D018664:Interleukin-12; D007371:Interferon-gamma