Definition and components of TLS

Numerous cytokines, growth factors, extracellular matrix and different types of cells including endothelial cells, immune cells, etc, are present in tumour immune microenvironments. They surround tumour cells and are nourished by blood vessels. It has a substantial effect on the therapeutic effectiveness of tumours. Immune cell aggregates in non-lymphoid tissues and generate TLS. Presently, they are associated with chronic inflammation, including autoimmunity, chronic infections and cancer. TLS is prevalent in the inner regions of B cells and T cells in the periphery. HEVs are speculated to promote lymphocyte recruitment as the particular vascular system in TLS. Dendritic cells, macrophages and other immune cells may also be incorporated. T cells in TLS are primarily CD3 + T cells, including CD4 + T cells and CD8 + T cells. In 2011, L de et al. showed that most T cells were CD62L + , primarily CD4 + memory phenotype in TLS in human lung cancer (Ref. Reference de Chaisemartin59). Furthermore, TLS contains CD103 + T cells (Refs Reference Mori60, Reference Mori61). Germain et al. demonstrated that the density of follicular B cells and mature dendritic cells can assess a patient's optimal clinical prognosis (Ref. Reference Germain62). TLS composition may differ based on location. It was found that the superficial layer of TLS contained substantially more T helper cells and early TLS than the deep layer (Ref. Reference van Dijk14).

TLS has been found in various human tumours including lung, melanoma, breast and colorectal cancers, however, it is uncommon in mouse tumour models. In patients with grade 1 or 2 non-functional pancreatic neuroendocrine tumours, TLS consists primarily of B cell follicles and T cell regions with dendritic cells (Ref. Reference Zhang63). A study revealed that CD3 + T cells, CD20 + B cells, CD8 + T cells, CD208 + dendritic cells and CD21 + follicular dendritic cells located in TLS of hepatocellular carcinoma (Ref. Reference Nie12). Moreover, some studies implied that antitumour plasma cells may be located in TLS (Ref. Reference Meylan64). TIM4 + macrophages (MΦ) are present in the cancer-associated T-cell region of TLS. TLSTIM4 + MΦ is enriched in tumours with elevated CD8 + T cell infiltration, associated with antitumour immunity (Ref. Reference Bugatti65). A TLS-related immune cell infiltration study found that CD3 + T, CD8 + T and CD20 + B cell infiltration elevates in patients with gastric cancer, whereas CD68 + cell infiltration is limited (Ref. Reference Yu66). TLS rich in CD20 + B cells, CD8 + T cells, CD4 + T cells and CD38 + plasma cells are found in endometrial carcinoma (Ref. Reference Qin13). TCF1/TCF7 + T cells showed a significant association with TLS in a model of oral cancer (Ref. Reference Peng67). CD20 + CD22 + ADAM28 + B cells are present in various tumour-associated TLS (Ref. Reference Wu68). The density of CD8 + T cells and CD20 + B cells was high in TLS-positive tissues (Ref. Reference Ahmed69). TGF-β-mediated SATB1 inhibition promotes T cell differentiation to Tfh and further promotes TLS formation in ovarian tumours (Ref. Reference Chaurio70). TLS mainly comprises B cells, T cells, macrophages, DCs, FDCs (follicular dendritic cells), and HEVs.

TME refers to the surrounding microenvironment in which tumour cells exist, including surrounding blood vessels, lymphatics, immune cells, fibroblasts, MDSC, various signalling molecules and extracellular matrix (ECM). TME is a complex environment that assists in the survival and development of tumour cells, particularly the immune cells. As a cluster of immune cells, TLS is an essential immune modulating component of the TME (Fig. 1). TLS regulation may play a significant role in TME reconfiguration.

Figure 1. TLS is a special structure in TME. TLS is mainly composed of B cells (native, mature, memory), T cells (CTL, native, Tfh), macrophage (M1, M2), DCs, FDCs and HEVs.

Role of TLS in tumour progression and prognosis

Reduced TLS formation in ALK + lung adenocarcinoma is closely correlated with tumour progression and may account for reduced immunotherapy response (Ref. Reference Zou71). The high-density of Treg cells in TLS correlates with decreased survival in NSCLC patients (Ref. Reference Devi-Marulkar72). Relapsed patients with advanced colorectal cancer have substantially elevated levels of Th (T helper)-type TLS (Ref. Reference Yamaguchi73). One study revealed that breast cancer patients with peritumoral TLS had worse DFS and OS than those with TLS (-) (Ref. Reference Sofopoulos74). TLS was associated with a poor prognosis in kidney clear cell carcinoma, however, it showed an improved prognosis in bladder cancer, according to a comparative analysis (Ref. Reference Masuda75). Early liver cancer can result in immature TLS formation and local immune activation, while promotes immune evasion and tumour progression (Ref. Reference Meylan76). Particular reasons require further investigation. Although there are few studies on TLS and tumour progression, TLS may play numerous functions in various cancer types, particularly in genetic mutations or advanced tumours, which may be one of our future research directions.

Previous research showed that TLS is an immune cell aggregation in the TME. Schumacher summarized that TLS possesses multiple advantages over peripheral circulation lymphocytes or tumour infiltration lymphocytes in the context of antitumour immunity (Ref. Reference Schumacher and Thommen10). First speed: priming T and B cells at the TLS may shorten the time required to generate immune responses because it bypasses the immune cells to and from the SLOs. Second efficiency: the formation of local germinal centres can enhance the efficiency of immunotherapy by initiating both humoral and cellular immune responses. Third control: direct exposure of TLS-associated immune cells to the TME may alter immune response reception of particular output signals. Fourth survival: the interaction between TLS-associated effector T cells and APCs promotes patients' survival. Some studies suggested a connection between profuse TILs and TLS (Refs Reference Acar77, Reference Cheng78). Researchers also noted that mature TLS may activate lymphocytes to perform an antitumour immune role (Refs Reference Byers79, Reference Wakasu80). A higher TILs number in TLS was associated with a better prognosis (Refs Reference Li81, Reference Li82, Reference Tanaka83). The number of TLS in a breast cancer metastatic site is proportional to the number of TILs (Ref. Reference Lee84). Thus, TILs were substantially correlated with TLS and had a favourable prognosis. When examining the relationship between various therapies and TLS, we can also investigate the relationship between TILs and multiple treatments.

Numerous studies examined the association between TLS and good tumour prognosis in melanoma, lung cancer and colorectal cancer. TLS is essential for initiating and maintaining local and systemic T and B cell antitumour responses, stimulating cellular and humoral immunity channels and increasing antitumour immune response. Currently, TLS has been identified in nearly all solid tumours and has been associated with clinical outcomes in patients with non-small cell lung cancer (Refs Reference Wakasu80, Reference Feng85, Reference Tamiya86), pancreatic neuroendocrine tumours (Ref. Reference Zhang63), breast cancer (Ref. Reference Wang87), gastric cancer (Refs Reference Mori60, Reference Cheng78), esophageal squamous cell carcinoma (Ref. Reference Li88), early oral tongue squamous cell carcinoma (Refs Reference Almangush89, Reference Li90, Reference Wang91), cutaneous angiosarcoma (Ref. Reference Magara92), epithelioid pleural mesothelioma (Ref. Reference Mannarino93), hepatocellular carcinoma (Refs Reference Jia94, Reference Li95, Reference Zhang96), perihilar cholangiocarcinoma (Ref. Reference Zhang97) and endometrial cancer (Ref. Reference Horeweg98), displayed in Table 1. Simultaneously, the prognosis of tumour patients changes according to the presence, maturity, location, and high or low TLS signature (Table 1). Most studies have found that TLS is associated with a favourable prognosis; however, TLS may play distinct roles in various regions of hepatocellular carcinoma. In intra-tumoral tissue, the prognosis of DFS in the TLS + group was better. However, in peritumoral tissues, the TLS-group had better OS and DFS outcomes than the TLS + group (Ref. Reference Zhang96). Another study demonstrated that intra-tumour TLS is substantially associated with improved RFS and DFS (Refs Reference Jia94, Reference Li95). Another study revealed that TLS did not affect the OS of patients with liver cancer but significantly affected RFS (Ref. Reference Li81).

Table 1. Initial studies discerning the effect of TLS on various cancer patients' outcomes

TLS can be categorized as mature TLS, immature TLS and negative TLS. Compared to immature TLS, the proportion of proliferating B cells, and CD4 + T cells increased in mature TLS, and B memory cells and Th17 cells increased in mature TLS. In esophageal cancer patients, mature TLS in conjunction with elevated CD8 + T cell infiltration is correlated with optimal survival, and the prevalence of mature TLS is an independent prognostic factor (Ref. Reference Ling99). In contrast, patients with mature TLS had prolonged survival, activated CD4 + memory cells, primitive B cells and NK cells than those with early-stage TLS (Ref. Reference Gunderson100). Similarly, patients with esophageal squamous cell carcinoma who received neoadjuvant chemotherapy had a superior prognosis in the mature TLS high-density group than in the mature TLS low-density group (Ref. Reference Deguchi101). In colorectal cancer, Helicobacter hepaticus-specific Tfh cells may facilitate TLS maturation, and further enhance antitumour immunity by promoting TLS maturation. This antitumour effect is surprisingly dependent on CD4 + T cells, B cells and natural killer cells, rather than CD8 + T cells (Ref. Reference Overacre-Delgoffe102). The analysis of 33 cases of colorectal cancer revealed that the expression of IL-36γ was correlated with the infiltration of CD4 + T cells and the enhancement in B cell density in TLS (Ref. Reference Weinstein103). It has been found that low-dose RT combined with PD-1 inhibitors increased both the quantity and maturity of TLS in patients with lung adenocarcinoma (Ref. Reference Wang104).

TLS can be classified as A, B and C grades based on the density of immune cells. The OS of patients with grades C or B improved significantly. TLS and CD20 + B cells were considerably higher in triple-negative breast cancer patients with high-density plasma cells than those with low-density plasma cells (Ref. Reference Seow105). TLS can also be divided into internal and peripheral tumours based on location. It has been revealed that intra-tumour TLS may have a better OS (Ref. Reference Yin106). Another study also showed that tumour-associated B cells are predominantly present in TLS (Ref. Reference Schlosser107). Both T follicular helper cells and regulatory T cells were significantly elevated in intra-tumoral TLS relative to the peritumoral area, and patients with intra-tumoral intrahepatic cholangiocarcinoma had a better prognosis (Ref. Reference Ding108).

TLS has the potential to stimulate antitumour immunity. TLS can improve the immune response in many solid tumours, and the level of TLS may be a predictor of immunotherapy efficacy. It has been suggested that B cells in TLS can act as APC to prompt cytotoxic T cells (Ref. Reference Yamakoshi109). In rhabdomyosarcoma, T cells in TLS may increase antitumour response (Ref. Reference Chen110). Intra-tumoral TLS was linked to a decreased risk of early recurrence in HCC patients undergoing surgery. It is suggested that TLS in tumours may indicate sustained and effective antitumour immunity (Ref. Reference Calderaro111). Studies also demonstrated that in hepatocellular carcinoma, peritumoral TLS showed significantly higher immune infiltration and positive immune response. Patients with a high peritumoral TLS density have excellent clinical outcomes (Ref. Reference Li112). Intra-tumour TLS is associated with the response to antitumour immunity in renal cell carcinoma (Refs Reference Meylan64, Reference Meylan113).

In summary, the presence of TLS in TME has a significant association with the clinical prognosis in solid tumours. We can see better OS, RFS and DFS in numerous solid tumours when TLS appears. These outcomes are based on the function of TLS in the TME. Similarly, the location, density and maturation of TLS also influence the outcomes. The relevant results need to be further studied.

RT regulates non-immune cells in TME

When tumour cells die following exposure to RT, they can change from non-immunogenic to immunogenic, prompting an antitumour immune response referred to as immunogenic cell death (ICD). ICD can produce new antigenic epitopes from dead tumour cells, generate damage-associated molecular patterns (DAMPs), and recruit antigen-presenting cells (APC). APC identifies antigens associated with tumour cells, presents them to T cells, activates adaptive immune responses, and produces durable antitumour immunity. RT induces the formation of reactive oxygen species (ROS), which facilitate the release or exposure of DAMPs (Ref. Reference Radogna and Diederich116). RT can induce ICDs and change surface markers of tumour cells, including ATP, CALR, HMGB1, Type 1 IFN and IL-1β (Ref. Reference Fucikova117). Extracellular ATP links to the purinergic receptor P2Y2 (P2RY2) to serve as a ‘find-me’ signal for dendritic cell precursors and macrophages, thus encouraging myeloid cell recruitment to the active ICD site (Ref. Reference Elliott118). In addition, amplifying the ‘eat me signal’ during ICD is crucial for cancer immunotherapy. Some treatments such as radiation therapy and photodynamic therapy can initiate or promote ICD and activate an antitumour immune response (Ref. Reference Xi119). CALR stimulates LDL receptor-associated protein 1 (LRP1, also known as CD91) on phagocytes to activate antigen-producing cells such as DC and macrophages that promote the clearance of tumour cells (Ref. Reference Gardai120). Extracellular HMGB1 can bind to numerous PRRs expressed by myeloid cells, with the most common mode being Toll-like receptor 4 (TLR4), which stimulates the release of pro-inflammatory cytokines (Refs Reference Apetoh121, Reference Park122). The immunogenicity of radiation induced ICDs relies on type I IFN signalling (Ref. Reference Vanpouille-Box123). Anthracyclines induce the endothelial cell pattern recognition receptor TLR3 to activate tumour cells to rapidly generate type I interferon and stimulate the release of CXCL10 (Ref. Reference Sistigu124). When ICD arises, the above DAMPs are released, APC (macrophages, DC) are activated and matured, and the secretion of IL-6, IL-1β, TNF-α and IFN-γ by mature DC promotes the differentiation of T cells into CD8 + phenotypes (Ref. Reference Kielbik, Szulc-Kielbik and Klink125). Cross-presentation of antigens by DC activates and transforms CD8 + T cells into cytotoxic T lymphocytes. TLS is considered ‘mature’ when at least one CD23 + dendritic cell is present (Ref. Reference Vanhersecke126). RT can promote the development of immature DC into mature DC, so we speculate that RT can also promote the development of immature TLS into mature TLS (Fig. 2A).

Different radiation dose caused varying degrees of damage to tumour vessels. At lower doses (6 or 12 Gy), the vascular bed recovered approximately six days after exposure. When a higher dose of 18 Gy was administered, the tumour's response time was substantially prolonged, and recovery did not commence until the 10th day (Ref. Reference Orlova127). Another study found that irradiation higher than 10 Gy /F may cause significant vascular damage, resulting in decreased blood perfusion, and damage to the microenvironment within the tumour (Ref. Reference Park128). High-dose radiotherapy (HDRT) promotes Notch1 signalling pathways and Notch1 expression, and Notch1 activation might defend tumour vessels from HDRT-induced damage (Ref. Reference Banerjee129). RT can promote the death of vascular endothelial cells, producing chemokines such as TNF-α and HMGB-1 (Ref. Reference Smith130). RT upregulates adhesion molecules (VCAM-1/ICAM-1 and p/e selection), normalizing the aberrant vascular system and facilitating the recruitment of circulating immune cells (Ref. Reference Portella and Scala115). RT might help initiate and maintain vascular normalization in TME (Refs Reference Ganss41, Reference Spiotto, Yang-Xin and Weichselbaum131). However, lactic acid accumulation in hypoxia/HIF-1α-driven TME can inhibit T cell proliferation, tumour infiltration and cytokine production, suppress the cytotoxic activity of NK and CD8 + T cells, and enhance the number of MDSCs (Refs Reference Brand132, Reference Husain133, Reference Potzl134).

RT and various immune cells in the TLS

RT eliminates tumour cells and induces the release of pro-inflammatory molecules. Necrotic tumour cells generate antigens associated with the tumour. APCs acquire tumour-associated antigens, migrate to draining lymph nodes, and present these antigens to specific T cells via MHC Class I molecules, enhancing T cell activation and proliferation. CD8 + T cells that have been activated can prevent the production of immunosuppressive cells, produce perforin and granzyme, and exert an antitumour effect. What impact does RT have on various immune cell types?