Nature | Vol 577 | 30 January 2020 | 689Article
VEGF-C-driven lymphatic drainage enables
immunosurveillance of brain tumours
Eric Song^1 , Tianyang Mao^1 , Huiping Dong^1 , Ligia Simoes Braga Boisserand^2 , Salli Antila^3 ,
Marcus Bosenberg1,4,5, Kari Alitalo^3 , Jean-Leon Thomas2,6,8* & Akiko Iwasaki1,3,7,8*Immune surveillance against pathogens and tumours in the central nervous system is
thought to be limited owing to the lack of lymphatic drainage. However, the
characterization of the meningeal lymphatic network has shed light on previously
unappreciated ways that an immune response can be elicited to antigens that are
expressed in the brain^1 –^3. Despite progress in our understanding of the development
and structure of the meningeal lymphatic system, the contribution of this network in
evoking a protective antigen-specific immune response in the brain remains unclear.
Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic
vasculature can be manipulated to mount better immune responses against brain
tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is
very limited when the tumour is confined to the central nervous system, resulting in
uncontrolled tumour growth. However, ectopic expression of vascular endothelial
growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining
deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance
of the glioblastoma and a long-lasting antitumour memory response. Furthermore,
transfection of an mRNA construct that expresses VEGF-C works synergistically with
checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal
the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a
new therapeutic approach to treat brain tumours.We used the C57BL/6 syngeneic cell line GL261 to model glioblastoma
in mice. Mice injected orthotopically with luciferase-expressing GL261
cells (GL261-Luc) showed cell-number-dependent growth kinetics and
lethality (Extended Data Fig. 1a–c), demonstrating that intracranial
injection of GL261-Luc is not sufficient to promote tumour rejection in
the central nervous system (CNS). To evaluate the effects of enhanced
lymphangiogenesis, we used two modes of delivery to express the
lymphangiogenesis-promoting factor VEGF-C in mice: an adeno-associ-
ated viral vector (AAV9) and an mRNA delivery vector. Consistent with
previous reports^1 ,^4 ,^5 , injection of AAV-VEGF-C into the cerebrospinal
fluid (CSF) of mice through the cisterna magna remodelled meningeal
lymphatic vessels in the dural confluence of sinuses (Fig. 1a, b) and in
the sagittal sinuses (Extended Data Fig. 1h, i), and did not affect the
integrity of the blood–brain barrier^4 (Extended Data Fig. 1d, e). In mice
that were prophylactically treated with AAV-VEGF-C, we observed a
near-complete rejection of tumours (Fig. 1c, Extended Data Fig. 1j).
VEGF-C-mediated protection requires T cells
Previous studies have shown that deep cervical lymph nodes are the
primary draining lymph nodes of the CNS, and that mandibular and
superficial cervical lymph nodes contribute to antigen sampling
in the CNS^1 ,^3 ,^6 ,^7. Thus, we surgically ligated the afferent lymphatic
vessels that drain to both deep cervical lymph nodes in mice treated
with AAV-VEGF-C. Although AAV-VEGF-C-treated mice showed
prolonged survival compared to control mice (Fig. 1d), after ligation of
the deep cervical lymph nodes the majority of the AAV-VEGF-C-treated
mice succumbed to the tumour. This indicates that VEGF-C-mediated
protection against glioblastoma requires lymph drainage to the deep
cervical lymph nodes.
The requirement for the deep cervical lymph nodes suggests that the
immune system has a role in VEGF-C-mediated protection. Depletion
of CD4 or CD8 T cells negated the protection that was conferred by
VEGF-C (Fig. 1e, Extended Data Fig. 1k). By contrast, B-cell-deficient
(μMT) mice that were treated with VEGF-C were protected from glioblas-
toma (Extended Data Fig. 1l). We examined the durability of this immune
response against glioblastoma in mice that were treated with VEGF-C,
and found that mice that rejected the intracranial tumour showed long-
term systemic memory responses; rechallenge with GL261-Luc in the
flank resulted in no detectable tumours (Fig. 1f, Extended Data Fig. 1m).
Together, these data demonstrate that by increasing lymphangiogen-
esis in the meninges, prophylactic treatment with VEGF-C can evoke
a robust and long-lasting T-cell-dependent immune response against
brain neoplasms.https://doi.org/10.1038/s41586-019-1912-x
Received: 16 January 2019
Accepted: 21 November 2019
Published online: 15 January 2020
(^1) Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. (^2) Department of Neurology, Yale University School of Medicine, New Haven, CT, USA. (^3) Translational
Cancer Medicine Program and Wihuri Research Institute, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland.^4 Department of Dermatology, Yale University School of Medicine,
New Haven, CT, USA.^5 Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.^6 Institut du Cerveau et de la Moelle Épinière, INSERM U1127, CNRS UMR 7225,
GH Pitié-Salpêtrière, Sorbonne Université, Paris, France.^7 Howard Hughes Medical Institute, Chevy Chase, MD, USA.^8 These authors jointly supervised this work: Jean-Leon Thomas,
Akiko Iwasaki. *e-mail: [email protected]; [email protected]