Orchestration of tertiary lymphoid structures: decoding developmental mechanisms for next-generation cancer immunotherapies

Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that form in non-lymphoid organs. TLSs are associated with cancer, autoimmune diseases, transplant rejection, and chronic inflammation. TLSs are primarily composed of B cells and T cells which make up the bulk of TLS-associated immune cells, antigen-presenting DC-LAMP⁺ mature dendritic cells, as well as Gfibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs) that provide structural and functional support for lymphocyte aggregates [1].The maturation of TLSs undergoes three stages: the initial formation of unstructured lymphocyte aggregates, the gradual emergence of distinct T and B cell zones, and the eventual formation of germinal centers with FDCs [2].Clinical studies have shown that TLSs are closely associated with tumor progression, immune responses, and patient prognosis. The correlation between TLSs and patient prognosis depends on the maturity level and distribution patterns of TLSs, as well as types of cancers. In the most cases, TLSs support in situ antibody production within tumor tissues, thereby enhancing both humoral and cellular immune responses. Patients with tumors containing a higher number of mature TLSs often exhibit better clinical outcomes [3]. However, it has also been reported that ectopic lymphoid structures form cytokine-enriched microniches that promote tumor progression in malignancies such as hepatocellular carcinoma [4].

Due to the anatomical resemblances to secondary lymphoid organs (SLOs), the formation process and drivers of TLSs are fundamentally similar to those of SLOs. In SLOs, inducer cells express lymphotoxin(LT) that binds to LTβR on organizer cells to stimulate the expression of chemokines and adhesion molecules, and coordinate lymphoid neogenesis. A recent study showed that IL-33 produced in pancreatic ductal adenocarcinoma (PDAC) can activate group 2 innate lymphoid cells (ILC2s) expressing LT and initiate TLSs formation through LTβR⁺ myeloid organizer cells [5]. They identified a previously undescribed pathway that can be engineered to treat pancreatic cancer and reveal a pro-lymphoid neogenic function for IL33 and ILC2s [5]. Within SLOs, stromal cells direct B cell migration into germinal centers through chemokine secretion, including CCL19, CCL21, CXCL12, and CXCL13 [1]. Recent studies have revealed that chronically activated tumor-reactive T cells function as TLS organizers through CXCL13 production, a process critical for effective immunotherapy responses in lung cancer [6]. In contrast to SLOs which form in normal tissues, the formation of TLSs within tumors is influenced by tumor-specific factors. These driving factors may originate from tumor cells or from the unique microenvironment shaped by the tumor cells. A study on nasopharyngeal carcinoma demonstrated that DNA fragments induced by chemotherapy activates the STING-dependent IFN-Ⅰ pathway to increase MHC-Ⅰ expression in cancer cells, and simultaneously induced innate-like B cell(ILB) aggregations, which support the formation of TLSs [7]. Beyond tumor cells, the tumor microenvironment also plays a pivotal role in the formation of TLSs. Stromal cells influence immune cell survival and aggregation in TLSs through inflammatory cytokines and chemokines. In high grade serous ovarian cancers(HGSOC), normal mesenchymal stem cells (nMSC) are reprogrammed by tumor cells into tumor-promoting cancer-educated mesenchymal stem cells(CA-MSCs), which are less ready to differentiate into lymphoid-supporting stromal cells [8]. CA-MSCs upregulate immunosuppressive factors such as WT1 and CD200, while downregulating TLS-promoting molecules, including CXCL12, IL7, PDGFRβ and FCεR1G [9].In vitro FDCs differentiation experiments also reveal that CA-MSCs are less ready to differentiate into lymphoid-supporting stromal cells [9]. Tumor cells possess the capacity to reprogram MSCs, thereby inhibiting the formation of TLSs.

In the majority of tumors, the presence of TLSs within tumors correlates positively with patient prognosis. However, the mechanism of their anti-tumor activity is still -under investigation. B cells and T cells serve as the principal effector populations within TLSs that mediate antitumor immunity. It has been identified that TLS⁺ tumors are enriched with IgG⁺ plasma cells(PCs) in colorectal cancer liver metastasis, which support antibody-dependent cellular cytotoxicity(ADCC) and antibody-dependent cellular phagocytosis(ADCP) activity through interaction with macrophages and promote tumor apoptosis [10]. TLS-associated PC-derived IgG isotypes was also observed in pancreatic cancer [11]. These studies dissected the pivotal role of B cell responses in mediating the anti-tumor functionality of TLSs. The spatial proximity to tumor cells significantly enhances the probability of B cells receiving antigenic stimulation. Besides, T cells are identified as the main lymphocytic cluster in mediating tumor cell cytotoxicity. Recent researches demonstrated that the LTα/TNFR2 axis-triggered mTOR pathway is crucial for effector function of CXCL13⁺CD103⁺CD8⁺ tissue-resident memory T (Trm) cells by enhancing glycolysis, a known subset of memory T cells within TLSs which contributed to a favorable response to anti–PD-1 therapy, shedding light on the role of CXCL13⁺CD103⁺CD8⁺ Trm cells in promoting cancer immunotherapy [12] (Figure 1).

Figure 1:

The drivers of TLSs formation and the anti-tumor function of TLSs.nMSCs, normal mesenchymal stem cells; CA-MSC, cancer-educated mesenchymal stem cell; ILB, innate-like B cell; TLR9, Toll-like receptor 9; PC, plasma cell; ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; ILC2, group 2 innate lymphoid cells; LT, lymphotoxin; TNFR2, tumor necrosis factor receptor2; ICI, Immune checkpoint inhibitors; DC, dendritic cell; FRC, fibroblastic reticular cell; FDC, follicular dendritic cell; VEC, vascular endothelial cell.

While TLSs are increasingly recognized as critical determinants of clinical prognosis, with higher TLS density broadly associated with improved therapeutic response and survival outcomes across multiple malignancies, the correlation between TLSs and tumor development is still ambiguous. Firstly, the precise stage of tumorigenesis at which TLS formation initiates remains unclear. and their dynamic evolution throughout tumor progression has yet to be elucidated. Secondly, beyond numerical abundance, the location and maturation status of TLSs also exhibit a significant correlation with tumor aggressiveness, metastatic potential, and therapeutic response. Thirdly, the prognostic value of TLSs exhibits inherent heterogeneity depending on the tumor type and molecular classification. In-depth exploration of the interactions between TLSs formation and tumor development may facilitate early tumor diagnosis and prognosis prediction through TLSs monitoring.

Therapeutic induction of TLSs has become a pivotal area of investigation in immuno-oncology, driven by their prognostic and immunomodulatory potential. Recent studies have revealed novel potential strategies for TLSs induction as mentioned above; however, significant translational gaps must be addressed before clinical implementation. Conventional clinical interventions, such as chemotherapy and radiotherapy [13], have been observed to induce TLSs formation in experimental settings, yet the underlying mechanisms remain obscure.

Due to the structural and functional complexity of TLSs, research in this area faces numerous challenges. First, conventional short-term murine tumor models (such as subcutaneous xenografts and orthotopic models) rarely develop TLSs, thus compelling researchers to predominantly rely on patient samples. Secondly, most current studies on TLSs rely on a combination of microdissection and sequencing analysis, but the spatial information obtained is limited. The diverse cellular components and intricate interactions within TLSs, as well as their scattered distribution within the tumor stroma surrounded by cancer-associated fibroblasts, pose difficulties in isolating TLSs and obtaining relevant information. Therefore, the development and application of high-resolution spatial multi-omics technologies, combined with big data analysis, is important for deciphering the cellular localization and histological function within TLSs [14], 15]. It will enable researchers to obtain a deeper understanding of the complex tumor microenvironment and develop novel immunotherapeutic strategies.