Fig 2: (A–D) Representative target cell aggregates of the conditions Hep (A), Hep Mo (B), Hep Ts (C), and Hep Ts Mo (D), in which the presence of dead target cells is DRAQ7+ (in red), HBV-specific TCR T cells are labeled with Cell tracker violet dye (in white), while monocytes are unlabeled. (E) Box plot of the percentage of dead target (HepG2-Pres1-GFP) volume after 24 h of co-culture with Tdx HBV-specific TCR T cells. Data in terms of a “Dead Target Index” are also shown where the percentage of dead target volume is normalized to the percentage of TCR+ T cells. (F) Box plot of the percentage of dead target volume after 24 h of treatment with Tdx HBV-specific TCR T cells with or without PD-L1- or PD-1-blocking antibody or their respective isotype (IgG) controls. (G) Box plot of the percentage of dead target (HepG2-Pres1-GFP) volume after 24 h of co-culture with EP HBV-specific TCR T cells. Data in terms of a “Dead Target Index” are also shown. Data points reflect the measured values of individual target cell aggregates and collectively represent the pooled results of three donors, where the 25th, 50th, and 75th percentiles as well as minimum and maximum values are indicated. Bars are drawn where a comparison was made with *P = 0.05, **P = 0.01, ***P = 0.001, and ****P = 0.0001. Statistical significance was evaluated by a one-way ANOVA with Holm-Sidak’s multiple comparisons test. EP, mRNA-electroporated; HBV, hepatitis B virus; Hep, HepG2-preS1-GFP; Mo, monocyte; n.s., not significant; Tc, control T cell; TCR T cells, T cell receptor-redirected T cells; Tdx, transduced; Ts, HBV-specific T cell.

Fig 3: Blockade of IFN-?, TNF-a, or CD81 does not affect PD-L1 and PD-L2 up-regulation by HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) as indicated. Graphs (A) show IFN-? and TNF-a levels in culture supernatants (mean ± SEM; n = 4). The dashed red line indicates the lowest value (15.6 pg/ml) of the dynamic range for the ELISA assays used. Neutralizing antibodies anti-IFN-? (B,C), anti-TNF-a (D,E), or anti-CD81 (F,G) were added to the cultures as indicated. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 or with conditioned medium from HPV-positive SCC154 alone (red histograms) or in the presence of blocking antibodies (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on fibroblasts for the indicated treatments. ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Fig 4: GSK3ß binds to and phosphorylates PD-L1 protein in vitro and in vivo.(a) Schematic diagram of GSK3ß phosphorylation and ß-TrCP-binding motifs and various mutants of PD-L1 expression constructs. PD-L1 was separated into ECD and ICD. SP, signal peptide; TM, transmembrane domain. The numbers indicate amino-acid positions. (b) In vitro GST pull-down assay of non-glycosylated PD-L1 and GSK3ß. (c) Co-immunoprecipitation (co-IP) measuring the interaction of GSK3ß and PD-L1 4NQ. Schematic diagram of PD-L1 4NQ deletion or truncation mutants showing on the left. Positions of glycosylation sites were labelled with red colour. The numbers indicate amino-acid positions. (d) Immunocomplex kinase assay measuring PD-L1 phosphorylation by GSK3ß. Coomassie blue staining showing equal loading amount of GST-PD-L1. CA, constitutive activation mutant (S9A); KD, kinase dead (K85A) mutant; WT, wild type. (e) Western blot analysis of phosphorylation of PD-L1 protein at T180 and S184 sites by phospho-T180 and -S184 PD-L1 antibodies, respectively. EV, empty vector. (f) Time-lapse microscopy image (at 12 h) showing the dynamic interaction between PD-L1 and PD-1 at the last time point. The kinetic graph showed the quantitative binding of green fluorescent labelled PD-1/Fc protein on PD-L1 WT, 3SA or 4NQ expressing BT549 cells at every hour time point (right). Scale bar, 100 µm. (g) T-cell-meditated tumour cell-killing assay in PD-L1 WT or 3SA-expressing BT549 cells. Representative phase, red fluorescent (nuclear-restricted RFP), and/or green fluorescent (Caspase 3/7 substrate)-merged images of PD-L1 WT- or PD-L1 3SA-expressing cells and activated T-cell co-cultures at 96 h. Green fluorescent cell was counted as dead cell. The quantitative ratio of dead cells showed in bar graph (right). Scale bar, 100 µm. (h) The tumour growth of mouse PD-L1 WT- or PD-L1 3SA-expressing 4T1 cells in BALB/c mice. Quantification of tumour volume is shown on the right and representative images of tumours are shown on the left. n=7 mice per group. Con, vector control; WT, PD-L1 WT; 3SA, PD-L1 3SA. *P<0.05 is statistically significant as shown by Student's t-test. All error bars are expressed as mean±s.d. of three independent experiments.

Fig 5: (A–C) Representative images of CTCs isolated from NSCLC patients stained by antibodies against cytokeratin (red), a leukocyte marker CD45 (green), and a nuclear stain (DAPI). (A) CK (+)/PD-L1 (+) CTC, (B) CK (+)/PD-L1 (-) CTC, (C) CTC cluster with WBC. Scale bar is 10 µm. (D) CTC enumeration from blood samples of healthy donors (n = 6) and from blood samples of NSCLC patients (n = 38), (***)P-value < 0.001. (E) Number of CTCs isolated by GO chip from blood samples from different visits of 13 NSCLC patients (P1 through P13). ‘P1’ stands for patient 1. Blue bar represents the number of CK (+)/PD-L1 (-) CTCs. Red bar represents the number of CK (+)/PD-L1 (+) CTCs.