Cross-presentation is more efficient than direct presentation of IAV.

<p>(<b>A</b>) DCs were exposed to infectious IAV, HI IAV or left untreated for 4 hr, washed and co-cultured with autologous CFSE labeled CD8 T cells at 1∶30 DC∶T cell ratio. After 10 days of co-culture, cells were harvested and stained with an HLA-A2 Influenza A M1 pentamer to detect M1-specific CD8 T cells by flow cytometry. T cell proliferation was detected by CFSE dilution. Dot plots show live CD8 T cells and numbers indicate frequency of positive CD8 T cells in each quadrant. One representative donor of four is shown. (<b>B</b>) Frequency of live CFSE^low CD8 T cells co-cultured with mDCs (blue) and pDCs (red) for 10 days, as described in (A), as a measurement of the total CD8 T cell response to IAV. Graph shows mean±SD from four donors. Differences were assessed using paired t test: n.s. no significant difference, * p<0.05, ** p<0.01. (<b>C</b>) DCs were exposed to increasing doses of pre-processed CMV pp65 peptide for 3 hr, washed and co-cultured with autologous CFSE-labeled CD8 T cells. After 10 days of co-culture, cells were harvested and stained with a CMV pp65 pentamer to detect CMV pp65-specific CD8 T cells by flow cytometry. The graph shows frequency of CMV pentamer+ CFSE^low CD8 T cells after co-culture with mDCs (blue) or pDCs (red) from one representative donor of five. (<b>D</b>) After 4 hr of exposure to infectious IAV or HI IAV, the expression of Influenza A protein was assessed using an anti-IAV polyclonal antibody (green) in HLA-DR (red) expressing MDCs and PDCs by confocal microscopy. Nuclei were stained using DAPI (blue). Scale bar 5 µm. (<b>E</b>) mDCs were exposed to infectious IAV or HI IAV for 8 hr and IAV+ mDCs were measured by MFI. The graph represents average MFI ± SD (n = 3) where 50–100 mDCs were analyzed for each donor. (<b>F</b>) mDCs were exposed to infectious IAV or HI IAV for 8 hr. Cells were lysed and analyzed by Western blot for viral protein content using a rabbit anti-IAV polyclonal antibody. The relative expression of IAV HA (51 kDa) and IAV M1 (28 kDa) was determined using ImageJ after normalizing the samples to GAPDH.