Phenotypic and functional characterization of pDCs before and after activation with CpG or heat-inactivated influenza virus. (A) Expression of CD40, CD86, and K^b molecules on CD11c^loB220⁺ spleen cells after in vitro activation. CD11c^+/lo were isolated from naive 129sv mice and cultured in CM alone or with CpG (top) or heat-inactivated influenza virus (bottom). Histograms compare the expression of each marker by live gated CD11c^loB220⁺ untreated (- -) or activated (—) cells. Labeling with isotype control mAbs is represented in gray. (B) IL-12p40 and IFN-α production by purified cDCs (□), pDCs (▪), and total DCs (▦) isolated from the spleens of 129sv mice after in vitro activation. Sorted cDCs or pDCs (1.5 × 10⁵) or total DCs (10⁵) were cultured in the following conditions: medium, CpG, inactivated influenza virus, or equivalent dilution of AL as a control. (C) Peptide presentation to B3Z T-cell hybridoma by pDCs (circles), cDCs (squares), or CD11c^– spleen cells (triangles). OVA-specific B3Z hybridoma T cells were cocultured with graded numbers of purified pDCs, cDCs, or CD11c^– spleen cells previously loaded (filled symbols) or unloaded (open symbols) with the OVA_257-264 peptide. The amount of IL-2 secreted by B3Z was determined by a CTL-L assay. (D) Proliferative response of naive CD8⁺ T cells from LNs of HY transgenic mice to unstimulated (left), CpG-activated (middle), and virus-activated (right) pDCs (•) or cDCs (▪) loaded with the HY peptide. Unloaded pDCs (○) or cDCs (□) were used as a specificity control. The right panel also shows the proliferative response of HY T cells to DCs incubated with control AL (gray lines and symbols). (E) pDCs were purified from the spleens of untreated or CpG-injected 129sv mice and labeled. Plasmacytoid DCs were analyzed by gating on live CD11c^loB220⁺ cells. Histograms compare the expression of each marker by untreated spleen pDCs (dashed line) or CpG-activated pDCs (solid line). Labeling with isotype control mAbs is represented in gray. (F) IFN-α production by spleen pDCs and cDCs purified from mice injected with heat-inactivated influenza virus or control AL. Purified pDCs (▪) and cDCs (□) were incubated for 18 hours in SFEM StemSpan medium and IFN-α secretion was assessed in culture supernatants by ELISA. All data are representative of 2 experiments. Error bars in panels C and D represent SDs from the means of duplicate experiments.