Fig 1: 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-? relative to untreated cells. Data are calculated with the ??CT 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 2: Effect of anthocyanins and delphindin chloride (DC) on programmed death-ligand 1 (PD-L1) localization in the cell and fluorescence intensity. Examples of confocal microscopy pictures of cells. (a) Untreated (UNT), (b) Cyanidin-3-O-glucoside (C3G), (c) Delphinidin-3-O-glucoside (D3G). (d) DC. (e) The relative fluorescence intensity of PD-L1 for treatments of C3G, D3G, and DC compared to the untreated control. The results are expressed as mean ± SEM. Different letters indicate statistical difference between treatments and the untreated control (100%) as determined by Student’s T-test (p < 0.05). At least two independent experiments were run. At least three sections of a well were used to quantify the expression of PD-L1 in Zen 2 (blue edition) software. White arrows indicate visualization of membrane localization of the protein.
Fig 3: sPD-L1 splicing variants attenuate the neutralizing activity of aPD-L1 by trapping the antibody. (A and B) Flow cytometric analysis of aPD-L1 antibody binding to PD-L1 (A) and aPD-1 antibody binding to PD-1 (B) in the presence of indicated sPD-L1 variants molar ratio. (C) Fc-tagged PD-L1 and aPD-L1 antibodies (0.75 µg/ml) preincubated with or without PD-L1v242 as indicated were added to PD-1 precoated 96-well ELISA plates. The binding of Fc-tagged PD-L1 to PD-1 was detected based on absorbance at 450 nm; 0.2 µg/ml PD-L1v242 is equal to 0.75 µg/ml aPD-L1 antibody in molar ratio. n = 3. Results are expressed as mean ± SD. Paired two-tailed Student t test was used. *, P < 0.05; ***, P < 0.001. (D) Schematic diagram illustrating the NFAT-luc assay for evaluating TCR-mediated NFAT transduction. (E) The neutralizing activity (EC50) of aPD-L1 and aPD-1 antibodies was determined using NFAT-luc assay in the presence of various molar ratios of PD-L1v242 to the antibody (n = 3). Results are expressed as mean ± SD. (F) The correlation between PD-L1v242 and the EC50 of the antibody according to the Lineweaver–Burk plot was analyzed based on the results of (E), which suggested that the sPD-L1 splicing variants reduce the aPD-L1 inhibitory activity competitively. Each experiment was independently performed twice, yielding similar results.
Fig 4: sPD-L1 splicing variants contribute the resistance to PD-L1 blockade in WT-1 tumor antigen–specific iPSC-derived CD8 T cell model. (A) Schematic illustration of apoptosis assay for testing whether PD-L1v242 attenuates the blockade effect of aPD-L1 antibody. (B) iPSC-derived WT-1–specific T cells overexpressing PD-1 were cocultured with THP-1 cells overexpressing PD-L1 for 18 h in the presence of aPD-L1 antibody (1 µg/ml) or PD-L1v242 (2 µg/ml). The dead cell ratio was flow cytometrically measured using propidium iodide staining, and bars represent the proportion of live T cells in comparison with those before coculture. 2 µg/ml of PD-L1v242 was approximately eight times more than 1 µg/ml aPD-L1 antibody in molar ratio. The results are representative from three independent experiments and are shown as mean ± SD (n = 3). Paired two-tailed Student t test was used. ***, P < 0.001. The experiment was independently performed twice, yielding similar results.
Fig 5: Box plot of changes in serum PD-L1 levels in the placebo group and the vitamin D group compared (A) for the subgroup of 1st quintile of PD-L1 levels (Quintile 1), (B) for the subgroup of 2nd quintile of PD-L1 levels (Quintile 2), (C) for the subgroup of 3rd quintile of PD-L1 levels (Quintile 3), (D) for subgroup of 4th quintile of PD-L1 levels (Quintile 4), (E) for subgroup of 5th quintile of PD-L1 levels (Quintile 5), (F) Sum of 1st–5th quintiles of PD-L1 levels. Pre = after the surgery and just before starting supplements; Post = 1 year after starting supplementation. Changes between pre and post were evaluated with the Wilcoxon signed-rank test.
Supplier Page from Abcam for Human PD-L1 ELISA Kit [28-8]