Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma

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-viralrelated 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 (T REG) and CD8+ resident memory T cells (T RM) were enriched in HBV-related HCC, whereas Tim-3+ CD8+ T cells and CD244+ natural killer cells were enriched in non-viralrelated HCC. NGS of isolated T REG and T RM from HBVrelated 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. T REG and T RM 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+ T REG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that T REG and T RM contributed to overall patient survival: T REG were associated with a poor prognosis and T RM 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.