For positive controls, HeLa/DC co-cultures were pulsed with EαGFP or EαRFP protein for 16 h. Cells were harvested, stained for CD11c and Y-Ae or CD11c and the Y-Ae isotype control (mouse IgG2b) and analysed by flow cytometry. DCs pulsed with EαGFP were Y-Ae+ (surface Eα peptide:MHC ClassII complex) ( Fig. 4B, black check details histogram), whereas both unpulsed DCs (blue histogram) and isotype controls (grey shading) show minimal staining. Flow cytometric analysis of CD11c+ cells from
plasmid-transfected HeLa/DC cultures, revealed Y-Ae+ DCs when DCs were co-cultured with pCI-EαGFP-transfectants ( Fig. 4C, black histogram) but not with pCIneo (blue histogram) or pCI-OVAeGFP (red histogram) control transfectants. Isotype controls showed little staining (grey shading). Flow cytometry results for pCI-EαRFP were similar to those for pCI-EαGFP and are not shown. Immunofluorescence staining of EαRFP protein-pulsed HeLa/DCs grown in chamber slides, clearly
demonstrated the presence of both Ag-laden cells (red) and pMHC+ (Y-Ae+) cells (green) ( Fig. 4D). Some unprocessed EαRFP can be seen in the cytosol of the Y-Ae+ cell (indicated by arrow). We also demonstrated pMHC+ cells (green) in pCI-EαRFP-transfected HeLa monolayers co-cultured with BMDCs ( Fig. 4E). In this example pCI-EαRFP-transfected HeLa cells expressing the EαRFP protein (red) can be seen adjacent to a Y-Ae+ cell (green), suggesting that the Y-Ae+ cell had acquired Ag or Eα peptide from another cell (i.e. cross-presentation). These results indicate that our Eα-based DNA vaccine constructs, PFT�� mouse in combination with the pMHC Ab Y-Ae, may be useful tools for identifying cells presenting DNA-encoded Ag in vivo. We prepared fluorescently labelled plasmid according to standard protocols,
injected labelled plasmid and attempted to identify its distribution and the phenotype of associated cells. Tissues including the TA muscle, draining popliteal and inguinal LNs, distal cervical and brachial LNs, spleen, peripheral blood and bone marrow, were collected 1 h and 24 h after however intramuscular injection of Cy5-labelled plasmid (pDNA-Cy5) or unlabelled control plasmid (pDNA). Cell suspensions and tissue sections were examined for the presence pDNA-Cy5 by flow cytometry and fluorescence microscopy (data not shown), respectively. We detected extensive Cy5+ signal in muscle 1 h after injection using fluorescence microscopy (data not shown). The signal was predominantly between muscle bundles and within myocytes, as has been shown by others previously [19]. During the preparation of the labelled pDNA we removed any unbound Cy5 by extensive washing and thus we are confident that Cy5 signal distribution corresponds with pDNA distribution. 1 h post-pDNA-Cy5 injection, we observed cell-associated pDNA-Cy5 in popliteal, inguinal and distal peripheral LNs by flow cytometry with the largest numbers found in the local muscle-draining popliteal LNs (Fig.