The mechanisms of TLO development share many similarities with those of lymph node development (**Figure 1**). Perhaps the most prominent example is the establishment of a chemokinedirected positive feedback loop that orchestrates lymphocyte recruitment and organization (4, 5). However, TLOs can form in the absence of LTi cells. For example, mice deficient in the nuclear hormone receptor retinoic acid-related orphan receptorγt (RORγt) and the transcriptional repressor Id2 still retain the capacity to develop TLOs at inflammatory sites, despite lacking LTi cells (16–19). This highlights one of the most striking differences between TLO and SLO development. While both rely on homeostatic chemokines (e.g., CXCL13, CCL19, CCL21, and CXCL12) and lymphoneogenic cytokines (e.g., LTαβ) for

their development, the initiation of TLO development relies on an inducible inflammatory trigger, while SLOs are developmentally pre-programed. In this regard, immune cells may substitute for LTi cells and act as primary orchestrators of tertiary lymphoneogenesis.

#### T HELPER 17 TYPE RESPONSES AND PLASTICITY DRIVE TLO DEVELOPMENT IN CHRONIC INFLAMMATION

T helper cells and their effector cytokines, particularly IL-17 secreting CD4+ (Th17) cells have recently emerged as key initiators of TLO development in inflammatory diseases (**Figure 2**) (20). For example, in a model of lipopolysaccharide (LPS)-driven pulmonary inflammation, neonatal mice developed inducible (i) BALT associated with heightened CXCL13 and CCL19 expression (19). Despite the presence of CD3−CD4+ LTi cells, iBALT development was dependent on IL-17 production by CD4+ T cells. Interestingly, antibody blockade revealed that IL-17 was important for initiation of iBALT development, but was dispensable for the maintenance of established lymphoid clusters. The development of iBALT has previously been shown in response to viral challenge (21, 22), bacterial infection (23), cigarette smoke (24), and protein nanoparticles (25). Notably, in response to infection with a replication-deficient poxvirus modified vaccinia virus Ankara, iBALT development was independent of both IL-17A and another Th17 effector cytokine, IL-17F (26). Therefore, while common mechanisms involving IL-17 may promote pulmonary TLO development in response to various inflammatory stimuli, it is also important to note that iBALT formation can occur independently of IL-17/Th17 involvement.

Recent studies have also highlighted roles for Th17 cells in TLO development in the central nervous system (CNS), inflamed joint tissues, and salivary glands. Peters et al. demonstrated a role for Th17 cells in promoting TLOs in an experimental model of

dendritic cell.

characteristics.

multiple sclerosis (27). Here, adoptive transfer of *in vitro* generated Th17 cells induced TLOs, which was partly IL-17 dependent. Only Th17 cells differentiated in the presence of IL-23, which maintains Th17 effector function (28, 29), were capable of inducing TLOs. Notably, the adoptive transfer of Th1, Th2, and Th9 cells failed to induce this phenotype. Interestingly, the development of TLOs in this model was also partly dependent on the expression of podoplanin (gp38) on transferred Th17 cells. While an appreciation of a role for podoplanin in regulating T cell responses is only now emerging (30), there is significant evidence for a role in regulating tertiary lymphoneogenesis. For example, we recently described IL-27 as a negative regulator of TLO development in experimental inflammatory arthritis (31). Here, synovial TLO development in IL-27R-deficient mice was associated with an increased number of peripheral podoplanin-expressing Th17 cells and the local recruitment of podoplanin-positive T cells to synovial lymphoid aggregates. The Th17 axis and podoplanin have also been linked with TLO development in human diseases, including rheumatoid arthritis, multiple sclerosis, renal allograft rejection, and giant-cell arteritis (31–35). Therefore, consistent with a key role for podoplanin

and its ligand CLEC-2 in lymph node development (11, 27, 36), podoplanin expression on T cells may support the recruitment and retention of leukocytes within TLOs.

While IL-17 stands as the "signature" cytokine for Th17 cells, these cells also produce IL-17F, IL-22, and IL-21. Recently, IL-17 and IL-22 have been shown to induce stromal production of homeostatic chemokines resulting in TLO development in mucosal tissues (37, 38). For example, IL-22 promotes TLO development in salivary glands following local adenovirus delivery (37). Here, the major source of IL-22 was αβ+ T cells and γδ+ T cells, which induced the expression of CXCL13 in podoplanin+ stromal cells and CXCL12 in epithelial cells. Therapeutic blockade of IL-22 activity inhibited TLO development and maintenance, thus highlighting IL-22-targeted therapies as a novel approach for the treatment of conditions featuring TLOs and autoantibodydriven disease.

IL-21 plays a central role in Th17 and T follicular helper (Tfh) cell differentiation, the development of naïve B cells into plasma cells or GC B cells, and the generation of high-affinity antibodies (39). Therefore Th17 or Tfh cell-derived IL-21 has potential to play an important role in TLO development and function. Elevated expression of IL-21 has been observed in tissues containing TLOs in experimental and clinical rheumatoid arthritis (31, 32), a model of salivary gland inflammation with Sjögren's syndrome-like characteristics (40) and in human renal grafts undergoing terminal failure (34). A recent study also described the development of TLOs in the retina during experimental uveitis, where TLOs were associated with heightened expression of Tfh cell markers (41). Interestingly, T helper cell plasticity may contribute to the development of TLOs, where T cells may transiently or fully acquire effector characteristics that support tertiary lymphoneogenesis. For example, Th17 cells that migrate and support the development of IgA-producing GC B cells in Peyer's patches acquire a Tfh-like phenotype, including the expression of IL-21, Bcl-6, CXCR5, and PD-1 (42). Similarly, Th17 cells that promote TLO development in the CNS during experimental autoimmune encephalomyelitis develop Tfh-like effector characteristics (27). While the development of TLOs in this latter model was independent of IL-21, the contribution of other Tfh effector characteristics cannot be excluded. Therefore, plasticity among T helper cell subsets may allow for the acquisition of Tfh-like effector characteristics (43, 44) that can support GC reactions and the development of TLOs.

#### REGULATORY T CELLS AS SUPPRESSORS OF TLO DEVELOPMENT AND FUNCTION

In chronic inflammatory disorders, TLOs are generally considered perpetuators of adaptive immune responses that contribute to pathology. A recent study describing a protective role for TLOs in atherosclerosis (45) raises an interesting question: *How can TLOs in the majority of chronic inflammatory disorders be damaging, yet in some be protective?*

In aged apolipoprotein E (ApoE)-deficient mice with atherosclerosis, smooth muscle cells beneath intimal plaques take on LTo-like properties and secrete CXCL13 and CCL21 to drive tertiary lymphoneogenesis (46). Engagement of the LTβR on smooth muscle cells played a central role in the induction of lymphoneogenic chemokines and the development of aortic TLOs, which were atheroprotective. These TLOs represent principal sites for the regulation of atherosclerotic T cell responses, including the development and activation of anti-inflammatory regulatory T (Treg) cells (45). In these aged mice, highly activated Treg cells within aortic TLOs may skew the local immune response toward anti-atherogenic outcomes by restricting the activation of effector and central memory T cells. Here, communication between aortic TLOs and vascular smooth muscle cells expressing the LTβR may be important in maintaining the structure, size, and composition of TLOs associated with protection from atherosclerosis (45). Notably, another recent study in ApoE-deficient mice outlines a role for regulatory CD8+ T cells in limiting the development of aortic TLOs during atherosclerosis (47). However, this study described a pro-atherogenic role for TLOs in both mouse and human atherosclerosis, where Tfh cells support GC reactions and the local maturation of potentially pathogenic B cells. Therefore, while Treg cells can prevent the development of aortic TLOs, further investigation is needed to determine the precise role played by TLOs during atherogenesis. The relative proportions of pro-inflammatory and Treg cell populations within aortic TLOs may be influenced by factors, such as age, stage of disease, and environmental factors, and may ultimately determine the impact of TLOs on disease progression.

Immunotherapies that inhibit Treg cell development or functionality have the potential of supporting antigen-specific responses against tumor antigens and prevent immune evasion by cancer cells. In this regard, many cancer immunotherapies focus on counteracting the immunosuppressive tumor environment to support tumor-specific T cell responses. The presence of tumor-infiltrating Treg cells is linked with immunosuppression and often correlates with poor patient prognosis (48), while TLOs have been associated with improved patient outcomes in certain cancers (5). This raises an interesting question: *Do therapies targeting Treg activities hold potential for supporting local anti-cancer responses at tumour-associated TLOs?* TLO's have been described in melanoma, mucosal-associated lymphoid tissue lymphoma, and non-small cell lung carcinoma (NSCLC), as well as breast, colorectal, rectal, ovarian, and germ cell cancers [see Dieu-Nosjean et al. (5) for a comprehensive review of TLOs in cancer]. Studies have reported a correlation between the number of TLOs, T/B cell infiltration into tumors and improved patient survival (49–52). In experimental mouse models and human cancers, there is significant evidence that TLOs are functional. For example, in a mouse model of melanoma, tumor-infiltrating lymphocytes developed TLOs and displayed clonal expansion of T cells that are reactive to tumor antigens on melanoma cells and inhibit tumor growth through the release of IFNγ (53, 54). Similar activities were also observed in LTαdeficient mice that lack peripheral lymph nodes, suggesting that T cell responses are primed locally within the tissue (54). In human NSCLC, TLOs are associated with improved patient survival. Here, enhanced anti-tumor immunity is associated with an increased frequency of follicular B cells and plasma cells that display antibody specificity to tumor-associated antigens (50). Therefore, TLO and HEV development in tumors may allow the recruitment of T and B cells that promote GC reactions and anti-tumor immunity.

Some recent studies support the hypothesis that targeting Treg cells can help establish anti-tumor immune responses locally at tumor-associated TLOs. In a mouse model of lung adenocarcinoma, TLOs that included activated Treg cells were observed in ~90% of tumors, where Treg cells suppressed anti-tumor responses (55). In this study, depletion of Treg cells resulted in enhanced expression of costimulatory molecules on dendritic cells (DCs), T cell proliferation, and anti-tumor responses leading to tumor destruction. The development of HEVs, a common feature of TLOs, is also linked with longer remission, reduced metastasis, and improved patient survival (5, 56, 57). Here, HEVs allow for the infiltration of naïve, central memory and effector Th1 cells that support the local priming of antigen-specific tumor responses (53, 58, 59). In a model of carcinogen-induced fibrosarcoma, Treg cell depletion resulted in the formation of HEVs and reduced tumor growth associated with an increase in T cell infiltration into tumors (60). Similarly improved outcomes were seen in a murine model of pancreatic cancer following TGFβ blockade, a cytokine involved in both Treg cell development and effector function (61).

In cancerous tissues, Treg cell populations suppress the proliferative expansion of CD8+ T cells responsible for delivering anti-tumor immunity. For example, studies have revealed that depletion of Treg cells contributes to CD8+ T cell proliferation and the development of enhanced anti-tumor responses (55, 60). These findings are highly relevant to human forms of cancer where CD8+ T cells, which are often found in tumor-associated TLOs, are linked with improved patient outcomes. Interestingly, NSCLC patients with a high frequency of CD8+ T cell infiltrates together with a high density of TLOs are associated with significantly improved survival compared to patients with high cytotoxic T cell infiltration without TLOs (51). Thus, tumor-associated TLOs enhance the prognostic value of tumor-infiltrating CD8+ T cells. While such studies demonstrate a correlation between CD8+ T cell infiltration, TLO densities, and patient survival, they also raise an interesting question regarding the function of cytotoxic T cells: *Are tumour infiltrating CD8+ T cells better equipped to control cancer due to their education within tumour-associated TLOs?* While further research is still needed in this area, a recent study of ovarian cancer showed that CD8+ tumor-infiltrating lymphocyte responses were increased in the presence of TLOs containing dense accumulations of plasma cells (62). Here, plasma cells expressed markers of active antigen-specific responses and were associated with heightened expression of cytotoxicity-related genes in tumors. TLOs may, therefore, support robust anti-tumor responses, where cytotoxic T cell activity and antibody-secreting plasma cells cooperate to improve patient survival.

Therapeutic strategies that support the development of tumorassociated TLOs and cytotoxic T cell responses may, therefore, prove beneficial for patient treatment. These may include antagonists of Treg cell development, maintenance, or activity. Blockade of TGFβ has been shown to inhibit Treg cells in experimental cancer and autoimmunity (61, 63). Treg cells can also be selectively depleted by metronomic low-dose cyclophosphamide, which improved tumor-specific T cell responses in cancer patients (64, 65). Further strategies may include antibodies, such as ipilimumab that target the CTLA-4 immune-checkpoint receptor, which has been shown to effectively deplete intratumoural Treg cells (66–68). Novel concepts currently in pre-clinical development may also inform next-generation approaches for inducing TLOs in cancer. For example, engineered adjuvant vector cells have been shown to promote the development of TLOs resulting in enhanced antigen-specific T cell responses in pre-clinical cancer models (69). While the majority of studies relating to Treg cells in TLOs are in the cancer field, these cells have also been implicated in suppressing iBALT development in LPS-challenged mice (70) and TLOs in atherosclerosis (45, 47). Where TLOs offer the potential of disease protection, interventions that block Treg cell activities or target their selective depletion may support the development of TLOs and promote anti-cancer or anti-pathogen responses. Such approaches may represent novel routes to patient treatment. However, such immunotherapeutic strategies must delicately balance establishing strong anti-cancer responses with minimizing the development of autoimmunity. This highlights the need to identify approaches for targeting Treg cells within the tumor microenvironment without compromising their role in maintaining immune tolerance.

#### INNATE IMMUNE CELLS ASSOCIATED WITH THE DEVELOPMENT OF TLOs

Innate leukocyte subsets have also been implicated in TLO regulation (**Figure 2**). These include roles for neutrophils (70) and innate lymphoid cells (ILCs) (71–73). Innate lymphocytes have recently emerged as important effector cells with roles in host defense and chronic inflammatory diseases. These cells have been termed ILCs and include cytotoxic ILCs represented by conventional NK cells and three new ILC groups that parallel T helper cell subsets in their cytokine-producing capacity and transcriptional programs (ILC1, ILC2, and ILC3) (74). Of these, ILC3s (which includes both prenatal and adult CCR6+ LTi cells) mirror Th17 cells in their expression of the master transcriptional regulator RORγt; the chemokine receptor CCR6; secretion of IL-17, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF); and responsiveness to IL-23 and aryl hydrocarbon receptor (Ahr) ligands (18, 75–77). It is perhaps these similarities in effector characteristics with Th17 cells that result in ILC3s being involved in TLO development (78). For example, adoptive transfer of adult CD4+CD3− LTi cells into newborn *Cxcr5*−/− mice, which phenotypically lack Peyer's patches and isolated lymphoid follicles (ILFs), promoted the development of intestinal lymphoid tissues (73). Here, IL-7 supported the *de novo* generation, proliferation, and survival of LTi cells. In another study, the same group demonstrated that transgenic overexpression of IL-7 supported the development of LTi cells that formed Peyer's patches, cecal patches and TLOs that displayed functional T cell-dependent B cell responses and GC reactions (72).

Recently, ILC3s have also been associated with tumorassociated TLOs (71). In NSCLC, natural cytotoxicity receptor (NCR)-expressing ILC3s localized to the edge of TLOs and produced IL-22, TNF, IL-8, and IL-2. These NCR+ ILC3s interacted with tumor cells and tumor-associated fibroblasts via their NKp44 receptor to trigger production of LTαβ, which resulted in the activation of endothelial cells and mesenchymal stem cells, including upregulation of ICAM-1 and VCAM-1. Thus, ILC3s correlate with the presence of TLOs in NSCLC and may drive the development of lymphoid structures linked with improved patient survival (49).

Innate lymphoid cells have now been associated with TLO development in a number of experimental models of inflammation or infection. While their presence and generation of prolymphoneogenic cytokines at TLOs is undisputed, their precise role in tertiary lymphoneogenesis remains to be fully elucidated. For example, ILCs and NK cells contribute to an early production of IL-22 that supports TLO development in salivary glands (37). However, while ILCs may contribute to TLO development in this context, the predominant source of IL-22 in this model were αβ and γδ T cells. Likewise, LTi cells have been found in inflamed lungs that develop iBALT. However, development of these lymphoid aggregates was not reliant on LTi cell activity (19). Intestinal TLOs also develop in response to microbiota in RORγt-deficient mice that lack LTi cells (17). Thus, despite mounting evidence for the presence of ILC3s at TLOs, further research is required to determine a precise role in the initiation of tertiary lymphoneogenesis, where tissue- and disease-specific factors may affect their input. Nevertheless given their presence at TLOs, ILC3s may contribute to the function of TLOs where crosstalk with LTβR-expressing stromal cells and the stimulation of B cells *via* B cell-activation factor (BAFF) and the ligand of costimulatory receptor CD40 (CD40L) has been shown to support antibody production (79–81).

In a similar fashion to ILC3s, γδ T cells can also share effector characteristics with activated Th17 cells, including the secretion of IL-17A, IL-17F, IL-22, IL-21, and GM-CSF (78). While the precise role of γδ T cells in TLO development is also unclear, there is evidence that they contribute to early tertiary lymphoneogenesis. In mice displaying iBALT in response to *Pseudomonas aeruginosa* infection, IL-17 triggered stromal cell differentiation into podoplanin+ follicular cells that express CXCL12 (38). Here, γδ T cells were the main source of IL-17. Similarly, development of iBALT in LPS-challenged neonatal mice is also IL-17 dependent, where both γδ and αβ T cells secrete IL-17 (19). Adoptive transfer experiments revealed that while γδ T cells facilitated the development of iBALT, αβ T cells formed larger areas of lymphoid aggregates. It has, therefore, been proposed that an early innate γδ T cell response initiates the development of iBALT, which is later maintained by infiltrating αβ T cells (19, 82). A similar role for γδ T cells in TLO development may occur in salivary glands, where an early prominent IL-22-producing γδ T cell response is later replaced by αβ T cells (37).

Fat-associated lymphoid clusters (FALCs) are TLO-like structures that develop within adipose tissues, including the mesentery, pericardial fat, and milky spots of the omentum (3). Interestingly, milky spots of the omentum increase in number and size during peritoneal dialysis (83, 84). During treatment, catheter insertion, exposure to peritoneal dialysis solution and particularly peritonitis associated with infection results in the expansion and alteration of the cellular composition of milky spots. This suggests an active role for milky spots in peritoneal immunity. Studies in mice demonstrate that the omentum senses peritoneal antigens and represents a site for generating adaptive T cell (CD4+ and CD8+) and B cell responses, including antibody class switching and somatic hypermutation (85, 86). In these studies, the development of milky spots was independent of LTi/ILC3s and LTαβ, but required stromal CXCL13 for the recruitment of B-1 cells. Interestingly, TNF-expressing myeloid cells, NKT cells, and IL-4R signaling were required for FALC formation following inflammatory challenge (86). This study and others have also demonstrated that FALCs can contain ILC2 cells. For example, during helminth infection, Lin−c-Kit+Sca-1+ cells produce Th2-type cytokines that support B-1 cell proliferation and drive goblet cell hyperplasia (87). Therefore, FALCs, including milky spots, play a role in regulating local immune responses. However, while they display similarities to TLOs, they are often less organized structures with fewer T cells and fDCs and are highly enriched for B-1 cells (3, 85).

Dendritic cells prime adaptive immune responses via antigen processing and presentation to T cells. While DCs are a common feature of TLOs, relatively little is known regarding their precise role in tertiary lymphoneogenesis. This gap in knowledge was addressed by two investigations. First, CD11chi DCs were identified as being essential for the long-term maintenance and function of iBALT following influenza virus infection (22). Here, depletion of DCs resulted in a loss of LTβ, CXCL13, CCL21, CCL19, and CXCL12 expression that disrupted the structural integrity and cellular organization of iBALT. This was associated with a reduced number of class-switched plasma cells in the lung and a lowering of antiviral serum IgG titers. In a similar approach, Halle et al. used a replication-deficient modified vaccinia virus Ankara to demonstrate that antigen-loaded DCs migrate into iBALT to support the activation of antigen-specific T cells (21). Thus, TLO-associated DCs are a major source of homeostatic chemokines and lymphoid cytokines that support the long-term maintenance of TLOs and encourage the generation of adaptive immune responses through local T cell priming and control of GC reactions.

#### FOLLICULAR B CELLS DRIVE AUTOANTIBODY-MEDIATED DISEASES AT TLOs

For TLOs to fully recapitulate the function of SLOs they must provide an environment for B cells to undergo affinity maturation and differentiation into memory B cells and antibody-secreting plasma cells (**Figure 3**). Active GCs express activation-induced cytidine deaminase (AID; also known as AICDA), which promotes somatic hypermutation and class-switch recombination to fine-tune antibody specificity and expand antibody-mediated effector functions (88, 89). In support of TLOs being factories for the development of adaptive immune responses, AID is expressed at TLOs in autoimmunity (7, 90–92), infection (93), and transplant rejection (34). Indeed, analysis of the variable (V)-gene repertoires in TLOs from inflamed tissues reveals a restricted profile of encoded sequences, indicating a clonal expansion of antigen-specific B cells within these lymphoid aggregates (91, 94–96). Furthermore, analysis of Iγ–Cμ and Iα–Cμ circular transcript expression reveals on-going class-switch recombination from IgM to IgG and IgA respectively at TLOs (7, 91, 97). Therefore, significant evidence exists to demonstrate that TLOs can contain functional GCs.

While TLOs may generally be considered protective in infection and cancer, GC activity at TLOs in autoimmunity can result in the local generation of disease-specific autoantibodies that perpetuate disease progression. For example, autoreactive plasma cells release autoantibodies, such as anti-citrullinated protein antibodies (ACPA/anti-CCP) in rheumatoid arthritis (7); antibodies targeting ribonucleoproteins Ro (Sjögren's syndrome antigen A; SSA) and La (Sjögren's syndrome antigen B; SSB) in Sjögren's syndrome (98); anti-thyroglobulin and thyroperoxidase antibodies in Hashimoto's thyroiditis (99); and nicotinic acetylcholine receptor-specific antibodies in myasthenia gravis (100). The mechanisms that allow autoreactive B cells to accumulate

within TLOs (101), when they are efficiently eliminated from GCs in SLOs are currently unclear. In SLOs autoreactive B cells become anergic and are excluded from follicular entry through downregulation of CXCR5, the receptor for CXCL13 (102). Emerging evidence points to a potential role for latent Epstein– Barr virus (EBV) in the development of autoimmunity at TLOs. EBV is a life-long infection, and infected B cells display increased proliferation and survival (103). EBV-infected cells are often observed within TLOs in the inflamed tissues of patients affected by autoimmunity, including rheumatoid arthritis, Sjögren's syndrome, multiple sclerosis, and myasthenia gravis (92, 104–106). For example, infected B cells display autoreactivity toward citrullinated fibrinogen and ribonucleoprotein Ro, the disease-specific autoantigens for rheumatoid arthritis and Sjögren's syndrome, respectively (105, 106). Therefore, EBVinfected autoreactive B cells may migrate to target tissues where they differentiate into autoantibody secreting plasma cells and perpetuate autoimmunity.

In addition to their classical role in generating antibody responses, B cells also support local immune cell activation within inflamed tissues. Using an experimental model where synovial biopsies comprising TLOs from rheumatoid arthritis patients are transplanted into severe combined immunodeficient mice (the HuRA-SCID model), Takemura and co-workers demonstrated that depletion of B cells from grafted synovial tissue resulted in a reduction in T cell derived IFNγ production and IL-1β secretion (107). Hence, synovial B cells contribute to T helper cell effector responses in rheumatoid synovitis to influence disease progression.

Typically when addressing the role of B cells in TLOs, the development of antigen-specific antibody responses is the primary consideration. However, B cells have long been known to produce LTαβ, which suggested a potential role in lymphoid neogenesis or the maintenance of lymphoid organs (108). More recently, a role for B cells in promoting the development of ILFs in the small intestine was described (109). Studies using bone marrow chimeric mice demonstrated that LTαβ-producing B cells were required for the development of these TLO-like structures. Similarly, Lochner et al. describe a LTαβ-dependent LTi-like role for B cells in the development of TLOs in dextran sulfate sodium (DSS)-induced colitis (17). This suggests that deploying B cell targeted therapies for the treatment of TLO-associated autoimmune diseases, such as rheumatoid arthritis has the potential to interfere with both early initiation of TLO development and the long-term maintenance of autoantibody responses and local T cell priming. However, in this context, an improved understanding of B cell targeted therapies (e.g., rituximab) is needed, where studies evaluate the peripheral effect on SLOs as well as the local impact on TLOs in inflamed tissues.

#### IMMUNE CELL–STROMAL CELL CROSSTALK IS CENTRAL TO TLO DEVELOPMENT AND FUNCTION

Our understanding of immune cells in the development, function, and maintenance of TLOs has greatly expanded in recent years. While this review is primarily focused on the role of immune cells, it is also important to emphasize the importance of immune cell–stromal cell crosstalk in the development and function of TLOs [comprehensively reviewed elsewhere (110)]. While immune cells adopt LTi-like functions to drive the development of TLOs during inflammation, activated resident stromal cells must phenotypically respond like LTo cells. For example, lung inflammation resulting in the development of iBALT in mice is dependent on immune cell derived IL-17, which triggers CXCL13 and CCL19 expression to recruit and organize lymphocytes (19). While early induction of CXCL13 and CCL19 expression was LTα-independent, once established, homeostatic chemokines and engagement of the LTβR was required for the maintenance of iBALT. Such observations suggest that the dual targeting of both IL-17 and LT signaling may be beneficial for the management of diseases where TLOs are a feature of local pathology. Similar studies using an influenza infection model revealed PNAd+ HEVs and stromal cells are the primary source of CXCL13, CCL21, and CCL19 during iBALT development (111). In other infection models that feature TLOs, effector cytokines, such as IL-17 and IL-22, have been shown to drive stromal cell differentiation toward podoplanin-positive CXCL12 and CXCL13 expressing cells (37, 38). Such experimental observations also translate into human disease, where the stromal cell response to inflammation contributes to TLO development. For example, in rheumatoid arthritis patients who display TLOs in inflamed joint tissue, synovial fibroblasts display LTo-like properties including the production of homeostatic chemokines and the induction of BAFF, which supports synovial B cell responses (112–114). Recently, several illustrative examples have further emphasized the significance of immune cell–stromal cell interactions. A highly novel role for TLOs as niches for the maturation of malignant hepatocellular carcinoma progenitor cells was recently described (115). Here, carcinoma progenitor cells exiting TLOs supported tumor growth and outline a detrimental role for TLOs in cancer. Consequently, further work is required to establish the context in which TLOs contribute to the tumor microenvironment. Crosstalk between Th17 cells and stromal cells was also recently shown to be important in an experimental model of multiple sclerosis, where stromal LTβR signaling promoted extracellular matrix deposition, T cell effector cytokine responses, and chemokine production that supported leukocyte retention in the meninges (116). Similarly, in experimental atherosclerosis, vascular smooth muscle cells provided LTo-like function within atherosclerotic aortas to support the development and maintenance of protective TLOs (45). Thus, while immune cells provide a trigger for the development of TLOs in inflamed tissues, the response of stromal cells is equally important in providing an environment conducive to lymphoid neogenesis.

## CONCLUDING REMARKS

Studies in recent years have increasingly highlighted potent roles for TLOs in regulating local immune responses in conditions featuring chronic inflammation. Experimental models of disease have provided mechanistic insight and identified novel immune cell subsets involved in TLO regulation, as well as verifying a role for TLOs in pathologic processes. Similarly, studies in human diseases have clearly demonstrated the presence, function, and correlative associations of TLOs with disease severity. However, further research is required to better define the precise role of TLOs in these clinical conditions. Here, it will be important to evaluate the prognostic and diagnostic potential of TLOs in specific diseases, their potential as novel therapeutic targets, as well as to determine how diseases with significant TLO involvement respond to the current arsenal of biologic interventions used in routine clinical practice. In this regard, research into the impact of TLOs in rheumatoid arthritis has provided valuable insight. For example, evidence suggests that rheumatoid arthritis patients with TLOs in inflamed joint tissues display an inferior response to frontline biological therapies that target TNF (8). Therefore, patients with synovial TLOs may be managed better using alternative biological therapies. Given the central role played by TLO B cells in the production of disease-specific autoantibodies, rituximab (an anti-CD20 B cell depleting antibody) may be a better alternative. While further research is needed in this area, two studies oppose this prediction. The first demonstrates that while rituximab treatment results in a reduction of disease-specific autoantibodies (rheumatoid factor and ACPA/anti-CCP) in patient serum, this treatment failed to reduce the local production of these autoantibodies in lymphoid aggregate-containing joint tissue (9). The second study in chronic renal allograft rejection similarly demonstrates that while rituximab treatment depleted peripheral B cells, surprisingly, intragraft B cells in TLOs evaded depletion (117). Thus, B cells residing within the TLO microenvironment may receive signals that allow them to survive rituximab treatment (117). Given the recent emergence of IL-17, and Th17- and Tfh-associated cytokines in the development of TLOs, it will now be interesting to see how targeting these axes fares in the management of TLO-associated diseases. While IL-17-targeted modalities have shown limited clinical efficacy in inflammatory arthritis (118, 119), these clinical trials did not stratify patients for the presence of synovial TLOs. In inflammatory arthritis, sampling of synovial tissue through ultrasound-directed biopsies has become more common in clinical trial design, and have provided new insight into the impact of intervention on TLOs (120). Such approaches may pave the way to identifying optimal strategies for the clinical management of TLO-associated diseases. The success of such approaches may prove important is shaping how clinicians evaluate the diagnosis and treatment of other chronic conditions.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

GJ, DH, and SJ co-wrote the manuscript and prepared the figures.

#### FUNDING

GJ and SJ are supported by career development fellowship and programme grant funding from Arthritis Research UK (20305, 20770). DH is funded by a Medical Research Council Ph.D. studentship.

distinguishes colorectal cancer patients from healthy controls and provides a target for *in vivo* therapy. *Cancer Immunol Res* (2013) 1(6):416–25. doi:10.1158/2326-6066.CIR-13-0035

and Ig class-switching in B cells. *Ann Rheum Dis* (2011) 70(10):1857–65. doi:10.1136/ard.2011.150219

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Jones, Hill and Jones. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Model-Driven Experimentation: A New Approach to Understand Mechanisms of Tertiary Lymphoid Tissue Formation, Function, and Therapeutic Resolution

*James A. Butler1,2,3† , Jason Cosgrove1,2,3† , Kieran Alden2,3 , Jon Timmis2,3 and Mark Christopher Coles1,3 \**

*1Centre for Immunology and Infection, Department of Biology, Hull York Medical School, York, UK, 2Department of Electronics, University of York, York, UK, 3York Computational Immunology Laboratory, University of York, York, UK*

#### *Edited by:*

*Andreas Habenicht, Ludwig Maximilian University of Munich, Germany*

#### *Reviewed by:*

*Jorge Caamano, University of Birmingham, UK Neil Macritchie, University of Glasgow, UK*

*\*Correspondence:*

*Mark Christopher Coles<EMAIL_ADDRESS>
*†*

*Co-first authors.*

*Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 26 August 2016 Accepted: 16 December 2016 Published: 04 April 2017*

#### *Citation:*

*Butler JA, Cosgrove J, Alden K, Timmis J and Coles MC (2017) Model-Driven Experimentation: A New Approach to Understand Mechanisms of Tertiary Lymphoid Tissue Formation, Function, and Therapeutic Resolution. Front. Immunol. 7:658. doi: 10.3389/fimmu.2016.00658*

The molecular and cellular processes driving the formation of secondary lymphoid tissues have been extensively studied using a combination of mouse knockouts, lineage-specific reporter mice, gene expression analysis, immunohistochemistry, and flow cytometry. However, the mechanisms driving the formation and function of tertiary lymphoid tissue (TLT) experimental techniques have proven to be more enigmatic and controversial due to differences between experimental models and human disease pathology. Systemsbased approaches including data-driven biological network analysis (gene interaction network, metabolic pathway network, cell–cell signaling, and cascade networks) and mechanistic modeling afford a novel perspective from which to understand TLT formation and identify mechanisms that may lead to the resolution of tissue pathology. In this perspective, we make the case for applying model-driven experimentation using two case studies, which combined simulations with experiments to identify mechanisms driving lymphoid tissue formation and function, and then discuss potential applications of this experimental paradigm to identify novel therapeutic targets for TLT pathology.

Keywords: multi-scale modeling, tertiary lymphoid tissue, systems immunology, mechanistic modelling, modeldriven experimentation

#### FORMATION AND FUNCTION OF SECONDARY AND TERTIARY IMMUNE MICROENVIRONMENTS

Lymphoid tissues are responsible for the orchestration of functional immune responses. This is achieved through the development and maintenance of niches that support the retention, activation, and proliferation of adaptive immune cells in response to antigenic stimulation. Adult lymphoid tissue architecture is organized by an underlying network of stromal cells that produce extracellular matrix (e.g., collagens) and provide survival (e.g., BAFF, IL-7), migratory (CCL19/21, CXCL13), and immune activation [the storage and presentation of immune complexes by follicular dendritic cell (FDC)] signals (1). Distinct stromal subsets with unique secretion profiles (chemokines, other cytokines, and survival factors) develop in response to signaling from lymphocytes with a key role for TNF superfamily receptors; this stromal– lymphocyte cross talk ensures the correct cell type is stimulated (or regulated) at the right time and place. Sustained cross talk between mesenchymal stroma and lymphocyte subsets is a core feature of lymphoid tissue formation and maintenance and occurs irrespective of the tissue type or anatomical location.

Formation of lymphoid tissues can occur by different cellular and molecular mechanisms. During fetal development, secondary lymphoid tissues form in a process dependent on the RARrelated orphan receptor gamma transcription factor expressing lymphoid tissue inducer cells (LTi) responding to localized chemotactic gradients leading to the formation of lymph nodes (LN) and Peyer's patches (PPs) in a lymphotoxin β (LTβ) dependent process (2). Localized mesenchyme, lymphoid tissue organizer (LTo) cells differentiate into adult marginal reticular cells, fibroblastic reticular cells, and FDCs (3). Likewise, in the adult, innate lymphoid cells type 3, the adult equivalent of LTi cells, have a key role in regulating cryptopatches that can mature into isolated lymphoid follicles (4). These specialized lymphoid structures contain predominantly B cells and often contain germinal center (GC) reactions.

In humans, tertiary lymphoid tissues (TLTs) are found in inflammatory immune responses associated with chronic pathology from hip joint replacements, keloids, tissues in autoimmune disease (e.g., the salivary gland in Sjogren's syndrome, multiple sclerosis, and rheumatoid arthritis) to solid tumors and follicular lymphomas in the bone marrow (5–9). Although the role of specific cell types has been controversial, there is an emerging paradigm of a multistep process where localized inflammation induces stromal cell activation in a lymphocyte independent process, leading to localized microenvironments permissive for T and B cells entry (10). These lymphocytes have the potential to drive the formation of organized tertiary tissue in an autocrine-dependent process. This process closely resembles the capacity of naive B cells to drive B cell follicle formation in secondary lymphoid tissues in a TNFα- and LTβ-dependent process and the capacity of activated B cells to generate the GC, a transient microenvironment that drives high-affinity immune responses in a self-regulating autocrinedependent process. In both secondary immune tissues (LN, PPs, and spleen) and TLTs including ILFs and TLT, activated B cells prime the formation of the GC reaction. This specialized microenvironment contains both activated and proliferating B cells and different stromal compartments of CXCL12-secreting stroma (dark zone) and CXCL13-secreting FDCs (light zone). This facilitates the cyclic selection and expansion of antigenspecific B cells (11).

Non-lymphoid inflammatory immune structures, granulomas, can form in the liver, intestine, adipose tissue (crown-like structures), and lung induced by chronic infection/inflammation associated with tuberculosis, sarcoidosis leishmaniasis, schistosomiasis, cell death, and Crohn's disease (12–14). The formation of these highly dynamic microenvironments superficially resembles TLT; however, their formation and organization is driven by activated macrophages rather than by the mesenchymal–lymphocyte cross talk observed in lymphoid tissues thus do not exhibit lymphocyte compartmentalization. Granuloma structures are very heterogeneous in presentation within individual patients in a continuum between early macrophage centric granulomas, self-resolving granulomas, and fibroblastic structures, these often being fibrotic rather than taking on a supportive stromal network phenotype. The triggers that drive granuloma formation instead of TLT formation appear not to be due to differences in the different chemotactic cues delivered by activated macrophages compared to those delivered by activated stromal fibroblasts, leading to a very different cellular make up to the inflammatory foci of leukocytes [primarily myelomonocytic (granuloma) vs. lymphocytic (TLT)].

#### CURRENT APPROACHES TO STUDYING LYMPHOID TISSUE FORMATION: LIMITS, CHALLENGES, AND NEW APPROACHES

Experimental studies, principally performed in gene knockout, lineage-specific fluorescent protein, and Cre reporter mouse lines have contributed significant insights into the roles of multiple different cell types and molecules in lymphoid tissue formation and function. This has been further validated using histology and flow cytometry analysis on human secondary lymphoid tissues. However, in contrast to secondary lymphoid tissues, there are some distinct differences in human tissue pathologies to those found in mice including the cellular composition of TLTs, granulomas, and other inflammatory tissues. This arises in part from genetic and physiological differences between human and mice including the timing and duration of the immune response (chronic vs. acute inflammation), the inflammatory triggers (infection, autoimmunity, and cancer), and transcriptional differences in immune cells in the different species. In general, mouse models of immune-mediated inflammatory disease are acute and fail to replicate the chronic human disease characterized by disease flairs followed by remission, limiting their translational capacity to human disease. Infection and tumor models in mice either rapidly resolve (too quickly for chronic pathology to establish) or lead to the mouse having to by euthanized for health and welfare prior to tertiary lymphoid pathology occurring. In comparison, humans may live the rest of their life with the disease pathology, particularly in the context of treatment with biologics and small molecules; thus, pathology has the opportunity to evolve from localized inflammation to fibrotic tissue failure, systemic inflammation, and autoimmunity working together to prevent disease resolution. Increasingly, human 3-dimensional tissue culture models containing both stroma and lymphocytes have become increasingly common and useful in understanding underlying molecule mechanisms of TLT formation. However, it is not currently possible to represent the full complexity of chronic human pathology *in vitro*.

Experimental systems (*in vivo* and *in vitro*) to date have proven limited in their ability to explain chronic clinical pathology and resolve established Sjogren's pathology, although TNF has an important role in FDC differentiation and B cell organization, anti-TNF fails to induce resolution disease (15). To better understand the form and function of TLTs, current knowledge of stromal regulation through molecular signals and immune cell behavior within lymphoid tissue must be consolidated and considered in a quantitative, systems-based approach. The development of systems-level stochastic computational models can bring together a broad understanding across spatiotemporal scales of how genetic and molecular factors relate to cellular and tissue level form and function and give rise to the complex, functional architectures observed in secondary lymphoid organs and disease-specific TLTs. These models permit *in silico* experimentation providing a unique platform driving further experimentation and assessing novel mechanistic targets and intervention strategies where *in vivo* observed heterogeneity can be replicated.

Alan Turing (of code breaking fame) in seminal early work in mathematical biology (16) noted that gastrulation arose from symmetry breaking, and this leads to fundamental insights and principles that drive modern mathematical and computational biology: the notion that chaotic, non-linear behavior of individual biological processes, including the self-organization of complex biological structures (e.g., TLT), can result in emergent properties that cannot be understood from consideration of each individual component in isolation. The development of models that capture the essential, emergent behavior of specific biological processes, with extraneous components excluded, enables understanding of how complex molecular and cellular interactions govern complex, emergent biological processes and can therefore lead to new insights and quantitative predictions (17). Emergent properties in a TLT model would include stromal networks, lymphocyte organization, migration and interactions with antigen-presenting cells, and localized cytokine/chemokine production.

#### APPLICATION OF MODEL-DRIVEN EXPERIMENTATION (MDE) TO UNDERSTAND MECHANISMS OF LYMPHOID TISSUE DEVELOPMENT AND FUNCTION

Advances in computing resources and computational modeling technology have provided the capacity to generate complex *in silico* models of lymphoid tissues that incorporate space, time, and cellular heterogeneity found in immune tissues including TLT. Applying *in silico* approaches to understand secondary lymphoid tissue formation and function requires the integration of experimental data across cellular, molecular, and tissue levels of organization. Ensuring that the biological processes are appropriately described requires a fine balance between model abstraction and interpretation (quantitative and qualitative) of experimental data. A number of different modeling approaches may be utilized (summarized in **Table 1**), increasingly, integration of different mathematical/computational techniques into a hybrid model is a common strategy to address the limitations of using each technique in isolation. This approach also facilitates the consolidation of data across different levels of organization (molecular, cellular, tissue, and patient) into a single multiscale model. For example, an agent-based model can capture an individual cell, which in turn incorporates a differential equation-based model capturing a "lower-level" aspect of that individual's behavior, such as surface expression of a receptor (42). Adopting an *in silico* approach provides a platform that can

TABLE 1 | Mathematical and computational techniques for modeling immune processes.

provide insights and generate predictions that can be verified *in vivo*: verification that can lead to increased biological understanding and incrementally improved *in silico* models for further experimentation. This iterative approach of combining *in vivo*, *in vitro*, and *in silico* approaches has been termed "model-driven experimentation" (18).

#### CASE STUDY 1: INSIGHTS FROM MDE TO SECONDARY LYMPHOID TISSUE FORMATION

Peyer's patches are specialized secondary lymphoid tissues of the intestine that develop during a fixed window in fetal development and have an essential role in maintaining intestinal immunity. PPs form stochastically along the midgut, with mice developing 8–12 patches; however, as the absence of or reduction in the number of PPs is observed in several different gene knockouts, the molecular process that triggers patch formation was unclear (19). Using an MDE-based approach had the potential to provide new insight into how different signaling pathways (RET, chemokine receptors, cytokine receptors, TNF superfamily, and adhesion molecules) might integrate to induce PP development *in silico* and to subsequently design key experiments to test hypotheses *in vivo*. PPSim is an agent-based PP simulator that captures key processes during the 72-h period of tissue development in prenatal mice and replicates (statistically similar) emergent cell behaviors found *in vivo,* specifically populations of hematopoietic cells, known as lymphoid tissue initiator (LTin) and lymphoid tissue inducer (LTi) cells, migrates into the developing gut, with data from laboratory observations suggesting these cells follow a random motion. Both cell populations express receptors for the adhesion molecule VCAM-1, expressed by stromal LTo cells residing in the gut wall (20, 21). In this computational model, LTi and LTin are captured as individual entities that migrate into the developing midgut serosa and undergo a random walk, interacting with their localized simulated environment through signaling pathways including GDRFs/Ret signaling pathways, adhesion molecules, and chemokine receptors, as is observed *in vivo*. On ensuring PPsim adequately represented individual cell responses, statistical analysis techniques, specifically sensitivity analyses, were used to explore mechanisms driving prenatal lymphoid organ formation (22, 23). This exploration of the simulated biological pathways revealed which pathways had significant impacts on simulated cell behavior at different time points during PP development. By examining correlations in the level of activity of simulated pathways and cell behavior, the hypothesis was derived that contact between LTin and LTo cells that leads to the localized upregulation of VCAM-1 on stromal cells was the key triggering event that determined the site of PP formation on the midgut (21). Utilizing this prediction, an *in vitro* assay imaging fetal midgut explants incubated in the presence or absence of anti-VCAM-1 antibodies was developed. Using this assay, it was verified that early upregulation of VCAM-1 was the triggering event that was essential for the initiation of LTi and LTin cell clustering. The model simulation results, supported by replicated experimentation and safety-critical systems-based fitness-forpurpose argumentation that details the knowledge integration in model composition, provide evidence that the simulation was fit for the purpose of aiding exploration of this specific research question: understanding the triggering of lymphoid tissue development, which was not possible by conventional genetic approaches (24, 25).

#### CASE STUDY 2: APPLYING MDE TO UNDERSTAND GC DYNAMICS AND FUNCTION

The GC reaction is a transient microenvironment in which affinity maturation occurs in response to immunization and infection, bearing key similarities to TLT in its evolution in the role of lymphocytes in inducing highly organized stromal networks, the essential role of TNF superfamily members in regulating its induction and the induction of chemokine gradients (10, 26). However, in comparison to TLT, the GC is a self-resolving tertiary lymphoid microenvironment. Recent technological advances, particularly the advent of intravital multiphoton imaging including photo-activated fluorescent proteins has led to the unprecedented availability of data on the dynamics B-cell migration and selection (27–30). However, imaging datasets provide a narrow window of insight into a process that occurs over a timescale of days and weeks. Furthermore, as imaging techniques are optimized for a given time and length scale, they are limited in their ability to link molecular, cellular, and tissue level processes. This has made the interpretation of imaging datasets in the context of the wider literature challenging. To address this issue, modeling approaches have been used to test the validity of different hypotheses of mechanisms controlling B-cell migration and selection within the GC (31–34).

In the GC reaction, model-derived insights have proved useful not only in the analysis of existing datasets but also as a driver for further experimentation. Specifically, an MDE approach has been used to examine the effects of antibody feedback on the process of affinity maturation (35). Analysis of an *in silico* GC reaction yielded the prediction that GC B-cells, which require antigen on FDCs for positive selection, were competing for antigen by early low-affinity antibodies. Only higher affinity B-cells were able to outcompete for antigen to receive the necessary survival signals. To experimentally validate this prediction, the authors manipulated the GC response with monoclonal antibodies of defined affinities and were able to confirm that antibody feedback provides a dynamic selection threshold to maximize Ig affinities (35). A similar approach was employed to investigate the role of toll-like receptor 4 (TLR4) on the GC where an iterative cycle of *in silico* and *in vivo* experimentation dissected the importance of TLR4 signaling on the maturation of FDCs, key regulators of B-cell selection in the light zone of the GC (36). Both of these MDE examples highlight the use of *in silico* experimentation as a means of refining experimental design through the identification of key time points and conditions to test *in vivo*. These case studies together provide example of how theoretical models can consolidate data from different

#### TABLE 2 | Key questions on tertiary lymphoid tissue (TLT) formation and maintenance that can be address in hybridized TLT models.

#### Formation

What are the minimum cellular requirements to initiate TLT formation? Is this driven by different types of stroma, lymphocytes, dendritic cells, or tissueresident macrophage?

What is the relative importance of inflammation and antigen in TLT induction? Is autoantigen required for induction or just an outcome of the pathology? What is the role of different cytokines and chemotactic signals on TLT formation?

#### Maintenance

What is the relative role of inflammatory cytokines, lymphocyte—stromal cross talk, immune cell entry, cell death, antigenic stimulation on TLT maintenance?

What are the key signaling pathways required to maintain TLT once it has formed? Can these pathways be targeted to induce TLT resolution?

Can TLT self-resolve in humans? If so, what is the balance between new TLT induction and resolution of existing structures?

sources as a platform for the development novel hypotheses and a driver for further experimentation.

#### PERSPECTIVE ON MDE AS APPLIED TO TLT FORMATION, FUNCTION, AND THERAPEUTIC RESOLUTION

When computational modeling is combined with knowledge derived from imaging, multi-dimensional cytometry, and gene expression analysis of human TLT pathology, MDE has the potential to provide novel insights to key questions on molecular and cellular mechanisms involved in TLT formation, maintenance, and function similar to its capacity to impact on our understanding of lymphoid stromal network and granuloma dynamics (**Table 2**) (37–40). One of the key advantages of applying multi-scale modeling is it permits capture of a wide range of different phenomena that occur on different orders of magnitude in terms of time and length scales that are critical in the stochastic processes involved in TLT induction. These include different cell types, states and interactions, inflammatory molecules, extracellular matrix, adhesion molecules, and chemotactic signals all in the context of an evolving tissue microenvironment. Developing *in silico* models permits temporal inhibition of different signaling pathways and cellular depletions during different stages of TLT pathology using statistical tools (**Figure 1**). This permits identification of key pathways that could be targeted to induce resolution of pre-existing TLT rather than inhibiting its formation as has been used to make *in silico* predictions for the treatment of tuberculosis (41). A large number of novel antibody therapies, biologics, and small molecular inhibitors have been developed to target immune function for the treatment of immune-mediated inflammatory diseases. These therapies are unlikely to show maximal efficacy against existing tissue pathology when used as monotherapies, rather it is more likely that use of therapeutic combinations that is most likely to show clinical efficacy. The clinical challenge is that there are already over 20,000 possible different

combinations using existing therapeutics that would need to be trialed to find optimal targeting strategy to resolve TLT pathology. Thus, MDE-based approaches provide a rational approach to identify novel combination therapeutic regimes that have a best potential in clinical trials (42).

Although the adoption of MDE has only recently started to impact on immunology research, it is starting to have a very significant impact on other areas of biology. We propose that the increased accessibility of computational models, the highperformance computing resources, the increased familiarity and understanding of simulations as tools to understand immune function, and the capacity to apply *in silico* approaches to identify potential therapeutic approaches and disease biomarkers will accelerate the application of MDE as a methodology understand and target disease resolution.

#### AUTHOR CONTRIBUTIONS

MC wrote this paper and is the corresponding author. JB, JC, KA and JT contributed to the writing of this perspective.

#### FUNDING

JC is supported by Wellcome Trust (Computational Approaches in Translational Science) WT095024MA. Research in MC providing a basis for this perspective is supported by MRC MR/ K021125/1.

#### REFERENCES

42. Cosgrove J, Butler J, Alden K, Read M, Kumar V, Cucurull-Sanchez L, et al. Agent-based modeling in systems pharmacology. *CPT Pharmacometrics Syst Pharmacol* (2015) 4(11):615–29. doi:10.1002/psp4.12018

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Butler, Cosgrove, Alden, Timmis and Coles. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Early IL-1 Signaling Promotes iBALT Induction after Influenza Virus Infection

*Katrijn Neyt1,2 , Corine H. GeurtsvanKessel3 , Kim Deswarte1,2 , Hamida Hammad1,2 and Bart N. Lambrecht1,2,4 \**

*1 Laboratory of Immunoregulation, VIB Inflammation Research Center, Ghent, Belgium, 2Department of Respiratory Medicine, Ghent University, Ghent, Belgium, 3Department of Viroscience, Erasmus MC, Rotterdam, Netherlands, 4Department of Pulmonary Medicine, Erasmus MC, Rotterdam, Netherlands*

Inducible bronchus-associated lymphoid tissue (iBALT) is a long lasting tertiary lymphoid tissue that can be induced following influenza A virus (IAV) infection. Previous studies have shown that iBALT structures containing germinal center (GC) B cells protect against repeated infection by contributing locally to the cellular and humoral immune response. If we are to exploit this in vaccination strategies, we need a better understanding on how iBALT structures are induced. One hypothesis is that the strength of the initial innate response dictates induction of iBALT. In the present study, we investigated the role of interleukin (IL)-1 and IL-1R signaling on iBALT formation. Mice lacking the IL-1R had a delayed viral clearance and, thus, a prolonged exposure to viral replication, leading to increased disease severity, compared to wild-type mice. Contradictorily, iBALT formation following clearance of the virus was heavily compromised in *Il1r1*−/− mice. Quantification of gene induction after IAV infection demonstrated induction of IL-1α and to a much lesser extent of IL-1β. Administration of recombinant IL-1α to the lungs of wild-type mice, early but not late, after IAV infection led to more pronounced iBALT formation and an increased amount of GC B cells in the lungs. Bone marrow chimeric mice identified the stromal compartment as the crucial IL-1 responsive cell for iBALT induction. Mechanistically, Q-PCR analysis of lung homogenates revealed a strongly diminished production of CXCL13, a B cell-attracting chemokine, in *Il1r*−/− mice during the early innate phase of IAV infection. These experiments demonstrate that appropriate innate IL-1α–IL-1R signaling is necessary for IAV clearance and at the same time instructs the formation of organized tertiary lymphoid tissues through induction of CXCL13 early after infection. These findings are discussed in the light of recent insights on the pathogenesis of tertiary lymphoid organ formation in the lung in various diseases where the IL-1 axis is hyperactive, such as rheumatoid arthritis and COPD.

Keywords: influenza, innate immunity, TLO, IL-1, iBALT, CXCL13

## INTRODUCTION

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal or pandemic outbreaks with severe outcome in elderly and immune compromised patients. Epithelial cells are the first target cells for IAV infection (1, 2) and also coordinate the innate immune defense to prevent spreading of the virus, *via* production of type I interferons (IFNs). Interleukin (IL)-1α and IL-1β are among the first

#### *Edited by:*

*Andreas Habenicht, Ludwig Maximilian University of Munich, Germany*

#### *Reviewed by:*

*Fiona Jane Culley, Imperial College London, UK Cecilia Johansson, Imperial College London, UK Thomas Tschernig, Saarland University, Germany*

> *\*Correspondence: Bart N. Lambrecht<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 10 May 2016 Accepted: 02 August 2016 Published: 16 August 2016*

#### *Citation:*

*Neyt K, GeurtsvanKessel CH, Deswarte K, Hammad H and Lambrecht BN (2016) Early IL-1 Signaling Promotes iBALT Induction after Influenza Virus Infection. Front. Immunol. 7:312. doi: 10.3389/fimmu.2016.00312*

cytokines that are secreted by epithelial cells and macrophages at sites of IAV replication (3). Secretion of IL-1β requires activation of the Nlrp3 inflammasome that leads to activation of caspase-1 and cleavage of pro-IL-1β into IL-1β. Infection with IAV leads to activation of the Nlrp3 inflammasome in a process requiring the type I IFN-induced RNAse L/OAS system, while the virus actively suppresses IL-1β production and Nlrp3 activation *via* the NS1 protein (4–6). IL-1 induces the expression of endothelial adhesion molecules that promote the entry of innate inflammatory cells like neutrophils, NK cells, dendritic cells (DCs), and monocytes resulting in a double effect on the host. On the one hand, it promotes survival by killing virus-infected cells, clearing debris, and alarming the adaptive immune response. On the other hand, overzealous neutrophil recruitment can also cause inflammatory pathology that can ultimately lead to diffuse alveolar damage and death (6–10). Not surprisingly, the outcome of genetic deficiency of key components in IL-1 generation or signaling has been very different depending on the severity of the IAV infection (2, 9, 11).

Simultaneously with the activation of the innate immune response, adaptive immune responses are initiated in the draining lymph nodes by antigen-presenting migratory DCs. The architecture of lymph nodes promotes contact between antigen-presenting DCs and rare antigen-specific T cells and B cells of the adaptive immune system to maximize the immune response against a certain antigen. Antigen-specific T lymphocytes undergo clonal proliferation upon encounter with antigen presented by antigen-presenting cells and migrate back to the site of inflammation as T effector memory (TEM) cells or become central memory T cells (TCM) or T resident memory (TRM) cells (12). Antibody production is initiated from B lymphocytes that differentiate into plasmablasts, immediately, or become plasma cells after going through a germinal center (GC) reaction that promotes somatic hypermutation and affinity maturation of B cells (13).

The coordinated events of T and B cell activation induced by virus-laden DCs mainly occur in secondary lymphoid organs (SLOs) like lymph nodes and spleen that develop during embryogenesis at predefined areas, often at the crossroads of lymphatic vessels (14). However, highly organized structures of T and B cells can also be formed in the lung after birth as an adaptation to the increased demand for a localized immune response. Various names, such as lymphoid tissue neogenesis, ectopic lymphoid tissue, and tertiary lymphoid structures have been used to describe these structures. Furthermore, they are often named after the anatomical region in which they occur. In the lungs, for example, lymphoid aggregations can often be found in close proximity to bronchi, and hence, these are called inducible bronchus-associated lymphoid tissues (iBALT). As they resemble SLOs anatomically and functionally, yet, only develop after birth as a result of chronic immune stimulation, they can also be called tertiary lymphoid organs (TLOs), even when found within the boundaries of another organ.

Tertiary lymphoid organs have been implicated in protection against IAV. Mice that lack SLOs can mount a rapid CD8 T cell response during IAV infection due to the induction iBALT (15), induced after clearance of IAV infection. Such TLO structures are generally formed in close proximity to bronchi after IAV infection, but can also be observed in the lung interstitium and are fully formed 17 dpi. iBALT structures that are induced in mice with functional lymph nodes can serve as an additional priming site for T cells (16) and can also contribute to the humoral immune responses (17). Once formed, iBALT structures can mount high affinity immune responses to other antigenic stimuli than the initiating stimulus due to the presence of GCs that allow somatic hypermutation and affinity maturation, complementing the immune response in the draining lymph nodes (16, 18). Furthermore, iBALT structures could be the perfect environment for depots of viral antigen that were described long after viral clearance and recovery from viral infection and possibly related to the induction or maintenance of virus-specific TRM cells (19, 20).

Despite morphological and functional similarities between SLOs and TLOs, the pathways that control formation and maintenance of TLOs are less clear. Generally, the molecular pathway that organizes T and B cells in discrete areas resembles the highly regulated inductive pathway for SLO development. Production of CCL19, CCL21, CXCL12, and CXCL13 by stromal cells and B cells helps to organize and retain T and B cells in discrete areas (21–26). Also IL-7 seems required for TLO formation, e.g., in joints of rheumatoid arthritis patients and mouse models (27–30) and lungs of idiopathic pulmonary hypertension (IPAH) patients (31). It is more controversial which cells give the initial instruction for stromal cells to produce these chemokines. During SLO development in the fetal period, this is the distinct task of lymphoid tissue inducer (LTi) cells, a cell type that develops from Flt3+ and Flt3− α4β7 integrin-positive common progenitors that also forms innate lymphoid cells (ILC), and get expanded in response to Flt3L injections (32, 33). During formation of the lymph nodes and spleen, LTi cells provide signals for lymphoid organogenesis like lymphotoxinbeta (LTβ) acting on the LTβR on stromal cells, but the precise signals might differ from organ to organ. *Flt3l*−/− mice for example have reduced LTi cells and lack Peyer's patches, but still have lymph nodes (33). However, research in *Id2*−/− and *Rorc*−/− mice, which lack LTi cells, showed that LTi cells were dispensable for the initial TLO induction after IAV infection or other forms of TLO induction (15, 34–38). Although LTi cells seem not strictly necessary for TLO induction, an instructive LTβ–LTβR signal remains essential for proper TLO development (25, 39). B cells, T cells, and DCs are heavily induced during inflammatory processes and all express LTα1β2 on their cell surface (17, 35, 40, 41); therefore, they are perfect candidates to function as a substitute for LTi cells.

Whatever the precise molecular mechanisms of TLO induction might be, these TLO structures are virtually always seen at sites of inflammation. Yet, which inflammatory cytokines contribute to TLO induction is currently unknown. After many insults to the lung, including viral or bacterial infection, IL-1α and IL-1β are among the first cytokines to be secreted (3). IL-1 secretion induces the expression of endothelial adhesion molecules that promote the entry of innate and adaptive immune cells and could, thus, promote TLO formation. On the other hand, it is also known that IL-1R−/− mice have a delayed viral clearance and, thus, a longer exposure to viral particles (7). Chronic immune stimulation is often assumed to lead to TLO formation (42). If IL-1 limits viral replication, it could reduce the trigger for TLO induction.

In this paper, we addressed the role of IL-1 and IL-1R in TLO formation in the lung. We show that the iBALT-inducing events are initiated early after infection, long before the virus is cleared. More specifically, we show that early IL-1R signaling is necessary for proper IAV-associated iBALT and GC induction and that prolonged viral presence does not automatically lead to iBALT induction.

## MATERIALS AND METHODS

#### Ethics Statement

All experiments were approved by the independent animal ethics committee "Ethische Commissie Proefdieren – faculteit Wetenschappen Universiteit Gent en VIB-site Ardoyen" (identification number: EC 2013\_070). Animal care and used protocols adhere to the Belgian Royal Degree of 29th May, 2013 for protection of experimental animals. European guideline 2010/63/EU is incorporated in this Belgian legislation.

#### Mice

C57Bl/6 mice (8–10 weeks) were purchased from Harlan Laboratories. IL-1R−/− mice were bred in-house and housed in specific pathogen-free housing.

To create chimeric mice, IL-1R−/− or wild-type acceptor mice were irradiated sublethally (9 Gy) and reconstituted with 2 × 106 bone marrow cells i.v. from wild-type or IL-1R−/− donor mice respectively 4 h after irradiation. Mice were used for experiment at least 10 weeks after reconstitution.

#### Influenza Virus Infection

Mice were infected intranasally with 105 TCID50 H3N2 influenza virus X-31 (Medical Research Council) or mock (allantoic fluid of uninfected eggs); diluted in 50 μl PBS. Weight loss was monitored daily. For suppletion assays, mice were treated intratracheally with 80 μg carrier-free recombinant IL-1α (R&D) at 2 or 10 dpi.

#### TCID50 Assay Viral Titers

Lungs were homogenized with a tissue homogenizer in 1 ml PBS and centrifuged (5 min, 400 *g*) to remove cellular debris before storage at −80°C. Titers of infectious virus were determined in triplicate by titration on MDCK cells in serum-free TPCKtreated trypsin-containing medium. Viral titers were determined by measuring chicken red blood cell agglutination activity in the cell supernatant after 7 days of infection of MDCK cells by using the calculation method of Reed and Muench.

#### Isolation of Lung Cells

Mice were sacrificed and the lungs were removed. Single-cell suspensions were prepared by digestion in collagenase/DNase solution for 30 min at 37°C. After digestion, the suspension was filtered over a 100 μm filter and red blood cells were lysed with osmotic lysis buffer (10 mM KHCO3, 155 mM NH4Cl, 0.1 mM EDTA in ddH2O).

#### Flow Cytometry

Lung cells were stained extracelullarly with anti-CD3 (17A2, conjugated to AF700, eBioscience), anti-CD19 (1D3, conjugated to APC, BD Bioscience), anti-IgM (R6-60.2, conjugated to PerCp-Cy5.5, BD Bioscience), anti-IgD (11-26c.2a, conjugated to PE, BD Bioscience), anti-CD95 (Jo2, conjugated to PE-Cy7, BD Bioscience), anti-GL7 (GL7, conjugated to Fitc, BD Bioscience), and a fixable live-dead marker (conjugated to eFl506, eBioscience).

Acquisition of samples was performed on a LSR II or Fortessa cytometer equipped with FACSDiva software (BD biosciences). GC B cells were defined as CD3−CD19+IgM−IgD−CD95+GL7+. Final analysis and graphical output were performed using FlowJo software v9 (Tree Star, Inc.).

#### Real-time Quantitative RT-PCR

Quantitative RT-PCR for IL-1α, IL-1β, LTβ, CCL19, CCL21, CXCL12, and CXCL13 were performed on RNA obtained from whole lung homogenates. Total RNA was extracted using Tripure reagent (Roche) according to the manufacturer's protocol. RNA was resuspended in diethyl-polycarbonate (DEPC, Sigma)-treated water. A total of 1 μg RNA was used for reverse transcription using the Transcriptor High Fidelity Reverse Transcriptase kit (Roche) according to the manufacturer's protocol. The subsequent target amplification on triplicates of each cDNA sample was performed using the Universal Probe Library system from Roche, which contains fluorescent hydrolysis probes of eight locked nucleic acids (LNA). Primers were designed with the help of the webbased application Probefinder (https://qpcr.probefinder.com), and a minimum of two primer pairs per target were analyzed. Primers were validated first using the LC480 SybrGreenI Master (Roche) with melting curve analysis (TM calling) in the LC480 Software and then using the LC480 Probes Master. Aspecific primer pairs were discarded. **Table 1** shows a comprehensive view of the primer/probe combinations chosen. PCR conditions were: 5′ pre-incubation at 95°C followed by 45 amplification cycles of 10″ at 95°C, 10″ at 60°C, and 20″ at 72°C using a Lightcycler 480 (Roche). PCR amplifications for the housekeeping genes encoding *Hprt* or *L27* were performed during each run for each sample to allow normalization between samples.

#### Histology

Lungs were inflated with 1 ml 1:1 PBS-OCT (Tissue Tec), snap frozen in liquid nitrogen and stored at −80°C. Frozen 8 μm sections were fixed in 4% PFA and blocked in a 1% blocking buffer (Roche).

Immunofluorescence staining was performed by staining for B220 (RA3-6B2, Rat-a-mB220 conjugated to PE, BD Bioscience + Goat-a-Rat conjugated to AF555, Invitrogen), CD4 (RM4-5, Rat-a-CD4 conjugated to Fitc, eBioscience + Rab-a-Fitc conjugated to AF488, Invitrogen), CD8 (53-6.7, Rat-a-CD8 conjugated to Fitc, BD bioscience + Rab-a-Fitc conjugated to AF488, Invitrogen), CD11c (N418, Hamster-a-mCD11c conjugated to AF647, eBioscience), and GL7 (GL7, Rat-a-mGL7 Fitc, BD bioscience + a-Fitc conjugated to HRP, Jackson + a-HRP conjugated to Fitc, Jackson). Where necessary, slides were incubated with 10% normal rat serum to prevent aspecific binding of antibodies.

TABLE 1 | Q-PCR primers.

Slides were counterstained with Dapi and digitized on a LSM710 microscope (Zeiss). All depicted pictures are representative of at least five mice per group. Images were analyzed using Imaris software (Bitplane).

#### Statistical Analysis

All experiments were performed using three to six animals per group. All experiments were performed at least two times. The difference between groups was calculated using the Student's *t* test for unpaired data (Prism version 6; GraphPad Software, Inc.). Data are depicted as mean ± SEM. Differences were considered significant when *p* < 0.05.

Analysis of the repeated relative body weight data was performed using the residual maximum likelihood (REML) as implemented in Genstat v17 (43). The following linear mixed model (random terms underlined) was fitted to the data: *Y*ijkt = μ + genotypej + treatmentk + timet + (genotype⋅treatment)jk+ (genotype⋅time)jt+ (treatment⋅time)kt+ (genotype⋅treatment⋅time)jkt+ (mouse⋅time)it+*residual*ijkt, where *Y*ijkt is the relative body weight of i-th mouse having genotype j, k-treated, and measured at time point *t* (*t* = 3–17 days; unequally spaced) and μ is the overall mean calculated for all mice considered across all time points. A first order antedependence covariance structure was used to model the within-subject correlation. The significance of the comparison between WT-X31 and KO-X31 across time was assessed by an *F*-test.

A log-linear model (Poisson distribution and log link) was fitted to the amount of viral particles measured by hemagglutination inhibiton assay. The dispersion parameter was set as free. Significance of main effects GENOTYPE and TIME and the GENOTYPE⋅TIME interaction effect was assessed by an *F*-test.

#### RESULTS

#### *Il1r1***−**/**−** Mice Have Prolonged Viral Load but Are Unable to Induce iBALT

To assess the immune response to IAV infection in mice lacking signaling *via* IL-1R, we infected *Il1r1*−/− mice and monitored weight loss and viral load in the lungs. Wild-type mice showed maximum weight loss around 6 dpi, and bodyweight was fully restored around 10 dpi. *Il1r1*−/− mice lost weight with slower and prolonged kinetics, and reaching a nadir at 8 dpi. Like wild-type mice they did manage to gain weight again, but did not recover to their starting bodyweight before 17 dpi (**Figure 1A**). This difference in the weight loss curve was also reflected in the viral load in the lungs (**Figure 1B**). Viral load in wild-type mice peaked around 6 dpi, yet was cleared at 8 dpi. *Il1r1*−/− mice had systematically higher viral loads during the entire course of infection and did not clear the infection completely at 8 dpi as an estimated remaining titer of 100,000 viral particles is detected at this time point.

Viral clearance depends on induction of adaptive immunity by DCs that activate CD8 and CD4 T cells and a humoral immune response by B cells. Total numbers and kinetics of increase of T and B cells and conventional DCs were not altered in *Il1r1*−/− mice in response to IAV infection. Lung conventional DCs consist of various subsets that have different functions and can be discriminated based on cell surface markers CD11b and CD103 (44, 45). As soon as there is inflammation in the lung, monocytes can also be recruited and these can rapidly differentiate into a MHCII+CD11c+ cell type (so called monocyte-derived DC, MoDC) that also expresses the macrophage marker CD64 (46). In contrast to the conventional CD103+ and CD11b+ DCs, the accumulation of CD11c+CD64+ MoDCs was reduced in the lungs of *Il1r1*−/− mice (**Figure 1C**).

To evaluate the effect of the higher and prolonged viral exposure on IAV-associated iBALT formation, we visualized iBALT structures in the lungs by hematoxilin staining. Generally, clusters of cells near the bronchi were more readily detected in wild-type mice than in *Il1r1*−/− mice (**Figure 1D**). Because a hematoxilin stain did not allow us to evaluate if the inflammatory clusters of cells were organized and immunologically active iBALT structures, we stained frozen lung sections for B cells, T cells, DCs, and GC B cells and analyzed them by confocal microscopy. In wildtype mice, we could easily detect organized structures composed of B cells, T cells, and DCs and B cell aggregates that contain GC B cells, but we were unable to detect similar infiltrates in *Il1r1*−/− mice (**Figure 1E**). To quantify the presence or absence of iBALT structures we measured the proportion of GC B cells in the lungs at 17 dpi by flow cytometry as a measure for biologically active iBALT. As GL7+ GC B cells are not found in the lungs of mice in the absence of iBALT, we believe this is a good approximation of the amount of iBALT formed (17). Wild-type mice showed an induction of GC B cells upon IAV infection, but this induction was absent in *Il1r1*−/− mice (**Figure 1F**). Taken together, these data suggest that although *Il1r1*−/− mice had a higher and prolonged exposure to viral particles in the lung, they were unable to form organized iBALT structures in the lung, pointing to an essential role for IL-1 cytokines in TLO induction following influenza virus infection.

#### IL-1**α** Administration Promotes Induction of iBALT Structures in the Lung

As the mere presence of viral particles was not enough to trigger the iBALT initiation and IL-1R signaling was necessary, we

quantified expression of IL-1α and IL-1β, which signal both *via* IL-1R, in the lungs after IAV infection. Both cytokines were induced after infection in a bimodal curve with a first peak around 4 dpi and a second, but smaller, peak around 10 dpi (**Figure 2A**). In general, the induction of mRNA for IL-1α was more pronounced compared with IL-1β.

We, next, addressed if administration of IL-1 cytokine would be enough to further boost iBALT induction in IAV-infected mice. Since IL-1α and IL-1β have similar effects on the IL-1R, and as induction of IL-1α was more pronounced after IAV infection, we chose to only administer IL-1α. When recombinant IL-1α was administered i.t. 2 days post IAV infection in wild-type mice, clustering of inflammatory cells could be detected more readily on lung sections compared to PBS-treated IAV-infected mice (**Figure 2B**). To quantify biologically active iBALT, we again quantified the proportion of GC B cells by flow cytometry. Administration of recombinant IL-1α at 2 dpi resulted in a higher proportion of GC B cells in the lungs 17 dpi compared to PBS

administration (**Figure 2C**). However, when the administration of recombinant IL-1α was only initiated at 10 dpi, no differences in the proportion of GC B cells could be observed between IL-1α and PBS-treated groups (**Figure 2C**). This suggests that early, but not late, IL-1R signaling is necessary and sufficient to promote GC B cell positive iBALT structures.

#### IL-1R Signaling on Stromal Cells Is Necessary to Induce GC B Cells

As early IL-1R signaling is necessary to induce iBALT structures in the lung, we sought to identify the cell type that is responsive to IL-1 signals. Therefore, we constructed bone marrow chimeric mice in which either the radiosensitive hematopoietic or the radioresistant stromal compartment was deficient for *Il1r1*. As a control, we also reconstituted *Il1r1*−/− mice with *Il1r1*−/− bone marrow as a substitute for intact *Il1r1*−/− mice and control for irradiation effects. *Il1r1*−/− mice that received wild-type bone marrow followed a weight loss curve characterized by a longer weight loss and slower recovery, as observed in *Il1r1*−/− mice. In contrast, wild-type mice that were reconstituted with *Il1r1*−/− bone marrow cells followed a weight loss curve that resembled the one observed in wild-type mice with a maximum of approximately 15% weight loss. Surprisingly, *Il1r1*−/− mice that were reconstituted with IL-1R sufficient bone marrow had a tendency to lose more weight than the *Il1r1*−/− mice that were reconstituted with IL-1R-deficient bone marrow (**Figure 3A**). We also counted the proportion of GC B cells in the lung. Wild-type mice with IL-1R-deficient hematopoietic cells were able to induce a higher proportion of GC B cells than *Il1r1*−/− mice with normal hematopoietic cells (**Figure 3B**), suggesting that IL-1 boosts the formation of iBALT structures by signaling to radioresistant stromal cells.

#### CXCL13 Expression Is Reduced in *Il1r1***−**/**−** Mice

To define which downstream iBALT-instructive signals are induced by IL-1R signaling in radioresistant cells, we measured the expression level of the chemokines CXCL12, CXCL13, CCL19, and CCL21, which all have been implicated in iBALT formation. The chemokines CCL19 and CCL21 instruct the organization of T cell zones in iBALT structures, but were not impaired in *Il1r1*−/− mice (**Figure 4**). The chemokine CXCL12 is important for B cell lymphopoiesis, but expression of this chemokine was only slightly reduced early after infection in *Il1r1*−/− mice. The chemokine CXCL13 functions as an LTi and B cell chemoattractant, and its expression was reduced 4 dpi in *Il1r1*−/− mice (**Figure 4**). None of the chemokines involved in iBALT formation and organization was differentially expressed at 17 dpi, when iBALT had fully developed in wild-type mice, showing that the instructive chemokine signals are given early after infection when the virus is not yet cleared from the lungs. Although CXCL13 expression was impaired early after infection, the total amount of B cells in the lungs was not significantly altered at the time iBALT was present (**Figure 1C**), suggesting that it is not recruitment of B cells to the lungs that is impaired in *Il1r1*−/− mice but that the B cells in the lung fail to cluster into organized iBALT structures. Whether this is a direct or indirect effect of defective IL-1R signaling on stromal cells remains a subject for future experiments.

#### DISCUSSION AND REVIEW OF THE LITERATURE

The IL-1 axis has been described previously to be responsible for inflammatory pathology in the lung, resulting in increased mortality, increased viral titers, and neutrophil recruitment following IAV infection (7). *Il1r1*−/− mice indeed suffered more from the mild X31 IAV infection and displayed a tendency to higher viral titers in the lung, but did manage to clear infection with delayed kinetics, leading to a presumably higher viral exposure over time. Despite this increased viral exposure, they hardly formed iBALT structures in the lung. Conversely, when recombinant IL-1 was administered early after IAV infection to wild-type mice, the formation of iBALT structures was facilitated. Mechanistically, we found impaired CXCL13 chemokine induction early after infection in *Il1r1*−/− mice. Later, at the time when iBALT was fully formed in wild-type mice, we could not detect any differences in CXCL13 levels, suggesting that the instructive signals that condition the lung for clustering adaptive immune cells are given very early (2–4 dpi) after infection. By studying iBALT formation in chimeric mice, we found that

FIGURE 3 | IL-1R signaling on stromal cells is necessary to induce germinal center B cells in the lung. (A) Weight loss curve after infection with X31 virus as percentage of the initial bodyweight for wild-type (squares) and IL-1R−/− (circles) mice reconstituted with wild-type (black) or IL-1R−/− bone marrow (white). (B) Proportion of germinal center B cells 17 dpi in the lungs of wild-type (squares) and IL-1R−/− (circles) mice reconstituted with wild-type (black) or IL-1R−/− bone marrow (white). ns, not significant. \**p* < 0.05. \*\**p* < 0.01.

IL-1R expression on stromal cells is necessary for proper iBALT formation. The exact cell type of stromal cells that is needed to induce GC B cells, however, still needs to be defined. Whether this stromal cell type is directly responsible for the CXCL13 production needed to initiate iBALT formation or an intermediate cell type is involved remains a matter of debate.

mice per group. ns, not significant. \**p* < 0.05.

As we observed a decreased induction in MoDCs and these cells are previously described as being major cytokine and chemokine producers (46), it is a possibility that these cells are involved in the CXCL13 induction. Alternatively, LTβ-sufficient B cells can support the progression toward mature, fully structured TLOs (35, 47) most likely *via* a positive feedback loop of CXCL13 production and LTβ expression (24). According to this hypothesis B cells are activated *via* TLR signaling, induce expression of LTβ on their surface, and interact on its turn with LTβR bearing B cells. This LTβ signaling will induce CXCL13 production and release, which attracts more B cells and upregulates LTβ expression. It is an attractive hypothesis that IL-1 might also induce LTβ expression on B cells, although this is hard to reconcile with our observation that a radioresistant cell type responds to IL-1 in our model.

We can only speculate about the source of IL-1α. Previous research has shown that IL-1α can be released by dying cells (48). In this respect, virus-infected lung epithelial cells might be a possible source of IL-1α as it has been observed that IL-1α exerts feedback on epithelial cells and induces a second cytokine and chemokine wave during innate immune responses in the lung (3, 49). We have only measured the mRNA for IL-1β. Secretion of bioactive IL-1β requires activation of the Nlrp3 inflammasome that leads to activation of caspase-1 and cleavage of pro-IL-1β into IL-1β. Infection with IAV leads to activation of the Nlrp3 inflammasome in a process requiring the type I IFN-induced RNAse L/ OAS system, while the virus actively suppresses IL-1β production and Nlrp3 activation *via* the NS1 protein (4, 5). Although others have shown that the NLRP3 inflammasome controls severity of infection (9, 11, 50, 51), future studies will have to address if lack of key components of this inflammasome also leads to reduced iBALT formation.

Generally, TLOs are absent in the lungs of healthy adults (52), but bronchus-associated lymphoid tissue can be observed in the lungs of children who are frequently infected by respiratory viruses (53) and in the lungs of adults who suffer from rheumatoid arthritis (28, 54), transplant rejection (55), COPD (56), and IPAH (31). We can only speculate that IL-1 might also be involved in the formation of these TLO structures. IL-1 is certainly a cytokine that has been implicated in the pathogenesis of rheumatoid arthritis, and targeting the IL-1 pathway *via* IL1RA (anakinra) has been used as an alternative biological treatment in patients failing therapy on TNFα blockade. A very common risk factor for rheumatoid arthritis development is smoking, which also leads to COPD. End-stage COPD is also accompanied by TLO formation in the lungs, and these can be sites of production of antibodies to citrullinated antigens, typical of RA patients. In a preclinical model of smoking-induced TLO formation, the production of autoantibodies and TLO structures was reduced in *Il1r1*−/− mice, accompanied by a reduced CXCL13-production in the lungs (57).

During development, neuronal cells give an LTα1β2 independent instructive signal to local fibroblasts to produce CXCL13 and, hereby, attract CD3−CD4+CD45+ LTi cells (58, 59). The crucial step for SLO development is the interaction of LTi cells with stromal lymphoid tissue organizer (LTo) cells. This process happens *via* interaction of LTβ expressed on LTi cells and the LTβR expressed on LTo cells. Upon this interaction, LTo cells produce homeostatic chemokines that drive the recruitment of lymphocytes. T cells and DCs are attracted by chemokine CC ligand (CCL)19 and CCL21; B cells are attracted by chemokine CXC ligand (CXCL)13. Expression of vascular cell adhesion molecule (VCAM)1, intercellular adhesion molecule (ICAM)1, and mucosal addressin cell adhesion molecule (MADCAM)1 allow the attracted cells to cluster together. IL-1 has been very well known for its effects of stimulating adhesion molecules on endothelial cells (60). It is tempting to speculate that the effects of IL-1 on radioresistant cells is *via* induction of the crucial adhesion molecules that initially tether a LTi-like cell to the circulation and subsequently to initiate a communication between stromal cells and lymphoid cells, that initiates the CXCL13 production.

The LTi-potential of T cells was first addressed in a model of thyroid overexpression of CCL21, where it was shown that CD3+CD4+-activated T cells interacted with DCs at sites of chronic inflammation and subsequently acted as LTi cells in the absence of Id2 activity (37). It has also been suggested that IL-17 signaling is involved during the initiation phase of iBALT formation by inducing CXCL13, but this role for IL-17 remains controversial (34). In two studies on neonatal mice exposed to endotoxin inhalation and on mice with experimental autoimmune encephalomyelitis, respectively, an activated Th17 CD4 T cell population was found to be involved in inducing TLO structures (34, 61). RORC+ IL-17-producing cells were also found inside lung TLOs of patients with IPAH. In humans, Th17 cells express the CCR6 receptor, and in the bloodstream of IPAH patients circulating CCR6+ cells were fewer, while the ligand CCL20 was produced in the perivascular TLOs (31). However, TLOs seem to develop normally in *Ccr6*−/− mice (34). The induction of TLOs by Th17 cells was dependent on expression of podoplanin, but why this is the case remains unknown. One possibility is that podoplanin is required for retention of Th17 cells at sites of TLO formation (34, 61). The role of Th17 as LTi-like cells is still under debate, and 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, while *Pseudomonas aeruginosa*-induced iBALT is dependent on IL-17 signaling (35, 62). As IL-17 production by γδ T cells and Th17 cells can be induced by IL-1 (63–66), we also considered the possibility that IL-17 is part of the cascade leading to IL-1α driven iBALT formation. In our hands, IAV infection indeed gave rise to a higher amount of IL-17+ CD4 T cells, but treatment with IL-1α 2 dpi could not increase the amount of IL-17+ CD4 T cells in the lung, and experiments in which we administered IL-1 to IAV-infected *Il17ra*−/− mice were inconclusive (data not shown). This suggests that, in contrast to SLO formation, the instructive signals can differ depending on the source of initiating antigen or the inflammatory stimulus that is elicited by the used model.

In almost all TLO structures that have been described, the T cell area contained antigen-presenting DCs (17, 31). As DCs activate T cells, it has been suggested that DCs are sufficient for TLO induction (67). This hypothesis is supported by the observation that repeated injection of DCs into the lungs of mice is sufficient for induction of iBALT structures accompanied by induction of myofibroblast differentiation (17, 68). During formation of Peyer's patches, a CD11c+ cell type expressing LTαβ accumulates at the LN anlagen and is necessary for instruction of stromal cells (59). DCs might also directly instruct stromal cells irrespectively of their effects on T cells. In TLO structures induced in the thymus, DCs were specifically necessary for induction of lymph angiogenesis from stromal cells (69), but how DCs induce TLOs is less clear. In virus-induced iBALT, mainly CD11b+ DCs or monocyte-derived cells accumulate; these cells express instructive LTα1β2 while also producing the homeostatic chemokines CXCL13 and CCL19/CCL21 (17). However, in some models, mostly pDCs accumulate, suggesting a functional role for type I IFN. As is the case in TLOs found in end-stage COPD patients and in a murine SLE model (56, 70). 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, most likely through trans-presentation of chemokines, or by providing a continuous source of antigen presentation to T cells (16, 17, 69). We did observe reduced numbers of monocyte-derived DCs in *Il1r1*−/− mice, but have not performed experiments in which only DCs lacked IL-1R to study if the effects of IL-1 were cell-intrinsic or resulting from effects of IL-1 on epithelial cells. Indeed, IL-1R triggering on lung epithelial cells is a very well-known trigger for the production of GM-CSF, one of the major cytokines driving activation of monocytes to adopt a DC-like phenotype (49).

In conclusion, we have described a novel role for early IL-1 production in IAV infection to control the formation of iBALT structures *via* induction of CXCL13 in a stromal cell compartment. Future studies will have to address if this effect of one of the

#### REFERENCES

best-known proinflammatory and innate cytokines is a general feature of TLO formation at sites of acute and chronic immune stimulation such as infectious disease and autoimmune pathologies and if this can be exploited to induce iBALT formation as part of a mucosal vaccination strategy (71).

#### AUTHOR CONTRIBUTIONS

KN was responsible for conceptualization of mouse experiments, experimentation, data analysis, and preparation of the manuscript. HH and KD provided experimental support for confocal imaging. CG, HH, and BL assisted in conceptualization of experiments and discussion of data and provided feedback for the manuscript. All authors read and approved the final manuscript.

#### ACKNOWLEDGMENTS

We thank the technical and logistic support of Sofie De Prijck, Manon Vanheerswynghels, Gert Van Isterdael, Justine Van Moorleghem, and Karl Vergote and the statistical support of Marnik Vuylsteke.

#### FUNDING

KN is supported by a grant from FWO Flanders. CG is supported by an ESMCID grant. BL is supported by Project grants of FWO Flanders, by an ERC Consolidator grant, by an Interuniversity Attraction Pole grant, and by a Ghent University MRP Grant (Group-ID consortium).

nanoparticle enhances protection in mice against diverse respiratory viruses. *PLoS One* (2009) 4(9):e7142. doi:10.1371/journal.pone.0007142

in the absence of ROR gamma t and LTi cells. *J Exp Med* (2011) 208(1):125–34. doi:10.1084/jem.20100052

T cells, amplifying Th17 responses and autoimmunity. *Immunity* (2009) 31(2):331–41. doi:10.1016/j.immuni.2009.08.001

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Neyt, GeurtsvanKessel, Deswarte, Hammad and Lambrecht. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Lymphoid Neogenesis and Tertiary Lymphoid Organs in Transplanted Organs

#### *Alice Koenig1,2,3 and Olivier Thaunat1,2,3 \**

*1Service de Transplantation, Néphrologie et Immunologie Clinique, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France, 2 INSERM UMR1111, Lyon, France, 3Université de Lyon, Lyon, France*

The progressive organization of immune effectors into functional ectopic lymphoid structures, named tertiary lymphoid organs (TLO), has been observed in many conditions in which target antigens fail to be eliminated by the immune system. Not surprisingly, TLO have been recurrently identified in chronically rejected allografts. Although significant progress has been made over the last decades in understanding the molecular mechanisms involved in TLO development (a process named lymphoid neogenesis), the role of intragraft TLO (if any) in chronic rejection remains elusive. The prevailing dogma is that TLO contribute to graft rejection by generating and propagating local humoral and cellular alloimmune responses. However, TLO have been recently observed in long-term accepting allografts, suggesting that they might also be able to regulate alloimmune responses. In this review, we discuss our current understanding of how TLO are induced and propose a unified model in which TLO can play deleterious or regulatory roles and therefore actively modulate the kinetics of chronic rejection.

#### *Edited by:*

*Andreas Habenicht, Ludwig Maximilian University of Munich, Germany*

#### *Reviewed by:*

*Matthias Lochner, Center for Experimental and Clinical Infection Research, Germany Megan K. L. MacLeod, University of Glasgow, UK*

#### *\*Correspondence:*

*Olivier Thaunat<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

> *Received: 03 October 2016 Accepted: 13 December 2016 Published: 27 December 2016*

#### *Citation:*

*Koenig A and Thaunat O (2016) Lymphoid Neogenesis and Tertiary Lymphoid Organs in Transplanted Organs. Front. Immunol. 7:646. doi: 10.3389/fimmu.2016.00646*

Keywords: transplantation, lymphoid neogenesis, tertiary lymphoid organs, chronic rejection, tolerance

#### INTRODUCTION: THE CHALLENGE OF CHRONIC REJECTION IN TRANSPLANTATION

Vital organ failure is a life-threatening condition where a vital organ (i.e., kidney, heart, liver, or lung…) does not perform its expected function. Recent lifestyle changes in developed countries, and the increased incidence of chronic diseases such as hypertension, obesity, and diabetes, have set the stage for accelerated risk for, and the occurrence of, vital organ failure. As a result, vital organ failure is currently recognized as the leading cause of debility and premature death worldwide (www.who. int). In France alone, the personal, societal, and economic consequences of vital organ failure have a cost of more than €70 billion a year (25% of total health expenditures).

Transplantation consists in the restoration of vital physiologic functions through the surgical substitution of a defective organ by a functioning graft retrieved from a donor. Patients with endstage vital organ failure depend on solid organ transplantation, which is their best (often their only) therapeutic option.

In clinical transplantation, the donor is from the same species but genetically different. Consequently, the immune system of the recipient inevitably recognizes the antigenic determinants (alloantigens) that differ between the recipient and the donor, particularly the highly polymorphic molecules from the major histocompatibility complex [i.e., human leukocyte antigen (HLA)] in humans. The alloimmune response that develops against the donor-specific HLA molecules is responsible for tissue damage, which leads to the failure of the transplanted organ, a process named "rejection."

In the absence of a clinically applicable protocol able to induce the specific tolerance of the allogenic transplant by the recipient's immune system (1, 2), the prevention of rejection is currently dependent upon immunosuppressive drugs (3). These drugs produce generalized immunosuppression, which means that any reduction in immune responsiveness to the allograft is accompanied by reduced immunity to infections and malignant diseases. Chronic immune injuries that result from the incomplete blockade of the recipient's alloimmune response (i.e., chronic rejection) are currently the main factor limiting graft function duration (4). No significant progress has been made on this issue over the last decades as highlighted by the stagnation of graft halflife (5). A better understanding of the pathophysiology of chronic rejection is therefore a mandatory step in identifying innovative approaches that would prolong graft function duration.

#### INTRAGRAFT TERTIARY LYMPHOID ORGANS (TLO)

Rejected grafts are characterized by interstitial infiltration of cellular effectors, mainly T cells and macrophages, but also dendritic cells, NK cells, B cells, and plasma cells.

In contrast with acute rejection, where infiltrates exhibit no particular spatial organization, during chronic rejection immune cells tend to organize themselves in structures that morphologically resemble the secondary lymphoid organs.

Analyzing all sorts of human kidney grafts removed for terminal chronic rejection, we and others showed that in the majority of chronically rejected grafts the immune cells were grouped, conferring a nodular organization to the infiltrate (6, 7). These nodules exhibited a highly organized microarchitecture with clear cell subset segregation: the core, made of the B cells intermingled with a network of follicular dendritic cells, was surrounded by T cells and mature dendritic cells. CD138-expressing plasma cells were found within or in close vicinity to TLO, suggesting that part of these cells differentiated locally. As in canonical secondary lymphoid organs the compartmentalization of the different cell subsets appeared to be mediated by gradients of homeostatic chemokines CCL21 (in the T cell area) and CXCL13 (in the B cell area). Furthermore, neolymphatic vessels and PNAd-expressing high endothelial venules (HEVs) were observed in the periphery of the nodules (8).

The structural organization of immune effectors observed in chronically rejected renal grafts (**Figure 1**) does not seem specific of this type of transplant since similar lymphoid structures have been observed in chronically rejected pancreas, livers, hearts (7, 9–11), lungs (12), and even composite transplants (13–15). This phenomenon is not specific of the alloimmune setting either, since the very same lymphoid structures have previously been observed in various inflammatory conditions, including chronic infections, autoimmune diseases, and cancers (16, 17). Structural organization of immune effectors therefore appears as a generic response of the chronically stimulated immune system that cannot eradicate targeted antigens.

transplant. Biopsy of a renal transplant was performed for progressive deterioration of graft function, suggestive of chronic rejection. (A) HES staining revealed nodular infiltrates of mononuclear cells within graft parenchyma (original magnification: left panel, ×20; right panel, ×200). (B) Immunostainings unraveled the organized distribution of T cells (CD3+, left panel) and B cells (CD20, right panel). Original magnification: ×200.

Because the microarchitecture of organized immune infiltrates is highly reminiscent of that of secondary lymphoid organs, these lymphoid structures have been named TLO.

#### MOLECULAR MECHANISMS INVOLVED IN THE DEVELOPMENT OF SECONDARY LYMPHOID ORGANS

Primary immune responses are initiated in secondary lymphoid organs, which are located at strategic sites where antigens are most likely to be encountered.

The development of secondary lymphoid organs, a process named lymphoid organogenesis, is initiated during embryogenesis independently of antigen recognition at predetermined sites as a result of complex interactions between hematopoietic, mesenchymal, and endothelial cells (18, 19). Lymphoid organogenesis can be schematically divided into two consecutive steps: first the induction, then the organization phase.

The induction phase depends on lymphoid-tissue inducer cells, which arise in the fetal liver. Under the influence of TRANCE (at sites of peripheral lymph node development) or IL-7 (at mucosal sites) lymphoid-tissue inducer cells express membranebound lymphotoxin: a heterotrimer containing lymphotoxin α and lymphotoxin β that allow lymphoid-tissue inducer cells to interact with the lymphotoxin β receptor (LTβR) of stromal cells. Signaling through the LTβR initiates NFκB signaling in stromal cells, which promotes the production of homeostatic chemokines (18, 19).

Homeostatic chemokines are crucial for the organization phase. CXCL13 recruits circulating B cells to what becomes the B cell area of lymphoid tissues, and the T zone chemokines (CCL19 and CCL21) attract T and dendritic cells to shape the T cell area (18, 19). The lymphotoxin signaling pathway is also crucial in promoting the differentiation of HEVs, which are postcapillary venules expressing specific adhesion molecules (known as addressins) that have a crucial role in lymphocyte trafficking to secondary lymphoid organs (18, 19).

#### MOLECULAR MECHANISMS OF LYMPHOID NEOGENESIS IN TRANSPLANTATION

Chronic rejection provides optimal conditions for studying the molecular mechanisms involved in the development of TLO (**Figure 2**). Indeed, (i) TLO have systematically been detected in chronically rejected grafts; (ii) the antigens targeted by the immune system are known (recipient-mismatched HLA antigens of the transplanted tissues); and (iii) chronically rejected grafts are sometimes removed, providing a large amount of diseased tissue, which can be comprehensively analyzed.

In-depth analysis of a series of detransplanted human renal grafts revealed the heterogenous nature of the cellular composition of TLO (8). Two types of B cell nodules could be identified: nodules composed of a uniform CD20pos B cell population expressing IgD and Bcl-2 were similar to primary follicles, while nodules with a core of CD20posIgDnegBcl-2neg B cells, highly expressing Bcl-6 that had pushed aside the CD20posIgDposBcl-2pos B cells, resembled secondary follicles, i.e., germinal centers (8). The ratio between these two types of structures differed between samples, and the number of ectopic germinal centers did not increase with the quantity of primary nodules. The phenotypic heterogeneity of TLO correlated more with the expression profile of a set of genes (*CCL19, CCL21, CXCL12, CXCL13*, CCR7, *CXCR4*, and *CXCR5*)

involved in the formation and the maintenance of canonical secondary lymphoid organs (i.e., the lymphoid organogenesis described in the previous section) (18, 19). The complete recapitulation of this genetic program in chronically rejected grafts resulted in the generation of fully functional ectopic germinal centers that allowed for the efficient maturation of B cells into memory B cells and plasma cells (**Figure 2**). In contrast, when this recapitulation was incomplete, local B cell maturation was impeded (8). These results highlighted the similarity between the molecular processes involved in the development of canonical secondary lymphoid organs and those involved in the organization of immune effectors during chronic inflammation, a process named lymphoid neogenesis.

If the molecular mechanisms responsible for the organization and maintenance of secondary lymphoid organs and TLO appear similar, the initiation of the cascade is likely to be different (**Figure 2**). The formation of secondary lymphoid organs in the embryo is developmentally programed and results from the interaction between lymphotoxin-α1β2-expressing lymphoid-tissue inducer cells and lymphotoxin-β receptor-expressing stromal organizer cells (18, 19). In contrast, TLO development seems independent of lymphoid-tissue inducer cells (20, 21). Yet, several studies have documented the importance of the lymphotoxin pathway in lymphoid neogenesis (21, 22), including in a transplantation setting (23), by demonstrating that the development of TLO was abolished by treatment with inhibitory LTβR–Ig fusion protein. We must then ask who provides lymphotoxin signaling in the chronic rejection setting. Beyond lymphoid-tissue inducer cells, lymphotoxin-α and lymphotoxin-β are also expressed by activated lymphocytes (24). It is therefore conceivable that activated T and/or B cells replace lymphoid-tissue inducer cells to initiate lymphoid neogenesis in rejected grafts (**Figure 2**) as already demonstrated for the induction of TLO in the gut (21). Another possibility is that lymphotoxin is dispensable for the formation of TLO. Lymphotoxin-α and lymphotoxin-β are two related members of the large TNF ligand family (25). Since homologous genes and gene products often have redundant physiological functions, it seems reasonable to propose that other ligands and/or receptors of the TNF superfamily could act as alternative pathways for TLO induction (**Figure 2**). In line with this hypothesis, the provision of the alternative LTβR ligand LIGHT (aka tumor necrosis factor superfamily member 14) by activated T cells infiltrating inflamed pancreas have been shown to be crucial for the formation of TLO (26). Furthermore, TNF-α, which is produced within rejected grafts (27), has been shown to be critical for the development of TLO in a murine model of atherosclerosis (20). TNF-α does not bind to LTβR but to distinct TNF receptors (25). Using apolipoprotein E-deficient mice, which spontaneously develop atherosclerotic lesions in their aorta, the Antonino Nicoletti's group recently demonstrated that the blockade of LTβR signaling had no effect, whereas that of TNFR1/2 signaling reduced the expression of homeostatic chemokines and the subsequent development of TLO (20). Finally, it has recently been reported that IL-17 produced by CD4+ T cells (i.e., Th17 cells) was essential for the formation of both (i) TLO in the central nervous system of mice during experimental autoimmune encephalomyelitis (the animal model of multiple sclerosis) (28) and (ii) the development of inducible bronchus-associated lymphoid tissue, an ectopic lymphoid tissue that forms in the lungs after pulmonary inflammation (29, 30). In the latter setting, IL-17 acted by triggering the expression of homeostatic chemokines independently of lymphotoxin signaling (**Figure 2**). If this hypothesis was proven true in transplantation, initiation of lymphoid neogenesis in chronically rejected grafts could therefore be totally independent of both lymphoid-tissue inducer cells and the lymphotoxin/TNF pathway. Interestingly, we have recently reported that a Th17 polarization of CD4+ T cells infiltrating the graft was associated with increased TLO development during clinical chronic rejection (31).

It is conceivable that instead of conflicting with each other, these different works reveal the fact that several pathways can promote the initiation of TLO depending on the initiating events. This hypothesis was recently substantiated by the demonstration that the development of bronchus-associated lymphoid tissue was triggered by different pathways according to the pathogen responsible for lung inflammation (29).

While significant progress has been made in the identification of the molecular mechanisms that participate to the development of TLO, the endogenous signals capable of inhibiting the lymphoid neogenesis are far more elusive. Through evaluation of synovial tissues from rheumatoid arthritis patients it has been recently reported that low interleukin-27 (IL-27) expression corresponds with an increased incidence of TLO and gene signatures associated with their development and activity. The presence of synovial TLO was also noted in mice deficient in the IL-27 receptor after the onset of inflammatory arthritis (32). IL-27 might therefore represent a negative regulator of TLO development. Whether this is also true for chronic rejection remains to be demonstrated.

#### DO INTRAGRAFT TLO PROMOTE CHRONIC REJECTION?

Tertiary lymphoid organs differ from canonical secondary lymphoid organs inasmuch as they develop in an inflammatory milieu (31, 33), enriched in neoantigens released from injured tissue and trapped by defective lymphatic drainage (34). Comparing the cellular composition of TLO of chronically rejected grafts with one of the secondary lymphoid organs, we observed a drastic increase in the percentage of activated and memory CD4+ T cell in intragraft TLO and a symmetric decrease in T regulatory subsets (IL-10-producing Tr1 cells and Foxp3pos Tregs) in both a murine experimental model and human samples (33, 35).

These peculiarities suggest that the local immune response that develops in intragraft TLO might be less tightly regulated than in secondary lymphoid organs and are therefore more aggressive. In line with this hypothesis, we (33) and others (23) have shown that intragraft TLO are a major site where B cell tolerance breakdown occurs during chronic rejection (**Figure 2**). Interestingly, the generation of autoantibodies following solid organ transplantation has long been reported to correlate with chronic rejection, and the deleterious impact of some autoantibodies on graft survival has been demonstrated (36, 37). Furthermore, comparing the alloimmune responses elicited in intragraft TLO, spleen, and draining lymph nodes in a rat model of chronic rejection, our group observed increased production of anti-HLA antibodies in TLO as compared with canonical secondary lymphoid organs (35). Not only were the humoral alloimmune responses elicited in TLO quantitatively enhanced but they also displayed a more diverse repertoire, a finding that we confirmed in the clinical setting by the analysis of chronically rejected human kidney allografts (8).

Tertiary lymphoid organs could also contribute to chronic destruction of the graft through antibody-independent functions of B cells. B cells are indeed unique antigen-presenting cells because (i) they have an antigen-specific receptor (B cell receptor), which when engaged by surface-tethered antigens leads to the formation of an immunological synapse that coordinates cell signaling events and promotes antigen uptake for presentation on MHC class II molecules (38), even when the antigen is membrane-tethered or is present in limiting quantities and (ii) B cells have the capacity to clonally expand, thereby becoming the numerically dominant antigen-presenting cells. Interestingly, it has been reported that the presence of B cell clusters within the graft during rejection was associated with reduced graft survival and resistance to steroid therapy, independently of C4d (a breakdown product generated during classical complement pathway activation) deposition or alloantibody detection (39). Some authors have proposed that this could be due to the local presentation of antigen to effector T cells by intragraft B cells (40). This hypothesis is supported by experimental data from the group of Fadi Lakkis, who showed that in a murine skin graft model, TLO perpetuate the rejection process by supporting naïve T cell activation within the graft (41). Strikingly, the same authors also demonstrated that TLO generate T cell memory immune responses (41).

In addition to presenting antigen, B cells can also enhance T cell-mediated immune responses through the secretion of cytokines and chemokines. Studies from the group of Frances Lund (42) have shown that B cells can be functionally subdivided based on their cytokine profile. B cells activated in the presence of TH1-type cytokines (referred to as Be-1 cells) secrete IFNγ and IL-12 but not IL-4, IL-13, or IL-2. By contrast, B cells activated in the presence of TH2-type cytokines (Be-2 cells) secrete IL-2, lymphotoxin, IL-4, and IL-13 but make minimal amounts of IFNγ and IL-12. Both Be-1 and Be-2 cells seem able to produce IL-10, TNFα, and IL-6. The importance of B cell cytokines in promoting T cell responses has been illustrated in several models. For example, *in vitro* generated effector B cells that produced either TH1- or TH2-type cytokines were shown to promote the activation and differentiation of naïve T cells into effector TH1 and TH2 cells, respectively (43). The importance of B cell cytokines in promoting T cell responses has been confirmed *in vivo*. In a murine model of *Toxoplasma gondii* infection, TNF production by B cells was shown to be required for the generation of an optimal TH1 cell protective response (44). In another set of experiments, the generation of a protective TH2 memory response to *H. polygyrus* was shown to depend on IL-2-producing B cells (45). The exact role of cytokine-producing B cells in enhancing intra-TLO T cell responses remains to be evaluated.

Since grafts in which TLO were harboring germinal center reactions had a shorter life expectancy (**Figure 2**), we have proposed that lymphoid neogenesis could play a detrimental role during chronic rejection (8). However, the validity of this conclusion is limited by the fact that only explanted grafts have been analyzed, i.e., organs displaying extreme rejection damage that are sometimes (notably in the case of renal grafts) removed after immunosuppressive therapy withdrawal. The definitive demonstration that TLO are involved in the pathophysiology of chronic rejection would require selectively impairing the development of intragraft TLO while leaving the rest of the recipient's immune system unaffected. Addressing this issue is not trivial because, as discussed above, TLO share many biological pathways with canonical lymphoid tissue, and hence an adequate experimental model is not currently available. Therefore, most of the attempts to validate the data obtained in murine experimental models and in human detransplanted grafts have relied on graft biopsies. The identification of TLO within the grafts before the development of the lesions indeed appears as a prerequisite for confirming the role of lymphoid neogenesis in chronic rejection. This implies a study of protocol biopsies, which has long been introduced as standard follow up in transplantation (46). Unfortunately, the numerous studies aiming at evaluating the correlation between the presence of TLO in protocol biopsies and the later development of chronic rejection have reached conflicting conclusions (**Table 1**).

The absence of an unequivocal deleterious role for B cell clusters has led to the conclusion that these structures could be like "fish in a sunken ship," i.e., although fish are frequently seen in a sunken boat, they play no role in the process responsible for the shipwreck.

#### INTRAGRAFT TLO: FRIENDS AND FOES?

An alternative explanation could reconcile these apparently conflicting results. As discussed above, the proportion of B cells that infiltrate chronically rejected kidney grafts does not correlate with the functionality of intragraft TLO (8). The attraction of B cells within inflamed tissue appears therefore to be a generic phenomenon with no intrinsic deleterious consequences on the graft. However, when intragraft B cells meet the appropriate microenvironment, and upon the complete recapitulation of the lymphoid organogenesis program, B cell nodules organize themselves into functional ectopic germinal centers, which harbor the development of a local aggressive immune response. Because graft biopsies provide only a very limited amount of tissue (which is already an important limitation for evaluation in a patchy process such as lymphoid neogenesis), they do not allow for functional analysis of the ectopic lymphoid organs and are therefore inappropriate for analyzing the role of B cell clusters in rejected grafts.

Another layer of complexity has recently been brought into the picture by experimental evidence that certain B cell subsets are endowed with an immune regulatory role (47). For instance, IL-10-producing B cells have been shown to efficiently prevent the induction of autoimmune disease in several mouse models (48–50). Tolerance in transplantation is defined as the maintenance of graft function in the absence of therapeutic immunosuppression for at least 12 months. About 100 tolerant patients have been identified among renal transplant recipients over the last decade (51). These patients, defined as "operationally tolerant,"

#### TABLE 1 | Summary of biopsy-based studies evaluating the role of graft-infiltrating B cells.

*DSA, donor-specific antibodies; HLA, human leukocyte antigen; HPF, high power field; PNAd, peripheral lymph node addressin; Tx, transplantation.*

are healthy, do not exhibit more infections or malignancies than healthy volunteers, and do not display clinical evidence of immune incompetence (51). When compared with transplanted patients with stable graft function under pharmacologic immunosuppression, operationally tolerant patients exhibited an increase in both absolute number and frequency of total B cells (52). Furthermore, two independent microarray analyses of PBMC revealed a higher expression of B cell-related genes and their associated molecular