LAUSR

BACKGROUND & AIMS Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TIL) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TIL. METHODS We isolated TIL from resected tumors of cholangiocarcinoma patients and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TIL, and determined whether targeting these molecules improved ex vivo functions of TIL. RESULTS Proportions of cytotoxic T cells and NK cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margin, and NK cells were excluded from the tumors. Co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TIL from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated to differences in TIL immune phenotype. CONCLUSIONS Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells with over-expression of co-inhibitory receptors in tumors suggest that tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for cholangiocarcinoma patients. LAY SUMMARY The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in cholangiocarcinoma patients.