Download Ectopic lymphoid-like structures in infection, cancer and autoimmunity

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Ectopic lymphoid-like structures in
infection, cancer and autoimmunity
Costantino Pitzalis1, Gareth W. Jones2, Michele Bombardieri1 and Simon A. Jones2
Abstract | Ectopic lymphoid-like structures often develop at sites of inflammation where they
influence the course of infection, autoimmune disease, cancer and transplant rejection.
These lymphoid aggregates range from tight clusters of B cells and T cells to highly organized
structures that comprise functional germinal centres. Although the mechanisms governing
ectopic lymphoid neogenesis in human pathology remain poorly defined, the presence
of ectopic lymphoid-like structures within inflamed tissues has been linked to both
protective and deleterious outcomes in patients. In this Review, we discuss investigations in
both experimental model systems and patient cohorts to provide a perspective on the
formation and functions of ectopic lymphoid-like structures in human pathology, with
particular reference to the clinical implications and the potential for therapeutic targeting.
Centre for Experimental
Medicine and Rheumatology,
William Harvey Research
Institute, Barts and The
London, School of Medicine
and Dentistry, Queen Mary
University of London,
Charterhouse Square,
London EC1M 6BQ, UK.
2
Cardiff Institute for Infection
and Immunity, The School of
Medicine, Cardiff University,
The Tenovus Building,
Heath Campus, Cardiff
CF14 4XN, Wales, UK.
Correspondence to S.A.J.
e‑mail: [email protected]
doi:10.1038/nri3700
Published online 20 June 2014
1
Inflammation supports immunological defence against
infection, trauma, injury and cancer. The regulation of
inflammation is governed by cellular communication
between non-haematopoietic stromal cells, resident
leukocytes and infiltrating immune cells1,2. Optimal
control of this process ensures competent host defence,
the elimination of the initiating antigen (or antigens) and
limited tissue damage1,3. However, repeated, persistent
or non-resolving episodes of inflammation lead to the
inappropriate regulation of this response and promote
autoimmunity, chronicity and tissue pathology1,3,4. The
mechanisms that control these events are often diverse
and they contribute to clinical differences in disease
presentation. For example, patients who have the same
underlying clinical condition invariably show differences
in disease severity, rate of disease onset and response to
treatment4. Although sex, age, genetics, metabolic factors and the environment are major determinants that
affect the propensity to develop chronic disease, these
indicators provide minimal information on the molecular or cellular pathways that drive the type of disease that
is observed.
Cytokines (including interleukins, interferons and
growth factors), chemokines, lipid mediators and innatesensing mechanisms control inflammation and they have
important roles in the pathology of various inflamma­
tory diseases and cancer3–5, thus representing major
targets for clinical intervention. For example, biological
therapies that inhibit pro-inflammatory cytokines — for
example, the inhibition of tumour necrosis factor (TNF)
by the monoclonal antibodies infliximab, adalimumab,
golimumab and certolizumab — or their receptors (for
example, the inhibition of interleukin‑6 (IL‑6) receptor
by tocilizumab) are now widely used in routine clinical practice for the treatment of rheumatoid arthritis3–5.
These therapies display specific modes of action and have
been developed on the basis of a better understanding of
cytokine involvement in inflammation. Although these
agents reduce the symptoms and progression of disease,
not all patients respond equally to the same therapy.
Indeed, 30–40% of patients with rheumatoid arthritis do
not respond to any treatments and some biological therapies are efficacious in certain clinical conditions but not
in others4. Such differences in therapeutic efficacy suggest that distinct inflammatory mechanisms must steer
the course of disease in any one individual. Typically, the
sooner after diagnosis appropriate therapy commences,
the better the clinical outcome and the increased likelihood of achieving remission6. Thus, cell­ular events that
are triggered early in the inflammatory process must
influence the course of disease.
A central feature of local inflammation is the inter­
action between the stromal tissue compartment and
immune cells1,2. Inflammatory mediators that are produced by stromal cells and tissue-resident monocytic
cells control the recruitment, activation and survival of
leukocytes. Cellular infiltration is traditionally viewed as
the diffuse influx of immune cells, with the cells being scattered throughout the inflamed tissue. However, infiltrating
leuko­cytes often form more organized aggregates and these
promote antigen-specific adaptive immune responses that
exacerbate chronic disease. Indeed, B cells, T cells, resident
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Table 1 | Clinical and experimental conditions that feature ELSs
Condition
Location
Species
Antigen recognized by
cells in ELSs
Other known functions of ELSs in
disease
Refs
Rheumatoid arthritis
Synovial tissue
•Human
•Mouse
•Rheumatoid factor
•Citrullinated proteins
•Histones?
•In situ B cell differentiation
•Ongoing CSR
•Autoantibody secretion
Sjögren’s syndrome
•Salivary glands
•Lacrimal glands
•Human
•Mouse
•SSA/Ro
•SSB/La
Unknown
39,73,74,157
Multiple sclerosis (and
EAE)
CNS
•Human
•Mouse
Myelin and other neuronal
antigens proposed
Unknown
29,158
Diabetes
Pancreatic islet
parenchyma
Mouse
Plasma cell anti-insulin
reactivity observed
Unknown
159,160
Hashimoto’s thyroiditis
Thyroid gland
Human
•Thyroglobulin
•Thyroperoxidase
Unknown
85
Primary sclerosing
cholangitis and primary
biliary cirrhosis
Liver
Human
Unknown
•Cellular organization
•CCL21 and MADCAM1 expression
Myasthenia gravis
Thymus
Human
Nicotinic acetylcholine
receptor
Unknown
SLE
Tubulointerstitium
of the kidneys
•Human
•Mouse
Atherosclerosis
Aorta
•Human
•Mouse
Inflammatory bowel
disease
Gut
COPD
Autoimmune disease
25,67,156
161–163
94,164
Immunoglobulin repertoire analysis
reveals B cell clonal expansion and
ongoing somatic hypermutation
130,165
Unknown
•Cellular organization
•LTβR, CXCL13 and CCL21 expression
166,167
•Human
•Mouse
Unknown
•Cellular organization
•CCL19 and CCL21 expression
168–172
Lung
•Human
•Mouse
Unknown
•Cellular organization
•Role for LTα, CXCL13, CCL19 and pDCs
173–175
Primary cancers
•Lung
•Breast
•Colon
•Human
•Mouse
TAA
In situ antigen-driven B cell
proliferation, somatic hypermutation
and affinity maturation
Germ cell
Ovarian cancer
Human
Unknown
None reported
Influenza virus
Lung (iBALT)
Mouse
Unknown
•Germinal centre reactions
•Class-switched plasma cells
•Antiviral immunoglobulin generated
30,59,79
HCV
Liver
Human
Unknown
•B cell clonal expansion
•Elevated CXCL13 expression
systemically and locally
120,121
MCMV
Salivary gland
Mouse
Unknown
•Local AID expression
•Ongoing CSR and somatic
hypermutation
•CXCL13 and BLIMP1 expression
Vaccinia virus Ankara
Lung (iBALT)
Mouse
Unknown
Local priming of T cell responses
Intestinal microbiota
Intestine
•Human
•Mouse
Unknown
•Development is LTi cell independent
•Presence of activated mucosal
γδ T cells
33,179
Helicobacter pylori
Gastric mucosa
•Human
•Mouse
Unknown
•CXCR5–CXCL13 dependent
•Local T cell priming and support of
TH17 cells
•IgG and IgA immune response
76,180
Helicobacter hepaticus
Liver
Mouse
Unknown
CCL21 and CXCL13 expression
181
Borrelia burgdorferi
(which causes chronic
Lyme arthritis)
Synovium
Human
Unknown
•Immunoglobulin V regions show
evidence of CSR
•Antigen-driven selection
182
Chronic inflammation
Cancer
10,40,
99–103
103,176–178
Infection
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Table 1 (cont.) | Clinical and experimental conditions that feature ELSs
Condition
Location
Species
Antigen recognized by
cells in ELSs
Other known functions of ELSs in
disease
Refs
Mycobacterium
tuberculosis
Lung (iBALT)
•Human
•Mouse
Unknown
•Priming of antigen-specific TH1 cells
•CCL19 and CCL21 expression
•Accumulation of CXCR5+CD4+
TFH-like cells
38,75,
78,118
Repeated intranasal LPS
challenge
Lung (iBALT)
Mouse
(neonatal)
Unknown
•LTi cell independent
•IL‑17 expression (potential link to
TH17 cells)
•CXCL13 expression
Infection (cont.)
30
Renal failure and transplantation
Allograft transplants
For example,
kidney, lungs and
heart
•Human
•Mouse
Unknown
Germinal centre reactions resulting in
anti-HLA-producing plasma cells and
memory B cells
Peritoneal dialysis
Peritoneal omental •Human
milky spots
•Mouse
Unknown
•Plasma cell responses to
T cell-dependent antigen delivered
intraperitoneally
•Dependent on LT and CXCL13
95,96
183–185
Environmental, degenerative and idiopathic conditions
Cigarette smoke
Lung
Mouse
Unknown
•Dependent on LTαβ and LTβR
•CXCL13 and CCL19 expression
173
Diesel exhaust particles
Lung (iBALT)
Mouse
Unknown
Unknown
186
Metal prosthetic joints
Joint
(periprosthetic
soft tissue)
Human
Unknown
Formation may involve cytotoxic and/or
delayed hypersensitivity response to
cobalt-chrome wear particles
187
Pristane adjuvant
Peritoneal
membrane
Mouse
Unknown
Lipogranuloma formation with
homeostatic chemokine expression
165
IPAH
Perivascular lung
tissue
Human
Autoantibodies against
vascular wall components
generated
•Expression of IL‑7, LTαβ, CCL19,
CCL20, CCL21 and CXCL13
•IL‑21+ TFH cells and FDCs
•AID expression suggests ongoing CSR
188
AID, activation-induced cytidine deaminase; BLIMP1, B lymphocyte-induced maturation protein 1; CCL, CC‑chemokine ligand; CNS, central nervous system;
COPD, chronic obstructive pulmonary disease; CSR, class-switch recombination; CXCL13, CXC-chemokine ligand 13; CXCR5, CXC-chemokine receptor 5;
EAE, experimental autoimmune encephalomyelitis; ELS, ectopic lymphoid structure; FDC, follicular dendritic cell; HCV, hepatitis C virus; iBALT, inducible
bronchus-associated lymphoid tissue; IL, interleukin; IPAH, idiopathic pulmonary hypertension; LPS, lipopolysaccharide; LTi cell, lymphoid tissue inducer cell;
LT, lymphotoxin; LTβR, lymphotoxin‑β receptor; MADCAM1, mucosal vascular addressin cell adhesion molecule 1; MCMV, murine cytomegalovirus;
pDC, plasmacytoid dendritic cell; SLE, systemic lupus erythematosus; SSA/Ro, Sjögren’s syndrome antigen A (ribonucleoprotein autoantigen); SSB/La, Sjögren’s
syndrome antigen B (autoantigen La); TAA, tumour-associated antigen; TFH cells, T follicular helper cells; TH cell, T helper cell.
Secondary lymphoid organs
(SLOs). In contrast to primary
(central) lymphoid organs,
where lymphocytes are
generated from immature
progenitor cells, SLOs — that
is, the spleen, Peyer’s patches
and lymph nodes — maintain
mature naive lymphocytes and
are sites of lymphocyte
activation by antigen.
Lymphoid neogenesis
The de novo development and
cellular organization of lymphoid
tissue into distinct anatomical
and functional compartments.
monocytic cells, macrophages and dendritic cells can form
discrete clusters within the inflamed tissue. These regions
can exist as simple lymphoid aggregates or as more sophisticated structures that histologically resemble secondary
lymphoid organs (SLOs)7,8 (TABLE 1). The spatial organization of leukocytes into defined, compartmentalized B cellrich and T cell-rich zones is termed lymphoid neogenesis.
These structures direct various B cell and T cell responses,
including the induction of effector functions, antibody
generation, affinity maturation, class switching and clonal
expansion. As a consequence, they are referred to as ectopic
lymphoid-like structures (ELSs) or tertiary lymphoid organs
(TLOs)7,8. In certain autoimmune conditions, patients who
have ELSs in the inflamed tissue often respond poorly to
standard biological therapy and thus remain a challenging treatment group9. However, in cancer (for example,
solid tumours such as colorectal carcinoma) the presence
of tumour-associated ELSs correlates with a better prognosis and they may coordinate endogenous antitumour
immune responses that improve patient survival10.
In this Review, we explore the molecular and genetic
basis for the formation of ELSs during infection, inflammation, autoimmune disease and cancer, and we discuss
the clinical significance of ELSs in terms of prognosis
and response to current biological therapies. With
insight from both experimental animal models and
human disease settings, we also consider the current
state of ELS-directed therapies and the rationale for targeting alternative molecular pathways that are associated
with ELS formation.
ELS development and function
Although ELSs display an architecture that resembles the
follicular compartments that are typically seen in SLOs
(BOX 1), their organization ranges from simple aggregates
of B cells and T cells through to highly ordered structures.
Much of our understanding of ELS formation originates
from findings that describe the generation of encapsulated
SLOs (as reviewed in REF. 11). Despite structural differences between SLOs and ELSs, many of the mechanisms
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Box 1 | Cellular activation events associated with lymphoid tissues
In the T cell zones of lymphoid tissues, dendritic cells (DCs) present peptide antigens on MHC class II molecules to naive
CD4+ T cells. The recognition of the peptide–MHC class II complexes by defined T cell receptors (TCRs) promotes the
activation and proliferation of CD4+ T cells, and their differentiation into specialized effector subsets. These effector
T cell subsets include CD4+ T follicular helper (TFH) cells, which express CXC-chemokine receptor 5 (CXCR5) and migrate
into the B cell zone in response to CXC-chemokine ligand 13 (CXCL13; see the figure). At the interface between the B cell
and T cell zones, CXCR5+ TFH cells support the activation of antigen-specific B cells. The provision of B cell help by TFH cells
drives B cell differentiation into plasmablasts or their migration into follicles to form germinal centres. Persistent antigen
presentation by B cells contributes to the full activation of TFH cells, which continue to provide B cell help. Thus, TFH cells
support the maintenance of germinal centres and promote the generation of both long-lived plasma cells and memory
B cells (see the figure). In this respect, TFH cells contribute to the control of antibody production and the fine-tuning of
mechanisms such as class switching, affinity maturation and somatic hypermutation.
Ectopic lymphoid-like
structures
(ELSs). Highly organized
lymphoid aggregates that form
in tissue sites that are not
typically associated with
lymphoid neogenesis.
T cell zone
B cell zone
Germinal centre
Class switching,
affinity maturation
and somatic
hypermutation
Encapsulated SLOs
Organized secondary lymphoid
organs (SLOs) that are encased
in a connective tissue capsule
that contains blood vessels.
Lymphoid tissue inducer
cells
(LTi cells). These cells are
present in developing lymph
nodes, Peyer’s patches and
nasopharynx-associated
lymphoid tissue (NALT) and
they are required for the
development of these
lymphoid organs. The inductive
capacity of LTi cells for the
generation of Peyer’s patches
and NALT has been shown by
adoptive transfer and it is
generally assumed that they
have a similar function in the
formation of lymph nodes.
T follicular helper cells
(TFH cells). Antigen-experienced
CD4+ T cells that are present
in B cell-rich regions of
structurally organized
lymphoid aggregates or
organs.
Lymphoid tissue organizer
cells
These cells are of
mesenchymal origin that are
activated by lymphoid cells
through lymphotoxin‑β
receptor signalling to express
adhesion molecules and
chemokines that regulate
lymphoid tissue development.
High endothelial venules
(HEVs). These are specialized
venules that occur in
secondary lymphoid organs,
except the spleen. HEVs allow
the continuous transmigration
of lymphocytes as a
consequence of the
constitutive expression of
adhesion molecules and
chemokines at their luminal
surface.
CXCR5
MHC
class II
TCR
DC
Peptide
T cell
CXCL13dependent
TFH cell
B cell
Memory
B cell
Plasmablast
that control the initial development, cellular composition and functional maintenance of these structures are
shared. However, ELS formation is distinct from the preprogrammed ontogenic processes that are associated with
secondary lymphoid organo­genesis and it does not occur
in all patients. Consequently, the generation of ELSs in
inflamed tissues — as opposed to a more typical diffuse
pattern of inflammatory infiltrate — must be governed by
a defined set of inflammatory signals. The mechanisms
that trigger these events are poorly defined.
Initiation of ectopic lymphoid neogenesis. Various
transgenic mouse models have emphasized the role of
inflamma­tory cytokines in lymphoid neogenesis. For
example, mice that are deficient in lymphotoxin‑α (LTα;
which is encoded by Lta) completely lack peripheral lymphoid organs12. Conversely, tissue-specific expression of
a transgene that encodes Lta in the kidney and pancreas
caused severe chronic inflammation with the accompanying formation of ELSs that were capable of promoting
antigen-specific responses and antibody class switching13.
Moreover, the overexpression of both Lta and Ltb (which
encodes LTβ) results in more prominent ELS formation compared with when only Lta is overexpressed14.
Thus, in combination with LTβ, LTα enhances ectopic
lymphoid neogenesis14. The activity of lymphotoxin is
associated with increased expression of the homeostatic
chemokines CXC-chemokine ligand 13 (CXCL13),
CC‑chemokine ligand 19 (CCL19) and CCL21, and
the increased infiltration of T cells that express CD62L
Plasma cell
Nature Reviews | Immunology
(also known as L-selectin)14
. Such findings indicate that
the propagation of ELSs within inflamed tissue is driven
by communication between local stromal cells, tissuespecific resident mononuclear cells and infiltrating
immune cells15,16. Although the actual cells that are
responsible for the positioning of these structures within
the inflamed tissue remain undefined, several new candidates have recently been proposed. These include lymphoid
tissue inducer cells (LTi cells), IL‑17‑secreting CD4+ T cell
populations and T follicular helper cells (TFH cells) (FIG. 1).
The accumulation of CD4+CD3−CD45+ LTi cells at
sites of lymph node development is an early event in
secondary lymphoid organogenesis17–19. This is coordin­
ated by the expression of CXCL13, receptor activator of
NF‑κB ligand (RANKL; also known as TNFSF11) and
IL‑7, which control the recruitment, survival and activation of LTi cells that express CXCR5 and IL-7 receptor
(IL-7R; also known as CD127)20. Central to this process is
the interplay between LTi cells and stromal mesenchymal
cells — known as lymphoid tissue organizer cells — which
act as the anchor for lymph node development11. The
release of IL‑7 and RANKL by activated lymphoid tissue organizer cells promotes the expression of LTα1β2 by
LTi cells, which, in turn, engages the LTβ receptor (LTβR)
on lymphoid tissue organizer cells that also express vascular cell adhesion molecule 1 (VCAM1) and inter­
cellular adhesion molecule 1 (ICAM1). This promotes
homeostatic chemokine release and vascularization by
high endothelial venules (HEVs), which also contribute to
lymphoid neogenesis11,15,20,21.
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a Initiation of ectopic lymphoid
b Cell recruitment to
inflamed site
tissue neogenesis
c Maintenance of ELSs
Inflamed tissue
IL-7R and RANK
signalling increase
LTα1β2 expression
IL-7
RANK
HEV
RANKL
VCAM1
IL-7Rα
TH2 cell
TH1 cell
ICAM1
CD62L
Development
of TFH-like cells
ELS
PD1
Accumulation
of LTi cells
B cell
RORγt
LTo cell
CD4+ LTi cell
BCL6
MAF
T cell
LTα1β2
LTβR signalling
promotes chemokine
release and HEV
development
CCL21 and
CXCL13
FDC
TH17 cell
TH17 cell
ICOS
CXCR5
TFH-like cell
LTi cell
Production of
CCL19, CCL21,
CXCL12 and CXCL13
maintains ELS
LTo cell
T cell
Figure 1 | Mechanisms that control the induction and maintenance of ectopic lymphoneogenesis. a | Various cell
Nature Reviews
| Immunology
types have been implicated as potential initiators of ectopic lymphoid-like structure (ELS) formation.
Although
the precise
mechanisms require further clarification, the cell types that are associated with this process may include interleukin‑17
(IL‑17)-secreting CD4+ T helper (TH) cells (not shown) and CD4+ lymphoid tissue inducer (LTi) cells. b | These cell populations
are attracted to inflammatory sites by certain chemokine signals, including CXC-chemokine ligand 13 (CXCL13) and
CC‑chemokine ligand 21 (CCL21). Within these inflamed lesions, resident stromal cells contribute to the cellular
organization of lymphoid aggregates. Pro-inflammatory mediators — including IL‑7 and lymphotoxin α1β2 (LTα1β2) —
regulate processes that affect the chemokine expression profile that is necessary for further recruitment of B cells and
T cells, the spatial arrangement of these cells into defined clusters and the control of angiogenesis. c | Although persistent
antigen presentation by follicular dendritic cells (FDCs) and B cells supports the long-term maintenance of these
structures, cell types such as CD4+ T follicular helper (TFH) cells are essential for relaying immunological instructions to
the B cells, which, in turn, ensure the continued action of TFH cells. Of potential relevance to the development of ELSs in
inflamed tissues is the ability of defined CD4+ TH effector subsets to acquire TFH cell-like characteristics. Commitment of
cells towards a TFH cell-like phenotype in inflamed tissues may aid the development or the activities that are associated
with ELSs. BCL6, B cell lymphoma 6; HEV, high endothelial venule; ICAM1, intercellular adhesion molecule 1;
ICOS, inducible T cell co‑stimulator; IL-7R, IL-7 receptor; LTβR, lymphotoxin‑β receptor; LTo cell, lymphoid tissue
organizer cell; PD1, programmed cell death protein 1; RANK, receptor activator of NF‑κB; RANKL, RANK ligand;
RORγt, retinoic acid receptor-related orphan receptor-γt; VCAM1, vascular cell adhesion molecule 1.
Innate lymphoid cell
(ILC). A type of innate
immune cell that is lymphoid
in morphology and
developmental origin but that
lacks properties of adaptive
B cells and T cells, such as
recombined antigen-specific
receptors. These cells regulate
immunity, tissue homeostasis
and inflammation in response
to cytokine stimulation.
Although stromal lymphoid tissue organizer cells and
LTi cells are important in secondary lymphoid organogenesis, their involvement in ELS formation is less clear.
However, stromal tissue cells may acquire lymphoid
tissue organizer cell-like properties in ELSs21–23. For example, synovial fibroblasts from the joints of patients with
rheumatoid arthritis release homeostatic chemokines and
cytokines that may contribute to ectopic lymphoid neogenesis within the inflamed synovium2,24–26. Moreover,
gene expression profiling of synovial tissue from patients
with rheumatoid arthritis has identified an IL7 signature
in ELS-associated synovitis26.
The recent discovery of innate adult LTi cells that
are part of the innate lymphoid cell (ILC) family18,19 raises
the possibility that these cells also contribute to inflammation-associated ELS development. For example, the
adoptive transfer of adult LTi cells into Cxcr5−/− mice
induces the formation of intestinal lymphoid tissues19.
Ectopic lymphoid tissues that develop in response to
transgenic overexpression of the Il7 gene also require
LTi cells 17. Adult LTi cells express the transcriptional regulator retinoic acid receptor-related orphan
receptor‑γt (RORγt) and can produce IL‑17, which are
characteristics of group 3 ILCs18,19. This pheno­type suggests an ancestral relationship between adult LTi cells
and IL‑17‑producing CD4+ T helper (TH17) cells18,27.
Furthermore, various studies have now linked IL‑17
and TH17 cells with ectopic lymphoid neogenesis, where
they have a role in chronic allograft rejection, experimental autoimmune encephalomyelitis (EAE) and the
development of inducible bronchus-associated lymphoid
tissue (iBALT)28–30. For example, ELS formation in the
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central nervous system is associated with TH17 cells
that express the lymphoid tissue-associated glycoprotein podoplanin (also known as gp38 in mice)29.
Podoplanin-deficient mice display defective development of lymph nodes and germinal centre structures,
and podoplanin expression by fibroblastic reticular
cells is associated with the development of the microarchitecture of the T cell zones29,31. Thus, podoplanin
expression is a defining feature of tissues that display
active lymphoid neogenesis. Although the precise
function of podoplanin is unclear, it may support the
retention of TH17 cells within these sites29,30. In addition
to these studies, various reports have also noted roles
for B cells and TNF-secreting F4/80+ myeloid cells in
ELS generation32,33. The formation of ELSs in different
tissues and in response to different forms of immuno­
logical activation suggests that the mechanisms
driving ELS expansion are complex. Inherent similarities in effector functions or cellular plasticity may
render various cell types able to promote tissue‑specific
ELS development.
Germinal centre
Located in peripheral lymphoid
tissues (for example, the spleen
or lymph nodes), these are
sites in which B cells proliferate
and clones that produce
antigen-specific antibodies of
higher affinity are selected.
Angiogenesis
The development of
new blood vessels from
existing blood vessels. It is
frequently associated with
tumour development and
inflammation.
Organization of cells within ELSs. Inflamed tissues
displaying ELS-associated pathology have increased
expression of homeostatic chemokines that govern
the cellular composition and functional properties of
ELSs34–37. These include CXCL12, CXCL13, CCL19
and CCL21. In such tissues, cellular communication between local stromal cells, tissue-specific resident mononuclear cells and infiltrating immune cells
propagates the development of ELSs and enables them
to function in a similar manner to germinal centres
(BOX 1). Such interactions affect leukocyte trafficking
and angiogenesis but are also instrumental in governing the organization of cells within these structures. For
example, CXCL13 and CCL21 control the segregation
of B cells and T cells in ELSs34,35. By contrast, CCL19
and CXCL12 promote lymphocyte infiltration and the
positioning of dendritic cells, B cells and plasma cells
within these aggregates 34 (FIG. 2). These specialized
properties probably reflect differences in the ability
of individual homeostatic chemokines to promote the
expression of LTα and LTβ by B cells and T cells34. For
example, transgenic expression of Cxcl13 in pancreatic tissue promotes the LTα1β2-mediated formation of
ELSs that display defined lymphoid zones, HEVs and
stromal reticulum35. Thus, homeostatic chemokines
affect the size, cellular composition and organization
of ELSs. Such features affect the functional properties of
ELSs and their impact on pathology.
However, the contribution of individual homeostatic
chemokines to ELS formation may also depend on the
anatomical site or ongoing disease processes. Although
transgenic expression of Ccl21 in the pancreas drives
pancreatic lymphoid neogenesis, the ectopic expression of Ccl21 in the skin does not36,37. Whether these
differences reflect a hierarchy of chemokine-mediated
outcomes that affects the degree of architectural organization displayed by ELSs or whether they reflect specific characteristics that are associated with ‘permissive’
versus ‘non-permissive’ tissues remains unknown.
Maintenance of ELSs within tissues. Considerable attention has been given to the development of lymphoid
structures, however, the presence of ELSs is often a transient feature of inflamed tissue. Thus, the mechanisms
that control the maintenance of these aggregates during
disease may have greater clinical significance. Recent
studies have linked TFH cells — or markers of TFH cell
activity — to the formation, maintenance and function
of ELSs29,38–40.
TFH cells promote B cell activities and high-affinity
antibody generation in germinal centres41,42. Recent studies of T cell plasticity in mice suggest that TH1, TH2 and
TH17 cells may also acquire TFH cell-like characteristics
and effector functions during their differentiation29,43–47.
For example, TH17 cells can display TFH cell-like characteristics during their differentiation, including the secretion of IL‑21 and the expression of TFH cell-associated
molecules, such as signal transducer and activator of
transcription 3 (STAT3), interferon-regulatory factor 4 (IRF4), MAF and inducible T cell co‑stimulator
(ICOS)48–53. These observations may help to explain the
ability of TH17 cells to promote ectopic lymphoid neogenesis28–30 and may also explain the regulation of ELS
activity in diseases that are associated with TH1, TH2 or
TH17 cell responses. Memory and effector TH cells that
lack typical TFH cell-like features are, nevertheless, also
capable of providing B cell help as they express CXCL13,
IL‑4, IL‑21 and CD40L54–56. Consequently, both positive
and negative regulators of effector T cell responses may
influence the maintenance of ELS activity (FIG. 2).
The CCR7‑dependent recruitment of dendritic cells
to developing ELSs represents an important homeostatic event that drives the initial propagation of adaptive immune responses within these sites. For example, infiltrating CD4+ T cells form tight clusters with
dendritic cells and this promotes T cell proliferation57.
As ELSs mature, dendritic cells within these structures
continue to support the efficient priming of T cell
responses through antigen presentation and they contribute to class-switch recombination, antibody generation and the formation of new lymphatic vessels58–60.
The importance of dendritic cells in ELSs is best illustrated by studies of mucosal immunity following viral
infection. The depletion of dendritic cells during viral
lung infection leads to impaired germinal centre reactions and a disruption of ELS architecture 59,60. The
sustained activation of dendritic cells within ELSs is
therefore required for both their formation and their
functional maintenance.
The function of ELSs as germinal centres. There is
conclusive evidence that ELSs not only recapitulate
the cellular, molecular and structural organization
of SLOs but that they can also support the function of
germinal centres. In the germinal centres of SLOs,
B cells undergo affinity maturation and differentiation
to memory B cells and plasma cells via antigen-driven
selection 61 (BOX 1). This process includes antibody
fine-tuning through somatic hypermutation and class
switching, both of which affect antigen recognition and
the effector capacity of the antibody62–65. Consistent with
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Classical lymphoid neogenesis
Putative regulators of ELS neogenesis
Non-inflamed tissue
LTi cell
Inflamed tissue
LTo cell
LTi cell
LTo cell
Initiators
• IL-7
• LTα1β2
• RANKL
Initiators
• IL-7
• LTα1β2
• RANKL
Initiators
• IFNs • IL-17
• IL-4 • IL-21
• IL-5 • TNF
T cell
FDC
B cell
Propagators
• CCL19
• CCL21
• CXCL12
• CXCL13
Propagators
• CCL19
• CCL21
• CXCL12
• CXCL13
Propagators
• IL-6
• IL-17
• IL-21
Inhibitors
• IL-2
• IL-27
Figure 2 | Cytokines and chemokine regulate ELS formation and function. The inducible formation of ectopic
Nature Reviews
| Immunology
lymphoid structures (ELSs) mimics the ontogenic process of secondary lymphoid organ (SLO) development,
whereby
the cytokines interleukin‑7 (IL‑7), lymphotoxin‑α (LTα), LTβ and receptor activator of NF‑κB ligand (RANKL) have a direct
role in initiating chemokine-directed lymphoid organogenesis. It is becoming clear that, during ELS formation, effector
cytokines that are produced in response to chronic inflammation, infection, autoimmunity and cancer are indirect
regulators that substitute for the cytokines that are involved in SLO formation. Recent studies have highlighted novel
roles for immune cell subsets in ectopic lymphoid neogenesis. For example, T helper 17 (TH17) cells and T follicular helper
(TFH)-like cells are associated with ELS development in the central nervous system and the lungs29,30,38. Various TH cell
subsets can also acquire TFH cell-like characteristics and effector functions29,43–47, and these cells may contribute to ELS
development in autoimmune and infectious diseases. The discovery of a role for these novel cell types brings with it a
host of regulators that may positively and negatively regulate ELS formation and function. These include factors that
determine the commitment or effector characteristics of defined T cell populations — for example, the control of
TH17 cells and TFH-like cells by IL‑2, IL‑6, IL‑21, IL‑27 and type I interferons (IFNs) — and mediators that have altered
expression in ELSs within defined anatomical locations (for example, IL‑4, IL‑5, IL‑17, IL‑21 and IL‑27). Potentially, these
may serve as alternative therapeutic targets or agents for ELS-associated pathology. CCL, CC-chemokine ligand;
CXCL, CXC-chemokine ligand; FDC, follicular dendritic cell; LTi cell, lymphoid tissue inducer cell; LTo cell, lymphoid
tissue organizer cell; TNF, tumour necrosis factor.
Activation-induced cytidine
deaminase
(AID). An enzyme that is
required for two crucial events
in the germinal centre —
somatic hypermutation and
class-switch recombination.
the role of ELSs as functional ectopic germinal centres,
activation-induced cytidine deaminase (AID) is expressed
at the mRNA and protein level within these structures,
and studies in patients and animal models support the
involvement of AID in autoimmunity66–69, infection70
and allograft rejection28. For example, AID controls
local antibody affinity maturation (including somatic
Permissive
tissue
b
hypermutation),
as evidenced
by a restricted profile
of variable (V)-gene repertoire usage, highly mutated
V regions and the
oligoclonal diversification of infiltratPancreas
ing B cells and plasma cells68,71–73. Active class switching
is also confirmed by the detection of Iγ–Cμ and Iα–Cμ
circular transcripts within ELSs, which marks on­going
class-switch recombination from IgM to IgG and IgA,
respectively67,68,74. Thus, ELSs in inflamed tissues retain
the necessary
machinery to support
in situ
Ectopicmolecular
CCL21-expression
ELS formation
antibody diversification, isotype switching, B cell
differ­entiation and
oligoclonal expansion. However,
Lung
as reviewed below, although the final outcome of lymphoid neogenesis is generally protective in the context
of infection and cancer, it is often deleterious in the
setting of autoimmunity and graft rejection.
ELSs in protective immunity and disease
ELSs as sites of anti-pathogen immune responses. InfecNon-permissive
tion with
bacteria (suchtissue
as Mycobacterium tuberculosis
and Helicobacter pylori) and viruses — such as influSkin
enza virus, hepatitis C virus (HCV) and several sialotropic viruses — has been associated with the formation
of ELSs in animal models39,59,74,75 and in humans76–78.
These ELSs resemble highly organized SLOs. Most
remarkably, in mice, infection-triggered lymphoid neogenesis seems to be preferentially induced at permissive mucosal
and results in the de novo
Ectopic sites,
CCL21-expression
Noformation
ELS formation
of iBALT and of lymphoid tissues associated with the
salivary glands79,80.
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Consistent with their immunological functions,
infection-associated mucosal ELSs can mount protective immune responses in situ. Examples of this include
influenza virus-induced and M. tuberculosis-induced
lymphoid neogenesis in the lungs of rodents. Following
influenza virus infection of the upper respiratory tract,
mice develop functional ELSs79 that are maintained by
lymphoid tissue-associated chemokines (for example,
CXCL13 and CCL21) and LTβ, which are produced by
infiltrating CD11b+CD11c+ dendritic cells59. These ELSs
promote the in situ differentiation of antiviral plasma
cells that are specific for the nucleoprotein of influenza
virus59. Notably, in LTα-deficient mice — in which SLOs
do not develop — reconstitution with Lta+/+ bone marrow
allows for ELS development in response to influenza
virus infection81. These bronchial ELSs can maintain
antiviral immunity and memory recall responses in
the absence of SLOs81. A role of ELSs in antimicrobial
immunity is further illustrated by their involvement in
M. tuberculosis infection, where they promote granuloma
formation and prevent the dissemination of infection38,75.
Although these studies demonstrate a non-redundant
role for ELSs in certain infections, they also indicate that
pathogen recognition by innate-sensing mechanisms
may contribute to the formation of these structures.
Overall, these data suggest an evolutionary role of
ELSs at sites of infection where they coordinate protective
immunity in addition to, and often independently from,
SLOs. However, a failure to eradicate a pathogen has
considerable clinical implications and can lead to the
development of autoimmunity (for example, in chronic
HCV infection) and lymphoma (for example, in H. pyloriassociated chronic gastritis). Thus, ectopic lymphoid
neogenesis in response to microbial challenge influences the natural history and clinical course of chronic
infection and associated complications.
Granuloma
A chronic inflammatory tissue
response that occurs at the
site of implantation of certain
foreign bodies and, in
particular, at sites of long-term
microbial persistence.
Granulomas are typically
well-organized structures that
are composed of T cells and
macrophages, some of which
fuse to form giant cells.
Severe combined
immunodeficiency mice
(SCID mice). A strain of mice
that possesses a genetic
defect in DNA recombination
that leads to severe
immunodeficiency. SCID mice
lack B cells and T cells, and
they are incompetent at
rejecting tissue grafts from
allogeneic and xenogeneic
sources.
ELSs in autoimmunity and the emerging role of Epstein–
Barr virus. The presence of ELSs with the appearance
of fully functional ectopic germinal centres has long
been described in the inflamed target organs or tissues
of patients who are affected by autoimmune diseases,
including the synovial tissue in rheumatoid arthritis (as
reviewed in REF. 82), the meninges in multiple sclerosis69,
the salivary glands in Sjögren’s syndrome (as reviewed in
REF. 83), the thymus in myasthenia gravis (as reviewed
in REF. 84) and the thyroid gland in Hashimoto’s thyroiditis85. In these clinical disorders, ELSs develop in response
to disease-specific autoantigens, which also ensure
the long-term maintenance of these structures within the
inflamed tissue.
The presence of ELSs in autoimmune conditions
perpetuates autoimmunity towards disease-specific
antigens. In this respect, many of the regulatory mechanisms that govern tolerance within SLOs are not seen
in autoimmune disease-associated ELSs. For example, in
SLOs, autoantigen-binding B cells may be excluded from
entering germinal centres and they lack responsiveness to
CXCL13 owing to a downregulation of CXCR5 (REF. 86).
However, autoimmune disease-associated ELSs permit
the entry of autoreactive B cells87. This allows for the
differentiation of the B cells into high-affinity auto­reactive
plasma cells that release disease-specific autoantibodies,
such as anti-citrullinated protein antibodies (ACPAs) in
rheumatoid arthritis67, and antibodies against the ribo­
nucleoproteins Ro and La (also known as Sjögren’s syndrome antigens A and B) in Sjögren’s syndrome88, against
thyroglobulin and thyroperoxidase in Hashimoto’s
thyroiditis85, and against the nicotinic acetylcholine
receptor in patients with myasthenia gravis84.
Although the mechanisms that are responsible for
the preferential accumulation of autoreactive B cells
in ELSs are not fully understood, a direct role has been
proposed for Epstein–Barr virus (EBV) in the development of autoimmunity. EBV is a γ‑herpesvirus that is
retained throughout the life of an infected individual and
that promotes the survival and proliferation of B cells89,90.
Emerging evidence suggests that ectopic follicles in
the target organs of patients with multiple sclerosis,
myasthenia gravis, rheumatoid arthritis and Sjögren’s
syndrome frequently harbour latent EBV infection69,91–93.
In these sites, EBV-transformed B cells and plasma cells
display autoreactivity to disease-specific autoantigens,
such as citrullinated fibrinogen in rheumatoid arthritis92
and the ribonucleoprotein Ro in Sjögren’s syndrome93.
Autoreactive EBV-infected B cells are therefore predicted
to transit from the peripheral compartment into ectopic
germinal centres where they undergo differentiation to
high-affinity autoreactive plasma cells. Thus, niches of
EBV colonization within ELSs might be considered as a
hallmark of organ-specific autoimmunity.
EBV infection may also explain another remarkable feature of ELSs, which is their ability to persist
in an activated state for several weeks in the absence
of recirculating immune cells from the periphery.
This is best demonstrated in chimeric severe combined
immuno­deficiency mice that are engrafted with synovial
or thymic tissue from patients with rheumatoid arthritis or myasthenia gravis, respectively. ELSs within the
transplanted tissue maintain their follicular organization
and auto­antibody generation despite both the absence of
‘new’ cells infiltrating the grafts and the impaired host
immune response67,94. This is of crucial importance, as
ELS neutralization is probably fundamental to avoiding
the re‑emergence of autoreactive clones that are capable
of driving disease relapse or resistance to therapy.
ELSs in transplantation. ELSs have been observed in
almost all types of human grafts that have been removed
owing to chronic rejection, including kidneys, lungs and
hearts95. Transplant-associated ELS formation seems to
recapitulate the molecular pathways that are triggered
during the ontogeny of SLOs. These ELSs harbour
ectopic germinal centres in which naive B cells differ­
entiate into anti-HLA-producing plasma cells and
memory B cells96. However, ELS formation in allografts
differs from that of canonical SLOs in a number of ways:
first, the considerable amounts of TH17 cell-associated
cytokines and growth factors — such as B cell-activating
factor (BAFF; also known as TNFSF13B) — that facilitate autoreactive B cell survival; second, the continuous
release of alloantigens from the injured tissue that are
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trapped locally by defective lymphatic drainage; and
third, the apparent defect of local immunoregulatory
mechanisms. As a consequence, there is an excessive
immune response within the ELSs that contributes to
allograft rejection95. However, two recent publications
in distinct experimental models (renal and cardiac
allografts) have reported that the presence of ELSs
promotes allograft tolerance and graft function97,98. On
this basis, it is postulated that under certain conditions,
ELSs may amplify beneficial immune responses —
that are associated with B cells and T cells displaying a
regulatory profile — to promote graft tolerance.
ELSs in cancer. In cancer, ELSs have been documented
in many tumours, including those that are associated
with lung99,100, colon10,101, breast40,102,103 and germ cell
cancers104,105. However, not every patient with a specific
type of cancer develops ELSs and when they do occur,
their contribution to disease varies considerably. Some
tumour types are more likely to induce ELS formation than others, which indicates that certain tumours
provide a microenvironment that is conducive to lymphoid neogenesis. Given the stark difference between
the immunosuppressive nature of the tumour microenvironment compared with chronic inflammatory
foci, it is perhaps surprising that ELSs develop at all in
tumours104. Although CXCL13, CCL19 and CCL21 have
a crucial role in the formation of cancer-associated ELSs,
LTi cells seem to be dispensable as ELSs can form in their
absence100,104,105. Permissive tumours probably induce the
production of lymphoid chemokines not only through
the expression of LTα1β2 but also through the expression
of pro-inflammatory cytokines — such as TNF, IL‑1β
and IL‑6 — which contribute to the transcriptional regulation of CXCL13, CCL19 and CCL21. However, the
precise stimuli that initiate the development of cancerassociated ELSs within the immunosuppressive tumour
microenvironment require further investigation.
Clinical implications
As discussed above, ELSs are found in patients with
diverse medical conditions but invariably, in each condition, ELSs form in some patients but not in others
(TABLE 1). Therefore, important questions relating to this
phenomenon remain to be answered. For instance, what
determines the formation of ELSs in some individuals but
not in others? Is ELS development typical of different disease subtypes (ab initio) or is ELS formation the inevitable
result of persistent inflammation — that is, are ELSs the
cause or the consequence of chronicity? To what extent
do ELSs contribute to ongoing inflammation and tissue
damage (disease prognosis)? In this section, we address
these questions in turn by providing recent data from the
literature, as well as from our own personal experience.
What governs ELS formation in some individuals
but not in others? This question still remains mostly
unanswered but it is likely that genetic and/or environmental factors shape the inflammatory response to give
rise to diverse structural outcomes within individual
tissue microenvironments.
Considering the genetic influence first, evidence is
beginning to emerge from various genetic studies that
have found associations between specific pathways that
are involved in ELS formation and/or function and
susceptibility to autoimmune diseases. For example, four
single nucleotide polymorphisms (SNPs) have been identified in the IL2–IL21 locus at chromosome region 4q27
that are associated with several autoimmune diseases106–108
and that can influence circulating levels of IL-21 (REF. 109).
Additionally, SNPs in loci that are near to the CXCR5 gene
have been linked with susceptibility to primary biliary
cirrhosis 110, Sjögren’s syndrome 111, systemic lupus
erythematosus (SLE)112 and multiple sclerosis113.
Although no sub-analysis with stratification of
patients for the presence or absence of ELSs has been
carried out in the above studies, the increased genetic
susceptibility to autoimmunity that is associated with loci
near to both the IL21 and CXCR5 genes corresponds with
the crucial role of IL‑21‑producing CXCR5+ TFH cells
in the formation and function of ELSs. This is also potentially of crucial relevance as novel strategies to specifically
target TFH cells are being developed for therapeutic use in
autoimmune conditions (as discussed below). It is likely
that the influence of genetic factors will become clearer
as larger scale analyses of stratified disease subsets are
carried out according to pathobiology (for example, the
presence or absence of ELSs).
With regard to the environmental factors that influence ELS formation, diverse host immune responses to
infection lead to different outcomes. Thus, it is possible
that in ‘permissive’ patients, an active EBV infection is
established in the diseased tissue and may drive ELS formation through its unique ability to infect and activate
B cells114,115. This hypothesis was recently supported by
our own work, in which we found a high frequency of
EBV-infected B cells and plasma cells in ELS-containing
synovia from patients with rheumatoid arthritis92. Notably, EBV was not detected in control synovia from patients
with osteoarthritis or in patients with rheumatoid arthritis
whose synovia showed diffuse infiltration of immune cells
but lacked ELSs92. Similar results were seen in the salivary
glands of patients with Sjögren’s syndrome93.
Is ELS formation pre-determined or a result of persistent
inflammation? The frequency at which ELSs occur in
patients with chronic inflammation typically varies from
20–40%7,82. However, it is unknown whether ELSs are
a typical feature of different disease subtypes from the
beginning (ab initio) or whether they form as a result of
persistent inflammation. The reason this this remains
unknown is twofold: first, in humans, clinical presentation is often delayed and it is thus impossible to precisely
determine the early events in the pathological process;
and second, there are an insufficient number of studies
(and an insufficient number of large cohort studies) in
which serial biopsies have been taken to establish the
‘pre-determined’ versus the ‘evolutionary’ nature of
ELSs. Nonetheless, data is beginning to emerge from
a cohort of 300 patients with rheumatoid arthritis who
had shown symptoms for less than 12 months; biopsies
were taken at the time of disease presentation and 70% of
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individuals were also biopsied 6 months post-treatment116.
Approximately 40% of patients displayed histo­logical
evidence of ELSs before receiving any disease-modifying
therapy. Thus, ELSs seem to be present ab initio and
to define a specific disease subset from the beginning.
However, in another early arthritis cohort of 93 patients
with only 17 biopsy repeats at 6 months, ELSs have been
reported as being related to the degree of inflammation
and not related to specific disease subtypes117. No serial
biopsy data is available from other diseases and, therefore, further work is required in this area to establish
the precise ontogeny of ELSs in the context of chronic
inflammation.
Cryoglobulinaemia
An autoimmune, extra-hepatic
manifestation that is
associated with certain viral
infections and chronic
inflammatory conditions,
whereby antigen–antibody
complexes are deposited in
capillaries and arterioles (and
occasionally small arteries)
causing vasculitis, renal
disease, arthralgias or arthritis.
To what extent do ELSs control disease progression and
outcome? From the above discussion, it is clear that ELSs
are dynamic structures, the main function of which is to
potentiate the immune response at sites of disease. The
clinical presentation of ELSs may therefore influence disease severity, the rate of disease progression and clinical
outcome. Ultimately, this will depend on the ability of the
host to clear triggering antigens, as well as the stage and
the nature of the disease in question. In the subsequent
paragraphs, we provide some examples of the influence
of ELSs on the progression and outcome of infection,
autoimmune conditions, transplantation and cancer.
On the basis of the evidence from animal models
showing a protective role of mucosal ELSs in pathogen
control, it is anticipated that the presence of ELSs in
infected humans could alter the natural history and
clinical manifestations of chronic infections. Accordingly, in M. tuberculosis infection, ELS formation in the
peripheral rim of lung granulomas is typically associated
with the latent, asymptomatic form of tuberculosis118.
Conversely, sites of active infection that have cavitary
tuberculous lesions lack B cell follicles118. Thus, ELSs
coordinate the local host response to pulmonary tuberculosis118. ELSs probably exert similar mechanisms of
pathogen containment in other chronic infections, such
as H. pylori and HCV76,77. However, it is now clear that
in the absence of pathogen eradication, ELSs also act as
a double-edged sword by contributing to autoimmunity
and lymphomagenesis. Although tuberculosis has long
been associated with the development of various auto­
immune phenomena119, the association with ELSs has
not been investigated. Conversely, in chronic HCVrelated hepatitis, elevated CXCL13 levels in liver biopsies
identify patients that develop mixed cryoglobulinaemia120.
Together with the clonal analysis of intrahepatic B cells
in portal tracts121, these data indicate that intraportal
lymphoid follicles contribute to the evolution from
polyclonal to oligoclonal to monoclonal autoreactive
B cell activation that occurs as a result of chronic HCV
infection. Pathogen-driven chronic B cell activation
within ELSs is also associated with lymphoproliferative
disorders, as evident in HCV-associated B cell lympho­
proliferation122 and H. pylori-driven gastric mucosaassociated lymphoid tissue (MALT) B cell lymphomas76.
The development of gastric (and salivary gland) MALT
lymphomas has been linked to the genetic instability
that is associated with the process of aberrant somatic
hypermutation, whereby dysregulated expression of AID
leads to mutations in the regulatory and coding sequences
of genes that regulate B cell survival and proliferation
(such as PAX5 and MYC)123. Interestingly, AID expression in gastric and salivary gland MALT lymphomas is
confined to ELSs and is not found in malignant marginal
zone B cells66,123, which suggests that lymphoma­genesis
is dependent on sustained antigen stimulation within
ELSs. Accordingly, gastric MALT lymphomas are crucially dependent on H. pylori-specific T cells124 and the
eradication of H. pylori results in both ELS resolution and
tumour regression125. Although in this context lympho­
magenesis is ultimately driven by chronic infection, a
striking and unique feature of MALT lymphomas is that
malignant B cells frequently originate from precursors
that express an autoreactive B cell receptor with homology to rheumatoid factor126. Thus, autoimmunity and
MALT lymphomagenesis can represent a continuum of
the same antigen-driven response in which ELSs have a
central role.
In chronic inflammatory and autoimmune conditions, the B cell-rich inflammatory infiltrate that
is typically seen in ELSs is associated with a strong
potential for local immune activation and cellular differentiation. This promotes autoantibody production,
complement activation and pro-inflammatory cytokine
release, which drives the inflammatory cascade, auto­
immunity and tissue destruction. For example, studies
in rheumatoid arthritis have reported worse disease outcomes in patients with ELSs116. In these cases, increased
joint destruction is associated with the production of
pro-inflammatory cytokines127, as well as osteoclastactivating molecules such as RANKL 128 and the
RANKL-inducing molecule TNF-related weak inducer
of apoptosis (TWEAK; also known as TNFSF12)129. In
autoimmune thyroiditis, most ELS B cells recognize the
autoantigens thyroglobulin and thyroperoxidase, and
this leads to glandular destruction85. In SLE, a restricted
repertoire of isotype-switched antibodies are deposited
in the glomerular basement membrane of the kidneys
with consequent organ damage130. Finally, the presence
of ELSs in the inflamed meninges of patients with progressive multiple sclerosis can be determined through
analyses of B cell markers, and the occurrence of ELSs is
associated with a gradient of cortical neurodegeneration
and a more aggressive course of disease131.
ELSs are thought to drive chronic transplant rejection
by enhancing intra-graft allogeneic responses, as grafts
in which the ELSs are most functional have a shorter
life expectancy28,96. However, the validity of this conclusion has been questioned by the fact that, by definition,
only de‑transplanted, failed grafts were included in the
analysis. In addition, as discussed above, there is evidence from experimental transplant models that regulatory B cells and ELSs may govern graft tolerance. Further
work is therefore required to clarify the contribution of
ELSs to graft rejection versus survival.
As discussed above, different cancers are more or
less immunogenic and hence, are more or less prone to
induce ELSs. Analyses of the cytokine and chemokine
gene signatures, the number of tumour-infiltrating
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lymphocytes and the level of ELS organization have
been used to stratify patients and types of cancer as
‘immune response positive’ and ‘immune response negative’. Although these classification criteria are not purely
based on ELSs, there is a general consensus that patients
who have immune-response-positive tumours (for example, breast cancer tumours) have a better prognosis than
those with immune-response-negative tumours102,132–134.
Similar results have been described in colon cancer,
where immune response positivity was associated with
the increased survival of patients independently of
tumour stage, previous treatment or microsatellite instability10. As expected, CXCL13 and CCL19 expression
was also associated with a good prognosis10. Finally, gene
expression profiling of immune-response-positive and
immune-response-negative human melanomas identified CXCL13 and CXCL8 as components of a discrete
group of 12 genes that were found to be diagnostic of
immune response status135. In another study, the mean
survival of patients with melanoma was 55 weeks in
the immune-response-positive group compared with
18 weeks in the immune‑response-negative group136.
In summary, ELS formation in peripheral tissues
seems to follow normal pathophysiological programmes
in response to the need to increase local immune
responses and clear exogenous antigens, alloantigens
and/or autoantigens. When such effector programmes
are successful, they lead to improvements in disease
pathology and a better prognosis, whereas their failure
or ineffectiveness leads to persistent inflammation, tissue damage and worse outcomes. This bivalent functionality has substantial clinical implications when it comes
to targeting ELSs for therapy.
ELSs as therapeutic targets
The modulation of ELSs for therapeutic purposes has
attracted marked interest both from the scientific community and from industry. On the basis of the functional and clinical data discussed above, it would be
desirable to potentiate the activity of ELSs in infection
and cancer but to inhibit their activity in the context of
transplantation and chronic inflammatory conditions.
Current cancer trials are testing whether ELS-related
pathways can be exploited to enhance antitumour
immunity. Although cytokine and chemokine administration, and tumour antigen vaccination represent
promising therapeutic approaches to target ELSs, the
long-term efficacy of these strategies remains debatable137,138. Similar vaccination approaches are also being
considered as adjunct therapies that promote local
antimicrobial immunity in the lungs139.
By contrast, there is a strong rationale for therapeutic inhibition of ELSs in graft rejection, and in chronic
inflammatory and autoimmune conditions. In a study
of patients showing chronic renal allograft rejection,
treatment with anti‑CD20 (rituximab) failed to cause
regression of ELSs despite the successful depletion of
B cells from the peripheral blood140. This suggests that
the local inflammatory microenvironment in ELSs may
facilitate B cell survival and allow evasion of rituximabmediated depletion140. In the context of autoimmunity,
biological interventions that promote the clearance or
inhibition of autoreactive lymphocytes have the potential to disrupt ELS involvement and restrict deleterious adaptive immune responses. In this regard, understanding the impact of TNF inhibitors on synovial ELSs
is of major importance. In a recent study, patients with
rheumatoid arthritis who had synovial ELSs displayed
an inferior response to TNF inhibitors compared with
patients who did not have synovial ELSs9. In addition,
the presence of ELSs in synovial tissue before treatment
was reported to be an independent negative predictor
of the response to TNF inhibitors9. Furthermore, high
protein-level expression of IL‑7R, which is normally
associated with ELSs, was reported to be associated
with a poor response to TNF inhibitors141. Therefore,
the development of selective ELS-targeted therapies
has attracted marked interest both from the scientific
community and from industry.
Currently licensed therapies for inflammatory diseases, including CD20‑specific (for example, rituximab) and IL‑6R‑specific (for example, tocilizumab)
monoclonal antibodies, oral Janus kinase inhibitors (for example, tofacitinib) and T cell activation
blockers (for example, abatacept) probably target
processes that are associated with ELS activities 4.
However, these drugs also possess broader modes of
action3–5. Given the prominent role of lymphotoxin
and homeostatic chemokines in ELS formation, initial
approaches targeted this axis. Pharmacological inhibition of LTβ using an LTβR–immunoglobulin fusion
protein resulted in disease amelioration in animal
models of arthritis142, autoimmune sialoadenitis143 and
type 1 diabetes144. Although, baminercept (which is a
humanised LTβR–IgG1 fusion protein) failed to show
clinical efficacy in a Phase II clinical trial in patients
with rheumatoid arthritis, its utility in Sjögren’s
syndrome is currently under investigation (TABLE 2).
Recently, a new LTα-specific monoclonal antibody
has completed a Phase I clinical trial in patients with
rheumatoid arthritis145 but the results of the Phase II
study have not yet been published. It is noteworthy
that none of the above studies have stratified patients
for the presence of ELSs in the disease-affected tissue,
which would probably influence the reported extent
of clinical responses. However, the baminercept trial
in Sjögren’s syndrome includes a salivary gland biopsy
sub-study, which will inform on ELS modulation after
treatment.
Blockade of CXCL13, CCL19 and CCL21 or their
receptors CXCR5 and CCR7 is another potential therapeutic strategy that has shown some promise in animal
models. CXCL13 blockade ameliorated collageninduced arthritis146 and autoimmune sialoadenitis147 but
not autoimmune diabetes148. Of note, although CXCL13
inhibition altered the structure of ELSs, it did not influence their function or the incidence of diabetes148. To
date, no developmental compound targeting this pathway has been tested in human clinical trials. However,
there is evidence that blocking immune cell recirculation to ELSs in the context of autoimmune disease is a
promising therapeutic strategy.
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Table 2 | Novel therapies targeting ELSs in ELS-positive autoimmune diseases
Pathway inhibited
Developmental compound
Target or class of drug
Completed and ongoing clinical trials
in ELS-positive autoimmune diseases*
IL‑17
Secukinumab
IL‑17A‑specific monoclonal antibody
•NCT01377012 (RA, Phase III, recruiting)‡
•NCT01350804 (RA, Phase III, recruiting)‡
•NCT01874340 (MS, Phase II, recruiting)
•NCT02044848 (T1D, Phase II, recruiting)
Ixekizumab
IL‑17A‑specific monoclonal antibody
NCT00966875 (RA, Phase II, completed)
Brodalumab
IL‑17RA‑specific monoclonal antibody
•NCT00950989 (RA, Phase II, completed)
•NCT01059448 (RA, Phase II, terminated)
ABT‑122
TNF and IL‑17 bispecific antibody
NCT01853033 (RA, Phase I, ongoing)
CNTO-6785
IL‑17A‑specific monoclonal antibody
NCT01909427 (RA, Phase III, recruiting)
IL‑21
NNC0114‑0006
IL‑21‑specific monoclonal antibody
•NCT01647451 (RA, Phase II, completed)
•NCT01689025 (SLE, Phase I, completed)
LTα/β
Baminercept
LTβR–IgG1 fusion protein
•NCT00664573 (RA, Phase II, completed)
•NCT01552681 (SS, Phase II, recruiting)§
Pateclizumab
LTα-specific monoclonal antibody
NCT01225393 (RA, Phase II, completed)
ICOS–ICOSL
AMG557 (also known as mAb-3B3)
ICOSL-specific monoclonal antibody
NCT01683695 (SLE, Phase I, recruiting)
S1PR1
Fingolimod (also known as FTY720)
S1PR1 antagonist
NCT01310166 (MS, Phase IV, recruiting)
BAFF
Belimumab
BAFF-specific monoclonal antibody
•NCT01008982 (SS, Phase II, completed)§
•NCT00071812 (RA, Phase II, completed)
•NCT01480596 (MG, Phase II, recruiting)
•NCT01639339 (SLE, Phase III, recruiting)
Tabalumab
BAFF-specific monoclonal antibody
NCT01202760 (RA, Phase III, completed)
BAFF, B cell-activating factor; ICOS, inducible T cell co‑stimulator; ICOSL, ICOS ligand; IL, interleukin; IL-17RA, IL-17 receptor A; LT, lymphotoxin; LTβR, LTβ receptor;
MG, myasthenia gravis; MS, multiple sclerosis; RA, rheumatoid arthritis; S1PR1, sphingosine-1‑phosphate receptor 1; SLE, systemic lupus erythematosus;
SS, Sjögren’s syndrome; T1D, type 1 diabetes; TNF, tumour necrosis factor. *Information from the ClinicalTrials.gov website, accurate as of May 2014. ‡NCT01377012 is
recruiting patients with RA who failed to respond to conventional disease-modifying anti-rheumatic drugs (DMARDs), whereas NCT01350804 enrols patients with
RA who are inadequate responders to anti-TNF therapy. § These studies include a salivary gland biopsy sub-study, pre-treatment and post-treatment.
Non-obese diabetic mice
(NOD mice). These mice
spontaneously develop
diabetes as a result of
autoreactive T cell-mediated
destruction of pancreatic
β-islet cells.
Fingolimod (also known as FTY720) — which
blocks the egress of lymphocytes from SLOs via functional antagonism of the sphingosine-1‑phosphate
receptor — has shown favourable efficacy in relapsing–
remitting multiple sclerosis149 and has received approval
from the US Food and Drug Administration (FDA) for
this indication. Fingolimod selectively reduces the frequency of circulating CCR7+CD4+ T cells (that is, naive
and central memory T cells) and traps this subset of
T cells in SLOs, which prevents their migration into the
central nervous system150.
Given the role of TFH cells in ELSs, there is also growing
interest in blocking key TFH cell-related signatures — for
example, the co-stimulatory molecules ICOS and ICOS
ligand (ICOSL) or the key TFH cell-related cytokine IL‑21.
Blockade of IL‑21–IL‑21R signalling ameliorated disease
in animal models of arthritis151 and SLE152, and an antihuman IL‑21 monoclonal antibody (NNC0114-0006) has
recently completed Phase II clinical trials in rheumatoid
arthritis and a Phase I clinical trial in SLE, but no published
data are currently available (TABLE 2). Pharmaco­logical
ICOS blockade has also shown benefit in experimental
arthritis153 and a mouse model of lupus154 via inhibition of
TFH cell responses. Similarly, the concomitant inhibition
of ICOS and CD40L co‑stimulation protects non-obese
diabetic mice (NOD mice) from diabetes155. As a consequence, an anti-ICOSL monoclonal antibody (AMG557;
also known as mAb‑3B3) has recently entered Phase I
clinical trials in SLE (TABLE 2). The co‑stimulatory
signalling that is mediated by ICOS–ICOSL and
CD40–CD40L is also required for TH17 cell survival48,153
and, as such, may contribute to the formation or maintenance of ELSs within inflamed tissue. Thus, it will be
extremely interesting to investigate whether directly targeting the IL‑17 pathway with novel therapeutics (such
as secukinumab, ixekizumab and brodalumab) that are
currently in late-stage clinical development (TABLE 2) will
modulate ELSs in the context of autoimmune disease.
Finally, it remains to be established whether therapeutics
that target factors that are associated with B cell survival
and proliferation disrupt ELSs or impair their role as functional niches for autoimmune B cell activation. In this
regard, data are eagerly anticipated from a biopsy-based
Phase II clinical trial in Sjögren’s syndrome with belimumab, which is an anti-BAFF monoclonal antibody that
has received FDA approval for SLE (TABLE 2). Such studies should elucidate whether BAFF inhibition is sufficient
to disrupt ELS functionality in the salivary glands.
Concluding remarks and future directions
An increasing number of clinical investigations emphasize that ectopic lymphoid neogenesis is a common
occurrence at sites of inflammation, whereby ELSs form
an integral part of the immune response to infections,
tumours and autoantigens. Although experimental animal models have defined certain mechanistic aspects
relating to ELS development, function and maintenance,
parallel investigations in human conditions remain
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REVIEWS
relatively sparse by comparison. It is important to determine whether the clinical consequences of ELS activities are beneficial (for example, immunity to infections
or cancer) or detrimental (for example, the promotion
of auto­immunity or graft rejection). Clinical trials in
conditions that are typically associated with ectopic lymphoid neogenesis may broaden our understanding of ELS
involvement in these diseases. An increasing number of
novel biologics that target mediators that are central to
ELS biology are now entering the clinical arena. However,
the key goal is to identify specific molecular signatures
that will predict at an early stage of the disease process
whether ELSs will form and that can be used to inform
decisions regarding the most appropriate therapy for individual patients. For example, there may be little point in
targeting pathways that promote ELS development if these
structures have already become a prominent feature of the
underlying pathology by the time a patient is diagnosed.
Instead, the use of agents that block the long-term maintenance of ELSs within these inflamed tissues may prove
to be a more suitable approach in this scenario. To realize
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Acknowledgements
G.W.J. is funded by Arthritis Research UK as a non-clinical
Career Development Fellow.
Competing interests statement
The authors declare no competing interests.
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