Tertiary lymphoid structural heterogeneity determines tumour immunity and prospects for clinical application

Effective anti-tumour immunity necessitates the presence of tumour-infiltrating lymphocytes (TILs). This has been particularly evidenced by the identification of tertiary lymphoid structures (TLS), representing well-organized clusters of TILs that elicit delayed immune responses [1]. TLS, immune cell clusters akin to secondary lymphoid organs (SLOs), emerge postnatally within non-lymphoid tissues, typically absent under normal physiological conditions, and predominantly manifest in chronic inflammatory conditions, including cancer, chronic infections, and autoimmune diseases [1, 2]. The context of chronic inflammation is strongly associated with tumour development. Analyses of lymphocyte infiltration and the spatial aggregation of TLS within the tumour microenvironment (TME) of patients with colorectal cancer (CRC), non-small-cell lung cancer (NSCLC), breast cancer (BC), and melanoma indicate that a patient’s prognosis correlates with a higher degree of lymphocyte infiltration, density, and maturation [3‐6]. Evaluating the maturity of TLS across different tumour types is critical for establishing TLS as a reliable prognostic tool in oncology. In addition, inducing a higher density and maturity of TLS in cancer patients contributes to the enhancement of the body’s anti-tumour immunity. Furthermore, promoting a greater density and maturity of TLS in cancer patients significantly enhances the body’s anti-tumour immune response.

In recent decades, immunotherapy targeting immune checkpoints—specifically programmed cell death protein 1 (PD-1), and its ligands programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)—has emerged as a pivotal strategy against various solid tumours. Nevertheless, immune checkpoint blockade (ICB) faces challenges, including clinical application limitations and safety concerns, potentially resulting in inflammatory side effects or immune-related adverse events (irAEs) [7, 8]. Intratumoral TLS has gained recognition as a biomarker capable of predicting and potentially enhancing ICB therapy efficacy [9, 10]. Analyses of intratumoral TLS have demonstrated a positive correlation with clinical benefits in patients, irrespective of PD-L1 expression status, facilitating new markers’ use in ICB therapy patient selection [11]. The distinct features of nanoparticles in multimodal and molecular imaging enable them to be utilized for extremely delicate non-invasive multimodal imaging of biological occurrences in vivo [12]. Developing more efficient, non-invasive techniques for visualizing and inducing TLS represents a novel research direction [13].

In this review, we outline the tissue and cellular structure of TLS and explore the pathways and molecular mechanisms involved in TLS formation and maturation. The dual effects of immune cell crosstalk within TLS on anti-tumour immunity are described, and the potential of TLS as a cancer biomarker is highlighted. Finally, the current induction strategies for TLS are summarized, and the dilemmas and solutions for its future development are discussed.

Based on the impact of mature TLS on tumour prognosis and treatment, it is important to understand the mechanisms of immune cell crosstalk within the internal tissue and cellular structure of mature TLS in the subsequent anti-tumour immune response. It is also essential to understand the important mechanisms of action during TLS formation and maturation.

Previous studies have highlighted that elevated densities of TILs are associated with favorable clinical outcomes across a wide array of solid tumours, underscoring the therapeutic promise of TIL-based approaches [51, 52]. Within the milieu of TIL-positive tumours, mature TLS emerge as pivotal arenas for intricate immune cell interactions [53]. These interactions within an active TLS microenvironment not only facilitate the (re)activation of initial or memory immune cells but also promote the expansion of effector cells, a process that surpasses the capabilities of TILs navigating the tumour bed without directional cues. Despite these beneficial dynamics, the accumulation of regulatory cells within TLS may dampen immune responses, leading to the potential inactivation or dormancy of TLS [54]. Nonetheless, when immune cells within TLS work collaboratively, particularly when B cells produce tumour-specific antibodies and cytotoxic T lymphocytes are activated, they orchestrate a potent anti-tumour immunity, thereby significantly improving the patient’s prognosis (Fig. 2) [19].

In the cancer immunity cycle, the balanced ratio of effector T cells to follicular regulatory T cells (Tfr) is critical, determining the outcome of cancer immunity. Tfr cells, characterized by the CD25 + CXCR5 + GARP + FOXP3 + phenotype and a demethylated forkhead box protein 3 (FOXP3) gene, undergo differentiation through multiple signaling pathways, primarily regulated by FOXP3 and Bcl-6 [50, 84]. The presence of Bcl-6 in Tfr cells, a key transcription factor also found in Tfh cells, suggests a functional link between Tfr and Tfh cells. Recent studies demonstrate that activation of IL-2, STAT3, and STAT5 pathways facilitates the conversion of memory Tfh cells into functional Tfr cells, targeting the FOXP3 and BCL6 genes [85]. Tfr cells produce high levels of TGF-β, which significantly inhibit Tfh cell expansion, GC activation of self-reactive B cells, and autoantibody production [86, 87]. However, mouse studies have demonstrated that IL-21 impedes Tfr cell development by inhibiting protein kinase B (Akt) phosphorylation and diminishing TGF-β and Foxp3 expression [85]. Consequently, the ratio of functional Tfh to Tfr cells dictates the varied impacts on tumour immunity [88]. In BC, Tfr cells have been observed to inhibit Tfh cell function within TLS [54]. Another study identified a significant presence of tumour-infiltrating regulatory T (Ti-Treg) cells around BC tumours, and these Ti-Treg cells were associated with recurrence and death in patients [89]. In NSCLC patients, Tregs migrating within TLS exhibited poor prognostic value, indicating that these TLS-associated Treg actively suppress the immune functions of certain cells such as DCs, T and/or B cells, mirroring findings in lung tumour models [90]. A negative correlation was observed in NSCLC between TLS-B cell density and DPP4 gene expression, a receptor implicated in TCR-mediated T-cell co-activation and Treg-mediated immunosuppression on TIL CD4 + T cells [67]. In early-stage lung cancer, an enrichment and infiltration of Tfr cells within intratumoral TLS were noted, with Tfr cells showing a negative correlation to CD8 + T cells [88].

The ultimate effect of B cells within a tumour is influenced in many ways, within the TLS, by their own phenotype, secreted cytokines, the type of antibody produced by differentiation into PCs, the presence and activation of Treg and activated macrophages [91, 92]. The presence of PD-1 in activated B cells inhibits BCR-mediated signalling, affecting self-maturation and antibody production. In turn, anti-PD-L1 antibodies acting on PD-L1 + Breg cells can impair their inhibitory effect on CD8 + and CD4 + T cells [93]. Although both IgG- and IgA-secreting PCs have been observed in RCC, only a small fraction of IgA is present, and effective anti-tumour activity is mainly associated with IgG [94]. IgA production may be associated with immunosuppressive TGF-β, as well as the secretion of inhibitory cytokines PD-L1 and IL-10 [52, 95]. This suggests that they are usually associated with a poor prognosis (Fig. 2b) [96]. Additionally, a high proportion of intratumoral IgA was observed to be associated with poor prognosis in LUAD [65]. Breg subsets, a subtype of B cells, can induce Treg cell differentiation and are associated with poor clinical outcomes in cancer [97]. Cytokines such as IL-10, IL-35, and TGF-β produced by Breg cells may indirectly inhibit the anti-tumour immune response on the one hand, and on the other hand, may also impair the effector function of T cells, tilting the macrophage towards an immunosuppressive phenotype [1]. This ultimately leads to an inefficient anti-tumour immune response. The presence of Breg cells in E-TLS-like lymphoid aggregates containing Treg cells was observed in BC, and their coexistence resulted in shorter metastasis-free survival compared to Treg cells alone [98].

Tumour-associated myeloid cells have been reported to be associated with poor prognosis. Tumour-associated macrophage-expressing PD-L1 can directly or indirectly suppress T cell responses by recruiting Tregs [99]. Additionally, macrophage-produced indoleamine 2,3-dioxygenase (IDO) has an inhibitory effect on immune cells [100]. M2-polarized macrophages are associated with poor cancer prognosis in BC, liposarcoma, and gastrointestinal stromal tumours [101‐103]. The analysis of TLS in patients with colorectal liver metastases revealed that the density of Treg cells, M2 macrophages, and Tfh cells in intratumoral TLS was significantly higher than that in peritumoral TLS and positively correlated with poor survival [104]. In a rat model of lymphatic malformation (LM), M2 polarized macrophages may accumulate in infected LM via TLS and contribute to disease progression by secreting VEGF [105].

Numerous studies have demonstrated a correlation between TLS and heightened objective response rates, indicating that TLS is a promising marker for immunotherapy. In light of TLS’s contribution to tumour immunity, researchers have increasingly explored the nature of TLS’s existence and its genetic signature to develop precise and dependable predictive biomarkers for cancer patients’ prognosis and treatment (Table 2).

The presence of TLS and B cells across a range of tumour types, including carcinomas, melanomas, soft tissue sarcomas, and RCC, is predictive of a positive response to ICB therapy, indicating the potential for ICB to assume an enhanced role in the treatment of tumours rich in B cells and TLS [11, 17, 57, 135, 136]. Furthermore, plasmacytoid B-cell profiles and TLS-associated B-cell profiles have been found to have a positive correlation with ICB responses in melanoma and LUAD [107, 137‐139]. To improve clinical outcomes post-ICB, therapies supporting TLS induction and activating B cells are necessary and require further research.

In mouse models of cancer and in patients with cancer, cytokines and chemokines, chemotherapy, radiotherapy, cancer vaccines and ICB treatments have demonstrated the ability to induce intra-tumour formation of TLS and increase the infiltration of immune cells (Table 3). In pancreatic cancer, targeted LIGHT localizes to the tumour vasculature via vascular-targeting peptide (VTP), inducing vascular normalization and the formation of HEV and TLS through the self-amplifying cycle of pancreatic cancer. The mechanism may be that LIGHT stimulates macrophages to express the inflammatory cytokines CXCL13, CXCL21, TNF, and IL-6, which recruits T-cells and thus promotes TLS formation [140, 141]. Low doses of STING agonists enhance T-cell and DC infiltration, foster an inflammatory TME, and stimulate DC maturation, which leads to the local secretion of CCL19, CCL21, LTα, LTβ, and LIGHT, thereby promoting vascular normalization and TLS formation [142]. In a trial of 39 patients with resected PDAC treated with a GM-CSF vaccine, 33 exhibited TLS aggregates within two weeks of vaccination. And inhibition of the Treg signalling pathway and enhancement of Th17 signalling within these TLS aggregates were found to be associated with increased survival [143]. Analysis of TLS in pancreatic cancer patients undergoing preoperative neoadjuvant chemoradiotherapy (NAC) through IHC revealed altered TME compositions, increased immune cell proportions, and favorable prognoses [144]. However, a study on lung squamous cell carcinoma (LSCC) patients indicated that corticosteroid use during chemotherapy impacted GC and TLS formation [47]. It is crucial to acknowledge that definitive evidence supporting the capacity of certain immunotherapies to initiate TLS in cancer is presently insufficient, necessitating further empirical research. Specifically, although radiotherapy has been observed to trigger the formation of intratumoral TLS, this phenomenon primarily results from the immediate depletion of local immune cells caused by low-dose radiation, accompanied by a diminution in the size of pre-existing TLS, which typically recuperates within a fortnight [145, 146]. Hence, induction of TLS by radiotherapy is currently not supported by sufficient evidence. Importantly, conventional cancer immunotherapies exhibit limitations, such as varying systemic toxicities and heterogeneous therapeutic outcomes, depending on the type of immunotherapy employed [7, 147]. Recent advancements in nanotechnology and biomaterials have unveiled significant potential for innovation in cancer immunotherapy (Fig. 4) [148].

The spatial structure of TLS (presence of GCs), its cellular composition (high-density TIL infiltration and differences in immune cell phenotypes), and its relationship to tumour location (peri-tumour or intra-tumour) affect the prognosis of tumour patients [3, 4, 25, 48, 175]. Notably, the dual role of TLS in tumour immunity—termed the “double-edged sword” effect—may hinge more on the proportions of various immune cell subpopulations within the TLS, particularly given the observed variability in T and/or B cell phenotypes. Among T cell subsets, CD4 + T follicular helper (Tfh) cells, expressing high levels of CXCR5 and Bcl6, along with CD8 + T cells, are the primary contributors to anti-tumour immunity among T cell subsets. But the precise mechanism underlying the cytotoxic effect of TLS-modified CD8 + T cells on tumour cells remains unclear. Tfh cells facilitate B cell recruitment through the high expression of co-stimulatory receptors (ICOS and CD153), thereby slowing tumour progression. Tfr cells, characterised as CD25CXCR5GARPFOXP3, inhibit Tfh cell activity with the involvement of TGF-β. Despite the lack of consensus on markers for Breg cells, their secretion of immunosuppressive cytokines such as IL-10, IL-35, and TGF-β can inhibit effector T cell activity and promote the expansion of Treg cells, thereby facilitating tumour growth. The characterization of B cell subpopulations through RNA sequencing (RNA-seq) or specific antibodies revealed that, with the exception of Breg cells, all B cell subpopulations generally correlate with positive clinical outcomes. The in situ activation of GC-B cells within TLS for differentiation and antibody production further underscores the significance of B cells within the cancer immune cycle. The phenotype of different immune cell subpopulations is largely dependent on cytokines with different immune properties in the TME, as well as on the influence of cell signalling pathways, which are not yet fully understood.

As a novel specific biomarker to influence and determine the prognosis of cancer patients, the future development of TLS involves two main aspects. Although biomarkers of TLS have been widely studied, there is no established uniform standard for quantification of TLS in different patients and different cancers. First, we need to focus on the difficulties that the heterogeneity of TLS poses for the quantification and assessment of different cancer types. The combined application of specific TLS markers, imaging technologies and AI in histopathology may open up a new path for TLS detection and quantitative analysis.

Second, we need to effectively induce the occurrence and maturation of TLS in tumours. Traditional induction methods through injection of cytokines or chemokines, radiotherapy, chemotherapy, ICB therapy and cancer vaccines have successfully induced TLS in mouse models or humans, but novel biomaterials such as collagen mechanisms, hydrogels, nanomaterials as well as the development of organoid and 3D technologies have reinvigorated TLS induction and enhanced the induction efficiency. The possibility of using complex technologies, including novel biomaterials, to generate inducible TLS, is being investigated with very promising results in preclinical models [176]. Nevertheless, the translation of these technological tools into the clinic still needs to address issues of tissue safety, cost control and selection of appropriate humanised in vivo models. This is essential to guide further precision and efficiency of TLS in immunotherapy and prognosis.