Article Text

Download PDFPDF

Original article
Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma
  1. Chun Jye Lim1,
  2. Yun Hua Lee1,
  3. Lu Pan1,
  4. Liyun Lai1,
  5. Camillus Chua1,
  6. Martin Wasser1,
  7. Tony Kiat Hon Lim2,3,
  8. Joe Yeong2,4,
  9. Han Chong Toh3,5,
  10. Ser Yee Lee3,5,6,
  11. Chung Yip Chan3,5,6,
  12. Brian KP Goh3,5,6,
  13. Alexander Chung3,5,6,
  14. Mathias Heikenwälder7,
  15. Irene OL Ng8,
  16. Pierce Chow3,5,6,
  17. Salvatore Albani1,
  18. Valerie Chew1
  1. 1 Translational Immunology Institute (TII), SingHealth-DukeNUS Academic Medical Centre, Singapore, Singapore
  2. 2 Department of Pathology, Singapore General Hospital, Singapore, Singapore
  3. 3 Duke-NUS Medical School, Singapore, Singapore
  4. 4 Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
  5. 5 National Cancer Centre, Singapore, Singapore
  6. 6 Department of Hepatopancreatobiliary and Transplant Surgery, Singapore General Hospital, Singapore, Singapore
  7. 7 Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
  8. 8 Department of Pathology and State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, Hong Kong
  1. Correspondence to Dr Valerie Chew, Translational Immunology Institute, SingHealth- DukeNUS Academic Medical Centre, Singapore 169856, Singapore; valerie.chew.s.p{at}


Background and aims Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics.

Methods We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays.

Results In-depth interrogation of the immune landscapes showed that regulatory T cells (TREG) and CD8+ resident memory T cells (TRM) were enriched in HBV-related HCC, whereas Tim-3+CD8+ T cells and CD244+ natural killer cells were enriched in non-viral-related HCC. NGS of isolated TREG and TRM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. TREG and TRM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1+ TREG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that TREG and TRM contributed to overall patient survival: TREG were associated with a poor prognosis and TRM were associated with a good prognosis in HCC.

Conclusion We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.

  • hepatitis B
  • hepatocellular carcinoma
  • immunotherapy
  • cancer immunobiology
  • immunoregulation

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:

View Full Text

Statistics from


  • CJL and YHL contributed equally.

  • SA and VC contributed equally.

  • Contributors YHL and CJL performed most experiments, analysed the data and prepared the manuscript; LP analysed the NGS data; LL assisted in CyTOF data acquisition; CC performed RNA sequencing experiments; JY assisted in immune subsets sorting; MW performed univariate and multivariate analyses; TKHL and JY (pathologists) prepared and provided tissue samples; HCT (oncologist) advised on patient recruitment and clinical analysis; SYL, CYC, BKPG and AC (surgeons) assisted in patient recruitment and provided samples; MH (Zurich) and IOLN (Hong Kong) provided samples from overseas cohorts; PC provided clinical advice, recruited and provided samples for most patients; SA designed the study pipeline and assisted in writing the manuscript; VC designed the studies, analysed the data and wrote the manuscript.

  • Funding This work was supported by the National Medical Research Council, Singapore (ref numbers: MOHIAFCAT2001, TCR15Jun006, CIRG16may048, CSAS16Nov006, NMRC/STaR/020/2013, CIRg13nov032, NMRC/MOHIAFCAT1-6003 and NMRC/MOHIAFCAT2/005/2015), the A*STAR Biomedical Research Council-Economy Development Board (BMRC-EDB IAF: IAF311020 and SPF2014/005) and Duke-NUS and SingHealth. MH was supported by an ERC CoG grant (HepatoMetaboPath) and the SFB 179 and 209.

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval Centre IRB.

  • Provenance and peer review Not commissioned; externally peer reviewed.

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.