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. 2021 Aug 9;13(16):4011.
doi: 10.3390/cancers13164011.

IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells

Grazia Carbotti  1 , Beatrice Dozin  2 , Stefania Martini  3 , Chiara Giordano  1 , Francesca Scordamaglia  4 , Michela Croce  1 , Gilberto Filaci  1   5 , Silvano Ferrini  1   6 , Marina Fabbi  1
Affiliations

IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells

Grazia Carbotti et al. Cancers (Basel). .

Abstract

Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Keywords: IL-27; IL-6; PD-L1; STAT1/3; mesothelioma; microenvironment; overall survival; pleural effusion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
IL-27 predominantly mediates STAT1 phosphorylation in MM cell lines. Western blot analysis of tyrosine phosphorylated (P)-STAT1, P-STAT3, total STAT1 and total STAT3 proteins (A) in MPP89 cells stimulated for 20 min with medium (CTR) or the indicated cytokines of the IL-12 family and (B) in MSTO, MPP89, and IST-MES1 cells stimulated for 20 min with medium (CTR), IL-6, sIL-6R/IL-6 chimera, and IL-27. Total STATs and α-tubulin were used as loading controls.
Figure 2
Figure 2
IL-27 upregulates PD-L1 molecule expression and release by MM cells. (A) Flow cytometry analysis of membrane PD-L1 expression in MSTO, MPP89, and IST-MES1 MM cell lines cultured with medium alone (baseline), IL-6, sIL-6R/IL-6 chimera, or IL-27 (induced). Dotted line shows isotype-matched Ig control. Numbers in brackets represent Median Fluorescence Intensity (MFI) values calculated as median anti-PD-L1 mAb minus median Ig control. Data are representative of two independent experiments yielding similar results. (B) qRT-PCR analysis of CD274 (PD-L1) mRNA expression in the same three MM cell lines stimulated with IL-6, sIL-6R/IL-6 chimera, IL-27, or IFN-γ relative to untreated cells. Data are calculated with the ΔΔCT method and expressed as fold change versus untreated control. Error bars represent SD of triplicates. LGALS3 (Galectin 3) and GBP1 (Guanylate Binding Protein 1) mRNA levels are shown, respectively, as negative and positive controls of IL-27 activity. (C) Evaluation by ELISA of soluble (s)PD-L1 release in the conditioned media of the indicated MM cells treated with IL-6, sIL-6R/IL-6 chimera, or IL-27. Data are expressed as a percent of untreated control and are the mean of two independent experiments, run in duplicates. Error bars represent the minimum and maximum values (* p < 0.001, Student’s t-test versus untreated control).
Figure 3
Figure 3
IL-27 is detectable in MM pleural exudates and correlates with sPD-L1 expression. (A) Concentration (pg/mL) of sPD-L1, IL-27, and IL-6 in pleural effusion. Bars indicate median values. (B) Correlation between IL-27 and sPD-L1 levels in MM pleural fluids. Spearman’s rank correlation coefficient (R) and p-value (P) are indicated. Lines represent the best fit linear regression analysis with the 95% confidence interval.
Figure 4
Figure 4
Kaplan–Meier curves assessing overall survival according to sPD-L1, IL-27, and IL-6 concentrations in the pleural effusion, as dichotomized around their respective median value. Analyses with respect to all cases (n = 77) (panels A–C) and the subgroup with the epithelioid histotype (n = 55) (panels D–F) are shown.
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