Autocrine activation of the IFN signaling pathway may promote immune escape in glioblastoma

Abstract

Background: Interferons (IFNs) are cytokines typically induced upon viral infection but are constitutively expressed also in the absence of acute infection. The physiological role of autocrine and paracrine IFN signaling, however, remains poorly understood, and its function in glioblastoma has not been explored in depth.

Methods: Using RNA interference-mediated gene silencing, we characterized constitutive type I IFN signaling and its role in human glioma cells.

Results: We observed constitutive expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and myxovirus resistance protein A (MxA), a classical IFN-response marker, in the absence of exogenous IFN-β. In vivo, we found higher MxA expression in gliomas than in normal tissue, suggesting that IFN signaling is constitutively active in these tumors. To demonstrate the presence of an autocrine type I IFN signaling loop in glioma cells in vitro, we first confirmed the expression of the type I alpha/beta receptor (IFNAR)1/2, and its ligands, IFN-α and IFN-β. Small interfering RNA-mediated receptor gene silencing resulted in reduced expression of MxA at mRNA and protein levels, as did gene silencing of the ligands, corroborating the hypothesis of an autocrine signaling loop in which type I IFNs induce intracellular signaling through IFNAR1/2. On a functional level, following IFNAR1 or IFNAR2 gene silencing, we observed reduced programmed death ligand 1 (PD-L1) and major histocompatibility complex (MHC) class I and II expression as well as an enhanced susceptibility to natural killer immune cell lysis, suggesting that autocrine IFN signaling contributes to the immune evasion of glioma cells.

Conclusions: Our findings point to an important role of constitutive IFN signaling in glioma cells by modulating their interaction with the microenvironment.

Keywords: PD-L1; STAT1; glioma; immunogenicity; interferon.

Fig. 1

IFN-β target genes are expressed in glioma cells. (A) The human LTC LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, and the GICs T-325, T-269, ZH-161, ZH-305, and S-24 were cultured without or with IFN-β (150 IU/mL) for 48 h and MxA mRNA expression levels were determined using real-time PCR (median expression levels ± SE are shown from 3 independent experiments). (B) The cells were treated as in (A). Whole cell lysates were subsequently analyzed for pSTAT1, STAT1, and MxA protein levels by immunoblot using actin as a loading control (1 out of 2 independent experiments is shown). (C) LN-308 or ZH-161 cells, untreated or exposed to IFN-β (150 IU/mL) for 48 h, were analyzed for MxA protein levels by immunofluorescence (scale bar, 50 µm). (D) LN-308, LN-229, T-325, or ZH-161 cells were cultured in various medium conditions as indicated for 48 h (SF, serum-free DMEM; NB, Neurobasal medium). Whole cell lysates were assessed for MxA expression levels using immunoblot. Actin was used as loading control.

Fig. 2

MxA is expressed in gliomas in vivo. (A) MxA mRNA expression levels in gliomas of different WHO grades were analyzed using data from the database of TCGA (left). Overall survival analysis within the TCGA database for glioblastoma patients with high versus low MxA expression was performed by Kaplan–Meier analysis. The median was used as cutoff (right). (B) MxA protein levels were assessed by immunohistochemistry on a glioma tissue microarray and quantified by H scoring (left). Phospho-STAT1 protein levels were analyzed by immunohistochemistry on a TMA and quantified by H scoring. A correlation analysis of pSTAT1 H scores with MxA H scores is shown (right). (C) Representative images of normal brain and glioblastoma specimens with low, intermediate, and high MxA levels are shown (scale bar, 100 µm or 10 µm for 20x or 40x magnification, respectively). (D) MxA/CD45 costaining was performed on a glioma TMA and the number of double-positive cells was counted.

Fig. 3

MxA expression in human GICs depends on signaling through IFNAR1 and IFNAR2. (A) Basal expression levels of IFNAR1 and IFNAR2 were assessed in LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells by real-time PCR (left) (median expression levels ± SE are shown from 2 independent experiments). Cell surface IFNAR2 protein was analyzed by flow cytometry (1 out of 2 independent experiments is shown). Isotype control antibody (gray) and specific antibody (black) are shown in the histograms (right). (B) SiRNA-mediated gene silencing of IFNAR1 (siIFNAR1), IFNAR2 (siIFNAR2), or IFNAR1 and IFNAR2 in parallel (siIFNAR1/2) in T-325 or ZH-161 cells was performed by electroporation and confirmed for IFNAR1, IFNAR2, and MxA by real-time PCR 24 h posttransfection (median expression levels ± SE are shown from 3 independent experiments) and for IFNAR2 by flow cytometry at 48 h following transfection. Isotype control antibody (gray) and specific antibody (black) are shown in the histograms. (C) Phospho-STAT1, STAT1, and MxA levels of control, siIFNAR1, siIFNAR2, or double knockdown cells (siIFNAR1/2) were determined 48 h after transfection by immunoblot (1 out of 3 independent experiments is shown).

Fig. 4

Glioma-derived IFN-α or IFN-β induces autocrine signaling. (A) IFN-α or IFN-β mRNA expression levels were determined in LN-308, LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells using real-time PCR (median expression levels ± SE are shown from 2 independent experiments). (B, C) SiRNA-mediated gene silencing of IFN-α (si_IFN-α) or IFN-β (si_IFN-β) was performed using electroporation in T-325 or ZH-161 cells. MxA mRNA expression was determined 24 h posttransfection by real-time PCR (median expression levels ± SE are shown from 2 independent experiments) (B), while pSTAT1, STAT1, and MxA protein levels were assessed at 48 h following transfection by immunoblot. Actin was used as a loading control (1 out of 3 independent experiments is shown) (C).

Fig. 5

Constitutive IFN signaling impairs the immunogenicity of glioma cells. (A) SiRNA-mediated silencing of IFNAR1/2 or transfection with control oligonucleotides was performed in T-325 or ZH-161 using electroporation (top), or the cells were exposed to 150 IU/mL IFN-β or not (bottom). Cell surface PD-L1 protein levels were assessed after 72 h by flow cytometry (1 out of 3 independent experiments is shown). Isotype control antibody (gray) and specific antibody (black) are shown in the histograms. (B) Correlation analysis of MxA with PD-L1 mRNA expression was performed for glioma patients within the database of TCGA. Two-tailed Pearson test coefficients (r) and significances (P) are indicated. (C, D) SiRNA-mediated gene silencing of IFNAR1/2 in T-325 or ZH-161 cells was performed by electroporation. MHC class I or II cell surface protein levels were determined 48 h posttransfection by flow cytometry (median expression levels ± SD are shown from 8 independent experiments) (C). ZH-161 cells were used as target cells in a 4 h NKL cell lysis assay at various effector:target (E:T) ratios as indicated 72 h after transfection. The percentage of target cell lysis corrected for spontaneous background lysis is shown (left) (1 out of 4 independent experiments is shown). The knockdown efficiency for IFNAR1 and IFNAR2 gene silencing was confirmed by real-time PCR (right) (D).