Fig 2: PD-L1 abundance regulates PLC-?1 phosphorylation, PKC-a activation, and calcium fluxes(A and B) PC9ER cells were pretransfected with either pGIPZ- EGFP or pGIPZ-PD-L1-EGFP (A). Alternatively, we used PD-L1 KO cells and parental cells (B). Cells were incubated for 30 min with Calcium Orange (2 µM) and stimulated with EGF 2 min after initial recording. The quantification of time-lapse fluorescence was performed over 17 min.(C) PC9ER, KO, and cells transiently expressing hemagglutinin (HA)-tagged PD-L1 were preloaded with Indo-1. EGF was added 1 min after the beginning of the experiment, and cells were subjected to flow cytometry. Calcimycin (A23187) was added after 180 s (arrows). Ca2+ influx was followed by measuring changes in the mean fluorescence ratio of Indo-1 at violet (405 nm) to blue laser (510 nm).(D) PC9ER cells were transfected with the pGIPZ-EGFP (control) or PD-L1-GFP plasmids. Alternatively, we used PD-L1 KO cells (1.4 × 107). Steady-state DAG levels were measured (4 biological repeats).(E) Control PC9ER cells and KO cells were serum starved, treated with EGF, and then fixed. Thereafter, cells were incubated with a PKC-a-specific antibody, followed by a secondary, FITC-conjugated antibody. Images were quantified in 2 experiments. Arrows mark PKC-a recruited to the plasma membrane.(F) The endogenous activity of RhoA was determined. PC9ER cells (0.5 × 106) were transfected with a PD-L1 plasmid (3 µg). After 48 h, cells were serum starved overnight and later preincubated for 60 min with either U73122 (15 µM), a PLC inhibitor, or PP2 (50 µM), a SRC inhibitor. This was followed by stimulation for 30 min at 37°C with either EGF (30 ng/mL) or PMA. Cleared cell extracts were assayed for RhoA GTPase levels. Statistical analyses were performed using 1-way ANOVA.(G) The model depicts the herein reported interactions between a short cytoplasmic segment of PD-L1 and PLC-?1. Formation of this complex enhances the activation of PLC-?1 by ligand- or mutation-activated EGFRs. Once stimulated by means of phosphorylation, PLC-?1 degrades phosphatidylinositol 4,5-bisphosphate (PIP2) to generate both diacylglycerol (which activates PKC) and inositol triphosphate (which elevates cytoplasmic Ca2+). The direct interaction between PD-L1 and PLC-?1 may explain the herein reported involvement of PD-L1 in chemotaxis and metastasis.

Fig 3: M1-like macrophages infiltration was associated with PD-L1 expression in HCC cells (A) The relationship between enrichment of macrophages or M1 macrophages and PD-L1 expression in HCC samples from GEO database. (B) The distribution of CD68+ HLA-DR+ M1 macrophage in HCC tissue. Double immunohistochemical staining was performed by using anti-CD68 and anti-HLA-DR. The black arrow points to CD68+ macrophages while the white arrow points to CD68+ HLA-DR+ M1-like macrophages. (C) The association of PD-L1 expression with infiltration of M1 macrophages in HCC tissues. PD-L1 expression and M1-like macrophage distribution were detected in serial sections from the same HCC paraffin tissue. The number of CD68+ HLA-DR+ macrophages in the PD-L1+ region was higher than that in PD-L1- region. (D) Positive and negative controls. A placental chorionic tissue was used as positive control (left). Negative controls were performed by omitting the primary antibodies (right). The data are presented as mean±SD. ** p < 0.01.

Fig 4: Immunoprecipitation analysis to detect the protein–protein interactions between programmed cell death ligand-1 (PD-L1) and ezrin/radixin/moesin (ERM) in HeLa cells. The whole-cell lysates of HeLa cells were immunoprecipitated with antibodies against (a) PD-L1, (b) ezrin, (c) radixin, (d) moesin, or control antibody. Western blot images of ezrin, radixin, and moesin as well as PD-L1, in addition to ß-actin, in the whole-cell lysates (input) and those co-immunoprecipitated (IP) with a control IgG or each antibody, are shown. Molecular weights are indicated in kDa.

Fig 5: ITGA2 transcriptionally increases PD-L1 expression in solid cancer cells. a and b. Volcano plot (a) and heatmap (b) to show the differential expressed genes of PANC-1 cells infected by si-Control or si-ITGA2. The blue points represented the downregulated genes, while red points represented the upregulated genes. c. Heatmap to show a subset of ITGA2 knockdown regulated genes participated in PD-L1 expression and PD-1 checkpoint pathway of PANC-1 cells. d-f. Forty-eight hours postinfection, PANC-1, HepG2, SGC-7901, and MDA-MB-231 cells infected with sh-Control or sh-ITGA2s were harvested for RT-PCR analysis (d), Western blotting analysis (e), and FACS analysis (f). Data are shown as means ± SD (n = 3). Each sh-ITGA2 group was compared with sh-Control group. Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparison’s tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. g-h. Forty-eight hours postinfection, PANC-1, HepG2, SGC-7901, and MDA-MB-231 cells infected with pcDNA3.1 or ITGA2 plasmids were harvested for RT-PCR analysis (g) and Western blotting analysis (h). Data are shown as means ± SD (n = 3–5). ITGA2 overexppression groups were compared with pcDNA 3.1 transfection group. Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparison’s tests. Compared groups were shown in the figures. **, P < 0.01; ***, P < 0.001