Analysis of reporter gene expression in ovine dermis and efferent lymph dendritic cells in vitro and in vivo

Plasmid DNA administration has revolutionised approaches to vaccination, an d many studies have demonstrated the generation of both humoral and cytotox ic T cell responses which confer protection against live pathogen challenge . However, the mechanisms underlying DNA vaccination are poorly understood. Several studies have suggested the involvement of professional antigen pre senting cells such as dendritic cells (DC), but direct evidence for this is lacking. We have used the pseudoafferent lymphatic cannulation model in sh eep to study the expression of a plasmid encoding enhanced green fluorescen t protein (EGFP) by afferent lymph DC following administration to skin. The cells were analysed by flow cytometry. Preliminary studies were carried ou t to determine if the pEGFP would function in sheep cells in vitro. The res ults showed that electroporation of sheep skin fibroblasts, primary macroph ages, and afferent lymph DC with 30 mu g pEGFP resulted in varying degrees of fluorescence in these cells e.g. 35% of skin cells examined at 48 h, and 7% of afferent lymph DC examined after 4 h. Following intradermal injectio n of 120 mu g of pEGFP, small numbers of fluorescent DC (1-5%) were evident by flow cytometry after 1-4 h. The fluorescent DC continued to drain into the lymphatics over a period of 24 h. Analysis by PCR showed that free pEGF P appeared in the afferent lymph plasma within 1 h of injection, peaking at 2 h and becoming undetectable after 6 h. The results suggest that primary immune responses may be initiated by uptak e of soluble protein antigen by afferent lymph DC and by free plasmid rapid ly draining to the lymphatics where it may be taken up by DC in the lymph p lasma and the local lymph node. (C) 1999 Elsevier Science B.V. All rights r eserved.