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ObjectiveChronic pancreatitis (CP) is a potentially fatal disease of the exocrine pancreas, with no specific or effective approved therapies. Due to difficulty in accessing pancreas tissues, little is known about local immune responses or pathogenesis in human CP. We sought to characterize pancreas immune responses using tissues derived from patients with different etiologies of CP and non-CP organ donors in order to identify key signaling molecules associated with human CP.DesignWe performed single-cell level cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) and T cell receptor sequencing of pancreatic immune cells isolated from organ donors, hereditary, and idiopathic CP patients who underwent total pancreatectomy. We validated gene expression data by performing flow cytometry and functional assay in the second CP patient cohort.ResultsDeep single-cell sequencing revealed distinct immune characteristics and 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. Shared TCR clonotype analysis among T cell lineages 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 CCR6 expression in CD4+ T cells was confirmed by flow cytometry and chemotaxis assay.ConclusionSingle-cell sequencing with pancreatic immune cells in human CP highlights pancreas-specific immune crosstalk through the CCR6-CCL20 axis that might be leveraged as a potential future target in human hereditary CP.Significance of this studyWhat is already known about this subject?Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease and remains a major source of morbidity among gastrointestinal diseases with no active approved therapy.Inflammation is a known hallmark and contributor to CP pathogenesis. However, little is known about local immune responses in human CP especially with different etiologies.What are the new findings?Single-cell RNA sequencing of pancreatic immune cells from CP patients and organ donors revealed distinct immune transcriptomic features in CP versus non-diseased controls and hereditary versus idiopathic CP.Single-cell T cell receptor sequencing unveiled pancreas-specific clonal expansion in CD8+ T cells and CD4+ T cells-driven unique TCR repertoire changes in hereditary CP.We identified that the CCR6-CCL20 axis was significantly upregulated in hereditary CP compared with controls or idiopathic CP suggesting a potential future target for human hereditary CP.How might it impact on clinical practice in the foreseeable future?The results of this study will improve our understanding of CP heterogeneity and identify distinct immune responses in different types of human CP that could provide novel conceptual directions for therapeutic strategies in treating hereditary and idiopathic CP.The CCR6-CCL20 axis found in hereditary CP could be a potential novel targetable signaling pathways in the treatment of hereditary CP.

G protein-coupled receptor 15 (GPR15) is a chemoattractant receptor which in response to its ligand, C10orf99/GPR15L, promotes colon homing of T cells in health and colitis. The functional role of GPR15 in colon cancer is largely unexplored, motivating our current studies using murine colon cancer models and human colorectal cancer (CRC) tissues. Our initial analysis of human CRC specimen revealed significant reduction in GPR15 expression and frequency of GPR15+immune cells in tumors compared to ‘tumor-free’ surgical margins. In the AOM/DSS murine model of colitis associated colon cancer (CAC), we observed increased colonic polyps/tumor burden and lower survival rate inGpr15-deficient (KO) compared toGpr15-sufficient (Het) mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+T cells and increased IL-17+CD4+and IL-17+CD8+T cells inGpr15-KO than in Het mice. GPR15 deficiency thus alters the immune environment in colonic polyps to mitigate T cell-mediated anti-tumor responses resulting in severe disease. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38 CRC mouse model increased CD45+cell infiltration, enhanced TNFαexpression on CD4+and CD8+T cells at the tumor site and dramatically reduced tumor burden. Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.