Immunomodulatory TGF-β Signaling in Hepatocellular Carcinoma
Authors: Jian Chen, Julian A. Gingold, Xiaoping Su
Journal: Trends in Molecular Medicine (2019)

Highly activated TGF-β in hepatocellular carcinoma (HCC) is significantly associated with immune cell exhaustion, while inactivated TGF-β is correlated with impaired DNA repair.The combination of TGF-β inhibitors and immune-checkpoint inhibitors successfully induced complete responses in mouse cancer models, highlighting the key immunosuppressive role of TGF-β signaling in regulating HCC immunotherapy.A novel immune-based classification identified three HCC tumor classes in the tumor microenvironment: the immune class, the intermediate immune class, and the immune exclusion class. Treatment of tumors in each class is likely to require specific immunotherapeutic approaches.Deep single-cell RNA sequencing of tumor-infiltrating lymphocytes identified molecular characteristics in the microenvironment that may guide future therapeutic strategies. Hepatocellular carcinoma (HCC) is an inflammation-induced and chemotherapy-resistant cancer. Dysregulated signaling in the transforming growth factor beta (TGF-β) pathway plays a central role in inflammation, fibrogenesis, and immunomodulation in the HCC microenvironment. This review dissects the genetic landscape of the TGF-β superfamily genes in HCC and discusses the essential effects of this pathway on the tumor immune microenvironment. We highlight the TGF-β signature as a potential biomarker for identifying individualized immunotherapeutic approaches in HCC. An improved understanding of the detailed mechanisms of liver cancer immunogenicity and the specific role of TGF-β in mediating immunotherapy resistance in HCC will provide important insights into HCC immune escape and promote the development of biomarker-derived combination immunotherapies for HCC. Hepatocellular carcinoma (HCC) is an inflammation-induced and chemotherapy-resistant cancer. Dysregulated signaling in the transforming growth factor beta (TGF-β) pathway plays a central role in inflammation, fibrogenesis, and immunomodulation in the HCC microenvironment. This review dissects the genetic landscape of the TGF-β superfamily genes in HCC and discusses the essential effects of this pathway on the tumor immune microenvironment. We highlight the TGF-β signature as a potential biomarker for identifying individualized immunotherapeutic approaches in HCC. An improved understanding of the detailed mechanisms of liver cancer immunogenicity and the specific role of TGF-β in mediating immunotherapy resistance in HCC will provide important insights into HCC immune escape and promote the development of biomarker-derived combination immunotherapies for HCC. HCC is the solid tumor type with the fastest-rising incidence in the USA and is the second-leading cause of cancer-related death worldwide [1Kulik L. El-Serag H.B. Epidemiology and management of hepatocellular carcinoma.Gastroenterology. 2019; 156: 477-491.e1Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 2Llovet J.M. et al.Molecular therapies and precision medicine for hepatocellular carcinoma.Nat. Rev. Clin. Oncol. 2018; 15: 599-616Crossref PubMed Scopus (906) Google Scholar]. Attributable HCC risk factors are highly heterogeneous across patients and regions but include chronic hepatitis C virus (HCV) or hepatitis B virus (HBV), alcoholic cirrhosis, obesity/diabetes, and nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). While HCV can now be cleared in almost all patients using antiviral therapies, patients infected with HBV typically remain infected for life [3European Association for the Study of the Liver EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.J. Hepatol. 2017; 67: 370-398Abstract Full Text Full Text PDF PubMed Scopus (2952) Google Scholar]. Additionally, the dramatic rise in obesity, and with it diabetes, NAFLD/NASH, and chronic liver inflammation, is expected to be the primary driver of increasing HCC incidence globally [4Marrero J.A. et al.Diagnosis, staging, and management of hepatocellular carcinoma: 2018 Practice Guidance by the American Association for the Study of Liver Diseases.Hepatology. 2018; 68: 723-750Crossref PubMed Scopus (2036) Google Scholar]. Regardless of the cause, HCC remains a highly lethal malignancy, particularly for the 40% of affected patients diagnosed with advanced-stage disease. Sorafenib is a multitarget tyrosine kinase inhibitor and was the only systemic therapy with FDA approval for the treatment of advanced HCC prior to 2016 [5Llovet J.M. et al.Sorafenib in advanced hepatocellular carcinoma.N. Engl. J. Med. 2008; 359: 378-390Crossref PubMed Scopus (8967) Google Scholar]. Recently, three new multikinase inhibitors, lenvatinib [6Kudo M. et al.Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised Phase 3 non-inferiority trial.Lancet. 2018; 391: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (2446) Google Scholar], regorafenib [7Bruix J. et al.Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, Phase 3 trial.Lancet. 2017; 389: 56-66Abstract Full Text Full Text PDF PubMed Scopus (2159) Google Scholar], and cabozantinib [8Abou-Alfa G.K. et al.Cabozantinib in patients with advanced and progressing hepatocellular carcinoma.N. Engl. J. Med. 2018; 379: 54-63Crossref PubMed Scopus (1176) Google Scholar], and an antibody-based VEFGR2 antagonist, ramucirumab, have received FDA approval for advanced HCC [9Zhu A.X. et al.Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased alpha-fetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, Phase 3 trial.Lancet Oncol. 2019; 20: 282-296Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar], although the median overall survival for patients treated with them remains under 15 months. Several immune-checkpoint inhibitors (see Glossary) have recently shown promising results in select patients with advanced HCC. Two monoclonal antibodies targeting the programmed cell death 1 (PD-1) pathway, nivolumab [10El-Khoueiry A.B. 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: 2492-2502Abstract Full Text Full Text PDF PubMed Scopus (2467) Google Scholar] and pembrolizumab [11Zhu A.X. 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: 940-952Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar], were recently approved by the FDA for treating patients with advanced-stage HCC previously treated with sorafenib and early-stage results have been reported with the anti-CTLA-4 therapy astremelimumab [12Sangro B. et al.A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C.J. Hepatol. 2013; 59: 81-88Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar]. However, tumor response rates for these immune-checkpoint inhibitors are reported in the 14–17% range. Thus, there remains a large unmet medical need for novel and effective (immuno)therapeutic strategies against HCC [13Greten T.F. et al.Targeted and immune-based therapies for hepatocellular carcinoma.Gastroenterology. 2019; 156: 510-524Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar]. It is thought that the complexity of HCC treatment and its high therapeutic resistance may arise from the multifactorial process that leads to its development, often involving the interaction of two or more risk factors including cirrhosis, NASH, viral infection, and impaired hepatic function. The lack of understanding of the heterogeneous mechanisms of HCC tumorigenesis and progression, the complex interactions of liver tumors with their immune microenvironment, and the lack of validated diagnostic and prognostic biomarkers have markedly hindered the development of clinically validated agents against HCC. In this review, we provide an overview of the immune landscape in HCC with a particular focus on signaling from TGF-β, a master immune regulator. We review the interaction of the TGF-β pathway with the HCC tumor immune microenvironment and discuss the TGF-β signature as a potential biomarker for identifying the 'exhausted' immune signature in HCC. Finally, we discuss opportunities and challenges for combination therapy for HCC targeting both TGF-β and immune checkpoints. The TGF-β superfamily of ligands includes TGF-βs, bone morphogenetic proteins (BMPs), inhibins, and nodal and growth and differentiation factors (activins), the receptors of which activate SMAD-dependent and SMAD-independent pathways to regulate transcriptional/translational cascades (Box 1). The TGF-β pathway plays a context-dependent role in cell processes, including promotion of cell differentiation and proliferation, control of cell apoptosis and cell cycle, regulation of the epithelial–mesenchymal transition (EMT), angiogenesis, extracellular matrix (ECM) formation, suppression of immune response, and maintenance of genomic stability and stem cell homeostasis. In addition, TGF-β signaling acts as a master regulator for immune cell proliferation, differentiation, development, and survival [14Li M.O. Flavell R.A. TGF-β: a master of all T cell trades.Cell. 2008; 134: 392-404Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 15Chen W. Ten Dijke P. Immunoregulation by members of the TGFβ superfamily.Nat. Rev. Immunol. 2016; 16: 723-740Crossref PubMed Scopus (224) Google Scholar].Box 1The TGF-β Superfamily in HCCTGF-β signal transduction is one of the major signaling pathways regulating cell homeostasis and embryo development [76Massague J. TGFβ signalling in context.Nat. Rev. Mol. Cell Biol. 2012; 13: 616-630Crossref PubMed Scopus (2142) Google Scholar, 77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar]. The TGF-β pathway plays a complex role in liver disease and liver cancer, on which it exerts fibrogenic/proinflammatory, tumor-suppressive, and/or prometastatic effects [78Principe D.R. et al.TGF-β: duality of function between tumor prevention and carcinogenesis.J. Natl. Cancer Inst. 2014; 106: djt369Crossref PubMed Scopus (349) Google Scholar]. Clinically, high levels of TGF-β are associated with a favorable prognosis in early-stage cancers but are associated with increased tumor invasiveness and dedifferentiation in advanced tumors, suggesting that TGF-β initially restrains liver cancers through its tumor-suppressor functions but may later exacerbate the malignancy through its pro-oncogenic activities [17Chen J. et al.Analysis of genomes and transcriptomes of hepatocellular carcinomas identifies mutations and gene expression changes in the transforming growth factor-beta pathway.Gastroenterology. 2018; 154: 195-210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Majumdar A. et al.Hepatic stem cells and transforming growth factor beta in hepatocellular carcinoma.Nat. Rev. Gastroenterol. Hepatol. 2012; 9: 530-538Crossref PubMed Scopus (119) Google Scholar].The TGF-β superfamily proteins mainly include TGF-βs, activins, BMPs, growth and differentiation factors, and cofactors/adaptors. The ligands, TGF-βs, activins, and BMPs act as dimers to bind with cell surface type 1 and type 2 receptors, leading to the activation of gene responses by SMAD-dependent and SMAD-independent cascades (Table S1, Table S2 in the supplemental information online). The details of the canonical SMAD-dependent pathway and noncanonical SMAD-independent pathway were reviewed recently [44Zhang Y.E. Non-Smad signaling pathways of the TGF-β family.Cold Spring Harb. Perspect. Biol. 2017; 9a022129Crossref PubMed Scopus (381) Google Scholar, 77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar].The complex set of interactions between these signaling pathways imposes significant challenges for attempts at cancer therapeutics targeting the TGF-β superfamily. It remains unknown whether blocking the canonical SMAD-dependent pathway will lead to a reduction or an increase in signaling via the noncanonical SMAD-independent pathway. Likewise, it is difficult to anticipate the effects of targeted inhibitors against crosstalk pathways on TGF-β signaling. The development of comprehensive, dynamic, and individualized genomic analyses will be essential to define the activities of TGF-β signaling for use in future HCC therapeutics. TGF-β signal transduction is one of the major signaling pathways regulating cell homeostasis and embryo development [76Massague J. TGFβ signalling in context.Nat. Rev. Mol. Cell Biol. 2012; 13: 616-630Crossref PubMed Scopus (2142) Google Scholar, 77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar]. The TGF-β pathway plays a complex role in liver disease and liver cancer, on which it exerts fibrogenic/proinflammatory, tumor-suppressive, and/or prometastatic effects [78Principe D.R. et al.TGF-β: duality of function between tumor prevention and carcinogenesis.J. Natl. Cancer Inst. 2014; 106: djt369Crossref PubMed Scopus (349) Google Scholar]. Clinically, high levels of TGF-β are associated with a favorable prognosis in early-stage cancers but are associated with increased tumor invasiveness and dedifferentiation in advanced tumors, suggesting that TGF-β initially restrains liver cancers through its tumor-suppressor functions but may later exacerbate the malignancy through its pro-oncogenic activities [17Chen J. et al.Analysis of genomes and transcriptomes of hepatocellular carcinomas identifies mutations and gene expression changes in the transforming growth factor-beta pathway.Gastroenterology. 2018; 154: 195-210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Majumdar A. et al.Hepatic stem cells and transforming growth factor beta in hepatocellular carcinoma.Nat. Rev. Gastroenterol. Hepatol. 2012; 9: 530-538Crossref PubMed Scopus (119) Google Scholar]. The TGF-β superfamily proteins mainly include TGF-βs, activins, BMPs, growth and differentiation factors, and cofactors/adaptors. The ligands, TGF-βs, activins, and BMPs act as dimers to bind with cell surface type 1 and type 2 receptors, leading to the activation of gene responses by SMAD-dependent and SMAD-independent cascades (Table S1, Table S2 in the supplemental information online). The details of the canonical SMAD-dependent pathway and noncanonical SMAD-independent pathway were reviewed recently [44Zhang Y.E. Non-Smad signaling pathways of the TGF-β family.Cold Spring Harb. Perspect. Biol. 2017; 9a022129Crossref PubMed Scopus (381) Google Scholar, 77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar]. The complex set of interactions between these signaling pathways imposes significant challenges for attempts at cancer therapeutics targeting the TGF-β superfamily. It remains unknown whether blocking the canonical SMAD-dependent pathway will lead to a reduction or an increase in signaling via the noncanonical SMAD-independent pathway. Likewise, it is difficult to anticipate the effects of targeted inhibitors against crosstalk pathways on TGF-β signaling. The development of comprehensive, dynamic, and individualized genomic analyses will be essential to define the activities of TGF-β signaling for use in future HCC therapeutics. The liver is the largest peripheral immunomodulatory organ and is filled with a multitude of innate and adaptive immune cells, including the largest population of resident macrophages (Kupffer cells) and a high density of natural killer (NK) cells, NK T (NKT) cells, and liver-transiting and/or -resident CD8+ T cells and CD4+ T cells [16Jenne C.N. Kubes P. Immune surveillance by the liver.Nat. Immunol. 2013; 14: 996-1006Crossref PubMed Scopus (644) Google Scholar]. TGF-β critically regulates immune cells in the liver to maintain a balance between immune tolerance and activation. TGF-β plays a central role in regulating immune cells including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), NK cells, and dendritic cells (DCs) (Box 2 and Figure 1).Box 2Regulation of TGF-β in Immune CellsTGF-β activates SMAD2/3 and cooperates with IL-21 activity to promote naïve CD4+ T cell generation of Th17 cells, a subtype of proinflammatory Th cells that expresses IL-17 and contributes to NAFLD-associated liver inflammation and development of HCC [43Gomes A.L. et al.Metabolic inflammation-associated IL-17A causes non-alcoholic steatohepatitis and hepatocellular carcinoma.Cancer Cell. 2016; 30: 161-175Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 79Martinez G.J. et al.Smad2 positively regulates the generation of Th17 cells.J. Biol. Chem. 2010; 285: 29039-29043Crossref PubMed Scopus (73) Google Scholar]. TGF-β also inhibits Th1 and Th2 lineages by downregulating T-bet and Sox4 expression, respectively, and probably by promoting a shift of Th1 towards Th2 cell differentiation [77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar, 80Flavell R.A. et al.The polarization of immune cells in the tumour environment by TGFβ.Nat. Rev. Immunol. 2010; 10: 554-567Crossref PubMed Scopus (667) Google Scholar]. In CD8+ cytotoxic T cells, TGF-β cooperates with the transcription factor ATF1 to suppress the expression of IFN-γ to inhibit its antitumor activity [77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar]. Intratumoral TGF-β suppresses NKT cells, a population responsible for recruiting effector immune cells to the tumor through the production of large amounts of IFN-γ.During B cell maturation, TGF-β cooperates with runt-related transcription factor 3 (RUNX3) to promote naïve B cell class switching to IgA-producing (IgA+) cells, which are involved in NAFLD-associated HCC, by coexpression of PD-L1 and IL-10 and by suppressing liver cytotoxic CD8+ T lymphocytes [81Shalapour S. et al.Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.Nature. 2017; 551: 340-345Crossref PubMed Scopus (267) Google Scholar]. TGF-β promotes the differentiation of M2-type macrophages, which suppress the activity of CD8+ T cells, NK cells, and DCs and increase the activity of CD4+ Treg cells [82de Gramont A. et al.Novel TGF-β inhibitors ready for prime time in onco-immunology.Oncoimmunology. 2017; 6e1257453Crossref PubMed Scopus (141) Google Scholar].Although TGF-β promotes chemotaxis in eosinophils and mast cells, it inhibits cytotoxicity and cytokine production in NK cells and inhibits antigen presentation on DCs [15Chen W. Ten Dijke P. Immunoregulation by members of the TGFβ superfamily.Nat. Rev. Immunol. 2016; 16: 723-740Crossref PubMed Scopus (224) Google Scholar]. TGF-β1 enhances antigen-induced PD-1 expression through SMAD3-dependent transcriptional activation in antigen-specific T cells, suggesting that the TGF-β pathway directly participates in immune-checkpoint regulation [29Park B.V. et al.TGFβ1-mediated SMAD3 enhances PD-1 expression on antigen-specific T cells in cancer.Cancer Discov. 2016; 6: 1366-1381Crossref PubMed Scopus (160) Google Scholar]. TGF-β activates SMAD2/3 and cooperates with IL-21 activity to promote naïve CD4+ T cell generation of Th17 cells, a subtype of proinflammatory Th cells that expresses IL-17 and contributes to NAFLD-associated liver inflammation and development of HCC [43Gomes A.L. et al.Metabolic inflammation-associated IL-17A causes non-alcoholic steatohepatitis and hepatocellular carcinoma.Cancer Cell. 2016; 30: 161-175Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 79Martinez G.J. et al.Smad2 positively regulates the generation of Th17 cells.J. Biol. Chem. 2010; 285: 29039-29043Crossref PubMed Scopus (73) Google Scholar]. TGF-β also inhibits Th1 and Th2 lineages by downregulating T-bet and Sox4 expression, respectively, and probably by promoting a shift of Th1 towards Th2 cell differentiation [77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar, 80Flavell R.A. et al.The polarization of immune cells in the tumour environment by TGFβ.Nat. Rev. Immunol. 2010; 10: 554-567Crossref PubMed Scopus (667) Google Scholar]. In CD8+ cytotoxic T cells, TGF-β cooperates with the transcription factor ATF1 to suppress the expression of IFN-γ to inhibit its antitumor activity [77David C.J. Massague J. Contextual determinants of TGFβ action in development, immunity and cancer.Nat. Rev. Mol. Cell Biol. 2018; 19: 419-435Crossref PubMed Scopus (411) Google Scholar]. Intratumoral TGF-β suppresses NKT cells, a population responsible for recruiting effector immune cells to the tumor through the production of large amounts of IFN-γ. During B cell maturation, TGF-β cooperates with runt-related transcription factor 3 (RUNX3) to promote naïve B cell class switching to IgA-producing (IgA+) cells, which are involved in NAFLD-associated HCC, by coexpression of PD-L1 and IL-10 and by suppressing liver cytotoxic CD8+ T lymphocytes [81Shalapour S. et al.Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.Nature. 2017; 551: 340-345Crossref PubMed Scopus (267) Google Scholar]. TGF-β promotes the differentiation of M2-type macrophages, which suppress the activity of CD8+ T cells, NK cells, and DCs and increase the activity of CD4+ Treg cells [82de Gramont A. et al.Novel TGF-β inhibitors ready for prime time in onco-immunology.Oncoimmunology. 2017; 6e1257453Crossref PubMed Scopus (141) Google Scholar]. Although TGF-β promotes chemotaxis in eosinophils and mast cells, it inhibits cytotoxicity and cytokine production in NK cells and inhibits antigen presentation on DCs [15Chen W. Ten Dijke P. Immunoregulation by members of the TGFβ superfamily.Nat. Rev. Immunol. 2016; 16: 723-740Crossref PubMed Scopus (224) Google Scholar]. TGF-β1 enhances antigen-induced PD-1 expression through SMAD3-dependent transcriptional activation in antigen-specific T cells, suggesting that the TGF-β pathway directly participates in immune-checkpoint regulation [29Park B.V. et al.TGFβ1-mediated SMAD3 enhances PD-1 expression on antigen-specific T cells in cancer.Cancer Discov. 2016; 6: 1366-1381Crossref PubMed Scopus (160) Google Scholar]. Chronic inflammation plays a well-established role in the development of HCC, often in association with liver fibrosis and cirrhosis. The activity of TGF-β has been established as essential for HCC pathogenesis, including the activation of cancer-associated fibroblasts (CAFs) [17Chen J. et al.Analysis of genomes and transcriptomes of hepatocellular carcinomas identifies mutations and gene expression changes in the transforming growth factor-beta pathway.Gastroenterology. 2018; 154: 195-210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Achyut B.R. Yang L. Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment.Gastroenterology. 2011; 141: 1167-1178Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 19Majumdar A. et al.Hepatic stem cells and transforming growth factor beta in hepatocellular carcinoma.Nat. Rev. Gastroenterol. Hepatol. 2012; 9: 530-538Crossref PubMed Scopus (119) Google Scholar]. Liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) are major producers of TGF-β, which contribute to hepatic regulatory T (Treg) cell induction [20Carambia A. et al.TGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells.J. Hepatol. 2014; 61: 594-599Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 21Zaiss D.M. et al.Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.Immunity. 2013; 38: 275-284Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar]. These data suggest an immunosuppressive role for TGF-β in HCC and imply that targeting the TGF-β pathway in hepatic immune cells might enhance antitumor immunity in HCC patients. Although current HCC immunotherapies modulate immune effector cell functions rather than the tumor immune microenvironment, clinical and preclinical data highlight that the highly immunosuppressive tumor environment in advanced HCC contributes to impaired effector T cell recruitment and immunotherapy response [22Prieto J. et al.Immunological landscape and immunotherapy of hepatocellular carcinoma.Nat. Rev. Gastroenterol. Hepatol. 2015; 12: 681-700Crossref PubMed Scopus (359) Google Scholar]. The inflammation and tumor microenvironment that are regulated by TGF-β signaling are therefore critical to HCC progression. The potent immune inhibitory function of Treg cells is a major obstacle to generating an effective antitumor response in HCC [23Ringelhan M. et al.The immunology of hepatocellular carcinoma.Nat. Immunol. 2018; 19: 222-232Crossref PubMed Scopus (486) Google Scholar]. Tumor cells and all stromal cells, including TAMs, HSCs, LSECs, and MDSCs, modulate Treg cell activity in HCC via the TGF-β pathway (Figure 1, top center). The Smad2/3-dependent TGF-β pathway upregulates the transcription factor FoxP3, which is typically expressed on Treg cells [24Chen W. et al.Conversion of peripheral CD4+CD25- naïve T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3.J. Exp. Med. 2003; 198: 1875-1886Crossref PubMed Scopus (3797) Google Scholar]. HCC patients demonstrate high levels of FoxP3+ Treg cells in peripheral blood and markedly increased tumor-infiltrating Treg cells [25Chen K.J. et al.Selective recruitment of regulatory T cell through CCR6–CCL20 in hepatocellular carcinoma fosters tumor progression and predicts poor prognosis.PLoS One. 2011; 6e24671Crossref PubMed Scopus (175) Google Scholar]. Treg CD4+ cells also secrete TGF-β, as well as adenosine and IL-10, to suppress effector T cells such as CD8+ cytotoxic T lymphocytes (CTLs) [22Prieto J. et al.Immunological landscape and immunotherapy of hepatocellular carcinoma.Nat. Rev. Gastroenterol. Hepatol. 2015; 12: 681-700Crossref PubMed Scopus (359) Google Scholar], which are the main subset of tumor-infiltrating lymphocytes in HCC responsible for secreting antitumor effector molecules such as interferon-gamma (IFN-γ), IL-2, and TNFα [26Flecken T. et al.Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma.Hepatology. 2014; 59: 1415-1426Crossref PubMed Scopus (229) Google Scholar]. Treg cells also inhibit immune responses through their membrane-bound CTLA-4 immune-checkpoint molecules to compete with costimulatory CD28, which interacts with the CD80/CD86 complex on antigen-presenting cells (APCs), to facilitate downregulation of the CD80/CD86 complex via CTLA-4-mediated transendocytosis [27Josefowicz S.Z. et al.Regulatory T cells: mechanisms of differentiation and function.Annu. Rev. Immunol. 2012; 30: 531-564Crossref PubMed Scopus (1961) Google Scholar, 28Sharma P. Allison J.P. The future of immune checkpoint therapy.Science. 2015; 348: 56-61Crossref PubMed Scopus (3023) Google Scholar]. Therefore, targeting Treg cells – for example, by blocking the TGF-β pathway or the membrane-bound receptor CTLA-4 – may be a particularly effective immune-based approach for improving the immune response against HCC. TGF-β secreted by tumor cells directly upregulates the transcriptional expression of PD-1 in cancer cells (Figure 1, bottom center) [29Park B.V. et al.TGFβ1-mediated SMAD3 enhances PD-1 expression on antigen-specific T cells in cancer.Cancer Discov. 2016; 6: 1366-1381Crossref PubMed Scopus (160) Google Scholar]. PD-1 is expressed by activated CD8+ cells and binds with its ligands, programmed death ligand 1 and 2 (PD-L1/2), on APCs or tumor cells. The interaction of PD-1 and PD-L1 is well known as one of major immunosuppressive mechanisms exploited by virally induced cancers. PD-1 engagement by PD-L1 blocks T cell antigen receptor (TCR) signaling and inhibits T cell proliferation and secretion of cytotoxic mediators, including Granzyme A (GZMA), Granzyme B (GZMB), TNFα, and IFN-γ, which collectively lead to T cell exhaustion (Figure 1, upper-right corner) [30Barber D.L. et al.Restoring function in exhausted CD8 T cells during chronic viral infection.Nature. 2006; 439: 682-687Crossref PubMed Scopus (3009) Google Scholar, 31Zheng C. et al.Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing.Cell. 2017; 169: 1342-1356.e16Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar]. Thus, increased TGF-β signaling may cause T cell exhaustion by upregulation of PD-1 signaling and inhibition of TGF-β signaling may directly enhance antitumor immunity in HCC [29Park B.V. et al.TGFβ1-mediated SMAD3 enhances PD-1 expression on antigen-specific T cells in cancer.Cancer Discov. 2016; 6: 1366-1381Crossref PubMed Scopus (160) Google Scholar]. Genomic alterations in the TGF-β signaling pathway, inc