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Abstract
Objective To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity.
Design We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight 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 harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-β may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment 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.
- IMMUNE RESPONSE
- PANCREATIC CANCER
Data availability statement
Data are available in a public, open access repository. Raw and processed gene expression data generated in this study are available at the Gene Expression Omnibus database (accession GSE226840). We collected publicly available scRNA-seq data from human PDAC,14 20–22 metastatic melanoma,26 non-small cell lung cancer,28 colorectal cancer29 and hepatocellular carcinoma,31 as well as paired scRNA-seq and scTCR-seq data from human endometrial cancer,27 non-small cell lung cancer,27 renal cell cancer,27colorectal cancer,27 squamous cell carcinoma30 and basal cell carcinoma.30
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Data availability statement
Data are available in a public, open access repository. Raw and processed gene expression data generated in this study are available at the Gene Expression Omnibus database (accession GSE226840). We collected publicly available scRNA-seq data from human PDAC,14 20–22 metastatic melanoma,26 non-small cell lung cancer,28 colorectal cancer29 and hepatocellular carcinoma,31 as well as paired scRNA-seq and scTCR-seq data from human endometrial cancer,27 non-small cell lung cancer,27 renal cell cancer,27colorectal cancer,27 squamous cell carcinoma30 and basal cell carcinoma.30
Footnotes
Twitter @Pedrohbarbosa01, @ClaudioVladmir
Contributors GSK and TSM conceived and designed the study. GSK, GAFV, MPCP, PHBP, MLRC, WASF, ASC, AR and AFG processed patient samples and performed in vitro experiments. ABD performed immunofluorescence staining. ACL and LMRBA performed Nanostring nCounter experiments. GSK analyzed scRNA-seq, flow cytometry, Nanostring and bulk RNA-seq data. AD assembled TCRs/BCRs and JLF modeled tumour progression from TCGA bulk RNA-seq data. TMB, RTN, AMP, AM and FJFC collected surgical specimens. TMB and VHFJ collected clinical data. GOS, WAN, LCLC, VHFJ, FJFC and VCCL provided clinicopathological guidance to the study. GOS, WAN and LCLC performed histological analyses. RW provided resources and experimental support. GSK, GAFV, VCCL and TSM interpreted the results. GSK, GAFV and TSM wrote the manuscript with input from all other authors. TSM is the guarantor of this study.
Funding The work was funded by the São Paulo Research Foundation (FAPESP, grant 18/14034-8 to TSM), the National Council for Scientific and Technological Development (CNPq, grant 465682/2014-6 to TSM) and the National Institute of Science and Technology in Oncogenomics and Therapeutic Innovation (INCITO, grant 14/50943-1 to TSM). GSK, GAFV, MLRC and LMRA were supported by fellowships from FAPESP (19/25129-2, 20/10299-7, 21/00643-5 and 21/04100-6, respectively). PHBP, ASC and AR were supported by fellowships from the Coordination for the Improvement of Higher Education Personnel (CAPES).
Competing interests VCCL: honoraria (educational presentations and participation in scientific events) from Astra-Zeneca, MSD, BMS, Roche, Amgen, GSK, Lilly; advisory board from Astra-Zeneca, MSD, BMS, Pfizer, Janssen, Amgen.
Provenance and peer review Not commissioned; externally peer reviewed.
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