Abstract
On September 9th, 2025, the 3rd Workshop on Tertiary lymphoid structures (TLS) took place in Utrecht as pre-meeting to CICON2025. Advances in TLS biology, diversity, and clinical significance are rapidly reshaping the field. The studies discussed here reveal the heterogeneity in TLS architecture, cellular composition, functional states, and developmental trajectories, shaped by tumor-specific chemokine gradients. Spatial multi-omics revealed that TLS appearing histologically similar can contain T cells with distinct functional profiles, influencing clinical outcomes independently of conventional immune markers. Efforts toward TLS standardization are gaining attraction through pathology-based algorithms capable of reliably identifying mature TLS across cancer types, supporting reproducible stratification and prediction of immunotherapy response. However, challenges remain in glioblastoma and ovarian cancer, where TLS are rare, anatomically constrained, and strongly influenced by local tissue niches. Experimental models demonstrate that vascular-targeted cytokine delivery can induce TLS formation and promote T cell–dependent tumor control, supporting novel therapeutic avenues. Technological advances, including spatially resolved antigen receptor sequencing, now allow high-resolution mapping of B- and T-cell clonotypes, clonal evolution, and early antigen discovery within TLS. Across cancers, TLS enriched in activated B cells, memory B cells, and plasma cells emerge as key drivers of sustained immune activation. Spatial analyses also reveal interactions between TLS and specific fibroblast or mesenchymal subsets that modulate immunotherapy response. Finally, integrative studies identify tumor-intrinsic metabolic pathways, such as GABA production, that suppress TLS activity and contribute to immunotherapy resistance. Collectively, these findings establish TLS as dynamic, spatially organized immune hubs with broad implications for cancer prognosis and therapy.