Figure 5.

Programmed death 1 ligand (PD‐L1) blockade stimulated the production of monocyte chemoattractant protein‐1 (MCP‐1) from activated peripheral blood mononuclear cells (PBMCs). (a,b) PBMCs co‐cultured with rhabdomyosarcoma (RD) cells [enterovirus A71 (EV‐A71⁺)] in the Transwell system. (a) Histograms showing representative results for enzyme‐linked immunosorbent assay (ELISA) antigen determination for MCP‐1, interleukin (IL)‐6, IFN‐γ and IL‐1β in supernatants of co‐cultured PBMCs and RD (EV‐A71 infected for 2 h) for 12 h. Samples of Transwell are shown as a schematic diagram in the right side of (b). (b) Histograms showing representative results for EV‐A71 replication influenced by anti‐PD‐L1 blockade, isotype antibody (negative control) and medium (blank). (c) PD‐L1 blockade was shown to increase intracellular MCP‐1 production of CD14⁺ monocytes in PBMCs activated by R848 or EV‐A71 virus; 2·5 × 10⁶ PBMCs were settled in 96‐well plates with R10 medium and incubated with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) + R848 ⁺ anti‐PD‐L1 antibody [or mouse immunoglobulin (Ig)G1κ isotype antibody] or GM‐CSF + EV‐A71 + anti‐PD‐L1 antibody (or isotype antibody) for 6 h. Cells performed intracellular cytokine staining with the following monoclonal antibodies: CD3‐AF700, CD14‐ef450 and MCP‐1‐PE. U.D. = undetectable. The experiments were repeated for four times. Differences between groups were assessed using the non‐parametric Wilcoxon matched‐pairs test.