Typical inhibitory receptors correlated with T-cell exhaustion

There were significant differences in IRs expression on Tex cells in different types and stages of liver cancer, suggesting that there might be multiple molecular mechanisms of T-cell exhaustion in liver cancer. A summary of molecule target therapies in liver cancer (Table 1).

Programmed cell death-1(PD-1)

PD-1, also known as CD279, is an immunosuppressive molecule (Zhang et al. 2004). PD-1 is mainly expressed on activated T-cells, B cells, and macrophages, and negatively regulates immune responses more extensively than other immune checkpoints. The ligands of PD-1 are programmed cell death ligand 1 (PD-L1) (also called B7-H1 or CD274) and PD-L2(also called B7-DC or CD273) (Wu et al. 2019). In HCC, PD-1 bond to PD-L1 to inhibit T-cell activation and cytokine release. It also promoted self-tolerance by down-regulating the immune system response and inhibiting the inflammatory activity of T-cells (Kotanides et al. 2020). Studies showed that high expression of PD-1 led to T-cell exhaustion in HCC. Blocking PD-1/PD-L1 increased the frequency of tumor-specific T-cells in HCC patients, but did not restore T-cell function (Gehring et al. 2009), indicating that other IRs might be present on T-cells. Studies found that low expressions of GZMA and F2R in liver cancer tissues are positively correlated with PD-1 and PD-L1, and inefficient GZMA-F2R communication impaired anti-PD-1’s ability to inhibit tumors through JAK2/STAT1 signal. It also indicated aggressive clinicopathologic features and poor prognosis (Gao et al. 2022) (Fig. 2). Studies showed that exhausted PD-1Hi CD8+T-cells in liver cancer tissues were increased, which showed the expression of IRs (including TIM-3, CTLA-4, etc.) and transcription factors (Eomes, BATF, etc.), the level of cytotoxic molecules decreased, the production capacity of pro-inflammatory cytokines was impaired, and the expression of anti-inflammatory cytokine IL-10 was up-regulated (Ma et al. 2019). In addition, the up-regulation of PD-L1 expression in HCC cells and the effect of PD-1 on tumor-infiltrating lymphocyte (TILs) further led to T-cell exhaustion (Bai et al. 2022). Therefore, tumor patients with high PD-L1 expression need to improve T-cell exhaustion from the perspective of reducing PD-L1. Moreover, PD-L1 positive HCC subsets mutated in the PI3K-Akt pathway showed an inflammatory phenotype, and PD-L1 positive tumor tissues were mostly exhausted CD8 cells, while PD-L1 negative HCC showed a mutation that led to β-catenin activation and non-inflammatory characteristics (Nishida et al. 2020) (Fig. 2). Studies indicated that patients with low levels of PD-L1 and high levels of miR-200c survive longer in HBV-related HCC. Expression of miR-200c by directly targeting 3’-UTR of CD274 (coding PD-L1) antagonist mediated PD-L1 expression of HBV, and reverse CD8+ T-cell exhaustion (Sun et al. 2018). Therefore, interventions targeting the PD-1/PD-L1 axis might be insufficient for an anti-tumor effect in such cases.

Fig. 2

T-cells promoted the expression of PD-L1 in cancer cells. T-cells, especially CD8 T-cells, secrete IFN-γ after recognizing tumor antigen, then bind to corresponding receptors and promoted the expression of PD-L1 through JAK2-STAT1 pathway or PI3K/Akt pathway

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)

CTLA-4 is mainly expressed in Tregs cells and plays a negative regulatory role in the immune system. When T-cells were activated, the expression of CTLA-4 was up-regulated and the degree of T-cell inflammatory response was reduced (Xu et al. 2020). In HCC, transforming growth factor beta 1 (TGF-β1) upregulated the expression of CTLA-4 and PD-1 through the TGF-βR/CaN/ NFATc1 pathway, enhancing tumor immune escape (Bao et al. 2021). CD28 and CTLA-4 are homologous glycoproteins of the immunoglobulin superfamily. In contrast to CTLA-4, B7 activates the CD28 signaling pathway to promote T-cell proliferation (Linsley et al. 1991). CTLA-4 mediated immunosuppression by competing with CD28 for the B7 protein and disrupting the CD28 signaling pathway. B7/BB1 is a cell surface protein called CD80, and B7-2 (CD86) is another CTLA-4 ligand. These ligands are highly expressed in antigen-presenting cells (APCs) (Liu and Zheng 2020). CD8+T-cell exhaustion with different phenotype, functional state and underlying mechanism was observed in HCC and chronic hepatitis B (CHB). Moreover, CD8+ T-cells from HCC showed higher expression levels of exhaustion markers (including CTLA-4, etc.), and lower proliferation, cell activity and effector cytokine production levels (Wang et al. 2019b).

T-cell Ig and ITIM domain (TIGIT)

TIGIT is a potential new target for cancer immunotherapy in addition to CTLA-4 and PD-1 (Khan et al. 2020). The expression of TIGIT on TILs was closely related to the expression of PD-1, and TIGIT expression was detected in CD8+ T-cells with high expression of PD1 from all tumor sources (Ma et al. 2019). In HCC, compared with single blocking of PD-1, blocking of TIGIT and PD-1 significantly enhanced proliferation, cytokine production, and cytotoxicity of CD8+TILs (Ge et al. 2021). TIGIT has two main ligands: CD155 (PVR) and CD112 (PVRL2, nectin-2). Among the two ligands, TIGIT has the highest affinity with CD155 (Chauvin and Zarour 2020). CD155 was significantly up-regulated in tumor cells of a variety of cancers and was associated with poor prognosis (Kučan Brlić, et al. 2019). It was found that in mouse liver cancer models and patients with liver cancer, the expression of TIGIT in Tex cells was significantly increased, and the combined blocking of PD-1 and TIGIT could synergistically inhibit the growth of liver cancer in mice with normal immune function (Ostroumov et al. 2021). Studies suggested that in HBsAg+ hepatocyte (HBs-HepR)-related HCC mouse models, TIGIT blocking reactivated CD8+T-cells, increased the production of TNF-α and IFN-γ, and increased the number of CD8+T-cells in tumor, slowing the development of HCC in HBs-HepR mice (Wu et al. 2023). HBV-related HCC benefited from blocking TIGIT expression by CD8+ T-cells (Liu et al. 2019a). In addition, CD155 was overexpressed in HCC, and CD155 HCC cells upregulated TIGIT on CD8 T-cells reduced the secretion of IFN-γ, tumor necrosis factor-α (TNF-α) and IL-17A, and increased the secretion of IL-10 in effector cells. TIGIT blocking or CD155 knockdown reversed the inhibition of liver cancer cells on CD8+ T-cells effector function (Zhang et al. 2020a).

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)

Tim-3, a member of the TIM family, is a checkpoint receptor. And inhibition of TIM-3 enhances the antitumor effects of PD-1 inhibitors (Wolf et al. 2020). TIM-3 has four ligands, including galactose lectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box-1 protein (HMGB1) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) (Jin et al. 2022a). According to TIM-3 expression, CD8+T-cells could be divided into distinct subpopulations: PD1-high (PD-1Hi), PD1-intermediate  (PD-1Int) and PD1-low (PD-1−) (Ma et al. 2019) but the expression of TIM-3 was limited to PD-1Hi CD8+T-cells. In addition, PD-1Hi CD8+T-cells were in a state of T-cell exhaustion, which showed that the production frequency of IL-2, IFN-γ and TNF-α were down-regulated, and the production frequency of anti-inflammatory IL-10 was increased. The metabolic activity of cells decreased and the production of proinflammatory cytokines decreased (Ma et al. 2019). Studies showed that the soluble TIM-3 (sTIM-3) level was rising steadily from asymptomatic HBV carriers, chronic hepatitis, cirrhosis to HCC. In HBV-related HCC, the level of TIM-3 expression on T-cells was associated with the severity of T-cell exhaustion and risk. The higher TIM-3 levels were associated with poorer overall survival (Li et al. 2018a). Lnc-TIM-3 was up-regulated in CD8 T-cells infiltrated by HCC patients and was negatively correlated with the production of IFN-γ and IL-2. Lnc-TIM-3 specifically bond to TIM-3 and blocks its interaction with Bat3, inhibiting downstream Lck/NFAT1/AP-1 signaling pathway and leading to nuclear localization of Bat3. And enhanced RelA transcriptional activation of p300-dependent p53 and anti-apoptotic genes, including MDM2 and Bcl-2, leading to T-cell exhaustion (Ji et al. 2018). Studies showed that 1516G/T polymorphisms in the TIM-3 gene promoter region influenced disease susceptibility and HCC characteristics associated with HBV infection, suggesting that TIM-3 played an important role in T-cell dysfunction and exhaustion involved in HCC and HBV development (Li et al. 2012a).

Lymphocyte-activation gene 3 (LAG-3)

LAG-3 is a potential cancer immunotherapy target that negatively regulates T-cells. It binds to PD1 to mediate an exhausted state (Andrews et al. 2017). Fibrinogen-like protein 1 (FGL-1) was found to be another major functional ligand of LAG-3. FGL-1 was a protein secreted by liver cells to promote liver cancer cells proliferation. Targeting LAG-3/FGL-1 pathways as potential targets for immune escape and cancer immunotherapy (Wang et al. 2019c). Increased expression of FGL1 in HCC led to the exhaustion of CD8+TRM cells in the tumor through binding with LAG3 on the cell membrane, resulting in tumor immune escape (Yang 2023). In hepatitis virus-related HCC, T-cells with high IRs expression were found to be more prone to dysfunction. In TCGA, it was found that compared with non-HBV or HCV-related liver cancer, exhausted cytotoxic T lymphocytes (CTLs) infiltration was significantly higher, and exhaustion markers were consistently higher, including PD-1, CTLA-4, CD27, CD52, ICOS, etc. (Lu et al. 2022).

Multiple inhibitory receptors

In addition, there are two or more IRs co-expression in liver cancer. It was found that PD-1 bond to the TIM-3 ligand Gal-9, which contributed to the persistence of PD-1+TIM-3+ T-cells and attenuated Gal-9/TIM-3 induced cell death. The combination of anti-Gal-9 and anti-GITR inhibited Tregs cells and induced synergistic anti-tumor activity (Yang et al. 2021). Studies found that the proportion of TIM-3+TIGIT+ in peripheral blood of CD4+ T-cells and CD8+ T-cells in HCC patients was higher, and the content of IL-10 was increased, which was associated with accelerated progression of HCC disease and poor prognosis (Yuan et al. 2021). Studies showed that PD-1HiTIGIT+ CD8+TILs decreased the ability to produce IFN-γ and TNF-α, and could co-express a variety of IRs including TIM3, LAG3 and TOX, etc. Compared with PD-1 alone, combined TIGIT/PD-1 blocking in vitro improved the proliferation, cytokine production and cytotoxicity of CD8+TILs (Ge et al. 2021). It was found that the co-expression of PD-1 and TIGIT on CD4+ and CD8+T-cells of HBV-related HCC patients was significantly up-regulated. Moreover, in advanced and advanced HBV-related HCC patients, the population of PD-1+ TIGIT+ CD8+ T-cells was elevated and T-cell exhaustion was present. T-cell exhaustion was characterized by high expression of other IRs (including CTLA-4, 2B4, and LAG-3, etc.), high susceptibility to apoptosis, decreased cytokine secretion capacity, and transcription factor expression consistent with exhaustion, which was associated with poor overall and progression-free survival (Liu et al. 2019a).

Furthermore, it was found that CD8+T-cells were enriched in HCC tissues, and CD8+T-cells showed various high levels of exhaustion markers (PD-1, TIM-3, CTLA-4 and LAG-3) (Yang et al. 2020; Wang et al. 2019d), and decreased proliferation (Ki67) and cell activity (CD69), reduced production of effector cytokines (IFN-γ, IL-2, and TNF-α) (Wang et al. 2019b), which was associated with poor progression-free and overall survival (Barsch et al. 2022). Studies indicated that the expression of C-C motif chemokine ligand 14 (CCL-14) or LPAR6 was significantly decreased in HCC, which was negatively correlated with a variety of exhaustion T-cell markers (PD-1, TIM-3, CTLA-4, etc.). This was associated with poor overall survival, disease-specific survival, progression-free survival, and relapse-free survival (Gu et al. 2020; He et al. 2022). Studies found that protein-tyrosine phosphatase 1B (PTP1B) (Zhu and Zu 2023), MKI67 (Wu et al. 2021a), DHX37 (Xu 2020), sperm-associated antigen 5 (SPAG5) (Chen et al. 2020b), karyopherin α4 (KPNA4) (Xu et al. 2021), TANK-Binding Kinase 1 (TBK1) (Jiang 2021), YTH domain family (YTHDF) 2 (Shao et al. 2020), angiotensin II receptor-associated protein (AGTRAP) (Liu 2021) were upregulated in liver cancer tissues, which was positively correlated with T-cell exhausted-related genes (TIM-3, TIGIT, PD-1, LAG-3) and immune cell infiltration, and was also associated with poor overall survival. Studies suggested that total CD8+ T and CD4+T-cells tumors expressed higher levels of IRs, including PD-1, LAG-3, B4 and TIGIT, in HCC (Liu et al. 2018). Studies found that CD8+T-cells infiltrated were in a state of severe functional exhaustion in chronic HBV infection of liver cancer, which was manifested as increased expression of various IRs (PD-1, TIM-3, CTLA-4, LAG-3), decreased cell proliferation ability (Ki67), and increased apoptosis level (Caspase-3). The ability of CD8+T-cells to secrete effector cytokines (IFN-γ, TNF-α, IL-2) was weakened (Wang and C. 2019).

Immune cells and cytokines and T-cell exhaustion

In the TME, non-malignant cells can help tumor cells proliferate, invade and metastasize. The immunosuppressive properties of tumor lesions are not only one of the main factors inducing tumor progression but also a challenge to effective immunotherapy. It has been found that stromal cells and tumor cells can produce immunosuppression related to chronic inflammatory factors such as growth factors, cytokines, chemokines and so on (Liu et al. 2019b). Multiple immune cells coexist and interact in a complex series of pathways that eventually lead to cancerous tumors.

Immune cells

Immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), regulatory T-cell (Tregs) and tumor-associated macrophages (TAMs) have been demonstrated in many solid tumors, and these immunosuppressive cells also contribute to ICIs resistance in liver cancer therapy. Targeting immune cells in liver cancer (Table 2).

Table 2 Targeting immune cells in liver cancerMyeloid-derived suppressor cells (MDSCs)

MDSCs are heterogeneous immature and immunosuppressed bone marrow cells that regulate immune response in cancer (Gabrilovich and Nagaraj 2009). MDSCs play an important role in tumor immunity, inhibiting effector T-cell immune responses in the tumor microenvironment by inducing T-cell exhaustion and tumor microenvironment dysfunction (Gabrilovich and Nagaraj 2009; Kumar et al. 2016). Patients with high MDSCs had significantly higher frequency of intrahepatic metastasis and vascular infiltration, and shorter relapse-free survival. High infiltration of MDSCs in HCC was an independent prognostic factor for overall survival and relapse-free survival, and patients with high infiltration of MDSCs and low infiltration of T-cells in HCC had poor prognoses (Tomiyama et al. 2022). In HCC-TME, MDSCs inhibited T-cell activation and infiltration by reducing the secretion of IL-6 and other cytokines (Xu et al. 2017). Myeloid cells are a group of heterogeneous innate immune cells including MDSCs, monocytes/macrophages and dendritic cells. HCC with low (MRSlo), medium (MRSint), and high myeloid response scores (MRS) (MRShi) corresponded to tumors with immune-active, immune-deficient, and immune-suppressive TME, respectively. The clinical prognosis of MRShi and MRSlo HCC was different. Although they have similar levels of CD8+T-cell infiltration, the CD8+T-cells in MRShi tumors showed a distinct pattern of exhaustion (Wu et al. 2020).

Regulatory T-cells (Tregs)

Tregs cells can be divided into two groups: “natural Tregs cells” that spontaneously arise in the thymus, and “induced Tregs cells” that arise in peripheral tissues stimulated by cytokines such as TGF-β, retinoic acid, and IL-2. Tumor hypoxia promoted the recruitment of Tregs cells by inducing the expression of chemokine CCL28 (Facciabene et al. 2011). It was found that a variety of cells including Tregs were enriched in the TME. Compared with Tregs isolated from the nontumor microenvironment (NTME), Tregs isolated from TME expressed multiple T-cell exhaustion markers, including PD-1, LAG-3, and TIM-3 (Chew et al. 2017). The infiltration of Tex cells, including regulatory T-cells (FOXP3+-Tregs), CD4+-Tex and CD8+-Tex, was found in HCC tissues. In addition, FOXP3+-Tregs were more predictive of early recurrence, and infiltration of CD8+T or CD8+-Tex cells was closely correlated with overall survival or recurrence-free survival (Liu 2020). Single-cell RNA sequencing results showed that Tregs and exhausted CD8+ T-cells were preferentially enriched and amplified in HCC, and layilin (LAYN) expression was increased in Tregs and activated CD8+ T cells and the function of CD8+ T-cells was inhibited in vitro (Zheng et al. 2017).

Tumor-associated macrophages (TAMs)

It is clear that bone marrow-derived cells, including TAMs, tumor-associated neutrophils (TANs), and MDSCs, contribute to tumor progression (Kitamura et al. 2015). TAMs, an important component of the HCC microenvironment, inhibited anti-tumor immunity and promote tumor progression by expressing cytokines and chemokines, and were associated with poor prognosis in HCC patients (Dong 2016). Studies showed that the repolarization of TAMs to antitumor phenotypes promoted tumor regression. In general, macrophages polarize into M1 or M2 macrophages. M1-polarized macrophages can be activated by Th1 cytokines such as IFN-γ and TNF-α. M1-polarization produces immune stimulators that promote inflammation, such as IL-12 or TNF-α (Qian and Pollard 2010). Moreover, studies showed that M2-polarized macrophages are more similar to TAMs and are activated by the Th2 cytokines IL-13 and IL-4, which have anti-inflammatory effects and produce TGF-β and IL-10 (Yang et al. 2018) (Fig. 3). Studies also indicated that tumor-derived Wnt ligands promoted M2-polarized macrophages through the Wnt/β-catenin pathway, promoting the growth, metastasis and immunosuppression of liver cancer (Yang et al. 2018). In the HCC environment, the infiltration of TAMs was proportional to that of CD4+ CD25+ FoxP3+Tregs cells. IDO or colony-stimulating factor 1 (CSF-1), which was produced by tumor cells, promoted the recruitment of TAMs and MDSCs to TME. TAMs produced a variety of chemokines, such as CCL22, which attracted Treg cells to cancer sites, blocking cytotoxic T-cell activation (Wang et al. 2019e; Mamrot et al. 2019). At the same time, prostaglandin E-2 (PGE-2) and TGF-β secreted by TAMs further aggravated immunosuppression (Li et al. 2012b). In HCC, TAMs also induced T-cell apoptosis by secreting PD-L1 and binding to PD-1 receptors expressed on effectors T-cells, resulting in the production of Arginase I (ARG1), which inhibited the production of L-arginine by T-cells necessary for their own activation (Liu and C. 2020). Studies showed that high expression of cyclooxygenase-2 (COX-2) in liver cancer reduced the secretion of Granzyme B and IFN-γ by M2 macrophage polarization and TGF-β pathway, leading to activated CD8+ T-cell exhaustion (Xun et al. 2021).

Fig. 3

Macrophage polarization. Macrophages polarize into M1 or M2 macrophages. M1-polarized macrophages are activated by Th1 cytokines such as IFN-γ and TNF-α. M1 polarization produces pro-inflammatory substances such as IL-12 or TNF-α. M2-polarized macrophages are activated by Th2 cytokines IL-13 and IL-4, have anti-inflammatory effects, and produce TGF-β and IL-10

Other immune cells

Neutrophil extracellular traps (NETs) had a direct impact on T-cells by promoting T-cell phenotype and functional failure, resulting in T-cell expression of multiple IRs, reduced cytokine production, reduced proliferation ability, and metabolic changes, which promoted tumor growth within TME. Targeting NETs containing PD-L1 with DNAse or anti-PD-L1 reduced tumor growth and T-cell exhaustion (Yazdani and Tohme 2019; Kaltenmeier 2021). The study showed that HCC tumors with high expression of xanthine dehydrogenase (XDH) contained abundant invasive CD8+T-cells in the microenvironment without T-cell exhaustion (Lin et al. 2021). The study found that 36 key prognostic genes in HCC were significantly associated with T-cell exhaustion markers, including PD-1, CTLA-4, TIM-3 and TIGIT, suggesting that these genes were closely related to T-cell exhaustion (Deng et al. 2021). Studies also found that overexpression of interferon regulatory factor 8 (IRF8) in HCC significantly enhanced the anti-tumor effect and improved T-cell exhaustion in the tumor microenvironment, which was conducive to the prognosis of patients (Wu et al. 2022). Studies showed that different CD8+ TRM cell populations existed in HCC patients, the high frequency of end-exhaustion T-cells in tumor was uncommon, and non-end-exhaustion HBV-specific CD8+ TRM cells were characterized by active participation and effective anti-tumor response (Cheng et al. 2021). Moreover, studies indicated that TRM was rich in the high expression of PD-1 in HBV-related HCC tissues, and was functionally more inhibitory and exhausted (Lim et al. 2019). It was found that tumor-specific CD8+T-cells (TST) became dysfunctional in the early stage of liver cancer, exhibiting phenotypic, functional, and transcriptional characteristics similar to those of late dysfunctional T-cells. Thus, T-cell dysfunction observed in advanced liver cancer might have been established early in tumorigenesis (Schietinger et al. 2016). Studies suggested that partial depletion of Id3 in CD8 T-cells promoted the development of exhausted CD8 T lymphocytes. And high expression of Id3 in CD8 T-cells inhibited the exhaustion development of anti-tumor CTLs, which was conducive to better tumor control (Jin et al. 2022b). PRDM1/BLIMP1 induced cancer immune evasion by modulating the USP22-SPI1-PD-L1 axis. Overexpression of Prdm1 could increase the number and activity of infiltrated CD8+T-cells (GZMB+) and reduce CD8+T-cell exhaustion (Li et al. 2022). Studies showed that RIN1, a transcriptional complex inhibitor of recombination signal binding protein for immunoglobulin kappa J region (RBPJ), inhibited the expression of exhaustion-related transcription factors and receptors, enhanced CD8+ T-cell response to IFN-α/β, alleviated CD8+ T-cell exhaustion, and inhibited HCC cells by inhibiting mTOR pathway (Pan et al. 2023). The exhaustion state of CAR T-cells was increased in CD39+CAR-T-cells, but shRNAs knockdown of PD-1, TIM-3, and LAG-3 further enhanced the antitumor activity of CD39+CAR-T-cells and increased IFN-γ secretion (Zou et al. 2021). Studies showed that the number of CD8+T-cells decreased and exhaustion increased in mice liver cancer induced by BNL-T combined with tumor endothelial cells (TECs). Inhibiting the expression of glycoprotein nonmetastatic melanoma protein B (GPNMB) could inhibit tumor growth and T-cell exhaustion (Sakano et al.