Article

a

SSC-AFSC-A FSC-WFSC-H SSC-WSSC-H

LivCD45-PE-Cy7

e/dead-AmCya

n

CD3-BCD45-PE-Cy7

UV737

CD4-BV496CD8-BUV395

CD3-BCD44-A700

UV737

CD44-A700FOXP3-BV421 PD-1-APC-Cy7TIM3-BV605

CD8-BRORt-APC

UV395

CD4/CD8 CD44+

TCF1-A488TIM3-BV605

CD8 CD44+

CD4 CD44+ CD4/CD8

CD44+

Naive

CD4+

CD8+

CD3+

CD45 CD3+

CD45

Single cell-2

Single cell-1 Single cell-2

Single cell-1

(^10379111315)
104
105
Days
FOXP3+ CD4
T cells
Brain
P=0.049P=0.043
(^10379111315)
104
105
Days
FOXP3+ CD4
T cells
Lymph node
P=0.0004P=0.02
(^10379111315)
104
105
106
Days
TCF7+ CD8
T cell
s
Lymph node
P=0.02P=0.01
(^10379111315)
104
105
106
Days
TCF7+ CD8
T cells
Brain
P=0.03P=0.05
(^10279111315)
103
104
105
Days
TCF7+ CD8
T cell
s
Meninges
P=0.02
20
40
60
80
%T-bet+ (CD4+Foxp3+)
P=0.016Brain
P=0.013P=0.01
Day 9Day 11 Day 14^0
10
20
30
40
50
%RORgT
(CD4+Foxp3+
) Brain
Day 9Day 11 Day 14
0
20
40
60
80
100
%T-bet+ (CD4+Foxp3+)
Meninges
Day 9Day 11 Day 14^0
10
20
30
40
%RORgT
(CD4+Foxp3+)
Meninges
Day 9Day 11 Day 14
0
20
40
60
80
% PD1+ (CD8+CD44+
) Tumor
Day 9Day 11 Day 14
P=0.047
0
20
40
60
80
100
% Tim3+ (CD8+CD44+
) Tumor
(^10279111315) Day 9Day 11 Day 14
103
104
105
106
FOXP3+ CD4
T cells
Meninges
Days
(^200)
40
60
80
(^100) Luc-mRNA
VEGFC-mRNA
TCF7-A488
b
f
SSC-AFSC-A FSC-WFSC-H SSC-WSSC-H LiveCD45-BUV737
/Dead-AmCy
an
CD3-APC-Cy7CD19 F4/80 NK1.1-BV605 CD4-BV496CD8-BUV395 CD44-A700CD3-APC-Cy7 CD44-A700CD3-APC-Cy7
IFN-CD3-APC-Cy7
BV711
IL2-APCCD3-APC-Cy7 GZMB-A488CD3-APC-Cy7 TNF-CD3-APC-Cy7
PE-Cy7
IFN-CD3-APC-Cy7
BV711
IL2-APCCD3-APC-Cy7 GZMB-A488CD3-APC-Cy7 TNF-CD3-APC-Cy7
PE-Cy7
Cells
Cells
Single cell-1
Single cell-1
Single cell-2
Single cell-2
CD45+
CD45+
CD3+
CD3+
CD4+ CD44+ CD44+
CD8+
CD8+ CD4+
CD8+ CD44+
CD4+ CD44+
IFN+ IL2+ GZMB+ TNF+
IFN+ IL2+ GZMB+ TNF+
CD8 T cells Meninges # of Cytokines
produced
0
(^12)
(^34)
Luc-mRNAVEGF-C-mRNA Luc-mRNAVEGF-C-mRNA
CD 8 T cells Brain CD8 T cells Lypymph node
Luc-mRNAVEGF-C-mRNA
Da
y^7
Day 11
Day
14
CD4 T cells Meninges # of Cytokines
produced
(^01)
(^23)
Luc-mRNAVEGF-C-mRNA Luc-mRNAVEGF-C-mRNA
CD4 T cells Brain CD4 T cells Lymph node
Luc-mRNAVEGF-C-mRNA
Day
7
Da
y^11
Da
y^14
g h
i
Tumor Bulk
Pe
ripher
y
Tumor
Normal Normal
CD3Luc-mRNAVCD31DAPI EGF-C-mRNA
Contralateral
Tumor
Lyve1CD3CD31DAPILyve1
c
FOXP3+
FOXP3+
RORgT+ TBET+
FOXP3+
CD8-BTBET-BV711
UV395
e
(^0) Luc-mRNAVEGF-C-mRNA
20
40
60
80
100
% TCF7+ (CD8+CD44+
d )
0
5000
10000
15000
20000

of Cells producing

four cytokines
Luc-mRNAVEGF-C-mRNA
P < 0.0001

P = 0.11
Extended Data Fig. 9 | VEGF-C treatment changes T cell phenotypes and
functionality. Mice bearing 7-day-tumours were treated with Luc mRNA or
VEGFC mRNA and evaluated for changes. a, Mouse brains were collected two
days after treatment with VEGFC mRNA, cryosectioned and analysed by
immunof luorescence microscopy. b, Gating strategy for f low cytometry
analysis of T cells. c, Example of TCF7 staining in CD3+CD8+CD44+ cell
populations after VEGFC mRNA treatment. The experiment was repeated twice
independently with similar results. d, Percentage of TCF7+ T cells in the
CD3+CD8+CD44+ population in the brain (Luc mRNA, n = 14; VEGFC mRNA, n = 9;
data pooled from 3 independent experiments). e, Number of CD3+CD8+CD44+
cells producing IFNγ, TNF, IL-2 and GZMB in the brain (n = 3; 3 mice were pooled
for each sample). Violin plots display quartiles (dotted horizontal lines),
median (dashed line) and minimum and maximum values (solid lines).
f, Quantification of cell counts in different compartments after treatment with
VEGFC mRNA. Percentage of cells expressing specific transcription factors or
immune checkpoint inhibitors after treatment with VEGFC mRNA. g, Gating
strategy for cytokine production in T cells. h, i, Quantification of T cells
expressing multiple cytokines (n = 3, 3 mice were pooled for each replicate).
Data are mean ± s.e.m. P < 0.05; P < 0.01; P < 0.001; ****P < 0.0001
(two-tailed unpaired Student’s t-test).