Introduction

Primary liver cancer is the second leading cause of cancer mortality and the fifth most common cancer worldwide. HCC is the most well-studied subtype and accounts for 85–90% of all primary liver cancers. Most HCCs are associated with a cirrhotic liver, with the primary cause being a chronic hepatitis B (HBV) or C (HCV) virus infection, followed by other etiologies, including alcohol consumption and fatty liver disease associated with metabolic syndrome [1]. In particular, alcoholic cirrhosis may become a leading cause of HCC in the future since it occupies approximately 47% of liver cirrhosis worldwide. Approximately 383,000 individuals die from liver cancer each year in China, which accounts for 51% of the deaths caused by liver cancer worldwide. In addition, as many as 80% of HCC cases in China are attributed to chronic HBV infection [2]. While patients in the early stages of the disease have a relatively good prognosis with a 5-year survival greater than 70%, the majority of HCC patients are diagnosed with late stage disease resulting in an overall 5-year survival rate less than 16% [3]. Immunological pathogenesis has raised additional concerns in HCC and immune therapy has gradually become a potentially powerful treatment for such advanced HCC patients.

Traditional and other therapeutic methods (Fig. 1)

According to the treatment guidelines, surgical resection, orthotopic liver transplantation, and percutaneous ablation can only be applied to less than 30% of early stage HCC patients [4]; however, most HCC patients are usually too late to see doctors when they are at an advanced status due to difficulties in making an early diagnosis. The molecularly targeting drug, sorafenib, a multiple tyrosine kinase inhibitor, was the first systemic agent approved by the FDA associated with the first-line treatment of patients with unresectable HCC [5]. Lenvatinib, an oral tyrosine kinase inhibitor, subsequently received FDA approval for the treatment of chemotherapy-naive HCC patients in July 2018 as another first-line treatment strategy [6]. In 2017, regorafenib, an oral kinase inhibitor that targets multiple protein kinases was approved by the FDA as a second-line treatment for advanced HCC after failing to respond to or tolerate sorafenib [7]. Other treatment regimens, including gene therapy, cytotoxic chemotherapy, radiotherapy, hormonal therapy, and Chinese herbal therapy [8] remain unsatisfactory for the treatment of HCC. Recently, immune therapy has been considered a potentially powerful treatment for such advanced HCC patients.

Fig. 1
figure 1

From the recommended guidelines to ongoing novel immunotherapy methods for advanced HCC patients. By following the recommended guidelines, less than 30% of HCC patients benefit from surgical resection, orthotopic liver transplantation, or percutaneous ablation. Other treatment regimens, including cytotoxic chemotherapy, radiotherapy, hormonal therapy, and herbal therapy remain unsatisfactory treatments for the disease. Recently, immune therapy has been considered to be a potentially powerful treatment for advanced HCC patients

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Immune disorder in HCC: rationale for immunotherapy (Fig. 2)

The liver is the largest immune organ in human body; under physiologic conditions, it plays a protective role by promoting immunotolerance [9]. However, the exacerbation of immunotolerant signals or escaping from immunosurveillance, inevitably leads to the development of HCC [10]. Therefore, immunotherapies appear to be an appropriate method of activating latent anticancer immunity.

Fig. 2
figure 2

Immune disorder in HCC. Escaping from immunosurveillance, such as changes in the number and/or function of immune cells (innate and adaptive immunity), cytokine/chemokine levels, the expression of inhibitory receptors or their ligands and microRNA inevitably leads to the development of HCC

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Innate and adaptive immune cells

The immune system can be divided into innate and adaptive immune responses, which can identify and destroy nascent tumor cells in a process termed cancer immune surveillance, which functions as an important defense against cancer; however, the immunosuppressive cancer environment (including HCC) substantially impacts the frequency and function of innate and adaptive immune cells, which finally lead to progression and metastasis in cancer patients. Since some reviews [11, 12] have already summarized the relationship between innate and adaptive immune cells and HCC, this review does not repeat such discussion here.

Cytokines and chemokines

Cytokines comprise an extremely important part of the immune system as a response to foreign pathogens, which can regulate the growth, differentiation, and activation of immune cells. Since previous reviews provide a comprehensive description regarding the relationship between cytokines and hepatocarcinogenesis [13], we only mention cytokines that have been in the spotlight within the past 2 years. Transforming growth factor beta (TGF-β) can cause a migratory stemness phenotype, and a higher expression of TGF-β is predictive of poor disease prognosis in HCC patients [14]. Interleukin (IL)-6 could also enhance cancer stemness and promote HCC metastasis [15]. IL-22 plays a dual role shifting from hepatoprotective to carcinogenic functionality [16]. Other cytokines, such as IL-15, IL-35, and IL-18 have also been reportedly connected with the outcome of HCC patients [17].

Chemokines and their receptors have also received increased attention. Previous studies have reported higher levels of expression and a significant correlation between CXCL12–CXCR4 expression and tumor progression, metastasis, and a decreased survival rate in HCC patients [18]. A recent study reported that both CXCL12 and CXCR4 polymorphisms are associated with increased susceptibility to HCC development [19]. Other chemokine axes, including CXCL8 [interleukin 8 or chemokine (C-X-C motif) ligand 9], XCR1, CCR1, CXCL5, CXCR3, CCL3, CX3CL1 and CCL20 have also been reportedly involved in the development of HCC [20].

Immune checkpoint molecules

Programmed cell death protein-1(PD-1)/PD-1 ligand (PD-L1) The first protein identified as an immune checkpoint molecule was PD-1, which was discovered in 1992 by Tasuku Honjo [21]. In 2009, Gao et al. [22] first found that PD-L1 overexpression was significantly associated with tumor aggressiveness and postoperative recurrence in HCC patients. In 2011, Shi [23] found that the upregulation of PD-1 and PD-L1 promoted CD8(+) T-cell apoptosis and postoperative recurrence in HCC patients. Thereafter, the impaired function of CD8+ T cells, CD4+ T cells, and myeloid-derived suppressor cells (MDSCs)/macrophages in HCC patients were also reported to correlate with PD-1/PD-L1 expression [24]. In addition, several clinical studies have found that the over-expression of PD-1/PD-L, both in the circulation [25] and the liver tissue [26], are associated with a poor prognosis in HCC patients.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) The relationship between CTLA-4 and HCC was first noted by its genetic susceptibility [27]. Further studies subsequently showed that tumor-derived Treg cells could inhibit DC function by CTLA-4 in HCC patients [28]. More recent studies have found that effector Tregs and CD8+ T cells strongly express CTLA-4 in NASH-related HCC, whereas PD-1 was only highly expressed in CD8+ T cells in HBV-related HCC patients [29].

T-cell immunoglobulin and mucin domain-3 (TIM-3) In 2002, TIM-3 was first identified as a molecule selectively expressed on IFN-γ-producing CD4+ T helper 1 (Th1) and CD8+ T cytotoxic 1 (Tc1) cells [30]. In 2012, Li et al. [31] were the first to report that the TIM-3/galectin-9 signaling pathway mediates T-cell dysfunction and could predict a poor prognosis in patients with hepatitis B virus-associated HCC (HBV-HCC) patients. In 2015, Yan et al. [32] found that TIM-3 expression was significantly increased in both peripheral blood monocytes and tumor-associated macrophages (TAMs) in patients with HCC, which was strongly correlated with higher tumor grades and the poor survival of patients with HCC. In 2018, Li et al. [33] found that highly elevated levels of soluble TIM-3 correlated with an increased HCC risk and poor survival of HBV-HCC patients.

MicroRNAs (miRNAs)

With the exception of functioning as oncogenes or tumor suppressors genes, miRNAs also play an immune modulatory role during HCC progression [34]. MiR-615-5p, miR-889 and miR-146a have been found to negatively regulate NK cell functions [35, 36], whereas miR-152 and miR-182 play the opposite roles for NK cells in HCC [37, 38]. MiR-214, miR-28-5p, and miR-98 regulate macrophage polarization, which was correlated with tumor metastasis, recurrence, and poor survival in HCC patients [39, 40]. In addition, miR-34a, hsa-miR-182-5p, hsa-miR-214-3p, and miR-125b have all been reported to regulate Treg cell function in HCC patients [41]. Moreover, miR-197 and miR-451 were reported to target the IL-6/STAT3 inflammatory signaling pathway [42, 43], while miR-449 and miR-542-3p targeted the TGF-β/Smad signaling pathway [44, 45], all of which were involved in the development of HCC.

Cancer immune subsets

The systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancer. Recently, Thorsson and his colleagues performed an extensive analysis of 10,000 tumors comprising 33 diverse cancer types, and revealed six immune subsets associated with various cancers: wound healing; IFN-γ-dominant; inflammatory; lymphocyte-depleted; immunologically quiet and TGF-β-dominant [46], while the majority of HCCs are characterized as a lymphocyte-depleted immune subset. Together, these data provide a resource for understanding immune–tumor interactions, with implications for identifying methods of advancing research on immunotherapy.

Current immunotherapeutic methods for HCC patients under clinical trial (Fig. 3)

Immune-based approaches focused on tumor vaccination, cytokines, non-specific T-cell activation, or adoptive cell transfer (e.g., DC and NKT) have been tested in HCC patients, however, the results were largely disappointing [47]. During the past few years, several novel immunotherapeutic methods, including immune checkpoint inhibitors used as monotherapy or combination therapy, new types of immune cell adoption, such as CAR-T, TCR gene-modified T cells and stem cells have been used in clinical trials for the treatment of HCC.

Fig. 3
figure 3

Clinical trials for immune therapy in HCC patients. Several novel immunotherapy methods, including immune checkpoint inhibitors that can be used as monotherapy or combination therapy, new types of immune cell adoption, such as chimeric antigen receptor T cells (CAR-T), TCR gene-modified T cells and stem cells, and microRNAs have been used in clinical trials for the treatment of HCC, some of them are even got permitted from FDA

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Immune checkpoint inhibitor (Table 1)

Anti-CTLA-4 antibodies

In 2013, tremelimumab [48] was first used to treat HCC patients and its safety profile and anti-tumor activity supported further investigation. In 2017, Duffy et al. [49] found that tremelimumab used in combination with tumor ablation is a potential new treatment for patients with advanced HCC; however, only a small subset of patients responded to treatment. Tremelimumab plus durvalumab combination therapy in a phase III trial (NCT03298451) is currently underway to evaluate treatment efficacy. Ipilimumab, another anti-CTLA-4 antibody, is also being assessed in clinical trials for HCC patients (Table 1).

Table 1 Ongoing clinical trials studying immune checkpoint inhibitors in HCC patients
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Anti-PD-1 antibodies

The positive results of CheckMate 040 (nivolumab) in HCC patients were published in The Lancet [50]. Primarily based on the data from this paper, the United States Food and Drug Administration approved nivolumab as second-line treatment agent for HCC on September 22, 2017. One case report suggested that metastatic HCC was responsive to pembrolizumab (a PD-1 inhibitor) following the failure of sorafenib [51]. Recently, combination therapies (e.g., nivolumab combined with Y-90 radioembolization [52]) may further enhance the anti-tumoral effects. A phase III Keynote-240 trial (NCT02702401) has recently found that the therapy did not meet its co-primary endpoints compared with the placebo. Other approaches involving combination therapy with a PD-1 blockade are currently ongoing (Table 1).

Anti-PD-L1 antibodies

The safety and activity of anti-PD-L1 antibody treatment has been proven in patients with advanced cancer [53]. In 2018, Liu et al. [54] provided a novel methodology to evaluate PD-L1 expression in the tumor microenvironment, which may help to select patients who would benefit from anti-PD-1/PD-L1 immunotherapies. All types of anti-PD-L1 antibodies (durvalumab, atezolizumab, and avelumab) are under investigation for their application in HCC patients (Table 1).

Other potential antibodies

Recently, a phase II clinical trial assessing the use of TSR-022 (anti-TIM3 antibodies) plus anti-PD-1 antibodies to treat advanced HCC has been registered (NCT03680508). Other inhibitory checkpoint molecules [e.g., lymphocyte-activation gene 3 (LAG-3), B- and T-lymphocyte attenuator (BTLA), and glucocorticoid-induced tumor necrosis factor receptor (GITR)] have also been found to be closely correlated with HCC progression, which may provide novel targets for the treatment of liver cancer [55].

Adoptive cellular transfusion

Cytokine-induced killer cells (CIKs)

In 2000, autologous lymphocytes activated in vitro were capable of lowering the frequency of recurrence following surgery for HCC [56]. From 2002, Prof. Wang focused on the anti-tumor activity of CIKs, and was the first to initiate CIK therapy for primary HCC patients in a phase I clinical trial [57], and found that the symptoms and characteristics of HCC patients were relieved without any major side effects. Thereafter, a series of classic randomized controlled trials for CIK were initiated, the results of which further confirmed the high treatment efficacy of CIK for HCC patients [58].

Antigen-specificity of HCC immunity

Several tumor-associated antigen (TAA)-specific T cells that targeted alpha-fetoprotein, glypican-3 (GPC3), melanoma-associated gene-A1, and New York-esophageal squamous cell carcinoma-1, respectively, have been regarded as potential therapeutic methods for HCC [59]. However, immunosuppressive mechanisms (e.g., immune checkpoint inhibitory molecules) lead to the above-mentioned specific T-cell exhaustion. Thus, blocking PD-L1, TIM3, and LAG3 might boost TAA-specific T-cell responses for the treatment of HCC [60]. Furthermore, some immunotherapies used to treat HCC patients have been found to favor the development of neoantigen-specific T cells that further enhance the anti-tumor response, leading to tumor shrinkage [61].

CAR-T cells

CAR-T cells are genetically engineered T cells achieved through the introduction of a chimeric antigen receptor. In 2014, GPC3-targeted CAR-T cells were first reported to specifically kill GPC3-positive HCC cells in vitro, and could significantly prolong the survival of HCC xenograft model [62]. In 2017, dual-targeted CAR-T cells co-expressing GPC3 and asialoglycoprotein receptor 1 (ASGR1), could exert superior anticancer activity [63]. Recently, CAR-T-directed CD133 (CART-133) was first tested in a phase I clinical study. The results showed the feasibility, controllable toxicity, and effective activity for its use in treating CD133-postive and late-stage metastasis of HCC patients [64]. Other targeted CAR-T therapies against liver cancer are currently under investigation [65].

TCR gene-modified T cells

In 2015, HBV-specific T-cell receptor (TCR)-modified T cells were constructed from Prof. Antonio’s lab, and a case report confirmed their feasibility and anti-tumor efficacy against HCC [66]. Thereafter, two clinical trials in China were initiated that involved the transfusion of escalating doses of HBV/TCR-T to treat (NCT02719782) and prevent (NCT02686372) the recurrence of HCC. In 2016, Spear et al. [67] found that hepatitis C-associated HCC could be efficiently treated by TCR gene-modified T cells. Additionally, in a recent study, Zhu et al. [68] identified AFP158-specific TCRs to have a substantial potential to treat HCC tumors. A Phase I open label clinical trial (NCT03132792) evaluating the safety and anti-tumor activity of AFPc332-specific TCRs in HLA-A2-positive subjects with advanced HCC patients is currently recruiting subjects.

Stem cells

In 2012, Ning et al. [69] reported that cancer stem cell (CSC)-vaccinated hosts were capable of killing CSCs in vitro. Based on these findings, a clinical trial (NCT02089919), termed the CSC Vaccine Therapy for treating HCC patients was performed between February 2014 and February 2015 without publishing the results. In 2015, Wang et al. [70] conducted a phase I trial of autologous DCs pulsed with autologous irradiated tumor stem cells (TSC) to treat HBV-HCC patients; autologous DC-TSC was found to be safe and did not exacerbate HBV in these HCC patients. In addition, genetically engineered mesenchymal stem cells co-expressing IFN-γ and IL-10 [71] or GPC3/CD3 bispecific T-cell engager (GPC3-ENG) [72] could inhibit HCC both in vitro and in vivo.

Other potential immunotherapy targets

Increased regulatory T cells (Tregs) represented both a potential prognostic marker and a therapeutic target for HCC [73]. Thereafter, decreasing the frequency and suppressor function of circulating Tregs with cyclophosphamide can increase tumor-specific immune responses in patients with advanced HCC (NCT00396682) [74]. Other targets, including microRNAs (e.g., MRX34), natural killer cells, cell cycle inhibitors, and exosomes are currently undergoing investigation as novel immunotherapeutic targets for HCC treatment [75,76,77].

Perspective

Although most of novel immune therapeutic methods are currently in clinical trials, some exhibit substantial potential anti-tumor efficacy. The following crucial issues must be considered: (1) standardization in the preparation of immune products. There are several different cell-based immunotherapies, and the standard operating procedure (e.g., source of the cells, culture methods, administration route, dosage, treatment cycle, stability indicating assay, and storage conditions) must be considered for each. In addition, the method of achieving individualized treatment should also be established based on each specific product; (2) a standardized clinical immunotherapy protocol. The timing of immunotherapy initiation and optimal patients who might benefit from such novel immunotherapeutic methods are current issues. Moreover, which target or combination of targets is the best strategy remains a concern for physicians; and (3) further study is necessary to elucidate the related mechanisms that are involved in the tumor microenvironment, gut microbiome, and HCC genomic features, which may influence the failure of immunotherapy. Moreover, safety issues should also be closely monitored. Finally, armed with a new understanding and unprecedented opportunities, the field of immunotherapy is now standing on the threshold of great advances in the fight against HCC.