Tertiary Lymphoid Structures in Cancers: Prognostic Value, Regulation, and Manipulation for Therapeutic Intervention

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that reflect lymphoid neogenesis occurring in tissues at sites of inflammation. They are detected in tumors where they orchestrate local and systemic anti-tumor responses. A correlation has been found between high densities of TLS and prolonged patient’s survival in more than 10 different types of cancer. TLS can be regulated by the same set of chemokines and cytokines that orchestrate lymphoid organogenesis and by regulatory T cells. Thus, TLS offer a series of putative new targets that could be used to develop therapies aiming to increase the anti-tumor immune response.

In this mini review, we summarize the available data in the literature regarding the prognostic value of TLS in human cancers, and discuss how these structures are controlled and could be manipulated in order to increase anti-tumor immune responses.

TLS AnD PROGnOSiS in CAnCeRS
In recent years, numerous publications have assessed the prognosis associated with the presence of TLS in different types of tumors. Several strategies for their quantification have been used. Historically, the first method to measure the densities of TLS was the quantification of mature DCs (DC-Lamp + ) within CD3 + T cell aggregates (1,20). Although relatively challenging due to the relative low number of DC-Lamp + DCs in some tumors (as compared to other immune populations), our group has described it as the most accurate marker for quantifying TLS (28,29). Up-to-date, eight publications have found a positive association between increased densities of DC-Lamp + DCs and prognosis, in several types of tumors, including NSCLC (1,2,9), melanoma (20), renal cell carcinoma [RCC (24)], breast (11), and colorectal cancer (15) (Table 1 and Figure 1).
The analysis of expression levels of TLS-related genes gives the possibility to rapidly assess the prognostic impact of these immune aggregates in large retrospective cohorts of tumors. So far, six studies have evaluated the prognostic impact of increased expression of TLS-related genes in cancer. Despite heterogeneity in the TLS-signatures, a significant correlation with good prognosis has been found in melanoma (21), colorectal (3,18), and gastric (19) cancers (Table 1). Interestingly, TLS found in inflammatory zones from hepatocellular carcinoma (HCC) correlate with increased risk for late recurrence and a trend toward decreased overall survival after HCC resection. This result could reflect an unexpected role for TLS, serving as niche for HCC progenitor cells via local production of Lymphotoxin (LT)-β (25,30).
Another approach that has been used to estimate the densities of TLS in cancers is the quantification of B-cell aggregates by immunohistochemistry (IHC) (CD20 + B-cell aggregates or islets). The majority of publications measuring CD20 + aggregates (four out of five), accounting for more than 349 analyzed tumors, has determined that increased densities of this population correlate with good prognosis in several neoplasias, such as NSCLC (2), colorectal cancer liver metastasis (27), gastric (19), and oral (22) cancer (Table 1 and Figure 1). Most of the studies quantifying the CD3 + T cell aggregates and immune-cell aggregates (after hematoxylin counterstaining) have also found a positive impact on patient's prognosis. However, high numbers of B cell or T cell aggregates were found to have no impact on prognosis in biliary tract cancer and in stage III colorectal cancer, respectively. Further studies are needed to investigate whether it reflects that cell aggregates counting is not an accurate method to quantify TLS, or a functional impairment of TLS in these two cancer types ( Table 1 and Figure 1).
Overall, despite the heterogeneity of methods used for quantifying TLS, most of the studies have consistently found a correlation between high densities of TLS and prolonged patient's survival in more than 10 different types of cancer ( Table 1).  Main immune functions and process  Cluster of gene related  to TLS presence   Reference   CCL2  CCL2, MCP-1,  MCAF   Monocyte, immature DC and T cell chemotaxis, G-protein-coupled receptor signaling  pathway, cell adhesion, JAK-STAT cascade, MAPK cascade, cellular calcium ion  homeostasis, cellular response to IFN-γ, IL-1, and IL-6 Chemotaxis (  Chemotaxis, chemotaxis/ Th1/cytotoxicity/ activation (3,9) (Continued) T cell apoptotic process, activation of cysteine-type endopeptidase activity involved in apoptotic process, inflammatory cell apoptotic process, necroptotic signaling pathway, positive regulation of I-kappaB kinase/NK-kappaB signaling, positive regulation of cell proliferation, response to growth factor, transcription and DNA-templated      Further efforts should be made to optimize TLS-quantifying methods. Indeed the use of multicolor IHC will facilitate their characterization, by allowing the simultaneous detection of all major cell types and providing an extensive analysis of their cellular complexity.

TLS neOGeneSiS
The cellular composition and spatial organization of TLS share many similarities with those of SLO. Indeed, an increasing number of studies performed in a large variety of inflammatory disorders, in mice and in humans, suggest that their formation and regulation involve the same set of chemokines than those acting in lymphoid organogenesis.

Positive Regulators
Lymphotoxin, CCL21, and CXCL13 were shown to play a major role during TLS neogenesis, and are related to TLS presence in human tumors (  (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)(35)(36)(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)(39)(40)(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.

negative Regulators
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.

MAniPULATiOn OF TLS FOR A THeRAPeUTiC inTeRvenTiOn in CAnCeR
A series of studies suggest that TLS are sites for generation and maintenance of adaptive anti-tumor responses (10). Therefore, TLS induction could be used as a therapeutic intervention for a better tumor control and prolonged survival of cancer patients. Since LN and TLS share many similarities in terms of cellular composition and organization, deciphering the mechanisms of lymphoid organogenesis enables to first highlight some putative key molecules that can support TLS neogenesis.

Targeting Molecules involved in Lymphoid Organogenesis
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.

Modulation of Chemokine and Cytokine networks
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)(65)(66). A Phase I clinical trial is currently under investigation in NSCLC patients receiving intra-tumoral injections of CCL21transduced 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.

COnCLUSiOn AnD PeRSPeCTiveS
By facilitating the direct entry of CCR7 + naïve T cells and CXCR5 + B cells into tumors through HEVs, TLS allow T cells to differentiate into effector cells upon contact with mature DCs and B cells to form GC, protected from the immunosuppressive milieu of the tumor microenvironment. Therefore, TLS represent sites for the induction and maintenance of the local and systemic anti-tumor responses, which confer long-term protection against metastasis and, hence, correlate with good prognosis for the patients. Indeed, therapies aiming to increase TLS formation may allow generating anti-tumor responses directly in situ and would be beneficial in patients with high mutational load. TLS may also constitute biomarkers of anti-tumor response in patients undergoing immunotherapies. Thus, TLS induction was observed in cervical cancer patients vaccinated with HPV DNA (69) or with G-VAX (70), and one may speculate that TLS signature could be used to evidence response to therapies that unlock the adaptive immune responses.

AUTHOR COnTRiBUTiOnS
ML, NG, HK, CG, and CSF wrote and revised the paper. WF and MCDN revised the paper.

ACKnOwLeDGMenTS
We thank all members of the Laboratory "Cancer, Immune control and Escape, " pathologists and clinicians who have participated in these studies, for their help and valuable discussions, as well as cancer patients.