Bedeutung von Toll-Rezeptoren und Typ I Interferonen für die Stimulation von CD8+ CTL und die Entstehung von Autoimmunität in der Haut
Zusammenfassung der Projektergebnisse
Ad 1) Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9- dependent maturation and IFN-a production: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. We explored the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). DC were derived from bone marrow precursors in vitro with the help of FLT3-Ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-a as well as lL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-a upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches. Ad 2) Langerin-expressing cells contribute to the induction of cytotoxic CD8+ T cell responses following gene gun immunization with replicase-based DNA expression plasmids: Particle-bombardment of the skin with plasmid DNA using the gene gun represents a novel vaccine approach which is able to induce antigen-specific CD8+ cytotoxic T cells important for protection against viral diseases and cancer. Here we further investigated the use of DNA expression plasmids encoding a Sindbis virus replicon to increase the efficacy of cutaneous gene gun immunization. We found that particle-mediated direct in vivo transfection of the skin with replicase-based expression plasmids led to increased mRNA expression but decreased protein expression when compared with conventional DNA expression plasmids. Replicasebased expression plasmids promoted significantly stronger systemic expansion and cytotoxic activity of antigen-specific CD8+ T lymphocytes. Both conventional and replicase-based expression plasmids critically required the presence of CD11c+ antigen-presenting dendritic cells for the induction of cytotoxic CD8+ T cell responses. The superior ability of replicasebased expression plasmids to stimulate cytotoxic CD8+ T cells was lost after removal of Langerin-expressing subpopulations of dendritic cells in the skin which include epidermal Langerhans cells. Taken together our experimental results provide evidence that Langerin-expressing skin dendritic cells contribute to the induction of cytotoxic CD8+ T cell responses following gene gun immunization with replicase-based expression plasmids.
Projektbezogene Publikationen (Auswahl)
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Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9- dependent maturation and IFNa production. J Gene Med 8; 1300-1306, 2006
Basner-Tschakarjan E, Gaffal E, O'Keeffe M, Tormo D, Limmer A, Wagner H, Hochrein H, Tüting T
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Evaluation of DNA vaccination with recombinant adenoviruses using bioluminescence imaging of antigen expression: Impact of application routes and delivery with dendritic cells. J Gene Med 8: 1046-1050, 2006
Schweichel D, Steitz J, Tormo D, Gaffal E, Ferrer A, Büchs S, Speuser P, Limmer A, Tüting T
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Attenuation of allergic contact dermatitis through the endocannabinoid system. Science 316: 1494-1497, 2007
Karsak M, Gaffal E, Date R, Wang-Eckhardt L, Rehnelt J, Petrosino S, Starowicz K, Steuder R, Schlicker E, Cravatt B, Mechoulam R, Buettner R, Werner S, Di Marzo V, Tüting T, Zimmer A
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Comparative evaluation of CD8+ CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells. Eur J Cell Biol 86:817-26, 2007
Gaffal E, Schweichel D, Tormo D, Steitz J, Lenz J, Basner-Tschakarjan E, Limmer A, Tüting T
