Nature | Vol 577 | 30 January 2020 | 693tumours (Fig. 4a, b). Similar results were observed with the B16 cell
line (Extended Data Fig. 10c–e). These results are consistent with the
idea that T cell priming—either through expression of VEGF-C in the
CSF or through a flank tumour—enables efficient checkpoint inhibitor
responses in the CNS. However, in the case of a tumour that is confined
to the CNS at steady state, immune checkpoint inhibitors alone do not
confer notable benefits, regardless of tumour type.
Finally, we sought to understand whether the antitumour effects of
VEGF-C are due to (i) the expansion and differentiation of T cells that
are capable of trafficking to the brain (that is, T-cell-intrinsic); or (ii)
changes in the brain environment that cause an increase in the recruit-
ment of T cells (that is, T-cell-extrinsic). We performed adoptive transfer
of leukocytes from the draining lymph nodes of tumour-bearing mice
into recipient mice that also had tumours (Fig. 4d, e). The infiltration
of T cells into the brain was slightly increased (T-cell-intrinsic; 1.6-fold
increase) if the T cells were from mice treated with VEGFC mRNA. How-
ever, T cells from tumour-bearing control mice were able to migrate
into glioblastoma-affected brains to a greater extent if the recipient
was treated with the VEGFC mRNA construct (T-cell-extrinsic; 3.1-fold
increase) (Fig. 4e). Moreover, donor T cells from mice that were
treated with VEGFC mRNA, transferred into a host that was also treated
with VEGFC mRNA, were able to infiltrate the tumour most efficiently
(combinatorial; 4.2-fold increase). Increases in the numbers of T cells
in the lymph nodes were dependent on the recipient mice having been
treated with VEGFC mRNA—consistent with the notion that antigen-
specific T cells proliferate only after increased drainage of tumour
antigens from the CNS. These results show that VEGF-C provides an
antitumour environment through a combination of its effect on the
meningeal lymphatic vasculature and the changes that it induces in
T-cell-intrinsic properties.
Discussion
We have demonstrated that ectopic expression of VEGF-C has the
potential to manipulate meningeal lymphatics, and thereby enables
immune surveillance and T-cell-mediated immunity against brain
tumours (Extended Data Fig. 10g). These results support a growing
body of work that highlights the importance of the meningeal lym-
phatic network in controlling immune responses in the CNS^3 –^5. The lack
of immune responses against tissue grafts^32 in the CNS has led to the
idea that the brain has an immune-privileged status, and a similar phe-
nomenon of immune privilege may allow primary brain tumours such
as glioblastoma to grow unhindered in the CNS^33. Together with these
previous reports, our study suggests that tumours confined within
the CNS may be inaccessible for immune priming, and that exogenous
expression of VEGF-C potentiates endogenous immune responses. We
considered the possibility that immune-cell- or tumour-cell-intrinsic
VEGF-C signalling causes this phenomenon, but found that immune
and tumour cells showed no changes after direct stimulation with
VEGF-C. Instead, our data suggest that VEGF-C (and its relationship
with its main receptor partner VEGFR-3) leads to an increase in lym-
phatic drainage^34 , which is necessary for immunosurveillance against
glioblastoma—similar to the way in which melanoma-intrinsic overex-
pression of VEGF-C enhances antitumour T cell responses^8. It is worth
noting that the immune-privileged status of the CNS does not apply to
all brain antigens^6 , and future studies are needed to define the rules of
CNS antigen sampling. In summary, our study shows that increasing
lymphatic drainage can overcome the immune ignorance of glioblas-
toma. This lays the foundation for a new strategy in which modulation of
the lymphatic vasculature is used to increase the effects of checkpoint
inhibitor therapy against tumours in immune-privileged sites.05 × 10410 × 10415 × 10420 × 10425 × 104Brain*********1.6×3.1×4.2×^15 ×^105
12 × 105
9 × 105
6 × 105
3 × 105
CD45.2+CD3+ cell countLymph node****
1.6×6.1×6.9×abc(^0010203040)
20
40
60
80
100
Days
01020
IC anti-PD-1 + anti-CTLA4 VEGFC mRNA +
IC anti-PD-1 + anti-CTLA4VEGFC mRNA +
(^03040)
20
40
60
80
100
Days
**
- Ligation FT IC VEGFC mRNA +
anti-PD-1 + anti-CTLA4
(^0010203040)
20
40
60
FT IC FT IC VEGFCGFP mRNA mRNA
FT IC FT IC GFPVEGFC mRNA + anti-PD-1 + anti-CTLA4 mRNA + anti-PD-1 + anti-CTLA4
80
100
Days
Survival (%)
**
YUMMER1.7 YUMMER1.7 YUMMER1.7
de
IC IC GFPVEGFC mRNA mRNA
IC IC GFPVEGFC mRNA + anti-PD-1 + anti-CTLA4 mRNA + anti-PD-1 + anti-CTLA4
Ligation IC VEGFC mRNA + anti-PD-1 + anti-CTLA 4
Survival (%) Survival (%)
NS
NS
NS
CD45.2^0
+CD3
- cell count
Donor: GFP
Recipient: GFPDonor: VEGF-
C
Recipient: GFP
Donor: GFP
Recipient: VEGF-CDonor: VEGF-
C
Recipient: VEGF-C
Donor: GFP
Recipient: GFPDonor: VEGF-
C
Recipient: GFP
Donor: GFP
Recipient: VEGF-CDonor: VEGF-
C
Recipient: VEGF-C
inductionTumour VEGFC mRNA
0 d 7 d 14 d
19 d
- Tdraining lymph nodeake CD45.2
2. Adoptive transfer to CD45.1
tumour-bearing mice (7 d) with or
without VEGFC mRNA treatment
3. Collect lymph nodes and
brain to evaluate in ltration
Fig. 4 | Therapeutic delivery of VEGF-C mediates protection against
intracranial melanoma when combined with checkpoint inhibitor blockade
and is equivalent to peripheral priming. a–c, Mice were given either only
YUMMER1.7 intracranial tumours (IC) (a) or both a YUMMER1.7 intracranial
tumour and a YUMMER1.7 f lank tumour (FT) (b), and treated with GFP mRNA or
VEGFC mRNA on day 7, and anti-PD-1 (RMP1-14) and anti-CTLA4 (9H10) on days 7,
9 and 11. Two groups (ligation IC VEGFC mRNA + anti-PD-1 + anti-CTLA4 and
ligation FT IC VEGFC mRNA + anti-PD-1 + anti-CTLA4) had lymph nodes ligated
7 days before tumour implantation (a, n = 12 for all groups except ligation IC
VEGFC mRNA + anti-PD-1 + anti-CTLA4, n = 7; b, n = 12 for all groups except
ligation FT IC VEGFC mRNA + anti-PD-1 + anti-CTLA4, n = 7). P values: a, P = 0.13
(NS) for IC VEGFC mRNA; *P = 0.001 for IC GFP mRNA + anti-PD-1 + anti-CTLA4;
**P < 0.0001 for IC VEGFC mRNA + anti-PD-1 + anti-CTLA4; P = 0.069 for
ligation IC VEGFC mRNA + anti-PD-1 + anti-CTLA4 (all versus IC GFP mRNA);
b, *P = 0.013 for FT IC VEGFC mRNA; P = 0.0029 for FT IC GFP mR NA + anti-
PD-1 + anti-CTLA4; ***P = 0.0003 for FT IC VEGFC mR NA + anti-PD -1 + anti-
CTL A4; P = 0.051 (NS) for ligation FT IC VEGFC mRNA + anti-PD-1 + anti-CTLA4
(all versus FT IC GFP mRNA); c, P = 0.72 (NS) for IC VEGFC mR NA + anti-
PD-1 + anti-CTLA4 versus FT IC VEGFC mRNA + anti-PD-1 + anti-CTLA4.
d, Schematic of experimental design for the results shown in e. Congenic
CD45.2 mice were injected with GL261 tumours. Seven days later, mice were
treated with GFP mRNA or VEGFC mRNA. Seven days after the mRNA treatment
(14 days after tumour inoculation) leukocytes from deep cervical lymph nodes
were transferred into congenic CD45.1 mice bearing 7-day tumours. Five days
after transfer, the deep cervical lymph nodes and brain tissues were collected
to analyse T cell infiltration. e, Quantification of brain-infiltrating T cells and
T cells in lymph nodes; numbers denote the fold change in the number of cells
over donor GFP and recipient GFP groups (n = 5 mice per group). Data are
mean ± s.e.m. For P values in e, *P < 0.05; ****P < 0.0001. P values were calculated
by two-tailed unpaired Student’s t-test or two-sided log-rank Mantel–Cox test.