Fig 2: EGCG inhibited mouse melanoma growth in vivo. (A) Flow cytometry data depicting the cell surface expression of PD-L1 in B16F10 cells treated with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-? (IFN-?) or a combination of IFN-? and EGCG (IFN-? + EGCG). Histogram (left panel) and quantification of mean fluorescent intensity (MFI) (right panel). (B) qRT-PCR analysis of PD-L1 after treatment with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-? (IFN-?) or a combination of IFN-? and EGCG (IFN-? + EGCG) in B16F10 cells. GAPDH served as a control. (C) Immunoblot analysis of p-STAT1, STAT1 and IRF1 in B16F10 cells treated with 0.1% DMSO (control), 10 µM EGCG (EGCG), 10 ng/mL IFN-? (IFN-?) or a combination of IFN-? and EGCG (IFN-? + EGCG). GAPDH served as a control. The band densities of proteins were quantified with image J and normalized to GAPDH. p-STAT1 to STAT1 ratio was calculated and normalized to control. (D) Tumor growth curve of B16F10 cells injected subcutaneously in C57BL/6 mice. Mice were treated from day 7 with saline daily intraperitoneally (control), 50 mg/kg EGCG daily intraperitoneally (EGCG), or 12.5 mg/kg anti-PD-1 antibody intraperitoneally (anti-PD-1) every 3 days. (E) Ki-67 staining of B16F10 tumors (derived from D) treated with saline (control), 50 mg/kg EGCG (EGCG), or 12.5 mg/kg anti-PD-1 antibody (anti-PD-1). Representative images (left panel) and quantification of positive cells (right panel). Ki-67-positive cells were counted in the whole field under a microscope and presented as % positive tumor cells. Bar = 50 µm. Data are representative of 2 independent experiments and expressed as the mean ± S.D., n = 3 (A,B), 10 (D), or 3 (E). * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 3: XVir-N-31-anti-PD-L1 expresses a functional PD-L1 antibody. (a) HEK293 cells were infected with 30 MOI of XVir-N-31 or XVir-N-31-anti-PD-L1, or were left untreated (control). Lysates or supernatants were collected at the indicated time points. The production and secretion of anti-PD-L1 were analyzed by immunoblot using an anti-HA antibody, GAPDH served as the loading control. (b) HEK293 cells were infected with 50 MOI of XVir-N-31, XVir-N-31-anti-PD-L1, or were left untreated. The supernatants were collected 48 h after infection and were subsequently used in an PD1/PD-L1 blocking assay. Increased luminescence indicates the PD-1/PD-L1 blockade. PBS served as the negative control, 10 µg/mL anti-PD-L1 antibody for the positive control (XVir-PD-L1: XVir-N-31-anti-PD-L1; n = 3; SEM; n.s.: not significant; * p < 0.05, ** p < 0.01).

Fig 4: Reduction of tumor growth. (a) Representative pictures of brain sections of the U87MG tumor bearing, immuno-humanized NSG mice. The ipsilaterally growing tumor was injected with either PBS (sham), 3 × 108 IFU Ad-WT, XVir-N-31, or XVir-N-31-anti-PD-L1. One group additionally received repeated systemic injections with nivolumab, as indicated in the Methods section. (b) Quantification of the tumor volume (mm3; XVir-PD-L1: XVir-N-31-anti-PD-L1; n = 7–8 mice per group, SEM; n.s.: not significant; * p < 0.05, ** p< 0.01, *** p < 0.001).

Fig 5: EGCG inhibited JAK-STAT signaling and increased granzyme B expression in CD8+ cells in the B16F10 tumor microenvironment. (A,B) qRT-PCR analysis of STAT1 (A) and IRF1 (B) in B16F10 tumors derived from Figure 3D, after the treatment with saline (control), 50 mg/kg EGCG (EGCG) or 12.5 mg/kg anti-PD-1 antibody (anti-PD-1) (n = 3). GAPDH served as a control. (C–E) Immunofluorescence study of B16F10 tumors derived from Figure 3D, treated with saline (control), 50 mg/kg EGCG (EGCG), or 12.5 mg/kg anti-PD-1 antibody (anti-PD-1). (C) Left, overlay images of tissue sections stained with CD8 (green) and DAPI (blue). Bar = 50 µm. Right, quantification of CD8+ cells per field (n = 17). Each symbol represents an individual field. (D) Left, overlay images of tissue sections stained with CD8 (green), PD-L1 (red) and DAPI (blue). Inset: higher magnification of the dotted-line rectangular area. Right, quantification of PD-L1 expression around CD8 cells. PD-L1 mean fluorescent intensity (MFI) was calculated by measuring 90 µm2 area around randomly selected six CD8 cells. (E) Overlay images of tissue sections stained with CD8 (green), granzyme B (GZB, red) and DAPI (blue). Arrows depict CD8/GZB double-positive cells. Inset: images of the dotted-line rectangular area with CD8 only (green, upper), GZB only (red, middle) and overlay of CD8 and GZB (merged, bottom). Bar = 50 µm. (F) Quantification of CD8/GZB double-positive cells per field (n = 7) (left) and % of GZB expression in CD8-positive cells (n = 7) (right). Each symbol represents an individual field. Data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001.