Post on 07-Aug-2020
immunology.sciencemag.org/cgi/content/full/4/41/eaay0555/DC1
Supplementary Materials for
VEGF-A drives TOX-dependent T cell exhaustion in anti–PD-1–resistant
microsatellite stable colorectal cancers
Chang Gon Kim, Mi Jang, Youngun Kim, Galam Leem, Kyung Hwan Kim, Hoyoung Lee, Tae-Shin Kim, Seong Jin Choi, Hyung-Don Kim, Ji Won Han, Minsuk Kwon, Jong Hoon Kim, Andrew J. Lee, Su Kyung Nam, Seok-Joo Bae, Sat Byol Lee, Sang Joon Shin, Sung Ho Park, Joong Bae Ahn, Inkyung Jung, Kang Young Lee, Su-Hyung Park,
Hoguen Kim*, Byung Soh Min*, Eui-Cheol Shin*
*Corresponding author. Email: ecshin@kaist.ac.kr (E.-C.S.); bsmin@yuhs.ac (B.S.M.); hkyonsei@yuhs.ac (H.K.)
Published 8 November 2019, Sci. Immunol. 4, eaay0555 (2019)
DOI: 10.1126/sciimmunol.aay0555
The PDF file includes:
Fig. S1. Relative numbers of tumor-infiltrating T cells to tumor cells in MSS and MSI CRC. Fig. S2. Expression of immune checkpoint inhibitory receptors in CD8+ T cells from the peripheral blood, adjacent normal mucosa, and tumors of patients with CRC. Fig. S3. Expression of immune checkpoint inhibitory receptors in CD8+ T cells from the peripheral blood and adjacent normal mucosa of patients with MSS and MSI CRC. Fig. S4. Expression of CTAG1B in normal adjacent mucosa and tumor tissues. Fig. S5. Production of IFN-γ and TNF in CD8+ TILs upon anti-CD3 and anti-CD28 stimulation. Fig. S6. Expression of upstream regulators of wound healing signature genes in CRC. Fig. S7. Correlation of VEGF-A levels between plasma and tissue homogenates. Fig. S8. Representative histograms for the expression of immune checkpoint receptors on CD8+ T cells stimulated with anti-CD3 antibodies and VEGF-A. Fig. S9. Expression of immune checkpoint receptors on CD8+ T cells treated with VEGF-A in the absence of anti-CD3 stimulation. Fig. S10. Correlation between VEGF-A expression and T cell infiltration in MSS CRC. Fig. S11. Effects of NFATc1 inhibition on CD8+ T cells. Fig. S12. H3K27ac ChIP-seq analysis for control siRNA– or TOX siRNA–transfected CD8+ T cells after anti-CD3 and VEGF-A treatment. Fig. S13. GSEA analysis of tumor-infiltrating CD8+ T cell transcriptomes. Fig. S14. Expression of TOX in CD8+ T cells from the peripheral blood and adjacent normal mucosa of MSS and MSI CRC patients. Fig. S15. Characteristics of NY-ESO-1157-165–specific CD8+ T cell lines. Fig. S16. Effects of the blockade of PD-1 and VEGF-A on the function of tumor-infiltrating CD8+ T cells. Fig. S17. Effects of the blockade of PD-1, VEGFR2, and VEGF-A on the phenotype of tumor-infiltrating CD8+ T cells.
Fig. S18. Expression of wound healing signature genes and VEGF-A in MC38-OVA tumor tissues. Fig. S19. Effects of T cell depletion in vivo. Fig. S20. Expression of VEGFR2 in tumor-infiltrating CD8+ T cells from wild-type and T cell–specific VEGFR2 conditional knockout mice. Fig. S21. Effects of in vivo blockade of PD-1 and VEGFR2 on the phenotype of OVA257-265-specific, tumor-infiltrating CD8+ T cells.
Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/4/41/eaay0555/DC1)
Table S1. Raw data (Excel). Table S2. List of transcription factors up-regulated by VEGF-A treatment in CD8+ T cells during antigen recognition [Log2(fold change) > 2 and adjusted P < 0.05; Excel]. Table S3. List of patients (Excel). Table S4. Key resources (Excel).
Supplementary Figures
Fig. S1. Relative numbers of tumor-infiltrating T cells to tumor cells in MSS and MSI
CRC. Relative numbers of tumor-infiltrating CD3+ T cells (A) or CD8
+ T cells (B) was
calculated based on the ratio of CD3+ T cells (CD45
+CD3
+ cells) or CD8
+ T cells
(CD45+CD3
+CD8
+ cells) to tumor cells (CD45
-EpCAM
+ cells). Bars represent mean ± SEM;
*p < 0.05;
**p < 0.01.
MSS(N=15)
MSI(N=3)
0.04
0.08
0.16
0.00
CD
45
+C
D3
+
/CD
45
- EpC
AM
+
*
0.12
MSS(N=15)
MSI(N=3)
0.02
0.03
0.05
0.00
CD
45
+C
D3
+C
D8
+
/CD
45
- EpC
AM
+
**
0.04
0.01
A B
Fig. S2. Expression of immune checkpoint inhibitory receptors in CD8+ T cells from the
peripheral blood, adjacent normal mucosa, and tumors of patients with CRC. The
percentages of PD-1high
, TIM-3+, LAG-3
+, and TIGIT
+ cells among CD8
+ T cells from the
peripheral blood, adjacent normal mucosa, and tumors of CRC patients (n=50) were analyzed
by flow cytometry. Data are presented as fold change relative to the percentage in the
peripheral blood. Bars represent mean ± SEM; **
p < 0.01; ***
p < 0.001; ****
p < 0.0001.
Peripheral blood
(N=50)
Normal mucosa(N=50)
Tumor(N=50)
Fold
change
of
PD
-1h
igh
50
100
150
250
0
200
*** ****
Peripheral blood
(N=50)
Normal mucosa(N=50)
Tumor(N=50)
Fo
ld c
ha
nge
of
TIM
-3+
5
10
20
0
15
**** ****
Peripheral blood
(N=50)
Normal mucosa(N=50)
Tumor(N=50)
Fold
change o
f LA
G-3
+
10
20
30
50
0
40
** ****
Peripheral blood
(N=50)
Normal mucosa(N=50)
Tumor(N=50)
Fold
change o
f T
IGIT
+
1
2
0
**** **
Fig. S3. Expression of immune checkpoint inhibitory receptors in CD8+ T cells from the
peripheral blood and adjacent normal mucosa of patients with MSS and MSI CRC. The
percentages of PD-1high
, TIM-3+, LAG-3
+, and TIGIT
+ cells among CD8
+ T cells from the
peripheral blood (A) or adjacent normal mucosa (B) of MSS (n=110 for peripheral blood;
n=45 for normal mucosa) and MSI (n=14 for peripheral blood; n=5 for normal mucosa) CRC
patients were analyzed. Bars represent mean ± SEM; NS, not significant.
A
MSS(N=110)
MSI(N=14)
10
15
0
% o
f LA
G-3
+
NS
5
MSS(N=110)
MSI(N=14)
20
40
60
100
0
% o
f T
IGIT
+
NS
80
MSS(N=110)
MSI(N=14)
4
6
8
10
0
% o
f P
D-1
hig
h
NS
2
MSS(N=110)
MSI(N=14)
5
10
20
0
% o
f T
IM-3
+
NS
15
B
MSS(N=45)
MSI(N=5)
2
4
6
0
% o
f LA
G-3
+
NS
MSS(N=45)
MSI(N=5)
20
40
60
100
0
% o
f T
IGIT
+
NS
80
MSS(N=45)
MSI(N=5)
5
10
15
20
0
% o
f P
D-1
hig
h
NS
MSS(N=45)
MSI(N=5)
6
8
10
0
% o
f T
IM-3
+
NS
4
2
Fig. S4. Expression of CTAG1B in normal adjacent mucosa and tumor tissues.
Quantitative reverse transcriptase PCR (qRT-PCR) was performed to examine the expression
of CTAG1B (gene name for NY-ESO-1) in normal adjacent mucosa and tumor tissues (n=110).
The mRNA levels of CTAG1B were normalized by the mRNA levels of ACTB (gene name for
β-actin). Testis tissue was used as a positive control. Bars represent mean ± SEM; ****
p <
0.0001.
0.16
0.04
0.08
0.12
0.00Normal mucosa(N=110)
Tumor(N=110)
Testis(N=1)
****
3
4
Copy
num
ber
of
CT
AG
1B
/C
opy
num
ber
of
AC
TB
Fig. S5. Production of IFN-γ and TNF in CD8+ TILs upon anti-CD3 and anti-CD28
stimulation. Single cells from MSS (n=8) and MSI (n=3) CRC were stimulated with anti-
CD3 and anti-CD28 antibodies and intracellular cytokine staining was performed for IFN-
and TNF production from tumor-infiltrating CD8+ T cells. Bars represent mean ± SEM; NS,
not significant; **
p < 0.01.
MSS(N=8)
MSI(N=3)
2
4
6
10
0
% o
f IF
NG
+T
NF
+
**
8
Fig. S6. Expression of upstream regulators of wound healing signature genes in CRC.
Expression of upstream regulators of wound healing signature genes identified by Ingenuity
pathway analysis were analyzed in MSS CRC (n=320) and MSI CRC (n=55) from TCGA
CRC cohort. (A) Gene expression is presented as row-wise z-scores of normalized RSEM. (B)
Gene expression is presented as differences of normalized RSEM between MSS and MSI
CRC. Genes were arranged according to the degree of differences of expression between
MSS and MSI CRC and location of product of gene was indicated. RSEM, RNA-sequencing
by expectation-maximization.
MSS MSI
-8000 -4000 0 4000 8000
RSEM differences(MSS-MSI)
ERBB2: plasma membraneHNF4A: nucleusJUN: nucleusMYC: nucleusVEGFA: extracellular space
-4.0 -2.0 0.0 4.0
Z-score
2.0
MSS (n=320)
MSI (n=55)
ERBB2
HNF4AJUN
MYCVEGFA
BA
Fig. S7. Correlation of VEGF-A levels between plasma and tissue homogenates.
Supernatants from tumor tissue homogenates were collected, and ELISA was performed to
measure the concentration of VEGF-A (n=30). The correlation of VEGF-A levels between
plasma and tissue homogenates was analyzed. ****
p < 0.0001.
4.5
5.0
4.0
3.5
3.02.0 2.5 3.0 3.5
Pearson r2=0.5361 (****)
Plasma VEGF-A(Log pg/mL)
Tum
or
tissue h
om
ogenate
s
VE
GF
-A(L
og
pg/m
L)
1.5
Fig. S8. Representative histograms for the expression of immune checkpoint receptors
on CD8+ T cells stimulated with anti-CD3 antibodies and VEGF-A. PBMCs from normal
donors were stimulated with anti-CD3 antibodies and VEGF-A for 84 h. The percentage of
PD-1+, TIM-3
+, LAG-3
+, and TIGIT
+ cells among CD8
+ T cells was indicated.
48.7 51.1 58.3 86.0 89.9
26.7 29.7 38.4 77.1 77.6
23.9 24.5 31.7 63.4 61.9
32.8 32.3 33.9 42.9 43.7
% o
f m
ax
PD-1
TIM-3
LAG-3
TIGIT
CD8+ T cells
VEGF-A 0 ng/mL VEGF-A 5 ng/mL VEGF-A 20 ng/mL VEGF-A 50 ng/mL VEGF-A 100 ng/mL
Fig. S9. Expression of immune checkpoint receptors on CD8+ T cells treated with
VEGF-A in the absence of anti-CD3 stimulation. PBMCs from normal donors were
stimulated with VEGF-A for 84 h in the absence of anti-CD3 stimulation. The percentages of
PD-1+, TIM-3
+, LAG-3
+, and TIGIT
+ cells among CD8
+ T cells were analyzed by flow
cytometry (n=8). Bars represent mean ± SEM; NS, not significant.
60
80
100
0
40
20
% o
f P
D-1
+
0 5 20 50 100
60
80
100
0
40
20% o
f T
IM-3
+
0 5 20 50 100
60
80
0
40
20% o
f LA
G-3
+
0 5 20 50 100
30
40
50
0
20
10% o
f T
IGIT
+
0 5 20 50 100
VEGF-A (ng/mL)
NS NSNSNS
Fig. S10. Correlation between VEGF-A expression and T cell infiltration in MSS CRC.
The expression of VEGF-A and the number of tumor-infiltrating CD3+ or CD8
+ T cells at the
invasive margin and tumor center was analyzed by immunohistochemical staining of tumor
tissues from MSS (n=89). *p < 0.05;
**p < 0.01;
****p < 0.0001.
2000
3000
1000
CD
3+
T c
ells
/mm
3
(invasiv
e m
arg
in)
0100 200 300 400
Pearson r2=0.1522 (****)
400
600
800
0
200
CD
3+
T c
ells
/mm
3
(tum
or
cente
r)
100 200 300
Pearson r2=0.0901 (**)
400
1500
2000
0
1000
CD
8+
T c
ells
/mm
3
(invasiv
e m
arg
in)
100 200 300 400
Pearson r2=0.0448 (*)
300
400
500
0
200
100
CD
8+
T c
ells
/mm
3
(tum
or
cente
r)
100 200 300 400
Pearson r2=0.0840 (**)
VEGF score
0 000
Fig. S11. Effects of NFATc1 inhibition on CD8+ T cells. PBMCs from normal donors (n=6)
were stimulated with anti-CD3 antibodies and VEGF-A for 84 h (A and B) or 6 h (C) in the
absence or presence of CsA. (A) The expression of PD-1, TIM-3, LAG-3, TIGIT, and
NFATc1 among CD8+ T cells was analyzed by flow cytometry (A). The proliferation of CD8
+
T cells was analyzed by cell trace violet (CTV) dilution (B). Intracellular cytokine staining
was performed for IFN- and TNF (C). Representative histograms or plots are presented at
the top or on the left side. CsA, cyclosporine A. MFI, mean fluorescence intensity. Bars
represent mean ± SEM; ****
p < 0.0001.
A
Control CsA
500
1000
1500
2000
0
MF
I of
PD
-1
****
Control CsA
5000
10000
15000
0
MF
I of
TIM
-3
****
Control CsA
2000
4000
6000
8000
0
MF
I of
LA
G-3
****
Control CsA
MF
I of
TIG
IT
1000
2000
4000
0
3000
****
Control CsA
2000
4000
8000
0
MF
I of
NF
AT
c1
6000
****
% o
f m
ax
CD8+ T cells
PD-1 TIM-3 LAG-3 TIGIT NFATc1
1749440
10443772
5852400
31801488
68882414
Control
CsA
B
Control CsA
50
100
150
200
0
Pro
lifera
tion s
core
****
CTV
% o
f m
ax
CD8+ T cells
Control
CsA
C
Control
2
4
8
0
% o
f IF
NG
+T
NF
+
6
CsA
****
IFNG
TN
F
Control
0.62 7.04
13.079.4
CsA
0.07 0.02
0.5699.4
CD8+ T cells
Fig. S12. H3K27ac ChIP-seq analysis for control siRNA– or TOX siRNA–transfected
CD8+ T cells after anti-CD3 and VEGF-A treatment. Purified CD8
+ T cells from normal
donors were treated with anti-CD3 antibodies and VEGF-A for 84 h. Twenty-four hours after
starting the treatment, CD8+ T cells were transfected with TOX siRNA or control siRNA. (A)
Number of downregulated (n=7,327) or upregulated (n=2,049) peaks by TOX siRNA
transfection compared to control siRNA transfection. ChIP-seq signal intensity based on
normalized read counts was presented on the right side. (B) Distribution of downregulated
peaks by TOX siRNA transfection. (C to F) Gene track view of H3K27ac ChIP-seq peaks
around PDCD1 (C), HAVCR2 (D), LAG3 (E), and TIGIT (F).
TOX siRNA
peaks
19,3017,327 2,049
Control
siRNA
TOX
siRNA
-2Kb 0Kb 2Kb -2Kb 0Kb 2Kb
Peak summit Peak summit
Normalized
read counts
0 25
Co
ntr
olsiR
NA
sp
ecific
pe
aks
(7,3
27
)
Control siRNA
peaks
A
Intron
(55.89%)
Intergenic
(24.73%)
Promoter
(11.17%)
Exon (3.24%)
TTS (1.97%)
5UTR (1.35%)
3UTR (0.96%)
pseudo (0.095%)
miRNA (0.013%)
Control siRNA-specific peaks
Control siRNA
LAG3
Pri
mary
CD
8T
cell
H3K
27ac
TOX siRNA
Control siRNA-specific peaks
RefGenePLEKHG6SCNN1A CD27-AS1MRPL51 NOP2 LPAR5ING4 PIANP PTMS CD4 USP5 C12orf57 EMG1 C1S C1R C1RL RBP5 PEX5
LTBR GAPDH CHD4 COPS7A P3H3 LRRC23
100 kb
HAVCR2
chr12:6,381,678-7,381,678
LPCAT3
7,122 kb 7,124 kb 7,126 kb 7,128 kb
Pri
mary
CD
8T
cell
H3K
27ac Control siRNA
TOX siRNA
Control siRNA-specific peaks
RefGeneSGCD PPP1R2P3 TIMD4 HAVCR1 MED7 ITK CYFIP2 NR_136205NIPAL4 ADAM19 SOX30
100 kbchr5:156,069,880-157,069,880
HAVCR1
156,450 kb 156,460 kb 156,470 kb
FARP2 MIR3133 BOK-AS1 THAP4 ATG4B ING5 GAL3ST2
D2HGDH
PDCD1LINC01237 LINC01880 LINC01881
100 kb242,726 kb 242,728 kb 242,730 kbchr2:242,301,060-243,301,060
Pri
mary
CD
8T
cell
H3K
27ac Control siRNA
TOX siRNA
Control siRNA-specific peaks
RefGene GAL3ST2
ATP6V1A GRAMD1C CCDC191 QTRT2 DRD3 ZNF80
ZDHHC23
TIGITZBTB20
chr3:113,495,760-114,495,760 114,100 kb100 kb
Control siRNA-specific peaks
RefGeneZBTB20
114,103 kb 114,106 kb
Pri
mary
CD
8T
cell
H3K
27ac Control siRNA
TOX siRNA
C
D
E
F
B
Fig. S13. GSEA analysis of tumor-infiltrating CD8+ T cell transcriptomes. (A) GSEA of
gene sets upregulated (left) or downregulated (right) in exhausted CD8+ T cells from the
chronic LCMV infection model was performed using the transcriptome of tumor-infiltrating
CD8+ T cells from MSS CRC versus those from MSI CRC. (B) GSEA of gene sets
upregulated (left) or downregulated (right) in VEGF-A-treated CD8+ T cells was performed
using the transcriptome of tumor-infiltrating CD8+ T cells from MSS CRC versus those from
MSI CRC. (C) GSEA of gene sets downregulated (left) or upregulated (right) in TOX siRNA-
transfected CD8+ T cells was performed using the transcriptome of tumor-infiltrating CD8
+ T
cells from MSS CRC versus those from MSI CRC. NES, normalized enrichment score.
AE
nrich
ment
score
0.70.60.50.40.30.20.10.0
Gene set: chronic LCMV infection (up)
P<0.001NES=1.817
Enrich
ment
score
0.0-0.1-0.2-0.3-0.4-0.5-0.6-0.7
Gene set: chronic LCMV infection (down)
P<0.001NES=-1.771
-0.8
B
Enrich
ment
score 0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
Gene set: VEGF-A treatment (down)
P<0.001NES=-2.308
Enrich
ment
score 0.7
0.60.50.40.30.20.10.0
Gene set: VEGF-A treatment (up)
P<0.001NES=1.999
0.8
C
Enrich
ment
score
0.1
0.0
-0.1
-0.2
-0.3
-0.4
Gene set: TOX siRNA transfection (up)
P<0.001NES=-1.631
Enrich
ment
score
0.400.350.300.250.200.150.050.00
Gene set: TOX siRNA transfection (down)
P<0.001NES=1.563
0.45
Fig. S14. Expression of TOX in CD8+ T cells from the peripheral blood and adjacent
normal mucosa of MSS and MSI CRC patients. The expression of TOX in CD8+ T cells
from the peripheral blood (A) and adjacent normal mucosa (B) of MSS (n=110 for peripheral
blood; n=45 for normal mucosa) and MSI (n=14 for peripheral blood; n=5 for normal mucosa)
CRC patients was analyzed. MFI, mean fluorescence intensity. Bars represent mean ± SEM;
NS, not significant.
A
MSS(N=110)
MSI(N=14)
1000
1500
0M
FI
of
TO
X
NS
500
B
MSS(N=45)
MSI(N=5)
500
1000
1500
0
MF
I of
TO
X
NS
Fig. S15. Characteristics of NY-ESO-1157-165–specific CD8+ T cell lines. (A) CD8
+ T cell
lines that recognize an HLA-A2-restricted NY-ESO-1157-165 were established with 99.1%
purity. (B) The expression of PD-1 and VEGFR2 was upregulated by anti-CD3 stimulation.
A
HL
A-A
2-N
Y-
ES
O-1
15
7-1
65
CD8
0.08 99.1
0.740.08
% o
f m
ax
HLA-A2-NY-ESO-1157-165-specific CD8+ T cells
PD-1 VEGFR2
Isotype control
No stimulation
Stimulation
B
Fig. S16. Effects of the blockade of PD-1 and VEGF-A on the function of tumor-
infiltrating CD8+ T cells. Single cells from MSS CRC were stimulated with anti-CD3
antibodies in the absence or presence of anti-PD-1 and/or anti-VEGF-A for 84 h (A; n=12) or
36 h (B; n=10). Proliferation of CD8+ T cells was analyzed by cell trace violet (CTV) dilution
(A). Intracellular cytokine staining was performed for IFN- and TNF production from CD8+
T cells following addition of brefeldin A and monensin 24 h after stimulation (B). Data are
presented as fold change relative to isotype controls. Bars represent mean ± SEM; *p < 0.05;
**p < 0.01;
***p < 0.001;
****p < 0.0001.
A BCD8+ TILs
Fold
change o
f C
TV
low 3.0
2.0
2.5
1.0
1.5
******
********
CD8+ TILs
Fold
change o
f IF
NG
+T
NF
+
2.0
1.0
1.5
3.0 ****
***
2.5
Fig. S17. Effects of the blockade of PD-1, VEGFR2, and VEGF-A on the phenotype of
tumor-infiltrating CD8+ T cells. Single cells from MSS CRC (n=8) were stimulated with
anti-CD3 antibodies in the absence or presence of anti-PD-1 (A and B), anti-VEGFR2 (A) or
anti-VEGF-A (B) for 84h. Expression of TOX, PD-1, TIM-3, LAG-3, and TIGIT in tumor-
infiltrating CD8+ T cells was evaluated. ∆MFI is defined as a difference of MFI of the target
protein and isotype control (MFI of the target protein – MFI of isotype control). MFI, mean
fluorescence intensity. Bars represent mean ± SEM; NS, not significant; *p < 0.05;
**p < 0.01;
***p < 0.001;
****p < 0.0001.
A
B
CD8+ TILs
25000
10000
15000
20000
0
∆ M
FI
of
PD
-1
****
***
*
**
5000
20000
10000
15000
0
∆ M
FI
of
TIG
IT
NS
NS****
****
5000
16000
4000
8000
12000
0
∆ M
FI
of
TO
X
*NS
***
***40000
20000
30000
0
∆ M
FI
of
TIM
-3
****NS
****
****
10000
12000
9000
0
∆ M
FI
of
LA
G-3
*NS
**
**
6000
3000
CD8+ TILs
16000
4000
8000
12000
0
∆ M
FI
of
TO
X
*NS
***
***40000
20000
30000
0
∆ M
FI
of
TIM
-3
****NS
****
****
10000
20000
10000
15000
0
∆ M
FI
of
TIG
IT
NS
NS****
****
5000
25000
10000
15000
20000
0
∆ M
FI
of
PD
-1
****
**
*
**
5000
12000
9000
0
∆ M
FI
of
LA
G-3
*NS
**
***
6000
3000
Fig. S18. Expression of wound healing signature genes and VEGF-A in MC38-OVA
tumor tissues. Mice were inoculated with MC38-OVA MSS CRC cells, and tumor tissue was
harvested on day 14. Expression of wound healing signature genes (A) and VEGF-A (B to D)
was analyzed. (A and B) RNA-seq was performed using MC38-OVA tumor tissue and normal
colon tissue (n=3). GSEA of gene sets constituting wound healing signature was performed
using the RNA-seq data from MC38-OVA tumor tissue versus normal colon tissue (A).
Expression of VEGFA mRNA was analyzed. The log2 (RPKM+1) value of VEGFA mRNA is
presented. RPKM, reads per kilobase per million (B). (C and D) Expression of VEGF-A
protein was analyzed in tissue lysates from MC38-OVA tumor and normal colon by Western
blot (C). Raw image data are presented (D).
A
VEGF-A
β-actin
Normal colon
TumorMC38-OVA
245180
140
100
75
60
45
35
25
15
10
5
Anti-VEGF-A antibody
kDa
Normal colon
TumorMC38-OVA
245180
140
100
75
60
45
35
25
15
10
5
Anti-β-actinantibody
kDa
Normal colon
TumorMC38-OVA
Normal colon(N=3)
MC38-OVA(N=3)
VEGFA, mRNA
3.0
3.5
4.5
2.5
Log
2(R
PK
M+
1)
**
4.0
P<0.001NES=1.302
Gene set: wound healing signature
Enrichm
ent
score 0.5
0.3
0.0
0.4
0.2
0.1
B C
D
Upregulated in MC38-OVA Downregulated in MC38-OVA
Fig. S19. Effects of T cell depletion in vivo. Anti-CD3 antibody was administered three
times at three-day interval before tumor inoculation. (A) T-cell depletion was confirmed by
flow cytometry analysis before tumor inoculation. (B and C) Wild-type (WT) or T cell-
specific VEGFR2 conditional knock-out (cKO) mice (n=12 for each group) were inoculated
with MC38-OVA MSS CRC cells after T-cell depletion and the tumor growth kinetics
analyzed (B). Estimated tumor volume is presented. Overall survival was analyzed with a
Kaplan-Meier survival curve (C). Bars represent mean ± SEM.
19.4 0.14 11.9 0.15
FS
C
CD3
CD45+ lymphocytes from WT
No depletion CD3 depletion
CD45+ lymphocytes from cKO
No depletion CD3 depletion
100
25
50
0
Overa
ll surv
ival(%
)
75
16 20 24 28 32 36 40Days
cKO
WT
cKO, T-cell depleted
WT, T-cell depleted
12
C4000
2000
0
Tum
or
volu
me (
mm
3)
4 8 12 16 20 24 28 32 36Days
3000
1000
40
cKO
WT
cKO, T-cell depleted
WT, T-cell depleted
B
0 0
A
Fig. S20. Expression of VEGFR2 in tumor-infiltrating CD8+ T cells from wild-type and
T cell–specific VEGFR2 conditional knockout mice. Wild-type (WT) or T cell-specific
VEGFR2 conditional knock-out (cKO) mice were inoculated with MC38-OVA MSS CRC
cells and TILs harvested 14 days after tumor cell inoculation. The expression of VEGFR2 in
tumor-infiltrating CD8+ T cells was analyzed by flow cytometry. Representative data are
presented.
VEGFR2
Isotype control
WT
cKO
% o
f m
ax
CD8+ TILs
Fig. S21. Effects of in vivo blockade of PD-1 and VEGFR2 on the phenotype of OVA257-
265-specific, tumor-infiltrating CD8+ T cells. Mice were inoculated with MC38-OVA cells.
During 21 days, anti-PD-1 and/or anti-VEGFR2 antibodies were administered three times,
and single cells were harvested from MC38-OVA tumors. The expression of PD-1, TIM-3,
LAG-3, and TIGIT in tumor-infiltrating OVA257-265-specific CD8+ T cells was analyzed (n=6
for each group). ∆MFI is defined as a difference of MFI of the target protein and isotype
control (MFI of the target protein – MFI of isotype control). MFI, mean fluorescence
intensity. Bars represent mean ± SEM; NS, not significant; *p < 0.05;
**p < 0.01;
****p <
0.0001.
OVA257-265-specific CD8+ TILs
40000
10000
20000
30000
0
∆ M
FI
of
PD
-1
****
**
**
****6000
3000
4500
0
∆ M
FI
of
TIM
-3
*NS
****
*
1500
25000
10000
20000
0
∆ M
FI
of
LA
G-3
****NS
****
****
15000
5000
50000
10000
30000
40000
0
∆ M
FI
of
TIG
IT
****NS
****
****
20000