TDO2-Associated Tryptophan Metabolism Correlates with Impaired Tertiary Lymphoid Structure Maturation and Reduced B Cell Class Switching in Breast Cancer

Background: Tertiary lymphoid structures (TLSs) promote antitumor immunity and predict favorable immunotherapy outcomes in breast cancer. The study aimed to investigate how Tryptophan 2,3-dioxygenase (TDO2)-associated tryptophan metabolism influences TLS maturation and B cell class switching in breast cancer. Methods: Bulk transcriptomic data from The Cancer Genome Atlas-Breast Invasive Carcinoma (TCGA-BRCA, n = 1055) were analyzed using Gene Set Variation Analysis (GSVA)–based metabolic scoring, immune deconvolution, and TLS quantification. Single-cell RNA sequencing (scRNA-seq, n = 26) and spatial transcriptomics (n = 1) were applied to map TDO2 expression and TLS spatial organization. Validation was performed by immunohistochemistry (n = 38) and multiplex immunofluorescence (n = 12). Results: We identified that elevated tryptophan metabolism was predominantly enriched in the Luminal A subtype and delineates an immune-cold phenotype with less immunogenicity, associated with a distinct immune-dominant cellular microenvironment, particularly enriched in T and plasma cells. High expression of the tryptophan-metabolizing enzyme TDO2 was significantly enriched in TLS-low tumors and negatively correlated with TLS maturation signatures. Functional enrichment revealed suppressed B cell class switching and attenuated C-X-C motif chemokine ligand 9 (CXCL9) expression in TLS-deficient tumors. Spatial transcriptomics and hotspot analysis demonstrated an inverse spatial correlation between TDO2 expression and TLS core components. Tumors with high tryptophan metabolism showed decreased cluster of differentiation 20 (CD20)+ and CXCL9+ cell infiltration within TLS zones. Tumors with strong TDO2–kynurenine activity displayed impaired TLS organization and attenuated humoral immunity. Conditional spatial co-occurrence modeling confirmed reduced proximity between tryptophan metabolism hotspots and TLS-related immune features. Conclusion: In conclusion, our findings suggest that TDO2-associated tryptophan metabolism is linked to impaired TLS maturation and suppressed B cell class switching in breast cancer. Targeting the TDO2-kynurenine axis may represent a promising strategy to restore TLS formation and enhance immunotherapy responsiveness in breast cancer.