1. 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
Log-rank p = 0.20
HR, 0.57
95% CI
Log-rank p = 0.07
HR, 0.98
95% CI
Log-rank p = 0.95
HR, 0.61
95% CI
Log-rank p = 0.12
HR, 0.99
95% CI
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.

2. 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
PPV: 98.5%
C-index: 1.000
95% C.I -6 -4 -2 2 4 6 -4 -2 2 4
Predicted
cluster B
cluster A
Predicted
cluster B
cluster A

3. 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.

4. 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
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
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
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
p = 0.01 72 84 96
108
120
Supplementary Figure S3
Ohue et al. Survival prediction in lung cancer patients

5. 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).

6. 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+

7. 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.

8. 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.

9. 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.