Germinal Center-Like Tertiary Lymphoid Structures Mark Immune Responsiveness and Enable Checkpoint Immunotherapy in...

Backgrounds: Tertiary lymphoid structures (TLSs) are increasingly recognized as modulators of anti-tumor immunity, yet their clinical relevance in bladder cancer remains incompletely understood, partly owing to heterogeneity in their maturation states. Here, we demonstrate that germinal center (GC)-like TLS maturity, rather than TLS presence alone, is closely associated with immune activation and therapeutic response to Programmed Death-Ligand 1 (PD-L1) blockade in bladder cancer. The objective of this study was to systematically investigate the clinical significance, biological function, and therapeutic potential of tertiary lymphoid structure (TLS) maturation in bladder cancer. Specifically, we aimed to determine whether GC-like TLS maturity provides prognostic and predictive value beyond TLS presence alone, to elucidate the immune programs and tumor microenvironment remodeling associated with TLS maturation, and to explore whether TLS maturation can be therapeutically induced to enhance responsiveness to PD-L1 blockade. Methods: We performed an integrative analysis combining multi-cohort transcriptomics, spatially resolved histopathology, single-cell RNA sequencing, and functional murine experiments. TLS maturation states were defined using gene-expression-based GC-like TLS signatures and validated through multiplex immunohistochemistry. Clinical relevance was assessed in public immunotherapy cohorts and an independent neoadjuvant PD-L1-treated muscle-invasive bladder cancer (MIBC) cohort. Tumor immune microenvironment remodeling and chemokine-mediated cellular crosstalk were analyzed using deconvolution, Weighted Gene Co-expression Network Analysis (WGCNA), and CellChat. The therapeutic inducibility of TLS maturation was examined using a lymphotoxin-β receptor (LTβR) agonist in combination with PD-L1 blockade in a syngeneic bladder cancer model. Results: Across multiple transcriptomic cohorts, tumors enriched for GC-like TLS signatures exhibited significantly prolonged survival and higher objective response rates to anti-PD-L1 therapy, whereas less mature TLS phenotypes showed no consistent association with clinical association. These observations were independently validated in a neoadjuvant PD-L1-treated muscle-invasive bladder cancer cohort, in which high mature TLS density was associated with major pathological response and prolonged event-free survival, outperforming PD-L1 expression. Integrative histopathological and transcriptomic analyses indicated that GC formation marks a functional transition linking humoral immune programs with cytotoxic effector activity and shaping a memory-prone, pro-inflammatory tumor immune microenvironment. Chemokine signaling via the CC chemokine ligand 21 (CCL21)-C-C chemokine receptor type 7 (CCR7) and C-X-C motif chemokine ligand 12 (CXCL12)-C-X-C chemokine receptor type 4 (CXCR4) axes was strongly associated with TLS maturation and spatial organization. Finally, in a syngeneic bladder cancer model, pharmacological activation of lymphotoxin-β receptor signaling promoted TLS maturation and enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Together, these findings suggest that GC-like TLS maturity represents a clinically relevant biomarker and a potential therapeutic entry point for precision immunotherapy in bladder cancer. Therapeutic strategies that promote TLS maturation may convert immune-cold tumors into checkpoint-responsive states, providing a mechanistically grounded precision immunotherapy approach.