title: Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.
abstract: Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.
authors.affiliation: Department of Surgery, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC 27710, USA.
authors.forename: Robin M
authors.initials: RM
authors.lastname: Cisco
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Abdel-Wahab
authors.forename: Jens
authors.initials: J
authors.lastname: Dannull
authors.forename: Smita
authors.initials: S
authors.lastname: Nair
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Tyler
authors.forename: Eli
authors.initials: E
authors.lastname: Gilboa
authors.forename: Johannes
authors.initials: J
authors.lastname: Vieweg
authors.forename: Yehia
authors.initials: Y
authors.lastname: Daaka
authors.forename: Scott K
authors.initials: SK
authors.lastname: Pruitt
mesh_terms: D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology / Q000502:physiology*; D006098:Granulocytes; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis; D007519:Isoantigens / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D012313:RNA / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: C531098:HNA-4A antigen; D007519:Isoantigens; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D012313:RNA