Effect of cytokine treatment on PD-1, PD-L1, and PD-L2 expression by BCWM.1 and MWCL-1 cell lines. BCWM.1 and MWCL-1 cell lines were starved overnight and then treated with complete media containing cytokines, including IL-21 (100 ng/mL), IL-6 (50 ng/mL), IFNγ (100 ng/mL), CCL5 (100 ng/mL), and G-CSF (100 ng/mL) for 72 hours. (A-B) Cells were used for mRNA extraction, cDNA synthesis, and RT-PCR analysis. Bar graphs represent relative gene expression of PD-1, PD-L1, PD-L2 normalized to housekeeping gene (RPLP0) in response to cytokine treatment. Data are shown as the mean ± SE of the mean (SEM) of 3 separate experiments (n = 3). Significant differences and the P values are depicted on each graph. (C) Cells were stained with fluorochrome-conjugated anti-human PD-L1 antibody. Histograms represent flow cytometry analysis of PD-L1 surface expression by BCWM.1 and MWCL-1 cell lines (red, isotype control; blue, control untreated; and orange, cytokine-treated cells).