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The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

  • Jiajie Hou
    Affiliations
    Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China

    Department of Hepatobiliary Surgery, Sun Yat-sen University Cancer Center, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou 510060, China
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  • Haiyan Zhang
    Affiliations
    State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou 510060, China
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  • Beicheng Sun
    Correspondence
    Corresponding authors. Addresses: Laboratory of Gene Regulation and Signal Transduction, Leichtag Biomedical Research Building Room 216, Departments of Pharmacology and Pathology, University of California San Diego, School of Medicine, La Jolla, CA 92093, USA; Tel.: +1 (858)534 1361 (M. Karin), or Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China (B. Sun).
    Affiliations
    Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China

    Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
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  • Michael Karin
    Correspondence
    Corresponding authors. Addresses: Laboratory of Gene Regulation and Signal Transduction, Leichtag Biomedical Research Building Room 216, Departments of Pharmacology and Pathology, University of California San Diego, School of Medicine, La Jolla, CA 92093, USA; Tel.: +1 (858)534 1361 (M. Karin), or Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China (B. Sun).
    Affiliations
    Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, University of California San Diego, School of Medicine, La Jolla, CA 92093, USA
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Published:August 23, 2019DOI:https://doi.org/10.1016/j.jhep.2019.08.014

      Summary

      Basic and clinical studies have demonstrated the efficacy of immunotherapy, a technical and conceptual breakthrough that has revolutionised cancer treatment. Hepatocellular carcinoma (HCC), a deadly malignancy with aetiologic diversity and a chronic course, is strongly influenced by the immune system, and was recently found to partially benefit from immune-checkpoint inhibitor therapy. Notably, HCC onco-immunology depends on diverse genetic and environmental factors that together shape cancer-promoting inflammation and immune dysfunction – critical processes that control HCC malignant progression and response to therapy. Herein, we summarise the current understanding of liver and HCC onco-immunology obtained through basic studies with mouse models and clinical practice in humans. In particular, we discuss preclinical and clinical findings that implicate immunomodulation as a major factor in HCC development and explain the basis for HCC-targeting immunotherapy.

      Keywords

      Linked Article

      • Preoperative immunotherapy for resectable hepatocellular carcinoma: Toward a paradigm shift?
        Journal of HepatologyVol. 73Issue 6
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          As extensively reviewed by Hou and colleagues, the role of immunotherapy in the management of hepatocellular carcinoma (HCC) is being widely investigated through numerous ongoing clinical trials.1 To date, immune checkpoint inhibitors (ICIs) such as PD-1 and PD-L1 inhibitors have been associated with promising response rates, improved survival and acceptable safety profiles in patients with advanced HCC.2,3 In contrast, few clinical trials investigating the role of perioperative immunotherapy in the setting of resectable HCC are accruing.
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      Introduction

      Hepatocellular carcinoma (HCC), the major primary liver malignancy, is the fourth-leading cause of cancer-related deaths worldwide.
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
      • Torre L.A.
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      HCC can be caused by chronic infection with hepatitis B (HBV) or C (HCV) viruses or different metabolic and inflammatory disorders related to non-alcoholic (NASH) and alcohol-related (ASH) steatohepatitis. Most HCCs occur in patients with long-term inflammatory liver damage and cirrhosis, and are usually diagnosed at an advanced stage by which point treatment options are limited. Even early HCC cases that received surgical resection or percutaneous ablation are still subject to frequent relapse, rendering it one of the most lethal cancers. Currently, over 50% of patients with HCC are given systemic therapies that are barely effective and cause considerable toxic damage to the remaining normal liver, further limiting clinical outcomes. Given the poor safety profile of multi-kinase inhibitors, such as sorafenib,
      • Nault J.C.
      • Galle P.R.
      • Marquardt J.U.
      The role of molecular enrichment on future therapies in hepatocellular carcinoma.
      and their limited success, clinicians and researchers have begun a search for drastically new alternatives to HCC therapy. In particular, the recent clinical success of immune-checkpoint inhibitors and various immune cell therapies and vaccines in other treatment-refractory cancers has sparked an interest in application of immunotherapeutics to HCC.
      • Worns M.A.
      • Galle P.R.
      Immune oncology in hepatocellular carcinoma-hype and hope.
      This translational interest had also revitalised basic HCC immunobiology research.
      • Ma C.
      • Kesarwala A.H.
      • Eggert T.
      • Medina-Echeverz J.
      • Kleiner D.E.
      • Jin P.
      • et al.
      NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      • Inarrairaegui M.
      • Melero I.
      • Sangro B.
      Immunotherapy of hepatocellular carcinoma: facts and hopes.
      An early and highly encouraging outcome of these efforts is the recent clinical approval of antibodies to the inhibitory receptor programmed cell death protein-1 (PD-1) or its ligand PD-L1, that were found to elicit durable responses in up to 25% of patients with advanced HCC in 2 early trials.
      • 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.
      • Zhu A.X.
      • Finn R.S.
      • Edeline J.
      • Cattan S.
      • Ogasawara S.
      • Palmer D.
      • et al.
      Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial.
      However, in 2 more recent trials, anti-PD-1 monotherapy did not reach the pre-specified primary endpoints.
      • Green J.E.
      • Choi J.W.
      • Boukai A.
      • Bunimovich Y.
      • Johnston-Halperin E.
      • DeIonno E.
      • et al.
      A 160-kilobit molecular electronic memory patterned at 10(11) bits per square centimetre.
      • Finn R.S.
      • Ryoo B.-Y.
      • Merle P.
      • Kudo M.
      • Bouattour M.
      • Lim H.-Y.
      • et al.
      Results of KEYNOTE- 240: phase 3 study of pembrolizumab (Pembro) vs best supportive care (BSC) for second line therapy in advanced hepatocellular carcinoma (HCC).
      Mounting new evidence supports the notion that the activation of tumour-specific T cells can benefit patients with HCC. However, HCC progression and therapy are strongly affected by the ongoing inflammation that characterises this malignancy, resulting in the formation of a tumour immune microenvironment (TIME) that is immunosuppressive in nature.
      • Grivennikov S.I.
      • Greten F.R.
      • Karin M.
      Immunity, inflammation, and cancer.
      • Shalapour S.
      • Karin M.
      PAS DE DEUX: control of tumor immunity by cancer-associated inflammation.
      The HCC TIME involves a variety of non-cancerous cell types, including normal hepatocytes, endothelial cells, pericytes, hepatic stellate cells (HSCs), peri-portal fibroblasts and distinct subsets of immune cells. In the past decade, molecular biology and “omics” technologies, as well as studies conducted in genetically modified mice, have highlighted the contributions of different immune cells, cytokines, chemokines and growth factors to cancer-promoting inflammation and immunosuppression in numerous malignancies, including HCC.
      • Pai C.S.
      • Huang J.T.
      • Lu X.
      • Simons D.M.
      • Park C.
      • Chang A.
      • et al.
      Clonal deletion of tumor-specific T cells by interferon-gamma confers therapeutic resistance to combination immune checkpoint blockade.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      Meanwhile, high-throughput and single cell sequencing are being used to better define the HCC-specific TIME.
      • Zheng C.
      • Zheng L.
      • Yoo J.K.
      • Guo H.
      • Zhang Y.
      • Guo X.
      • et al.
      Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.
      Here, we provide a summary of current knowledge regarding the immunobiology of mouse and human HCC and discuss the roles of immune- and non-immune-related factors and the pathogenic and immunoregulatory mechanisms that influence HCC development and its response to immunotherapy.

      Composition and function of immune cells in HCC

      While immune cells are prevalent in the non-cancerous liver, an important immune organ,
      • Makarova-Rusher O.V.
      • Medina-Echeverz J.
      • Duffy A.G.
      • Greten T.F.
      The yin and yang of evasion and immune activation in HCC.
      the HCC TIME differs from its benign counterpart, not only in composition, but also in function. The impact of the ongoing changes in TIME composition and function is manifested throughout malignant progression, from initiation to metastatic spread and growth.
      Analysis of human clinical specimens indicates that the HCC tumour immune microenvironment (TIME) contains different cell populations, mainly associated with chronic inflammation and immune dysfunction.

      Cohort-based TIME data

      To date, a large body of moderate-resolution data have been generated in respect to TIME composition through histochemical analysis of tissue microarrays and flow cytometry of lymphocyte infiltrates. Early studies showed that intratumoural lymphocytes are distributed either sporadically throughout the cancer or are aggregated within lymphoid clusters, with the frequency of different immune cell subsets showing great variation amongst individual patients.
      • 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.
      Integrative analysis indicates that regulatory T cell (Treg) numbers positively correlate with HCC invasiveness, and that the intratumoural balance between regulatory and cytotoxic T cells (CTLs) predicts recurrence and survival after curative resection.
      • 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.
      During the past decade, different CD4+ T helper (Th) subpopulations have been identified in both tumoural and peritumoral HCC tissue. Notably, Th17, Th22, T follicular helper (Tfh) cells and recently described Th9 cells, were found to exert pro-tumorigenic functions.
      • Zhang J.P.
      • Yan J.
      • Xu J.
      • Pang X.H.
      • Chen M.S.
      • Li L.
      • et al.
      Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients.
      • Jiang R.
      • Tan Z.
      • Deng L.
      • Chen Y.
      • Xia Y.
      • Gao Y.
      • et al.
      Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3.
      • Kuang D.M.
      • Xiao X.
      • Zhao Q.
      • Chen M.M.
      • Li X.F.
      • Liu R.X.
      • et al.
      B7-H1-expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets.
      • Chen M.M.
      • Xiao X.
      • Lao X.M.
      • Wei Y.
      • Liu R.X.
      • Zeng Q.H.
      • et al.
      Polarization of tissue-resident TFH-Like cells in human hepatoma bridges innate monocyte inflammation and M2b macrophage polarization.
      • Tan H.
      • Wang S.
      • Zhao L.
      A tumour-promoting role of Th9 cells in hepatocellular carcinoma through CCL20 and STAT3 pathways.
      Importantly, inhibitory molecules such as PD-1, T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activating-3 (LAG-3) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) are upregulated on both CD4+ and CD8+ T cells in HCC tissue and peripheral blood, indicating prevalent T cell exhaustion (exhaustion is a state of dysfunction commonly associated with chronic T cell stimulation by viral or tumour antigens) in human HCC.
      • Li H.
      • Wu K.
      • Tao K.
      • Chen L.
      • Zheng Q.
      • Lu X.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      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.
      • 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.
      CD68+ macrophages are also elevated in tumoural and peritumoral tissue relative to the non-tumour liver. High macrophage density correlates with large tumour size, high TNM stage, and intrahepatic metastasis, all of which predict likely recurrence and poor survival after hepatectomy.
      • Zhu X.D.
      • Zhang J.B.
      • Zhuang P.Y.
      • Zhu H.G.
      • Zhang W.
      • Xiong Y.Q.
      • et al.
      High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.
      One canonical function of tumour-associated macrophages (TAMs) is to regulate angiogenesis. Specifically, a subtype of CD14+CD16+TIE2+ myeloid cells in the blood or tumours of patients with HCC was found to associate with intratumoural micro-vessel density.
      • Matsubara T.
      • Kanto T.
      • Kuroda S.
      • Yoshio S.
      • Higashitani K.
      • Kakita N.
      • et al.
      TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.
      Most intratumoural macrophages are interleukin-10 (IL-10)-positive with peritumoral macrophages expressing high levels of HLA-DR and PD-L1, which allow these cells to extinguish antitumour immune responses.
      • Kuang D.M.
      • Wu Y.
      • Chen N.
      • Cheng J.
      • Zhuang S.M.
      • Zheng L.
      Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes.
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • Xu J.
      • Zhang J.P.
      • Wu C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      However, a recent study suggested that PD-L1 expression by macrophages positively correlates with T cell infiltration and patient survival.
      • Liu C.Q.
      • Xu J.
      • Zhou Z.G.
      • Jin L.L.
      • Yu X.J.
      • Xiao G.
      • et al.
      Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.
      Peritumoral macrophages were also demonstrated to foster Th17 expansion.
      • Kuang D.M.
      • Peng C.
      • Zhao Q.
      • Wu Y.
      • Zhu L.Y.
      • Wang J.
      • et al.
      Tumor-activated monocytes promote expansion of IL-17-producing CD8+ T cells in hepatocellular carcinoma patients.
      Mouse studies indicate that Th17 and other IL-17 producing cells, first shown to drive colon cancer development,
      • Grivennikov S.I.
      • Wang K.
      • Mucida D.
      • Stewart C.A.
      • Schnabl B.
      • Jauch D.
      • et al.
      Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.
      support the pathogenesis of liver fibrosis,
      • Meng F.
      • Wang K.
      • Aoyama T.
      • Grivennikov S.I.
      • Paik Y.
      • Scholten D.
      • et al.
      Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.
      NASH and HCC.
      • Gomes A.L.
      • Teijeiro A.
      • Buren S.
      • Tummala K.S.
      • Yilmaz M.
      • Waisman A.
      • et al.
      Metabolic inflammation-associated IL-17A causes non-alcoholic steatohepatitis and hepatocellular carcinoma.
      In HBV-associated HCC, both Kupffer cells (KCs) and dendritic cells (DCs) express galectin-9, the ligand for TIM-3; but, in HBV-negative patients, galectin-9 expression by KCs and DCs is negligible.
      • Li H.
      • Wu K.
      • Tao K.
      • Chen L.
      • Zheng Q.
      • Lu X.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      A subset of CD14+CTLA-4+ regulatory DCs, which represent ~13% of peripheral blood mononuclear cells (PBMCs) from patients with HCC, can limit T cell-mediated antitumour responses via IL-10 and indoleamine 2,3-dioxygenase (IDO) production.
      • Han Y.
      • Chen Z.
      • Yang Y.
      • Jiang Z.
      • Gu Y.
      • Liu Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      CD15+ neutrophils are highly represented in the peritumoral stroma of HCC, whereas CD66b+ neutrophils were predominantly detected in tumour areas.
      • Li Y.W.
      • Qiu S.J.
      • Fan J.
      • Zhou J.
      • Gao Q.
      • Xiao Y.S.
      • et al.
      Intratumoral neutrophils: a poor prognostic factor for hepatocellular carcinoma following resection.
      . Both of these cell types are harbingers of poor survival prospects. In the presence of abundant IL-17, neutrophils could secrete matrix metalloproteinase (MMP)-9 and stimulate the proangiogenic activity of HCC cells.
      • Kuang D.M.
      • Zhao Q.
      • Wu Y.
      • Peng C.
      • Wang J.
      • Xu Z.
      • et al.
      Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma.
      The frequency of CD14+HLA-DR−/low myeloid-derived suppressor cells (MDSCs) in PBMCs from patients with HCC was significantly increased in comparison to healthy controls, suggesting dampening of anti-HCC immunity.
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      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.
      • Hoechst B.
      • Ormandy L.A.
      • Ballmaier M.
      • Lehner F.
      • Kruger C.
      • Manns M.P.
      • et al.
      A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
      The accumulation and function of MDSCs in diverse mouse HCC models have been well summarised in a previous review.
      • Kapanadze T.
      • Gamrekelashvili J.
      • Ma C.
      • Chan C.
      • Zhao F.
      • Hewitt S.
      • et al.
      Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma.
      Functional natural killer (NK) cells in tumour tissues predict improved survival, but in advanced HCC NK cells are decreased in number and show impaired cytokine production. Infiltration of peritumoral macrophages was positively correlated with intratumoural NK cell dysfunction.
      • Wu Y.
      • Kuang D.M.
      • Pan W.D.
      • Wan Y.L.
      • Lao X.M.
      • Wang D.
      • et al.
      Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.
      Frequencies of Vα24/Vβ11 invariant natural killer T (iNKT) cells are also increased in HCC. However, the predominant CD4+ iNKT cell exhibits high Th2 cytokine production and lower cytolytic activity, which could perturb tumour-specific CD8+ CTL expansion.
      • Bricard G.
      • Cesson V.
      • Devevre E.
      • Bouzourene H.
      • Barbey C.
      • Rufer N.
      • et al.
      Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.
      Mouse studies indicate that the liver is particularly enriched in NKT cells, which could be involved in NASH pathogenesis,
      • Syn W.K.
      • Oo Y.H.
      • Pereira T.A.
      • Karaca G.F.
      • Jung Y.
      • Omenetti A.
      • et al.
      Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
      but their role in HCC is yet to be determined.
      Peritumoral tissues are highly infiltrated with B cells, whose CXCR3+ subpopulation boosted M2b macrophage polarization and positively correlated with early HCC recurrence.
      • Liu R.X.
      • Wei Y.
      • Zeng Q.H.
      • Chan K.W.
      • Xiao X.
      • Zhao X.Y.
      • et al.
      Chemokine (C-X-C motif) receptor 3-positive B cells link interleukin-17 inflammation to protumorigenic macrophage polarization in human hepatocellular carcinoma.
      About 10% of peritumoral B cells exhibited a PD-1-high phenotype, while over 50% are FcgRIIlow/− B cells, both of which acquired regulatory functions that blunted tumour-specific T cell immunity.
      • Xiao X.
      • Lao X.M.
      • Chen M.M.
      • Liu R.X.
      • Wei Y.
      • Ouyang F.Z.
      • et al.
      PD-1hi identifies a novel regulatory B-cell population in human hepatoma that promotes disease progression.
      • Ouyang F.Z.
      • Wu R.Q.
      • Wei Y.
      • Liu R.X.
      • Yang D.
      • Xiao X.
      • et al.
      Dendritic cell-elicited B-cell activation fosters immune privilege via IL-10 signals in hepatocellular carcinoma.
      By contrast, intratumoural B cells were found to be in close contact with CD8+ T cells and correlate with elevated levels of granzyme B and IFN-γ as well as reduced cancer cell viability.
      • Garnelo M.
      • Tan A.
      • Her Z.
      • Yeong J.
      • Lim C.J.
      • Chen J.
      • et al.
      Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.
      A different population of B cells with immunosuppressive properties that are characterised by production of immunoglobulin A (IgA) and high expression of PD-L1 and IL-10, were described in pre-malignant NASH tissue as well as in NASH-related HCC in humans and mice.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      This particular B cell subset plays a critical role in HCC progression.
      Mucosal-associated invariant T (MAIT) cells, an innate immune subset that is liver enriched, manifest lower densities in human HCC and display a typical memory phenotype.
      • Duan M.
      • Goswami S.
      • Shi J.Y.
      • Wu L.J.
      • Wang X.Y.
      • Ma J.Q.
      • et al.
      Activated and exhausted MAIT cells foster disease progression and indicate poor outcome in hepatocellular carcinoma.
      Intratumoural MAIT cells are reprogrammed toward a tumour-promoting direction, with upregulated expression of immune-checkpoint and inflammatory mediators, but minimal production of granzyme B, IFN-γ and perforin. High MAIT cell infiltration correlates with an unfavourable clinical outcome.
      • Duan M.
      • Goswami S.
      • Shi J.Y.
      • Wu L.J.
      • Wang X.Y.
      • Ma J.Q.
      • et al.
      Activated and exhausted MAIT cells foster disease progression and indicate poor outcome in hepatocellular carcinoma.
      Peritumoral mast cells and HSCs were found to correlate with high Treg density and early HCC recurrence after curative resection.
      • Ju M.J.
      • Qiu S.J.
      • Gao Q.
      • Fan J.
      • Cai M.Y.
      • Li Y.W.
      • et al.
      Combination of peritumoral mast cells and T-regulatory cells predicts prognosis of hepatocellular carcinoma.
      • Ju M.J.
      • Qiu S.J.
      • Fan J.
      • Xiao Y.S.
      • Gao Q.
      • Zhou J.
      • et al.
      Peritumoral activated hepatic stellate cells predict poor clinical outcome in hepatocellular carcinoma after curative resection.
      Interestingly, a group of IL-21-producing CXCR5PD-1lo/BTLACD69hi Tfh-like cells were found to promote plasma cell differentiation and IgA production.
      • Chen M.M.
      • Xiao X.
      • Lao X.M.
      • Wei Y.
      • Liu R.X.
      • Zeng Q.H.
      • et al.
      Polarization of tissue-resident TFH-Like cells in human hepatoma bridges innate monocyte inflammation and M2b macrophage polarization.
      High Tfh density was found to correlate with the generation of immunosuppressive IgA-producing plasma cells, most likely due to their ability to support the IgA class switch event.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      Such cohort-based human studies have estimated the proportions and phenotypes of distinct immune cells and provided important clues as to how TIME composition and status affect HCC prognosis (Table 1). Additional gene expression data obtained from bulk tissue specimens interrogated via methods such as CIBERSORT
      • Newman A.M.
      • Liu C.L.
      • Green M.R.
      • Gentles A.J.
      • Feng W.
      • Xu Y.
      • et al.
      Robust enumeration of cell subsets from tissue expression profiles.
      and TIMER
      • Li B.
      • Severson E.
      • Pignon J.C.
      • Zhao H.
      • Li T.
      • Novak J.
      • et al.
      Comprehensive analyses of tumor immunity: implications for cancer immunotherapy.
      can be used to further investigate how the composition of the HCC and HCC-adjacent immune infiltrates changes in a longitudinal manner during tumour progression. Another computational method has recently been developed to infer the immune cell types that contribute to inflammation-to-cancer transition in human liver.
      • Chen X.
      • Xu C.
      • Hong S.
      • Xia X.
      • Cao Y.
      • McDermott J.
      • et al.
      Immune cell types and secreted factors contributing to inflammation-to-cancer transition and immune therapy response.
      Additionally, integrating immunohistochemistry and transcriptomic data from a training cohort of patients with HCC can produce an “immunoscore” that could be used to stratify a validation cohort of patients and predict disease outcome.
      • Kurebayashi Y.
      • Ojima H.
      • Tsujikawa H.
      • Kubota N.
      • Maehara J.
      • Abe Y.
      • et al.
      Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification.
      Amongst 956 patients subjected to such analysis, approximately 1/4 of HCCs displayed an inflammatory TIME classification, with high PD-1 and PD-L1 expression.
      • Sia D.
      • Jiao Y.
      • Martinez-Quetglas I.
      • Kuchuk O.
      • Villacorta-Martin C.
      • Castro de Moura M.
      • et al.
      Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features.
      Interestingly, HBV infection and alcohol abuse were more prevalent in patients negative for the immune classifier, while HCV infection was significantly associated with a positive immune classifier. However, to enlarge the validation window, HCC TIME data should be correlated with other aetiological factors, including NASH, liver fibrosis, alcohol consumption and HBV or HCV infection. It is also worth mentioning that PD-L1 expression in HCC correlates with serum alpha-fetoprotein levels, satellite nodule numbers, vascular invasion, poor differentiation, progenitor subtype and eventually poor survival.
      • Liu C.Q.
      • Xu J.
      • Zhou Z.G.
      • Jin L.L.
      • Yu X.J.
      • Xiao G.
      • et al.
      Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.
      • Calderaro J.
      • Rousseau B.
      • Amaddeo G.
      • Mercey M.
      • Charpy C.
      • Costentin C.
      • et al.
      Programmed death ligand 1 expression in hepatocellular carcinoma: relationship with clinical and pathological features.
      Both HCC-intrinsic and -extrinsic factors shape the HCC TIME and dictate its role in tumour development and progression.
      Table 1Roles of immune subtypes in human HCC based on moderate-resolution data.
      Cell typesIntratumour/peritumour/bloodAntitumour/tumour-promotingCommentsRef
      Granzyme B+ CD8+ TIntratumourAntitumourThe frequency of intratumourally activated CTLs is an independent predictor for overall survival of HCC
      • 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.
      TregIntratumourTumour-promotingIntratumoural Treg frequency positively correlates with HCC invasiveness, and is negatively associated with CTL number and patient survival
      • 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.
      Th17IntratumourTumour-promotingTh17 cell density correlates with poor survival in HCC
      • Zhang J.P.
      • Yan J.
      • Xu J.
      • Pang X.H.
      • Chen M.S.
      • Li L.
      • et al.
      Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients.
      Th22IntratumourTumour-promotingIL-22 producing CD4+ T cells promote HCC growth
      • Jiang R.
      • Tan Z.
      • Deng L.
      • Chen Y.
      • Xia Y.
      • Gao Y.
      • et al.
      Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3.
      • Kuang D.M.
      • Xiao X.
      • Zhao Q.
      • Chen M.M.
      • Li X.F.
      • Liu R.X.
      • et al.
      B7-H1-expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets.
      TfhIntratumourTumour-promotingIL-21-producing CXCR5-PD-1lo/−BTLA-CD69hi Tfh-like cells promote IgA+ plasma cell differentiation and M2 polarization
      • Chen M.M.
      • Xiao X.
      • Lao X.M.
      • Wei Y.
      • Liu R.X.
      • Zeng Q.H.
      • et al.
      Polarization of tissue-resident TFH-Like cells in human hepatoma bridges innate monocyte inflammation and M2b macrophage polarization.
      Th9IntratumourTumour-promotingHCC patients with higher tumour-infiltrating Th9 frequency have significantly shorter disease-free survival
      • Tan H.
      • Wang S.
      • Zhao L.
      A tumour-promoting role of Th9 cells in hepatocellular carcinoma through CCL20 and STAT3 pathways.
      CD68+Intratumour/peritumourTumour-promotingHigh MΦ density correlates with large tumour size, high TNM stage and intrahepatic metastasis
      • Zhu X.D.
      • Zhang J.B.
      • Zhuang P.Y.
      • Zhu H.G.
      • Zhang W.
      • Xiong Y.Q.
      • et al.
      High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.
      CD14+CD16+ TIE2+Intratumour/bloodTumour-promotingCD14+CD16+TIE2+ MΦ are associated with intratumoural micro-vessel density
      • Matsubara T.
      • Kanto T.
      • Kuroda S.
      • Yoshio S.
      • Higashitani K.
      • Kakita N.
      • et al.
      TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.
      IL10+IntratumourTumour-promotingIL10+ MΦ inhibit antitumour immune responses
      • Kuang D.M.
      • Wu Y.
      • Chen N.
      • Cheng J.
      • Zhuang S.M.
      • Zheng L.
      Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes.
      HLA-DR+PD-L1+Intratumour/bloodTumour-promotingPD-L1 expression on MΦ correlates with T-cell infiltration and patient survival
      • Kuang D.M.
      • Zhao Q.
      • Peng C.
      • Xu J.
      • Zhang J.P.
      • Wu C.
      • et al.
      Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
      • Liu C.Q.
      • Xu J.
      • Zhou Z.G.
      • Jin L.L.
      • Yu X.J.
      • Xiao G.
      • et al.
      Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.
      CD14+CTLA-4+ DCIntratumourTumour-promotingCD14+CTLA-4+ DCs limit T cell mediated anti-tumour responses via IL-10 and IDO production
      • Han Y.
      • Chen Z.
      • Yang Y.
      • Jiang Z.
      • Gu Y.
      • Liu Y.
      • et al.
      Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
      CD15+ NeutrophilPeritumourTumour-promotingA poor prognostic factor
      • Li Y.W.
      • Qiu S.J.
      • Fan J.
      • Zhou J.
      • Gao Q.
      • Xiao Y.S.
      • et al.
      Intratumoral neutrophils: a poor prognostic factor for hepatocellular carcinoma following resection.
      CD66b+ NeutrophilIntratumourTumour-promotingA poor prognostic factor
      • Li Y.W.
      • Qiu S.J.
      • Fan J.
      • Zhou J.
      • Gao Q.
      • Xiao Y.S.
      • et al.
      Intratumoral neutrophils: a poor prognostic factor for hepatocellular carcinoma following resection.
      CD14+HLA-DR−/low MDSCBloodTumour-promotingThe frequency of circulating CD14+HLA-DR−/low MDSC in HCC patients is significantly increased in comparison to healthy controls
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      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.
      • Hoechst B.
      • Ormandy L.A.
      • Ballmaier M.
      • Lehner F.
      • Kruger C.
      • Manns M.P.
      • et al.
      A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
      CD4+ iNKTIntratumourTumour-promotingCD4+ iNKT cells perturb tumour-specific CD8+ CTL expansion
      • Bricard G.
      • Cesson V.
      • Devevre E.
      • Bouzourene H.
      • Barbey C.
      • Rufer N.
      • et al.
      Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.
      CXCR3+ B cellPeritumourTumour-promotingCXCR3+ B cells positively correlate with early HCC recurrence
      • Liu R.X.
      • Wei Y.
      • Zeng Q.H.
      • Chan K.W.
      • Xiao X.
      • Zhao X.Y.
      • et al.
      Chemokine (C-X-C motif) receptor 3-positive B cells link interleukin-17 inflammation to protumorigenic macrophage polarization in human hepatocellular carcinoma.
      PD-1high B cellPeritumourTumour-promotingPD-1high B cells suppress tumour-specific T cell immunity via IL-10 signalling
      • Xiao X.
      • Lao X.M.
      • Chen M.M.
      • Liu R.X.
      • Wei Y.
      • Ouyang F.Z.
      • et al.
      PD-1hi identifies a novel regulatory B-cell population in human hepatoma that promotes disease progression.
      FcgRIIlow/− B cellPeritumourTumour-promotingFcgRIIlow/− B cells blunt tumour-specific T cell immunity
      • Ouyang F.Z.
      • Wu R.Q.
      • Wei Y.
      • Liu R.X.
      • Yang D.
      • Xiao X.
      • et al.
      Dendritic cell-elicited B-cell activation fosters immune privilege via IL-10 signals in hepatocellular carcinoma.
      CD20+ B cellIntratumourAntitumourIntratumoural B cells correlate with elevated levels of granzyme B and IFN-γ as well as reduced cancer cell viability
      • Garnelo M.
      • Tan A.
      • Her Z.
      • Yeong J.
      • Lim C.J.
      • Chen J.
      • et al.
      Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.
      IgA+PD-L1+IL-10+ B cellIntratumourTumour-promotingIgA+PD-L1+IL-10+ B cells directly suppress liver CD8+ CTL
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      MAITIntratumourTumour-promotingHigh MAIT cell infiltration correlates with an unfavourable clinical outcome
      • Duan M.
      • Goswami S.
      • Shi J.Y.
      • Wu L.J.
      • Wang X.Y.
      • Ma J.Q.
      • et al.
      Activated and exhausted MAIT cells foster disease progression and indicate poor outcome in hepatocellular carcinoma.
      Mast cellPeritumourTumour-promotingPeritumoural mast cells correlate to high Treg density and early HCC recurrence after curative resection
      • Ju M.J.
      • Qiu S.J.
      • Gao Q.
      • Fan J.
      • Cai M.Y.
      • Li Y.W.
      • et al.
      Combination of peritumoral mast cells and T-regulatory cells predicts prognosis of hepatocellular carcinoma.
      CTL, cytotoxic T lymphocyte; DC, dendritic cell; HCC, hepatocellular carcinoma; IDO, indoleamine 2,3-dioxygenase; IL, interleukin; iNKT, invariant NKT; MΦ, macrophage; MDSC, myeloid-derived suppressor cell; MAIT, mucosal-associated invariant T cell; NK, natural killer; Th, T helper cell; Treg, regulatory T cell.

      High-resolution TIME data

      Despite the results described above, the fundamental roles of many HCC-infiltrating leukocytes remain controversial with contradictory conclusions often being reached. Deciphering the functional heterogeneity of immune infiltrates can be improved by using high-resolution techniques. An integrative investigation using time-of-flight mass cytometry (CyTOF), multiplex immunofluorescent staining, and NanoString analysis confirmed that human HCC is abundantly infiltrated with Tregs, resident memory CD8+ T cells (TRMs), NK cells, and macrophages.
      • Chew V.
      • Lai L.
      • Pan L.
      • Lim C.J.
      • Li J.
      • Ong R.
      • et al.
      Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses.
      In addition, intratumoural Tregs and TRMs were confirmed to express multiple T cell exhaustion markers, including PD-1, LAG-3, and TIM-3. Notably, high PD-1 expression was detected in HBV-positive patients compared to uninfected individuals, suggesting a role for viral antigens in TRM exhaustion and offering novel therapeutic opportunities.
      • Chew V.
      • Lai L.
      • Pan L.
      • Lim C.J.
      • Li J.
      • Ong R.
      • et al.
      Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses.
      Furthermore, deep single cell RNA sequencing has been used to interrogate the molecular and functional properties of T cell subsets within the HCC TIME and delineate their developmental trajectories.
      • Zheng C.
      • Zheng L.
      • Yoo J.K.
      • Guo H.
      • Zhang Y.
      • Guo X.
      • et al.
      Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.
      Signature genes for each of the 11 T cell clusters were described, including those that mark exhausted CD8+ T cells and Tregs, which were preferentially expanded in HCC. One such gene, LAYN coding for layilin, exhibited a highly specific expression pattern in exhausted T cells and was correlated with reduced disease-free survival.
      • Zheng C.
      • Zheng L.
      • Yoo J.K.
      • Guo H.
      • Zhang Y.
      • Guo X.
      • et al.
      Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.
      Besides, resting digitally defined clusters further confirmed the presence of naïve CD4+ and CD8+ T cells, effector CD4+ and CD8+ T cells, MAIT and exhausted CD4+ T cells in the HCC TIME.
      • Zheng C.
      • Zheng L.
      • Yoo J.K.
      • Guo H.
      • Zhang Y.
      • Guo X.
      • et al.
      Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.
      To date, HCC-infiltrating leukocytes other than T cells, especially B lymphocytes and myeloid cells, still await high-resolution single cell RNA sequencing. Meanwhile, the single cell RNA sequencing data from myeloid cells in the normal liver
      • MacParland S.A.
      • Liu J.C.
      • Ma X.Z.
      • Innes B.T.
      • Bartczak A.M.
      • Gage B.K.
      • et al.
      Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.
      or obesity-related steatohepatitis
      • Krenkel O.
      • Hundertmark J.
      • Abdallah A.T.
      • Kohlhepp M.
      • Puengel T.
      • Roth T.
      • et al.
      Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.
      provide a basis for understanding disease-related changes in the major drivers of pro-tumorigenic chronic inflammation. Next-generation platforms and improved cell isolation and preservation techniques should generate transcriptomic profiles of individual immune cells and further define the cellular heterogeneity and dynamic trajectory of the HCC TIME. Together with proteomic and epigenomic scales, these single cell-based technologies are expected to provide an unprecedented view of the HCC immune landscape. The availability of high-resolution TIME data should improve the classification of tumour-infiltrating lymphocytes and illuminate future strategies for overcoming the immunosuppressive and dysfunctional nature of the HCC immune system. Despite the incremental progress in defining the HCC TIME, much improvement is needed to speed up the analyses, thereby allowing treatment decisions to be made within a reasonable time scale following HCC diagnosis or surgical resection. The reliability of such analyses also needs substantial improvement, allowing the surgeon or the oncologist to make unequivocal choices that should enhance patient survival.
      Improved “omics”-based immunoprofiling and sophisticated computational tools will be instrumental in improving the efficacy of HCC immunotherapy.

      Mutational burden and immunogenicity of human HCC

      Recent studies have demonstrated that T cell-based immunotherapies, including immune-checkpoint blockade (ICB) and adoptive T cell transfer, are effective in a wide range of human malignancies.
      • Sahin U.
      • Tureci O.
      Personalized vaccines for cancer immunotherapy.
      • Ribas A.
      • Wolchok J.D.
      Cancer immunotherapy using checkpoint blockade.
      . Currently, the overall mutational load, an objective but indirect measure of tumour immunogenicity, is considered to be an important predictor of tumour-specific T cell immunity.
      • Blankenstein T.
      • Coulie P.G.
      • Gilboa E.
      • Jaffee E.M.
      The determinants of tumour immunogenicity.
      Indeed, microsatellite instability high (MSI-H) colon cancers, which display a high rate of nonsynonymous single-nucleotide polymorphisms that lead to expression of many neoepitopes (or so-called “neoantigens”), exhibit higher intratumoural T cell content than MSI-low (MSI-L) colon cancers. Although most intratumoural T cells express markers of exhaustion, they can be re-invigorated by ICB therapy and therefore their frequency often predicts ICB responsiveness.
      • Yarchoan M.
      • Johnson 3rd, B.A.
      • Lutz E.R.
      • Laheru D.A.
      • Jaffee E.M.
      Targeting neoantigens to augment antitumour immunity.
      Correspondingly, the majority of colon cancers, which are MSI-L, show very poor response to ICB. Such examples support the notion that mutational load or neoantigen fitness predict patient survival after immunotherapy.
      • Samstein R.M.
      • Lee C.H.
      • Shoushtari A.N.
      • Hellmann M.D.
      • Shen R.
      • Janjigian Y.Y.
      • et al.
      Tumor mutational load predicts survival after immunotherapy across multiple cancer types.
      Consistent with this notion, neoantigen-based personal vaccines or cell therapy are being developed to treat ICB refractory cancers.
      • Stronen E.
      • Toebes M.
      • Kelderman S.
      • van Buuren M.M.
      • Yang W.
      • van Rooij N.
      • et al.
      Targeting of cancer neoantigens with donor-derived T cell receptor repertoires.
      • Ott P.A.
      • Hu Z.
      • Keskin D.B.
      • Shukla S.A.
      • Sun J.
      • Bozym D.J.
      • et al.
      An immunogenic personal neoantigen vaccine for patients with melanoma.
      • Zacharakis N.
      • Chinnasamy H.
      • Black M.
      • Xu H.
      • Lu Y.C.
      • Zheng Z.
      • et al.
      Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.
      • Keskin D.B.
      • Anandappa A.J.
      • Sun J.
      • Tirosh I.
      • Mathewson N.D.
      • Li S.
      • et al.
      Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.
      However, there are many exceptions to this rather simplistic view.
      Importantly, standardised neoantigen prediction for human HCC must be reported before it can serve as a reliable predictor of ICB responsiveness. Nonetheless, the HCC genomic landscape has been extensively investigated. In an early study of 10 HBV-related HCC specimens, a total of 356 nonsynonymous somatic mutations were identified within 347 genes by exome sequencing. Individually, the numbers of mutations ranged from a minimum of 8 to a maximum of 99 per tumour.
      • Huang J.
      • Deng Q.
      • Wang Q.
      • Li K.Y.
      • Dai J.H.
      • Li N.
      • et al.
      Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.
      Putative driver mutations in the TERT, TP53, ARID1A, ARID2, CTNNB1, AXIN1 and PIK3CA genes were detected and were confirmed to be of rather frequent occurrence in several large cohorts of HCC with different aetiologies.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      Cancer Genome Atlas Research Network
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      However, it has been suggested that neoantigens derived from driver mutations generally result in profound immune tolerance and T cell exhaustion due to long-term antigen exposure (Fig. 1).
      • Turajlic S.
      • Litchfield K.
      • Xu H.
      • Rosenthal R.
      • McGranahan N.
      • Reading J.L.
      • et al.
      Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis.
      • Schietinger A.
      • Philip M.
      • Krisnawan V.E.
      • Chiu E.Y.
      • Delrow J.J.
      • Basom R.S.
      • et al.
      Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis.
      The impact of such mutations on the response to ICB therapy is not entirely clear and likely to depend on multiple factors. Moreover, some driver mutations can switch-on biological circuits of cancer-driven immune evasion (discussed below). Of note, in a cohort of 956 HCCs, the mutational burden was comparable between the immune-specific class and other HCCs.
      • Sia D.
      • Jiao Y.
      • Martinez-Quetglas I.
      • Kuchuk O.
      • Villacorta-Martin C.
      • Castro de Moura M.
      • et al.
      Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features.
      Another study showed that the mutational load of human HCC was negatively correlated with PD-L1 expression and T cell cytolytic activity, indicating a TIME classification of “immunological ignorance”.
      • Chen Y.P.
      • Zhang Y.
      • Lv J.W.
      • Li Y.Q.
      • Wang Y.Q.
      • He Q.M.
      • et al.
      Genomic analysis of tumor microenvironment immune types across 14 solid cancer types: immunotherapeutic implications.
      Aflatoxin exposure is another aetiological factor that increases HCC risk. The co-occurrence of mutation-associated neoantigens and higher expression of PD-L1 were observed in patients with aflatoxin-associated HCC, suggesting that these patients may be more responsive to T cell-based and ICB immunotherapies than other HCC aetiologies.
      • Zhang W.
      • He H.
      • Zang M.
      • Wu Q.
      • Zhao H.
      • Lu L.L.
      • et al.
      Genetic features of aflatoxin-associated hepatocellular carcinoma.
      Figure thumbnail gr1
      Fig. 1Chronic antigen exposure can induce immune tolerance or dysfunction. Neoantigens encoded by HCC-associated mutant alleles may prime tumour-specific T cells during the pre-malignant stage, but continuous antigen encounter renders antitumoural T cells dysfunctional. This may cause an immune ignorant phenotype and primary resistance to ICB therapy due to long-term antigen exposure. HCC, hepatocellular carcinoma; ICB, immune-checkpoint blockade.
      Despite the relatively large number of driver mutations in human HCCs, somatic insertion and/or deletion (indel) mutations were found to occur with an average frequency.
      • Huang J.
      • Deng Q.
      • Wang Q.
      • Li K.Y.
      • Dai J.H.
      • Li N.
      • et al.
      Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.
      • Schumacher T.N.
      • Schreiber R.D.
      Neoantigens in cancer immunotherapy.
      Such mutations may also encode neopeptides and elicit immune recognition.
      • Turajlic S.
      • Litchfield K.
      • Xu H.
      • Rosenthal R.
      • McGranahan N.
      • Reading J.L.
      • et al.
      Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis.
      However, it remains to be determined whether successful antitumour immune responses can target indel-generated neoepitopes. Bioinformatic algorithms and actual identification of neoantigens that are responsible for tumour rejection remain major future challenges. Special attention needs to be given to multicentric or multifocal tumours, in which genomic heterogeneity may contribute to a variation in the set of neoantigens expressed by each HCC clone.
      • Xue R.
      • Li R.
      • Guo H.
      • Guo L.
      • Su Z.
      • Ni X.
      • et al.
      Variable intra-tumor genomic heterogeneity of multiple lesions in patients with hepatocellular carcinoma.
      The involvement of immune dysfunction and CD8+ T cell exhaustion in malignant progression makes HCC potentially susceptible to immune-checkpoint inhibitors.

      The paradox of cancer-promoting inflammation and anti-HCC immunity

      The liver is populated by multiple immune cell subtypes; however, the role of certain inflammatory and innate immune populations in HCC development remains controversial. Much of our current knowledge is derived from studies in mouse models. The relevance and the quality of information derived from such studies is very much dependent on the particular mouse model being used. Macrophages were found to preferentially accumulate in the liver of diethylnitrosamine (DEN)-treated mice, where they facilitate tumour initiation by producing cytokines such as TNF and IL-6 that activate NF-kB and STAT3 in HCC progenitor cells.
      • He G.
      • Dhar D.
      • Nakagawa H.
      • Font-Burgada J.
      • Ogata H.
      • Jiang Y.
      • et al.
      Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
      • He G.
      • Yu G.Y.
      • Temkin V.
      • Ogata H.
      • Kuntzen C.
      • Sakurai T.
      • et al.
      Hepatocyte IKKbeta/NF-kappaB inhibits tumor promotion and progression by preventing oxidative stress-driven STAT3 activation.
      In contrast, CD8+ CTLs, which are also elevated in this model, can eliminate neoplastic cells bearing neoantigens.
      • Schneider C.
      • Teufel A.
      • Yevsa T.
      • Staib F.
      • Hohmeyer A.
      • Walenda G.
      • et al.
      Adaptive immunity suppresses formation and progression of diethylnitrosamine-induced liver cancer.
      However, it is questionable whether the immunobiology of DEN-induced HCC is of much relevance to the immunobiology of human HCC. Much better evidence for CD8+ T cell-mediated immunosurveillance was seen in the MUP-uPA + high-fat diet (HFD) model of NASH-driven HCC,
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      which was shown to be quite relevant to human NASH-induced HCC.
      • Febbraio M.A.
      • Reibe S.
      • Shalapour S.
      • Ooi G.J.
      • Watt M.J.
      • Karin M.
      Preclinical models for studying NASH-driven HCC: how useful are they?.
      CD8+ T cell ablation or immunodepletion in MUP-uPA mice resulted in much faster and more strongly enhanced HCC development. Oddly, however, in a different mouse model of diet-induced NASH and in a hereditary tyrosinemia model, it was found that activation of CD8+ T and NKT cells contribute to chronic liver damage and spontaneous HCC onset,
      • Wolf M.J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      • Endig J.
      • Buitrago-Molina L.E.
      • Marhenke S.
      • Reisinger F.
      • Saborowski A.
      • Schutt J.
      • et al.
      Dual role of the adaptive immune system in liver injury and hepatocellular carcinoma development.
      results that are inconsistent with the success of ICB therapy in human HCC. The presence of intratumoural ectopic lymphoid-like structures (ELSs), which reflect an interaction between tumour cells and the immune system, was used as evidence for the existence of anti-HCC immunity,
      • Finkin S.
      • Yuan D.
      • Stein I.
      • Taniguchi K.
      • Weber A.
      • Unger K.
      • et al.
      Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.
      but non-tumoural liver ELSs were found to support growth of HCC progenitors.
      • Finkin S.
      • Yuan D.
      • Stein I.
      • Taniguchi K.
      • Weber A.
      • Unger K.
      • et al.
      Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.
      • Calderaro J.
      • Petitprez F.
      • Becht E.
      • Laurent A.
      • Hirsch T.Z.
      • Rousseau B.
      • et al.
      Intra-tumoral tertiary lymphoid structures are associated with a low risk of early recurrence of hepatocellular carcinoma.
      HBV is one of the major causes of human inflammatory liver diseases and HCC. Unfortunately, however, HBV-induced HCC is difficult to study in mice, and most of our knowledge has been derived from analysis of human specimens. Although innate and adaptive immune responses are required to restrict HBV replication, inflammation and collateral damage mediated by virus-directed T cells collectively trigger a cancer-promoting program in the HBV-exposed liver. Curiously, in a recently established HBV-transgenic (HBs-Tg) mouse model, HCC incidence was 16.7%, but administration of a small dose of polyinosinic:polycytidylic acid [poly (I:C)], which activates NK cells, increased HCC incidence to 100%.
      • Chen Y.
      • Hao X.
      • Sun R.
      • Wei H.
      • Tian Z.
      Natural killer cell-derived interferon-gamma promotes hepatocellular carcinoma through the epithelial cell adhesion molecule-epithelial-to-mesenchymal transition axis in hepatitis B virus transgenic mice.
      Under these conditions, infiltrating lymphocytes directed against viral antigens caused hepatocyte apoptosis and compensatory proliferation along with epithelial-to-mesenchymal transition. Conversely, depletion of NK cells or IFN-γ markedly alleviated hepatitis and reduced HCC incidence in this model.
      • Chen Y.
      • Hao X.
      • Sun R.
      • Wei H.
      • Tian Z.
      Natural killer cell-derived interferon-gamma promotes hepatocellular carcinoma through the epithelial cell adhesion molecule-epithelial-to-mesenchymal transition axis in hepatitis B virus transgenic mice.
      Meanwhile, targeting TIGIT, an immune-checkpoint receptor that is highly expressed on hepatic CD8+ T cells, resulted in a HBsAg-specific immune response and concurrent liver fibrosis in the HBV-Tg model. In the absence of TIGIT, HBsAg vaccination failed to control disease and caused unresolved inflammation that led to enhanced HCC development.
      • Zong L.
      • Peng H.
      • Sun C.
      • Li F.
      • Zheng M.
      • Chen Y.
      • et al.
      Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice.
      Accordingly, HBV infection is likely to trigger cancer-promoting inflammation, which can conceal effective anti-HCC immunity. Furthermore, chronic viral infections are well established inducers of PD-L1 expression in hepatocytes and are therefore capable of triggering CD8+ T cell exhaustion, thereby supporting HCC development.
      Other biological forces in pre-malignant or malignant cells, such as DNA damage,
      • Grivennikov S.I.
      • Greten F.R.
      • Karin M.
      Immunity, inflammation, and cancer.
      necrosis/necroptosis
      • Sakurai T.
      • He G.
      • Matsuzawa A.
      • Yu G.Y.
      • Maeda S.
      • Hardiman G.
      • et al.
      Hepatocyte necrosis induced by oxidative stress and IL-1 alpha release mediate carcinogen-induced compensatory proliferation and liver tumorigenesis.
      • Seehawer M.
      • Heinzmann F.
      • D'Artista L.
      • Harbig J.
      • Roux P.F.
      • Hoenicke L.
      • et al.
      Necroptosis microenvironment directs lineage commitment in liver cancer.
      and endoplasmic reticulum (ER) stress,
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      all of which were suggested to stimulate immune surveillance in other types of cancer, were found to promote HCC development and progression in an inflammation-dependent manner (Fig. 2). In addition, the gut microbiome, whose products reach the liver via the portal circulation, participates in inflammation-driven HCC, despite its often-documented favourable impact on ICB therapeutics in other cancers.
      • Dapito D.H.
      • Mencin A.
      • Gwak G.Y.
      • Pradere J.P.
      • Jang M.K.
      • Mederacke I.
      • et al.
      Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4.
      • Sears C.L.
      • Pardoll D.M.
      The intestinal microbiome influences checkpoint blockade.
      Another mechanism that favours HCC development is the ability of chronic liver inflammation to promote the generation of immunosuppressive IgA+ plasmocytes through production of transforming growth factor-β (TGF-β) and Tfh-related mechanisms.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      IgA+ plasma cells, which express both PD-L1 and IL-10, inhibit CTL-mediated immunosurveillance and favour HCC malignant progression.
      Mouse models of HCC have been useful for studying the role of antitumour immunity and pro-tumorigenic inflammation in HCC development and progression.
      Figure thumbnail gr2
      Fig. 2The immune system acts as a double-edged sword when it comes to HCC. Cancer-promoting inflammation, the undesirable facet of immune activation, is an important pathogenic factor in HCC development, which invariably occurs in the chronically inflamed liver. Cellular processes including DNA damage, necrosis and ER stress can affect both immunosurveillance and cancer-promoting inflammation. Chronic liver inflammation can also promote immunosuppression leading to inactivation of antitumour immunity. ER, endoplasmic reticulum; HCC, hepatocellular carcinoma.

      Negative regulation of anti-HCC immunity

      Cancer-cell-intrinsic mechanisms

      Bioinformatic and mechanistic studies have highlighted the impact of oncogenic signalling cascades on immune inhibitory pathways and cell populations in a broad spectrum of cancers.
      • Spranger S.
      • Gajewski T.F.
      Impact of oncogenic pathways on evasion of antitumour immune responses.
      Although endogenous p53 or oncogenic N-Ras can trigger a “senescence surveillance” program that activates immune-mediated clearance of malignant or pre-malignant hepatocytes,
      • Xue W.
      • Zender L.
      • Miething C.
      • Dickins R.A.
      • Hernando E.
      • Krizhanovsky V.
      • et al.
      Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas.
      • Kang T.W.
      • Yevsa T.
      • Woller N.
      • Hoenicke L.
      • Wuestefeld T.
      • Dauch D.
      • et al.
      Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.
      other cancer-cell-intrinsic genetic alterations have been linked to immune dysfunction. Of note, Wnt/CTNNB1 pathway alterations, which are quite common in HCC, characterise immune-excluded tumours and cause primary resistance to ICB in different human cancer types, including HCC.
      • Pinyol R.
      • Sia D.
      • Llovet J.M.
      Immune exclusion-Wnt/CTNNB1 class predicts resistance to immunotherapies in HCC.
      • Anson M.
      • Crain-Denoyelle A.M.
      • Baud V.
      • Chereau F.
      • Gougelet A.
      • Terris B.
      • et al.
      Oncogenic beta-catenin triggers an inflammatory response that determines the aggressiveness of hepatocellular carcinoma in mice.
      • Ruiz de Galarreta M
      • Bresnahan E
      • Molina-Sanchez P
      • Lindblad KE
      • Maier B
      • Sia D
      • et al.
      beta-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma.
      • Calderaro J.
      • Couchy G.
      • Imbeaud S.
      • Amaddeo G.
      • Letouze E.
      • Blanc J.F.
      • et al.
      Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification.
      Specifically, CTNNB1-mutated HCC cells impede immune surveillance by reducing CCL5 production.
      • Ruiz de Galarreta M
      • Bresnahan E
      • Molina-Sanchez P
      • Lindblad KE
      • Maier B
      • Sia D
      • et al.
      beta-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma.
      Emerging evidence also suggests an interaction between cyclin-dependent kinases and tumour immunity. In the case of HCC, tumour-intrinsic cell cycle-related kinase (CCRK) activates an inflammatory signalling cascade that augments IL-6 production, thereby leading to polymorphonuclear (PMN) MDSC-mediated inhibitory signals.
      • Zhou J.
      • Liu M.
      • Sun H.
      • Feng Y.
      • Xu L.
      • Chan A.W.H.
      • et al.
      Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.
      c-MYC overexpression occurs in 70% of viral and alcohol-related HCCs and is critical for malignant transformation of hepatocytes. In a transgenic mouse model of HCC, c-MYC overexpression caused translational upregulation of PD-L1, thus enabling immune escape and lung metastasis.
      • Xu Y.
      • Poggio M.
      • Jin H.Y.
      • Shi Z.
      • Forester C.M.
      • Wang Y.
      • et al.
      Translation control of the immune checkpoint in cancer and its therapeutic targeting.
      Tumour-derived soluble factors mediate intercellular communication between cancer cells and immune cells. HCC-derived chemokines, such as CCL15 and CXCL5, recruit immunosuppressive neutrophils and monocytes, respectively,
      • Zhou S.L.
      • Dai Z.
      • Zhou Z.J.
      • Wang X.Y.
      • Yang G.H.
      • Wang Z.
      • et al.
      Overexpression of CXCL5 mediates neutrophil infiltration and indicates poor prognosis for hepatocellular carcinoma.
      • Liu L.Z.
      • Zhang Z.
      • Zheng B.H.
      • Shi Y.
      • Duan M.
      • Ma L.J.
      • et al.
      CCL15 recruits suppressive monocytes to facilitate immune escape and disease progression in hepatocellular carcinoma.
      whereas CXCL13 from activated HSCs may recruit B cells that are eventually converted to immunosuppressive IgA+ plasma cells in the fibrotic liver microenvironment.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      Tumour-secreted extracellular vesicles can also transmit oncogenic signals to local or distant immune cells. Exosomes released by HCV-infected cells promote Galectin-9 secretion by monocytes, resulting in TIM-3-mediated immune dysfunction.
      • Harwood N.M.
      • Golden-Mason L.
      • Cheng L.
      • Rosen H.R.
      • Mengshol J.A.
      HCV-infected cells and differentiation increase monocyte immunoregulatory galectin-9 production.
      By contrast, HBV-infected hepatocytes release exosomes that express interferon-induced transmembrane protein 2 (IFITM2) and inhibit the synthesis of endogenous IFN-α in DCs.
      • Shi Y.
      • Du L.
      • Lv D.
      • Li H.
      • Shang J.
      • Lu J.
      • et al.
      Exosomal IFITM2 transmitted to DCs inhibits IFNalpha pathway activation and blocks anti-HBV efficacy of exogenous IFNalpha.
      Moreover, HCC-derived exosomes could equip B cells with high expression of TIM-1 and inhibitory activity toward CD8+ T cells.
      • Ye L.
      • Zhang Q.
      • Cheng Y.
      • Chen X.
      • Wang G.
      • Shi M.
      • et al.
      Tumor-derived exosomal HMGB1 fosters hepatocellular carcinoma immune evasion by promoting TIM-1(+) regulatory B cell expansion.
      ER-stressed HCC cells release exosomal microRNAs which upregulate PD-L1 expression by TAMs (Fig. 3).
      • Liu J.
      • Fan L.
      • Yu H.
      • Zhang J.
      • He Y.
      • Feng D.
      • et al.
      Endoplasmic reticulum stress promotes liver cancer cells to release exosomal miR-23a-3p and up-regulate PD-L1 expression in macrophages.
      Figure thumbnail gr3
      Fig. 3Oncogenic signals promote evasion of anti-HCC immunity. Aberrant activation of the Wnt/CTNNB1 pathway within HCC cells unleashes production of LECT2 and accumulation of monocytes that promote immune evasion. Wnt/CTNNB1 signalling also inhibits CCL5 production, thereby interfering with DC recruitment. HCC-intrinsic CCRK increases IL-6 expression to promote MDSC infiltration. MYC overexpression upregulates PD-L1 at the translational level. HCC-secreted chemokines including CCL15 and CXCL5 and exosomes carrying IFITM2, galectin-9, miR-23a-3P or HMGB1 compromise anti-HCC immunity through recruitment of suppressive cells or engagement of inhibitory receptors. HIF-1α further upregulates CCL20 and CCL26, which can promote IDO expression by TAMs and recruitment of CX3CR1-positive MDSCs, respectively. CCL5, CC-chemokine ligand 5; CCRK, cell cycle-related kinase; CXCL5, CXC-chemokine ligand 5; DC, dendritic cell; HCC, hepatocellular carcinoma; HIF-1α, hypoxia-inducible factor-1α; HMGB1, high mobility group box 1; IDO, indoleamine 2,3-dioxygenase; IFITM2, interferon-induced transmembrane protein 2; LECT2; leukocyte cell-derived chemotaxin-2; MDSCs, myeloid-derived suppressor cells; TAMs, tumour-associated macrophages.

      Context-dependent mechanisms

      Pathological processes that accelerate carcinogenesis can also affect anti-HCC immunity. Liver fibrosis, a hallmark of human HCC, is associated with production of the immunosuppressive cytokine TGF-β and can hinder local immune surveillance by reducing the activity of NK and NKT cells, or by interfering with antigen recognition by intravascular CD8+ T cells.
      • Zhang D.Y.
      • Friedman S.L.
      Fibrosis-dependent mechanisms of hepatocarcinogenesis.
      • Guidotti L.G.
      • Inverso D.
      • Sironi L.
      • Di Lucia P.
      • Fioravanti J.
      • Ganzer L.
      • et al.
      Immunosurveillance of the liver by intravascular effector CD8(+) T cells.
      Notably, and consistent with myofibroblast activation in fibrosis, fibroblast-derived IL-6 and stromal cell-derived factor (SDF)-1a were found to facilitate MDSC generation and infiltration in HCC.
      • Deng Y.
      • Cheng J.
      • Fu B.
      • Liu W.
      • Chen G.
      • Zhang Q.
      • et al.
      Hepatic carcinoma-associated fibroblasts enhance immune suppression by facilitating the generation of myeloid-derived suppressor cells.
      Moreover, and as already mentioned, TGF-β triggers immune evasion and growth of both HCC and liver metastatic colon cancer.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      • Chen J.
      • Zaidi S.
      • Rao S.
      • Chen J.S.
      • Phan L.
      • Farci P.
      • et al.
      Analysis of genomes and transcriptomes of hepatocellular carcinomas identifies mutations and gene expression changes in the transforming growth factor-beta pathway.
      NASH increases HCC risk and modulates antitumour immunity mainly through the induction of immunosuppressive IgA+ plasma cells, which were detected in both patients with NASH and mouse models.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      In different models of liver metabolic inflammation based on a combination of DEN with HFD, or the consumption of a methionine-choline-deficient diet, intrahepatic CD4+ T cells were suggested to be depleted due to lipid-mediated mitochondrial dysfunction, leading to disruption of HCC surveillance.
      • Ma C.
      • Kesarwala A.H.
      • Eggert T.
      • Medina-Echeverz J.
      • Kleiner D.E.
      • Jin P.
      • et al.
      NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
      Despite being an ‘‘immune-privileged’’ organ, the liver frequently encounters immunostimulatory microbial products arriving from the gut via the portal vein. Obesity-associated gut microbial metabolites can accelerate HCC development by inducing a senescence-associated secretory phenotype (SASP) in HSCs, as well as through production of prostaglandin E2 (PGE2), both of which interfere with antitumour immunity.
      • Loo T.M.
      • Kamachi F.
      • Watanabe Y.
      • Yoshimoto S.
      • Kanda H.
      • Arai Y.
      • et al.
      Gut microbiota promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity.
      Given the contradictory roles of the adaptable gut microbiota,
      • Yoshimoto S.
      • Loo T.M.
      • Atarashi K.
      • Kanda H.
      • Sato S.
      • Oyadomari S.
      • et al.
      Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome.
      • Singh V.
      • Yeoh B.S.
      • Chassaing B.
      • Xiao X.
      • Saha P.
      • Aguilera Olvera R.
      • et al.
      Dysregulated microbial fermentation of soluble fiber induces cholestatic liver cancer.
      • Li J.
      • Sung C.Y.
      • Lee N.
      • Ni Y.
      • Pihlajamaki J.
      • Panagiotou G.
      • et al.
      Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice.
      depletion of commensal bacteria was found to exert potent immunomodulatory effects on intrahepatic tumour growth. The gut microbiome was also shown to hijack bile acids as messengers that reduce hepatic NKT cell infiltration and restrict liver antitumour immunity.
      • Ma C.
      • Han M.
      • Heinrich B.
      • Fu Q.
      • Zhang Q.
      • Sandhu M.
      • et al.
      Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells.
      Immune pressure from hepatic NKT cells could also be relieved by mucosal cell-derived exosome-like nanoparticles expressing PGE2
      • Deng Z.B.
      • Zhuang X.
      • Ju S.
      • Xiang X.
      • Mu J.
      • Liu Y.
      • et al.
      Exosome-like nanoparticles from intestinal mucosal cells carry prostaglandin E2 and suppress activation of liver NKT cells.
      (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Context-dependent immunosuppression in HCC. The fibrotic liver favours an immune compromised TIME by directly reducing cytotoxic cell (CD8+ T, NK and NKT) infiltration and providing TGF-β- or IL-6-mediated immunosuppressive signals. In a NASH-afflicted liver, lipid deposition can cause selective loss of CD4+ T cells, while the associated inflammation leads to accumulation of immunosuppressive IgA+ plasma cells that impede T cell immunity through PD-L1 and IL-10 expression. HFD-related microbial products impose a SASP on HSCs, leading to production of PGE2 and inhibition of NK and cytotoxic T cells. Gram-positive bacteria modulate the bile acid spectrum to decrease CXCL16 expression by LSECs and consequently limit CXCR6+ NKT cell function. Besides, hypoxia imposes macrophages with an M2 phenotype for immunosuppression and tumour angiogenesis. HCC, hepatocellular carcinoma; HFD, high-fat diet; HSCs, hepatic stellate cells; IL-, interleukin-; LSECs, liver sinusoidal endothelial cells; NASH, non-alcoholic steatohepatitis; NKT, natural killer T; PGE2, prostaglandin E2; SASP, senescence-associated secretory phenotype; TGF-β, transforming growth factor-β; TIME, tumour immune microenvironment; TAM, tumour-associated macrophages.
      Of further note, the hypoxic microenvironment, frequently present in most solid tumours, activates tumour-extrinsic and -intrinsic mechanisms that negatively regulate anti-HCC immunity. Notably, hypoxia directs TAMs toward an M2-like (pro-angiogenic and immunosuppressive) phenotype in HCC.
      • Zhang J.
      • Zhang Q.
      • Lou Y.
      • Fu Q.
      • Chen Q.
      • Wei T.
      • et al.
      Hypoxia-inducible factor-1alpha/interleukin-1beta signaling enhances hepatoma epithelial-mesenchymal transition through macrophages in a hypoxic-inflammatory microenvironment.
      Meanwhile, hypoxic HCC cells produce more CCL20
      • Ye L.Y.
      • Chen W.
      • Bai X.L.
      • Xu X.Y.
      • Zhang Q.
      • Xia X.F.
      • et al.
      Hypoxia-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma induces an immunosuppressive tumor microenvironment to promote metastasis.
      and/or CCL26,
      • Chiu D.K.
      • Xu I.M.
      • Lai R.K.
      • Tse A.P.
      • Wei L.L.
      • Koh H.Y.
      • et al.
      Hypoxia induces myeloid-derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C-C motif) ligand 26.
      which in turn upregulate IDO expression by TAMs or recruit CX3CR1-expressing MDSCs, respectively.

      Modulation and clinical application of anti-HCC immunity

      Immunology in the clinical setting

      Liver transplantation is often a curative treatment for early-stage HCC. Improved use of immunosuppressive drugs has prevented graft loss and rejection, but adversely increased the risk of HCC recurrence.
      • Khorsandi S.E.
      • Heaton N.
      Optimization of immunosuppressive medication upon liver transplantation against HCC recurrence.
      Previous clinical observations and animal studies demonstrated systemic antitumour immune responses after percutaneous radiofrequency ablation (RFA), which might be relevant to heat shock protein 70 (HSP70) induction and antigen-presenting cell activation.
      • Chu K.F.
      • Dupuy D.E.
      Thermal ablation of tumours: biological mechanisms and advances in therapy.
      Transcatheter arterial chemoembolisation (TACE) has also been suggested to increase tumour immunogenicity by releasing antigens and stimulating intratumoural CTL recruitment.
      • Kudo M.
      Immuno-oncology in hepatocellular carcinoma: 2017 update.
      However, the extent of immune cell infiltration into the microwave-ablated tissue seems to be inversely correlated with clinical outcome.
      • Dong B.W.
      • Zhang J.
      • Liang P.
      • Yu X.L.
      • Su L.
      • Yu D.J.
      • et al.
      Sequential pathological and immunologic analysis of percutaneous microwave coagulation therapy of hepatocellular carcinoma.
      More recently, high-dimensional analysis demonstrated local and systemic immune activation in response to Yttrium-90 (Y90)-radioembolisation (RE).
      • Chew V.
      • Lee Y.H.
      • Pan L.
      • Nasir N.J.M.
      • Lim C.J.
      • Chua C.
      • et al.
      Immune activation underlies a sustained clinical response to Yttrium-90 radioembolisation in hepatocellular carcinoma.
      In both patients and tumour-bearing mice, sorafenib was found to promote intratumoural infiltration with neutrophils, which further recruited macrophages and Tregs to the TIME and led to immune dysfunction and enhanced HCC progression.
      • Zhou S.L.
      • Zhou Z.J.
      • Hu Z.Q.
      • Huang X.W.
      • Wang Z.
      • Chen E.B.
      • et al.
      Tumor-associated neutrophils recruit macrophages and T-regulatory cells to promote progression of hepatocellular carcinoma and resistance to sorafenib.
      Orthotopic HCC models revealed that sorafenib treatment caused tumour hypoxia and PD-L1 overexpression.
      • Chen Y.
      • Ramjiawan R.R.
      • Reiberger T.
      • Ng M.R.
      • Hato T.
      • Huang Y.
      • et al.
      CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice.
      Guadecitabine (SGI-110), a second-generation DNA methyltransferase inhibitor (DNMTi), which is being evaluated for anti-HCC activity, was reported to activate endogenous retrovirus-elicited immune responses.
      • Liu M.
      • Zhang L.
      • Li H.
      • Hinoue T.
      • Zhou W.
      • Ohtani H.
      • et al.
      Integrative epigenetic analysis reveals therapeutic targets to the DNA methyltransferase inhibitor guadecitabine (SGI-110) in hepatocellular carcinoma.
      When testing other epigenetic drugs for anti-cancer effects in vivo, divergent results were obtained in different animal models. Small molecule inhibitors of the histone H3 lysine 27 methyltransferase EZH2 as well as DNMTi enhance CXCL10 production by cancer cells, leading to Th1 trafficking and subcutaneous tumour regression.
      • Peng D.
      • Kryczek I.
      • Nagarsheth N.
      • Zhao L.
      • Wei S.
      • Wang W.
      • et al.
      Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy.
      In an orthotopic mouse model of HCC, drug-induced CXCL10 production was found to cause accumulation of IgG-producing plasma cells, which counterbalanced antitumour immune responses by modulating TAM activity.
      • Wei Y.
      • Lao X.M.
      • Xiao X.
      • Wang X.Y.
      • Wu Z.J.
      • Zeng Q.H.
      • et al.
      Plasma cell polarization to the immunoglobulin G phenotype in hepatocellular carcinomas involves epigenetic alterations and promotes hepatoma progression in mice.

      Preclinical HCC immunotherapy

      Despite recent failures, ICB-based immunotherapy appears to be one of the most promising strategies for HCC treatment. However, response rates rarely exceed 20 to 25% and much effort is needed to identify strategies to overcome ICB resistance and enhance overall response rates. To this end, ICB agents are being tested in combination with other systemic treatments. Given the existence of IDO-mediated adaptive resistance, IDO inhibitors were found to increase the efficacy of ICB in a mouse model of HCC.
      • Brown Z.J.
      • Yu S.J.
      • Heinrich B.
      • Ma C.
      • Fu Q.
      • Sandhu M.
      • et al.
      Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma.
      The multikinase inhibitor lenvatinib was also found to enhance the therapeutic response to PD-1 blockade by changing monocyte-to-T cell ratios.
      • Kimura T.
      • Kato Y.
      • Ozawa Y.
      • Kodama K.
      • Ito J.
      • Ichikawa K.
      • et al.
      Immunomodulatory activity of lenvatinib contributes to antitumor activity in the Hepa1-6 hepatocellular carcinoma model.
      Inhibition of CXCR4 with AMD3100 prevented formation of an immunosuppressive TIME and facilitated anti-PD-1 immunotherapy in sorafenib-treated mouse HCC.
      • Chen Y.
      • Ramjiawan R.R.
      • Reiberger T.
      • Ng M.R.
      • Hato T.
      • Huang Y.
      • et al.
      CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice.
      In a subcutaneous murine HCC model, the histone deacetylase inhibitor (HDACi) belinostat improved the antitumour activity of CTLA-4 and PD-1 directed ICB and led to complete tumour rejection.
      • Llopiz D.
      • Ruiz M.
      • Villanueva L.
      • Iglesias T.
      • Silva L.
      • Egea J.
      • et al.
      Enhanced anti-tumor efficacy of checkpoint inhibitors in combination with the histone deacetylase inhibitor Belinostat in a murine hepatocellular carcinoma model.
      In concert with the ability of poly(IC) treatment to increase intratumoural NK and T cells, incorporating poly(IC) with anti-PD-L1 treatment led to robust immune elimination of mouse liver tumours.
      • Chew V.
      • Tow C.
      • Huang C.
      • Bard-Chapeau E.
      • Copeland N.G.
      • Jenkins N.A.
      • et al.
      Toll-like receptor 3 expressing tumor parenchyma and infiltrating natural killer cells in hepatocellular carcinoma patients.
      • Wen L.
      • Xin B.
      • Wu P.
      • Lin C.H.
      • Peng C.
      • Wang G.
      • et al.
      An Efficient Combination Immunotherapy for Primary Liver Cancer by Harmonized Activation of Innate and Adaptive Immunity in Mice.
      Notably, 2 recent reports highlighted the implications of targeting TAM in reversing the immunosuppressive TIME in HCC and reversing anti-PD-L1 resistance.
      • Zhu Y.
      • Yang J.
      • Xu D.
      • Gao X.M.
      • Zhang Z.
      • Hsu J.L.
      • et al.
      Disruption of tumour-associated macrophage trafficking by the osteopontin-induced colony-stimulating factor-1 signalling sensitises hepatocellular carcinoma to anti-PD-L1 blockade.
      • Wu Q.
      • Zhou W.
      • Yin S.
      • Zhou Y.
      • Chen T.
      • Qian J.
      • et al.
      Blocking triggering receptor expressed on myeloid cells-1-positive tumor-associated macrophages induced by hypoxia reverses immunosuppression and anti-programmed cell death ligand 1 resistance in liver cancer.
      Furthermore, unexplored ICB strategies such as blockade of LAG-3 or its ligand, fibrinogen-like protein 1 (FGL1), have recently been shown to be effective for the treatment of subcutaneously grown mouse HCC.
      • Wang J.
      • Sanmamed M.F.
      • Datar I.
      • Su T.T.
      • Ji L.
      • Sun J.
      • et al.
      Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3.
      In addition to ICB, other immunotherapies have also been studied in preclinical HCC models. Cytokine-induced killer (CIK) cell-based immunotherapy works as an effective adjuvant therapy against early-stage HCC, but becomes ineffective in treating advanced HCC, probably because of MDSC generation. In subcutaneous and orthotopic mouse HCC models, a Food and Drug Administration (FDA)-approved inhibitor of cGMP-specific phosphodiesterase type 5 (PDE5), tadalafil, was used to reverse MDSC suppressive function and maintain CIK-induced antitumour immunity.
      • Yu S.J.
      • Ma C.
      • Heinrich B.
      • Brown Z.J.
      • Sandhu M.
      • Zhang Q.
      • et al.
      Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma.
      Tadalafil may also interfere with the generation of suppressive IgA+ plasma cells through its ability to inhibit TGF-β production.
      • Ammirante M.
      • Shalapour S.
      • Kang Y.
      • Jamieson C.A.
      • Karin M.
      Tissue injury and hypoxia promote malignant progression of prostate cancer by inducing CXCL13 expression in tumor myofibroblasts.
      While immunisation with cell-, epitope- or exosome-based vaccines can prime antigen-specific T cell responses in murine HCC models,
      • Chen C.
      • Hou J.
      • Lin Z.
      • Yao M.
      • Jiang R.
      • Wang Y.
      • et al.
      A bystander cell-based GM-CSF secreting vaccine synergized with a low dose of cyclophosphamide presents therapeutic immune responses against murine hepatocellular carcinoma.
      • Hong Y.
      • Peng Y.
      • Guo Z.S.
      • Guevara-Patino J.
      • Pang J.
      • Butterfield L.H.
      • et al.
      Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.
      • Brinkhoff B.
      • Ostroumov D.
      • Heemcke J.
      • Woller N.
      • Gurlevik E.
      • Manns M.P.
      • et al.
      Microsphere priming facilitates induction of potent therapeutic T-cell immune responses against autochthonous liver cancers.
      • Wu Q.
      • Pi L.
      • Le Trinh T.
      • Zuo C.
      • Xia M.
      • Jiao Y.
      • et al.
      A novel vaccine targeting glypican-3 as a treatment for hepatocellular carcinoma.
      • Lu Z.
      • Zuo B.
      • Jing R.
      • Gao X.
      • Rao Q.
      • Liu Z.
      • et al.
      Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.
      adoptive transfer of chimeric antigen receptor (CAR) T cells targeting ɑ-fetoprotein or glypican-3 (GPC3) also manifested promising therapeutic effects.
      • Dargel C.
      • Bassani-Sternberg M.
      • Hasreiter J.
      • Zani F.
      • Bockmann J.H.
      • Thiele F.
      • et al.
      T cells engineered to express a T-cell receptor specific for glypican-3 to recognize and kill hepatoma cells in vitro and in mice.
      • Liu H.
      • Xu Y.
      • Xiang J.
      • Long L.
      • Green S.
      • Yang Z.
      • et al.
      Targeting alpha-fetoprotein (AFP)-MHC complex with CAR T-cell therapy for liver cancer.
      In addition, a chimeric oncolytic virus vector has been developed as a safe and efficient platform for boosting anti-HCC immunity.
      • Abdullahi S.
      • Jakel M.
      • Behrend S.J.
      • Steiger K.
      • Topping G.
      • Krabbe T.
      • et al.
      A novel chimeric oncolytic virus vector for improved safety and efficacy as a platform for the treatment of hepatocellular carcinoma.
      Interestingly, a recent report showed that nanoliposome-loaded C6-ceramide particles can permeate the fibrotic mouse liver, interfere with TAM-associated immunosuppression and trigger tumoricidal CD8+ T cell function.
      • Li G.
      • Liu D.
      • Kimchi E.T.
      • Kaifi J.T.
      • Qi X.
      • Manjunath Y.
      • et al.
      Nanoliposome C6-ceramide increases the anti-tumor immune response and slows growth of liver tumors in mice.
      Despite such promising preclinical results, further evidence supporting the clinical benefits of these regimens is much needed.
      Future efforts should be dedicated to the identification of combination therapies that will substantially enhance the efficacy of immune-checkpoint inhibitors.

      Trials of HCC immunotherapy

      Tremelimumab, a monoclonal antibody that blocks CTLA-4, was the first ICB drug tested in patients with HCC. In a phase II, non-controlled, multicentre trial, tremelimumab monotherapy had an acceptable safety profile and elicited 17.6% partial responses among 17 evaluable patients, with both antitumoural capacity against HCC and an antiviral effect against HCV.
      • Sangro B.
      • Gomez-Martin C.
      • de la Mata M.
      • Inarrairaegui M.
      • Garralda E.
      • Barrera P.
      • et al.
      A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C.
      In a phase I/II trial (CheckMate 040) involving patients with advanced HCC and poor survival prospects, treatment with nivolumab (anti-PD-1) led to an objective response rate (ORR) of 15–24% and promising survival durations, resulting in accelerated approval by the U.S. FDA.
      • 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.
      A phase III randomised trial has been setup to compare nivolumab with sorafenib as a first-line treatment for advanced HCC (NCT02576509). Recently, Bristol-Myers Squibb announced that this particular trial did not achieve statistical significance for its pre-specified primary endpoint. Still, the results did show a trend towards improved overall survival (OS) for patients treated with nivolumab compared to sorafenib,
      • Green J.E.
      • Choi J.W.
      • Boukai A.
      • Bunimovich Y.
      • Johnston-Halperin E.
      • DeIonno E.
      • et al.
      A 160-kilobit molecular electronic memory patterned at 10(11) bits per square centimetre.
      and even the first nivolumab CheckMate 040 trial did not find any differences in ORR between patients treated with nivolumab plus sorafenib and those treated with nivolumab alone.
      • 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.
      Another PD-1 antibody, pembrolizumab, was also found to be effective and tolerable, producing one complete response and 16 partial responses among 104 patients in a phase II trial, and resulting in FDA approval.
      • Zhu A.X.
      • Finn R.S.
      • Edeline J.
      • Cattan S.
      • Ogasawara S.
      • Palmer D.
      • et al.
      Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial.
      Pembrolizumab has been assessed in 2 randomised, phase III trials as a second-line HCC treatment.
      • Finn R.S.
      • Chan S.L.
      • Zhu A.X.
      • Knox J.J.
      • Cheng A.-L.
      • Siegel A.B.
      • et al.
      Phase 3, randomized study of pembrolizumab (pembro) vs best supportive care (BSC) for second-line advanced hepatocellular carcinoma (HCC): KEYNOTE-240.
      Most recently, however, a pivotal phase III trial of pembrolizumab (KEYNOTE-240) did not meet its co-primary endpoints of OS and progression-free survival (PFS) in advanced HCC (NCT02702401).
      • Finn R.S.
      • Ryoo B.-Y.
      • Merle P.
      • Kudo M.
      • Bouattour M.
      • Lim H.-Y.
      • et al.
      Results of KEYNOTE- 240: phase 3 study of pembrolizumab (Pembro) vs best supportive care (BSC) for second line therapy in advanced hepatocellular carcinoma (HCC).
      Consistent with tumoural PD-L1 levels, durvalumab (anti-PD-L1) monotherapy produced a more promising response rate in HCV-positive than in HBV-positive patients with HCC.
      • Abou-Alfa G.K.
      • Chan S.L.
      • Furuse J.
      • Galle P.R.
      • Kelley R.K.
      • Qin S.
      • et al.
      A randomized, multicenter phase 3 study of durvalumab (D) and tremelimumab (T) as first-line treatment in patients with unresectable hepatocellular carcinoma (HCC): HIMALAYA study.
      Completed or ongoing trials of ICB therapies against HCC are summarised in Table 2.
      Table 2Main clinical trials for immune-checkpoint inhibitors in hepatocellular carcinoma.
      ICBLines of therapyNCT numberStrategyPhasePatientsPopulationPrimary endpoint
      Anti-PD-1/PD-L11
      Nivolumab
      1L02576509Nivolumab vs. sorafenibIII725Advanced HCCOS
      03439891Nivolumab + sorafenibII40Advanced/metastatic HCCORR-1
      03695250Nivolumab + BMS-986205I/II23Advanced HCCORR-2
      01658878Nivolumab vs. nivolumab + ipilimumab vs. sorafenib vs. nivolumab + cabozantinib vs. nivolumab + ipilimumab + cabozantinibI/II620Advanced HCCORR-1
      Adj.03383458Nivolumab vs. placeboIII530HCC after resection or ablationRFS
      Neo.03510871Nivolumab + ipilimumab vs. placeboII40HCCORR-2
      Neo. + adj.03630640Nivolumab vs. placeboII50HCC eligible for electroporationRFS
      Tislelizumab
      1L03412773Tislelizumab vs. sorafenibIII660Unresectable HCCOS
      2L03419897TislelizumaII228Unresectable HCCORR-2
      Pembrolizumab
      1L03713593Lenvatinib + pembrolizumab vs. lenvatinib + placeboIII750Advanced HCCOS
      03347292Regorafenib + pembrolizumabI40Advanced HCCTEAE
      2L02702401Pembrolizumab vs. placeboIII414Previously treated advanced HCCPFS; OS
      03062358Pembrolizumab vs. placeboIII450Asian participants with previously treated advanced HCCOS
      03163992PembrolizumabII60Advanced HCC after failure of sorafenibORR-2
      Adj.03867084Pembrolizumab vs. placeboIII950HCC after surgeryRFS
      Neo. + adj.03337841PembrolizumabII50HCC before curative treatmentRFS
      SHR-1210
      1L03605706SHR-1210 + FOLFOX4 vs. FOLFOX4 or sorafenibIII448Advanced/ metastatic HCCOS
      2L02989922SHR-1210II/III220Advanced HCCOS
      03463876SHR1210 + apatinibII190Advanced HCCORR-2
      Adj.03722875SHR1210 + apatinibNA45HCC after surgeryRFS; OS
      Neo.03793725SHR1210 + apatinibII30Unresectable HCCRR-1
      Spartalizumab
      2L02795429INC280 + PDR001 vs. PDR001Ib/II87Advanced HCCORR-1
      Anti-PD-L1
      Durvalumab
      1L03298451Durvalumab vs. sorafenib vs. durvalumab + tremelimumabIII1,310Advanced HCCOSS
      2L03847428Durvalumab + bevacizumab vs. durvalumab + bevacizumab placebo vs. durvalumab placebo + bevacizumab placeboIII888High risk of recurrence HCC after curative treatmentRFS
      Atezolizumab
      1L03434379Atezolizumab + bevacizumab vs. sorafenibIII480Advanced/metastatic HCCPFS; OS
      03755791Atezolizumab + cabozantinib vs. sorafenib vs. cabozantinibIII640Advanced/metastatic HCCPFS; OS
      Avelumab
      2L03389126AvelumabII30Advanced HCCRR-2
      Anti-CTLA-4
      Tremelimumab
      1L03298451Durvalumab vs. sorafenib vs. durvalumab + tremelimumabIII1,310Advanced HCCOS
      2L01008358TremelimumabII20Advanced HCCRR
      02519348Tremelimumab vs. durvalumab vs. durvalumab + tremelimumab vs. durvalumab + bevacizumabII545Advanced HCCAE
      Ipilimumab
      Neo.03222076Nivolumab vs. nivolumab + ipilimumabII45Resectable HCCAE
      1L, first-line; 2L, second-line; Adj, adjuvant; AE, adverse events; neo, neoadjuvant; n.a., not applicable; ORR-1, overall response rate; ORR-2, objective response rate; OS, overall survival; PFS, progression-free survival; RFS, recurrence-free survival; RR-1, resection rate; RR-2, response rate; TEAE, treatment-emergent adverse event.
      Simultaneous blockade of both PD-1/PD-L1 and CTLA-4 has been proven to be an effective strategy in several advanced solid malignancies.
      • Silva I.P.
      • Long G.V.
      Systemic therapy in advanced melanoma: integrating targeted therapy and immunotherapy into clinical practice.
      According to the CheckMate 040 trial, the nivolumab + ipilimumab combination in sorafenib-treated patients with advanced HCC elicited an average ORR of 31% and an acceptable safety profile (data presented at the 2019 American Society of Clinical Oncology (ASCO) Annual Meeting, NCT01658878).
      • Yau T.
      • Kang Y.-K.
      • Kim T.-Y.
      • El-Khoueiry A.B.
      • Santoro A.
      • Sangro B.
      • et al.
      Nivolumab (NIVO) + ipilimumab (IPI) combination therapy in patients (pts) with advanced hepatocellular carcinoma (aHCC): results from CheckMate 040.
      A phase I/II trial of combination treatment with durvalumab and tremelimumab also achieved an ORR of 25% with a manageable toxicity profile. Of note, the most common grade 3 or greater treatment-related adverse event (AE) was asymptomatic increased aspartate aminotransferase (10%) (NCT02519348).
      • Kelley R.K.
      • Abou-Alfa G.K.
      • Bendell J.C.
      • Kim T.-Y.
      • Borad M.J.
      • Yong W.-P.
      • et al.
      Phase I/II study of durvalumab and tremelimumab in patients with unresectable hepatocellular carcinoma (HCC): phase I safety and efficacy analyses.
      In addition, a global phase III trial (NCT03298451) was designed to compare the efficacy of different regimens (durvalumab monotherapy, durvalumab + tremelimumab combination therapy and sorafenib monotherapy) as first-line treatments for HCC.
      • Abou-Alfa G.K.
      • Chan S.L.
      • Furuse J.
      • Galle P.R.
      • Kelley R.K.
      • Qin S.
      • et al.
      A randomized, multicenter phase 3 study of durvalumab (D) and tremelimumab (T) as first-line treatment in patients with unresectable hepatocellular carcinoma (HCC): HIMALAYA study.
      Since patients with HCC and Child-Pugh class B cirrhosis experienced high rates of AEs with nivolumab monotherapy,
      • Kambhampati S.
      • Bauer K.E.
      • Bracci P.M.
      • Keenan B.P.
      • Behr S.C.
      • Gordan J.D.
      • et al.
      Nivolumab in patients with advanced hepatocellular carcinoma and Child-Pugh class B cirrhosis: safety and clinical outcomes in a retrospective case series.
      extreme attention should be paid to potential side effects of combinational ICB therapy. Similarly, it is vitally important to determine whether ICB therapy aggravates NASH or ASH progression as a side effect.
      In line with an immunological rationale, synergistic effects are expected when immune-checkpoint inhibitors are combined with locoregional treatments. A pilot trial evaluating the cooperation of tremelimumab with local therapy (RFA or TACE) indicated that 26.3% of evaluable patients achieved partial responses outside of the areas treated with ablation or TACE without observed dose-limiting toxicities.
      • Duffy A.G.
      • Ulahannan S.V.
      • Makorova-Rusher O.
      • Rahma O.
      • Wedemeyer H.
      • Pratt D.
      • et al.
      Tremelimumab in combination with ablation in patients with advanced hepatocellular carcinoma.
      A phase II trial of nivolumab + TACE therapy and a different phase I/II trial of pembrolizumab + TACE were initiated to evaluate the safety and efficacy of these combinations. Notably, adjuvant or neoadjuvant immunotherapy accompanied with surgical resection are also being tested in several different clinical trials (Table 2). The combination of ICB therapy with targeted therapeutics, especially anti-angiogenic drugs, appears to be quite promising. A phase I trial of atezolizumab (anti-PD-L1) plus bevacizumab (anti-VEGF) therapy has resulted in durable responses and a tolerable AE profile.
      • Pishvaian M.J.
      • Lee M.S.
      • Ryoo B.-Y.
      • Stein S.
      • Lee K.-H.
      • Liu B.
      • et al.
      LBA26Updated safety and clinical activity results from a phase Ib study of atezolizumab + bevacizumab in hepatocellular carcinoma (HCC).
      Promising response rates and manageable AEs were reported for phase I trials of homologous combination regimens, such as pembrolizumab plus lenvatinib (a multi-VEGFR inhibitor) and SHR-1210 plus apatinib (a VEGFR2 inhibitor), at the 2018 ASCO Annual Meeting.
      • Ikeda M.
      • Sung M.W.
      • Kudo M.
      • Kobayashi M.
      • Baron A.D.
      • Finn R.S.
      • et al.
      A phase 1b trial of lenvatinib (LEN) plus pembrolizumab (PEM) in patients (pts) with unresectable hepatocellular carcinoma (uHCC).
      Most recently, a phase III study has been initiated to evaluate the safety and efficacy of lenvatinib + pembrolizumab vs. lenvatinib + placebo as first-line therapy for advanced HCC (NCT03713593).
      • Llovet J.M.
      • Kudo M.
      • Cheng A.-L.
      • Finn R.S.
      • Galle P.R.
      • Kaneko S.
      • et al.
      Lenvatinib (len) plus pembrolizumab (pembro) for the first-line treatment of patients (pts) with advanced hepatocellular carcinoma (HCC): phase 3 LEAP-002 study.
      Moreover, a global phase III trial (NCT03434379) has been launched to compare the survival outcomes of the atezolizumab + bevacizumab combination along with sorafenib monotherapy as first-line treatments for advanced or metastatic HCC.
      • Finn R.S.
      • Ducreux M.
      • Qin S.
      • Galle P.R.
      • Zhu A.X.
      • Ikeda M.
      • et al.
      IMbrave150: a randomized phase III study of 1L atezolizumab plus bevacizumab vs sorafenib in locally advanced or metastatic hepatocellular carcinoma.
      Several early-stage trials of ICB combined with radiotherapy, chemotherapy or local immunotherapy have been initiated, including: a phase II trial of nivolumab + yttrium-90 (NCT03033446), a phase II trial of durvalumab + tremelimumab + radiotherapy (NCT03482102), a phase II trial of SHR-1210 + FOLFOX4 (NCT03092895), and a phase I/II trial of pembrolizumab + talimogene + laherparepvec (NCT02509507).
      • Hecht J.R.
      • Prat A.
      • Pless M.
      • Cubillo A.
      • Calvo A.
      • Raman S.
      • et al.
      A phase 1b/2, multicenter, open-label trial to evaluate the safety of talimogene laherparepvec (T-VEC) injected into primary and metastatic liver tumors alone and in combination with pembrolizumab (pembro) (MASTERKEY-318).
      Preliminary results from a phase I study evaluating GPC3 CAR-T cells in refractory or relapsed GPC3+ patients showed that these cells slowed HCC progression.
      • Greten T.F.
      • Lai C.W.
      • Li G.
      • Staveley-O’Carroll K.F.
      Targeted and immune-based therapies for hepatocellular carcinoma.
      More phase I trials (NCT02905188, NCT02395250, NCT02723942) are now recruiting patients to evaluate CAR-T cells for the treatment of advanced HCC. Several oncolytic viruses have been tested in clinical trials of patients with HCC, including JX-594, dl1520, H101 (NCT01869088) and VSV-hIFN-β (NCT01628640).
      • Heo J.
      • Breitbach C.J.
      • Moon A.
      • Kim C.W.
      • Patt R.
      • Kim M.K.
      • et al.
      Sequential therapy with JX-594, a targeted oncolytic poxvirus, followed by sorafenib in hepatocellular carcinoma: preclinical and clinical demonstration of combination efficacy.
      • Habib N.A.
      • Mitry R.R.
      • Sarraf C.E.
      • Jiao L.R.
      • Havlik R.
      • Nicholls J.
      • et al.
      Assessment of growth inhibition and morphological changes in in vitro and in vivo hepatocellular carcinoma models post treatment with dl1520 adenovirus.
      JX-594 is the lead oncolytic virus, with a good safety profile and promising response rate in phase I-II trials.
      • Park B.H.
      • Hwang T.
      • Liu T.C.
      • Sze D.Y.
      • Kim J.S.
      • Kwon H.C.
      • et al.
      Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial.
      • Heo J.
      • Reid T.
      • Ruo L.
      • Breitbach C.J.
      • Rose S.
      • Bloomston M.
      • et al.
      Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.
      A recently initiated, open label, phase III trial (NCT02562755) is comparing JX-594 + sorafenib to sorafenib alone.

      Future perspectives

      Despite tremendous progress in applying immunomodulatory strategies to HCC, it appears that currently only a small portion of cancer patients benefit from immunotherapy. Accordingly, we need to improve our understanding of the HCC TIME and discover how to modulate it in order to improve ICB therapeutic outcomes. For each liver-resident cell type, (epi)genomic, transcriptomic and proteomic information needs to be collected and evaluated at the single cell level to obtain a detailed and HCC-specific view of the immune landscape. Especially important is to understand how the HCC genotype affects the balance between pro-tumorigenic inflammation and antitumour immunity. Hopefully, high-resolution and multidimensional immune profiles from different patients stratified according to aetiology and genotype at different disease stages, and before and after treatment, will reveal the actual mechanisms that dictate the evolution of natural and drug-stimulated anti-HCC immunity.
      It is also evident that the immune system plays a multifaceted role in HCC development, either accelerating its progression through pro-tumorigenic inflammation or containing its onset and growth through immunosurveillance. New and improved mouse models are needed for careful functional evaluation of the role played by different cell types and immunomodulatory molecules. Since HCC arises from multiple genetic and epigenetic alterations, multidimensional and longitudinal sequencing in patients with HCC and genetic manipulation in mouse models are needed to fully appreciate the impact of these alterations on the HCC onco-immunity cycle. An improved understanding of the mechanisms responsible for immune dysfunction at the pre-malignant stage will improve our understanding of the negative regulation of HCC immunosurveillance, allowing the oncologist to overcome primary resistance to immunotherapy. Integrating environmental signals with biological processes in immune cells, including epigenetic and metabolic rewiring, will offer new mechanistic perspectives and therapeutic opportunities.
      In the clinical setting, even in cancers with high mutational burdens and T cell infiltration, ICB has led to highly variable responses.
      • Schumacher T.N.
      • Schreiber R.D.
      Neoantigens in cancer immunotherapy.
      This variability and the high current cost of immunotherapy underscore the importance of finding new biomarkers and computational tools to optimise both patient selection and treatment regimens with the goal of improving clinical outcomes and minimising waste. Meanwhile, adjuvant or neoadjuvant immunotherapy schemes together with surgical resection or locoregional interventions are likely to become the pivotal treatment options in the near future. Beyond that, customised immunotherapies, such as neoantigen vaccines and tailor-made cytotoxic lymphocytes, will occupy the next stage of drug development for those patients who are deemed to be non-responsive to standard immunotherapy. The combination of ICB with other molecularly targeted or anti-inflammatory agents should further improve clinical outcome. Some of these combinations should be aimed at dismantling the immunosuppressive TIME, perhaps using inhibitors of TGF-β,
      • Tolcher A.W.
      • Berlin J.D.
      • Cosaert J.
      • Kauh J.
      • Chan E.
      • Piha-Paul S.A.
      • et al.
      A phase 1 study of anti-TGFbeta receptor type-II monoclonal antibody LY3022859 in patients with advanced solid tumors.
      arginase 1(ARG1),
      • Steggerda S.M.
      • Bennett M.K.
      • Chen J.
      • Emberley E.
      • Huang T.
      • Janes J.R.
      • et al.
      Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment.
      PGE2 receptor 4 (EP4),
      • O'Callaghan G.
      • Houston A.
      Prostaglandin E2 and the EP receptors in malignancy: possible therapeutic targets?.
      and adenosine receptors.
      • Vijayan D.
      • Young A.
      • Teng M.W.L.
      • Smyth M.J.
      Targeting immunosuppressive adenosine in cancer.
      We are also curious how current and upcoming anti-NASH therapies
      • Rotman Y.
      • Sanyal A.J.
      Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease.
      and other liver-targeted drugs will affect HCC immunotherapy. Not to be overlooked, proper dietary control and microbiota management may further benefit anti-HCC immunotherapy.
      • Zitvogel L.
      • Ma Y.
      • Raoult D.
      • Kroemer G.
      • Gajewski T.F.
      The microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies.
      Of course, all of these promising avenues will need to be evaluated in a large number of patients using well designed trials.

      Abbreviations

      AE, adverse event; CCRK, cell cycle-related kinase; CIK, cytokine-induced killer; CTL, cytotoxic T lymphocyte; DC, dendritic cell; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; HFD, high-fat diet; HSCs, hepatic stellate cells; ICB, immune-checkpoint blockade; IL-, interleukin-; iNKT, invariant NKT; LSECs, liver sinusoidal endothelial cells; MΦ, macrophage; MAIT, mucosal-associated invariant T cell; MDSC, myeloid-derived suppressor cells; NASH, non-alcoholic steatohepatitis; NK, natural killer; ORR, objective response rate; OS, overall survival; PFS, progression-free survival; PGE2, prostaglandin E2; RFA, radiofrequency ablation; SASP, senescence-associated secretory phenotype; TAM, tumour-associated macrophages; TEAE, treatment-emergent adverse event; TGF-β, transforming growth factor-β; Th, T helper cell; TIME, tumour immune microenvironment; Treg, regulatory T cell.

      Financial support

      This work was supported by grants from the National Key Research and Development Program of China ( 2016YFC0905900 to B.S.); the State Key Program of National Natural Science Foundation ( 81430062 to B.S., 81672801 to J.H. and 81700498 to H.Z.); Innovative Research Team Grant of Jiangsu Province ( 303073227 to B.S. & M.K.), and the National Institutes of Health ( U01AA027681 to M.K.).

      Conflict of interest

      J.H., H.Z. and B.S. declare no conflicts of interest. M.K. had received research support from Merck, USA.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      All authors contributed to the manuscript. J.H. and H.Z. contributed equally.

      Supplementary data

      The following are the Supplementary data to this article:

      References

      Author names in bold designate shared co-first authorship

        • Bray F.
        • Ferlay J.
        • Soerjomataram I.
        • Siegel R.L.
        • Torre L.A.
        • Jemal A.
        Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
        CA Cancer J Clin. 2018; 68: 394-424
        • Nault J.C.
        • Galle P.R.
        • Marquardt J.U.
        The role of molecular enrichment on future therapies in hepatocellular carcinoma.
        J Hepatol. 2018; 69: 237-247
        • Worns M.A.
        • Galle P.R.
        Immune oncology in hepatocellular carcinoma-hype and hope.
        Lancet. 2017; 389: 2448-2449
        • Ma C.
        • Kesarwala A.H.
        • Eggert T.
        • Medina-Echeverz J.
        • Kleiner D.E.
        • Jin P.
        • et al.
        NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
        Nature. 2016; 531: 253-257
        • Shalapour S.
        • Lin X.J.
        • Bastian I.N.
        • Brain J.
        • Burt A.D.
        • Aksenov A.A.
        • et al.
        Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
        Nature. 2017; 551: 340-345
        • Inarrairaegui M.
        • Melero I.
        • Sangro B.
        Immunotherapy of hepatocellular carcinoma: facts and hopes.
        Clin Cancer Res. 2018; 24: 1518-1524
        • 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
        • Zhu A.X.
        • Finn R.S.
        • Edeline J.
        • Cattan S.
        • Ogasawara S.
        • Palmer D.
        • et al.
        Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial.
        Lancet Oncol. 2018; 19: 940-952
        • Green J.E.
        • Choi J.W.
        • Boukai A.
        • Bunimovich Y.
        • Johnston-Halperin E.
        • DeIonno E.
        • et al.
        A 160-kilobit molecular electronic memory patterned at 10(11) bits per square centimetre.
        Nature. 2007; 445: 414-417
        • Finn R.S.
        • Ryoo B.-Y.
        • Merle P.
        • Kudo M.
        • Bouattour M.
        • Lim H.-Y.
        • et al.
        Results of KEYNOTE- 240: phase 3 study of pembrolizumab (Pembro) vs best supportive care (BSC) for second line therapy in advanced hepatocellular carcinoma (HCC).
        J Clin Oncol. 2019; 37 (abstr 4004)
        • Grivennikov S.I.
        • Greten F.R.
        • Karin M.
        Immunity, inflammation, and cancer.
        Cell. 2010; 140: 883-899
        • Shalapour S.
        • Karin M.
        PAS DE DEUX: control of tumor immunity by cancer-associated inflammation.
        Immunity. 2019;
        • Pai C.S.
        • Huang J.T.
        • Lu X.
        • Simons D.M.
        • Park C.
        • Chang A.
        • et al.
        Clonal deletion of tumor-specific T cells by interferon-gamma confers therapeutic resistance to combination immune checkpoint blockade.
        Immunity. 2019; 50 (477–492 e478)
        • Brown Z.J.
        • Heinrich B.
        • Greten T.F.
        Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
        Nat Rev Gastroenterol Hepatol. 2018; 15: 536-554
        • Zheng C.
        • Zheng L.
        • Yoo J.K.
        • Guo H.
        • Zhang Y.
        • Guo X.
        • et al.
        Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.
        Cell. 2017; 169 (1342–1356 e1316)
        • Makarova-Rusher O.V.
        • Medina-Echeverz J.
        • Duffy A.G.
        • Greten T.F.
        The yin and yang of evasion and immune activation in HCC.
        J Hepatol. 2015; 62: 1420-1429
        • 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
        • Zhang J.P.
        • Yan J.
        • Xu J.
        • Pang X.H.
        • Chen M.S.
        • Li L.
        • et al.
        Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients.
        J Hepatol. 2009; 50: 980-989
        • Jiang R.
        • Tan Z.
        • Deng L.
        • Chen Y.
        • Xia Y.
        • Gao Y.
        • et al.
        Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3.
        Hepatology. 2011; 54: 900-909
        • Kuang D.M.
        • Xiao X.
        • Zhao Q.
        • Chen M.M.
        • Li X.F.
        • Liu R.X.
        • et al.
        B7-H1-expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets.
        J Clin Invest. 2014; 124: 4657-4667
        • Chen M.M.
        • Xiao X.
        • Lao X.M.
        • Wei Y.
        • Liu R.X.
        • Zeng Q.H.
        • et al.
        Polarization of tissue-resident TFH-Like cells in human hepatoma bridges innate monocyte inflammation and M2b macrophage polarization.
        Cancer Discov. 2016; 6: 1182-1195
        • Tan H.
        • Wang S.
        • Zhao L.
        A tumour-promoting role of Th9 cells in hepatocellular carcinoma through CCL20 and STAT3 pathways.
        Clin Exp Pharmacol Physiol. 2017; 44: 213-221
        • Li H.
        • Wu K.
        • Tao K.
        • Chen L.
        • Zheng Q.
        • Lu X.
        • et al.
        Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
        Hepatology. 2012; 56: 1342-1351
        • Kalathil S.
        • Lugade A.A.
        • Miller A.
        • Iyer R.
        • Thanavala Y.
        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. 2013; 73: 2435-2444
        • 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; 153 (1107–1119 e1110)
        • Zhu X.D.
        • Zhang J.B.
        • Zhuang P.Y.
        • Zhu H.G.
        • Zhang W.
        • Xiong Y.Q.
        • et al.
        High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.
        J Clin Oncol. 2008; 26: 2707-2716
        • Matsubara T.
        • Kanto T.
        • Kuroda S.
        • Yoshio S.
        • Higashitani K.
        • Kakita N.
        • et al.
        TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.
        Hepatology. 2013; 57: 1416-1425
        • Kuang D.M.
        • Wu Y.
        • Chen N.
        • Cheng J.
        • Zhuang S.M.
        • Zheng L.
        Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes.
        Blood. 2007; 110: 587-595
        • Kuang D.M.
        • Zhao Q.
        • Peng C.
        • Xu J.
        • Zhang J.P.
        • Wu C.
        • et al.
        Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1.
        J Exp Med. 2009; 206: 1327-1337
        • Liu C.Q.
        • Xu J.
        • Zhou Z.G.
        • Jin L.L.
        • Yu X.J.
        • Xiao G.
        • et al.
        Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.
        Br J Cancer. 2018; 119: 80-88
        • Kuang D.M.
        • Peng C.
        • Zhao Q.
        • Wu Y.
        • Zhu L.Y.
        • Wang J.
        • et al.
        Tumor-activated monocytes promote expansion of IL-17-producing CD8+ T cells in hepatocellular carcinoma patients.
        J Immunol. 2010; 185: 1544-1549
        • Grivennikov S.I.
        • Wang K.
        • Mucida D.
        • Stewart C.A.
        • Schnabl B.
        • Jauch D.
        • et al.
        Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.
        Nature. 2012; 491: 254-258
        • Meng F.
        • Wang K.
        • Aoyama T.
        • Grivennikov S.I.
        • Paik Y.
        • Scholten D.
        • et al.
        Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.
        Gastroenterology. 2012; 143e763
        • Gomes A.L.
        • Teijeiro A.
        • Buren S.
        • Tummala K.S.
        • Yilmaz M.
        • Waisman A.
        • et al.
        Metabolic inflammation-associated IL-17A causes non-alcoholic steatohepatitis and hepatocellular carcinoma.
        Cancer Cell. 2016; 30: 161-175
        • Han Y.
        • Chen Z.
        • Yang Y.
        • Jiang Z.
        • Gu Y.
        • Liu Y.
        • et al.
        Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma.
        Hepatology. 2014; 59: 567-579
        • Li Y.W.
        • Qiu S.J.
        • Fan J.
        • Zhou J.
        • Gao Q.
        • Xiao Y.S.
        • et al.
        Intratumoral neutrophils: a poor prognostic factor for hepatocellular carcinoma following resection.
        J Hepatol. 2011; 54: 497-505
        • Kuang D.M.
        • Zhao Q.
        • Wu Y.
        • Peng C.
        • Wang J.
        • Xu Z.
        • et al.
        Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma.
        J Hepatol. 2011; 54: 948-955
        • Hoechst B.
        • Ormandy L.A.
        • Ballmaier M.
        • Lehner F.
        • Kruger C.
        • Manns M.P.
        • et al.
        A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.
        Gastroenterology. 2008; 135: 234-243
        • Kapanadze T.
        • Gamrekelashvili J.
        • Ma C.
        • Chan C.
        • Zhao F.
        • Hewitt S.
        • et al.
        Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma.
        J Hepatol. 2013; 59: 1007-1013
        • Wu Y.
        • Kuang D.M.
        • Pan W.D.
        • Wan Y.L.
        • Lao X.M.
        • Wang D.
        • et al.
        Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions.
        Hepatology. 2013; 57: 1107-1116
        • Bricard G.
        • Cesson V.
        • Devevre E.
        • Bouzourene H.
        • Barbey C.
        • Rufer N.
        • et al.
        Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.
        J Immunol. 2009; 182: 5140-5151
        • Syn W.K.
        • Oo Y.H.
        • Pereira T.A.
        • Karaca G.F.
        • Jung Y.
        • Omenetti A.
        • et al.
        Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
        Hepatology. 2010; 51: 1998-2007
        • Liu R.X.
        • Wei Y.
        • Zeng Q.H.
        • Chan K.W.
        • Xiao X.
        • Zhao X.Y.
        • et al.
        Chemokine (C-X-C motif) receptor 3-positive B cells link interleukin-17 inflammation to protumorigenic macrophage polarization in human hepatocellular carcinoma.
        Hepatology. 2015; 62: 1779-1790
        • Xiao X.
        • Lao X.M.
        • Chen M.M.
        • Liu R.X.
        • Wei Y.
        • Ouyang F.Z.
        • et al.
        PD-1hi identifies a novel regulatory B-cell population in human hepatoma that promotes disease progression.
        Cancer Discov. 2016; 6: 546-559
        • Ouyang F.Z.
        • Wu R.Q.
        • Wei Y.
        • Liu R.X.
        • Yang D.
        • Xiao X.
        • et al.
        Dendritic cell-elicited B-cell activation fosters immune privilege via IL-10 signals in hepatocellular carcinoma.
        Nat Commun. 2016; 7: 13453
        • Garnelo M.
        • Tan A.
        • Her Z.
        • Yeong J.
        • Lim C.J.
        • Chen J.
        • et al.
        Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.
        Gut. 2017; 66: 342-351
        • Duan M.
        • Goswami S.
        • Shi J.Y.
        • Wu L.J.
        • Wang X.Y.
        • Ma J.Q.
        • et al.
        Activated and exhausted MAIT cells foster disease progression and indicate poor outcome in hepatocellular carcinoma.
        Clin Cancer Res. 2019;
        • Ju M.J.
        • Qiu S.J.
        • Gao Q.
        • Fan J.
        • Cai M.Y.
        • Li Y.W.
        • et al.
        Combination of peritumoral mast cells and T-regulatory cells predicts prognosis of hepatocellular carcinoma.
        Cancer Sci. 2009; 100: 1267-1274
        • Ju M.J.
        • Qiu S.J.
        • Fan J.
        • Xiao Y.S.
        • Gao Q.
        • Zhou J.
        • et al.
        Peritumoral activated hepatic stellate cells predict poor clinical outcome in hepatocellular carcinoma after curative resection.
        Am J Clin Pathol. 2009; 131: 498-510
        • Newman A.M.
        • Liu C.L.
        • Green M.R.
        • Gentles A.J.
        • Feng W.
        • Xu Y.
        • et al.
        Robust enumeration of cell subsets from tissue expression profiles.
        Nat Methods. 2015; 12: 453-457
        • Li B.
        • Severson E.
        • Pignon J.C.
        • Zhao H.
        • Li T.
        • Novak J.
        • et al.
        Comprehensive analyses of tumor immunity: implications for cancer immunotherapy.
        Genome Biol. 2016; 17: 174
        • Chen X.
        • Xu C.
        • Hong S.
        • Xia X.
        • Cao Y.
        • McDermott J.
        • et al.
        Immune cell types and secreted factors contributing to inflammation-to-cancer transition and immune therapy response.
        Cell Rep. 2019; 26 (1965–1977 e1964)
        • Kurebayashi Y.
        • Ojima H.
        • Tsujikawa H.
        • Kubota N.
        • Maehara J.
        • Abe Y.
        • et al.
        Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification.
        Hepatology. 2018; 68: 1025-1041
        • Sia D.
        • Jiao Y.
        • Martinez-Quetglas I.
        • Kuchuk O.
        • Villacorta-Martin C.
        • Castro de Moura M.
        • et al.
        Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features.
        Gastroenterology. 2017; 153: 812-826
        • Calderaro J.
        • Rousseau B.
        • Amaddeo G.
        • Mercey M.
        • Charpy C.
        • Costentin C.
        • et al.
        Programmed death ligand 1 expression in hepatocellular carcinoma: relationship with clinical and pathological features.
        Hepatology. 2016; 64: 2038-2046
        • Chew V.
        • Lai L.
        • Pan L.
        • Lim C.J.
        • Li J.
        • Ong R.
        • et al.
        Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses.
        Proc Natl Acad Sci U S A. 2017; 114: E5900-E5909
        • MacParland S.A.
        • Liu J.C.
        • Ma X.Z.
        • Innes B.T.
        • Bartczak A.M.
        • Gage B.K.
        • et al.
        Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.
        Nat Commun. 2018; 9: 4383
        • Krenkel O.
        • Hundertmark J.
        • Abdallah A.T.
        • Kohlhepp M.
        • Puengel T.
        • Roth T.
        • et al.
        Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.
        Gut. 2019; (In Press)
        • Sahin U.
        • Tureci O.
        Personalized vaccines for cancer immunotherapy.
        Science. 2018; 359: 1355-1360
        • Ribas A.
        • Wolchok J.D.
        Cancer immunotherapy using checkpoint blockade.
        Science. 2018; 359: 1350-1355
        • Blankenstein T.
        • Coulie P.G.
        • Gilboa E.
        • Jaffee E.M.
        The determinants of tumour immunogenicity.
        Nat Rev Cancer. 2012; 12: 307-313
        • Yarchoan M.
        • Johnson 3rd, B.A.
        • Lutz E.R.
        • Laheru D.A.
        • Jaffee E.M.
        Targeting neoantigens to augment antitumour immunity.
        Nat Rev Cancer. 2017; 17: 209-222
        • Samstein R.M.
        • Lee C.H.
        • Shoushtari A.N.
        • Hellmann M.D.
        • Shen R.
        • Janjigian Y.Y.
        • et al.
        Tumor mutational load predicts survival after immunotherapy across multiple cancer types.
        Nat Genet. 2019; 51: 202-206
        • Stronen E.
        • Toebes M.
        • Kelderman S.
        • van Buuren M.M.
        • Yang W.
        • van Rooij N.
        • et al.
        Targeting of cancer neoantigens with donor-derived T cell receptor repertoires.
        Science. 2016; 352: 1337-1341
        • Ott P.A.
        • Hu Z.
        • Keskin D.B.
        • Shukla S.A.
        • Sun J.
        • Bozym D.J.
        • et al.
        An immunogenic personal neoantigen vaccine for patients with melanoma.
        Nature. 2017; 547: 217-221
        • Zacharakis N.
        • Chinnasamy H.
        • Black M.
        • Xu H.
        • Lu Y.C.
        • Zheng Z.
        • et al.
        Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.
        Nat Med. 2018; 24: 724-730
        • Keskin D.B.
        • Anandappa A.J.
        • Sun J.
        • Tirosh I.
        • Mathewson N.D.
        • Li S.
        • et al.
        Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.
        Nature. 2019; 565: 234-239
        • Huang J.
        • Deng Q.
        • Wang Q.
        • Li K.Y.
        • Dai J.H.
        • Li N.
        • et al.
        Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.
        Nat Genet. 2012; 44: 1117-1121
        • Schulze K.
        • Imbeaud S.
        • Letouze E.
        • Alexandrov L.B.
        • Calderaro J.
        • Rebouissou S.
        • et al.
        Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
        Nat Genet. 2015; 47: 505-511
        • Cancer Genome Atlas Research Network
        Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
        Cell. 2017; 169 (1327–1341 e1323)
        • Turajlic S.
        • Litchfield K.
        • Xu H.
        • Rosenthal R.
        • McGranahan N.
        • Reading J.L.
        • et al.
        Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis.
        Lancet Oncol. 2017; 18: 1009-1021
        • Schietinger A.
        • Philip M.
        • Krisnawan V.E.
        • Chiu E.Y.
        • Delrow J.J.
        • Basom R.S.
        • et al.
        Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis.
        Immunity. 2016; 45: 389-401
        • Chen Y.P.
        • Zhang Y.
        • Lv J.W.
        • Li Y.Q.
        • Wang Y.Q.
        • He Q.M.
        • et al.
        Genomic analysis of tumor microenvironment immune types across 14 solid cancer types: immunotherapeutic implications.
        Theranostics. 2017; 7: 3585-3594
        • Zhang W.
        • He H.
        • Zang M.
        • Wu Q.
        • Zhao H.
        • Lu L.L.
        • et al.
        Genetic features of aflatoxin-associated hepatocellular carcinoma.
        Gastroenterology. 2017; 153 (249-262 e242)
        • Schumacher T.N.
        • Schreiber R.D.
        Neoantigens in cancer immunotherapy.
        Science. 2015; 348: 69-74
        • Xue R.
        • Li R.
        • Guo H.
        • Guo L.
        • Su Z.
        • Ni X.
        • et al.
        Variable intra-tumor genomic heterogeneity of multiple lesions in patients with hepatocellular carcinoma.
        Gastroenterology. 2016; 150: 998-1008
        • He G.
        • Dhar D.
        • Nakagawa H.
        • Font-Burgada J.
        • Ogata H.
        • Jiang Y.
        • et al.
        Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
        Cell. 2013; 155: 384-396
        • He G.
        • Yu G.Y.
        • Temkin V.
        • Ogata H.
        • Kuntzen C.
        • Sakurai T.
        • et al.
        Hepatocyte IKKbeta/NF-kappaB inhibits tumor promotion and progression by preventing oxidative stress-driven STAT3 activation.
        Cancer Cell. 2010; 17: 286-297
        • Schneider C.
        • Teufel A.
        • Yevsa T.
        • Staib F.
        • Hohmeyer A.
        • Walenda G.
        • et al.
        Adaptive immunity suppresses formation and progression of diethylnitrosamine-induced liver cancer.
        Gut. 2012; 61: 1733-1743
        • Febbraio M.A.
        • Reibe S.
        • Shalapour S.
        • Ooi G.J.
        • Watt M.J.
        • Karin M.
        Preclinical models for studying NASH-driven HCC: how useful are they?.
        Cell Metab. 2019; 29: 18-26
        • Wolf M.J.
        • Adili A.
        • Piotrowitz K.
        • Abdullah Z.
        • Boege Y.
        • Stemmer K.
        • et al.
        Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
        Cancer Cell. 2014; 26: 549-564
        • Endig J.
        • Buitrago-Molina L.E.
        • Marhenke S.
        • Reisinger F.
        • Saborowski A.
        • Schutt J.
        • et al.
        Dual role of the adaptive immune system in liver injury and hepatocellular carcinoma development.
        Cancer Cell. 2016; 30: 308-323
        • Finkin S.
        • Yuan D.
        • Stein I.
        • Taniguchi K.
        • Weber A.
        • Unger K.
        • et al.
        Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.
        Nat Immunol. 2015; 16: 1235-1244
        • Calderaro J.
        • Petitprez F.
        • Becht E.
        • Laurent A.
        • Hirsch T.Z.
        • Rousseau B.
        • et al.
        Intra-tumoral tertiary lymphoid structures are associated with a low risk of early recurrence of hepatocellular carcinoma.
        J Hepatol. 2019; 70: 58-65
        • Chen Y.
        • Hao X.
        • Sun R.
        • Wei H.
        • Tian Z.
        Natural killer cell-derived interferon-gamma promotes hepatocellular carcinoma through the epithelial cell adhesion molecule-epithelial-to-mesenchymal transition axis in hepatitis B virus transgenic mice.
        Hepatology. 2019; 69: 1735-1750
        • Zong L.
        • Peng H.
        • Sun C.
        • Li F.
        • Zheng M.
        • Chen Y.
        • et al.
        Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice.
        Nat Commun. 2019; 10: 221
        • Sakurai T.
        • He G.
        • Matsuzawa A.
        • Yu G.Y.
        • Maeda S.
        • Hardiman G.
        • et al.
        Hepatocyte necrosis induced by oxidative stress and IL-1 alpha release mediate carcinogen-induced compensatory proliferation and liver tumorigenesis.
        Cancer Cell. 2008; 14: 156-165
        • Seehawer M.
        • Heinzmann F.
        • D'Artista L.
        • Harbig J.
        • Roux P.F.
        • Hoenicke L.
        • et al.
        Necroptosis microenvironment directs lineage commitment in liver cancer.
        Nature. 2018; 562: 69-75
        • Nakagawa H.
        • Umemura A.
        • Taniguchi K.
        • Font-Burgada J.
        • Dhar D.
        • Ogata H.
        • et al.
        ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
        Cancer Cell. 2014; 26: 331-343
        • Dapito D.H.
        • Mencin A.
        • Gwak G.Y.
        • Pradere J.P.
        • Jang M.K.
        • Mederacke I.
        • et al.
        Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4.
        Cancer Cell. 2012; 21: 504-516
        • Sears C.L.
        • Pardoll D.M.
        The intestinal microbiome influences checkpoint blockade.
        Nat Med. 2018; 24: 254-255
        • Spranger S.
        • Gajewski T.F.
        Impact of oncogenic pathways on evasion of antitumour immune responses.
        Nat Rev Cancer. 2018; 18: 139-147
        • Xue W.
        • Zender L.
        • Miething C.
        • Dickins R.A.
        • Hernando E.
        • Krizhanovsky V.
        • et al.
        Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas.
        Nature. 2007; 445: 656-660
        • Kang T.W.
        • Yevsa T.
        • Woller N.
        • Hoenicke L.
        • Wuestefeld T.
        • Dauch D.
        • et al.
        Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.
        Nature. 2011; 479: 547-551
        • Pinyol R.
        • Sia D.
        • Llovet J.M.
        Immune exclusion-Wnt/CTNNB1 class predicts resistance to immunotherapies in HCC.
        Clin Cancer Res. 2019; 25: 2021-2023
        • Anson M.
        • Crain-Denoyelle A.M.
        • Baud V.
        • Chereau F.
        • Gougelet A.
        • Terris B.
        • et al.
        Oncogenic beta-catenin triggers an inflammatory response that determines the aggressiveness of hepatocellular carcinoma in mice.
        J Clin Invest. 2012; 122: 586-599
        • Ruiz de Galarreta M
        • Bresnahan E
        • Molina-Sanchez P
        • Lindblad KE
        • Maier B
        • Sia D
        • et al.
        beta-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma.
        Cancer Discov. 2019; 9: 1124-1141
        • Calderaro J.
        • Couchy G.
        • Imbeaud S.
        • Amaddeo G.
        • Letouze E.
        • Blanc J.F.
        • et al.
        Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification.
        J Hepatol. 2017; 67: 727-738
        • Zhou J.
        • Liu M.
        • Sun H.
        • Feng Y.
        • Xu L.
        • Chan A.W.H.
        • et al.
        Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.
        Gut. 2018; 67: 931-944