Single-cell sequencing unveils distinct immune microenvironments with CCR6-CCL20 crosstalk in human chronic pancreatitis

Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease with no active FDA approved therapy. Due to difficulty in accessing pancreas tissues, little is known about local immune responses in human CP. Here we attempted to uncover the disease-specific immune responses in pancreata from two different etiologies of CP (hereditary and idiopathic CP) compared with those from non-diseased controls by using CITE-seq and scTCR-seq. Methods: We performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) and T cell receptor sequencing of pancreatic immune cells isolated from organ donors (n=3) and CP patients (Hereditary CP, n=5; Idiopathic CP, n=4) who underwent total pancreatectomy. Results: Deep single-cell sequencing revealed distinct immune characteristics and a significantly enriched CCR6+ CD4+ T cells in hereditary compared with idiopathic CP. In hereditary CP, a reduction in T cell clonality was observed due to the increased CD4+ T (Th) cells that replaced tissue resident CD8+ T cells. Lineage tracing analysis with scRNA/TCR-seq data also unveiled unique interactions between CCR6+ Th and Th1 subsets, and TCR clustering analysis showed unique common antigen binding motifs in hereditary CP. In addition, we observed a significant upregulation of the CCR6 ligand (CCL20) among monocytes in hereditary CP as compared with those in idiopathic CP. The functional significance of the CCR6 expression in CD4+ T cells was confirmed by flow cytometry and migration assay. Conclusions: Our approaches with integrative single-cell analyses unveiled distinct pancreatic immune signatures and pathways between different etiologies of CP. Our study specifically unveiled pancreas-specific immune crosstalks through a CCR6-CCL20 axis that might be leveraged as a potential future target in human hereditary CP Overall design: Simultaneous single-cell profiles of 13 feature protein expressions, transcriptomes, and TCR repertoires in pancreatic immune cells from humam donors and CP patients.

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