Immunometabolic Targets in CD8⁺ T Cells within the Tumor Microenvironment of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) accounts for approximately 90% of primary liver cancers (PLCs) and has shown no significant decrease in mortality rates over the past decade [1]. Currently, HCC ranks as the third leading cause of cancer-related deaths worldwide [2]. The primary pathophysiological mechanism underlying HCC involves chronic inflammation and necrosis of liver tissue, with hepatitis viruses and metabolic disorders being the major etiological factors [1]. Despite considerable advancements in various treatment modalities for HCC since the early 21st century, multikinase inhibitors have not yielded substantial improvements in overall survival rates for liver cancer in recent years [3, 4].

Research into immunotherapy has been ongoing for over a century, achieving success with interventions such as interleukins and interferons (IFNs) in cancer therapy [5]. Recently, focus has intensified on the tumor microenvironment (TME), composed of immune cells, tumor cells, cytokines, nutrients, and metabolites. This suppressive TME hinders the inherent functions of tumor-targeting cells, thereby facilitating tumor progression. Among the tumor-infiltrating immune cells, the presence of CD8⁺ T cells in tumor tissues has been identified as a crucial indicator correlated with the tumor-node metastasis classification system and HCC prognosis [6]. Reactivating immunosuppressed CD8⁺ T cells within the TME has shown potential to improve clinical outcomes [7]. In contrast, immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) exacerbate CD8⁺ T-cell exhaustion, further contributing to tumor progression [8‒10]. The elevated expression of immune checkpoints plays a critical role in mediating T-cell immunosuppression [11‒14]. The discovery of these immune checkpoints within TME-associated immune cells represents a significant advancement in cancer immunotherapy.

In addition to suppression from surrounding cells, CD8⁺ T cells in the TME face competition for limited nutrients and the accumulation of metabolites, which impedes their growth and functionality [15, 16]. This resource competition within the TME leads to T-cell exhaustion, characterized by the overexpression of immune checkpoints and dysregulated metabolism, ultimately compromising antitumor immune responses [17‒19]. The reprogramming of metabolic pathways in naïve T cells (Tn), effector T cells (Teff), and memory T cells (Tm) is intricately linked to T-cell differentiation, with energy metabolism playing a crucial role in this process [20‒22]. Therefore, immune studies that overlook metabolic considerations provide an incomplete understanding of the immune response in HCC. Additionally, nonalcoholic fatty liver disease (NAFLD), now recognized as metabolic-associated fatty liver disease, has increasingly emerged as a contributing factor in HCC etiology. Consequently, the reprogramming and metabolic disorders observed in HCC pose significant challenges for future research [23‒25].

Historically, research on tumor-infiltrating immune cells has predominantly focused on their functional characteristics. However, the development of the TME concept has redirected attention toward understanding the interactions among immune cells, immune molecules, and metabolites within the tumor environment. The TME includes various immune cell types, such as dendritic cells (DCs), TAMs, T cells, and MDSCs, all of which play critical roles in regulating tumor immunity [26]. Acting as a cellular and molecular landscape for immune responses, the TME reflects the immune and metabolic interplay between immune and tumor cells. The mammalian target of rapamycin (mTOR) serves as a central regulatory hub for T-cell metabolism, integrating signals from the TME to coordinate T-cell function and survival [27, 28].

In clinical settings, TME-associated therapies have encountered challenges, including resistance and non-responsiveness to immunotherapy, particularly following the introduction of sorafenib and immune checkpoint blockers (ICBs) in HCC treatment [29, 30]. Various combination therapies, including those incorporating metabolic strategies, have been developed to address these issues. Studies have unexpectedly revealed synergistic interactions between metabolic mechanisms and immunotherapeutic approaches [15, 31‒33]. Nevertheless, the limited focus on metabolic research related to immune cells has impeded progress in tumor immunotherapy development. Advancing tumor immunity requires the integration of studies on immunocyte metabolism and immune functionality. Therefore, this review provides an overview of recent research at the intersection of immunology and metabolism, specifically focusing on tumor-infiltrating CD8⁺ T cells within the HCC microenvironment. The goal was to highlight the importance of immunocyte metabolism while summarizing potential mechanisms, recent advancements, and future prospects in this evolving field.

Tumor-infiltrating CD8⁺ T cells are recruited and activated by specific chemokines to release cytotoxic mediators, enabling their tumor-killing functions. In HCC, CD8⁺ T-cell activation occurs exclusively through T-cell receptor (TCR) signaling, mediated by DCs [34]. However, with prolonged immune stimulation, cytotoxic CD8⁺ T cells can shift to an immunosuppressive state known as exhaustion (Fig. 1).

CD8⁺ T-cell exhaustion is marked by reduced functionality, often signaled by increased expression of inhibitory receptors and the activation of adverse regulatory pathways within these cells [17, 19]. In chronic liver disease, metabolic alterations in CD8⁺ T cells contribute to this exhaustion. While exhausted CD8⁺ T cells have limited function, the accompanying metabolic changes can extend their lifespan [28, 35]. The accumulation of exhausted T cells, combined with the apoptosis of effector T cells, reduces overall antitumor efficacy and promotes immune escape in HCC.

The clinical success of ICBs in HCC has heightened interest in immune checkpoint receptors. Overexpression of these receptors, such as programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin-domain-containing protein 3 (TIM-3), and lymphocyte-activation gene 3 (LAG-3), is documented in malignancies and correlates with the exhaustion of tumor-specific CD8⁺ T cells [36‒40]. This suggests substantial clinical potential for ICBs across malignancies. Strategies to block PD-1, TIM-3, and LAG-3 have shown synergistic effects on tumor-infiltrating CD8⁺ T cells in HCC patients [14].

The prominence of PD-1 and its ligand PD-L1 rose after the Nobel Prize in 2018. PD-L1 or PD-L2, expressed by tumor cells, binds to PD-1 on T cells, sending inhibitory signals that limit CD8⁺ T-cell proliferation. In HCC, PD-L1 expression by tumor cells and in exosomes significantly impairs CD8⁺ T-cell function, especially in hepatitis B virus (HBV)-related cases [41]. Blocking PD-L1 exposes cancer cells to immune detection, while PD-1 blockade reprograms CD8⁺ T-cell metabolic pathways, effectively reversing exhaustion. High PD-1 expression is a hallmark of early stage exhausted CD8⁺ T cells, known as progenitor exhausted CD8⁺ T cells [42]. These PD-1^hi CD8⁺ T cells often co-express other immune checkpoint molecules [43]. Notably, CD8⁺ T cells are primary targets for anti-PD-1 therapy, with tumor-infiltrating CD8⁺ T cells indicating a favorable therapeutic response [44]. The PD-1/PD-L1 interaction can lead to T-cell exhaustion by altering metabolism, shifting from glucose and amino acid usage to fatty acid metabolism [33, 45, 46].

However, a substantial percentage of patients exhibit resistance to anti-PD-1 therapy [30]. In solid tumors, the TME challenges therapy efficacy due to low antigen immunogenicity, which impairs antigen presentation and limits the effectiveness of PD-1 blockade in restoring antitumor immunity [47]. Enhancing T-cell energetic metabolism has emerged as a promising strategy to address the limitations of PD-1 blockade [48‒50].

The abnormal blood vessel architecture in tumors can restrict immune cell infiltration, thus reducing the effectiveness of PD-1 blockade [51]. Strategies to disrupt and normalize tumor vasculature have emerged as practical approaches to overcoming resistance to PD-1 blockade [52]. For instance, regorafenib, a multikinase inhibitor targeting vascular endothelial growth factor receptors, has shown promise in normalizing tumor vascular system and enhancing CD8⁺ T-cell recruitment when combined with PD-1 blockade, especially in Rag1-deficient mice lacking functional T cells in the liver [53]. Additionally, combining anti-PD-1 therapy with vascular endothelial growth factor receptor 2 inhibitors has produced promising results in clinical settings [54]. However, even in tumors where T-cell recruitment is adequate, the effectiveness of PD-1 blockade may be hindered by interactions between intratumoral DC-CD4⁺ T helper cell niches and CD8⁺ T cells [55].

TIM-3, encoded by the hepatitis A virus-cell receptor 2, is a type I membrane protein predominantly expressed in T cells. In HCC, TIM-3 and PD-1 are key markers of exhausted CD8⁺ T cells [56]. The interaction between TIM-3 and PD-1 plays an important role in suppressing immune responses to normal cells and inhibiting T-cell inflammatory activity, promoting immune tolerance. Within exhausted CD8⁺ T-cell subsets, TIM-3 serves as a marker of terminal exhaustion, signifying a further decline in antitumor immunity [42].

Previous studies have highlighted TIM-3’s role in HCC as a receptor for galectin-9 (Gal-9). Zhao et al. elucidated the mechanisms by which the miR-93-5p/Gal-9 axis regulates trimethylation at histone H3 lysine 27 (H3K27me3). This regulation is essential for reprogramming the TME and enhancing CD8⁺ T-cell apoptosis via TIM-3 signaling [57]. The TIM-3/Gal-9 pathway has been shown to inhibit both innate and adaptive immune cells, thereby weakening antitumor immunity [58]. Conversely, deficiency in the TIM-3/Gal-9 pathway may impair T cells’ immune regulatory capacity, particularly in autoimmune and viral hepatitis [59, 60].

In addition to its immunosuppressive effects, the interaction of Gal-9 with TIM-3 and PD-1 has been found to support the survival of exhausted CD8⁺ T cells [61]. This persistence of exhausted CD8⁺ T cells, combined with the depletion of effector T cells allows tumor cells to evade immune surveillance and survive.

Beyond PD-1 and TIM-3, several other immunotherapy targets are being investigated for HCC treatment. This section reviews additional immune targets with potential to advance HCC immunotherapy.

Thymocyte selection-associated high mobility group box protein (TOX) is induced by calcineurin and the nuclear factor of activated T cells 2, regulating T cells through a calcineurin-independent feed-forward loop that results in sustained stimulation and ultimately T-cell exhaustion [62]. TOX has been identified as a marker of effector CD8⁺ T cells, reflecting the degree of CD8⁺ T-cell dysfunction [63‒65]. Importantly, TOX also plays a key role in T-cell differentiation and lifecycle maintenance, which is essential for the survival of tumor-specific T cells [63, 66]. Research on TOX’s role in CD8⁺ T-cell activation suggests its potential to reverse T-cell exhaustion and enhance T-cell functionality in malignancies [67‒69]. Additionally, TOX shows promise as a predictive marker for the efficacy of PD-1 and T-cell immunoreceptors with Ig and ITIM domains (TIGIT) blockade in HCC treatment [70]. However, the clinical application of TOX in HCC is still limited by technical constraints.

Tumor necrosis factor receptor superfamily member 9, commonly known as 4-1BB or CD137, is another co-stimulatory molecule expressed in CD8⁺ T cells, operating independently of the CD28 signaling pathway. The interaction between the 4-1BB ligand (4-1BBL) on DCs and 4-1BB on T cells recruits TNFR-associated factors 1 and TNFR-associated factor 2, which subsequently upregulate nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinase, and B-cell lymphoma proteins to enhance T-cell function and survival [71]. The 4-1BB signaling pathway has been associated with improvements in CD8⁺ T-cell fatty acid oxidation (FAO) and mitochondrial biogenesis via mechanisms involving DNA methylation [72, 73]. Activation of 4-1BB has shown positive results in various cancers, hepatitis, and CAR-T-cell therapy [73‒77]. Recent research by Kim et al. [43] has emphasized the potential of 4-1BB co-stimulation to reverse the exhaustion of tumor-infiltrating CD8⁺ T cells within the HCC microenvironment [78]. Therefore, the role of 4-1BB in HCC immunometabolism merits further investigation.

In combination with PD-1 inhibitors, blocking LAG-3 and CTLA-4 has shown effectiveness in controlling various tumors [79, 80]. However, LAG-3 primarily relies on major histocompatibility complex class II signaling, which predominantly restores CD4⁺ T-cell immunity with less impact on CD8⁺ T cells [81]. Conversely, CTLA-4 blockade has been shown to restore the function of helper T cells (T_H cells) rather than enhancing the cytotoxicity of Teff [82]. Unfortunately, CTLA-4 blockade is associated with significant hepatotoxicity and a higher incidence of hepatitis when combined with PD-1 inhibitors, making it unsuitable for HCC treatment [83]. Similar to TIM-3, TIGIT has been identified as a marker of T-cell exhaustion in PLC and has demonstrated synergistic effects with PD-1 inhibitors [63, 64]. TIGIT enables HCC cells to enhance signaling through the poliovirus receptor (PVR) and its ligand PVRL1, which suppresses the cytotoxic response of T cells, thereby reinforcing the inhibitory PVRL1/PVR/TIGIT axis in HCC [62, 66]. While targeting TIGIT alone has uncertain clinical efficacy, it has been shown to improve antitumor immunity in HCC [84].

Lastly, we compiled recent clinical trials to evaluate the outcomes of CD8⁺ T cell-related immunotherapy and combination strategies in HCC treatment [85‒94] (Table 1). PD-1 blockade-based combinations have improved survival rates in patients with unresectable HCC, though this has come with an increased incidence of adverse events. Notably, the STRIDE combination surprisingly alleviated symptoms. However, most CD8⁺ T cell-related targets remain distant from routine clinical application.

In recent decades, ICB therapy has progressed significantly. However, despite these advancements, immunotherapy has not yet achieved satisfactory results for HCC patients [29]. Along with rising obesity rates and the increasing incidence of NAFLD contributing to HCC, metabolic changes in tumor-infiltrating immune cells and immunometabolic therapy have resurfaced as key areas of research. Immune cells have the capacity to adjust their metabolic pathways in response to the nutrient and metabolite composition of the TME. Consequently, cells within the TME often increase nutrient uptake to support growth or functional activities, leading to competition for available resources.

The prevalence of steatosis in hepatitis leads to lipid accumulation within the HCC microenvironment. This nutrient-rich environment causes alterations in glucose, lipid, and amino acid levels, characteristic of various HCC etiologies [95]. Among these, glucose and glutamine are the most influential nutrients driving resource competition [96, 97] (Fig. 2). Activated T cells increase their energetic metabolism, particularly through mitochondrial activity, to generate adenosine triphosphate (ATP), essential for biosynthesis and T-cell effector functions [98]. The TME opposes the effector functions of CD8⁺ T cells through immune checkpoint receptors, inhibitory cytokine production, and changes in extracellular metabolic conditions [99]. Additionally, a lower density of CD8⁺ T cells and a higher presence of regulatory T cells in the HCC TME correlate with poorer prognoses [31].

Changes in functionality reflect the metabolic alterations occurring in CD8⁺ T cells. The survival and immune efficacy of tumor-infiltrating CD8⁺ T cells are closely tied to their metabolic pathways and influenced by the concentrations of metabolic substrates in the TME [100]. Furthermore, metabolites can act as mediators of immune signaling and function as discrete signaling molecules for T cells [101]. Similar to tumor cells, the metabolic pathways involving glucose, lipids, and amino acids are crucial in determining immune cell functionality within the liver. In HCC patients, liver tissue typically exhibits lipid surplus and relative glucose and oxygen deficits, resulting from an imbalance between glucose and lipid synthesis and catabolism [95]. HBV induces persistent oxidative stress, while the hepatitis C virus promotes insulin resistance and lipid accumulation, worsening hepatitis severity and increasing the likelihood of developing PLC [102‒105]. Additionally, steatosis associated with advanced HBV-related HCC can lead to hepatic carnitine leakage, contributing to the exhaustion of HBV-specific CD8⁺ T cells [106]. Among various immunometabolic targets, mitochondrial metabolism and reactive oxygen species (ROS) pathways are particularly significant in regulating T-cell functionality (Fig. 3).

The activation of T cells initiates glycometabolism to meet the energy demands required for their development from Tn to Teff. This process includes the activation of mitochondrial biogenesis, enabling the catabolism of glucose and glutamine [107]. Within T cells, glucose is catabolized through multiple pathways, dictated by the concentrations of available metabolites and cytokines. The primary pathways for glucose utilization include the tricarboxylic acid (TCA) cycle and anaerobic glycolysis, both essential for energy production. Additionally, the pentose phosphate pathway (PPP) converts glucose-6-phosphate into reduced nicotinamide adenine dinucleotide phosphate (NADPH). While PPP activation has been linked to oxaliplatin resistance in HCC tumor cells, its specific role in tumor-infiltrating T cells remains unclear [108]. Furthermore, glucose metabolism interconnects with ROS, redox balance, and intracellular signaling, linking fatty acid, and amino acid catabolism with the TCA cycle [109].

Glucose availability is crucial for T-cell growth, with the glycolytic rate in CD8⁺ T cells serving as an indicator of their immunoactivity [33, 110, 111]. Conversely, the hypoxic environment characteristic of HCC can upregulate 4-1BB signaling via hypoxia-inducible factor-1α, enhancing glycolysis and subsequently boosting CD8⁺ T-cell immunity and survival [77, 112]. However, evidence indicates that 4-1BB activation in HBV-related HCC can lead to excessive cytokine secretion by memory CD8⁺ T cells, potentially resulting in chronic inflammation and fibrosis [78]. Additionally, in hepatitis B, mitochondrial metabolism in Teffs is often suppressed, leading to excessive ROS production and reduced ATP levels, both contributing to T-cell exhaustion [46]. Thus, targeting glucose metabolism presents a promising strategy for restoring CD8⁺ T-cell function.

Among histone modifications, methylation and acetylation have been shown to correlate with T-cell functions [113]. In a chronic inflammatory microenvironment, the TCA cycle in CD8⁺ T cells sustains high levels of acetyl-coenzyme A (CoA), supporting histone acetylation and enhancing IFN-γ transcription [114]. However, the therapeutic application of CD8⁺ T-cell histone acetylation in HCC has yet to be explored.

Glucose metabolism is critical for T-cell proliferation and functional maturation. The activation of glycolysis and oxidative phosphorylation (OXPHOS) within CD8⁺ T-cell mitochondria promotes naïve T-cell proliferation and enhances CD8⁺ T-cell functionality [48]. Glucose transport across the cell membrane relies on glucose transporter 1 (Glut-1) and the glucose concentration within the TME. Combined CD28 and B7 signaling enhances T-cell proliferation, development, and functionality by activating the serine/threonine protein kinase B (AKT/PKB) pathway, which increases glucose uptake via Glut-1 [115]. Upregulating Glut-1 expression independently enhances the glycolysis of tumor-infiltrating CD8⁺ T cells [116]. However, tumor cells in the TME efficiently consume glucose, limiting the glycometabolic and immune activity of T cells and leading to T-cell exhaustion [117]. Notably, IFN-α treatment can increase glucose levels in the TME by reducing glucose consumption by HCC tumor cells, enhancing CD8⁺ T-cell cytotoxicity and improving PD-1 blockade efficacy [32]. This underscores the therapeutic potential of targeting glycometabolism in HCC. Additionally, other nutrients act as regulators of glycolysis and OXPHOS [118]. The glycometabolism of tumor-infiltrating immune cells is intricately connected to that of tumor cells within the TME, yet T-cell glycometabolism is often suppressed due to competition from tumor cells and other infiltrating immune cells. Positron emission tomography tracers have shown that MDSCs and TAMs are more efficient than T cells in glucose uptake and utilization [97]. Hepatocarcinoma cells promote proliferation by reprogramming glucose metabolism, altering metabolite concentrations in the TME, and impacting tumor-infiltrating immune cells.

ROS are generated during glycolysis in the mitochondria as byproducts of ATP and guanosine triphosphate (GTP) synthesis. ROS play a role in cellular signaling and gene expression, influencing T-cell lifecycle and activation [119‒122]. However, excessive ROS accumulation due to ROS-induced ROS release can lead to oxidative stress and damage to cellular organelles [119, 120, 123]. Studies on HCC tumor cell mitochondria indicate that mitoribosome defects contribute to elevated lactate and ROS levels in the TME, ultimately exhausting tumor-infiltrating T cells [124]. The interplay between ROS and mitochondrial oxidative stress can both positively and negatively impact CD8⁺ T-cell functionality. Restoring mitochondrial adaptability in CD8⁺ T cells may disrupt this cycle and enhance therapies targeting these cells [50]. Additionally, ROS production is a common pathway through which chronic inflammation impairs DNA damage repair mechanisms [125], leading to cell cycle deregulation, a hallmark of cancer [126]. Excessive ROS can also induce endoplasmic reticulum stress and mitochondrial dysfunction, causing T-cell apoptosis [127]. The relationship between ROS and CD8⁺ T-cell exhaustion is well-established [128]. Interestingly, ROS can also induce apoptosis in HCC cells via the c-Jun N-terminal kinase pathway [129, 130]. However, HCC tumor cells often adopt autophagy mechanisms to eliminate ROS, increasing their tolerance to ROS compared to tumor-infiltrating T cells [131]. Overall, the role of ROS within the HCC TME exhibits a dual nature, intricately linking tumor cells and T cells.

Lipids are a fundamental component of the eukaryotic plasma membrane, essential for energy storage and playing vital roles in cellular physiological activities and functions. Cholesterol, sphingolipids, and proteins work together to stabilize lipid rafts, which are critical for membrane signaling and trafficking [132]. Fatty acids are transported into the mitochondrial matrix, where they undergo β-oxidation, breaking down free fatty acids into acetyl-CoA. This fuels the TCA cycle and generates reduced nicotinamide adenine dinucleotide, supporting OXPHOS [133]. Here, we focus on lipid metabolism within the unique HCC microenvironment.

Disruptions in FAO metabolism are particularly significant in HCC. Tumor cells in the HCC microenvironment modify fatty acid metabolism, impacting CD8⁺ T cells, particularly in the context of obesity [134]. Hepatocytic steatosis related to liver injury, whether due to viral hepatitis or NAFLD, is similarly accompanied by dysregulated lipometabolism [135]. The accumulation of free fatty acids in the TME can cause cellular damage and alter metabolic flux in immune cells, including T cells [25]. Excess lipids can also suppress T-cell immunity through interactions with liver cells and other immune cells associated with CD8⁺ T cells. Elevated fatty acid levels may prompt liver cells to secrete acetate, potentially inducing CD8⁺ T cell-mediated autoimmunity [136]. Lipid accumulation in MDSCs can hinder DC antigen presentation, subsequently suppressing T-cell activation [137]. Additionally, de novo fatty acid synthesis and lipid signaling enhance regulatory T cells functions, leading to increased immunosuppression within the TME [138]. Conversely, FAO can act as an alternative energy source, helping alleviate the burden of accumulated fatty acids on CD8⁺ T cells. Upregulating FAO may enhance CD8⁺ T-cell antitumor immunity within the TME [48]. While FAO can offset energy deficits in T cells when glycolysis is suppressed, an increased FAO flux in effector T cells may also indicate exhaustion linked to PD-1 signaling [33].

Peroxisome proliferator-activated receptors (PPARs) are a key family of nuclear receptors involved in fatty acid metabolism and cellular energy regulation, showing promise in metabolic disease research. In the context of HCC, PPAR-α has been observed to stimulate FAO and gluconeogenesis in CD8⁺ T cells [139]. In contrast, PPAR-γ has been shown to restore the metabolic function and effector capabilities of tumor-infiltrating T cells within the HCC microenvironment [140]. However, inhibiting PPAR-γ in HCC tumor cells can suppress oncogenesis and metastasis by regulating PPAR-γ-dependent metabolic activities in these cells [141‒143].

Beyond fatty acids, cholesterol also plays a crucial regulatory role in T-cell signal transduction pathways. The TCR complex of CD8⁺ T cells consists of α and β TCR chains that associate with CD3 to recognize major histocompatibility complex I molecules, mediating immune functions. Cholesterol association with TCR-β can modulate phosphorylation signaling through allosteric transformation and nanocluster formation, enhancing T-cell immunoactivity and potentially aiding cancer immunotherapy [144, 145]. For instance, dedicator of cytokinesis protein 2, a potential marker for T-cell infiltration, has been inhibited by cholesterol sulfate synthesis in HCC tumor cells and could serve as an additional target for PD-1 blockade [146]. Research into cholesterol metabolism and regulatory checkpoints in T cells has focused on liver X receptors (LXRs) and sterol regulatory element-binding proteins (SREBPs), which are essential for maintaining intracellular cholesterol homeostasis. SREBPs, including SREBP1 and SREBP2 located in the endoplasmic reticulum, regulate the synthesis of 3-hydroxy-3-methyl-glutaryl-CoA and the transport of low-density lipoproteins, promoting intracellular cholesterol biosynthesis. In livers affected by nonalcoholic steatohepatitis (NASH), SREBP2 can induce excessive cholesterol production and accumulation, impairing antitumor immune surveillance by T cells in HCC [147]. Additionally, SREBP signaling has been linked to the clonal expansion of CD8⁺ T cells during viral infections [148]. However, the role of SREBP in HCC associated with viral hepatitis remains underreported.

Conversely, LXRs regulate cholesterol metabolism, leading to reduced intracellular cholesterol levels. Oxysterols can enhance LXRβ signaling while suppressing the SREBP2 pathway, thereby decreasing cholesterol levels in tumor-infiltrating CD8⁺ T cells. This reduction may induce autophagy-mediated apoptosis of T cells, contributing to weakened antitumor immunity in PLC [149]. Additionally, LXRα activation of the signal transducer and activator of transcription 3 pathway promotes the accumulation of secondary bile acids and oxysterols, further intensifying innate immune suppression within HCC [150]. Notably, differences in LXR content between tumor cells and T cells can dysregulate cholesterol-related signaling pathways, leading to competition for cholesterol within the TME.

Multiple nuclear receptors regulate lipid metabolism and transport, affecting T-cell functions; however, clinical applications targeting these processes in HCC remain limited [151]. Lipid-based immunotherapy, particularly strategies enhancing T-cell antitumor immunity, presents a promising avenue for cancer treatment [152]. Therefore, further exploration of the interactions between lipid metabolism and current T cell-targeted immunotherapies is warranted.

Amino acid metabolism plays a critical role in the biosynthesis, antioxidant production, and energy supply of CD8⁺ T cells through various intermediate metabolites. Upon activation, T cells uptake amino acids to sustain high metabolic rates and support protein and nucleotide biosynthesis [153]. Concurrently, activated Tn undergo protein catabolism to meet the amino acid demands necessary for differentiation into Teffs [154]. Amino acid transporters, essential for amino acid uptake and function in T cells, are classified into six categories, including the solute carrier (Slc) family, based on sodium dependency and substrate specificity [155]. Hypoxia-inducible factor-1α and the transcription factor c-Myc are known to upregulate Slc transporters, enhancing amino acid transport [156, 157]. Among these, Slc7a5 and Slc1a5 are particularly significant in amino acid metabolism for activated CD8⁺ T cells, especially in HCC [153]. The activities of Slc7a5 and Slc1a5 also play roles in HCC progression [158, 159]. Notably, Slc7a5 is part of the transport system for large neutral amino acids such as methionine and leucine, while Slc1a5 transports neutral amino acids like glutamine in the liver [155, 160, 161] (Fig. 4).

Methionine, a key amino acid transported by Slc7a5, has gained attention for its role in maintaining T-cell functions within the HCC microenvironment, particularly regarding nucleotide methylation processes [162]. S-adenosylmethionine (SAM), a metabolite derived from methionine, serves as an essential substrate for biological methylation and antioxidant production. Abnormalities in hepatic SAM levels can lead to liver injury and fibrosis, contributing to the development of PLCs [163]. The methionine salvage pathway, which includes metabolites like 5′-methylthioadenosine and SAM, is crucial for maintaining cellular methionine levels [164]. Research by Hung et al. [165] revealed a positive correlation between the methionine salvage pathway and tumorigenesis in HCC, where oncogenic reprogramming of methionine recycling in HCC cells results in elevated methylthioadenosine and SAM levels, ultimately exhausting CD8⁺ T cells. Genetically, SAM plays a pivotal role in CD8⁺ T-cell development as a primary methyl donor for DNA and histone methylation, which may contribute to CD8⁺ T-cell exhaustion in HCC [21, 165, 166]. Methionine and its metabolite levels could serve as potential biomarkers predictive of survival in HCC patients.

Notably, intracellular methionine can be converted to cysteine through transsulfuration, providing a substrate for glutathione (GSH) synthesis, which is essential for counteracting ROS-induced apoptosis in naïve T cells [167]. Glutathione depletion in HCC cells significantly contributes to ferroptosis and oxidative damage in tumor cells [168‒171]. To protect against oxidative cellular damage from excessive ROS generated during mitochondrial metabolism, T cells utilize amino acid metabolism to produce antioxidants. Elevated ROS levels in T cells stimulate GSH synthesis, regulated by glutamate cysteine ligase and the substrates glutamate, glycine, and cysteine [172, 173]. Depletion of cystine, a precursor for GSH synthesis, can lead to ferroptosis and dysfunction in CD8⁺ T cells [173]. The drug lenvatinib has been reported to inhibit the xCT system, leading to the accumulation of lipid-derived ROS and inducing ferroptosis in hepatoma cells [174]. Research by Toshida et al. [175] suggested that combining lenvatinib with inhibition of TP53-induced glycolysis and apoptosis regulator could enhance ferroptosis in hepatoma cells, thereby reducing tumor growth and intrahepatic metastasis in PLC and improving postoperative prognosis for HCC patients. Consequently, cysteine and GSH represent key points of nutrient competition between T cells and tumor cells. However, contrary to previous assumptions about its necessity for T-cell growth and function, the xCT transporter system for cysteine and glutamate transport has been shown to be non-essential for HCC tumor control [156, 176].

The production of antioxidants such as GSH and NADPH is closely linked to one-carbon metabolism, which is associated with glycine [177]. Teff specifically utilize extracellular serine uptake to supply glycine and one-carbon units necessary for de novo nucleotide synthesis [178]. However, limited research has focused on the role of one-carbon units and nucleotide biosynthesis in antioxidant production and the behavior of HCC tumor-infiltrating T cells.

Branched-chain amino acids (BCAAs) – especially leucine – provide an additional energy source for the T-cell lifecycle. Since BCAAs cannot be synthesized de novo in animals, supplementation in cancer or chronic liver disease patients could potentially enhance immune function [179, 180]. Leucine contributes carbon groups to form acetyl-CoA, which is essential for mitochondrial oxidation and TCA cycle intermediates [181]. Consequently, leucine deprivation in HCC tissues can reduce the amino acid energy supply, suppressing tumor cell proliferation and invasive capabilities [182]. The catabolism of BCAAs and associated energy pathways in both tumor cells and immune cells presents a potential target for HCC treatment. Furthermore, research by Yao et al. [116] highlighted the role of BCAAs in promoting OXPHOS in tumor-infiltrating CD8⁺ T cells. Impaired BCAA catabolism has been linked to enhanced tumor growth in HCC [183]. Conversely, earlier studies suggested that BCAA intake might counteract insulin-induced tumor proliferation [184]. Thus, the precise roles of BCAAs within the TME of HCC warrant further investigation.

Glutamine is a unique neutral amino acid transported by both Slc7a5 and Slc1a5, fulfilling critical roles across various aspects of amino acid metabolism. Glutamine supplies glutamate for de novo GSH synthesis, aiding in ROS defense in T cells [172]. While T-cell function traditionally relies on glucose metabolism, glutamine consumption is essential for T-cell proliferation, providing carbon and nitrogen for biosynthesis and cellular maintenance; however, the specific intermediate metabolites involved require further study [185]. In glucose-deprived conditions, adenosine monophosphate can be salvaged from glutamine through the purine salvage pathway, underscoring glutamine’s importance in nucleotide synthesis and energy production for T cells.

Overall, amino acid metabolism plays a critical role in supporting various functions of CD8⁺ T cells, including protein biosynthesis, antioxidant defense, and energy production, all of which are highly relevant for understanding the immune landscape in HCC. Further research into amino acid metabolism dynamics could offer valuable insights into therapeutic strategies to enhance T-cell responses in HCC.

mTOR is a critical serine/threonine kinase from the phosphoinositide 3-kinase-related kinase family that supports biogenesis and inhibits catabolic processes. It integrates various extracellular signals from the TCR, co-stimulatory receptors, cytokines, and microenvironmental cues, reprogramming metabolism to regulate CD8⁺ T-cell proliferation, differentiation, and function [44, 186‒191]. Tumor-infiltrating CD8⁺ T cells exert robust antitumor effects through AKT pathway activation, which is linked to mTOR, impacting immune responses and tumor progression through both immune cells and tumor cells [192]. While extensive research has explored the mTOR signaling pathway and its mechanisms, this discussion focuses specifically on its roles in CD8⁺ T cells within the HCC microenvironment (Fig. 5).

mTOR functions through two primary complexes, mTORC1 and mTORC2, which are composed of mTOR, unique subunits (Raptor for mTORC1 and Rictor/Sin1 for mTORC2), and stabilizing proteins like mammalian lethal with Sec13 protein 8. Together, these complexes are essential for T-cell metabolic balance [193, 194]. mTORC2 can phosphorylate AKT, which subsequently activates mTORC1 as well as downstream targets, including forkhead box protein O (FOXO), ribosomal protein S6 kinase, and the tuberous sclerosis complex. Tuberous sclerosis complex serves as a negative regulator of mTORC1 through the GTPase signaling pathway involving Ras homolog enriched in the brain (Rheb) [195‒197]. Although their roles overlap, mTORC1 primarily governs growth and metabolism, while mTORC2 is more involved in cellular longevity and structural organization [35]. Consequently, mTORC1 and mTORC2 exhibit distinct effects on metabolic processes – especially glucose metabolism – that influence T-cell differentiation into Teff or Tm phenotypes. Generally, mTORC1 signaling promotes the differentiation of naïve CD8⁺ T cells into Teffs while enhancing metabolic pathways that support functional development [28, 154].

During Teff maturation, mTORC1 activation influences early differentiation stages and enhances c-Myc expression, which is crucial for T-cell clonal expansion [155, 157]. Additionally, mTORC1 plays an essential role in redistributing the use of nutrients like glucose and glutamine to mitigate nutrient competition within the TME, thereby protecting Teffs from metabolic stress [97]. A notable feature of mTORC1 signaling is its response to cholesterol deficiency; the AKT/mTORC1 pathway activates SREBP2 to enhance cholesterol uptake, establishing a feedback loop that counteracts functional impairments in tumor-infiltrating CD8⁺ T cells due to low cholesterol levels [149]. Conversely, mTOR inhibitors can suppress tumors by inhibiting hepatocellular cholesterol biosynthesis [147]. While mTORC1 enhances CD8⁺ T-cell activity, it often does so at the expense of stability, resulting in a terminally differentiated effector phenotype with a shorter lifespan and reduced immune activity due to the inhibition of memory state transitions.

In contrast, suppressing mTORC2 appears to promote differentiation toward a memory phenotype, which may reduce immediate cellular immunity but extend the longevity of the T-cell population [28]. During the early stages of CD8⁺ T-cell differentiation, mTORC2 supports TCR and CD28 signaling, facilitating early glycolytic processes through sustained AKT activation [20]. The loss of Rictor, a critical mTORC2 subunit, has been shown to enhance FOXO1 phosphorylation, thereby promoting memory responses by shifting the energetic metabolic pattern [28]. Importantly, deficiencies in both mTORC1 and mTORC2 have been associated with CD8⁺ T-cell exhaustion in HCC, highlighting the relevance of mTOR signaling in immunometabolic therapies for this condition [198].

mTOR serves as an integrative hub for microenvironmental signals, governing the lifecycle of T cells, with nutrients and metabolites emerging as pivotal regulatory factors [27]. mTORC1 activation depends on amino acid signaling mediated by Slc transporters for amino acid metabolism [157, 158, 160, 193]. Stimulation from amino acids and their metabolites activates mTORC1 through GTPase activity and engagement of Rags 1/2 and Rheb signals [27, 162, 193, 195]. Moreover, GSH deficiencies can inhibit mTOR activity, creating a negative feedback loop that exacerbates ROS accumulation and impairs naïve T-cell activation [199]. Conversely, amino acid-dependent GSH biosynthesis can activate mTOR and promote c-Myc expression [172]. Leucine catabolism, in particular, activates mTORC1, supporting T-cell proliferation [180, 181, 200]. While the pathways and targets of mTOR regulation on CD8⁺ T-cell functionality have been broadly elucidated, specific and nuanced mechanisms remain to be fully explored.

As research continues into mTOR’s role in T-cell metabolism and functionality, its potential as a therapeutic target in HCC and other malignancies becomes evident. Understanding the interplay between nutrient availability, mTOR signaling, and CD8⁺ T-cell responses could lead to novel strategies to enhance antitumor immunity while mitigating tumor-induced metabolic dysfunction.

Ferroptosis is a distinct form of programmed cell death characterized by iron dependence and alterations in cellular redox status. Key hallmarks of ferroptosis include the inactivation of glutathione peroxidase 4 and the accumulation of lipid peroxides, ultimately leading to oxidative cellular damage [201]. Recent studies have shown that IFN-γ secretion, triggered by co-stimulation, can disrupt tumor cell lipid metabolism, promoting ferroptosis in these cells [202]. Additionally, the lipid-rich environment typical of the TME influences cellular susceptibility to ferroptotic death [203].

In the context of HCC, bevacizumab, an antibody targeting VEGF, has been shown to directly impact HCC lipid metabolism. This intervention induces ferroptosis in tumor cells and activates tumor-infiltrating CD8⁺ T cells, thereby enhancing their antitumor activity [204]. Ferroptotic death in HCC cells has effects beyond individual cells, including recruiting immune cells into the TME and decreasing PD-L1 expression on tumors, which collectively enhances the antitumor responses of tumor-infiltrating CD8⁺ T cells [205].

However, the bidirectional nature of ferroptosis in the TME warrants careful consideration. While ferroptosis in tumor cells can foster an immune-stimulatory environment, similar processes can occur in tumor-infiltrating lymphocytes, particularly CD8⁺ T cells. Cystine deprivation and excessive ROS production can induce oxidative stress, leading to ferroptotic death in these immune cells [173, 206]. The mechanisms underlying these processes – especially the role of cellular redox imbalance and ferroptosis in tumor-infiltrating CD8⁺ T cells within HCC – remain largely undefined.

Understanding these dynamics is crucial, as they highlight the dual role of ferroptosis in promoting antitumor immunity while potentially compromising T-cell functionality. Further research is needed to clarify how ferroptosis shapes the immune landscape in HCC, particularly for tumor-infiltrating CD8⁺ T cells. Investigating interventions that fine-tune the balance between tumor cell ferroptosis and T-cell survival may offer new therapeutic avenues for enhancing immune responses in HCC.

For advanced unresectable HCC, there has been a shift toward using multitarget kinase inhibitors and ICBs as competitive alternatives to traditional chemotherapy and radiation. However, the efficacy of these therapies is often limited by drug resistance and variability in patient sensitivity, affecting a significant portion of advanced HCC cases [3]. Resistance to chemotherapy and immunotherapy has been linked to alterations in the TME, driven by metabolic reprogramming resulting from changes in gene expression and cytokine regulation [108, 207, 208].

The TME plays a critical role in shaping locoregional treatments and pharmacological strategies beyond the direct impact of surgical interventions. While standard chemotherapies, radiotherapies, and multikinase inhibitors like sorafenib primarily target tumor cells and the vascular system, immune cells – particularly tumor-infiltrating CD8⁺ T cells – are essential for determining treatment efficacy and patient prognosis [44, 48, 53‒55, 70, 209]. For example, metformin has been shown to restore the mitochondrial health of tumor-infiltrating CD8⁺ T cells, enhancing the efficacy of anti-PD-1 therapy in patients with NASH [50].

Cytokine levels are indicators of CD8⁺ T-cell functionality and present opportunities for combination therapies, such as with CXCR4 inhibitors, which have shown comparable efficacy to sorafenib and anti-PD-1 therapies [210]. While many chemokines are recognized for their relevance in HCC, few have been directly targeted as therapeutic options.

Another area of exploration is targeting epigenetic modifications to improve ICB efficacy. For instance, enhancer of zeste homolog 2 (EZH2) is involved in epigenetic regulation via trimethylation of H3K27me3, influencing gene expression related to T-cell activation and function. Although research on EZH2 has primarily focused on its expression in tumor cells – where it has been implicated in oncogenesis, glycometabolic adaptation, and chemosensitivity – its role in tumor-infiltrating T cells remains less understood. EZH2 expression in T cells is associated with immune evasion mechanisms, including modulation of the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway, increased CXCR4 expression, and PD-L1 expression, ultimately impacting ICB efficacy [211‒214]. The overall role of EZH2 in T cells is still debated, but targeting T-cell epigenetics could represent a novel strategy for enhancing T cell-related therapies.

The interplay between functional molecules and metabolic pathways in immune cells is complex and significantly influences TME dynamics. Targeting metabolic pathways may inhibit tumor proliferation in HCC, as demonstrated by drugs like metformin, which regulates both adenosine monophosphate-activated protein kinas signaling in tumor cells and immune checkpoints in exhausted CD8⁺ T cells, thereby inhibiting HCC progression [50, 215]. Additionally, 7-ketocholesterol has been shown to reduce the effectiveness of doxorubicin in hepatoma cells via the phosphatidylinositide 3-kinase (PI3K)/AKT/mTOR pathway, highlighting the importance of metabolic factors in therapeutic outcomes [216].

Leading immunotherapeutic strategies for HCC often target metabolic pathways within T cells, with energetic metabolism-targeted therapies showing promise as adjuncts to ICBs [11, 98]. BCAA-dependent glycolysis has been associated with improved responses to PD-1 blockade, with BCAA intake linked to enhanced recovery in patients [116, 180]. Disrupting amino acid metabolism in tumor cells is being explored as a synergistic strategy alongside ICBs and other treatments [217]. Regarding ROS, targeting glutamine metabolism has shown potential to alleviate ROS-induced dysfunction in exhausted CD8⁺ T cells, while cystine and glutamate transport system knockout has been linked to improved ICB efficacy [176, 218, 219].

Moreover, aberrant expression of inhibitor of differentiation 1 in HCC has been linked to elevated ROS production and chemotherapy resistance, suggesting a connection between oxidative stress and treatment failure [108]. ROS-responsive nanoparticles have been proposed to overcome resistance to anti-PD-1 therapies in other cancers, but similar strategies remain unexplored in HCC [220]. Additionally, the upregulation of PD-L1 and recruitment of MDSCs observed in oxaliplatin-resistant HCC correlates with c-Myc expression, acting as a key link between mTOR activity and amino acid metabolism regulation [209, 221, 222].

Interestingly, while CD8⁺ T cells are typically seen as protective against tumors, they may also contribute to carcinogenesis in chronic liver conditions. For instance, in the context of NASH, CD8⁺ T cells can upregulate CXCR6 expression, leading to hepatocyte-targeted autoimmunity and exacerbating NASH pathology [136, 215]. This dual role underscores the need to critically assess the phenotype of tumor-infiltrating T cells in relation to HCC etiology before initiating ICB therapy.

Overall, the nuances of T-cell metabolism, epigenetics, and immune responses within the TME emphasize the complexity of HCC treatment and the need for integrated and innovative therapeutic approaches. Further research is essential to delineate these interactions and develop strategies that enhance antitumor immunity while mitigating risks associated with chronic liver disease and tumor progression.

The challenge of treating HCC is significantly compounded by high rates of late-stage presentation and recurrence, which complicate therapeutic strategies [223]. In response to these challenges, immunotherapy has emerged as a promising treatment modality for unresectable and advanced HCC cases [224]. However, the exploration of combination therapies remains crucial due to the currently limited efficacy of monotherapies [1]. Although various targets have been proposed to enhance the effectiveness of these combination approaches, the concept of immunometabolic therapy focusing on CD8⁺ T cells has not received the attention it deserves.

T cells operate as an integrated system where functional characteristics and metabolic pathways involving glucose, lipids, and amino acids are interrelated. Metabolites and nutritional substrates serve as signaling molecules that influence T-cell differentiation, proliferation, and antitumor responses. T-cell metabolism is shaped by signals transmitted via the TCR and related pathways, directly impacting differentiation and the expression of various immune molecules, including checkpoints. This foundational understanding suggests that metabolism-targeted therapies could ideally complement ICB and other immunotherapies. For instance, synergy has been observed between energetic metabolism, cytokines, anti-PD-1 therapy, and glycometabolic remodeling, underscoring the potential of such integrative approaches [32].

Notably, the functionality of PD-1^hi CD8⁺ T cells has been linked to fatty acid and amino acid metabolism, highlighting the need to further explore these metabolic pathways [48, 116, 156]. Mitochondrial redox reactions, as well as the TCA cycle and OXPHOS pathways, play critical roles in providing the energy and metabolites required for T-cell function. Unlike tumor cells, which may tolerate ROS, T cells often suffer from exhaustion, which can be intensified by ROS-induced mitochondrial dysfunction [128, 131]. Although mitochondrial defects in tumor-infiltrating T cells are known to contribute to their exhaustion, the role of T-cell mitochondria and ROS as independent therapeutic targets remains understudied.

From a signaling perspective, the mechanisms involving mTOR and downstream pathways – such as AKT and c-Myc – in CD8⁺ T cells have been elucidated; however, the potential of mTOR signaling in CD8⁺ T cells as a target for combination therapies has yet to be fully explored [28, 100, 183, 189].

In conclusion, leveraging the immune surveillance system and the robust antitumor capabilities of tumor-infiltrating CD8⁺ T cells holds great promise for enhancing HCC treatment outcomes. Research has primarily focused on the functional aspects of these T cells without adequately addressing changes in their lifecycle and metabolic patterns. Exploring combination therapies to maximize the efficacy of existing immunotherapies is crucial for improving CD8⁺ T-cell responses. Additionally, metabolism-targeting therapies represent a potential clinical strategy that could either complement existing treatments or serve as standalone options. Given the unique TME of HCC, further investigation into the metabolic profiles of HCC-infiltrating CD8⁺ T cells is essential to refine and optimize therapeutic strategies for this challenging disease.

The authors have no conflicts of interest to declare.

This work was supported by the Open Project Fund for the State Key Laboratory of Central Asian High Disease Pathogenesis and Prevention (SKL-HIDCA-2023-2); the National Natural Science Foundation of China (82360111); the Xinjiang Uygur Autonomous Region University research project (XJEDU2021I016); and the Tianchi Talent Recruitment Program of the Xinjiang Uyghur Autonomous Region and the Young Researchers’ Start-up Fund of the First Affiliated Hospital of Xinjiang Medical University (2023YFY-QKMS-03).

Y.L. and R.R. conceived the review, performed the majority of the work, and wrote the manuscript. Y.L., R.Z., T.T., and M.W. were responsible for preparing all the figures. D.Z., A.T., and Z.Y. assisted in checking the format and content of the manuscript. T.J. contributed to enhancing the quality of the language. T.A., and Y.S. are responsible for the guidance and quality check. Additionally R.R. provided financial support for the work. All authors read and approved the final manuscript.

Yanze Lin and Rexiati Ruze contributed equally to this work.