Lymphotoxin, CCL21, and CXCL13 were shown to play a major role during TLS neogenesis, and are related to TLS presence in human tumors (Table 2). In a mouse model of atherosclerosis, the activation of LTβR⁺ medial smooth muscle cells in the abdominal aorta by LT produced by CD11c⁺ CD68⁺ Ly6C^lo monocytes leads to the expression of CCL19, CCL21, CXCL13, and CXCL16 chemokines, which in turn trigger the recruitment of lymphocytes to the adventitia and the development of TLS (31). The same observation was made by Thaunat et al. in a rat model of chronic allograft rejection, in which M1-macrophages behaved as LTi cells in diseased arteries by expressing high levels of LTα and TNF-α (32). In human NSCLC, a TLS-related gene signature was identified, including CCL19, CCL21, IL-16, and CXCL13 (5) (Table 2). Interestingly, Matsuda et al. recently suggested in a mouse intrapulmonary tracheal transplant model that lymphoid neogenesis was dependent on spleen tyrosine kinase (Syk)-signaling. Decreased expression of CXCL12, CXCL13, and VEGF-α, lower B-cell recruitment into allograft, and smaller lymphoid aggregate area were observed in Syk-deficient recipient mice as compared to controls (33).

The generation of HEVs is also a critical step in TLS neogenesis. HEV endothelial cells express LTβR, and the continuous engagement of LTβR on HEVs by LT⁺ CD11c⁺ DCs is critical for the induction and maintenance of the mature HEV phenotype required for the extravasation of blood lymphocyte into LNs (34–37). In addition, CD11c⁺ DCs can be sources of proangiogenic factors, such as VEGF, favoring the development of HEVs, and ultimately lymphocyte entry into LN (38–41). Consistently, LTβ expression correlates with that of HEV-associated chemokines in human breast cancer, and DC-Lamp⁺ DC density correlates with HEV density, lymphocyte infiltration, and favorable clinical outcome (11). Other cell types were shown to favor the development of HEV. For instance, ectopic expression of CCL21 in the thyroid gives rise first to the recruitment of CD3⁺ CD4⁺ T cells followed by DC, and this DC-T cross-talk is required for the local development of both TLS and mature HEV (42). Tumor-infiltrating CD8⁺ T cells and NK cells were also shown to drive the de novo development of PNAd⁺ TNFRI⁺ CCL21⁺ HEV-like blood vessels through the production of LT and IFN-γ (43).

Th17 cells share many developmental and effector markers with LTi cells, including the nuclear hormone receptor retinoic acid-related orphan receptor γt (RORγt), which promotes not only the production of IL-17 and IL-22 by Th17 cells, LTi cells, and other RORγt⁺ innate lymphoid cells (ILCs), but also cell membrane expression of LT [reviewed in Ref. (44)]. In mice lungs, the formation of TLS [called here induced-bronchus-associated lymphoid tissues (i-BALT)] following LPS sensitization was dependent of IL-17 production by T cells, including Th17 and γδ T cells (45). This observation was also observed in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (46). Similarly, IL-17α-deficient mice exposed to cigarette smoke displayed decreased number of ectopic lymphoid follicles and decreased expression of CXCL12 as compared to wild-type mice in a model of chronic obstructive lung disease (47). It has also been suggested that Th17 cells, and IL-17 and IL-21 secretion by these cells can promote TLS neogenesis within human renal grafts, and are associated with the presence of active GC B cells and fast chronic rejection (48).

Other inflammatory cytokines also seem to promote TLS neogenesis. In rheumatoid arthritis (RA), high protein levels of IL-23 and IL-17F were detected in the synovial fluid of patients displaying ectopic lymphoid follicles, and a positive correlation was observed between CD21L mRNA (as a TLS marker) and IL-23 but also IL-17F, IL-21, and IL-22 mRNAs (49). IL-22 was also proposed to favor TLS induction (50). In a mouse model of virus-induced autoantibody formation in the salivary glands, it was shown that the ligation of IL-22R expressed by epithelial cells and fibroblasts leads to CXCL12 and CXCL13 production, allowing B-cell recruitment and TLS organization. In that case, IL-22 was mainly produced by γδ T cells and to a lesser extent by ILCs and NK cells during the early phase post-infection, and then by αβ T cells later after infection.

On the opposite, IL-27, a cytokine known to inhibit effector Th17 responses was recently suggested to negatively regulate the development of ectopic lymphoid-like structures in the synovial tissues of RA patients. While patients having a high density of TLS displayed high synovial levels of IL-17 and IL-21, high levels of IL-27 were observed in patients devoid of any TLS, and IL-27 expression was inversely correlated with CD3⁺ and CD20⁺ infiltrates and with synovitis. This observation was confirmed in a mouse model of RA (51).

Among the immune cells infiltrating tumors are regulatory T cells (Tregs), which have been considered in many reports as a marker of poor prognosis in cancer (52, 53). Tregs have been reported to negatively interfere with BALT development. Indeed in CCR7-deficient mice, BALTs developed spontaneously in the absence of infection, an event that is directly reverted by the adoptive transfer of wild-type Tregs but not CCR7^−/− Tregs (54). In human breast cancer, Tregs were detected in lymphoid aggregates surrounding tumor nests, and their presence was linked with the poor clinical outcome of patients (55). In mice bearing breast tumors, Treg depletion led to an increased density of HEV within the tumor, facilitated T cell recruitment from the blood, and ultimately induced tumor destruction (56). This observation is in accordance with a human study showing that HEV^high breast tumors correlated with a high LT-β expression, a high density of tumor-infiltrating mature DC, and a decreased FoxP3⁺/CD3⁺ T cell ratio (11).

More recently, a new mechanism involving regulation of TLS formation by Tregs was found, by dampening neutrophilic inflammation (57). The presence of neutrophils seemed to be critical for the neogenesis and the humoral immune function of i-BALT by enhancing B-cell activation and survival, Ig class switching to IgA as well as plasma cell survival (57).

Regulatory T cells have been shown to dampen the effector T cell response promoted within tumor-associated TLS. Treg depletion causes immune-mediated tumor destruction associated with an increased expression of co-stimulatory ligands by DCs and proliferation of T cells in a murine model of lung adenocarcinoma (58). Further studies should be carried out to analyze the prognostic importance of Tregs and their immunosuppressive potential in cancer patients according to their localization.

Altogether, TLS neogenesis and lymphoid organogenesis share many common mechanisms. On the one hand, the production of inflammatory cytokines (LT, IL-17, IL-22, and IL-23) and lymphoid chemokines (CCL21, CXCL12, and CXCL13), HEV development as well as the activation of DCs, B, and effector cells seem to be crucial events leading to TLS neogenesis under inflammatory conditions, such as cancers. On the other hand, the presence of Tregs appears to negatively impact TLS formation and TLS-associated T cell responses.

The key cross-talk between LTi cells and lymphoid tissue organizer cells (LTo cells that are cells of mesenchymal origin) occurring during LN development involves several molecules along with RANK and its ligand, which lead to LTβR signaling (59). Therefore, targeting RANK/LT pathway may modulate TLS development through the activation of LTo cells. Currently, antagonists of LTα (Pateclizumab NCT01225393), LTβR (Baminercept, NCT01552681) and RANK signaling (NCT01973569) are under investigation in several inflammatory situations. A special attention should be made in cancer setting where these antagonists might block TLS formation and, hence, reduce survival. The use of agonists might rather present a benefit to cancer patients but no drugs have been developed so far.

Activation of LTβR signaling pathway in LTo cells induces VCAM-1 and ICAM-1 upregulation, and ultimately leukocyte infiltration (60). Because both molecules are known to be induced by inflammation, an ICAM-1 antagonist called Alicaforsen has been tested in autoimmune diseases (NCT00048113, NCT00063830). We can speculate that the development of VCAM-1/ICAM-1 agonists would promote LTi-like cells-LTo clusterings and improve the leukocyte recruitment in order to generate cancer-associated TLS.

IL-7 receptor (CD127) signaling has been reported as a key pathway for TLS neogenesis (61). IL-7 is not only crucial for the survival and proliferation of LTi cells but also for GC formation and Tfh differentiation (62). To date, only one pharmacologic agent (IL-7R) is under investigation in NOD mice to deplete autoreactive T cells and to regulate pro-inflammatory mediators (63).

Altogether, as a counterpart of autoimmune diseases, development of agonist molecules targeting lymphoid organogenesis might be a promising strategy for the initiation and the maintenance of TLS in cancers.

Lymphoid chemokines represent a good therapeutic target for the modulation of TLS (Table 2). The CCL19–CCL21/CCR7 and CXCL13/CXCR5 couples are induced after LT-βR signaling during lymphoid genesis (60). They are overexpressed in TLS of melanoma (21), colorectal (3), and lung (5) cancer patients. Using lymphoid chemokines or their agonists could be a promising strategy to induce TLS neogenesis in cancers. For example, CCL21 has been shown to attract circulating naïve T cells and DCs in tumors, and contribute to the control of tumor growth (64–66). A Phase I clinical trial is currently under investigation in NSCLC patients receiving intra-tumoral injections of CCL21-transduced autologous DCs (NCT00601094, NCT01574222). It is tempting to speculate that this vaccine therapy would boost TLS formation in tumors associated with an influx of lymphocytes, an effective anti-tumor immune response, and a reduction of tumor burden.

IL-21, which is mainly secreted by Th17 cells and neutrophils, represents also an interesting molecular target. First, this cytokine has been shown to promote TLS neogenesis in lungs after acute LPS exposure and IL-21^−/− mice exhibit fewer TLS in allografts than the control group (57). Second, IL-21 can enhance B and plasma cell survival as well as B-cell-dependent immunity, and induce conventional T cells to become refractory to Treg immunosuppression (48, 57, 67). Even if IL-21 can block IL-2 production with deleterious consequences in terms of Treg differentiation, IL-21 can substitute for IL-2 as a T cell growth factor (68). Recombinant IL-21 is currently tested in many clinical trials, alone or in combination with chemotherapy, therapeutic antibodies or tyrosine kinase inhibitors (e.g., NCT00617253, NCT00389285, NCT00095108, NCT01629758, NCT00336986, and NCT01489059). Altogether, it is likely that IL-21 could promote a robust anti-tumor immunity in a TLS-dependent manner.