Fig 2: Positive correlation between the expression of PD-L1 and HK2 in human lung cancer tissues. Immunohistochemistry (IHC) analyses for GLUT1, HK2, and PKM2 were performed using tumor tissues from NSCLC patients (N = 393). a The expression (H-score) of these molecules was compared between PD-L1negative and PD-L1positive NSCLCs and statistical differences were analyzed using Mann Whitney U-tests. The expression of these molecules was also compared between PD-L1negative and PD-L1positive cases in patients with b wild-type EGFR (N = 230) and c mutated EGFR (N = 126). Statistically differences were analyzed using Mann Whitney U-tests. The whiskers are drawn from the 10th percentile to the 90th percentile. The midline of the box is the median and “+” denotes the mean. Points below and above the whiskers are individual points. d Representative IHC images are as follows: D-1, a pSqCC case that is PD-L1negative and expresses low levels of GLUT1, HK2, and PKM2; D-2, a pSqCC case that is PD-L1positive and expresses high levels of GLUT1, HK2, and PKM2; D-3, a pADC case that is PD-L1negative and expresses low levels of GLUT1, HK2, and PKM2; D-4, a pADC case that is PD-L1positive and expresses low level of GLUT1 and high levels of HK2 and PKM2. Cases were separated into low and high expression group using the median H-score of each molecule as a cutoff. Cases with PD-L1 score 2 and 3 (moderate to strong membranous staining in ≥10% of tumor cells) was designated as PD-L1positive. (original magnification × 400, bar = 100 μm). Abbreviations: N, negative; P, positive; L, low; H, high

Fig 3: HK2 expression is inversely correlated with the expression of T-cell effector molecules in human lung cancers, particularly in those with high PD-L1 expression. a The expression levels of T-cell effector molecules, including CD4, CD8A, GZMA, GZMB, IFNG, CXCL9, CXCL10 and PRF1, were assessed according to CD274 (PD-L1) and HK2 expression status in NSCLCs from TCGA dataset (N = 1015). Cases were dichotomized into PD-L1low and PD-L1high groups based on median values and then trichotomized into HK2low (<25th percentile), HK2intermediate (25-75th percentile), and HK2high (>75th percentile) groups. Statistical differences were analyzed using Kruskal-Wallis tests. b and c The expression levels of T-cell effector molecules were compared according to HK2 expression status in NSCLCs with low CD274 (PD-L1) mRNA levels (b) and with high CD274 (PD-L1) mRNA levels (c) in NSCLCs from TCGA dataset. Statistical significance was calculated using Pearson’s correlation analyses. *p < 0.05; **p < 0.001. Abbreviations: L, low; I, intermediate; H, high

Fig 4: Reactivity and affinity of PD-1pos and PD-1neg T cell clones. (A). Flow cytometric analysis of PD-L1 expression on M113 (left panel) and T2 (right panel) cell lines wild type and stably transfected with a PD-L1 coding expression vector. The filled histograms represent negative control staining while the over-laid empty histograms show staining with a PE-conjugated anti-PD-L1 Ab (clone MIH1, BD Biosciences). (B). Expression of HLA-A2 (clone BB7.2, BD Biosciences), Melan-A (clone A103, Dako, Denmark), ICAM-1 (CD54, clone HA58, BD Biosciences) and LFA-3 (CD58, clone 1C3, BD Biosciences) on M113 (upper panel) and M113-PD-L1pos (lower panel) cell lines. All the antibodies, unless A103 Ab were PE-conjugated. For Melan-A intracellular staining, cells were fixed, permeabilized, incubated with A103 Ab, and stained with PE-conjugated goat Fab’2 anti-mouse IgG secondary Ab (Beckman Coulter, France). (C). IFNγ production of PD-1neg and PD-1pos T cell clones in response to melanoma cell lines. IFNγ production was measured by ELISA (n = 2) in supernatants of PD-1neg (left column, dotted lines) and PD-1pos (right column, solid lines) T cell clones after 6 h of activation with M113 melanoma cell line (white circles) or M113-PD-L1pos melanoma cell line (black circles). (D). IFNγ production of PD-1neg and PD-1pos MELOE-1-specific T cell clones in response to T2 cells loaded with the MELOE-136-44 peptide. IFNγ production was measured by ELISA (n = 2) in supernatants of PD-1neg (left panel, dotted lines) and PD-1pos (right panel, solid lines) MELOE-1-specific T cell clones after a 6-h-activation period with peptide-loaded T2 cells (white circles) or peptide-loaded T2-PD-L1pos cells (black circles). (E). Avidities of PD-1neg and PD-1pos Melan-A and MELOE-1-specific T cell clones. T cell clones' avidities (PD-1neg, dotted lines and PD-1pos, solid lines) were evaluated by measuring IFNγ (left panel) and TNF-α production (right panel) in response to T2 cells loaded either with a range of Melan-AA27L peptide or MELOE-136-44 peptides, at an E:T ratio of 1:2. Cytokine production was evaluated by intracellular labeling with antibodies specific for IFNγ or TNF-α (gated on CD8+ T cells).

Fig 5: High HK2 expression is inversely correlated with the number of CD8+ TILs in patients samples and high glycolysis signature is inversely related with CD8A expression in TCGA dataset. a The numbers of CD8+ TILs were evaluated according to PD-L1 and HK2 expression status using tumor tissues from patients with pSqCC. Statistical significance was calculated using Kruskal-Wallis tests. b HK2 expression was evaluated according to PD-L1 expression and the numbers of CD8+ TILs using tumor tissues from patients with pSqCC. Statistical significance was calculated using Kruskal-Wallis tests. c & d CD8A transcript levels were compared according to CD274 (PD-L1)/glycolysis signature and the glycolysis signature was compared according to the tumor microenvironment immune type (TMIT) based on CD274 (PD-L1)/CD8A expression status c in pADC and d in pSqCC from TCGA dataset. Statistical differences were analyzed using Kruskal-Wallis tests. *p < 0.05; **p < 0.001. Abbreviations: N, negative; P, positive; L, low; H, high