Role of lymphatic vessels in tumor immunity: passive conduits or active participants?

Abstract

Research in lymphatic biology and cancer immunology may soon intersect as emerging evidence implicates the lymphatics in the progression of chronic inflammation and autoimmunity as well as in tumor metastasis and immune escape. Like the blood vasculature, the lymphatic system comprises a highly dynamic conduit system that regulates fluid homeostasis, antigen transport and immune cell trafficking, which all play important roles in the progression and resolution of inflammation, autoimmune diseases, and cancer. This review presents emerging evidence that lymphatic vessels are active modulators of immunity, perhaps fine-tuning the response to adjust the balance between peripheral tolerance and immunity. This suggests that the tumor-associated lymphatic vessels and draining lymph node may be important in tumor immunity which in turn governs metastasis.

Full text

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Role of Lymphatic Vessels in Tumor Immunity: Passive
Conduits or Active Participants?
Amanda W. Lund & Melody A. Swartz
Received: 18 July 2010 /Accepted: 29 August 2010 /Published online: 11 September 2010
# Springer Science+Business Media, LLC 2010
Abstract Research in lymphatic biology and cancer im-
munology may soon intersect as emerging evidence
implicates the lymphatics in the progression of chronic
inflammation and autoimmunity as well as in tumor
metastasis and immune escape. Like the blood vasculature,
the lymphatic system comprises a highly dynamic conduit
system that regulates fluid homeostasis, antigen transport
and immune cell trafficking, which all play important roles
in the progression and resolution of inflammation, autoim-
mune diseases, and cancer. This review presents emerging
evidence that lymphatic vessels are active modulators of
immunity, perhaps fine-tuning the response to adjust the
balance between peripheral tolerance and immunity. This
suggests that the tumor-associated lymphatic vessels and
draining lymph node may be important in tumor immunity
which in turn governs metastasis.
Keywords Lymph node.Lymphangiogenesis.
Inflammation.Autoimmunity.Cancer
Abbreviations
APC
CCL19/21
CCR7
antigen‐presenting cell
C‐C chemokine ligand 19/21
C-C chemokine receptor 7
CXCL13
DC
FRC
LN
TLO
TRegcell
VEGF
C-X-C Chemokine ligand 13
dendritic cell
fibroblastic reticular cell
lymph node
tertiary lymphoid organ
regulatory T cell
vascular endothelial growth factor
Introduction
The lymphatic system is an extensive network of vessels
that function to regulate tissue fluid homeostasis, immune
cell trafficking and transport of dietary lipids [1]. Lymphat-
ic vessels bring peripheral antigens and antigen presenting
cells (APCs) like dendritic cells (DCs) to lymph nodes
(LNs) where adaptive immunity can be initiated. This
occurs either from lymph-borne antigen capture by B cells
and lymph node resident APCs [2–4], or by the more
classical route of peripherally-activated DC migration to the
LN and activation of resident T cells [5]. Lymph nodes are
also important centers for the maintenance of tolerance to
self-antigens [4, 6–9].
For such functions, lymphatic vessels are traditionally
considered to be passive conduits that deliver peripheral
antigens and cells to the LN. However, new evidence is
emerging that lymphatic vessels play very active roles in
directing the initial steps of the immune response and
sensing and responding to subtle changes in the peripheral
microenvironment that may in turn alter LN functions in
immunity and tolerance. Furthermore, evidence is emerging
that indicates a coupling between lymphatic vessel function
and LN function in tolerance and immunity.
A. W. Lund:M. A. Swartz
Institute of Bioengineering and Swiss Institute of Experimental
Cancer Research (ISREC),
École Polytechnique Fédérale de Lausanne,
Lausanne 1015, Switzerland
M. A. Swartz (*)
Institute of Bioengineering,
École Polytechnique Fédérale de Lausanne,
Station 15, Lausanne CH 1015, Switzerland
e-mail: melody.swartz@epfl.ch
J Mammary Gland Biol Neoplasia (2010) 15:341–352
DOI 10.1007/s10911-010-9193-x

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Adult lymphangiogenesis occurs during inflammation
and wound healing [1, 10], but is also strongly associated
with chronic inflammation, autoimmunity, and allograft
rejection [11–16] as well as with tumor invasion and
metastasis when initiated both around the tumor and in the
draining LN [17–21]. These are all associated with different
immunological outcomes: from Th1 and Th2 inflammatory
responses to immune escape and tolerance. Thus, the
specific and functional roles of lymphatics and lymphangio-
genesis in regulating the host immune response remain
obscure.
Lymphatics pervade the interlobular connective tissue of
the mammary gland during lactating periods and drain into
collecting lymphatics, which run along mammary ducts
[22–24]. During lactation the number of interlobular
lymphatics and interendothelial gaps increase to promote
transport [22]. Additionally, the lymphatics are the key
route for LN and distal metastases, have altered flow
patterns during cancer and increased levels of vascular
endothelial growth factor C (VEGF-C) and its receptor
VEGFR-3 lead to poor overall prognosis [25–27]. In this
way, the lymphatics are active components of both normal
tissue function and disease in the mammary gland;
however, little is known about the dynamics of their
function in this microenvironment.
In this review, we discuss recent insights into the
immunological relationships between lymphatic vessels,
their functional regulation, and the draining LN, with broad
application to the biology of the mammary gland and
tumors. We introduce new perspectives on how lymphatic
vessels may help fine-tune the immune response, and
present new perspectives on the potential role of tumor-
associated lymphatics on tumor immunity and tolerance.
Lymphatic Physiology and Neogenesis
Lymphatic vessels are found in all vascularized tissues,
with the exception of bone marrow and the central nervous
system. Interstitial fluid drains into lymphatic capillaries
(also known as initial lymphatics and terminal lymphatics)
that are blind-ended vessels with a discontinuous basement
membrane that lack pericytes. The interendothelial adhe-
sions are maintained by discontinuous, “button-like” junc-
tions serving to both freely drain interstitial proteins and
also facilitate immune cell transmigration [28–31]. These
overlapping cell-cell junctions serve as primary valves to
prevent backflow from the lymphatic vessel into the tissue
[32] and secondary valves prevent backflow within the
vessel [33]. These capillaries drain into precollecting and
collecting vessels that have continuous, “zipper-like”
interendothelial junctions [30] and are surrounded by
smooth muscle. Collecting vessels are organized into
contractile segments called lymphangions, separated by
bileaflet valves, that create the driving force for unidirec-
tional lymph propulsion [30, 31, 34–36]. Collecting
lymphatic vessels that carry lymph to and from the lymph
nodes are referred to as afferent and efferent lymphatic
vessels, respectively. Lymph typically passes through
several lymph nodes before collecting in the thoracic duct
where it is returned to the blood via connection with the
great veins of the neck. Thus, the lymphatic vascular
hierarchy is adapted to specifically promote the entrance of
APCs and antigen-rich lymph into blind-ended capillaries
and drive continuous, one-way movement of antigen and
cells towards the draining LNs.
The initial lymphatic vasculature can be subdivided into
the initial lymphatic capillaries and the precollectors, which
link the capillaries to the collecting vessels. Interestingly,
these segments express different levels of podoplanin that is
associated with differential chemokine expression [37].
Lymphatic capillaries express high levels of podoplanin as
well as the chemokine (C-C motif) ligand 21 (CCL21),
which may bind podoplanin and which attracts activated
(CCR7+) antigen-presenting cells; the coexpression of
podoplanin and CCL21 may have important implications
for their specific function. On the other hand, precollectors
express lower levels of podoplanin and secrete CCL27,
which recruits memory CCR10+T cells. In this way,
different immune cell types enter lymphatic capillaries at
specialized sites [37].
Enhanced vascular permeability and leakage is observed
during tissue injury and certain types of inflammation,
thereby increasing the fluid load on the draining lym-
phatics. Both the increase of interstitial pressure and flow,
as well as the change in cytokines and inflammatory
mediators, stand to influence this drainage, yet the latter is
only beginning to be explored [38, 39]. Transmural flow
itself can activate the lymphatic endothelium, increasing
fluid and solute permeability and uptake as well as
upregulating adhesion molecules required for immune cell
transmigration [40]. These changes can be observed at very
low fluid flows of 0.1-1dyn/cm2, which is well-correlated
to the slow interstitial flows observed in normal and
inflamed tissue. Flow also enhances the expression of
CCL21 both in lymphatic vessels [40] and in the lymph
node [41]. In addition to activating lymphatic endothelium,
lymphatic drainage also facilitates interstitial flow, directed
towards the lymphatics. This directional flow promotes cell
homing by biasing pericellular autocrine chemokine gra-
dients thereby driving DC and tumor cell chemotaxis
towards draining lymphatics [42, 43]. Thus, lymphatics
are dynamic sensors of acute biomechanical changes that
accompany the onset of tissue injury and inflammation and
are inherently coupled to immune cell trafficking and
antigen transport to and within the draining LN [44].
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In addition to regulating function, fluid flow is also an
important regulator of lymphatic morphogenesis or lym-
phangiogenesis, and has been shown to drive lymphatic
capillary organization in dermal wound healing models [45,
46] as well as in vitro culture models [47–49]. Without
flow, as in the case of lymphedema, lymphatic endothelium
becomes hyperplastic (i.e. the diameter of lymphatic
capillaries increases while their density remains unchanged)
[50]. In general, lymphatic hyperplasia and lymphangio-
genesis have not been carefully distinguished in the
literature, even though these are likely to have different
effects on tissue fluid clearance and drainage to the lymph
node[1, 28, 51]. Molecular mediators of lymphangiogenesis
are well described in several recent reviews [1, 52, 53] and
will not be discussed in detail here. Briefly, lymphangio-
genesis is mainly associated with vascular endothelial
growth factor (VEGF)‐C and ‐D, VEGFR‐3 ligands, and
contribution from other factors such as VEGF‐A and co‐
receptorsVEGFR‐2andneuropilin-2havealsobeenreported.
Blocking VEGFR-3 in adult mice, alone or in combination
with VEGFR-2 blockade, can specifically and completely
inhibit lymphangiogenesis [1, 52, 54, 55], and has been a
useful tool to determine the effects of lymphangiogenesis
on graft rejection, autoimmunity, and cancer metastasis, as
described later.
Secondary Lymphoid Organs
Transport of lymph to the draining LN is achieved in such
a way as to optimize the delivery of pathogenic signals,
antigens, and immune cells to promote antigen capture by
B cells and DCs, and facilitate T cell education and
priming [5, 51] (Fig. 1). Lymph enters the subcapsular
sinus of the draining LN through afferent vessels and
moves through the medullary sinus via a network of
conduits, formed by follicular dendritic cells in the B cell
zone and fibroblastic reticular cells (FRCs) in the T cell
zone, prior to leaving the LN via efferent vessels [56, 57].
FRCs express podoplanin (gp38) and the CCR7 ligands,
CCL21 and CCL19; CCL21 is readily immobilized into
the proteoglycan components of the extracellular matrix to
form solid-phase gradients upon which DCs and T cells
migrate [58, 59].
FRCs bundle collagen fibers to form 10–20 μm conduits
that are in close proximity to an extensive network of DCs
that directly sample antigen carried by the lymph for
presentation to naïve T cells [60]. These conduits direct
antigen and APCs towards high endothelial venules, a
specialized vasculature that promotes the delivery of naïve
T cells into the LN. This site of interaction between
extravasating lymphocytes and mature APCs is important
for initiating T cell‐specific immunity.
Fluid flow through this FRC/collagen network is required
for proper 3D organization in vitro [41]. During acute
inflammation or injury, lymph flow through the LN can be
increased, dramatically enhancing CCL21 expression by
FRCs [41] and accelerating the rate of APC and antigen
delivery to the draining LN. Occlusion of the afferent
lymphatic vessel, and consequent blockage of lymph flow
into the LN, results in alterations in high endothelial venules
(flattening of lumen and decreased luminal peripheral node
addressin expression) and a subsequent decrease in lympho-
cyte extravasation from the blood [61]. Thus, the drainage
function of peripheral lymphatic vessels is critical not only
for peripheral tissue homeostasis and fluid balance, but also
for proper LN organization and function.
Lymph node development and organization is accom-
plished mainly by the differential expression of CCL21 and
CXCL13 in the T and B cell zones, respectively [62–65].
CCL21 and CXCL13 are ligands for CCR7 and CXCR5
(expressed by B cells), respectively, and are secreted by
mesenchymal cells in the early developing lymph node to
drive lymphoid tissue formation through the attraction of
CD3ε−CD4+IL-7RαhiCCR7+and CXCR5+lymphoid
tissue inducer cells [62, 66]. The 3D organization of the
lymph node is also driven and maintained by these
cytokines; FRCs of the T cell zone express CCL21 and
CCL19, which guide the interactions between CCR7+T
cells and APCs, while follicular dendritic cells express
CXCL13 to define and maintain zones of CXCR5+B cells
[67]. Mice deficient in both CXCR5 and CCR7 lack
peripheral lymph nodes and exhibit abnormal architectures
of the deep mesenteric lymph nodes and the spleen [66].
LN architecture is conserved across anatomical loca-
tions, but the immune responses induced in different
LNs can differ [68] as the homing capacity of T cells is
dependent upon the draining LN in which they are
activated. For example, T cells activated in the mesenteric
lymph node express CCR9 and α4β7 integrin and
specifically home to the intestinal mucosa, whereas T
cells activated by peripheral LN home to skin [68]. This
specificity is unique to the LN stroma and cannot be
replicated by resident DCs; for example, DCs extracted
from the mesenteric LN can stimulate T cells in vitro to
home to the gut, but when injected subcutaneously they
instead direct T cell homing to the skin [69]. Therefore,
the induction of specific and directed immune responses is
attributable to innate functions of the LN stromal
compartments.
Lymphoid Organs in Peripheral Tolerance
The compartmentalized expression of CCL19 and CCL21
in the paracortex of the LN is not only critical for the
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correct positioning of APCs with T cells, but also required
for its tolerance-maintaining functions [62]. Mice lacking
CCR7 show impaired ability to maintain tolerance to
peripheral antigens as they develop signs of autoimmunity
[70, 71]: they exhibit lymphocyte infiltration in peripheral
organs, elevated levels of circulating antibodies towards
tissue-specific antigens, IgG deposition around the renal
glomeruli, and increased susceptibility to inducible diabe-
tes, and they can spontaneously develop chronic autoim-
mune renal disease [70]. These mice, as well as those
lacking CCL19 and CCL21 (plt mice), which lack
recognizable T cells zones within all LNs [72–76], can still
mount strong cellular immune responses [6].
This is consistent with the mounting evidence that
although B cell responses require their residence in
functional lymph nodes, T cell immunity apparently does
not, and T cells can be activated in the spleen or even liver
when lymph nodes are absent or dysfunctional (reviewed in
[77]). For this reason, proper LN function may be more
indispensible for peripheral tolerance than cellular immu-
nity. Unlike CD8+effector cells, FoxP3+TRegcells require
LN occupancy and CCR7 signaling for their activation and
function [78–80]. TRegcells sequentially migrate between
peripheral tissues and LNs to both inhibit DC migration from
the periphery as well as prevent effector T cell migration,
activation and proliferation [81]. In the inflamed peripheral
tissues, TRegcells are activated, secrete transforming growth
factor β and interleukin 10 (IL-10) and then migrate to the
draining LN in a CCR7-dependent manner [81]. Upon
reaching the LN, TReg cells preferentially migrate to the
paracortex where they interact with CD8α+DCs and tissue
derived CD11b−CD8α−DCs [82]. CD8α+DCs, which are
critical for peripheral cross-tolerance to soluble antigen,
cluster with TRegcells in the paracortical area of the LN
[60, 82]. The role of CCR7 in coordinating these interactions
is again highlighted by studies in CCR7−/−mice, which
exhibit impaired TRegcell positioning and loss of their ability
to promote tolerance and prevent autoimmune pathologies
[83, 84]. In addition, both CCR7+MHCII+CD86+DCs as
well as CCR7+TReg cells are required for the optimal
induction of a tolerance response, implying that CCR7
critically functions to bring these two cell types together
within the context of the LN stroma [84].
The tolerance-maintaining functions of the LN are
also facilitated by stromal cells, which express adhesion
molecules and chemokines that shape cell migration
routes, fluid distribution and tissue-specific immune
responses (Fig. 1). In LN transplantation experiments,
where the hematopoietic cells of the LN were completely
replaced by host cells while the stromal compartments
remained donor derived, the specific T cell homing
responses induced were those of the original location,
rather than the transplanted location [9, 69, 85]. Addition-
ally, while removal of the cervical LN blocked the
induction of musosal tolerance, rescue could be achieved
by transplanting a donor cervical LN but not mesenteric or
peripheral LNs [86]. The stromal cells maintain peripheral
tolerance functions through the constitutive expression of
relevant peripheral tissue antigens presented on MHC
class I molecules for CD8+T cell deletion [87]. This
mirrors the central tolerance maintaining functions of
medullary thymic epithelial cells, which exhibit promis-
cuous gene expression and thereby express antigen from
all tissues of the body to deactivate self-reactive T cells
through deletion (recessive tolerance) and TReg cell
induction (dominant tolerance) [88]. Thus, the LN stromal
cells hold intrinsic, tissue-specific capabilities that likely
play similar roles in peripheral tolerance as thymic
epithelial cells play in central tolerance [7, 89–91].
Recently, it has been shown that lymphatic endothelial
cells in the LN also present endogenous antigen on MHC
class I molecules (Fig. 1), and this tolerance function is
independent of the autoimmune regulator Aire [8]. Specif-
ically, peripheral LNs draining the skin were shown to
express the antigens tyrosinase and melanocytic differenti-
ation antigen, leading to deletional tolerance of autoreactive
CD8+Tcells. Thus, lymphatic vessels themselves are likely
to be important players in maintaining tolerance to self-
antigens, particularly after tissue injury. This is achieved
both through their control over peripheral drainage, which
provides constant antigen sampling to the largely immature
APC population that resides in the draining LN [44], as
well as their ability to directly present endogenous antigens
for deletional tolerance [8].
Together, these recent findings provide a new perspec-
tive on the role of LN lymphangiogenesis, which occurs in
Figure 1 Lymphatic-mediated antigen and cell transport to the
draining lymph node primes the resident stroma and lymphatic
endothelial cells for antigen presentation. Peripheral lymphatics
collect soluble antigen and secrete CCL21 to attract activated dendritic
cells (DCs), which use lymphatics for transport to the draining lymph
node (LN). Lymph is drained through the subcapsular lymphatics and
through the fibroblastic reticular cell conduit network bathing the
LN in antigens and peripherally expressed inflammatory cytokines.
Lymph exits the LN through efferent lymphatic vessels and passes
through several lymph nodes prior to recycling back into the blood
vasculature. Peripherally activated DCs migrate along the
CCL21+gp38+stromal conduit networks to educate naïve CCR7+T
cells. Resident immature DCs also lie close to this stromal network
and sample antigen draining through the LN. CCR7+regulatory T
(TReg) cells similarly require this CCL21+stroma to migrate through
the LN and become activated while efficient CD8+T cell responses
can occur outside of the LN. FRCs endogenously express peripheral
antigen on MHC class I molecules to promote tolerance through
CD8+T cell deletion. Similarly, CCL21+gp38+LYVE1+lymphatic
endothelial cells express endogenous antigen for T cell deletion.
Thus, the tolerance-maintaining functions of the draining lymph node
are dependent upon peripheral tissue drainage and local non-
hematopoeitic cell populations
R
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