Immune cell infiltration is crucial in cancer's tumor microenvironment (TME). Tertiary lymphoid structures (TLS) are well-organized ectopic immune cell compartments that develop in inflamed or tumoral tissues. TLS contain T and B cell zones, antigen-presenting cells (APCs), stromal cells, T follicular helper (Tfh) cells, and follicular dendritic cells (FDCs). Recent single-cell RNA sequencing and spatial transcriptomics studies have revealed specific TLS cell populations in various cancers, but their heterogeneity and composition warrant further investigation using larger sample sizes and advanced technologies. Germinal centers (GCs) are crucial features of mature TLS, playing a role in B cell clone activation and long-lived plasma cell production. Although GC B cells and tumor-infiltrating B or plasma cells coexist in multiple tumors, the dynamics of GC reaction and B cell development within tumors, especially in TLS, remain largely unknown. Multiple cellular components (LTi cells, FRCs, FDCs) and molecular processes (LTBR signaling, adhesion molecules, lymphoid chemokines) are known to drive TLS formation, but their roles in human tumors are less understood. TLS process tumor antigens, generating effector memory T cells, memory B cells, and plasma cells that participate in anti-tumor responses. TLS-associated plasma cells secrete antibodies that recognize tumor-associated antigens, promoting malignant cell apoptosis through ADCC and ADCP. This explains the positive association of TLS with tumor-infiltrating immune cells, favorable patient survival, and better responses to immune checkpoint blockade (ICB) therapy. However, the spatial interplay between TLS-associated effector T and B cells and tumor cells is not fully characterized. This study aims to uncover the cellular composition, formation, and function of TLS in nasopharyngeal carcinoma (NPC), a prevalent malignancy with intensive immune cell infiltration. The prognostic value of key TLS components for patient survival and immunotherapy response will also be evaluated.

The literature extensively documents the importance of tertiary lymphoid structures (TLS) in cancer, highlighting their complex interplay with tumor cells and the surrounding microenvironment. Studies have identified various key cell types within TLS, including T and B cells, follicular dendritic cells, and stromal cells, which collectively contribute to the anti-tumor immune response. However, the precise composition and functional dynamics of TLS vary across different cancer types, emphasizing the need for more detailed investigations into specific cancers such as nasopharyngeal carcinoma (NPC). The role of germinal centers (GCs) within TLS has been established, but the specific developmental trajectories of B cells within the tumor microenvironment, especially their relationship to plasma cells and antibody production, require further exploration. Moreover, the interaction of TLS with tumor cells and the implications for immunotherapy responses have been partially characterized, and this area would benefit from further investigation. The current literature review sets the stage for a detailed investigation into the cellular composition, spatial organization, and functional role of TLS in NPC, providing crucial insights into their impact on tumor progression and immunotherapy outcomes.

This study employed a multi-omics approach combining single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) to comprehensively characterize TLS in nasopharyngeal carcinoma (NPC). For scRNA-seq, 77 samples (56 tumors, 11 nasopharyngeal non-cancerous tissues, and 10 PBMCs) from 56 NPC patients were analyzed, yielding 343,829 cells after quality control. Immune cell receptor profiles were also generated. ST analysis was performed using two platforms: Stereo-seq (3 fresh-frozen tumors, 9563 data bins) and Visium (12 FFPE tumors, 21,753 data spots). Additionally, data from a previously published microdissection study (189 samples) were integrated. Multiplex immunohistochemistry (IHC) was used to validate findings. Data analysis included graph-based clustering, pseudotime trajectory analysis, differential gene expression analysis, gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), cell-cell interaction analysis using CellChat and CSOmap, and spatial proximity analysis using SpaGene. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards models. In vitro co-culture assays were conducted to validate the functional roles of identified cell populations, assessing B cell adhesion, migration, and antibody production. Finally, the EBV infection status was assessed using in situ hybridization of EBERs and gene expression analysis. Statistical analyses were performed using various methods, including t-tests, Wilcoxon tests, Pearson correlation tests, Chi-square tests, and Kruskal-Wallis tests. The significance level was set at P<0.05. Specific details on data processing and analysis methods are provided in the supplementary materials.

This study identified several key cell populations within tertiary lymphoid structures (TLS) in nasopharyngeal carcinoma (NPC) tumors. These include CXCL13+ cancer-associated fibroblasts (CAFs), stem-like CXCL13+CD8+ T cells, and B and T follicular helper cells. The germinal center (GC) reaction within TLS was shown to mature plasma cells, which then interact with tumor cell aggregates (TCA). These plasma cells promote apoptosis of Epstein-Barr virus (EBV)-related malignant cells, enhancing immunotherapy response. The CXCL13+ CAFs were found to promote B cell adhesion and antibody production, while activating CXCL13+CD8+ T cells that become exhausted within TCAs. The proportion of GC B cells decreased in advanced-stage NPC tumors, while plasma cell proportions remained relatively constant. This seeming contradiction was explained by a reduced transition of BCR clonotypes between GC B and plasma cells and lower immunoglobulin (Ig) transcription levels in advanced-stage tumors. The study found a positive correlation between plasma cell proportions and NK cell/macrophage proportions, suggesting a role for ADCC and ADCP. Tumors with high EBV load exhibited lower pan-Ig transcription in plasma cells. Two types of TCAs (EBVhigh and EBVlow) were identified, with EBVlow-TCAs negatively associated with high plasma cell proportions. EBVhigh-TCAs with plasma cells (EBVhigh-TCA-WP) showed higher apoptosis signatures than other TCA types. In immunotherapy responders, higher tumoral pan-Ig expression and more spots with plasma cell infiltration were observed, along with higher proportions of EBVhigh-TCA-WP and apoptosis signatures. CXCL13+ CAFs interacted with B cells via CXCL13-CXCR5, VCAM1-integrin, ICAM1-integrin, and TNFSF13B-related ligand-receptor pairs, promoting B cell adhesion, activation and maturation, and antibody production. This was confirmed through in vitro co-culture experiments. A developmental trajectory from CXCL13+CD8+ T cells in TLS to exhausted CD8+ T cells in TCAs was observed, marked by decreased stemness and TCR diversity. TLS-associated cell clusters (B lineage cells, CD4_C8_CXCR5 and CD8_C8_CXCL13 T cells, and CXCL13 CAFs) were strongly associated with better NPC prognosis. A high TLS cell signature (TLS-CS) was independently associated with better survival outcomes.

This study provides comprehensive insights into the intricate interplay between tertiary lymphoid structures (TLS) and tumor cell aggregates (TCA) in nasopharyngeal carcinoma (NPC). The identification of key cell populations within TLS, including CXCL13+ CAFs and stem-like CXCL13+CD8+ T cells, highlights their distinct roles in shaping the tumor microenvironment. The observed decrease in GC B cells in advanced-stage NPC, coupled with the sustained presence of plasma cells, underscores the dynamic nature of the GC reaction within the tumor. The findings regarding plasma cell-mediated apoptosis of EBV-related malignant cells, particularly in EBVhigh-TCA-WP, offer compelling evidence for the importance of humoral immunity in NPC. The functional validation of CXCL13+ CAFs in promoting B cell activity and antibody production reinforces their significance in supporting TLS formation and anti-tumor responses. The identification of a developmental trajectory of CD8+ T cells, progressing from stem-like cells within TLS to exhausted cells in TCAs, suggests a potential mechanism for immune cell dysfunction in advanced NPC. The strong association between TLS-related cell signatures and favorable prognosis and immunotherapy response highlights the potential for TLS-related biomarkers in predicting patient outcomes and guiding treatment strategies. The results underscore the importance of considering both the spatial organization and the functional interactions of immune cells within the TME for a comprehensive understanding of cancer progression and treatment response. These findings have significant implications for the development of novel therapeutic approaches targeting TLS components and cellular interactions.

This study comprehensively characterized the cellular composition and dynamic interactions within tertiary lymphoid structures (TLS) and tumor cell aggregates (TCA) in nasopharyngeal carcinoma (NPC). The findings reveal key cellular interactions driving anti-tumor responses and highlight the prognostic and predictive significance of TLS-related cell signatures. These insights offer novel therapeutic targets, particularly focusing on CXCL13+ CAFs and the potential for enhancing the anti-tumor activity of stem-like CD8+ T cells. Future research should focus on further elucidating the molecular mechanisms underlying TLS formation and function, validating the identified biomarkers in larger independent cohorts, and exploring the potential of targeted therapies that modulate TLS activity.

Several limitations should be considered. The small size of the immunotherapy response cohort limits the generalizability of those findings. The reliance on top 100 DEGs as a surrogate for EBV infection status in the Visium FFPE cohort introduces some uncertainty, although this was mitigated by cross-validation approaches. The 10x Visium platform's inability to capture EBV genes is another limitation. Microfluidic-based scRNA-seq potentially introduces a capture bias, resulting in fewer tumor cells compared to immune and stromal cells. Future research with larger and more diverse cohorts and additional functional validation is needed to confirm these findings and fully elucidate the complexities of TLS in NPC and other cancers.