Figure 3.

PD-L1 and IDO1 expression are both required for MDSC and NCM superinduction in response to IFN-γ-exposed GBM EVs. (A) dBT114, dBT116, dBT120, and dBT165 cells were transfected with either nontargeting (sicon), PD-L1 (80 nmol/L) or IDO1 (80 nmol/L). After 72 h, the transient transfectants were stimulated in the absence (−) or presence (+) of 100 ng/mL IFN-γ for 24 h and western blotted for PD-L1, IDO1, and HSP90 as a loading control. (B) Immunoblots from (A) were normalized to loading control (HSP90) and evaluated. Bar graphs showing mean frequency of MDSC (C) or NCM (D) induction in serum-free media, EVs, EVs from dBT cell lines, PD-L1 knockdown dBT cells or IDO1 knockdown dBT cells treated with (+) or without (−) IFN-γ (100 ng/mL) for 3 days. Note that both PD-L1 and IDO1 knockdown markedly reduced the superinduction of MDSC and NCM in response to IFN-γ. No synergy or additive effects are seen. *P < .05, **P<0.01, ***P < .001, ****P < .0001. EVs, extracellular vesicles; GBM, glioblastoma; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; MDSC, myeloid-derived suppressor cell; NCM, nonclassical monocyte; PD-L1, programmed cell death ligand 1.