Mature tertiary lymphoid structures are key niches of tumor-specific immune responses in pancreatic ductal adenocarcinomas

Objective: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalization into tertiary lymphoid structures (TLSs) for the generation of local antitumor immunity. Design: We characterized the functional states and spatial organization of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolor immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. Additionally, we performed a pan-cancer analysis of tumor-infiltrating T cells using scRNA-seq and single-cell T cell receptor sequencing (scTCR-seq) datasets from 8 cancer types. To evaluate the clinical relevance of our findings we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. Results: We found that a subset of PDACs harbors fully developed TLSs where B cells proliferate and differentiate to plasma cells. These mature TLSs also support T cell activity and are enriched with tumor-reactive T cells. Importantly, we showed that chronically activated, tumor-reactive T cells exposed to fibroblast-derived TGF-β may act as TLS organizers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13+ tumor-infiltrating T cells across multiple cancer types further indicated a conserved link between tumor-antigen recognition and the allocation of B cells within sheltered hubs in the tumor microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pre-treatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. Conclusion: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials. We evaluated the expression profile of immune-related genes in microdissected PDAC-associated TLSs in different maturation stages.