Tlrl lrna40

Enriched blood DCs or sorted cord blood cDCs were resuspended at 1×10⁶ cells/mL, PBMCs or tumor digests were resuspended at 5–10×10⁶ cells/mL in DC medium (RPMI GlutaMAX, Gibco, #72 400–021, 1% human serum, Sigma, #H4522, 1% Pen/Strep). 1×10⁵ cells (enriched blood DCs or sorted cord blood cDCs) or 0.5–1×10⁶ cells (PBMCs or tumor digests) in 200 µL/well were treated with the indicated stimuli in a 96-well plate at 37°C in a CO₂ incubator. For DC activation in the presence of tumor-conditioned medium, 1:1 diluted supernatant from a 6-hour culture of patient-derived tumor digest in DC medium or a 2-day culture of COR-L105 tumor cell line (ECACC, #92031918) in RPMI GlutaMAX, 10% FBS, 1% Pen/Strep was added to sorted cord blood cDCs during stimulation. The following concentrations were used: 10'000 U/mL huIFN-α (R&D, #11100–1), 10'000 U/mL huIFN-β (R&D, #8499-IF-010), 1 µg/mL huIFN-λ (R&D, #1598-IL-025), 50'000 U/mL huIFN-γ (PeproTech, #300–02), 1 µg/mL Pam3CSK4 (Invivogen, #vac-pms), 10 µg/mL Poly(I:C) (Invivogen, #vac-pic), 0.1 µg/mL LPS (Sigma, #L2880), 1 µM TL8-506 (Invivogen, #tlrl-tl8506), 10 µM CL075 (Invivogen, #tlrl-c75), 10 µM R848 (Invivogen, #vac-r848), 10 µg/mL ssRNA40 (Invivogen, #tlrl-lrna40), 10 µg/mL 2’3’-cGAM(PS)2(Rp/Sp) (Invivogen, #tlrl-nacga2srs).