Supplementary Figure S1 Ohue et al. Survival prediction in lung cancer patients Time after surgery (months) CD8 CD4 FoxP3 CD8/Foxp3 CD4/Foxp3 20 40 60 80 100 Overall survival (%) 12 24 36 48 TILpos TILneg HR, 0.67 95% CI 0.33-1.25 Log-rank p = 0.20 HR, 0.57 95% CI 0.27-1.03 Log-rank p = 0.07 HR, 0.98 95% CI 0.51-1.89 Log-rank p = 0.95 HR, 0.61 95% CI 0.32-1.13 Log-rank p = 0.12 HR, 0.99 95% CI 0.52-1.86 Log-rank p = 0.97 (n= 80) (n= 40) (n= 83) (n= 37) (n= 78) (n= 42) (n= 61) (n= 59) (n= 65) (n= 55) Supplementary Figure S1 OS of the 120 lung adenocarcinoma patients with positive and negative infiltration of TILs by the Kaplan-Meier method.

A B Supplementary Figure S2 Discriminant scores Validation cohort Ohue et al. Survival prediction in lung cancer patients Discriminant scores A B Cluster B Cluster A Cluster B Cluster A Study cohort Validation cohort PPV: 96.7% C-index: 0.996 95% C.I. 0.989-1.000 PPV: 98.5% C-index: 1.000 95% C.I. 1.000-1.000 -6 -4 -2 2 4 6 -4 -2 2 4 Predicted cluster B cluster A Predicted cluster B cluster A

Supplementary Figure S2 Ohue et al. Survival prediction in lung cancer patients Supplementary Figure S2 Discriminant scores of each patient belonging to clusters A and B in the study cohort (A) and in the validation cohort (B) calculated by the discriminant function. Positive predicted value (PPV) was calculated by the leave-one-out cross-validation strategy. Concordance (C)-index is shown with 95% C.I.

Supplementary Figure S3 Overall survival (%) 20 40 60 80 100 Study cohort: pStage I-IIIA (n=120) 5yr-OS 53.6% vs 29.2% HR, 0.48 95% CI 0.21-0.82 Log-rank p = 0.007 Predicted Cluster A Predicted Cluster B (n= 80; events, 24) (n= 40; events, 19) Study cohort: pStage I 85.0% vs 68.1% HR, 0.24 95% CI 0.03-0.90 Log-rank p = 0.04 (n=62) (n= 44; events, 3) (n= 18; events, 4) 3yr-OS 47.3% vs 26.2% HR, 0.59 95% CI 0.26-1.1 Wilcoxon p = 0.05 (n= 36; events, 21) (n= 22; events, 15) Study cohort: pStage II-IIIA (n=58) A B Time after surgery (months) 12 24 36 48 Validation cohort: pStage I (n=68) (n= 40; events, 0) (n= 28; events, 6) 10yr-OS 100% vs 53.8% HR, 0.0 95% CI 0.02-0.59 p = 0.01 72 84 96 108 120 Supplementary Figure S3 Ohue et al. Survival prediction in lung cancer patients

Supplementary Figure S3 Ohue et al. Survival prediction in lung cancer patients Supplementary Figure S3 OS rates of the patients belonging to the predicted clusters A and B classified by the discriminant scores calculated by the discriminant function are shown in A for the study cohort and in B for the validation cohort by the Kaplan-Meier method. OS rates of pStage I and pStage II-IIIA patients are also separately shown in A. The study cohort consisted of patients in Toyama University Hospital, and the validation cohort consisted of patients in Kawasaki Medical School Hospital (see Materials and Methods).

Supplementary Figure S4 Ohue et al. Survival prediction in lung cancer patients C A B 100 200 300 400 50 150 H-score of whole lung cancer tissue PD-L1 Gal-9 r=0.84 P=0.0011 CD4 T cells CD8 T cells H-score of PD-L1 on tumor cells 20 30 40 60 70 80 r=0.40 P=0.22 r=0.73 P=0.001 r=-0.72 P=0.012 r=-0.71 P=0.014 10 r=0.66 P=0.026 r=0.79 P=0.004 r=0.51 P=0.11 r=0.71 P=0.015 r=-0.69 P=0.018 r=-0.74 P=0.009 r=0.67 P=0.024 P=0.027 H-score of Gal-9 on tumor cells %cells TIL HE CD3 PD-1- TIM-3- PD-1+ TIM-3- PD-1+ TIM-3+

Supplementary Figure S4 Ohue et al. Survival prediction in lung cancer patients Supplementary Figure S4 Representative IHC staining of CD3, PD-L1, and Galectin-9 in 11 surgically resected lung adenocarcinoma specimens used in the experiment shown in Figure 5 (A). For correlation of PD-L1 and Galectin-9 expression (B), and expressions and T-cell infiltration (C) in the tumor, the IHC H-score analyzed by ScanScope was used. The H-score is the summation of the percentage of positive staining cells multiplied by an ordinal value corresponding to the intensity level (0, none; 1, weak; 2, moderate; 3, strong). The resulting score ranged from 0 (no staining in the tumor) to 300 (diffuse intense staining in the tumor) (see Materials and Methods). In B and C, a dot indicates each patient. r, the Spearman’s correlation coefficient. Bar in panel indicates 1 cm.

RT - PCR Supplementary Figure S5 M 6 1 2 3 4 5 9 7 8 346 bp XAGE1 Ohue et al. Survival prediction in lung cancer patients M 6 1 2 3 4 5 9 7 8 346 bp XAGE1 G3PDH RT - PCR Adenocarcinoma Squamous cell carcinoma 1. PC-9 2. II-18 3. A549 4. HLC-1 5. OU-LC-ON 6. OU-LC-SK 7. Sq-1 8. EBC-1 9. RERF-LC-AI M. Marker Supplementary Figure S5 RT-PCR analysis of XAGE1 mRNA in 9 lung cancer cell lines.

Supplementary Figure S6 Ohue et al. Survival prediction in lung cancer patients PD-L1 Surface Intracellular Galectin-9 Adenocarcinoma % of Max Isotype/non-treat/IFNγ treat PC-9 A549 HLC-1 OU-LC-ON OU-LC-SK Supplementary Figure S6 Effect of IFNγ on PD-L1 and Galectin-9 expression in lung cancer cell lines. The cells were treated with IFNγ for 48 hrs at a concentration of 10 μM and analyzed by FACS Canto II.