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: [email protected] (E.-C.S.); [email protected] (B.S.M.); [email protected] (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-1 157-165 –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.
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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,
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