FIGURE 4: TL-532 is a specific agonist of TLR3 and does not activate TLR7-8-9, RIG-I, and MDA5. All the wt and modified cell lines used are stably transfected with either SEAP-reporter gene under control of NF-κB promoter or Lucia-reporter gene under control of ISRE promotor. (A) To confirm the capacity of TL-532 to activate TLR3, wild-type HEK293-Dual, that do not express TLRs (HEK293 wt), and its re-expressed human TLR3 counterpart (HEK293 hTLR3), were treated with 100 µg/mL of the indicated TLR3-ligand for 24 hours. NF-κB activation was measured with SEAP reporter gene assay and expressed as fold increase of untreated cells. (B) In order to analyze TL-532 specificity with regards to the TLR7 and TLR9, TL-532 activity was compared in RAMOS wt versus RAMOS MyD88 KD (the common adaptor of all TLRs except for TLR3 that signals only through TRIF). Cells were treated with 10 µg/mL of TLR7-ligand (CL264), 5 µg/mL of TLR9-ligand (ODN2006), 10 µg/mL of TL-532, and 0.1 μg/mL of TNFα for 24 hours. NF-κB activation was measured with SEAP reporter gene assay and expressed as the percentage of SEAP over their relative TNFα activation. (C) To analyze TL-532 specificity with regards to the TLR8, HEK293-Blue wt was reexpressed with human TLR8 (HEK293 hTLR8). Cells were treated with the three TLR8-ligands R848 at 1 µg/mL – TL8-506 at 1 µg/mL – ssRNA Lyovec at 5 µg/mL, TL-532 at 2000 µg/mL, TLR4-ligand LPS at 0.1 µg/mL, TLR7-ligand CL-264 at 5 µg/mL, and 0.1 μg/mL of TNFα for 24 hours. NF-κB activation was measured with SEAP reporter gene assay and expressed as the percentage of SEAP over their relative TNFα activation. (D) To determine the TL-532 specificity with regards to MAVS-dependent cytosolic dsRNA helicases sensors RIG-I and MDA5, wild-type A549 cell line (A549 wt), known to express cytosolic sensors but weak TLR3 protein, has been knocked-out for the MAVS adaptor (A549 MAVS KO). Cells were treated with either 0.1 µg/mL of MDA5-ligand (Poly(I:C) HMW Lyovec – Poly(I:C)lyo), 1 µg/mL of RIG-ligand (pppRNA Lyovec – pppRNAlyo), 2000 µg/mL of TL-532, 30 µg/mL of STING-ligand (2'3'cGAMP), 0.1 µg/mL of TLR4-ligand (LPS), and 1000 IU/mL of IFNα for 24 hours. ISRE activation was measured with Lucia reporter gene assay and expressed as the percentage of Lucia over their relative IFNα activation. Data are the mean of three independent experiments. Results are expressed as mean ± SD. Unpaired Student's t-test: * = p<0.05; ** = p<0.01; *** = p<0.001; ns: not significant.