Figure 2.

Hypoxia up-regulates PD-L1 on mouse and human tumor cell lines and on macrophages and DCs from naive C57BL/6 mice. (A–H) Mouse and human tumor cell lines were cultured under normoxia and hypoxia (0.1% pO₂) at indicated times. Recombinant mouse or human IFN-γ–treated (10 ng/ml) cells were used as positive control for PD-L1 induction under normoxia for 24 h. Shown is the surface expression of PD-L1 and PD-L2 on B16-F10 (A), LLC (B), CT26 (C), 4T1 (D), T1 (E), M4T (F), IGR-Heu (G), and MCF-7 (H) tumor cells. Statistically significant differences (indicated by asterisks) between tumor cells cultured under normoxia or hypoxia are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). Three separate experiments with the same results were performed. Error bars indicate SD. (I and J) C57BL/6 naive mice spleens were used for preparing single-cell suspensions by mechanical dissociation, followed by removal of red blood cells with ammonium chloride lysis buffer (ACK). Macrophages and DCs were further isolated by cell sorting on FACS MoFlo or FACSAria (BD) after incubating with F4/80⁺ PE or CD11c⁺ APC-conjugated antibodies, respectively. The sorted F4/80⁺ PE (I) and CD11c⁺ APC (J) cells were incubated under normoxia or hypoxia (0.1% pO₂) for 24, 48, and 72 h, and PD-L1 and PD-L2 expression was evaluated by flow cytometry. Statistically significant differences (indicated by asterisks) between macrophages and DCs incubated under normoxia or hypoxia are shown (*, P < 0.05; **, P < 0.005). The experiment was performed with n = 3 mice and repeated twice with the same results. Error bars indicate SD.