Given its primary role as a barrier, the skin is the site of important immune functions. Invasive pathogens are contained locally, and also trigger the induction of protective adaptive immune responses. Genetic immunization with the gene gun aims to exploit the unique immune properties of the skin. Using this method, we observed that DNA plasmids encoding a Sindbis virus replicon induced significantly stronger cytotoxic T cell (CTL) responses compared to those achieved by conventional DNA expression plasmids. We found that this difference depended on the presence of langerin-expressing dendritic cells (DC) in the skin. Preliminary experiments support the hypothesis that sensing of cytosolic RNA replication by nucleic acid receptors in both keratinocytes and DC in the skin is critical for the generation of strong locoregional and systemic CTL responses. Both cell types are likely to promote antigen-presentation by activated DC in local draining lymph nodes and support peripheral effector and memory CTL functions in the skin. In our project we will evaluate how conventional and RNA replicon-based plasmid DNA vectors are recognized by innate immune receptors that detect foreign nucleic acids in keratinocytes and DC in the skin and how this influences antigen-processing and presentation by the different DC subpopulations to CD8+ T cells. Furthermore, we will investigate how activation of innate immune signalling pathways following gene gun immunization impacts on the expansion and functional differentiation of effector and memory CD8+ T cells in vivo, focussing on a localized, tissue-specific CTL response against a model self antigen expressed by melanocytes and melanoma cells in the skin. Our work will contribute to a better general understanding of the mechanisms governing the induction of CTL responses following gene gun-mediated direct in vivo transfection of the skin. This may help to design more effective genetic vaccine strategies in the future which imitate the immunogenicity of viral infection.