Nature 2020 01 30 Part.02

a

VEGF-C-mRNA/GFP-mRNA

Intracisternal injection

Lymph Node Tumor

GFP-mRNA

VEGF-C-mRNA

GFP-mRNA

VEGF-C-mRNA

103

104

105

106

107

# of cell / Brain hemisphere

or draining L

ymph nod

e

*

P=0.028

P=0.069ns

020406080

0

20

40

60

80

100

Days

Percent surviva

l

Naived100 rejected

P = 0.0049

**

0204060

0

20

40

60

80

100

Days

Percent surviva

l

WT

WT Naive T cell transfer

WT Memory T cell transfer

P = 0.26

P = 0.003

**

bc

020406080100

VEGF-C-mRNA

PD1

0

20

40

60

80

100

Days

Percent surviva

l

VEGF-C-mRNA + PD1

VEGF-C-mRNA + PD1 + CD 4

VEGF-C-mRNA + PD1 + CD 8

P = 0.14 GFP-mRNA + PD1

P = 0.005

P = 0.72

d **

e f

GFP-mRNA + PBS

GFP-mRNA + TIM3 + PD-1

VEGF-C-mRNA + PBS

VEGF-C-mRNA + TIM3 + PD1

P = 0.01

P = 0.50

P = 0.007

01020304050

0

20

40

60

80

100

Days

Percent surviva

kll

GFP-mRNA + PBS

GFP-mRNA + PD-1 + 4-1BB

VEGF-C-mRNA + PBS

P=0.005 VEGF-C-mRNA + PD1 + 4-1BB

0204060

0

20

40

60

80

100

Days

Percent surviva

l

**

CT2A-BFP

4-1BB

PD1

4-1BB

PD1

4-1BB + αPD1

GFP-mRNA + Isotype

GFP-mRNA +

GFP-mRNA +

VEGF-C-mRNA +

VEGF-C-mRNA +

VEGF-C-mRNA +

P = 0.02

P = 0.03

P = 0.003

P = 0.003

P = 0.001

0255075

0

20

40

60

80

100

Days

Percent surviva

l

**

**

**

*

*

CT2A

GL261

GL261-Luc

GL261-Luc GL261-Luc

GL261

020406080

0

20

40

60

80

100

Days

Percent surviva

l

GFP-mRNA + PBS

GFP-mRNA + CTLA4

VEGF-C-mRNA + CTLA4

VEGF-C-mRNA + PBS

P = 0.42

P = 0.002

P = 0.08

**

010203040

0

20

40

60

80

100

Days

Percent surviva

l

GFP-mRNA + PBS

GFP-mRNA + TIM3

VEGF-C-mRNA + TIM3

VEGF-C-mRNA + PBS

P = 0.50

P = 0.003

P = 0.08

**

GL261

020406080

0

20

40

60

80

100

Days

Percent surviva

l

GFP-mRNA + isotype

GFP-mRNA + PD1

VEGF-C-mRNA + PD1

VEGF-C-mRNA + isotype

P = 0.0001

P < 0.0001

P = 0.02

*

***

****

GL261

g

hij

Extended Data Fig. 5 | Therapeutic delivery of VEGF-C potentiates
checkpoint inhibitor therapy even at late stages of tumour development.
a, Related to Fig. 3c, d. Quantification of the number of cells per tumour-
bearing brain hemisphere or lymph node using CountBright absolute counting
beads and autocounter (see Methods for details) (lymph nodes: GFP mRNA,
n = 6; VEGFC mRNA, n = 12; tumour: GFP mRNA, n = 3; VEGFC mRNA, n = 5).
b, Mice that rejected tumours after combination therapy with VEGFC mRNA
and anti-PD-1 (RMP1-14) were rechallenged in the contralateral hemisphere and
observed for survival (naive, n = 5; day 100 rejected, n = 4). c, T cells from lymph
nodes and spleens from mice that rejected tumours after combination therapy
with VEGFC mRNA and anti-PD-1 (RMP1-14) or naive wild-type mice were
isolated and transferred into naive wild-type mice intravenously. After 24 h,
GL261 tumours were inoculated intracranially and the mice were observed for
survival (wild type, n = 5; wild type with transfer of naive T cells, n = 5; wild type
with transfer of memory T cells, n = 7). d, Mice inoculated with 50,000 GL261-
Luc cells were treated with VEGFC mRNA or GFP mRNA (day 7) and with either
anti-PD-1 (RMP1-14) antibodies or isotype antibodies (days 7, 9 and 11), and
monitored for survival. Mice were depleted of CD4 or CD8 T cells using anti-
CD4 (GK1.5) or anti-CD8 (YTS169.4) antibodies starting one day before tumour
inoculation and redosed every four days afterwards (VEGFC mRNA + anti-PD-1,

n = 6; GFP mRNA + anti-PD-1, n = 6; VEGFC mRNA + anti-PD-1 + anti-CD4, n = 5;

VEGFC mRNA + anti-PD-1 + anti-CD8, n = 5). e, Schematic of experimental design

for the results shown in f and g. f, g, Mice inoculated with 50,000 CT-2A–BFP

cells (f) or CT-2A cells (g) were treated with VEGFC mRNA or GFP mRNA (day 7)

and either with PBS or with anti-PD-1(RMP1-14) and/or anti-4-1BB (LOB12.3)

antibodies (days 7, 9 and 11), and monitored for survival (f, VEGFC mRNA + anti-

PD-1 + anti-4-1BB, n = 5; GFP mRNA + anti-PD-1 + anti-4-1BB, n = 5; VEGFC mRNA +

PBS, n = 4; GFP mRNA + PBS, n = 6; g, n = 5 for all groups except VEGFC mRNA +

anti-4-1BB + anti-PD-1, n = 7). h–j, Mice inoculated with 50,000 GL261 cells were

treated with VEGFC mRNA or GFP mRNA (day 7) and either with PBS or with anti-

PD -1 (R MP1-14) (h), anti-TIM3 (RMT3-23) (i) or anti-CTLA4 (9H10) antibodies (j)

(days 7, 9 and 11), and monitored for survival (n = 5). For i and j, the same control

mice were used for the GFP mRNA + PBS and VEGFC mRNA + PBS groups.

k, Schematic of experimental design for the results shown in l. l, Mice

inoculated with 50,000 GL261-Luc cells were treated with VEGFC mRNA or GFP

mRNA (day 20) and either with PBS or with anti-PD-1 (RMP1-14) and anti-TIM3

(RMT3-23) antibodies (days 20, 22 and 24), and monitored for survival (n = 5).

*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed unpaired Student’s

t-test or two-sided log-rank Mantel–Cox test).