Single-cell sequencing unveils distinct immune microenvironment in human chronic pancreatitis

Purpose: 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.

目的：慢性胰腺炎（Chronic pancreatitis, CP）是一种不可逆的纤维炎性胰腺疾病，目前尚无美国食品药品监督管理局（FDA）批准的有效治疗方案。由于胰腺组织获取难度较大，目前对人类慢性胰腺炎的局部免疫应答机制尚缺乏深入认知。本研究拟通过细胞索引转录组与表位测序（Cellular Indexing of Transcriptomes and Epitopes by Sequencing, CITE-seq）及单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCR-seq），对比非疾病对照样本，解析两种不同病因慢性胰腺炎（遗传性慢性胰腺炎与特发性慢性胰腺炎）患者胰腺组织中的疾病特异性免疫应答特征。
方法：我们对接受全胰切除术的器官捐献者（n=3）及慢性胰腺炎患者（遗传性CP组n=5，特发性CP组n=4）分离得到的胰腺免疫细胞，开展了细胞索引转录组与表位测序（CITE-seq）及T细胞受体测序分析。
结果：深度单细胞测序结果显示，相较于特发性CP，遗传性CP患者的胰腺免疫特征存在显著差异，且CCR6+ CD4+ T细胞群体显著富集。在遗传性CP中，因替换组织驻留CD8+ T细胞的CD4+ T辅助（Th）细胞数量增多，导致T细胞克隆性降低。基于scRNA/TCR-seq数据的谱系追踪分析进一步揭示了CCR6+ Th细胞与Th1亚群间独特的相互作用；T细胞受体聚类分析则在遗传性CP样本中发现了独特的共有抗原结合基序。此外，相较于特发性CP组，遗传性CP组单核细胞中CCR6的配体CCL20的表达水平显著上调。通过流式细胞术及迁移实验，我们验证了CD4+ T细胞中CCR6表达的功能意义。
结论：本研究通过整合性单细胞分析方法，揭示了不同病因慢性胰腺炎患者间独特的胰腺免疫特征与信号通路。本研究特别阐明了通过CCR6-CCL20轴介导的胰腺特异性免疫串扰，该轴或可作为未来人类遗传性慢性胰腺炎的潜在治疗靶点。
整体实验设计：本研究对人类器官捐献者及慢性胰腺炎患者的胰腺免疫细胞，同时开展了13种特征蛋白表达、转录组及T细胞受体库的单细胞图谱分析。