MSC were seeded at a density of 0.6‒6 × 104 cells/mL in 24-well plates. After 24 h, the cells were incubated with 10 μg/mL mitomycin C (Fujifilm) for 2 h at 37 °C to inhibit cell proliferation. Next, 6 × 105 PBMC/mL were added directly or indirectly using a transwell system (pore size, 0.4 μm; Corning, NY, USA) at a PBMC/MSC ratio of 10:1 or 100:1. The lymphocytes in PBMC were stimulated with 4 μg/mL PHA (Fujifilm) and 100 U/mL IL-2 (PeproTech, NJ, USA). Anti-human antibodies against PD-L1 and PD-L2 (AF156 and AF1224; R&D Systems, MN, USA) were used at 250 or 500 ng/mL to inhibit ligand function. PHA/IL-2-stimulated PBMC and MSC were cocultured for 3 days.
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