Skip to main content
SpringerLink
Log in

Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

ALT:

Alanine aminotransferase

BFA:

Brefeldin A

Con A:

Concanavalin A

CT:

Computer tomography

CTLA-4:

Cytolytic T lymphocyte-associated antigen

FACS:

Fluorescence-activated cell sorting

FMO:

Fluorescence minus one

Foxp3:

Forkhead box p3

GITR:

Glucocorticoid-induced TNFR family-related gene

HCC:

Hepatocellular carcinoma

HPF:

High power field

IFN:

Interferon

IL:

Interleukin

LDCC:

Lectin-dependent cellular cytotoxicity

MRI:

Magnetic resonance imaging

NASH:

Non-alcoholic steatohepatitis

NK cell:

Natural killer cell

PBMC:

Peripheral blood mononuclear cells

PBS:

Phosphate-buffered saline

PD-1:

Programmed death 1 receptor

PD-L1:

Programmed death ligand 1

PD-L2:

Programmed death ligand 2

TGF:

Transforming growth factor

Tim-3:

T cell immunoglobulin and mucin-domain containing-3

Treg:

Regulatory T cell

References

  1. Langhans B, Nischalke HD, Krämer B et al (2019) Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma. J Hepatol 70(Suppl):e377 (abstract number THU-492)

    Google Scholar 

  2. El-Serag HB (2011) Hepatocellular carcinoma. N Engl J Med 365:1118–1127

    CAS  PubMed  Google Scholar 

  3. Wallace MC, Preen D, Jeffrey GP et al (2015) The evolving epidemiology of hepatocellular carcinoma: a global perspective. Expert Rev Gastroenterol Hepatol 9:765–779

    CAS  PubMed  Google Scholar 

  4. Siegel RL, Miller KD, Jemal A (2018) Cancer statistics, 2018. CA Cancer J Clin 68:7–30

    PubMed  Google Scholar 

  5. Llovet JM, Zucman-Rossi J, Pikarsky E et al (2016) Hepatocellular carcinoma. Nat Rev Dis Primer 2:16018

    Google Scholar 

  6. Prieto J, Melero I, Sangro B (2015) Immunological landscape and immunotherapy of hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 12:681–700

    CAS  PubMed  Google Scholar 

  7. Elsegood CL, Tirnitz-Parker JE, Olynyk JK et al (2017) Immune checkpoint inhibition: prospects for prevention and therapy of hepatocellular carcinoma. Clin Transl Immunol 6:e161

    Google Scholar 

  8. Nishida N, Kudo M (2018) Immune checkpoint blockade for the treatment of human hepatocellular carcinoma. Hepatol Res Off J Jpn Soc Hepatol 48:622–634

    CAS  Google Scholar 

  9. Chen L, Flies DB (2013) Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 13:227–242

    PubMed  PubMed Central  Google Scholar 

  10. Freeman GJ, Long AJ, Iwai Y et al (2000) Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192:1027–1034

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Iwai Y, Ishida M, Tanaka Y et al (2002) Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci USA 99:12293–12297

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Popovic A, Jaffee EM, Zaidi N (2018) Emerging strategies for combination checkpoint modulators in cancer immunotherapy. J Clin Investig 128:3209–3218

    PubMed  PubMed Central  Google Scholar 

  13. Hargadon KM, Johnson CE, Williams CJ (2018) Immune checkpoint blockade therapy for cancer: an overview of FDA-approved immune checkpoint inhibitors. Int Immunopharmacol 62:29–39

    CAS  PubMed  Google Scholar 

  14. Shi F, Shi M, Zeng Z et al (2011) PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients. Int J Cancer 128:887–896

    CAS  PubMed  Google Scholar 

  15. Kim H-D, Song G-W, Park S et al (2018) Association between expression level of PD1 by tumor-infiltrating CD8+ T cells and features of hepatocellular carcinoma. Gastroenterology 155(6):1936–1950.e17

    CAS  PubMed  Google Scholar 

  16. Unitt E, Marshall A, Gelson W et al (2006) Tumour lymphocytic infiltrate and recurrence of hepatocellular carcinoma following liver transplantation. J Hepatol 45:246–253

    CAS  PubMed  Google Scholar 

  17. Budhu A, Forgues M, Ye Q-H et al (2006) Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell 10:99–111

    CAS  PubMed  Google Scholar 

  18. Brandacher G, Winkler C, Schroecksnadel K et al (2006) Antitumoral activity of interferon-gamma involved in impaired immune function in cancer patients. Curr Drug Metab 7:599–612

    CAS  PubMed  Google Scholar 

  19. Okazaki T, Honjo T (2007) PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol 19:813–824

    CAS  PubMed  Google Scholar 

  20. El-Khoueiry AB, Sangro B, Yau T et al (2017) Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet Lond Engl 389:2492–2502

    CAS  Google Scholar 

  21. Zhu AX, Finn RS, Edeline J et al (2018) Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. Lancet Oncol 19:940–952

    PubMed  Google Scholar 

  22. Kalathil S, Lugade AA, Miller A et al (2013) Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality. Cancer Res 73:2435–2444

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Yeung OWH, Lo C-M, Ling C-C et al (2015) Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma. J Hepatol 62:607–616

    CAS  PubMed  Google Scholar 

  24. Iwata T, Kondo Y, Kimura O et al (2016) PD-L1 + MDSCs are increased in HCC patients and induced by soluble factor in the tumor microenvironment. Sci Rep 6:39296

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Greten TF, Sangro B (2017) Targets for immunotherapy of liver cancer. J Hepatol 18:1–20

    Google Scholar 

  26. Shitara K, Nishikawa H (2018) Regulatory T cells: a potential target in cancer immunotherapy. Ann N Y Acad Sci 1417:104–115

    CAS  PubMed  Google Scholar 

  27. Shevach EM, Thornton AM (2014) tTregs, pTregs, and iTregs: similarities and differences. Immunol Rev 259:88–102

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Wang F, Wan L, Zhang C et al (2009) Tim-3-Galectin-9 pathway involves the suppression induced by CD4 + CD25 + regulatory T cells. Immunobiology 214:342–349

    CAS  PubMed  Google Scholar 

  29. Sakaguchi S, Miyara M, Costantino CM et al (2010) FOXP3 + regulatory T cells in the human immune system. Nat Rev Immunol 10:490–500

    CAS  PubMed  Google Scholar 

  30. Heimbach JK, Kulik LM, Finn RS et al (2018) AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatol Baltim Md 67:358–380

    Google Scholar 

  31. Perl A, González-Cabello R, Onody K et al (1986) Independence of depressed lectin-dependent cell-mediated cytotoxicity from interleukin 2 production in patients with systemic lupus erythematosus. Clin Exp Immunol 65:286–292

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Tsukishiro T, Shimizu Y, Higuchi K et al (2000) Effect of branched-chain amino acids on the composition and cytolytic activity of liver-associated lymphocytes in rats. J Gastroenterol Hepatol 15:849–859

    CAS  PubMed  Google Scholar 

  33. Mayer E, Bannert C, Gruber S et al (2012) Cord blood derived CD4 + CD25(high) T cells become functional regulatory T cells upon antigen encounter. PLoS One 7:e29355

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Llovet JM, Montal R, Sia D et al (2018) Molecular therapies and precision medicine for hepatocellular carcinoma. Nat Rev Clin Oncol 15(10):599–616

    PubMed  Google Scholar 

  35. Shrestha R, Prithviraj P, Anaka M et al (2018) Monitoring immune checkpoint regulators as predictive biomarkers in hepatocellular carcinoma. Front Oncol 8:269

    PubMed  PubMed Central  Google Scholar 

  36. Iñarrairaegui M, Melero I, Sangro B (2018) Immunotherapy of hepatocellular carcinoma: facts and hopes. Clin Cancer Res Off J Am Assoc Cancer Res 24:1518–1524

    Google Scholar 

  37. Semaan A, Dietrich D, Bergheim D et al (2017) CXCL12 expression and PD-L1 expression serve as prognostic biomarkers in HCC and are induced by hypoxia. Virchows Arch Int J Pathol 470:185–196

    CAS  Google Scholar 

  38. Zhou G, Sprengers D, Boor PPC et al (2017) Antibodies against immune checkpoint molecules restore functions of tumor-infiltrating T cells in hepatocellular carcinomas. Gastroenterology 153(1107–1119):e10

    Google Scholar 

  39. Calderaro J, Rousseau B, Amaddeo G et al (2016) Programmed death ligand 1 expression in hepatocellular carcinoma: relationship with clinical and pathological features. Hepatol Baltim Md 64:2038–2046

    CAS  Google Scholar 

  40. Langhans B, Braunschweiger I, Arndt S et al (2010) Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C. Clin Sci Lond Engl 119:97–109

    CAS  Google Scholar 

  41. Langhans B, Krämer B, Louis M et al (2013) Intrahepatic IL-8 producing Foxp3+CD4+ regulatory T cells and fibrogenesis in chronic hepatitis C. J Hepatol 59:229–235

    CAS  PubMed  Google Scholar 

  42. Kobayashi N, Hiraoka N, Yamagami W et al (2007) FOXP3 + regulatory T cells affect the development and progression of hepatocarcinogenesis. Clin Cancer Res 13:902–911

    CAS  PubMed  Google Scholar 

  43. Lin SZ, Chen KJ, Xu ZY et al (2013) Prediction of recurrence and survival in hepatocellular carcinoma based on two Cox models mainly determined by FoxP3 + regulatory T cells. Cancer Prev Res Phila 6:594–602

    CAS  PubMed  Google Scholar 

  44. Zhang A-B, Qian Y-G, Zheng S-S (2016) Prognostic significance of regulatory T lymphocytes in patients with hepatocellular carcinoma. J Zhejiang Univ Sci B 17:984–991

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ribas A, Shin DS, Zaretsky J et al (2016) PD-1 blockade expands intratumoral memory T cells. Cancer Immunol Res 4:194–203

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Toor SM, Syed Khaja AS, Alkurd I et al (2018) In-vitro effect of pembrolizumab on different T regulatory cell subsets. Clin Exp Immunol 191:189–197

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Ingrid Braunschweiger, Eva-Maria Schumacher and Susanne Steiner for their perfect technical assistance.

Funding

This work was supported by the German Center for Infection Research [to B. Langhans, L. Dold and U. Spengler (TTU 05.808)], the German Research Foundation (SFB/TRR57 to U. Spengler and J. Nattermann), and the Deutsche Krebshilfe [to U. Spengler and H.D. Nischalke (70112169), to M.A. Gonzalez-Carmona (109255)]. The study sponsors had no role in the study design, collection, analysis, and interpretation of data.

Author information

Authors and Affiliations

  1. Department I of Internal Medicine, University Hospital of Bonn (UKB), Venusberg-Campus-1, 53127, Bonn, Germany

    Bettina Langhans, Hans Dieter Nischalke, Benjamin Krämer, Leona Dold, Philipp Lutz, Raphael Mohr, Annabelle Vogt, Jacob Nattermann, Christian P. Strassburg, Maria Angeles Gonzalez-Carmona & Ulrich Spengler

  2. German Center for Infection Research (DZIF), Partner Site Cologne-Bonn, Bonn, Germany

    Bettina Langhans, Leona Dold, Philipp Lutz & Ulrich Spengler

  3. Department of Experimental Pathology, University Hospital of Bonn (UKB), Bonn, Germany

    Marieta Toma

  4. Institute of Virology, University Hospital of Bonn (UKB), Bonn, Germany

    Anna Maria Eis-Hübinger

Authors
  1. Bettina Langhans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hans Dieter Nischalke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benjamin Krämer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Leona Dold
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philipp Lutz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Raphael Mohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Annabelle Vogt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marieta Toma
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Anna Maria Eis-Hübinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jacob Nattermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Christian P. Strassburg
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Maria Angeles Gonzalez-Carmona
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ulrich Spengler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

BL, LD, PL, RM, AV, JN, CPS, MAGC and US were involved in the study concept and design as well as in patient acquisition. BL, HDN, BK, AV, MT, AMEH, MAGC and US were involved in the acquisition, analysis, and interpretation of the data. BL, LD, MAGC and US drafted the manuscript, and all authors were involved in critical revision of the manuscript.

Corresponding author

Correspondence to Bettina Langhans.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval and ethical standards

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board of the Bonn University Ethics Committee (Approval #150/15).

Informed consent

All patients and healthy blood donors gave written informed consent prior to blood sample collection for the use of biomaterials and clinical data for scientific purposes.

Cell line authentication

The mouse mastocytoma cell line P815 and the various hepatoma cell lines HepG2, Hep3B, and Huh7 were purchased from the American Type Culture Collection (ATCC®) TIB64™. Cell lines Huh4 and HepT1 were a kind gift of W. Caselmann and T. Pietsch, respectively. Cell line authentication for human HepG2, Hep3B, Huh7, Huh4, and HepT1 was not necessary, because these cell lines were excluded from our in vitro studies due to up-regulation of PD-L1/L2 in cell culture. Cell line authentication of murine P815 cells was performed by the Swiss DNA company Microsynth (Supplementary Table 3).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 359 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Langhans, B., Nischalke, H.D., Krämer, B. et al. Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma. Cancer Immunol Immunother 68, 2055–2066 (2019). https://doi.org/10.1007/s00262-019-02427-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-019-02427-4

Keywords

Search

Navigation