Figure 3.

HIF-1α binds directly to the HRE in the PD-L1 proximal promoter and up-regulates its expression under hypoxia. (A–F) Surface expression levels of PD-L1 and PD-L2 (A and B) on MSC-1 cells cultured under normoxia and hypoxia (0.1% pO₂) at indicated times as compared with isotype control (gray-shaded histogram). IFN-γ was used as a positive control for PD-L1 up-regulation. Statistically significant differences (indicated by asterisks) between MSC-1 cells cultured under normoxia or hypoxia are shown (**, P < 0.005; ***, P < 0.0005). Three separate experiments with the same results were performed. Error bars indicate SD. (C) Western blot was performed to show HIF-1α, HIF-2α, and PD-L1 protein levels. β-Actin was used as a control. Three separate experiments with the same results were performed. (D–F) SYBR Green RT-qPCR was used to monitor Ldha, Car-9, Pdl1, Pdl2, Pd1, and Ctla-4 expressions levels at indicated conditions in MSC-1 (D), B16-F10 spleen Gr1⁺ (E), and 4T1 spleen Gr1⁺ (F) cells. Expression level of 18S was used as endogenous control. Statistically significant differences (indicated by asterisks) between cells (MSC-1 or spleen Gr1⁺) cultured under normoxia or hypoxia are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). Three separate experiments (in triplicates) with the same results were performed. Error bars indicate SD. (G–K) MSC-1 cells were transfected with different siRNA targeting HIF-1α, HIF-2α, or scrambled control (CT) and cultured under normoxia or hypoxia for 48 h. Expression levels of Ldha, Car-9, HIF-1α, HIF-2α, and Vegfa (G) and Cd80, Cd86, Pdl1, Pdl2, Pd1, and Ctla-4 (H) were evaluated by SYBR Green RT-qPCR. (I) Western blot was performed to show HIF-1α, HIF-2α, and PD-L1 protein levels. β-Actin was used as a control. (J) Surface expression levels of PD-L1 and PD-L2 were determined by flow cytometry. (K) Surface expression levels of CD80, CD86, PD-1, and CTLA-4 were determined by flow cytometry. Statistically significant differences (indicated by asterisks) between MSC-1 cells transfected with either siRNA-CT and siRNA-HIF-1α are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). The experiment was repeated three times with the same results. Error bars indicate SD. (L) MSC-1 cells were cultured at normoxia or hypoxia (0.1% pO₂) and ChIP was performed using anti-HIF-1α antibody followed by SYBR Green RT-qPCR using Vegfa, Ldha, Slc2a1, and Pdl1 HRE sites (HRE-1, HRE-2/3, and HRE-4) and RPL13A primers. For each gene, the RT-qPCR signals were normalized to the normoxic condition. Statistically significant differences (indicated by asterisks) between normoxic and hypoxic conditions are shown (*, P < 0.05; **, P < 0.005). Two separate experiments (in triplicates) with the same results were performed. Error bars indicate SD. (M) Different HREs in mouse PD-L1 promoter (PD-L1 mRNA; NCBI reference sequence NM_021893.3) are shown. The numbering scheme is from the refseq RNA start. (N) MSC-1 cells were co-transfected with pGL4-hRluc/SV40 vector and pGL3 empty vector (pGL3 EV), pGL3 HRE-4, or pGL3 HRE-4 MUT vectors and grown under normoxia or hypoxia. After 48 h, firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega) and the ratio of firefly/Renilla luciferase was determined. Statistically significant differences (indicated by asterisks) between normoxic and hypoxic conditions are shown (**, P < 0.005; ***, P < 0.0005). The experiment was performed in triplicates and repeated three times with the same results. Error bars indicate SD.