title: Direct transfection and activation of human cutaneous dendritic cells.
abstract: Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.
authors.affiliation: Department of Dermatology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA.
authors.forename: A T
authors.initials: AT
authors.lastname: Larregina
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: G
authors.initials: G
authors.lastname: Erdos
authors.forename: L A
authors.initials: LA
authors.lastname: Spencer
authors.forename: W J
authors.initials: WJ
authors.lastname: Storkus
authors.forename: D
authors.initials: D
authors.lastname: Beer Stolz
authors.forename: L D
authors.initials: LD
authors.lastname: Falo
mesh_terms: D019470:Biolistics / Q000379:methods*; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology*; D004817:Epidermis / Q000276:immunology; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006046:Gold / Q000493:pharmacokinetics; D006801:Humans; D007801:Langerhans Cells / Q000276:immunology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection; D019076:Transgenes
chemical_list: D000939:Epitopes; C105787:MART-1-Melan-A(27-35) epitope; D009363:Neoplasm Proteins; D006046:Gold