Influenza virus and CL097 induce human pDC activation by triggering TLR7. (A, left) Flow cytometric evaluation of purity of unstimulated pDCs enriched from healthy donor blood labeled with anti-BDCA2 and anti-CD123 antibodies. Cells were untreated (medium) or stimulated with UV-formol–inactivated influenza virus (Flu) or synthetic TLR7 ligand (CL097). Expression of CD40 was measured by flow cytometry on CD123⁺ cells. Dot plots are shown, the percentage of CD40⁺ cells is indicated on each plot. Results shown are representative of 4 independent experiments. (B) GEN2.2 cells were untreated or stimulated with Flu or CL097 for 24 hours. CD40 expression was evaluated on forward scatter (FSC)/side scatter (SSC)–gated live cells by flow cytometry. Percentages indicated on dot correspond to the proportion of CD40⁺ cells. Results shown are representative of at least 5 independent experiments. (C) Expression levels of TLR7 and TLR9 in the GEN2.2 cell line and in lentiviral shRNA TLR7-transfected GEN2.2 cells (GENshTLR7) measured by real-time PCR. Expression levels are normalized to G6PDH. Data are shown as the mean and SD from duplicate values of 2 independent experiments. (D) GEN2.2 and GENshTLR7 cells were untreated or stimulated with Flu, CL097, or 2 different synthetic TLR9 ligands (CpG A and CpG B) for 24 hours. Expression of CD40 was measured by flow cytometry. The mean percentages and SDs from duplicate values of 3 independent experiments are shown.