Binding of dsRNA to APCs and the subsequent effect on adaptive immunity. (a) MACS-separated APCs were incubated with 10 μg/ml pA:pU-F, then washed and analyzed. Alternatively, APCs were preincubated with 20 or 100 μg/ml of nontagged pA:pU, pA, or pI:pC before staining. The profiles of stained (unshaded region) and nonstained (shaded region) cells and the percentage of highly stained APCs are represented in each panel, with a logarithmic x axis. (b) In vivo binding of pulmonary CD11c⁺ and CD11b⁺ APCs by tagged pA:pU. Animals were treated with pA:pU-F by intratracheal instillation, and lung interstitial cells separated. The cells were stained with PE-tagged isotype control, anti-CD11c, or anti-CD11b antibodies. The percentages of pA:pU-F–stained cells were estimated in the gated CD11c⁺, CD11c^–, CD11b⁺, and CD11b^– subpopulations. (c) Activation of CD11c⁺ APCs by pA:pU and viral dsRNA was assessed by measuring the cytokine production. MACS-separated CD11c⁺ or control CD11c^– cells were incubated with 25 μg/ml dsRNA, RNA from noninfected cells (ctrl RNA), or no RNA (Ctrl). The cytokines were expressed as mean ± SEM in pg/ml. The right y axis scale is for IL-12 (diamonds) and the left y axis scale is for TNF-α (squares). (d and e) The effect of dsRNA from influenza virus–infected cells on the response to an antigen (gp140) was measured subsequent to immunization with antigen + dsRNA. The specific IgG response (d) and the IgG1 and IgG2a components (e) were measured by ELISA. As controls, we used mice immunized with gp140 in PBS, gp140 + pA:pU, or gp140 + ctrl RNA from noninfected cells. Results are expressed as mean ± SEM (n = 3 mice/group) of OD (405 nm) at various serum dilutions (d) or diluted 100 times (e).