1. Patients’ characteristics
Supplementary Table S1
Ohue et al. Survival prediction in lung cancer patients
Patients’ characteristics
Characteristics
Total
Cluster A
Cluster B
p-value
n=120
n=76
n=44
Sex – no. (%)
　　Male
61（50.8）
35（46.1）
26（59.1）
0.19
　　Female
59（49.2）
41（53.9）
18（40.9）
Age – yr
Mean ± S.D.
64.1 ± 9.8
63.3 ± 10.2
65.3 ± 9.2
0.29
Smoking status – no. (%)
Never or light
71（59.2）
45（59.2）
1.00
Heavy
49（40.8）
31（40.8）
Histology differentiation – no. (%)
Well
64（53.3）
46（60.5）
0.06
Moderate – poor
56（46.7）
30（39.5）
TNM classification – no. (%)
T　1a - 1b
77（64.2）
53（69.7）
24（54.5）
0.12
　　 a – 3
43（35.8）
23（30.3）
20（45.5）
N　0 – 1
93（77.5）
63（82.9）
30（68.2）
0.07
2 – 3
27（22.5）
13（17.1）
14（31.8）
pStage - no. (%) I
62（51.7）
43（56.6）
19（43.2）
II – IIIA
58（48.3）
33（43.4）
25（56.8）
pStage, pathological stage.

2. Supplementary Table S2
Ohue et al. Survival prediction in lung cancer patients
Patients’ characteristics of study and validation cohorts
in pStage I lung adenocarcinoma
Characteristics
Study cohort
pStage I
Validation cohort
p-value
n=62
n=68
Sex – no. (%)
　　Male
14（22.6）
35（51.5）
0.001
　　Female
48（77.4）
33（48.5）
Age – yr
Mean ± S.D.
63.8 ± 9.2
67.3 ± 10.9
0.05
Smoking status – no. (%)
Never or light
30（48.4）
20（29.4）
0.03
Heavy
32（51.6）
48（70.6）
Histology differentiation – no. (%)
Well
42（67.7）
32（47.1）
0.02
Moderate – poor
20（32.3）
36（52.9）
pStage - no. (%) IA
52（86.7）
42（61.8）
0.006 IB
10（13.3）
26（38.2）
pStage, pathological stage.
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).

3. Supplementary Table S3 Cell lines
Ohue et al. Survival prediction in lung cancer patients
Cell lines
Cell line
Source
Lung adenocarcinoma
PC-9
Riken Bioresource Center, Ibaragi, Japan
II-18
A549
HLC-1
OU-LC-ON
Our laboratory
OU-LC-SK
Lung squamous cell carcinoma
Sq-1
EBC-1
RERF-LC-AI

4. Supplementary Table S4 Antibodies used
Ohue et al. Survival prediction in lung cancer patients
Antibodies used
Antibody
Clone
Source
Rabbit anti-human PD-L1 Ab ー
ProSci incorporated, Poway, CA, USA
Rabbit anti-human PD-L1 mAb
SP-142
Spring Bioscience, Pleasanton, CA, USA
Mouse anti-human Galectin-9 mAb
9M1-3
GalPharma Co,Ltd., Kagawa, Japan
Anti-XAGE1 (GAGED2a) mAb
USO 9-13
Our laboratory
Anti-human CD4
EPR6855
Abcam, Cambridge, UK
Anti-human CD8
C8/144B
Bio SB, Santa Barbara, CA, USA
Anti-human CD3
SP-7
Nichirei Biosciences Inc., Tokyo, Japan
Anti-human FoxP3
236A/E7
Anti-CD3-V450
UCHT1
BD Horizon™,BD Bioscience, San Jose, CA, USA
Anti-CD4-PerCP/Cy5.5
SK3
BioLegend, San Diego, CA, USA
Anti-CD8-APC/Cy7
SK1
BD Pharmingen™, San Jose, CA, USA
Anti-CD279 (PD-1)-PE/Cy7
EH12.2H7
Anti-CD366 (TIM-3)-APC
F38-2E2
eBioscience, San Diego, CA, USA
Anti-CD27-FITC
M-T271
Annexin-V-PE
Anti-Ki-67-FITC
B-56
Anti-CD274 (PD-L1)-Brilliant Violet 421™
29E.2A3
Anti-Galectin-9-APC
LEAF™ Purified anti-human Galectin-9 Antibody
LEAF™ Purified anti-human TIM-3 Antibody
F338-2E2
LEAF™ Purified Mouse IgG1, κ Isotype Ctrl Antibody
MOPC-21

5. Supplementary Table S5
Ohue et al. Survival prediction in lung cancer patients
Expression of PD-L1 and Galectin-9 in lung cancer
Localization
Adenocarcinoma
n=194
Squamous
cell carcinoma
n=112
Small cell carcinoma
n=12
Others
n=11
Lung metastasis
n=32
High 　
(%)
High 　
PD-L1
Membrane 46
(24) 7
(6) - 2
Cytoplasm 83
(43) 34
(30)
(58) 4
(36) 16
(50)
and
(18) 5
(4)
Membrane or Cytoplasm 95
(49) 36
(32)
Galectin-9 1
(1) 60
(31) 18
(16) 3
(25) 14
(44) 61
IHC Score = D x I
Distribution (D) : 0, 0%; 1, 1-50%; 2, %
Intensity (I) : 0, none; 1, weak; 2, moderate; 3, strong
High: IHC score ≧ 3

6. Expression of XAGE1 in lung cancer
Supplementary Table S6
Ohue et al. Survival prediction in lung cancer patients
Expression of XAGE1 in lung cancer
Adenocarcinoma
n=194
Squamous
cell carcinoma
n=112
Small cell carcinoma
n=12
Others
n=11
Lung metastasis
n=32
Positive 　
(%)
XAGE1 54
（28） 4
（3.6）
（0） 2
（18） 1
（3.1）
XAGE1-positive: ≧ 5% positive staining of cells in the tissue by IHC

7. Cut-off values for each T-cell parameter
Supplementary Table S7
Ohue et al. Survival prediction in lung cancer patients
Cut-off values for each T-cell parameter
determined at the inflection point on the trendline
Parameters
Cut-off value
CD4
1.3%
CD8
1.8%
T cells (CD4+CD8)
3.7%
FoxP3
0.1%
CD4/FoxP3 ratio
7.3
CD8/FoxP3 ratio
7.0

8. Supplementary Table S8
Ohue et al. Survival prediction in lung cancer patients
Standardized coefficients in the discriminant function
for predicting overall survival using multiple immune parameters
Parameters
Standardized coefficients
Cluster A vs. B
Wilks’ lambda　p-value
Bootstrap
95% C.I. lower, upper
PD-L1 score
0.66
<
0.44, 0.90
0.63
0.40, 0.87
Galectin-9 score
-0.93
-1.22, -0.60
-0.94
-1.23, -0.62
XAGE1 expression
-1.26
-1.43, -1.11
-1.27
-1.46, -1.12
Histology differentiation -
0.16
-0.08, 0.39
pStage
-0.17
-0.36, 0.05
pStage, pathological stage.
The significance of each parameter in the discrimination function was examined by calculating the standardized coefficient with the bootstrap method. Bootsrap was based on 1,000 bootstrap samples.
IHC scores of PD-L1 and Galectin-9 included the addition of the IHC scores of the cell membrane and cytoplasm. IHC score = D x I (see footnotes in Supplementary Table S5).
XAGE1 expression: 0, negative; 1, positive.
Histology differentiation: 0, poor; 1, moderate; 2, well.
pStage: 1, IA; 2, IB; 3, IIA; 4, IIB; 5, IIIA.

9. Univariate and multivariate Cox regression analysis
Supplementary Table S9
Ohue et al. Survival prediction in lung cancer patients
Univariate and multivariate Cox regression analysis
Factor
Univariate
Multivariate HR
95% C.I.
p-value
Sex, male
1.15
0.65 -
Age, < 65 yrs.
1.32
0.37
Smoking habit, light or never
0.35
0.34
Histology differentiation, well
0.28
< 0.001
0.44
0.02
TNM: T factor, 1a-1b
0.001
0.48
TNM: N factor, 0-1
0.40
0.005
0.89
0.74
pStage, I
0.24
0.41
0.03
Predicted Cluster A (discriminant score ≧ 0 )
0.006
0.20 – 0.80
0.01
XAGE1 expression, positive
2.16
PD-L1 expression, high
0.59
0.09
Gal-9 expression, high
1.34
0.36
CD4, positive
0.56
0.07
CD8, positive
0.98
0.96
T cells (CD4 + CD8), positive
0.58
FoxP3, positive
0.67
0.20
CD4/FoxP3, high
0.60
0.12
CD8/FoxP3, high
0.99
0.97
pStage, pathological stage.