Fig. 2

Mn²⁺ treatment upregulated PD-L1 and synergized YM101 in one-way mixed lymphocyte reaction (MLR) assay. a–b The effect of Mn²⁺ treatment on PD-L1 expression on dendritic cells (DCs). After 1 mM Mn²⁺ treatment for 24 h, the mean fluorescence intensity (MFI) of PD-L1 on MoDCs was measured. After 0.5 mM Mn²⁺ treatment for 24 h, MFI of PD-L1 on BMDCs was measured (n = 3 technical replicates). c–k MLR assay exploring the synergistic effect between Mn²⁺ treatment and YM101. BMDCs from BALB/c mice were used as stimulator cells, and splenocytes from C57BL/6 mice were used as responder cells. The immature BMDCs were treated with 0.5 mM Mn²⁺ or saline for 24 h. The ratio of responder to stimulator was 2:1. Then, TGF-β1 (20 ng/ml) and antibodies (10⁵ pM) were added to the mixed cells. After culture for 96 h, the ratio of daughter CD8⁺ T cell and the cytokine levels in the supernatant were measured (n = 4 technical replicates). The ratio of daughter CD8⁺ T cell by CFSE dilution assays (c) and the cytokine levels were measured by LEGENDplex™ MU Th Cytokine Panel. The cytokine levels were visualized by heatmap (d). The levels of IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-10, and IL-13 are shown (e–k). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 denote the significant difference relative to the last group when not marked with lines. MoDC: monocyte-derived dendritic cell; BMDC: bone marrow-derived dendritic cell; MFI: mean fluorescence intensity; MLR: mixed lymphocyte reaction