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.