Figure 2:

EVs containing PD-L1⁺ downregulate CD69 expression on effector CD8⁺ T cells. (A) The PD-L1 in SBC-3 and DMS273 cells was analyzed by Western blot analysis after transfection with NC or PD-L1 (upper). Relative mRNA amounts of PD-L1 in SBC-3-NC, SBC-3-PD-L1, DMS273-NC, and DMS273-PD-L1 cells. The amounts of mRNA from the gene of interest were normalized to GAPDH (lower). (B) The effect of PD-L1 overexpression on SBC-3 and DMS273 cell proliferation was determined by the cell viability assay. (C) Size distribution of SBC-3-PD-L1-derived EVs, as analyzed by nFCM. (D) Immunoblots for PD-L1 in purified EVs from SBC-3-NC, SBC-3-PD-L1, DMS273-NC, and DMS273-PD-L1 cells. The same amounts of proteins were loaded for each fraction. (E) The PD-L1 in EVs harvested from SBC-3-NC and SBC-3-PD-L1 cells were analyzed by nFCM. The preparation of EVs from culture medium was fluorescently labeled with PE-conjugated mAbs specific to PD-L1. The percentage of phenotype-positive EVs was provided in the plot.