Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-gamma through PD-L1 and IDO1

Background Glioblastoma (GBM), the most common primary brain tumor, has a median survival of 15-16 months. Immunotherapy is promising but GBM-mediated immunosuppression remains a barrier. GBMs express the interferon-gamma (IFN-gamma)-responsive immunosuppressive molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1). Extracellular vesicles (EVs) have also been implicated in GBM-mediated immunosuppression, in part through PD-L1. We therefore sought to determine if GBM IFN-gamma exposure increased GBM EV-mediated immunosuppression and mechanisms underlying this. Methods Human GBM-derived cells were cultured in the presence/absence of IFN-gamma. EVs were harvested. PD-L1, IDO1, and EV-associated protein expression was assessed. GBM EVs (+/-IFN-gamma) were cultured with healthy donor monocytes. Immunosuppressive myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) frequency was determined. Impact of GBM (+/-IFN-gamma) EV-treated monocytes on CD3/CD28-mediated T cell proliferation was assessed. The impact of PD-L1 and IDO1 knockdown in GBM EVs in this system was evaluated. Results IFN-gamma exposure increased PD-L1 and IDO1 expression in GBM cells and EVs without altering EV size or frequency. IFN-gamma -exposed GBM EVs induced more MDSC and NCM differentiation in monocytes and these monocytes caused more T cell inhibition than IFN-gamma -naive GBM EVs. PD-L1 and/or IDO1 knockdown in GBM cells abrogated the immunosuppressive effects of IFN-gamma -exposed GBM EVs on monocytes. Conclusions IFN-gamma exposure such as might occur during an antitumor immune response results in superinduction of GBM EVs' baseline immunosuppressive effects on monocytes. These effects are mediated by increased PD-L1 and IDO1 expression in GBM EVs. These data highlight mechanisms of GBM EV-mediated immunosuppression and identify therapeutic targets (PD-L1, IDO1) to reverse these effects.