Fig. 6.

Detection of pMHC complexes in lymphoid tissue following DNA injection. (A) Flow cytometric analysis of pooled peripheral lymph nodes collected 3 days after pCI-EαRFP injection, revealed a small population of Y-Ae⁺CD11c⁺ cells (upper right quadrant), representing approximately 0.3% of live cells. 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 immunisation with EαRFP protein, i.e. several days after the peak of pMHC complex display. pCIneo-immunised mice and isotype (mIgG2b) controls showed only background staining (typically <0.1%). Results from one experiment (n = 2) are shown in (B) 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. Analysis of CD11c⁺ gated cells (C) showed that approximately 14% and 12% of CD11c⁺ cells were also Y-Ae⁺ for pCI-EαRFP and EαRFP protein, respectively. Although the percentage of CD11c⁺ cells displaying pMHC 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 positive following pCI-EαRFP injection. There was little change in Y-Ae expression following pCIneo immunisation.