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