We

wanted to determine if this same strategy was sufficiently sensitive to detect pMHC+ cells following DNA injection where small amounts of antigen are produced in vivo, in contrast to bolus injection of protein Ag. We were specifically interested in both the kinetics of appearance and the anatomical distribution of pMHC complex-bearing cells following pDNA injection. Flow cytometric analysis of live cells from pooled peripheral lymph nodes collected 3 days after pCI-EαRFP injection, revealed a small population of Y-Ae+CD11c+ cells, representing 0.34% of live cells (Fig. 6A, upper right quadrants). pCIneo-immunised mice and isotype (mIgG2b) controls showed only background staining (0.03% and 0.11%, respectively). The proportion of Y-Ae+CD11c+ cells in pCI-EαRFP-immunised mice (i.e. 0.34%) is comparable to that seen 3 days after PLX4032 ic50 immunisation with EαRFP protein, i.e. several days after the peak of pMHC complex display. Results from one experiment (n = 2) LY294002 chemical structure are shown in Fig. 6B and other experiments (n = 3) showed a similar trend. The percentage of Y-Ae+CD11c+ cells is higher in pCI-EαRFP-immunised mice compared to both pCIneo-immunised mice and for isotype control staining.

The percentage of Y-Ae+CD11c− cells in pCI-EαRFP-immunised mice was no different to that observed for pCIneo-immunised mice ( Fig. 6A, upper left quadrants), suggesting that the only cells that display pMHC complexes in DNA immunised mice are CD11c+ cells, presumably dendritic cells. This is in contrast to what we observed following EαRFP and EαGFP protein immunisation, where about 1% of live cells are Y-Ae+CD11c− ( Fig. 6 and Fig. 1). When we gated on CD11c+ cells from draining lymph nodes of pCI-EαRFP- and EαRFP protein-immunised mice at day 3 following injection, we observed that approximately 14% and 12% respectively of these CD11c+ cells were Y-Ae+ ( Fig. 6C). Although the percentage of CD11c+ cells displaying pMHC complexes was similar, the pattern of Y-Ae expression was quite different. We observed

a shift in Y-Ae expression for the entire population following EαRFP protein immunisation, relative to its’ isotype control, whereas only a discrete population was below positive following pCI-EαRFP injection. These cells were RFP− (data not shown), suggesting that the EαRFP protein had already been processed or was below the level that we could detect by flow cytometry. There was little change in Y-Ae expression following pCIneo immunisation. We could detect antigen GFP expression at the muscle injection site, 24 h after pDNA injection by immunofluorescence microscopy. GFP+ muscle cells could be easily distinguished from the autofluorescent oxidative fibres [20] (Fig. 7A and B) and were predominantly found in the vicinity of the injection site, as evidenced by the inflammatory infiltrate at the needle trajectory (Fig. 7B).