Polystyrene dishes

Human cancer cell lines purchased from the ATCC (Manassas, VA, USA), JCRB Cell Bank (Osaka, Japan), or Riken BRC (Ibaraki, Japan) were cultured in polystyrene dishes (Becton Dickinson, Franklin Lakes, NJ, USA) in RPMI 1640 medium (Fujifilm Wako, Osaka, Japan) or DMEM (Fujifilm Wako) supplemented with 1% penicillin/streptomycin and 10% FBS. Human IFN‐α (1000 IU/ml; MSD, Tokyo, Japan), IFN‐β (10 ng/ml; Peprotech, East Windsor, NJ, USA), IFN‐γ (10 ng/ml; Wako), LPS (10 ng/ml; Sigma‐Aldrich, St. Louis, MO, USA), or T‐cell culture supernatant (described below) was added during the last 24 h of incubation to induce PD‐L1 expression.

RPMI 1640 medium (Fujifilm Wako) supplemented with 1% penicillin/streptomycin (Fujifilm Wako) and 10% FBS was used as the complete culture medium for all cells. PBMCs were isolated from peripheral blood obtained from healthy volunteer donors, who provided written informed consent, via density gradient centrifugation with Lymphoprep (Axis‐Shield, Oslo, Norway). Monocytes and T cells were isolated from the PBMCs using a magnetic bead‐based isolation procedure (MACS CD14 microbeads, Pan T cell Isolation Kit, columns and separators; Miltenyi Biotec, Bergisch Gladbach, Germany). The monocytes were cultured in polystyrene dishes (Becton Dickinson) in complete medium with M‐CSF (50 ng/mL; Fujifilm Wako) for 3–7 days to induce HMDM production. The T cells were cultured in the complete medium with immobilized anti‐CD3 (5 μg/ml; Invitrogen, Waltham, MA, USA) and anti‐CD28 antibodies (2 μg/ml; BioLegend, San Diego, CA, USA) for 3–7 days to obtain the culture supernatant. IFNs, LPS, or the T‐cell culture supernatant was added to the HMDMs to induce PD‐L1 expression. IAXO‐101, synthetic CD14/TLR4 antagonist (25 μM; Innaxon, Tewkesbury, UK), anti‐CD14 antibodies (1.5 μg/ml; R&D Systems, Minneapolis, MN, USA), or mouse IgG1 isotype control (1.5 μg/ml; MBL, Aichi, Japan) was added 30 min before adding LPS.