Fig 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).

Fig 3: 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.

Fig 4: 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.

Fig 5: Hyper-IL-6 treatment does not affect proliferation of iPSC-derived NPCs. (A) Representative western blot of p-vimentin (top), PAX6 (middle) and total protein (bottom) in iPSC-derived NPCs after 5 days of treatment with Hyper-IL-6 as well as with vehicle. (B) Quantification of signal intensity of p-vimentin relative to total protein from A. (C) Quantification of signal intensity of PAX6 relative to total protein from A. (D) Representative western blot of NR2F1 (top) and total protein (bottom) in iPSC-derived NPCs after 5 days of treatment with Hyper-IL-6 as well as with vehicle. (E) Quantification of signal intensity of NR2F1 relative to total protein stain from D. For B, C and E, each dot represents an individual protein sample from consecutive differentiation batches of iPSC-derived NPCs (n=3 batches). Data are mean±s.e.m. Comparisons were analyzed using paired two-tailed Student's t-test separately for D19 and D26: ns, P≥0.05; **P<0.01.