CD40 engagement through CD154⁺ T cells induces the antiviral innate immune surveillance system in ECs. (A) Validation of CD40-dependent induction of key antiviral innate immune response genes by RT-qPCR. Relative mRNA levels corresponding to TLR3, IFIH1, RIG-I, and RNASEL from msiRNA-2–treated (■) and siRNA-2–treated (□) HUVECs stimulated with (CD154⁺) T cells for 4 hours (IFIH1, RIG-I, RNASEL), or 4, 10, and 16 hours (TLR3), were assessed by real-time RT-qPCR using CYPA as a control, and normalized from the corresponding values obtained from unstimulated, msiRNA-2–treated, or siRNA-2–treated HUVECs. Results are mean values plus or minus SEM from 2 independent experiments performed in triplicate. (B) Functional validation of the antiviral surveillance pathways induced by CD40 signaling in HUVECs. msiRNA-2–treated (■) and siRNA-2–treated HUVECs were stimulated with Jurkat D1.1 (CD154⁺) T cells for 4 or 16 hours and either analyzed or further challenged with poly (I:C) (25 μg/mL) for 6 hours and analyzed. Relative mRNA levels of the proinflammatory chemokine IL8 and the type I IFNβ were assessed by real-time RT-qPCR using the housekeeping CYPA as a control. Results are mean values plus or minus SEM from 3 independent experiments performed in triplicate. (C) The relative amount of synthesized IFNβ was also evaluated at the protein level by immunoblotting using an anti-IFNβ polyclonal antibody, in fractionated cell extracts from msiRNA-2–treated (lanes 2 and 4) and siRNA-2–treated (lanes 1 and 3) HUVECs stimulated with Jurkat D1.1 (CD154⁺) T cells for 4 and 16 hours, and further challenged with poly (I:C) (25 μg/mL) for 6 hours. β-actin protein levels were assessed for normalization. A representative blot from 3 independent experiments is shown.