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

Abstract

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-γ)-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-γ exposure increased GBM EV-mediated immunosuppression and mechanisms underlying this.

Methods: Human GBM-derived cells were cultured in the presence/absence of IFN-γ. EVs were harvested. PD-L1, IDO1, and EV-associated protein expression was assessed. GBM EVs (+/-IFN-γ) were cultured with healthy donor monocytes. Immunosuppressive myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) frequency was determined. Impact of GBM (+/-IFN-γ) 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-γ exposure increased PD-L1 and IDO1 expression in GBM cells and EVs without altering EV size or frequency. IFN-γ-exposed GBM EVs induced more MDSC and NCM differentiation in monocytes and these monocytes caused more T cell inhibition than IFN-γ-naive GBM EVs. PD-L1 and/or IDO1 knockdown in GBM cells abrogated the immunosuppressive effects of IFN-γ-exposed GBM EVs on monocytes.

Conclusions: IFN-γ 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.

Keywords: IDO1; PD-L1; extracellular vesicles (EVs); glioblastoma; interferon-gamma (IFN-γ).

Figure 1.

IFN-γ increases PD-L1 and IDO1 expression in human glioblastoma cells and extracellular vesicles. (A) Glioblastoma cell lines dBT114 or dBT116 ± 100 ng/mL IFN-γ for 24 h and expression of indicated proteins in whole-cell lysate (WCL) and extracellular vesicles (EVs) was assessed by western blot. (B) Immunoblots from (A) were normalized to loading control (HSP90) by densitometry. Relative intensity compared to baseline (WCL without IFN-γ exposure) is shown (mean ± SEM; n = 3). (C) dBT114 or dBT116 cells ± 10 or 100 ng/mL IFN-γ for 24 h and analyzed by western blot for IDO1, PD-L1, and GAPDH. Bar graphs for PD-L1 show median fluorescence intensity on flow cytometry relative to EVs without IFN-γ exposure (median ± standard deviation, n = 3) while those for IDO1 show relative densitometry intensity compared to baseline (no IFN-γ) after normalization to GAPDH (mean ± standard deviation; n = 3). (D) Nanoparticle tracker analysis histograms and photomicrographs showing the size distribution and frequency of dBT114 and dBT116 EVs ± 100 ng/mL IFN-γ. *P < .05, **P < 0.01,***P < .001, ****P < .0001. IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; PD-L1, programmed cell death ligand 1.

Figure 2.

EVs from IFN-γ-treated glioblastoma cells cause superinduction of myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) formation. Representative dot plots showing MDSC frequency (CD14⁺/HLA-DR⁻) (A) and NCM frequency (CD14⁺/PD-1⁺/CD16 +) (C) in monocytes in serum-free media, EVs, IFN-γ- (100 ng/mL) treated EVs (left lane, middle lane, right lane) from dBT cell lines. Bar graphs showing a significant induction in the mean frequency of CD14⁺/HLA-DR⁻ cells (B) and CD14⁺/PD1⁺/CD16⁺ cells (D). *P < .05, **P<0.01, ***P < .001, ****P < .0001. EVs, extracellular vesicles; IFN-γ, interferon-gamma.

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.

Figure 4.

Superinduction of T cell inhibition by monocytes cultured with IFN-γ-exposed GBM EV depends on GBM PD-L1 and IDO1 expression. (A) Representative histograms showing proliferation of CFSE-stained T cells stimulated with or without anti-CD3/anti-CD28 in serum-free media alone, with naive GBM EV-treated monocytes, or with IFN-γ-treated GBM EVs. (B) Bar graphs showing mean T cell proliferation (from 4 donors) in response to anti-CD3/anti-CD28 antibodies in the conditions outlined. (C) Bar graphs showing mean T cell proliferation in response to anti-CD3/anti-CD28 antibodies in serum-free media alone or in the presence of monocytes exposed to GBM EVs, GBM EVs with PD-L1 knockdown, or GBM EVs with IDO1 knockdown. GBM cells treated with (+) or without (−) IFN-γ (100 ng/mL) for 3 days prior to EV harvest. *P < .05, **P<0.01, ***P < .001, ****P < .0001. EV, extracellular vesicle; GBM, glioblastoma; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-gamma; PD-L1, programmed cell death ligand 1.