Which cytokines and chemokines are involved in TLO formation?

Formation of TLOs at sites of chronic inflammation follows many of the pathways also used by lymphoid organogenesis before birth (schematically summarized in Figure 2 ). Indeed, when LTα, CCL21 or CXCL13 are overexpressed from a tissue-specific promoter (such as the rat insulin promoter; RIP), ectopic lymphoid tissues are induced before or soon after birth 9, 10, 11, 12, 13, 14, 15, 16. Conversely, in experiments in which either CXCL13, its receptor CXCR5, CCR7 or LTα was genetically absent or neutralized, TLO structures did not develop in various infectious and autoimmune models 17, 18, 19, 20, 21, 22. As in SLO formation, LTα seems to instruct stromal cells to develop into FDCs and HEVs, whereas CCL19 and CCL21 acting via CCR7 control the organization of the T cell zones of the TLO 17, 20, 23, 24.

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Figure 2

Development and structure of tertiary lymphoid structures. (a) During chronic immune responses or transplant rejection, DCs continuously present antigens to T and B cells. Activated B cells express LTβ and can act as potent LTi-like cells, to induce an LTo phenotype in local myofibroblasts. Alternatively, chronic antigen presentation by DCs might also lead to induction of a Th17 cell response that can also induce TLOs through unclear mechanisms. Th17 cells are held in place via interactions with podoplanin. (b) As in SLOs, TLOs are divided into discrete B and T cell areas. Although, fully formed TLO structures often contain only a single T cell area and a larger B cell area, in which GC reactions can be seen. These also contain DCs and FDCs. At the periphery, an elaborate network of lymphatics (Lyve1⁺ and Prox1⁺) is commonly found but it is currently unknown if these are afferent or efferent lymphatics.

The homeostatic cytokine interleukin (IL)-7 deserves separate mentioning. Il7 ^–/– mice lack LNs and Peyer's patches, whereas overexpression of IL-7 induces development of additional LNs. IL-7 is involved in stimulating LTi cells and in maintaining survival of T lymphocytes. IL-7 expression is augmented in the synovial tissue of rheumatoid arthritis (RA) patients, and there is a strong correlation between IL-7 expression and LTβ expression [25]. In the perivascular TLOs of patients with idiopathic pulmonary hypertension (IPAH), strong immunoreactivity for IL-7 very close to ER^–TR7⁺ FRCs was found [26].

Together these data illustrate that homeostatic chemokines and cytokines are sufficient and necessary for the induction of TLOs. Given this, it has been suggested that inflammation-induced TLOs might have developed during evolution before SLOs did.

Are LTi cells necessary for inducing TLO formation?

In contrast to the involvement of CD3^–CD4⁺CD45⁺ LTi cells in the formation of LNs, their necessity for TLO induction is controversial [2]. The differentiation of LTi cells from lymphoid progenitors involves the transcription factors Id2 and ROR-γt. Mice lacking these factors do not develop most LNs (except nasal associated mucosal tissues, NALT, and tear duct associated lymphoid tissue, TALT) 4, 27, 28, 29. When Id2 ^–/– or Rorc ^–/– mice, which are lacking LTi cells, are infected with influenza virus, they develop normal iBALT structures in the lung 23, 30, 31. Other phenomena that do not depend on LTi cells are postnatal development of omental milky spots (these are organized lymphomyelopoietic tissue in the peritoneal cavity), isolated lymphoid follicles and TLOs in the intestine caused by dextran sodium sulfate (DSS)-induced colitis, and TLOs in autoimmune thyroiditis or insulitis 12, 31, 32, 33. The controversy surrounding the role of LTi cells stems from three observations. First, adoptive transfer studies provide evidence that injection of LTi cells into normal skin is sufficient to induce TLO structures [34]. Second, LTi cells have been observed in models of spontaneous TLO development, such as the perivascular TLOs seen in hypercholesterolemic Apoe ^–/– mice and the iBALT structures seen in neonatal lungs exposed to lipopolysaccharide (LPS) [30]. Human TLOs, such as those seen in IPAH, also contain adult LTi-like cells that are CD4^–, cKit⁺ and OX40L positive, and also express RORc and Id2 [26]. Third, overexpression of homeostatic chemokines under a tissue-specific promoter to induce TLOs (e.g. RIP or the intestine-specific villin promoter driving expression of CXCL13) and transgenic overexpression of IL-7 both result in increased numbers of LTi cells and dependence of TLO formation on LTi cells [33]. However, in these systems, lymphoid organogenesis starts before birth, which means that the requirement for LTi cells in induction of TLO formation could be overestimated. Nevertheless, most groups agree that LTα1β2 is necessary for TLO induction 17, 21.

Which other cells can act as LT inducers in chronic inflammation?

In the models in which LTi cells have been shown to be redundant, which other LTα1β2-expressing cell type could be providing the instructive signal to activate stromal organizer cells and initiate TLO formation? It has been proposed that B cells, T cells or DCs could substitute for LTi cells in their inductive function, especially when they are activated and express LTα1β2 on their surface 31, 35, 36, 37. Progression towards mature, fully structured TLOs is dependent on LTβ-sufficient B cells [24]. In a model of thyroid overexpression of CCL21, it has been shown that CD3⁺CD4⁺ activated T cells interact with DCs at sites of chronic inflammation, and subsequently the activated T cells act as LTi cells in the absence of Id2 activity [12]. In two recent studies on neonatal mice exposed to endotoxin inhalation and mice with experimental autoimmune encephalomyelitis (EAE), an activated T helper (Th)17 CD4 T cell population was found to be involved in inducing TLO structures 30, 38. RORC⁺ IL-17-producing cells have also been found inside TLOs of patients with IPAH. In humans, Th17 cells express the CCR6 receptor, and in the bloodstream of IPAH patients, circulating CCR6⁺ cells are fewer, whereas the ligand CCL20 is produced in the perivascular TLOs [26]. However, TLOs seem to develop normally in Ccr6 ^–/– mice [30]. The induction of TLOs by Th17 cells is dependent on expression of podoplanin, but why this is so remains to be studied. One possibility is that podoplanin is required for retention of Th17 cells at sites of TLO formation 30, 38. It remains to be seen whether all forms of TLO depend on IL-17 production, and whether IL-17A and/or IL-17F is involved. In this regard, iBALT induced by infection with modified vaccinia virus Ankara or influenza virus is not affected by deficiency of IL-17A 31, 39. LTβ signaling is not triggered only via LTα1β2; LIGHT (TNFSF14) can also bind the LTβ receptor. In a model of nonobese diabetes (NOD), LIGHT expressed on T cells has been shown to be important in the development and maintenance of TLO structures [40].

In almost all TLO structures that have been described, the T cell area contained antigen-presenting DCs 26, 37. DCs activate T cells, therefore, it has been suggested that DCs are sufficient for TLO induction [41]. Repeated injection of DCs into the lungs of mice is sufficient for induction of iBALT structures accompanied by induction of myofibroblast differentiation 37, 42. During formation of Peyer's patches, a CD11c⁺ cell type expressing LTαβ accumulates at the LN anlagen and is necessary for stromal instruction [8]. DCs might also directly instruct stromal cells irrespective of their effects on T cells. In TLO structures induced in the thymus, DCs are specifically necessary for induction of lymphangiogenesis from stromal cells [43]. How DCs induce TLOs is less clear. In virus-induced iBALT, mainly CD11b⁺ DCs accumulate; these cells express instructive LTα1β2 and at the same time produce the homeostatic chemokines CXCL13 and CCL19/CCL21 [37]. However, in some models, mostly pDCs accumulate, suggesting a functional role for type I interferons. This is the case in TLOs found in end-stage chronic obstructive pulmonary disease (COPD) patients and in a murine systemic lupus erythematosus (SLE) model 44, 45. Three studies have shown that depletion of DCs leads to disappearance of existing TLO structures, suggesting that DCs are necessary for structural organization and maintenance of TLOs; probably through transpresentation of chemokines, or by provision of a continuous source of antigen presentation to T cells 37, 43, 46.

Overall, there is no strong evidence pointing towards one specialized LTi cell type in TLO induction. LTi cells, B cells, T cells and DCs remain candidate cell types, with an emerging role for Th17 cells.

TLOs as immune inductive sites for protective immunity to (re-)infection

Similar to SLOs that bring together naive T cells, B cells and DCs, TLOs have been shown to allow the activation of recirculating naive T cells and the activation of B cells within germinal centers (GCs). One caveat is that these studies were performed in mice lacking LNs, which could have led to overestimation of the potential of TLOs [23]. However, recent work has suggested that TLOs can be immune inductive even in mice with normal LN anatomy and a fully functional immune system. DCs in TLOs often have an activated phenotype, suggesting that they are the predominant APCs stimulating immunity [45]. The function of DCs in TLO biology has been studied mainly in iBALT structures induced by modified Ankara vaccine virus or influenza virus 37, 46. Preformed iBALT structures collect DCs that are injected into the trachea, but it is unclear how these DCs reach the T cell area of iBALT [46]. One possibility is that iBALT structures are connected to afferent lymphatics located immediately underneath the epithelial basement membrane [52]. In many instances, the TLOs are found immediately next to lymphatics, but no study so far has been able to determine if these lymphatics are afferent (bringing antigen to the TLO) or efferent (exporting lymphocytes). It is also possible that the remodeling around blood vessels and the subepithelial areas leads to formation of a conduit system that provides a path for the encountered antigens to reach the TLO. In support of this possibility is the close resemblance between the type of extracellular matrix found in atherosclerosis-associated or IPAH-associated TLOs and the one found in the FRC network and conduit system of LNs 19, 26. Whatever the mechanism, TLO structures do allow the extravasation of naive T cells and their differentiation into effector T cells upon contact with DCs [46].

A major constituent of TLOs are the B cells that accumulate on the FDC network. Almost all TLOs described exhibit signs of B cell class switching, as exemplified by the presence of high amounts of the Activation-induced cytidine deaminase (AID) enzyme and the presence of GC reactions 26, 37. Such B cells provide an important source of memory B cells that are activated upon reinfection 37, 55. Plasma cells are found in the immediate vicinity of the TLOs and secrete antibodies specific for the pathogen that induces the TLO, such as influenza virus or Helicobacter species 37, 56. Although some of the B cells generated in the TLO also seem to reach the bone marrow, where they can reside as long lived plasma cells, the iBALT system that forms after influenza virus infection and the ILFs that form in response to intestinal commensals mainly serve to produce local mucosal IgA 37, 57, 58.

It is not known why some infections leave behind a TLO structure whereas others do not. In a model of influenza, for T cell immunity to persist, there is continued dependency on lung DCs that take viral antigens from the lung to the mediastinal LNs, even long after the virus has been cleared, for presentation to memory T cells. CD11b⁺ DCs can be found mainly in iBALT in the lung long after virus is cleared, therefore, it is possible that they capture retained viral antigens from the FDC network [59].

TLOs in immunopathology

The presence of TLOs in models of chronic autoimmunity and in chronic transplant rejection and their association with tissue destruction (Table 1) has led to the suggestion that they are important inductive sites for self-reactive T lymphocytes and antibodies that contribute to pathology. However, the evidence for this is rather sparse. During progression of diabetes in NOD mice, there is progressive development of TLOs in the pancreas. In these TLOs, T lymphocytes are detected that destroy islets and plasma cells with specificity for insulin 40, 60. The expansion of B cells inside an individual TLO around islets is often oligoclonal, suggesting the presence of an autoantigen [61]. More severe pathology of the grey matter and earlier onset of disease has been noticed in cases of autoimmune encephalomyelitis in which ectopic B cell follicles are present [62].

In a model of DSS-induced colitis in mice lacking RORγT (and thus lacking LNs, LTi cells, Th17 cells and innate lymphoid cells), the induction of B-cell-rich TLOs was associated with more severe immunopathology, and the mice succumbed from a lethal wasting disease that could be prevented by treatment with LTβR immunoglobulin. Strikingly, the disease was also prevented by treatment with intravenous immunoglobulins (IVIGs) to saturate Fc receptors on effector cells. This finding shows that these TLOs contribute to immunopathology via a humoral mechanism [31]. Chronic alloimmune responses induce TLOs in mice and humans, and these TLOs could be involved in chronic rejection. Transplantation of skin harboring TLOs to mice of a different strain lacking all LNs and spleen leads to chronic rejection, whereas transplantation of skin lacking TLOs is not rejected [63].

Also in humans, TLO structures might contribute to disease progression in autoimmunity and transplant rejection 64, 65. In the lungs of patients with RA, B cells in the TLO produce rheumatoid factor (RF) and antibodies against citrullinated proteins; typical self antigens in this disease [52]. In human autoimmune thyroiditis, most B cells inside ectopic GCs bound the autoantigens thyroglobulin and thyroidal peroxidase [66]. A study on SLE patients has confirmed the restricted B cell repertoire, with formation of isotype-switched antibodies that are deposited in the glomerular basement membrane [67]. TLOs have also been found in the meninges of patients with secondary progressive multiple sclerosis; a severe feature of SLE. Notably, all meningeal B cell follicles are found in cerebral sulci adjacent to large subpial cortical lesions, suggesting that soluble factors diffusing from these structures have a pathogenic role [68].

However, it is less clear which antigens the lymphocytes might react to in more severely degenerative diseases such as COPD induced by cigarette smoke and atherosclerosis induced by western diet. Although it has been proposed that there is an autoimmune component in many chronic tissue-destructive diseases such as COPD, another explanation could be colonization of the lung by latent adenovirus or unsuspected commensals in the lung [50]. Until we know the antigen-specificity of the B and T cell responses in TLOs, we can only speculate on the nature of the antigens presented in diseases such as IPAH and primary biliary sclerosis. In any case, the extensive formation of myofibroblast-like cells that constitute part of the stromal organizing network might contribute to local fibrosis that could compromise organ function.

K.N. is supported by a grant from FWO Flanders. F.P. is supported by a grant from Fondation pour la Recherche Médicale (FRM), grant number DEQ20100318257, D.H.G. is supported by a ESCMID grant, H.H. is supported by grants from FWO Flanders, B.N.M. is supported by Odysseus and Project grants of FWO Flanders, by an ERC Consolidator grant, and by a Ghent University MRP Grant (Group-ID consortium).