Figure 4.

A, PD‐L1 expression on DAUDI (left) and pancreatic ductal adenocarcinoma (PDAC) cells (right) as determined by immunohistochemistry analysis with an anti‐PD‐L1 antibody; Burkitt lymphoma cells were utilized as the negative control. The upper panel shows 200× magnification, and the bottom panel shows 400× magnification. PD‐L1 expression in S2‐013 (B, D) and MIAPaCa2 (C, E) cells after cytokine assays, determined by real‐time PCR (B, C) or flow cytometry (D, E). The minus symbol indicates that no cytokines were added. Among the various cytokines (including LPS) analyzed, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ strongly induced PD‐L1 expression in both cell lines (S2‐013 and MIAPaCa2). F, Inhibitor assay with MIAPaCa2 cells using a nuclear factor‐kappa B (NF‐κB) inhibitor. The plus sign indicates that the inhibitor was added, whereas the minus sign indicates that the inhibitor was not added. PD‐L1 expression was increased in MIAPaCa2 cells after treatment with TNF‐α and was inhibited after treatment with both TNF‐α and NF‐κB inhibitors. G, Western blot analysis of proteins detected by probing with anti‐NF‐κB or anti‐phosphorylated NF‐κB antibodies. TNF‐α increased the level of phosphorylated NF‐κB. Full‐length gels are presented in Figure S4. H, Correlation between PD‐L1 expression and TNF‐α mRNA levels in frozen human PDAC tissues. Patients were sorted into a high or low PD‐L1 expression group based on analysis of frozen tissues, and the (H) PD‐L1 expression in paraffin sections and TNF‐α mRNA expression in frozen tissues harvested from the same PDAC patients (I) were compared. PD‐L1 expression was typically high in patients with high TNF‐α mRNA levels. Macrophage infiltration was also confirmed in consecutive paraffin sections using double immunostaining with PD‐L1 and Iba‐1 antibodies (J). The cells stained brown represent PD‐L1‐expressing PDAC cells, whereas those stained green are infiltrating macrophages