(A) Primary human pDCs were cocultured with uninfected Huh 7.5.1 cells (first bar), with infected Huh 7.5.1 cells (second and third bar), or with DENV alone (fourth bar). Supernatants were collected and IFN-α2a was measured. To assess the requirement for pDCs in IFN production, infected Huh 7.5.1 cells were cultured alone (seventh bar). To assess the requirement for cell-to-cell contact, pDCs were cultured with supernatant collected from infected Huh 7.5.1 cells (fifth bar). To demonstrate that TLR signaling is intact in the pDCs in the absence of hepatoma cells, pDC were cultured with 1 μg/mL TLR7 agonist resiquimod (REQ) (sixth bar). This figure represents 4 independent experiments with distinct pDC donors, with n ≥ 3 per condition. (B) Schematic representation of experimental design to follow: Huh 7.5.1 cells are infected with DENV at 0.1–1 MOI for 48 hours. At 48 hours, antibody treatment is added. pDCs are isolated and cocultured with treated Huh 7.5.1 one hour after antibody addition. Supernatants are collected after 24 hours and analyzed for IFN-α2a. (C and D) Primary human pDCs were cocultured with infected Huh 7.5.1 following 1 hour of treatment with anti–ICAM-1 antibody at 2–10 μg/mL (C) or anti–α_L-integrin antibody at 0.1–1 μg/mL (D). Supernatants were collected, and IFN-α2a was measured. (C) Seven independent experiments with distinct pDC donors (3–7 donors per condition) with n > 3 replicates per independent experiment. (D) Seven distinct pDC donor experiments (1–7 donors per condition) with n > 3 replicates per condition per experiment. The y axis represents IFN relative to baseline level generated after pDC + Huh coculture with 0.1 MOI DENV. Statistical significance was determined by 1-way ANOVA. *P < 0.05, ****P < 0.0001.