Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles

Plasmid-encoded GM-CSF (pGM-CSF) is an adjuvant for genetic vaccines; howev er, little is known about how pGM-CSF enhances immunogenicity. We now repor t that pGM-CSF injected into mouse muscle leads to a local infiltration of potential APCs, Infiltrates reached maximal size on days 3 to 5 after injec tion and appeared in several large discrete clusters within the muscle. Imm unohistological studies in muscle sections from mice injected with pGM-CSF showed staining of cells with the macrophage markers CD11b, Mac-3, IA(d)/E- d and to the granulocyte marker GR-1 from day 1 through day 14, Cells stain ing with the dendritic cell marker CD11c were detected only on days 3 to 5, Muscles injected with control plasmids did not stain for CD11c but did sta in for CD11b, Mac3, IA(d)/E-d and GR-1, No staining was observed with the A PC activation markers, B7,1 or CD40, or with markers for T or B cells. Thes e findings are consistent with the infiltrating cells in the pGM-CSF-inject ed muscles being a mixture of neutrophils, macrophages, and immature dendri tic cells and suggest that the i,m, APCs may be enhancing immune responses to coinjected plasmid Ags, This hypothesis is supported by data showing tha t 1) separation of injections with pGM-CSF and Ag-expressing plasmid into d ifferent sites did not enhance immune responses and 2) immune enhancement w as associated with the presence of CD11c(+) cells in the infiltrates. Thus, pGM-CSF enhancement may depend on APC recruitment to the i,m, site of inje ction.