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. 2019 Mar 6;9:3655. doi: 10.1038/s41598-019-40145-0

Figure 5.

Figure 5

BCG-mediated up-regulation of PD-L1 is diminished by inhibiting the interleukin-STAT3 axis. (A) Macrophages were pre-treated with 50 μg/mL blocking antibodies or isotype controls for ~2 h and infected with 0.1 MOI BCG for 24 h. PD-L1 expression was measured by flow cytometry. Left: Representative histogram of PD-L1 expression with MFIs depicted. Right: Pooled data from multiple experiments, with calculated percentage reduction in PD-L1 MFI. (B) Macrophages were pre-treated with 50 μg/mL IL-6R and IL-10R blocking antibodies and then infected with 0.1 MOI BCG for 18 h. Levels of p-STAT3 were determined by intracellular flow cytometry. Shown is a representative plot with depicted MFI for p-STAT3. (C) Macrophages were pre-treated with the indicated dose of Stattic or DMSO equivalent for 2 hours, and then infected with 0.1 MOI BCG or stimulated with 100 ng/mL LPS for 24 h. Levels of PD-L1 were measured by flow cytometry. Left: Representative histogram showing Stattic-mediated PD-L1 down-regulation. Right: Pooled data from multiple experiments showing percentage change from the vehicle control. Significance was tested by one-way ANOVA with Tukey’s post-test (A) or two-way ANOVA with Dunnett’s post-test vs the vehicle control (B). Bars depict means ± SEM. Data are derived from n = 3 experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.