IL-27 induces the expression of IDO and PD-L1 in human cancer cells - PubMed (original) (raw)

IL-27 induces the expression of IDO and PD-L1 in human cancer cells

Grazia Carbotti et al. Oncotarget. 2015.

Abstract

IL-27 is a member of the IL-12 family that is produced by macrophages and dendritic cells. IL-27 inhibits the growth and invasiveness of different cancers and therefore represents a potential anti-tumor agent. By contrast, it may exert immune-regulatory properties in different biological systems. We reported that IL-27 induces the expression of the IL-18 inhibitor IL-18BP, in human Epithelial Ovarian Cancer (EOC) cells, thus potentially limiting the immune response. Here, we tested whether IL-27 may modulate other immune-regulatory molecules involved in EOC progression, including Indoleamine 2,3-dioxygenase (IDO) and Programmed Death-Ligand (PD-L)1. IDO and PD-L1 were not constitutively expressed by EOC cells in vitro, but IL-27 increased their expression through STAT1 and STAT3 tyrosine phosphorylation. Differently, cells isolated from EOC ascites showed constitutive activation of STAT1 and STAT3 and IDO expression. These findings, together with the expression of IL-27 in scattered leukocytes in EOC ascites and tissues, suggest a potential role of IL-27 in immune-regulatory networks of EOC. In addition, IL-27 induced IDO or PD-L1 expression in monocytes and in human PC3 prostate and A549 lung cancer cells. A current paradigm in tumor immunology is that tumor cells may escape from immune control due to "adaptive resistance" mediated by T cell-secreted IFN-γ, which induces PD-L1 and IDO expression in tumor cells. Our present data indicate that also IL-27 has similar activities and suggest that the therapeutic use of IL-27 as anti-cancer agent may have dual effects, in some tumors.

Keywords: IDO; IL-27; Immune response; Immunity; Immunology and Microbiology Section; PD-L1; STAT.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflict of interest to disclose.

Figures

Figure 1

Figure 1. IL-27 induces IDO protein and mRNA expression in human EOC cells in vitro

A. Western blot analysis of IDO expression in six EOC cell lines stimulated with the indicated cytokines or medium only (CTR) for 48 hours. α-tubulin is used as loading control. A representative experiment out of two is shown. B. QRT-PCR analysis of IDO1 mRNA expression in EOC cells treated with IL-27 relative to untreated (Ctrl) cells. Data are the mean of three independent experiments and expressed as ΔΔCT-fold change. Error bars represent SD. C. Kynurenine production in the conditioned medium of IL-27, IFN-γ or untreated (CTR) EOC cells, as detected by HPLC analysis. Histograms represent mean values of three biological replicates and error bars are standard deviations.

Figure 2

Figure 2. IL-27 increases PD-L1 surface protein and mRNA expression in EOC cells in vitro

A. FACS analysis of surface PD-L1 expression in four EOC cell lines, cultured in the presence of medium (control), IL-27 or IFN-γ. Dotted lines are isotype-matched unrelated Ig staining controls. Numbers in brackets are Median Fluorescence Intensity (MFI) values calculated as median PD-L1 minus median Ig control. Data are representative of two independent experiments showing similar results. B. QRT-PCR analysis of PDL1 mRNA expression in five IL-27-stimulated EOC cells relative to untreated cells. Data are the mean (±SD) of three independent experiments. C. Comparative analysis of IDO1 and PDL1 mRNA up-regulation by IL-27 or IFN-γ in a representative EOC cell line (CAOV3). Data are the mean of two independent replicates and are expressed as ΔΔCT-fold change. Error bars represent the minimum and maximum.

Figure 3

Figure 3. IL-27R chains GP130 and WSX1/IL-27RA are expressed in EOC cell lines

FACS analysis of surface GP130 and WSX1/IL-27RA expression in six EOC cell lines. Dotted lines represent isotype-matched unrelated Ig staining controls. Numbers in brackets are MFI values calculated as median GP130 or WSX1/IL-27RA minus median Ig control. Data are representative of two independent experiments with similar results.

Figure 4

Figure 4. IL-27 induces STAT1 and STAT3 phosphorylation (P) in EOC cell lines in vitro

A. Western blot analysis of tyrosine phosphorylated (P)-STAT1, P-STAT3 and STAT3 proteins in A2774 EOC cells either cultured for 10 or 30 minutes with medium (CTR) or with IL-27 (20 ng/ml). Total STAT3 and α-tubulin served as controls. Similar kinetics of STAT1/3 phosphorylation were observed in another cell line (A2780, not shown). B. Analysis of P-STAT1, STAT1, P-STAT3 and STAT3 proteins in SKOV3 and OC316 cell lines cultured in medium alone (CTR), or with IFN-γ or IL-27 (20 minutes). Total STAT1 and STAT3 were used as controls. Data are representative of three independent experiments.

Figure 5

Figure 5. IDO and tyrosine-phosphorylated forms of STAT1 and STAT3 are constitutively present in cells from EOC ascites

A. Western blot analysis of IDO, P-STAT1 and P-STAT3 proteins in neoplastic cells isolated from the ascites of three ovarian cancer patients cultured with medium (CTR) or IL-27 for 20 minutes. β-actin was used as loading control. * indicate lanes from a blot re-probed for IDO and phosphorylated STAT3 for which STAT1 and actin were presented in a previous article [17]. B. Immunohistochemical analysis of IDO expression in ascites cells. Both tumor cell nests (arrow) and scattered reactive cells express IDO protein.

Figure 6

Figure 6. Silencing of STAT1 or STAT3 with siRNA effects IL-27-driven IDO or PD-L1 expression

A. Western blot analysis of STAT1 and STAT3 unphosphorylated and phosphorylated (P) proteins in STAT1- or STAT3-silenced or scrambled (NT) siRNA-transfected A2774 and OC316 EOC cells untreated or treated for 20 minutes with IL-27 (20 ng/ml). Total STAT3 or STAT1 served as controls for STAT1- or STAT3-silenced cells, respectively. B. Western blot analysis of IDO expression in STAT1- or STAT3-silenced or scrambled siRNA-transfected A2774 and OC316 EOC cells untreated or treated for 48 hours with IL-27 (50 ng/ml). α-tubulin is shown as loading control. A replicate experiment is shown in Figure S6A C. FACS analysis of PD-L1 surface expression in STAT1- or STAT3-silenced or scrambled siRNA-transfected OC316 or A2774 EOC cells untreated or treated for 48 hours with IL-27 (50 ng/ml). Numbers in brackets are MFI values calculated as median PD-L1 minus median Ig control. The results of different experiments are shown in Figure S6B.

Figure 7

Figure 7. IL-27 induces PD-L1 and/or IDO expression in human PC3 prostate and A549 lung cancer cells and adherent PBMC

A. Western blot analysis of IDO expression in human adherent PBMC, non-adherent PBMC and in PC3 and A549 cells treated with the indicated cytokines or medium only for 48 hours. α-tubulin is used as loading control. Similar results were observed in two additional experiments. B. QRT-PCR analysis of IDO1 and PDL1 mRNA expression in cytokine-stimulated adherent or non-adherent PBMC, PC3 and A549 cells relative to untreated cells. Data are expressed as ΔΔCT-fold change. Mean values of three independent experiments. Error bars represent SD. C. FACS analysis of surface PD-L1 in IL-27-treated or untreated PC3 and A549 cell lines and adherent PBMC. Dotted lines are isotype-matched unrelated Ig staining controls. Similar results were obtained in five different experiments (PC3: MFI 5.5 ± 2.7 vs 1.5 ± 0.6, mean ± SD in IL-27-treated vs un-stimulated cells, P = 0.03 by paired Student's t test; A549: MFI 2 ± 1 vs 0.43 ± 0.4, P = 0.01).

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