Tertiary Lymphoid Structures in Cancer: Advancing Therapeutic Implications

Introduction

The tumor microenvironment (TME) is a complex ecosystem comprising immune cells, stromal components, and vasculature that collectively influence cancer progression and response to therapy. Among the emerging players in tumor immunology are tertiary lymphoid structures (TLS)- ectopic lymphoid aggregates that form in non-lymphoid tissues, including tumors. These structures resemble secondary lymphoid organs (SLOs) such as lymph nodes and play a crucial role in shaping anti-tumor immunity. Recent research has highlighted their prognostic significance, association with improved immunotherapy responses and potential as therapeutic targets. This review explores the maturation, induction, and clinical relevance of TLS in cancer, providing insights for oncologists and immunologists.

What Are Tertiary Lymphoid Structures?

Tertiary lymphoid structures are organized lymphoid formations that develop in peripheral tissues under chronic inflammatory conditions, including cancer, autoimmune diseases, and chronic infections. Unlike SLOs, which form during embryogenesis, TLS arise de novo in response to persistent antigenic stimulation. Structurally, mature TLS contain distinct T-cell zones, B-cell follicles, germinal centers, and high endothelial venules (HEVs), facilitating local immune cell recruitment and activation. Their presence in tumors correlates with enhanced lymphocyte infiltration, improved patient survival, and better responses to immune checkpoint inhibitors (ICIs).

Maturation Stages of TLS in Cancer

The development of TLS follows a stepwise maturation process, categorized into three main stages: early, intermediate, and fully mature TLS. Early TLS are characterized by diffuse lymphocyte aggregates without clear organization. Intermediate TLS exhibit segregated T- and B-cell zones but lack germinal centers. Fully mature TLS display well-defined follicles with active germinal centers, dendritic cell networks, and HEVs, enabling efficient antigen presentation and adaptive immune responses.

The maturation of TLS depends on lymphoid chemokines (CXCL13, CCL19, CCL21), cytokines (lymphotoxin-α/β, IL-17), and stromal cell interactions. Tumor-associated fibroblasts and endothelial cells contribute to TLS formation by secreting these factors. Notably, the presence of mature TLS in tumors has been linked to prolonged survival in multiple cancers, including melanoma, lung adenocarcinoma, and colorectal carcinoma, making them a promising biomarker for immunotherapy efficacy.

Induction of TLS in the Tumor Microenvironment

Given their role in promoting anti-tumor immunity, strategies to induce TLS in cancer patients are being actively explored. Several approaches have shown promise in preclinical and clinical studies:

1. Chemokine-Based Therapies

CXCL13, CCL19, and CCL21 are critical for lymphocyte homing and TLS formation. Intratumoral delivery of these chemokines, either via gene therapy or recombinant proteins, has been shown to enhance TLS development in murine models. Clinical trials investigating oncolytic viruses engineered to express lymphoid chemokines are underway, with early data suggesting improved T-cell infiltration and tumor regression.

2. Immune Checkpoint Blockade and TLS Formation

Interestingly, responders to PD-1/PD-L1 inhibitors often exhibit pre-existing or therapy-induced TLS, indicating a synergistic relationship between TLS and ICIs. Checkpoint inhibitors may amplify TLS function by reinvigorating exhausted T cells within these structures. Combining ICIs with TLS-inducing agents (e.g., CD40 agonists, TLR ligands) could further enhance anti-tumor immunity.

3. Stromal Cell Reprogramming

Tumor-associated fibroblasts and endothelial cells can either support or inhibit TLS formation. Targeting inhibitory signals (e.g., TGF-β, VEGF) while promoting lymphoid chemokine secretion may facilitate TLS induction. Drugs such as VEGF inhibitors (bevacizumab) and FAK inhibitors are being tested for their ability to remodel the TME and promote TLS.

Clinical Implications and Prognostic Value

The presence of TLS has emerged as a favorable prognostic marker in multiple malignancies. In breast cancer, TLS density correlates with improved recurrence-free survival. In non-small cell lung cancer (NSCLC), patients with mature TLS show higher response rates to PD-1 blockade. Additionally, TLS-rich tumors exhibit increased tumor-infiltrating lymphocytes (TILs), tertiary lymphoid structure-associated B cells (TLS-B), and plasma cells, which contribute to antibody-mediated anti-tumor responses.

However, not all TLS are beneficial. In certain contexts (e.g., hepatocellular carcinoma, pancreatic ductal adenocarcinoma), TLS may harbor regulatory T cells (Tregs) or tumor-promoting B cells, suggesting functional heterogeneity. Further research is needed to decipher the mechanisms underlying TLS plasticity and their dual roles in cancer immunity.

Challenges and Future Directions

Despite their therapeutic potential, several challenges remain in harnessing TLS for cancer treatment. Standardized methods for TLS quantification and classification in clinical samples are lacking, complicating biomarker validation. Additionally, optimal strategies for pharmacological TLS induction without exacerbating autoimmunity require careful optimization. Emerging technologies, such as spatial transcriptomics and multiplex immunohistochemistry, may provide deeper insights into TLS biology and guide precision immunotherapy approaches.

Conclusion

Tertiary lymphoid structures represent a fascinating intersection of adaptive immunity and cancer biology, with significant implications for prognosis and treatment. Their ability to foster anti-tumor immune responses makes them attractive targets for next-generation immunotherapies. By elucidating the molecular pathways governing TLS maturation and induction, researchers can develop novel strategies to enhance immunotherapy efficacy and improve patient outcomes. As the field advances, integrating TLS biomarkers into clinical practice could pave the way for personalized cancer immunotherapy.