Fig 2: PD-L1 knockout does not generate a phenotype in vitro. (A) PD-L1 protein levels were assessed by flow cytometry after exposure to IFN-γ (150 U/ml, 24 h). Protein levels are expressed as SFI values relative to an isotype control antibody (black histograms). (B) PD-L1 protein induction was assessed by immunohistochemistry after exposure to IFN-γ (150 U/ml, 24 h). Photoghraphs were taken with 40× magnification. Inserts depict parallel samples stained with isotype control antibody. Scale bar represents 100 μm. (C) CRISPR control or PD-L1 KO cells were seeded in serial dilutions. The cells were incubated for 7–10 days to determine cell growth. At the end point, the wells were stained with crystal violet and absorbance was measured at 590 nm.

Fig 3: PD-L1 and PD-L2 expression in murine glioma models in vitro and in vivo. (A–D) Relative Pdl1 and Pdl2 mRNA expression were determined by RT-qPCR using Hprt1 as a reference gene (A, C). PD-L1 and PD-L2 surface protein levels were determined by flow cytometry. Black histograms represent isotype control antibody staining whereas gray represents experimental antibody staining. (B,D). E,F. PD-L1 (E) and PD-1 (F) protein was assessed by immunohistochemistry in brains of tumor-bearing mice. 10× magnification was used for an overview, stained cells are exemplified by arrows and a scale bar of 100 µm is included in the bottom right corner of the figure panel. 40× magnification was used for the inserts with a scale bar of 50 µm in the bottom right corner of the figure panel.

Fig 4: PD-L1 knockout confers a survival benefit in the CT-2A model. (A) CT-2A control (WT and CRISPR control grouped together) or PD-L1 KO (PD-L1 KO clone 1 or 2 grouped together) were orthotopically implanted in C57BL/6 mice (for each combined group n = 14). Survival data are presented as Kaplan–Meier plots (log-rank test,*p < .05). Median survival (days) is indicated within brackets in legend. (B) Tumor volumes were calculated on the basis of H&E sections of the whole tumor (n = 4). (C) Immunohistochemical staining of CT-2A control or PD-L1 KO tumors. Scale bar represents 100 µm. Quantification of DAB signal intensity was performed and represented in graphs to the right (n = 4).

Fig 5: PD-L1 tumor knockout abrogates response to anti-PD-L1 treatment, but the effect is T cell mediated. (A) CT-2A control or PD-L1 KO cells were orthotopically implanted in C57BL/6 mice (for each group n = 7). Tumor-bearing mice were treated 6 times with PBS or an anti-PD-L1 antibody (5 mg/kg, i.p., twice weekly, starting at day 5). (B) CT-2A WT, CRISPR control (grouped as control), PD-L1 KO clone 1 or 2 (grouped as PD-L1 KO) were orthotopically implanted in RAG−/− C57BL/6 mice (for each group n = 7). (C) CT-2A WT cells were implanted were orthotopically implanted in RAG−/− C57BL/6 mice (for each group n = 7). Tumor-bearing mice were treated 6 times with PBS or an anti-PD-L1 antibody (5 mg/kg, i.p., twice weekly, starting at day 5). Survival data are presented as Kaplan–Meier plots (log-rank test,*p < .05, ***p < .001). Median survival (days) is indicated within brackets in legend.