Cellular & Molecular Immunology (2017) 14, 809–818 & 2017 CSI and USTC All rights reserved 2042-0226/17 $32.00 www.nature.com/cmi

REVIEW

Lymphotoxin signalling in tertiary lymphoid structures and immunotherapy

Haidong Tang1, Mingzhao Zhu2, Jian Qiao1 and Yang-Xin Fu1,2

Tertiary lymphoid structures (TLS) often develop at sites of persistent inflammation, including cancers and autoimmune diseases. In most cases, the presence of TLS correlates with active immune responses. Because of their proximity to pathological loci, TLS are an intriguing target for the manipulation of immune responses. For several years, it has become clear that lymphotoxin (LT) signalling plays critical roles in lymphoid tissue organogenesis and maintenance. In the current review, we will discuss the role of LT signalling in the development of TLS. With a focus on cancers and autoimmune diseases, we will highlight the correlations between TLS and disease progression. We will also discuss the current efforts and potential directions for manipulating TLS for immunotherapies. Cellular & Molecular Immunology (2017) 14, 809–818; doi:10.1038/cmi.2017.13; published online 17 April 2017

Keywords: autoimmune disease; cancer; immunotherapy; lymphotoxin signalling; tertiary lymphoid structure

INTRODUCTION expression patterns and cellular distributions. LTα was origin- Inflammation is the immune response to various pathological ally identified because of its ability to induce tumour cell death conditions, such as cancer, autoimmune disease and infection.1 in vitro; thus, it was named TNFβ.7,8 Similar to TNF, LTα itself Inflammatory mediators, especially cytokines, chemokines and can form a homotrimer that binds to TNF receptor 1 (TNFR1) adhesion molecules, recruit and activate various types of innate and TNF receptor 2 (TNFR2; Figure 1).9 LTα can also form and adaptive immune cells to the site of inflammation, heterodimers together with LTβ as either LTα2β1orLTα1β2. including macrophages, DCs, mast cells as well as T and B Among them, LTα1β2 is the predominant form. LTα1β2binds cells.2 In the case of persistent inflammation, the migration and to lymphotoxin beta receptor (LTβR).10 LTβRisbroadly positioning of immune cells usually follow certain patterns, expressed by stromal cells in lymphoid tissues, epithelial cells, which are similar to those observed in the secondary lymphoid monocytes, DCs and other cell types (Table 1).11,12 LTβRcan organs (SLO). These structures have been named tertiary bind to another TNF superfamily member, LIGHT, which is lymphoid structure (TLS; also called tertiary lymphoid organs homologous to lymphotoxins. It has inducible expression and (TLO)).3,4 Several cytokines/chemokines and their correspond- competes with herpes simplex virus glycoprotein D for ing receptors have been shown to be involved in the formation herpesvirus entry mediator (HVEM), a receptor expressed on of TLS, including lymphotoxins (LTs), tumour necrosis factor T lymphocytes. LIGHT can also provide co-stimulatory signals (TNF), CCL21, CCL19, CXCL13 and others. Among them, the to T cells by interacting with HVEM.13 Together, these LT signalling pathway plays key roles.5,6 In this review, we will cytokines and receptors form a complex signalling network highlight the role of LT signalling in the formation of TLS and that plays important roles in several immunological processes. its application for drug design targeting cancers and auto- immune diseases. FROM SLO TO TLS: THE ROLE OF LYMPHOTOXIN SIGNALLING IN LYMPHOID TISSUE DEVELOPMENT LYMPHOTOXIN SIGNALING: AN OVERVIEW Although originally identified as cytotoxins, it later became LTs belong to the TNF superfamily. Two distinct forms of LTs, clear that LTs play a more important role in lymphoid tissue LTα and LTβ,havebeenidentified. They show different organogenesis and maintenance. The direct evidence came

1Department of Pathology, University of Texas, Southwestern Medical Center, Dallas, TX 75235, USA and 2IBP-UTSW Joint Immunotherapy Group, Chinese Academy of Science, Key Laboratory for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Correspondence: Dr H Tang, Phd or Dr Y-X Fu, MD, PhD, The Department of Pathology and Immunology, UT Southwestern Medical Center, University of Texas, 6000 Harry Hines, ND6, R200A, Dallas, TX 75235, USA. E-mail: [email protected] or [email protected] Received: 22 December 2016; Revised: 24 January 2017; Accepted: 24 January 2017 LT signalling in TLS HTanget al

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from genetically manipulated mice.14–17 Mice deficient in LTα induces the expression of many chemokines and adhesion show significant defects in lymphoid organ development. molecules, including CCL19, CCL21, CXCL12, CXCL13, Specifically, these mice are born without secondary lymphoid ICAM1, VCAM1 and MAdCAM1.23 These molecules coordi- organs, including lymph nodes (LNs) and Payer’s Patches (PPs; nate a positive feedback loop by recruiting more LTi cells. They Table 1). LTβ-deficient mice phenocopy what has been also recruit other immune cells such as B and T cells for further observed in LTα mice.18,19 Interestingly, although TNF- and lymphoid tissue maturation and organization. This function of TNFR-deficient mice lack PPs, they still have normal LTβR is true not only in LN/PP development but also in non- 17,20–22 β LN. These data indicate a more profound role of LT R lymphoid tissues. In fact, the exogenous expression of LT or signalling in lymphoid tissue organogenesis and maintenance. LIGHT, which activates LTβR signalling, is able to promote functional lymphoid neogenesis in non-lymphoid tissues.24–26 LTβR regulates mesenchymal stromal cells for immune cell The essential role of LTβR signalling in mesenchymal cells for clustering and patterning – TLS has also been reported.27 29 In these studies, an arterial Awell-defined role of LTβR signalling during SLO develop- TLS model in apoE− / − mice was examined. When LTβRwas ment is its ability to induce the expression of lymphoid conditionally deleted from vascular smooth muscle cells, chemokines and adhesion molecules for initial immune cell although aorta tertiary lymphoid organ (ATLO) neogenesis clustering. For lymph node/PP development, LTβR signalling triggered by LT from lymphoid tissue inducer (LTi) cells was not affected, aberrant ATLO structures, as indicated by reduced size, loose T- and B-cell infiltrates and segregation, were found. TNF LTα1β2 LIGHT

LTβR regulates mesenchymal stromal cell differentiation LTα3 Fibroblastic reticular cells (FRCs) and follicular dendritic cells

TNF LIGHT (FDCs) are important organizer stromal cells in T and B zones, respectively. During SLO development, FDCs and FRCs are differentiated from mesenchymal precursors. LTβR signalling was recently found to be important for their differentiation. LN FRCs are mainly derived from adipocyte precursors, and their differentiation is critically dependent on the LTβR-NFκB2-RelB signalling pathway.30 Furthermore, in vivo organogenesis assays showed that embryonic and adult adipocyte precursor cells can

TNFRI TNFRII LTβR HVEM migrate into newborn lymph nodes and differentiate into a variety of lymph node stromal cells. FDCs can also arise from Figure 1 The lymphotoxin signalling network. TNF and LTα3can α β β ubiquitous perivascular precursors (preFDCs) expressing bind to TNFRI and TNFRII. Both LT 1 2 and LIGHT bind to LT R. β β LIGHT can also interact with HVEM and deliver co-stimulatory platelet-derived growth factor receptor (PDGFR ). The signals to T cells. TNF and LIGHT can be either membrane-bound expansion of preFDCs requires both LTi cells and or soluble. TNF, tumour necrosis factor; LT, lymphotoxin; TNFRI, lymphotoxin.31 The ubiquity of preFDCs and their strategic TNF receptor I; TNFRII, TNF receptor II; LTβR, lymphotoxin beta location at blood vessels may explain the de novo generation of receptor; HVEM, herpesvirus entry mediator; LIGHT, homologous organized TLS at sites of lymphocytic inflammation. Whether a to lymphotoxins, inducible expression, competes with herpes β simplex virus glycoprotein for HVEM, a receptor expressed on T similar LT R-dependent mechanism controls TLS develop- lymphocytes. ment remains to be determined.

Table 1 Expression profile and phenotype of mice deficient in lymphotoxin-related genes

Molecule Cellular expression Deficiency in lymphoid neogenesis

LTα Primarily on T-, B-, NK and LTi cells Lack of lymph nodes and Payer’s patches, absence of T–B segregation in spleen LTβ Primarily on T-, B-, NK and LTi cells Lack of lymph nodes and Payer's patches, absence of T–B segregation in spleen LTβR Broadly expressed, except in mature lymphocytes Lack of lymph nodes and Payer's patches, absence of T–B segregation in spleen LIGHT Activated T- and NK cells, immature DCs, platelet Normal TNF Broadly expressed Lack Payer’s patches, absence of T–B segregation in spleen TNFRI Broadly expressed on most nucleated cells Lack Payer’s patches, absence of T–B segregation in spleen TNFRII Hematopoietic cells Normal

Abbreviations: NK cells, natural killer cells; TNF, tumor necrosis factor.

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LTβR regulates HEV for organized lymphocyte recruitment cancers, the LT inducer cells have not been reported. Since LT- The LTβR signalling pathway plays critical roles for high expressing DCs have been shown to be associated with TLS, it endothelial venule (HEV) differentiation and function.32,33 may be an interesting candidate for testing. HEVs are specialized blood vessels that are essential for the migration of lymphocytes into SLO.34 Compared to capillaries, TLS IN CANCER IMMUNOTHERAPY HEVs are distinct in their expression of peripheral node Correlation between TLS and cancer progression: Clinical addressins (PNADs). PNADs are a group of highly glycosylated study and sulphated forms of sialomucins, such as glycosylation- TLSs form and attract lymphocytes during chronic inflamma- dependent cell-adhesion molecule-1 (GlyCAM-1), CD34, tion. In the case of tumours, TLSs are formed adjacent to podocalyxin, endoglycan and endomucin.35,36 Some sialomu- malignant cells. Compared to draining lymph nodes (dLNs), cins are also expressed in capillaries, though with lower levels these structures are closer to tumour cells, which makes them of post-translational modifications. This may be due to the better places for T cells to meet tumour antigen-carrying DCs preferential expression of genes involved in glycoconjugate and become primed. Clinically, the presence and abundance of formation in HEVs.36 Besides PNADs, HEVs also express high TLSs in tumour patients usually correlate with active immune levels of lymphoid chemokines, such as CCL21.37 DCs promote responses and better prognosis.2,53,54 LT signalling through LTβR for HEV differentiation and One piece of evidence comes from studies in melanoma. The function.38 In addition, LTβR signalling is also involved in distribution of DCs follows a certain pattern in melanoma HEV network growth via regulating VEGF expression. FRCs tumour tissues.55 Specifically, DC-Lamp+ mature DCs are are the major source of VEGF in lymph nodes.39 LTβR mostly confined to peritumoural areas, and a higher frequency signalling in FRCs is important for VEGF production.40 of DC-Lamp+ mature DCs correlates with better prognosis in Alternatively, DCs have also been reported to express VEGF patients. The presence of TLS in metastatic melanoma was directly for initial HEV formation.40,41 further identified as lymphoid follicles containing HEVs and HEV-like structures are also found in TLS. Because of the clusters of B cells, T cells, FDCs and mature DCs.56 B cells similarity between SLO and TLS, it is speculated that LT inside these TLSs show signs of clonal amplification, somatic signalling is also involved in HEV differentiation and function mutation and isotype switching, indicating active immune in TLSs. In line with this notion, HEV-like structures are responses. Similar structures have also been confirmed by associated with LT-expressing DCs in human breast cancer.42 another group.57 Furthermore, a 12-chemokine gene expres- However, the exact roles of DC and LT signalling on HEV sion signature (GES) has been identified and can be used to differentiation, growth and function in TLSs remain to be predict the presence of TLSs. This 12-chemokine GES includes determined. CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11 and CXCL13. Most of these Different lymphoid tissue inducers for SLO and TLS cytokines have been shown to be critical for the formation of In addition to stromal cells and endothelial cells, LTi cells are SLO and TLS. Interestingly, many of them can be directly also critical for LN development, as they deliver the initial upregulated by LT signalling.58 triggering of stromal cells. For SLO development, the crucial In non-small cell lung cancer (NSCLC) patients, the − role of CD4+CD3 RORγt+ LTi cells has been well existence of TLSs has been shown in tumours but not normal documented.43–45 Whether LTi cells play a role in TLS tissues.59–61 These TLSs contained mature DC and T-cell development remains controversial. In a model where TLS clusters adjacent to B-cell follicles. The density of mature was induced by ectopic expression of CCL21 in the thyroid DCs is associated with favourable clinical outcomes. Intrigu- gland, the formation of TLS was independent of LTi cells.46 ingly, the density of mature DCs also correlates with tumour However, in another study, IL-7 overexpression led to the infiltrating lymphocytes (TILs), which have been shown to formation of multiple organized ectopic lymph nodes and correlate with better prognosis in many different cancers.62 caecal patches because of the increased survival of LTi cells.47 A follow-up study identified that tumour infiltrating T cells are Mice overexpressing IL-7 but lacking either RORγ, a factor predominantly of the effector-memory phenotype, which is required for LTi cell generation, or LT had no ectopic lymph consistent with the important role of TLSs as a site for T-cell nodes. Even so, the data do not exclude the role of Th17 in TLS priming.63 development, since RORγt is also required for Th17 differ- Mature DCs are not the only cell type that correlates with entiation. In fact, several studies have linked IL-17 and Th17 to the presence of TLS and better prognosis. In colorectal the development of TLSs in various models.48–50 In addition, B carcinoma patients, a higher number of CD3+ T-cell infiltra- cells have also been reported to be involved in TLS formation tion into tumour-associated lymphoid nodules predicts in RA patients.51 Recently, an intriguing study showed that B improved survival.64–66 In a mouse model of colorectal cancer, cells can serve as LTi cells by providing LT signalling for the adoptive transfer of splenocytes results in the accumulation microbiota-induced tertiary lymphoid tissue formation, even in of lymphocytes at the TLS, suggesting an active role of TLSs in RORγt-deficient mice, which lack both LTi cells and Th17 recruiting lymphocytes to tumour tissues. Higher TILs and the cells.52 Therefore, different LT-producing cells may work as LTi presence of TLSs also correlate with disease-free survival in cells depending on the scenarios. For TLS development in patients with breast cancer42,67–71 and melanoma.55,57,64,72

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Taken together, these studies suggest a strong correlation prognosis, it has become an intriguing hypothesis that being between the presence of TLS and a better prognosis in cancer able to induce TLS formation might also be a potent strategy to patients. induce anti-tumour immunity (Figure 2).79 Several studies On the other hand, however, although the presence of TLS aiming at developing strategies to induce TLSs in order to usually correlates with positive clinical outcome, it could also enhance anti-tumour immune responses have been reported indicate an adverse prognosis in some scenarios. In breast (Table 2). cancer patients, a high number of tumour-infiltrating regula- Because of its central role during lymphoid tissue neogenesis, tory T (Ti-Treg) cells was found to be present in the LTβR has attracted the most attention. Early studies have peritumoural areas.73 These Ti-Treg cells are associated with shown that exogenous expression of LTβR ligands, either LT or relapse and death in patients. It has been suggested that Treg LIGHT, is able to promote functional lymphoid neogenesis in – cells are activated by mature DCs through tumour-associated non-lymphoid tissues.24 26 In a proof-of-concept study, the antigen presentation. Activated Treg cells inhibit the functions expression of LTβR was evaluated using an immunohistologic of effector T cells, leading to immune escape and tumour survey.81 Most samples (87% to 96%) showed at least 1+ progression. Interestingly, the specific recruitment of Treg to staining for LTβR in human tumours. The wide expression TLSs is not through CCL19 or CCL21 because the receptor of of LTβR in tumour tissues lays the foundation for activating both chemokines, CCR7, is equally expressed in effector T cells LTβR signalling for TLS induction. An agonistic antibody (Teff) and Treg. Instead, the authors suggested that Treg cells against LTβR has been developed. In both a human xenograft are more likely to be recruited by CCL22, as Treg has a much model and a mouse syngeneic model, the agonist anti-LTβR higher level of CCR4, a receptor for CCL22.73 antibody is able to induce anti-tumour immune responses and increase lymphocyte infiltration into tumour tissues.81 How- Targeting lymphotoxin signalling for cancer immunotherapy ever, the anti-tumour effects were less impressive, possibly Tumour microenvironments are usually inhibitory and prevent because of the wide expression of LTβR, which makes it hard effective lymphocyte priming.74,75 As TLSs are located adjacent to specifically activate LTβR signalling in tumour tissues. The to tumour cells, there could be several advantages to priming lack of tumour-specific targeting also raised concerns regarding T cells inside TLSs. First, it is less difficult for antigen- side effects. In fact, the systemic activation of LT signalling presenting cells (APCs) to take up tumour antigens and results in serous toxicity.101 migrate to TLSs. Second, the tumour antigen load in TLSs The other approach to activate LTβR signalling is through could be higher than tumour dLNs.76 Third, activated effector engagement with LIGHT. The expression of LIGHT in non- cells can target tumour cells and stromal cells with less lymphoid tissues, such as islets, is able to induce development migration.76,77 In fact, bystander elimination of stromal cells of TLS.25 The ectopic activation of LTβR signalling induces is essential to control tumour growth.78 Considering the diabetes as a result of autoimmune disease. This process can be significant correlation between the presence of TLSs and better blocked by a neutralizing recombinant LTβR fusion protein.

T cell zone LTβR

Antigen T 1 2 T DC DC DC T T

Tumor T T T Chemokines cells 3 (CCL19, CCL21, DC CXCL13, …

B B Stroma 4 B B B

B cell zone HEV

Figure 2 Immune responses mediated by tumour-related tertiary lymphoid structures (TLSs). Lymphocytes are recruited from HEV to TLSs, where they form T- and B-cell-rich zones (1). Dendritic cells (DCs) take up antigens and present them to T cells (2). After activation and proliferation, T cells can migrate to tumour cells for destruction (3). Antigens can also be transferred to B cells to induce antibody production (4). Lymphotoxin signalling plays important roles in lymphocyte recruitment, stromal and LTi cell differentiation, and HEV organization. HEV, high endothelial venules. LTβR, lymphotoxin beta receptor.

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Interestingly, forced expression of LIGHT in tumour cells profound role of Treg cells during the progression of mouse promotes the formation of lymphoid-like structures for direct melanoma, it is interesting to speculate that instead of T-cell sequestration and activation, leading to tumour promoting effector T-cell priming, TLS might facilitate the regression.77,84 Alternatively, mesenchymal stem cells engi- activation of Treg cells and inhibit effector T cells in tumours neered to express LIGHT can home into tumour tissues and or tissues where Treg play a dominant role. promote anti-tumour immunity.102 However, because of the CCL19 and CCL21 share the same receptor, CCR7. Although wide expression of LIGHT receptors, a major challenge in the they have a similar affinity to CCR7, the signalling induced by delivery of LT signalling for cancer immunotherapy is how to CCL19 and CCL21 is different.110 Specifically, the binding of specifically target to the tumour (Table 1). To overcome such a CCL19 to CCR7 induces internalization of the receptor, leading limitation, tumour-specific antibody targeting has been utilized to desensitization.111 Furthermore, binding CCL19 desensitized to deliver LIGHT specifically to tumour tissues.103 Further the receptor to subsequent responses to CCL21. Because of study suggested that LTβR signalling activated by LIGHT is able these reasons, it is likely that the anti-tumour effects of CCL19 to attract lymphocytes to the tumour tissues, resulting in in mouse tumour models are not as impressive as CCL21.112–115 tumour destruction. Overall, CCL19 represents a more moderate, self-limited, and As a ligand of LTβR, LTα is another promising molecule for tuneable stimulator for CCR7 activation. the activation of LTβR signalling. Several studies have been After induction, TLSs in tumour tissues are able to provoke carried out with recombinant LTα that is fused to an anti-GD2 anti-tumour immunity through several mechanisms that are antibody for specific tumour targeting.24,80 This fusion protein quite unique compared to other therapeutic approaches. First, is able to inhibit tumour growth and prolong survival in a most current immunotherapies aim to activate/reactivate T cells syngeneic mouse melanoma model. Intriguingly, seven days inside tumours or dLNs. However, T cells inside established after seven continually daily treatments with this fusion tumours are usually exhausted or anergic because of the protein, HEV-like structures could already be observed inside inhibitory or prolonged activating microenvironment and are tumours, supporting lymphocyte migration.24 These studies difficult to be activated.116 In significant contrast, HEVs in TLSs suggest that the tumour-specific targeting of LT is able to support the recruitment of naive T cells from the induce the formation of TLSs in a short period of time and periphery.77,117,118 Naive T cells or newly attracted T cells are exert anti-tumour effects.24 less exposed to the inhibitory microenvironment and have a The strategies discussed above aim to activate LTβRsignal- lower threshold for activation. Second, the microenvironment ling using either an agonist antibody or its ligands. On the in TLSs, including cytokines/chemokines and co-stimulatory other hand, another strategy is to deliver major effector signalling, promotes better priming of T cells and prevents them molecules of LT signalling to tumour tissues, such as CCL21 from reaching exhaustion or anergy. Third, as mentioned or CCL19. CCL21 is a ligand of CCR7. It is an important above, because of higher antigen loads and proximity to tumour effector chemokine that can mediate the homing of DCs and cells, TLSs provide a better place for T cells to be primed and to T cells to both lymphoid and non-lymphoid tissues.104,105 infiltrate inside tumour tissues. Fourth, in situ T-cell priming CCL21 overexpression in B16 melanoma results in tumour can also lead to a broader and more comprehensive response, rejection.106 Therapeutic application of CCL21 in tumour especially for antigens that are hard or unable to be cross- immunotherapy has also been reported.107 Recombinant presented by APCs. In summary, the successful induction of CCL21 is able to induce tumour regression. The anti-tumour TLSs in tumour tissues, either alone or combination with other effects were completely abolished in immunodeficient mice or therapies, may be a potent strategy for tumour immunotherapy. in the absence of T cells, suggesting an essential role of adaptive immunity. Alternatively, cells transduced with CCL21 cDNA TLS IN AUTOIMMUNE DISEASE can be used instead of recombinant CCL21 protein.108 Inter- Role of TLS in autoimmune diseases estingly, intratumoral injection of DCs expressing CCL21 TLS can be found in many autoimmune diseases, including showed robust anti-tumour activity and induced regression rheumatoid arthritis (RA), systemic lupus erythematosus in all tumours tested. In significant contrast, fibroblasts (SLE), and type 1 diabetes (T1D). However, unlike in tumours, expressing the same chemokine can only induce regression in the function and prognostic value of TLS in some autoimmune 25% of the mice. These results are consistent with the notion diseases are still controversial, depending on the disease and the that DC is the major target of CCL21.104 In another study, loci of TLS formation. CCL21 was knocked down in a mouse melanoma model and In RA, it has long been observed that tissue-infiltrating an opposite result was observed.109 Surprisingly, tumour cells lymphocytes can form ectopic follicles in some patients.4 These expressing higher levels of CCL21 established an immune- follicles are organized in a way that is similar to SLO.119,120 LTβ tolerant tumour microenvironment, which facilitates tumour and CXCL13 are characteristic cytokines/chemokines that can progression. The tumour microenvironment in tumours independently predict the presence of TLS in patients, which expressing high levels of CCL21 was characterized by a ;higher indicates a critical role of LT signalling during the formation of number of Treg cells. This phenomenon is similar to what has these follicles.51 There is also a correlation between TLS and been observed in some breast cancer patient samples, which disease progression in RA. Specifically, FDCs in TLS express also show a higher number of Treg in TLSs.73 Considering the activation-induced cytidine deaminase (AID), an enzyme that

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Table 2 Biologics targeting lymphotoxin signalling for the treatment of cancers and autoimmune diseases

Target Species Type of agent Disease/model Trade name Stage of clinical Reference molecule development

Cancer LTα Mouse Antibody-LTα fusion protein Melanoma —— 24,80 α-LTβR agonist antibody Colon carcinoma —— 81,82 LTβRMouse α-LTβR neutralizing antibody Sarcoma, Breast cancer —— 83 Adenovirus expressing LIGHT Fibrosarcoma, Colon carcinoma, Breast —— 77,84–86 cancer, Prostate cancer LIGHT Mouse Salmonella expressing LIGHT Lymphoma —— 87 Antibody-LIGHT fusion protein Fibrosarcoma, Colon carcinoma —— 103 Autoimmune disease α-LTα antibody RA, EAE —— 88 Mouse LTα/LTβ α-LTβ antibody EAE —— 89 Human α-LTα antibody RA Pateclizumab Phase 2 (discontinued) 90 Mouse LTβR-Ig fusion protein RA, IBD, EAE, T1D, SS —— 89,91–96 β LT R 97,98 Human LTβR-Ig fusion protein RA Baminercept Phase 2 (discontinued) SS Baminercept Phase 2 99 LIGHT Human α-LIGHT antibody Crohn's disease, Ulcerative colitis SAR252067 Phase 1 (discontinued) Sanofi 1Q 2015 report IBD SAR252067 Phase 1 100

Abbreviations: EAE, Experimental autoimmune encephalomyelitis; IBD, Inflammatory bowel disease; RA, Rheumatoid arthritis; SS, Sjogren’s syndrome; T1D, Type 1 diabetes.

is required for somatic hypermutation and the class-switch As LT signalling is critical for the development of TLS, many recombination of Ig genes.121 B cells producing a specific efforts have been focused on blocking this signalling as therapy marker of RA, anti-citrullinated protein/peptide antibodies, are for autoimmune diseases.100,125 found to be in close contact with FDCs. These data provide An interesting example is the development of the anti-LTα evidence that TLS in RA may support autoantibody production antibody for the treatment of arthritis. Collagen-induced and contribute to disease progression. However, although the arthritis (CIA) is a commonly used murine autoimmune presence of TLS is associated with a higher level of lymphocyte disease model in which the disease development depends on infiltration and systemic inflammation, it does not seem to T helper (TH) cells.126 In a survey of the gene expression correlate with the severity of the disease.122 A possible profile in immune cells in CIA, the expression of LTα was explanation is that clinical disease severity may be influenced found to be specifically in TH cells, especially TH1 and TH17 by factors other than synovial inflammation. cells.88 Treating CIA mice with an anti-LTα antibody signifi- Nephritis is the major cause of death for SLE. In the analysis cantly inhibited disease development. A mechanistic study of lupus nephritis biopsies, ectopic lymphoid structures were showed that the administration of anti-LTα antibody depletes found to be associated with nephritis.123 In those structures, T TH1 and TH17 cells, resulting in decreased IL-17, IFN-γ,and and B cells were well-organized together with FDCs. These TNFα production. These data led to the development of the samples were positive for many lymphoid chemokines that are drug Pateclizumab, a humanized anti-LTα antibody. In clinical negative in normal renal tissues, including CXCL12, BAFF, trials, Pateclizumab was used to treat RA and graft-versus-host CXCL13, CCL21 and CXCL10. The presence of lymphoid disease (GVHD).127,128 Unfortunately, although proven to be structures is associated with in situ B-cell clonal expansion and safe in a phase 1 trial, it failed the phase 2 trial for RA because somatic hypermutation.123 These data suggested a correlation therewerenosignificant benefits over placebo.90 Apossible between TLS and disease progression. Similar patterns of TLS explanation might be the tight regulation during TLS formation and chemokine expression were observed in a formation.129 mouse model.124 Furthermore, DCs in TLSs were shown to Although not successful in LTα blocking, probably because express a high level of type 1 interferon (IFN), which might be of less efficient blockade, blocking LT signalling with an responsible for the pathogenesis of SLE. effective LTβR-Ig130 has been shown to reduce the formation of TLS and inhibit autoimmune disease progression in many Lymphotoxin signalling as a therapeutic target in different disease models, including T1D, Hashimoto’sthyroi- autoimmune diseases ditis, and experimental autoimmune encephalomyelitis In many cases of autoimmune diseases, it is believed that TLSs (EAE).131–134 The potent effects of LTβR-Ig in mouse models play a positive role in disease progression, which is consistent have pushed a humanized version, Baminercept, into clinical with the notion that TLSs are a site of immune activation. trials.97 Its first clinical trial in RA was discontinued because no

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815 significant effect was observed.135 However, further analysis 9 Hohmann H-P, Remy R, Pöschl B, Van Loon A. Tumor necrosis showed that there were significant changes in some biomarkers factors-alpha and-beta bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor 98 and with clinical improvements in a subset of patients. NF-kappa B at low receptor occupancy and within minutes after A second clinical trial against Sjogren’s syndrome (SS) has also receptor binding. JBiolChem1990; 265:15183–15188. fi 10 Wang Y, Zhu M, Miller M, Fu YX. Immunoregulation by tumor necrosis reached phase 2. Although there were no bene ts for increasing factor superfamily member LIGHT. Immunol Rev 2009; 229: salivary flow or reducing ocular dryness, Baminercept signifi- 232–243. cantly improved the EULAR Sjogren’s syndrome Disease 11 Browning JL, French LE. Visualization of lymphotoxin-β and lympho- 99 toxin-β receptor expression in mouse embryos. JImmunol2002; 168: Activity Index (ESSDAI). 5079–5087. 12 Murphy M, Walter BN, Pike-Nobile L, Fanger NA, Guyre PM, CONCLUDING REMARKS Browning JL et al. Expression of the lymphotoxin receptor on follicular stromal cells in human lymphoid tissues. Cell Death Differ 1998; 5: Recent breakthroughs in immunotherapy have opened a new – 136 497 505. era of disease treatment. Clinical trials with checkpoint 13 Mauri DN, Ebner R, Montgomery RI, Kochel KD, Cheung TC, Yu G-L blockade and chimeric antigen receptor-T-cell therapies have et al. LIGHT, a new member of the TNF superfamily, and lymphotoxin fi α are ligands for herpesvirus entry mediator. Immunity 1998; 8: shown remarkable long-term ef cacy in patients with a variety – 137–139 21 30. of cancers. Although only a minority of the total treated 14 De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S patients respond to the current immunotherapy treatment, the et al. Abnormal development of peripheral lymphoid organs in mice fi – unprecedented durable responses in some patients have shown de cient in lymphotoxin. Science 1994; 264: 703 707. 15 Banks TA, Rouse BT, Kerley MK, Blair PJ, Godfrey VL, Kuklin NA that potent effects could be achieved with immunotherapies. et al. Lymphotoxin-α-deficient mice effects on secondary lymphoid Because of its close and active interactions with pathological organ development and humoral immune responsiveness. JImmunol 1995; 155:1685–1693. tissues, TLS has become an intriguing target not only for 16 Ryffel B, Di Padova F, Schreier MH, Le Hir M, Eugster HP, Quesniaux studying disease progression but also for manipulating immune VFJ. Lack of type 2T cell-independent b cell responses and defect in responses during immunotherapy. Studies during the past years isotype switching in TNF-lymphotoxin α-deficient mice. 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ACKNOWLEDGEMENTS 20 Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and fl α fi YXF holds the Mary Nell and Ralph B. Rogers Professorship in in ammatory responses in TNF -de cient mice: A critical require- ment for TNFα in the formation of primary B cell follicles, follicular Immunology. This work was supported in part by the US National dendritic cell networks and germinal centers, and in the maturation of Institutes of Health through National Cancer Institute grants the humoral immune response. J Exp Med 1996; 184:1397–1411. CA141975 and CA97296, CPRIT grant RR150072, grants from the 21 Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Richards E et al. fi Chinese Academy of Sciences (XDA09030303), and grants from the Characterization of tumor necrosis factor-de cient mice. Proc Natl Acad Sci USA 1997; 94:8093–8098. Chinese Ministry of Science and Technology (2012ZX10002006, 22 Neumann B, Luz A, Pfeffer K, Holzmann B. 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