(A) pDCs (red) and mDCs (blue) were continuously exposed to IAV for 1, 6 or 24 hr and the frequency of IAV+ DCs was measured by flow cytometry using a rabbit polyclonal antibody raised against IAV/X31. Graph shows mean±SD percent of IAV+ CD123+ CD14− pDCs and CD11c+ CD14− mDCs (n = 7). Differences between IAV infection of mDCs and pDCs were assessed using paired t test: n.s. no significant difference, ** p<0.01, *** p<0.001. (B) DCs were treated with NH₄Cl, then exposed to IAV for 24 hr at 4°C or 37°C. The frequency of IAV+ DCs and level of CD86 expression was determined by flow cytometry. One representative experiment of six is shown. Dot plots show live DCs and numbers in each quadrant depict the frequency of positive DCs. (C) DCs were exposed to IAV for 1 hr, washed 3 times to remove free virus and allowed to adhere to coverslips. Surface HLA-DR (red) was labeled before fixation to visualize the plasma membrane. After permeabilization, virus was stained using an anti-IAV antibody (green) and the nucleus was stained using DAPI (blue). The entire volume of each cell was analyzed using confocal microscopy (100× 1.47NA oil objective, 6× digital zoom) and one single optical slice through the center of the cell is shown with arrowheads pointing to virus structures. Scale bar 10 µm. (D) The frequency of IAV+ DCs after 1 hr of virus exposure was determined by analyzing entire z-stacks of DCs and counting the number of cells that had virus associated with them, either on the membrane (white) or intracellularly (black). The graph shows average frequency of IAV+ DCs±SD, with 100–300 DCs analyzed per donor and condition (n = 4). (E) DCs were treated with LPS, TLR7/8L, IFNα or nothing for 24 hr and the MxA expression was determined by flow cytometry using intracellular staining with a monoclonal anti-MxA antibody. Graph shows MFI±SD of MxA with isotype control subtracted (n = 3).

(A) 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) 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. (C) 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. (D) 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. (E) 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. (F) 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.

(A) mDCs were exposed to infectious IAV, HI IAV or left untreated for 4 hr and washed, then exposed to whole, γ-irradiated CMV for an additional 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 an HLA-A2 CMV pp65 pentamer to detect CMV pp65-specific CD8 T cells and analyzed by flow cytometry. Dot plots show live CD8 T cells and numbers indicate frequency of positive CD8 T cells. One representative experiment of five is shown. (B) Frequency of live CMV pentamer+ CFSE^low CD8 T cells co-cultured with uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) for 10 days, as described in (A) where mDCs were fed increasing doses of whole, γ-irradiated CMV before co-culture with CD8 T cells. (C) Frequency of live CMV pentamer+ CFSE^low CD8 T cells co-cultured with uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) for 10 days, as described in (A) after co-culture at different DC∶CD8 T cells ratios. (D) Uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) 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 for 10 days. The graph shows frequency of live CMV pentamer+ CFSE^low CD8 T. One representative experiment of five is shown. (E) mDCs were exposed to infectious IAV, HI IAV or left untreated for 4 hr, washed to remove non-cell associated virus and exposed to 100 µL whole, γ-irradiated CMV for an additional 3 hr. mDCs were then washed and co-cultured with autologous CD4 T cells at a 1∶30 DC∶T cell ratio. After 1 hr brefeldin A was added and the cells were further incubated overnight. Cells were harvested and stained with antibodies to detect cytokine-producing CD4 T cells and analyzed by flow cytometry. Dot plots show live CD4 T cells and numbers indicate frequency of IFNα, TNFα, IL-2+ CD4 T cells. One representative experiment of four is shown.

(A) mDCs were exposed to infectious IAV, HI IAV or untreated for 4 hr, washed and exposed to increasing doses of whole, γ-irradiated CMV for an additional 3 hr. DCs were then washed, adhered to coverslips and fixed with PFA. After permeabilization, samples were stained for CMV pp65 (green), IAV (red) and HLA-DR (blue). The entire volume of each cell was analyzed using confocal microscopy (100× 1.47NA oil objective, 7× digital zoom) and one single optical slice is shown through the center of the cell with arrowheads pointing to CMV pp65+ structures. Scale bar 5 µm. (B) The frequency of CMV pp65+ uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of cells that had virus associated with them. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3). (C) The average number of CMV pp65+ puncta per cell after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of green (CMV) puncta per nucleated cell, assessed by DAPI staining. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3).

mDCs (A) or pDCs (B) were exposed to infectious IAV or HI IAV in the absence or presence of NH₄Cl for 24 hr. The frequency of IAV+ DCs and their CD86 expression was determined. Dot plots show live DCs and numbers in each quadrant depict the frequency of positive DCs. One representative donor of eight is shown. (C) The surface expression of CD86, CD40, MHCI (HLA-ABC) and MHCII (HLA-DR) on mDCs (blue) and pDCs (red) was determined after 24 hr of exposure to infectious IAV, HI IAV or TLR7/8L in the absence or presence of NH₄Cl. Bar graphs show MFI±SD (n = 3). The levels of secreted IFNα from supernatants of pDCs (D) or mDCs (E) after 24 hr were determined by ELISA. The graphs show mean±SD (n = 9).

pDCs (red) and mDCs (blue) were exposed to infectious IAV, HI IAV or left untreated for 4 hours, washed and co-cultured with autologous CD4 T cells at a 1∶30 DC∶T cell ratio. After 1 hr of co-culture brefeldin A was added. After another 16 hr of co-culture, cells were harvested and stained with antibodies to detect cytokine-producing CD4 T cells and analyzed by flow cytometry. Graph shows mean±SD percent of live, IFNα, TNFα, IL-2+ CD4 T cells (n = 3).

(A) mDCs were exposed to infectious IAV, HI IAV or left untreated. mDCs were collected at different time points and stained with Annexin V to assess cell viability. Graph shows average frequency ± SD of Annexin V negative (live) mDCs treated with nothing (blue), infectious IAV (black) or HI IAV (white) (n = 3). (B) mDCs were infected or not with infectious IAV for 2 hr, washed and exposed to 700 µg/mL whole, γ-irradiated CMV for an additional 6 hr. mDCs were then washed, split in two, and half of the DCs were fixed with 0.5% PFA for 10 min and washed extensively to remove any residual PFA before co-culture with autologous CFSE-labeled CD8 T cells at a 1∶30 DC∶T cell ratio for 10 days. Cells were harvested and stained with an HLA-A2 CMV pp65 pentamer and analyzed by flow cytometry. Dot plots show live CD8 T cells and numbers indicate frequency of positive CD8 T cells. One representative experiment of two is shown. (C) Same experiment as described in (B) but uninfected or IAV infected mDCs were loaded with 25 ng/mL pre-processed CMV peptide before fixation (or not) and co-cultured with autologous CD8 T cells at a 1∶30 DC∶T cell ratio. Dot plots show live CD8 T cells and numbers indicate frequency of positive CD8 T cells. One representative experiment of two is shown.