Fig. 2

Hypoxia up-regulate PD-L1 expression via HIF-1α in glioma cell lines and combination treatment with HIF-1α inhibitor and anti–PD-L1 antibody can reduce tumor growth in murine model of glioma. a qPCR analysis of HIF-1α and PD-L1 mRNA expression in U251 and U343 lines with different treatments as indicated. The qPCR data were normalized to GAPDH. The data were presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01. b Western blot analysis of U251 and U343 cells with different treatments using indicated antibodies. c Chromatin immunoprecipitation (ChIP) analysis of the PD-L1 promoter in U251 cells using anti-HIF-1α mAb. The experiments were performed in triplicates and repeated three times. d Immunofluorescence staining of HIF-1α and PD-L1 expression in tumor cells analyzed by confocal microscopy. Representative images are shown. Scale bars, 50 μm. e Mice bearing GL261 cells were divided into the indicated treatment groups. The tumor volumes of mice treated with control, anti–PD-L1 monoclonal antibody, HIF-1α inhibitor (PX-478), or combined anti–PD-L1 antibody and PX-478 were measured and plotted (n = 5). Tumor volume was measured twice weekly. Data are presented as mean ± SEM. and the statistical significance was determined by two-way ANOVA. f Survival from mice receiving the indicated treatments as described in e. Statistical significance was determined by log-rank (Mantel-Cox) test. For (e) to (f) *P < 0.05, **P < 0.01. g The HE staining of intracranial tumor and immunohistochemistry analysis of CD8⁺ T cells in intracranial tumor from mice receiving control, anti–PD-L1 antibody, PX-478, or anti–PD-L1 antibody and PX-478. h Representative flow cytometry analysis and quantification of CD4⁺ T, CD8⁺ T, CD11c⁺ DC and CD11b⁺ myeloid cells populations in GL261 tumors with the indicated treatments (n = 5). i Quantification flow cytometry analysis of the PD-L1 expression on CD45⁻, CD3⁺, CD11c⁺ and CD11b⁺ cells (n = 5). j Representative flow cytometry analysis and quantification of CD8⁺ INF- γ⁺ T cells in GL261 tumors and the MFI of INF- γ in CD8⁺ T cells in U261 tumors at day 14 after treatment (n = 5). For (h) to (j), data are presented as means ± SEM. P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01