Gene Therapy (2001) 8, 608–617  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Direct transfection and activation of human cutaneous dendritic cells AT Larregina 1,2 , SC Watkins 2,3 , G Erdos 1,2 , LA Spencer 1,2 , WJ Storkus 4 , D Beer Stolz 3 and LD Falo Jr 1,2 1 Department of Dermatology, 2 University of Pittsburgh Cancer Institute, 3 Department of Cell Biology and Physiology, 4 Department of Surgery, Molecular Biology and Biochemistry and Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA USA 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 Keywords: genetic immunization; gene gun; human skin; dendritic cells; Langerhans cells; melanoma Introduction DNA vaccine technologies offer considerable promise as effective immunization strategies. 1,2 Cutaneous DC (epidermal Langerhans cells (LC) and dermal dendritic cells (DDC)) are potent antigen presenting cells (APC). 3,4 In the presence of the appropriate stimuli (tumor necrosis factor- (TNF-), IL-1, bacterial or viral components (lipopolysacharide, CpG DNA motifs, dsRNA)) cutaneous DC become activated and traffic to draining lymph nodes, where they can present antigenic peptides to naive T lymphocytes. 5–7 Taken together, these features suggest that skin DC may be ideal targets for the delivery of transgenic antigen for the purpose of genetic immunization. Using murine models our laboratory and others have demonstrated that after gene delivery to the skin, trans- fected DC are present in draining lymph nodes where they can induce specific immune responses. 8,9 Similar studies addressing the mechanism of genetic immuni- zation in human skin have not yet been reported. There are significant architectural differences between murine and human skin and considerable evidence that after skin transfection, the expression and distribution of transgenic proteins are different in human skin versus mouse skin. For example, in mice, following intradermal injection of Correspondence: LD Falo Jr, Department of Dermatology, 145 Lothrop Hall, 190 Lothrop St, Pittsburgh, PA, 15213, USA Received 28 September 2000; accepted 8 December 2000 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. Gene Therapy (2001) 8, 608–617. naked DNA, the expression of transgenic proteins is evi- dent in the epidermis, dermis, underlying fat tissue and muscle, while transgene expression in human skin is limited to the epidermis. 10 To analyze gene expression and the mechanisms of immune stimulation following gene delivery to human skin, we utilized human skin organ cultures. This experi- mental model of human skin remains viable for several days in culture, maintaining the anatomy and physiology of normal skin. 11,12 In the present work, we employed the gene gun to transfect human skin organ cultures. Our studies demonstrate that epidermal LC are transfected directly in situ. Biolistic delivery of DNA did not affect the viability, morphology or the immune phenotype of transfected DCs. Interestingly we find that the use of the gene gun induced a rapid mobilization of resident epi- dermal LC from transfected human skin. Importantly, when skin samples were transduced with a plasmid encoding the human melanoma Ag MART-1, migrating LC efficiently presented the MART1/Melan-A 27–35 pep- tide epitope to a human cytotoxic T cell (CTL) clone that recognizes the transgenic peptide sequence in the context of the MHC-class I HLA-A2 molecule. Results Gold particle delivery to human skin and epidermal dendritic cells in situ To evaluate gold particle delivery to human skin, and particularly to epidermal LC (resident in the basal