Inflammatory processes in the development of cholangiocarcinoma in Primary Sclerosing Cholangitis – Do the T cells play a role?

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Extrahepatic cholangiocarcinoma (CCA) is the most dreaded comorbidity in Primary Sclerosing Cholangitis (PSC). The prognosis is very poor and as of yet the pathogenesis is unknown, but there is evidence that the immune system and the microbiota contribute towards promoting this disease. One suggested prerequisite for carcinogenesis in PSC-associated CCA (PSC-CCA) is a strong association of chronic biliary inflammation with biliary metaplasia and dysplasia, typical of PSC. In addition, differences in the composition of the intestinal and biliary microbiota of patients with PSC have been shown. Finally, the impact of the microbiota on T cell function, which is often determined by the secretion of cytokines and the expression of co-inhibitory receptors, has recently been implicated in carcinogenesis. On the basis of all this, we hypothesize that the PSC specific microbiota favours the pro-tumorigenic function of T cells, ultimately leading to carcinogenesis in PSC patients. The first objective of our study is to define the functional phenotype of human tumour infiltrating T cells in PSC-CCA by using single-cell RNA/TCR sequencing in combination with Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) and RNA seq of total tissue. Results will allow us to identify new cell populations and their cell relationships with either a pro- or anti-tumorigenic functional phenotype. In addition, by defining the co-inhibitory receptor profile of these populations, we will set the basis for designing ad hoc immune therapies (e.g. anti-PD1). Finally, we will infer the potential interaction among the cells and the tissue by matching differentially expressed genes identified in the single cell RNA analysis to expression patterns of known interaction partners (e.g. cytokine-receptor pairings) in the surrounding tissue, identified by total tissue RNA sequencing. The second objective is to identify specific PSC-CCA microbiota able to shape the T cell response. Therefore, we will first sequence the faecal and biliary microbiota from PSC-CCA patients, and then test the microbiota - T cell interaction by transferring human microbiota into germ-free mice. The third objective is to develop a specific mouse model that exhibits a PSC phenotype and develops extrahepatic CCA. We will achieve this by using the bile duct injection technique to introduce carcinogenic substances directly into the biliary tree of Mdr2-/- mice. Additionally, using the Cre-Lox-system, we will develop a transgenic mouse model with a conditional overexpression of the oncogene kras and deletion of the tumour suppressor gene pten in cholangiocytes. In summary, the results of this project will contribute towards the understanding of the pathogenesis of PSC-associated CCA. They will reveal immunological mechanisms and will help the clinical translation of possible therapeutic targets in order to directly treat patients suffering from this disease.