Although plasmid DNA immunization provides an effective means of inducing CTL responses to an expressed Ag, the mechanism by which CTL precursors are activated remains to be established. Insights could be gained by identifying the cells responsible for Ag presentation when DNA is introduced into different tissue sites. By immunizing parent into F1 bone marrow chimeric mice with an influenza nucleoprotein-expressing plasmid, we have demonstrated that the key cells in this presentation process for both gene gun-mediated epidermal injection and needle intramuscular injection of plasmid DNA are bone marrow derived. Furthermore, as assessed by intramuscular injection, coexpression of nucleoprotein with the costimulatory molecule B7-2, or the cytokines granulocyte-macrophage CSF and IL-12, did not convert nonhemopoietic cells into APCs. Thus, for two distinctly different modes of DNA immunization, in one case with or without coexpressed immunostimulatory factors, the APCs were consistently found to be of hemopoietic origin.

Surgical ablations revealed different dependencies on the injection site tissue for i.m. needle and epidermal gene gun DNA immunizations. Excision of an injected muscle bundle within 10 min of DNA inoculation did not affect the magnitude or longevity of Ag-specific Ab responses. By contrast, biopsy of the skin target site up to 24 h after gene gun bombardment completely abrogated the Ab response in the majority of mice. The form of a DNA-expressed Ag (membrane bound, secreted, or intracellular) did not affect the temporal requirements for the skin and muscle target sites. Skin cells, but not muscle cells, were also required for DNA-induced CTL. We conclude that transfected cells in gene gun-bombarded skin, but not needle-injected muscle, play a central role in DNA-initiated Ab and CTL responses.