Advertisement
Research Article| Volume 71, ISSUE 4, P753-762, October 2019

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules

      Highlights

      • NK cells and cytotoxic T cells infiltrate poorly into cholangiocarcinoma.
      • Regulatory T cells accumulate in cholangiocarcinoma.
      • PD1, CTLA4 and GITR are over-expressed on tumor-infiltrating T cells in cholangiocarcinoma.
      • Blocking PD1 or CTLA4 or stimulating GITR enhances effector functions of tumor-infiltrating T cells in cholangiocarcinoma.

      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 (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs.

      Methods

      We isolated TILs from resected tumors of patients with cholangiocarcinoma 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 TILs, and determined whether targeting these molecules improved ex vivo functions of TILs.

      Results

      Proportions of cytotoxic T cells and natural killer 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 margins, and natural killer cells were excluded from the tumors. The 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 TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype.

      Conclusions

      Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the 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 patients with cholangiocarcinoma.

      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 patients with cholangiocarcinoma.

      Graphical abstract

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

      Author names in bold designate shared co-first authorship

        • Torre L.A.
        • Bray F.
        • Siegel R.L.
        • Ferlay J.
        • Lortet-Tieulent J.
        • Jemal A.
        Global cancer statistics, 2012.
        CA Cancer J Clin. 2015; 65: 87-108
        • Rizvi S.
        • Gores G.J.
        Pathogenesis, diagnosis, and management of cholangiocarcinoma.
        Gastroenterology. 2013; 145: 1215-1229
        • Razumilava N.
        • Gores G.J.
        Cholangiocarcinoma.
        Lancet. 2014; 383: 2168-2179
        • Bergquist A.
        • von Seth E.
        Epidemiology of cholangiocarcinoma.
        Best Pract Res Clin Gastroenterol. 2015; 29: 221-232
        • Zhou G.
        • Sprengers D.
        • Boor P.P.C.
        • Doukas M.
        • Schutz H.
        • Mancham S.
        • et al.
        Antibodies against immune checkpoint molecules restore functions of tumor-infiltrating T cells in hepatocellular carcinomas.
        Gastroenterology. 2017;
        • Zhou G.
        • Noordam L.
        • Sprengers D.
        • Doukas M.
        • Boor P.P.C.
        • van Beek A.A.
        • et al.
        Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver metastases of colorectal cancer.
        Oncoimmunology. 2018; 7e1448332
        • El-Khoueiry A.B.
        • Sangro B.
        • Yau T.
        • Crocenzi T.S.
        • Kudo M.
        • Hsu C.
        • et al.
        Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial.
        Lancet. 2017; 389: 2492-2502
        • Cabo M.
        • Offringa R.
        • Zitvogel L.
        • Kroemer G.
        • Muntasell A.
        • Galluzzi L.
        Trial watch: immunostimulatory monoclonal antibodies for oncological indications.
        Oncoimmunology. 2017; 6e1371896
        • Fontugne J.
        • Augustin J.
        • Pujals A.
        • Compagnon P.
        • Rousseau B.
        • Luciani A.
        • et al.
        PD-L1 expression in perihilar and intrahepatic cholangiocarcinoma.
        Oncotarget. 2017; 8: 24644-24651
        • Walter D.
        • Herrmann E.
        • Schnitzbauer A.A.
        • Zeuzem S.
        • Hansmann M.L.
        • Peveling-Oberhag J.
        • et al.
        PD-L1 expression in extrahepatic cholangiocarcinoma.
        Histopathology. 2017; 71: 383-392
        • Gani F.
        • Nagarajan N.
        • Kim Y.
        • Zhu Q.
        • Luan L.
        • Bhaijjee F.
        • et al.
        Program death 1 immune checkpoint and tumor microenvironment: implications for patients with intrahepatic cholangiocarcinoma.
        Ann Surg Oncol. 2016; 23: 2610-2617
        • Sabbatino F.
        • Villani V.
        • Yearley J.H.
        • Deshpande V.
        • Cai L.
        • Konstantinidis I.T.
        • et al.
        PD-L1 and HLA class I antigen expression and clinical course of the disease in intrahepatic cholangiocarcinoma.
        Clin Cancer Res. 2016; 22: 470-478
        • Ma K.
        • Wei X.
        • Dong D.
        • Wu Y.
        • Geng Q.
        • Li E.
        PD-L1 and PD-1 expression correlate with prognosis in extrahepatic cholangiocarcinoma.
        Oncol Lett. 2017; 14: 250-256
        • Tumeh P.C.
        • Harview C.L.
        • Yearley J.H.
        • Shintaku I.P.
        • Taylor E.J.
        • Robert L.
        • et al.
        PD-1 blockade induces responses by inhibiting adaptive immune resistance.
        Nature. 2014; 515: 568-571
        • Nakamura H.
        • Arai Y.
        • Totoki Y.
        • Shirota T.
        • Elzawahry A.
        • Kato M.
        • et al.
        Genomic spectra of biliary tract cancer.
        Nat Genet. 2015; 47: 1003-1010
        • Tran E.
        • Turcotte S.
        • Gros A.
        • Robbins P.F.
        • Lu Y.C.
        • Dudley M.E.
        • et al.
        Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer.
        Science. 2014; 344: 641-645
        • Chen D.S.
        • Mellman I.
        Elements of cancer immunity and the cancer-immune set point.
        Nature. 2017; 541: 321-330
        • Takagi S.
        • Miyagawa S.
        • Ichikawa E.
        • Soeda J.
        • Miwa S.
        • Miyagawa Y.
        • et al.
        Dendritic cells, T-cell infiltration, and Grp94 expression in cholangiocellular carcinoma.
        Hum Pathol. 2004; 35: 881-886
        • Goeppert B.
        • Frauenschuh L.
        • Zucknick M.
        • Stenzinger A.
        • Andrulis M.
        • Klauschen F.
        • et al.
        Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer.
        Br J Cancer. 2013; 109: 2665-2674
        • Goeppert B.
        • Frauenschuh L.
        • Zucknick M.
        • Roessler S.
        • Mehrabi A.
        • Hafezi M.
        • et al.
        Major histocompatibility complex class I expression impacts on patient survival and type and density of immune cells in biliary tract cancer.
        Br J Cancer. 2015; 113: 1343-1349
        • McCully M.L.
        • Ladell K.
        • Andrews R.
        • Jones R.E.
        • Miners K.L.
        • Roger L.
        • et al.
        CCR8 expression defines tissue-resident memory T cells in human skin.
        J Immunol. 2018;
        • Turner D.L.
        • Bickham K.L.
        • Thome J.J.
        • Kim C.Y.
        • D'Ovidio F.
        • Wherry E.J.
        • et al.
        Lung niches for the generation and maintenance of tissue-resident memory T cells.
        Mucosal Immunol. 2014; 7: 501-510
        • Gu F.M.
        • Gao Q.
        • Shi G.M.
        • Zhang X.
        • Wang J.
        • Jiang J.H.
        • et al.
        Intratumoral IL-17(+) cells and neutrophils show strong prognostic significance in intrahepatic cholangiocarcinoma.
        Ann Surg Oncol. 2012; 19: 2506-2514
        • Freud A.G.
        • Zhao S.
        • Wei S.
        • Gitana G.M.
        • Molina-Kirsch H.F.
        • Atwater S.K.
        • et al.
        Expression of the activating receptor, NKp46 (CD335), in human natural killer and T-cell neoplasia.
        Am J Clin Pathol. 2013; 140: 853-866
        • Peppa D.
        • Gill U.S.
        • Reynolds G.
        • Easom N.J.
        • Pallett L.J.
        • Schurich A.
        • et al.
        Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell-mediated deletion.
        J Exp Med. 2013; 210: 99-114
        • Halama N.
        • Spille A.
        • Lerchl T.
        • Brand K.
        • Herpel E.
        • Welte S.
        • et al.
        Hepatic metastases of colorectal cancer are rather homogeneous but differ from primary lesions in terms of immune cell infiltration.
        Oncoimmunology. 2013; 2e24116
        • Mlecnik B.
        • Bindea G.
        • Kirilovsky A.
        • Angell H.K.
        • Obenauf A.C.
        • Tosolini M.
        • et al.
        The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis.
        Sci Transl Med. 2016; 8: 327ra326
        • Mlecnik B.
        • Van den Eynde M.
        • Bindea G.
        • Church S.E.
        • Vasaturo A.
        • Fredriksen T.
        • et al.
        Comprehensive intrametastatic immune quantification and major impact of immunoscore on survival.
        J Natl Cancer Inst. 2018; 110
        • Bindea G.
        • Mlecnik B.
        • Tosolini M.
        • Kirilovsky A.
        • Waldner M.
        • Obenauf A.C.
        • et al.
        Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.
        Immunity. 2013; 39: 782-795
        • Mlecnik B.
        • Bindea G.
        • Angell H.K.
        • Maby P.
        • Angelova M.
        • Tougeron D.
        • et al.
        Integrative analyses of colorectal cancer show immunoscore is a stronger predictor of patient survival than microsatellite instability.
        Immunity. 2016; 44: 698-711
        • Gao Q.
        • Qiu S.J.
        • Fan J.
        • Zhou J.
        • Wang X.Y.
        • Xiao Y.S.
        • et al.
        Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.
        J Clin Oncol. 2007; 25: 2586-2593
        • Halama N.
        • Braun M.
        • Kahlert C.
        • Spille A.
        • Quack C.
        • Rahbari N.
        • et al.
        Natural killer cells are scarce in colorectal carcinoma tissue despite high levels of chemokines and cytokines.
        Clin Cancer Res. 2011; 17: 678-689
        • Bosma B.M.
        • Metselaar H.J.
        • Mancham S.
        • Boor P.P.
        • Kusters J.G.
        • Kazemier G.
        • et al.
        Characterization of human liver dendritic cells in liver grafts and perfusates.
        Liver Transpl. 2006; 12: 384-393
        • Pedroza-Gonzalez A.
        • Verhoef C.
        • Ijzermans J.N.
        • Peppelenbosch M.P.
        • Kwekkeboom J.
        • Verheij J.
        • et al.
        Activated tumor-infiltrating CD4+ regulatory T cells restrain antitumor immunity in patients with primary or metastatic liver cancer.
        Hepatology. 2013; 57: 183-194
        • Pedroza-Gonzalez A.
        • Zhou G.
        • Singh S.P.
        • Boor P.P.
        • Pan Q.
        • Grunhagen D.
        • et al.
        GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo.
        Oncoimmunology. 2015; 4e1051297
        • Legat A.
        • Speiser D.E.
        • Pircher H.
        • Zehn D.
        • Fuertes Marraco S.A.
        Inhibitory receptor expression depends more dominantly on differentiation and activation than “exhaustion” of human CD8 T cells.
        Front Immunol. 2013; 4: 455
        • Lim Y.J.
        • Koh J.
        • Kim K.
        • Chie E.K.
        • Kim S.
        • Lee K.B.
        • et al.
        Clinical implications of cytotoxic T lymphocyte antigen-4 expression on tumor cells and tumor-infiltrating lymphocytes in extrahepatic bile duct cancer patients undergoing surgery plus adjuvant chemoradiotherapy.
        Target Oncol. 2017; 12: 211-218
        • Wang L.
        • Dong H.
        • Ni S.
        • Huang D.
        • Tan C.
        • Chang B.
        • et al.
        Programmed death-ligand 1 is upregulated in intrahepatic lymphoepithelioma-like cholangiocarcinoma.
        Oncotarget. 2016; 7: 69749-69759
        • Shi X.L.
        • Mancham S.
        • Hansen B.E.
        • de Knegt R.J.
        • de Jonge J.
        • van der Laan L.J.
        • et al.
        Counter-regulation of rejection activity against human liver grafts by donor PD-L1 and recipient PD-1 interaction.
        J Hepatol. 2016; 64: 1274-1282
        • Sideras K.
        • Biermann K.
        • Verheij J.
        • Takkenberg B.R.
        • Mancham S.
        • Hansen B.E.
        • et al.
        PD-L1, galectin-9 and CD8(+) tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma.
        Oncoimmunology. 2017; 6e1273309
        • Pfirschke C.
        • Engblom C.
        • Rickelt S.
        • Cortez-Retamozo V.
        • Garris C.
        • Pucci F.
        • et al.
        Immunogenic chemotherapy sensitizes tumors to checkpoint blockade therapy.
        Immunity. 2016; 44: 343-354
        • Ariyan C.E.
        • Brady M.S.
        • Siegelbaum R.H.
        • Hu J.
        • Bello D.M.
        • Rand J.
        • et al.
        Robust antitumor responses result from local chemotherapy and CTLA-4 blockade.
        Cancer Immunol Res. 2018; 6: 189-200