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. 2010 Feb 10;28(6):1620–1634. doi: 10.1016/j.vaccine.2009.11.014

Fig. 4.

Fig. 4

DNA vaccine expression constructs used in this study. (A) Constructs were all based on the pCIneo (Promega). pCI-CytOVAeGFP encodes a cytosolic form of an OVAeGFP fusion protein for simultaneous detection of OVA and eGFP in vivo. pCI-EαGFP encodes the EαGFP fusion protein described above and is used for identifying cells expressing (GFP) and presenting (Eα) the Ag EαGFP. pCI-EαRFP is similar to pEαGFP except for the substitution of RFP. (B–E) In vitro Ag presentation assay to demonstrate that plasmid-expressed EαGFP and EαRFP is processed and the Eα peptide displayed on the surface of APCs in vitro. HeLa cells were transfected with the plasmid constructs pCIneo, pCI-OVAeGFP, pCI-EαGFP, or pCI-EαRFP and B6 bone marrow DCs (+LPS) were added and cells were co-cultured for 24 h to allow DCs to acquire, process and display plasmid-expressed Ag. For positive controls, HeLa/DC co-cultures were pulsed with EαGFP or EαRFP protein. Cells were harvested, stained for CD11c and pMHC complexes using Y-Ae or isotype control (mIgG2b) and analysed by flow cytometry. (B) DCs pulsed with EαGFP were Y-Ae+ (surface Eα peptide:MHC ClassII complex) (black), whereas both unpulsed DCs (blue) and isotype controls (grey shading) show minimal staining. (C) Y-Ae+ DCs were only present when DCs were co-cultured with pCI-EαGFP-transfectants (black) but not with pCIneo (blue) nor pCI-CytOVAeGFP (red) 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. (D) Immunofluorescence staining of cytospins of EαRFP protein-pulsed HeLa/DCs shows, EαRFP-containing monocytic cells (red) with a larger dendritic-like cell also staining with Y-Ae mAb (green). Intact EαRFP Ag is also visible within the Y-Ae+ cell (indicated by arrow). (E) Y-Ae+ cells are also present in pCI-EαRFP-transfected HeLa/DC cultures (green). A transfected HeLa cell (red) can be seen adjacent to the Y-Ae+ cell. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)