Rpmi 1640 medium

The isolated PBMCs or MAIT cells were cultured alone or cocultured with MSCs at a ratio of 5:1 (2.5 × 10⁵ PBMCs:0.5 × 10⁵ MSCs in 12-well plates, 1.25 × 10⁵ MAIT cells:0.25 × 10⁵ MSCs in 24-well plates) in RPMI-1640 medium containing 10% fetal bovine serum (Zhejiang Tianhang Biotechnology). For the Transwell coculture, 6.5 mm Transwell chambers with a 0.4 μm pore polycarbonate membrane insert (Corning) were used, with MSCs seeded in the upper insert and MAIT cells seeded in the lower chamber. In addition, PBMCs or MAIT cells were cultured with or without stimulation with a purified CD3 antibody (1 μg/mL, BD Pharmingen, 555336) and purified CD28 antibody (1 μg/mL, BD Pharmingen, 555725). For the blocking experiments, 20 μg/mL anti-human MR1 (BioLegend, 361103), 10 μg/mL anti-human TGF-β (R&D Systems, AF-246-NA), 1 μg/mL anti-human IL-6 (R&D Systems, MAB206-100), 10 μg/mL anti-human IL-7 (BioLegend, 501304), 10 μg/mL anti-human IL-15 (BioLegend, 515001), or the corresponding amount of isotype control (BioLegend, 400224) was added to the culture medium. For treatment with recombinant proteins, 20 ng/mL recombinant human TGF-β, 20 ng/mL recombinant human IL-6, 20 ng/mL recombinant human IL-7, and 50 ng/mL recombinant human IL-15 (all from R&D Systems) were used. Cells were treated with 500 nM rapamycin and 10 mM 3-MA to modulate autophagy.