Ectopic Lymphoid-Like Structures in Infection, Cancer and Autoimmunity

REVIEWS

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.

Inflammation supports immunological defence against golimumab and certolizumab — or their receptors (for infection, trauma, injury and cancer. The regulation of example, the inhibition of interleukin‑6 (IL‑6) receptor inflammation is governed by cellular communication by tocilizumab) are now widely used in routine clini- between non-haematopoietic stromal cells, resident cal practice for the treatment of rheumatoid arthritis3–5. leukocytes and infiltrating immune cells1,2. Optimal These therapies display specific modes of action and have control of this process ensures competent host defence, been developed on the basis of a better understanding of the elimination of the initiating antigen (or antigens) and cytokine involvement in inflammation. Although these limited tissue damage1,3. However, repeated, persistent agents reduce the symptoms and progression of disease, or non-resolving episodes of inflammation lead to the not all patients respond equally to the same therapy. inappropriate regulation of this response and promote Indeed, 30–40% of patients with rheumatoid arthritis do autoimmunity, chronicity and tissue pathology1,3,4. The not respond to any treatments and some biological thera- mechanisms that control these events are often diverse pies are efficacious in certain clinical conditions but not and they contribute to clinical differences in disease in others4. Such differences in therapeutic efficacy sug- presentation. For example, patients who have the same gest that distinct inflammatory mechanisms must steer underlying clinical condition invariably show differences the course of disease in any one individual. Typically, the 1Centre for Experimental in disease severity, rate of disease onset and response to sooner after diagnosis appropriate therapy commences, Medicine and Rheumatology, treatment4. Although sex, age, genetics, metabolic fac- the better the clinical outcome and the increased likeli- William Harvey Research tors and the environment are major determinants that hood of achieving remission6. Thus, cellular events that Institute, Barts and The affect the propensity to develop chronic disease, these are triggered early in the inflammatory process must London, School of Medicine and Dentistry, Queen Mary indicators provide minimal information on the molecu- influence the course of disease. University of London, lar or cellular pathways that drive the type of disease that A central feature of local inflammation is the inter Charterhouse Square, is observed. action between the stromal tissue compartment and London EC1M 6BQ, UK. Cytokines (including interleukins, interferons and immune cells1,2. Inflammatory mediators that are pro- 2Cardiff Institute for Infection and Immunity, The School of growth factors), chemokines, lipid mediators and innate- duced by stromal cells and tissue-resident monocytic Medicine, Cardiff University, sensing mechanisms control inflammation and they have cells control the recruitment, activation and survival of The Tenovus Building, important roles in the pathology of various inflamma leukocytes. Cellular infiltration is traditionally viewed as Heath Campus, Cardiff tory diseases and cancer3–5, thus representing major the diffuse influx of immune cells, with the cells being scat- CF14 4XN, Wales, UK. targets for clinical intervention. For example, biological tered throughout the inflamed tissue. However, infiltrating Correspondence to S.A.J. e‑mail: [email protected] therapies that inhibit pro-inflammatory cytokines — for leukocytes often form more organized aggregates and these doi:10.1038/nri3700 example, the inhibition of tumour necrosis factor (TNF) promote antigen-specific adaptive immune responses that Published online 20 June 2014 by the monoclonal antibodies infliximab, adalimumab, 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 Other known functions of ELSs in Refs cells in ELSs disease Autoimmune disease Rheumatoid arthritis Synovial tissue • Human • Rheumatoid factor • In situ B cell differentiation 25,67,156 • Mouse • Citrullinated proteins • Ongoing CSR • Histones? • Autoantibody secretion Sjögren’s syndrome • Salivary glands • Human • SSA/Ro Unknown 39,73,74,157 • Lacrimal glands • Mouse • SSB/La Multiple sclerosis (and CNS • Human Myelin and other neuronal Unknown 29,158 EAE) • Mouse antigens proposed Diabetes Pancreatic islet Mouse Plasma cell anti-insulin Unknown 159,160 parenchyma reactivity observed Hashimoto’s thyroiditis Thyroid gland Human • Thyroglobulin Unknown 85 • Thyroperoxidase Primary sclerosing Liver Human Unknown • Cellular organization 161–163 cholangitis and primary • CCL21 and MADCAM1 expression biliary cirrhosis Myasthenia gravis Thymus Human Nicotinic acetylcholine Unknown 94,164 receptor SLE Tubulointerstitium • Human Immunoglobulin repertoire analysis 130,165 of the kidneys • Mouse reveals B cell clonal expansion and ongoing somatic hypermutation Chronic inflammation Atherosclerosis Aorta • Human Unknown • Cellular organization 166,167 • Mouse • LTβR, CXCL13 and CCL21 expression Inflammatory bowel Gut • Human Unknown • Cellular organization 168–172 disease • Mouse • CCL19 and CCL21 expression COPD Lung • Human Unknown • Cellular organization 173–175 • Mouse • Role for LTα, CXCL13, CCL19 and pDCs Cancer Primary cancers • Lung • Human TAA In situ antigen-driven B cell 10,40, • Breast • Mouse proliferation, somatic hypermutation 99–103 • Colon and affinity maturation Germ cell Ovarian cancer Human Unknown None reported 103,176–178 Infection Influenza virus Lung (iBALT) Mouse Unknown • Germinal centre reactions 30,59,79 • Class-switched plasma cells • Antiviral immunoglobulin generated HCV Liver Human Unknown • B cell clonal expansion 120,121 • Elevated CXCL13 expression systemically and locally MCMV Salivary gland Mouse Unknown • Local AID expression 74 • Ongoing CSR and somatic hypermutation • CXCL13 and BLIMP1 expression Vaccinia virus Ankara Lung (iBALT) Mouse Unknown Local priming of T cell responses 60 Intestinal microbiota Intestine • Human Unknown • Development is LTi cell independent 33,179 • Mouse • Presence of activated mucosal γδ T cells Helicobacter pylori Gastric mucosa • Human Unknown • CXCR5–CXCL13 dependent 76,180 • Mouse • Local T cell priming and support of

TH17 cells • IgG and IgA immune response Helicobacter hepaticus Liver Mouse Unknown CCL21 and CXCL13 expression 181 Borrelia burgdorferi Synovium Human Unknown • Immunoglobulin V regions show 182 (which causes chronic evidence of CSR Lyme arthritis) • Antigen-driven selection

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Table 1 (cont.) | Clinical and experimental conditions that feature ELSs Condition Location Species Antigen recognized by Other known functions of ELSs in Refs cells in ELSs disease Infection (cont.)

Mycobacterium Lung (iBALT) • Human Unknown • Priming of antigen-specific TH1 cells 38,75, tuberculosis • Mouse • CCL19 and CCL21 expression 78,118 • Accumulation of CXCR5+CD4+

TFH-like cells Repeated intranasal LPS Lung (iBALT) Mouse Unknown • LTi cell independent 30 challenge (neonatal) • IL‑17 expression (potential link to

TH17 cells) • CXCL13 expression Renal failure and transplantation Allograft transplants For example, • Human Unknown Germinal centre reactions resulting in 95,96 kidney, lungs and • Mouse anti-HLA-producing plasma cells and heart memory B cells Peritoneal dialysis Peritoneal omental • Human Unknown • Plasma cell responses to 183–185 milky spots • Mouse T cell-dependent antigen delivered intraperitoneally • Dependent on LT and CXCL13 Environmental, degenerative and idiopathic conditions Cigarette smoke Lung Mouse Unknown • Dependent on LTαβ and LTβR 173 • CXCL13 and CCL19 expression Diesel exhaust particles Lung (iBALT) Mouse Unknown Unknown 186 Metal prosthetic joints Joint Human Unknown Formation may involve cytotoxic and/or 187 (periprosthetic delayed hypersensitivity response to soft tissue) cobalt-chrome wear particles Pristane adjuvant Peritoneal Mouse Unknown Lipogranuloma formation with 165 membrane homeostatic chemokine expression IPAH Perivascular lung Human Autoantibodies against • Expression of IL‑7, LTαβ, CCL19, 188 tissue vascular wall components CCL20, CCL21 and CXCL13 + generated • IL‑21 TFH cells and FDCs • AID expression suggests ongoing CSR 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.

monocytic cells, macrophages and dendritic cells can form In this Review, we explore the molecular and genetic discrete clusters within the inflamed tissue. These regions basis for the formation of ELSs during infection, inflam- can exist as simple lymphoid aggregates or as more sophis- mation, autoimmune disease and cancer, and we discuss ticated structures that histologically resemble secondary the clinical significance of ELSs in terms of prognosis lymphoid organs (SLOs)7,8 (TABLE 1). The spatial organiza- and response to current biological therapies. With tion of leukocytes into defined, compartmentalized B cell- insight from both experimental animal models and Secondary lymphoid organs rich and T cell-rich zones is termed lymphoid neogenesis. human disease settings, we also consider the current (SLOs). In contrast to primary These structures direct various B cell and T cell responses, state of ELS-directed therapies and the rationale for tar- (central) lymphoid organs, where lymphocytes are including the induction of effector functions, antibody geting alternative molecular pathways that are associated generated from immature generation, affinity maturation, class switching and clonal with ELS formation. progenitor cells, SLOs — that expansion. As a consequence, they are referred to as ectopic is, the spleen, Peyer’s patches lymphoid-like structures (ELSs) or tertiary lymphoid organs ELS development and function and lymph nodes — maintain 7,8 mature naive lymphocytes and (TLOs) . In certain autoimmune conditions, patients who Although ELSs display an architecture that resembles the are sites of lymphocyte have ELSs in the inflamed tissue often respond poorly to follicular compartments that are typically seen in SLOs activation by antigen. standard biological therapy and thus remain a challeng- (BOX 1), their organization ranges from simple aggregates ing treatment group9. However, in cancer (for example, of B cells and T cells through to highly ordered structures. Lymphoid neogenesis solid tumours such as colorectal carcinoma) the presence Much of our understanding of ELS formation originates The de novo development and encapsulated cellular organization of lymphoid of tumour-associated ELSs correlates with a better prog- from findings that describe the generation of tissue into distinct anatomical nosis and they may coordinate endogenous antitumour SLOs (as reviewed in REF. 11). Despite structural differ- and functional compartments. immune responses that improve patient survival10. ences 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 Ectopic lymphoid-like support the maintenance of germinal centres and promote the generation of both long-lived plasma cells and memory structures B cells (see the figure). In this respect, TFH cells contribute to the control of antibody production and the fine-tuning of (ELSs). Highly organized mechanisms such as class switching, affinity maturation and somatic hypermutation. lymphoid aggregates that form in tissue sites that are not T cell zone B cell zone typically associated with Germinal centre lymphoid neogenesis. Class switching, Encapsulated SLOs aﬃnity maturation Organized secondary lymphoid and somatic organs (SLOs) that are encased hypermutation in a connective tissue capsule MHC CXCR5 that contains blood vessels. class II

Lymphoid tissue inducer TCR cells T cell T cell B cell (LTi cells). These cells are CXCL13- FH Memory Peptide B cell present in developing lymph DC dependent nodes, Peyer’s patches and nasopharynx-associated lymphoid tissue (NALT) and they are required for the development of these Plasmablast Plasma cell lymphoid organs. The inductive capacity of LTi cells for the generation of Peyer’s patches and NALT has been shown by 14Nature Reviews | Immunology adoptive transfer and it is that control the initial development, cellular composi- (also known as L-selectin) . Such findings indicate that generally assumed that they tion and functional maintenance of these structures are the propagation of ELSs within inflamed tissue is driven have a similar function in the shared. However, ELS formation is distinct from the pre- by communication between local stromal cells, tissue- formation of lymph nodes. programmed ontogenic processes that are associated with specific resident mononuclear cells and infiltrating 15,16 T follicular helper cells secondary lymphoid organogenesis and it does not occur immune cells . Although the actual cells that are in all patients. Consequently, the generation of ELSs in responsible for the positioning of these structures within (TFH cells). Antigen-experienced CD4+ T cells that are present inflamed tissues — as opposed to a more typical diffuse the inflamed tissue remain undefined, several new candi- in B cell-rich regions of pattern of inflammatory infiltrate — must be governed by dates have recently been proposed. These include lymphoid structurally organized a defined set of inflammatory signals. The mechanisms tissue inducer cells (LTi cells), IL‑17‑secreting CD4+ T cell lymphoid aggregates or T follicular helper cells (FIG. 1) organs. that trigger these events are poorly defined. populations and (TFH cells) . The accumulation of CD4+CD3−CD45+ LTi cells at Lymphoid tissue organizer Initiation of ectopic lymphoid neogenesis. Various sites of lymph node development is an early event in cells transgenic mouse models have emphasized the role of secondary lymphoid organogenesis17–19. This is coordin These cells are of mesenchymal origin that are inflammatory cytokines in lymphoid neogenesis. For ated by the expression of CXCL13, receptor activator of activated by lymphoid cells example, mice that are deficient in lymphotoxin‑α (LTα; NF‑κB ligand (RANKL; also known as TNFSF11) and through lymphotoxin‑β which is encoded by Lta) completely lack peripheral lym- IL‑7, which control the recruitment, survival and activa- receptor signalling to express phoid organs12. Conversely, tissue-specific expression of tion of LTi cells that express CXCR5 and IL-7 receptor adhesion molecules and a transgene that encodes Lta in the kidney and pancreas (IL-7R; also known as CD127)20. Central to this process is chemokines that regulate lymphoid tissue development. caused severe chronic inflammation with the accompa- the interplay between LTi cells and stromal mesenchymal nying formation of ELSs that were capable of promoting cells — known as lymphoid tissue organizer cells — which High endothelial venules antigen-specific responses and antibody class switching13. act as the anchor for lymph node development11. The (HEVs). These are specialized Moreover, the overexpression of both Lta and Ltb (which release of IL‑7 and RANKL by activated lymphoid tis- venules that occur in secondary lymphoid organs, encodes LTβ) results in more prominent ELS forma- sue organizer cells promotes the expression of LTα1β2 by 14 except the spleen. HEVs allow tion compared with when only Lta is overexpressed . LTi cells, which, in turn, engages the LTβ receptor (LTβR) the continuous transmigration Thus, in combination with LTβ, LTα enhances ectopic on lymphoid tissue organizer cells that also express vas- of lymphocytes as a lymphoid neogenesis14. The activity of lymphotoxin is cular cell adhesion molecule 1 (VCAM1) and inter consequence of the associated with increased expression of the homeostatic cellular adhesion molecule 1 (ICAM1). This promotes constitutive expression of adhesion molecules and chemokines CXC-chemokine ligand 13 (CXCL13), homeostatic chemokine release and vascularization by chemokines at their luminal CC‑chemokine ligand 19 (CCL19) and CCL21, and high endothelial venules (HEVs), which also contribute to surface. the increased infiltration of T cells that express CD62L lymphoid neogenesis11,15,20,21.

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a Initiation of ectopic lymphoid b Cell recruitment to c Maintenance of ELSs tissue neogenesis inﬂamed site

Inﬂamed tissue IL-7R and RANK IL-7 signalling increase HEV T 1 cell T 2 cell T 17 cell LTα1β2 expression H H H RANKL ICAM1 RANK IL-7Rα VCAM1 CD62L ELS Development of TFH-like cells PD1 ICOS Accumulation RORγt B cell of LTi cells BCL6 CXCR5 T cell LTα1β2 MAF CCL21 and CD4+ LTi cell LTo cell T -like cell CXCL13 FDC FH LTβR signalling TH17 cell promotes chemokine LTi cell release and HEV development

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 types have been implicated as potential initiators of ectopic lymphoid-like structure (ELS) formation.Nature Reviews Although | Immunology 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.

Although stromal lymphoid tissue organizer cells and adoptive transfer of adult LTi cells into Cxcr5−/− mice LTi cells are important in secondary lymphoid organo- induces the formation of intestinal lymphoid tissues19. genesis, their involvement in ELS formation is less clear. Ectopic lymphoid tissues that develop in response to However, stromal tissue cells may acquire lymphoid transgenic overexpression of the Il7 gene also require tissue organizer cell-like properties in ELSs21–23. For exam- LTi cells17. Adult LTi cells express the transcrip- ple, synovial fibroblasts from the joints of patients with tional regulator retinoic acid receptor-related orphan Innate lymphoid cell rheumatoid arthritis release homeostatic chemokines and receptor‑γt (RORγt) and can produce IL‑17, which are (ILC). A type of innate cytokines that may contribute to ectopic lymphoid neo- characteristics of group 3 ILCs18,19. This phenotype sug- immune cell that is lymphoid 2,24–26 in morphology and genesis within the inflamed synovium . Moreover, gests an ancestral relationship between adult LTi cells + 18,27 developmental origin but that gene expression profiling of synovial tissue from patients and IL‑17‑producing CD4 T helper (TH17) cells . lacks properties of adaptive with rheumatoid arthritis has identified an IL7 signature Furthermore, various studies have now linked IL‑17 B cells and T cells, such as 26 in ELS-associated synovitis . and TH17 cells with ectopic lymphoid neogenesis, where recombined antigen-specific The recent discovery of innate adult LTi cells that they have a role in chronic allograft rejection, experi- receptors. These cells regulate innate lymphoid cell 18,19 immunity, tissue homeostasis are part of the (ILC) family raises mental autoimmune encephalomyelitis (EAE) and the and inflammation in response the possibility that these cells also contribute to inflam- development of inducible bronchus-associated lymphoid to cytokine stimulation. mation-associated ELS development. For example, the tissue (iBALT)28–30. For example, ELS formation in the

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central nervous system is associated with TH17 cells Maintenance of ELSs within tissues. Considerable atten- that express the lymphoid tissue-associated glyco- tion has been given to the development of lymphoid protein podoplanin (also known as gp38 in mice)29. structures, however, the presence of ELSs is often a tran- Podoplanin-deficient mice display defective develop- sient feature of inflamed tissue. Thus, the mechanisms ment of lymph nodes and germinal centre structures, that control the maintenance of these aggregates during and podoplanin expression by fibroblastic reticular disease may have greater clinical significance. Recent

cells is associated with the development of the micro- studies have linked TFH cells — or markers of TFH cell architecture of the T cell zones29,31. Thus, podoplanin activity — to the formation, maintenance and function expression is a defining feature of tissues that display of ELSs29,38–40.

active lymphoid neogenesis. Although the precise TFH cells promote B cell activities and high-affinity function of podoplanin is unclear, it may support the antibody generation in germinal centres41,42. Recent stud- 29,30 retention of TH17 cells within these sites . In addition ies of T cell plasticity in mice suggest that TH1, TH2 and

to these studies, various reports have also noted roles TH17 cells may also acquire TFH cell-like characteristics for B cells and TNF-secreting F4/80+ myeloid cells in and effector functions during their differentiation29,43–47. 32,33 ELS generation . The formation of ELSs in different For example, TH17 cells can display TFH cell-like charac- tissues and in response to different forms of immuno teristics during their differentiation, including the secre-

logical activation suggests that the mechanisms tion of IL‑21 and the expression of TFH cell-associated driving ELS expansion are complex. Inherent simi- molecules, such as signal transducer and activator of larities in effector functions or cellular plasticity may transcription 3 (STAT3), interferon-regulatory fac- render various cell types able to promote tissue‑specific tor 4 (IRF4), MAF and inducible T cell co‑stimulator ELS development. (ICOS)48–53. These observations may help to explain the

ability of TH17 cells to promote ectopic lymphoid neo- Organization of cells within ELSs. Inflamed tissues genesis28–30 and may also explain the regulation of ELS

displaying ELS-associated pathology have increased activity in diseases that are associated with TH1, TH2 or

expression of homeostatic chemokines that govern TH17 cell responses. Memory and effector TH cells that

the cellular composition and functional properties of lack typical TFH cell-like features are, nevertheless, also ELSs34–37. These include CXCL12, CXCL13, CCL19 capable of providing B cell help as they express CXCL13, and CCL21. In such tissues, cellular communica- IL‑4, IL‑21 and CD40L54–56. Consequently, both positive tion between local stromal cells, tissue-specific resi- and negative regulators of effector T cell responses may dent mononuclear cells and infiltrating immune cells influence the maintenance of ELS activity (FIG. 2). propagates the development of ELSs and enables them The CCR7‑dependent recruitment of dendritic cells to function in a similar manner to germinal centres to developing ELSs represents an important homeo- (BOX 1). Such interactions affect leukocyte trafficking static event that drives the initial propagation of adap- and angiogenesis but are also instrumental in govern- tive immune responses within these sites. For exam- ing the organization of cells within these structures. For ple, infiltrating CD4+ T cells form tight clusters with example, CXCL13 and CCL21 control the segregation dendritic cells and this promotes T cell proliferation57. of B cells and T cells in ELSs34,35. By contrast, CCL19 As ELSs mature, dendritic cells within these structures and CXCL12 promote lymphocyte infiltration and the continue to support the efficient priming of T cell positioning of dendritic cells, B cells and plasma cells responses through antigen presentation and they con- within these aggregates34 (FIG. 2). These specialized tribute to class-switch recombination, antibody genera- properties probably reflect differences in the ability tion and the formation of new lymphatic vessels58–60. of individual homeostatic chemokines to promote the The importance of dendritic cells in ELSs is best illus- expression of LTα and LTβ by B cells and T cells34. For trated by studies of mucosal immunity following viral example, transgenic expression of Cxcl13 in pancre- infection. The depletion of dendritic cells during viral

atic tissue promotes the LTα1β2-mediated formation of lung infection leads to impaired germinal centre reac- ELSs that display defined lymphoid zones, HEVs and tions and a disruption of ELS architecture59,60. The stromal reticulum35. Thus, homeostatic chemokines sustained activation of dendritic cells within ELSs is affect the size, cellular composition and organization therefore required for both their formation and their Germinal centre of ELSs. Such features affect the functional properties of functional maintenance. Located in peripheral lymphoid tissues (for example, the spleen ELSs and their impact on pathology. or lymph nodes), these are However, the contribution of individual homeostatic The function of ELSs as germinal centres. There is sites in which B cells proliferate chemokines to ELS formation may also depend on the conclusive evidence that ELSs not only recapitulate and clones that produce anatomical site or ongoing disease processes. Although the cellular, molecular and structural organization antigen-specific antibodies of higher affinity are selected. transgenic expression of Ccl21 in the pancreas drives of SLOs but that they can also support the function of pancreatic lymphoid neogenesis, the ectopic expres- germinal centres. In the germinal centres of SLOs, Angiogenesis sion of Ccl21 in the skin does not36,37. Whether these B cells undergo affinity maturation and differentiation The development of differences reflect a hierarchy of chemokine-mediated to memory B cells and plasma cells via antigen-driven new blood vessels from outcomes that affects the degree of architectural organ- selection61 (BOX 1). This process includes antibody existing blood vessels. It is frequently associated with ization displayed by ELSs or whether they reflect spe- fine-tuning through somatic hypermutation and class tumour development and cific characteristics that are associated with ‘permissive’ switching, both of which affect antigen recognition and inflammation. versus ‘non-permissive’ tissues remains unknown. the effector capacity of the antibody62–65. Consistent with

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Classical lymphoid neogenesis Putative regulators of ELS neogenesis

Non-inﬂamed tissue Inﬂamed tissue

LTi cell LTo cell LTi cell LTo cell Initiators Initiators Initiators • IL-7 • IL-7 • IFNs • IL-17 • LTα1β2 • LTα1β2 • IL-4 • IL-21 • RANKL • RANKL • IL-5 • TNF

T cell

FDC

B cell

Propagators Propagators Propagators Inhibitors • CCL19 • CCL19 • IL-6 • IL-2 • CCL21 • CCL21 • IL-17 • IL-27 • CXCL12 • CXCL12 • IL-21 • CXCL13 • CXCL13 Figure 2 | Cytokines and chemokine regulate ELS formation and function. The inducible formation of ectopic lymphoid structures (ELSs) mimics the ontogenic process of secondary lymphoid organ (SLO) development,Nature Reviews whereby | Immunology 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 29,30,38 (TFH)-like cells are associated with ELS development in the central nervous system and the lungs . Various TH cell 29,43–47 subsets can also acquire TFH cell-like characteristics and effector functions , 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.

the role of ELSs as functional ectopic germinal centres, as reviewed below, although the final outcome of lym- activation-induced cytidine deaminase (AID) is expressed phoid neogenesis is generally protective in the context at the mRNA and protein level within these structures, of infection and cancer, it is often deleterious in the and studies in patients and animal models support the setting of autoimmunity and graft rejection. involvement of AID in autoimmunity66–69, infection70 and allograft rejection28. For example, AID controls ELSs in protective immunity and disease local antibody affinity maturation (including somatic ELSs as sites of anti-pathogen immune responses. Infec- hypermutation),b Permissive as evidenced tissue by a restricted profile tion withNon-permissive bacteria (such tissue as Mycobacterium tuberculosis of variable (V)-gene repertoire usage, highly mutated and Helicobacter pylori) and viruses — such as influ- Skin V regions and thePancreas oligoclonal diversification of infiltrat- enza virus, hepatitis C virus (HCV) and several sialo- ing B cells and plasma cells68,71–73. Active class switching tropic viruses — has been associated with the formation is also confirmed by the detection of Iγ–Cμ and Iα–Cμ of ELSs in animal models39,59,74,75 and in humans76–78. Activation-induced cytidine circular transcripts within ELSs, which marks ongoing These ELSs resemble highly organized SLOs. Most deaminase class-switch recombination from IgM to IgG and IgA, remarkably, in mice, infection-triggered lymphoid neo- (AID). An enzyme that is respectively67,68,74. Thus, ELSs in inflamed tissues retain genesis seems to be preferentially induced at permis- required for two crucial events Ectopic CCL21-expression ELS formation Ectopic CCL21-expression No ELS formation in the germinal centre — the necessary molecular machinery to support in situ sive mucosal sites, and results in the de novo formation somatic hypermutation and antibody diversification, isotype switching, B cell of iBALT and of lymphoid tissues associated with the class-switch recombination. differentiation andLung oligoclonal expansion. However, salivary glands79,80.

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Infection with Mycobacterium Inducible BALT tuberculosis or inﬂuenza virus formation REVIEWS

Consistent with their immunological functions, differentiation of the B cells into high-affinity autoreactive infection-associated mucosal ELSs can mount protec- plasma cells that release disease-specific autoantibodies, tive immune responses in situ. Examples of this include such as anti-citrullinated protein antibodies (ACPAs) in influenza virus-induced and M. tuberculosis-induced rheumatoid arthritis67, and antibodies against the ribo lymphoid neogenesis in the lungs of rodents. Following nucleoproteins Ro and La (also known as Sjögren’s syn- influenza virus infection of the upper respiratory tract, drome antigens A and B) in Sjögren’s syndrome88, against mice develop functional ELSs79 that are maintained by thyroglobulin and thyroperoxidase in Hashimoto’s lymphoid tissue-associated chemokines (for example, thyroiditis85, and against the nicotinic acetylcholine CXCL13 and CCL21) and LTβ, which are produced by receptor in patients with myasthenia gravis84. infiltrating CD11b+CD11c+ dendritic cells59. These ELSs Although the mechanisms that are responsible for promote the in situ differentiation of antiviral plasma the preferential accumulation of autoreactive B cells cells that are specific for the nucleoprotein of influenza in ELSs are not fully understood, a direct role has been virus59. Notably, in LTα-deficient mice — in which SLOs proposed for Epstein–Barr virus (EBV) in the develop- do not develop — reconstitution with Lta+/+ bone marrow ment of autoimmunity. EBV is a γ‑herpesvirus that is allows for ELS development in response to influenza retained throughout the life of an infected individual and virus infection81. These bronchial ELSs can maintain that promotes the survival and proliferation of B cells89,90. antiviral immunity and memory recall responses in Emerging evidence suggests that ectopic follicles in the absence of SLOs81. A role of ELSs in antimicrobial the target organs of patients with multiple sclerosis, immunity is further illustrated by their involvement in myasthenia gravis, rheumatoid arthritis and Sjögren’s M. tuberculosis infection, where they promote granuloma syndrome frequently harbour latent EBV infection69,91–93. formation and prevent the dissemination of infection38,75. In these sites, EBV-transformed B cells and plasma cells Although these studies demonstrate a non-redundant display autoreactivity to disease-specific autoantigens, role for ELSs in certain infections, they also indicate that such as citrullinated fibrinogen in rheumatoid arthritis92 pathogen recognition by innate-sensing mechanisms and the ribonucleoprotein Ro in Sjögren’s syndrome93. may contribute to the formation of these structures. Autoreactive EBV-infected B cells are therefore predicted Overall, these data suggest an evolutionary role of to transit from the peripheral compartment into ectopic ELSs at sites of infection where they coordinate protective germinal centres where they undergo differentiation to immunity in addition to, and often independently from, high-affinity autoreactive plasma cells. Thus, niches of SLOs. However, a failure to eradicate a pathogen has EBV colonization within ELSs might be considered as a considerable clinical implications and can lead to the hallmark of organ-specific autoimmunity. development of autoimmunity (for example, in chronic EBV infection may also explain another remark- HCV infection) and lymphoma (for example, in H. pylori- able feature of ELSs, which is their ability to persist associated chronic gastritis). Thus, ectopic lymphoid in an activated state for several weeks in the absence neogenesis in response to microbial challenge influ- of recirculating immune cells from the periphery. ences the natural history and clinical course of chronic This is best demonstrated in chimeric severe combined infection and associated complications. immunodeficiency mice that are engrafted with synovial or thymic tissue from patients with rheumatoid arthri- ELSs in autoimmunity and the emerging role of Epstein– tis or myasthenia gravis, respectively. ELSs within the Barr virus. The presence of ELSs with the appearance transplanted tissue maintain their follicular organization of fully functional ectopic germinal centres has long and autoantibody generation despite both the absence of Granuloma been described in the inflamed target organs or tissues ‘new’ cells infiltrating the grafts and the impaired host 67,94 A chronic inflammatory tissue of patients who are affected by autoimmune diseases, immune response . This is of crucial importance, as response that occurs at the including the synovial tissue in rheumatoid arthritis (as ELS neutralization is probably fundamental to avoiding site of implantation of certain reviewed in REF. 82), the meninges in multiple sclerosis69, the re‑emergence of autoreactive clones that are capable foreign bodies and, in particular, at sites of long-term the salivary glands in Sjögren’s syndrome (as reviewed in of driving disease relapse or resistance to therapy. microbial persistence. REF. 83), the thymus in myasthenia gravis (as reviewed Granulomas are typically in REF. 84) and the thyroid gland in Hashimoto’s thyroidi- ELSs in transplantation. ELSs have been observed in well-organized structures that tis85. In these clinical disorders, ELSs develop in response almost all types of human grafts that have been removed are composed of T cells and to disease-specific autoantigens, which also ensure owing to chronic rejection, including kidneys, lungs and macrophages, some of which 95 fuse to form giant cells. the long-term maintenance of these structures within the hearts . Transplant-associated ELS formation seems to inflamed tissue. recapitulate the molecular pathways that are triggered Severe combined The presence of ELSs in autoimmune conditions during the ontogeny of SLOs. These ELSs harbour immunodeficiency mice perpetuates autoimmunity towards disease-specific ectopic germinal centres in which naive B cells differ (SCID mice). A strain of mice that possesses a genetic antigens. In this respect, many of the regulatory mech- entiate into anti-HLA-producing plasma cells and 96 defect in DNA recombination anisms that govern tolerance within SLOs are not seen memory B cells . However, ELS formation in allografts that leads to severe in autoimmune disease-associated ELSs. For example, in differs from that of canonical SLOs in a number of ways: immunodeficiency. SCID mice SLOs, autoantigen-binding B cells may be excluded from first, the considerable amounts of TH17 cell-associated lack B cells and T cells, and entering germinal centres and they lack responsiveness to cytokines and growth factors — such as B cell-activating they are incompetent at (REF. 86) rejecting tissue grafts from CXCL13 owing to a downregulation of CXCR5 . factor (BAFF; also known as TNFSF13B) — that facili- allogeneic and xenogeneic However, autoimmune disease-associated ELSs permit tate autoreactive B cell survival; second, the continuous sources. the entry of autoreactive B cells87. This allows for the release of alloantigens from the injured tissue that are

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trapped locally by defective lymphatic drainage; and Considering the genetic influence first, evidence is third, the apparent defect of local immunoregulatory beginning to emerge from various genetic studies that mechanisms. As a consequence, there is an excessive have found associations between specific pathways that immune response within the ELSs that contributes to are involved in ELS formation and/or function and allograft rejection95. However, two recent publications susceptibility to autoimmune diseases. For example, four in distinct experimental models (renal and cardiac single nucleotide polymorphisms (SNPs) have been iden- allografts) have reported that the presence of ELSs tified in the IL2–IL21 locus at chromosome region 4q27 promotes allograft tolerance and graft function97,98. On that are associated with several autoimmune diseases106–108 this basis, it is postulated that under certain conditions, and that can influence circulating levels of IL-21 (REF. 109). ELSs may amplify beneficial immune responses — Additionally, SNPs in loci that are near to the CXCR5 gene that are associated with B cells and T cells displaying a have been linked with susceptibility to primary biliary regulatory profile — to promote graft tolerance. cirrhosis110, Sjögren’s syndrome111, systemic lupus erythematosus (SLE)112 and multiple sclerosis113. ELSs in cancer. In cancer, ELSs have been documented Although no sub-analysis with stratification of in many tumours, including those that are associated patients for the presence or absence of ELSs has been with lung99,100, colon10,101, breast40,102,103 and germ cell carried out in the above studies, the increased genetic cancers104,105. However, not every patient with a specific susceptibility to autoimmunity that is associated with loci type of cancer develops ELSs and when they do occur, near to both the IL21 and CXCR5 genes corresponds with + their contribution to disease varies considerably. Some the crucial role of IL‑21‑producing CXCR5 TFH cells tumour types are more likely to induce ELS forma- in the formation and function of ELSs. This is also poten- tion than others, which indicates that certain tumours tially of crucial relevance as novel strategies to specifically

provide a microenvironment that is conducive to lym- target TFH cells are being developed for therapeutic use in phoid neogenesis. Given the stark difference between autoimmune conditions (as discussed below). It is likely the immunosuppressive nature of the tumour micro- that the influence of genetic factors will become clearer environment compared with chronic inflammatory as larger scale analyses of stratified disease subsets are foci, it is perhaps surprising that ELSs develop at all in carried out according to pathobiology (for example, the tumours104. Although CXCL13, CCL19 and CCL21 have presence or absence of ELSs). a crucial role in the formation of cancer-associated ELSs, With regard to the environmental factors that influ- LTi cells seem to be dispensable as ELSs can form in their ence ELS formation, diverse host immune responses to absence100,104,105. Permissive tumours probably induce the infection lead to different outcomes. Thus, it is possible production of lymphoid chemokines not only through that in ‘permissive’ patients, an active EBV infection is

the expression of LTα1β2 but also through the expression established in the diseased tissue and may drive ELS for- of pro-inflammatory cytokines — such as TNF, IL‑1β mation through its unique ability to infect and activate and IL‑6 — which contribute to the transcriptional reg- B cells114,115. This hypothesis was recently supported by ulation of CXCL13, CCL19 and CCL21. However, the our own work, in which we found a high frequency of precise stimuli that initiate the development of cancer- EBV-infected B cells and plasma cells in ELS-containing associated ELSs within the immunosuppressive tumour synovia from patients with rheumatoid arthritis92. Nota- microenvironment require further investigation. bly, EBV was not detected in control synovia from patients with osteoarthritis or in patients with rheumatoid arthritis Clinical implications whose synovia showed diffuse infiltration of immune cells As discussed above, ELSs are found in patients with but lacked ELSs92. Similar results were seen in the salivary diverse medical conditions but invariably, in each con- glands of patients with Sjögren’s syndrome93. dition, ELSs form in some patients but not in others (TABLE 1). Therefore, important questions relating to this Is ELS formation pre-determined or a result of persistent phenomenon remain to be answered. For instance, what inflammation? The frequency at which ELSs occur in determines the formation of ELSs in some individuals but patients with chronic inflammation typically varies from not in others? Is ELS development typical of different dis- 20–40%7,82. However, it is unknown whether ELSs are ease subtypes (ab initio) or is ELS formation the inevitable a typical feature of different disease subtypes from the result of persistent inflammation — that is, are ELSs the beginning (ab initio) or whether they form as a result of cause or the consequence of chronicity? To what extent persistent inflammation. The reason this this remains do ELSs contribute to ongoing inflammation and tissue unknown is twofold: first, in humans, clinical presenta- damage (disease prognosis)? In this section, we address tion is often delayed and it is thus impossible to precisely these questions in turn by providing recent data from the determine the early events in the pathological process; literature, as well as from our own personal experience. and second, there are an insufficient number of studies (and an insufficient number of large cohort studies) in What governs ELS formation in some individuals which serial biopsies have been taken to establish the but not in others? This question still remains mostly ‘pre-determined’ versus the ‘evolutionary’ nature of unanswered but it is likely that genetic and/or environ- ELSs. Nonetheless, data is beginning to emerge from mental factors shape the inflammatory response to give a cohort of 300 patients with rheumatoid arthritis who rise to diverse structural outcomes within individual had shown symptoms for less than 12 months; biopsies tissue microenvironments. were taken at the time of disease presentation and 70% of

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individuals were also biopsied 6 months post-treatment116. hypermutation, whereby dysregulated expression of AID Approximately 40% of patients displayed histological leads to mutations in the regulatory and coding sequences evidence of ELSs before receiving any disease-modifying of genes that regulate B cell survival and proliferation therapy. Thus, ELSs seem to be present ab initio and (such as PAX5 and MYC)123. Interestingly, AID expres- to define a specific disease subset from the beginning. sion in gastric and salivary gland MALT lymphomas is However, in another early arthritis cohort of 93 patients confined to ELSs and is not found in malignant marginal with only 17 biopsy repeats at 6 months, ELSs have been zone B cells66,123, which suggests that lymphomagenesis reported as being related to the degree of inflammation is dependent on sustained antigen stimulation within and not related to specific disease subtypes117. No serial ELSs. Accordingly, gastric MALT lymphomas are cru- biopsy data is available from other diseases and, there- cially dependent on H. pylori-specific T cells124 and the fore, further work is required in this area to establish eradication of H. pylori results in both ELS resolution and the precise ontogeny of ELSs in the context of chronic tumour regression125. Although in this context lympho inflammation. magenesis is ultimately driven by chronic infection, a striking and unique feature of MALT lymphomas is that To what extent do ELSs control disease progression and malignant B cells frequently originate from precursors outcome? From the above discussion, it is clear that ELSs that express an autoreactive B cell receptor with homol- are dynamic structures, the main function of which is to ogy to rheumatoid factor126. Thus, autoimmunity and potentiate the immune response at sites of disease. The MALT lymphomagenesis can represent a continuum of clinical presentation of ELSs may therefore influence dis- the same antigen-driven response in which ELSs have a ease severity, the rate of disease progression and clinical central role. outcome. Ultimately, this will depend on the ability of the In chronic inflammatory and autoimmune con- host to clear triggering antigens, as well as the stage and ditions, the B cell-rich inflammatory infiltrate that the nature of the disease in question. In the subsequent is typically seen in ELSs is associated with a strong paragraphs, we provide some examples of the influence potential for local immune activation and cellular dif- of ELSs on the progression and outcome of infection, ferentiation. This promotes autoantibody production, autoimmune conditions, transplantation and cancer. complement activation and pro-inflammatory cytokine On the basis of the evidence from animal models release, which drives the inflammatory cascade, auto showing a protective role of mucosal ELSs in pathogen immunity and tissue destruction. For example, studies control, it is anticipated that the presence of ELSs in in rheumatoid arthritis have reported worse disease out- infected humans could alter the natural history and comes in patients with ELSs116. In these cases, increased clinical manifestations of chronic infections. Accord- joint destruction is associated with the production of ingly, in M. tuberculosis infection, ELS formation in the pro-inflammatory cytokines127, as well as osteoclast- peripheral rim of lung granulomas is typically associated activating molecules such as RANKL128 and the with the latent, asymptomatic form of tuberculosis118. RANKL-inducing molecule TNF-related weak inducer Conversely, sites of active infection that have cavitary of apoptosis (TWEAK; also known as TNFSF12)129. In tuberculous lesions lack B cell follicles118. Thus, ELSs autoimmune thyroiditis, most ELS B cells recognize the coordinate the local host response to pulmonary tuber- autoantigens thyroglobulin and thyroperoxidase, and culosis118. ELSs probably exert similar mechanisms of this leads to glandular destruction85. In SLE, a restricted pathogen containment in other chronic infections, such repertoire of isotype-switched antibodies are deposited as H. pylori and HCV76,77. However, it is now clear that in the glomerular basement membrane of the kidneys in the absence of pathogen eradication, ELSs also act as with consequent organ damage130. Finally, the presence a double-edged sword by contributing to autoimmunity of ELSs in the inflamed meninges of patients with pro- and lymphomagenesis. Although tuberculosis has long gressive multiple sclerosis can be determined through been associated with the development of various auto analyses of B cell markers, and the occurrence of ELSs is immune phenomena119, the association with ELSs has associated with a gradient of cortical neurodegeneration not been investigated. Conversely, in chronic HCV- and a more aggressive course of disease131. related hepatitis, elevated CXCL13 levels in liver biopsies ELSs are thought to drive chronic transplant rejection identify patients that develop mixed cryoglobulinaemia120. by enhancing intra-graft allogeneic responses, as grafts Together with the clonal analysis of intrahepatic B cells in which the ELSs are most functional have a shorter in portal tracts121, these data indicate that intraportal life expectancy28,96. However, the validity of this conclu- lymphoid follicles contribute to the evolution from sion has been questioned by the fact that, by definition, Cryoglobulinaemia polyclonal to oligoclonal to monoclonal autoreactive only de‑transplanted, failed grafts were included in the An autoimmune, extra-hepatic B cell activation that occurs as a result of chronic HCV analysis. In addition, as discussed above, there is evi- manifestation that is associated with certain viral infection. Pathogen-driven chronic B cell activation dence from experimental transplant models that regula- infections and chronic within ELSs is also associated with lymphoproliferative tory B cells and ELSs may govern graft tolerance. Further inflammatory conditions, disorders, as evident in HCV-associated B cell lympho work is therefore required to clarify the contribution of whereby antigen–antibody proliferation122 and H. pylori-driven gastric mucosa- ELSs to graft rejection versus survival. complexes are deposited in associated lymphoid tissue (MALT) B cell lymphomas76. As discussed above, different cancers are more or capillaries and arterioles (and occasionally small arteries) The development of gastric (and salivary gland) MALT less immunogenic and hence, are more or less prone to causing vasculitis, renal lymphomas has been linked to the genetic instability induce ELSs. Analyses of the cytokine and chemokine disease, arthralgias or arthritis. that is associated with the process of aberrant somatic gene signatures, the number of tumour-infiltrating

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lymphocytes and the level of ELS organization have biological interventions that promote the clearance or been used to stratify patients and types of cancer as inhibition of autoreactive lymphocytes have the poten- ‘immune response positive’ and ‘immune response nega- tial to disrupt ELS involvement and restrict deleteri- tive’. Although these classification criteria are not purely ous adaptive immune responses. In this regard, under- based on ELSs, there is a general consensus that patients standing the impact of TNF inhibitors on synovial ELSs who have immune-response-positive tumours (for exam- is of major importance. In a recent study, patients with ple, breast cancer tumours) have a better prognosis than rheumatoid arthritis who had synovial ELSs displayed those with immune-response-negative tumours102,132–134. an inferior response to TNF inhibitors compared with Similar results have been described in colon cancer, patients who did not have synovial ELSs9. In addition, where immune response positivity was associated with the presence of ELSs in synovial tissue before treatment the increased survival of patients independently of was reported to be an independent negative predictor tumour stage, previous treatment or microsatellite insta- of the response to TNF inhibitors9. Furthermore, high bility10. As expected, CXCL13 and CCL19 expression protein-level expression of IL‑7R, which is normally was also associated with a good prognosis10. Finally, gene associated with ELSs, was reported to be associated expression profiling of immune-response-positive and with a poor response to TNF inhibitors141. Therefore, immune-response-negative human melanomas identi- the development of selective ELS-targeted therapies fied CXCL13 and CXCL8 as components of a discrete has attracted marked interest both from the scientific group of 12 genes that were found to be diagnostic of community and from industry. immune response status135. In another study, the mean Currently licensed therapies for inflammatory dis- survival of patients with melanoma was 55 weeks in eases, including CD20‑specific (for example, rituxi- the immune-response-positive group compared with mab) and IL‑6R‑specific (for example, tocilizumab) 18 weeks in the immune‑response-negative group136. monoclonal antibodies, oral Janus kinase inhibi- In summary, ELS formation in peripheral tissues tors (for example, tofacitinib) and T cell activation seems to follow normal pathophysiological programmes blockers (for example, abatacept) probably target in response to the need to increase local immune processes that are associated with ELS activities4. responses and clear exogenous antigens, alloantigens However, these drugs also possess broader modes of and/or autoantigens. When such effector programmes action3–5. Given the prominent role of lymphotoxin are successful, they lead to improvements in disease and homeostatic chemokines in ELS formation, initial pathology and a better prognosis, whereas their failure approaches targeted this axis. Pharmacological inhibi- or ineffectiveness leads to persistent inflammation, tis- tion of LTβ using an LTβR–immunoglobulin fusion sue damage and worse outcomes. This bivalent function- protein resulted in disease amelioration in animal ality has substantial clinical implications when it comes models of arthritis142, autoimmune sialoadenitis143 and to targeting ELSs for therapy. type 1 diabetes144. Although, baminercept (which is a humanised LTβR–IgG1 fusion protein) failed to show ELSs as therapeutic targets clinical efficacy in a Phase II clinical trial in patients The modulation of ELSs for therapeutic purposes has with rheumatoid arthritis, its utility in Sjögren’s attracted marked interest both from the scientific com- syndrome is currently under investigation (TABLE 2). munity and from industry. On the basis of the func- Recently, a new LTα-specific monoclonal antibody tional and clinical data discussed above, it would be has completed a Phase I clinical trial in patients with desirable to potentiate the activity of ELSs in infection rheumatoid arthritis145 but the results of the Phase II and cancer but to inhibit their activity in the context of study have not yet been published. It is noteworthy transplantation and chronic inflammatory conditions. that none of the above studies have stratified patients Current cancer trials are testing whether ELS-related for the presence of ELSs in the disease-affected tissue, pathways can be exploited to enhance antitumour which would probably influence the reported extent immunity. Although cytokine and chemokine admin- of clinical responses. However, the baminercept trial istration, and tumour antigen vaccination represent in Sjögren’s syndrome includes a salivary gland biopsy promising therapeutic approaches to target ELSs, the sub-study, which will inform on ELS modulation after long-term efficacy of these strategies remains debat- treatment. able137,138. Similar vaccination approaches are also being Blockade of CXCL13, CCL19 and CCL21 or their considered as adjunct therapies that promote local receptors CXCR5 and CCR7 is another potential thera- antimicrobial immunity in the lungs139. peutic strategy that has shown some promise in animal By contrast, there is a strong rationale for therapeu- models. CXCL13 blockade ameliorated collagen- tic inhibition of ELSs in graft rejection, and in chronic induced arthritis146 and autoimmune sialoadenitis147 but inflammatory and autoimmune conditions. In a study not autoimmune diabetes148. Of note, although CXCL13 of patients showing chronic renal allograft rejection, inhibition altered the structure of ELSs, it did not influ- treatment with anti‑CD20 (rituximab) failed to cause ence their function or the incidence of diabetes148. To regression of ELSs despite the successful depletion of date, no developmental compound targeting this path- B cells from the peripheral blood140. This suggests that way has been tested in human clinical trials. However, the local inflammatory microenvironment in ELSs may there is evidence that blocking immune cell recircula- facilitate B cell survival and allow evasion of rituximab- tion to ELSs in the context of autoimmune disease is a mediated depletion140. In the context of autoimmunity, 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.

Fingolimod (also known as FTY720) — which signalling that is mediated by ICOS–ICOSL and 48,153 blocks the egress of lymphocytes from SLOs via func- CD40–CD40L is also required for TH17 cell survival tional antagonism of the sphingosine-1‑phosphate and, as such, may contribute to the formation or main- receptor — has shown favourable efficacy in relapsing– tenance of ELSs within inflamed tissue. Thus, it will be remitting multiple sclerosis149 and has received approval extremely interesting to investigate whether directly tar- from the US Food and Drug Administration (FDA) for geting the IL‑17 pathway with novel therapeutics (such this indication. Fingolimod selectively reduces the fre- as secukinumab, ixekizumab and brodalumab) that are quency of circulating CCR7+CD4+ T cells (that is, naive currently in late-stage clinical development (TABLE 2) will and central memory T cells) and traps this subset of modulate ELSs in the context of autoimmune disease. T cells in SLOs, which prevents their migration into the Finally, it remains to be established whether therapeutics central nervous system150. that target factors that are associated with B cell survival

Given the role of TFH cells in ELSs, there is also growing and proliferation disrupt ELSs or impair their role as func-

interest in blocking key TFH cell-related signatures — for tional niches for autoimmune B cell activation. In this example, the co-stimulatory molecules ICOS and ICOS regard, data are eagerly anticipated from a biopsy-based

ligand (ICOSL) or the key TFH cell-related cytokine IL‑21. Phase II clinical trial in Sjögren’s syndrome with beli- Blockade of IL‑21–IL‑21R signalling ameliorated disease mumab, which is an anti-BAFF monoclonal antibody that in animal models of arthritis151 and SLE152, and an anti- has received FDA approval for SLE (TABLE 2). Such stud- human IL‑21 monoclonal antibody (NNC0114-0006) has ies should elucidate whether BAFF inhibition is sufficient recently completed Phase II clinical trials in rheumatoid to disrupt ELS functionality in the salivary glands. arthritis and a Phase I clinical trial in SLE, but no published data are currently available (TABLE 2). Pharmacological Concluding remarks and future directions ICOS blockade has also shown benefit in experimental An increasing number of clinical investigations empha- arthritis153 and a mouse model of lupus154 via inhibition of size that ectopic lymphoid neogenesis is a common

Non-obese diabetic mice TFH cell responses. Similarly, the concomitant inhibition occurrence at sites of inflammation, whereby ELSs form (NOD mice). These mice of ICOS and CD40L co‑stimulation protects non-obese an integral part of the immune response to infections, spontaneously develop diabetic mice (NOD mice) from diabetes155. As a conse- tumours and autoantigens. Although experimental ani- diabetes as a result of autoreactive T cell-mediated quence, an anti-ICOSL monoclonal antibody (AMG557; mal models have defined certain mechanistic aspects destruction of pancreatic also known as mAb‑3B3) has recently entered Phase I relating to ELS development, function and maintenance, β-islet cells. clinical trials in SLE (TABLE 2). The co‑stimulatory parallel investigations in human conditions remain

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relatively sparse by comparison. It is important to deter- this ambition, clinicians need to consider more precise mine whether the clinical consequences of ELS activi- diagnostic approaches in their patient management proto- ties are beneficial (for example, immunity to infections cols. The use of ultrasound-directed biopsy techniques has or cancer) or detrimental (for example, the promotion revolutionized oncology treatment and the introduction of autoimmunity or graft rejection). Clinical trials in of molecular pathology in autoimmune conditions is now conditions that are typically associated with ectopic lym- being used to enhance the mechanistic understanding of phoid neogenesis may broaden our understanding of ELS the crucial pathways that are driving disease. For exam- involvement in these diseases. An increasing number of ple, ultrasound-guided synovial biopsy studies in patients novel biologics that target mediators that are central to with rheumatoid arthritis have highlighted the clinical ELS biology are now entering the clinical arena. However, heterogeneity of synovitis within the disease-affected the key goal is to identify specific molecular signatures tissue116. The tremendous progress in miniaturized tech- that will predict at an early stage of the disease process nologies and high-throughput ‘multi-omic’ approaches whether ELSs will form and that can be used to inform will enable clinicians to move away from defining disease decisions regarding the most appropriate therapy for indi- mainly on the basis of symptoms and signs, and towards vidual patients. For example, there may be little point in a new taxonomy that integrates molecular signatures into targeting pathways that promote ELS development if these systematic algorithms to map the observed pathology structures have already become a prominent feature of the onto existing disease classifications. This information will underlying pathology by the time a patient is diagnosed. lead to improved patient stratification, better and more Instead, the use of agents that block the long-term main- appropriate clinical management and an increased likeli- tenance of ELSs within these inflamed tissues may prove hood of remission, and will eventually fulfil the ‘promise to be a more suitable approach in this scenario. To realize of personalized medicine’.

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