Abstract
T cell–based immunotherapies have transformed cancer treatment, yet their efficacy in solid tumors is constrained by the nutrient-poor and oxidative tumor microenvironment (TME). Accumulating evidence indicates that reactive oxygen species (ROS), methionine metabolism, and the amino acid stress sensor general control nonderepressible 2 (GCN2) are tightly interconnected regulators of T cell activation, differentiation, and effector function. In this review, we detail how these pathways form an integrated redox–metabolic circuit that dynamically tunes T cell responses to environmental stress. Physiological ROS are essential for T cell receptor signaling, glycolytic reprogramming, and cytotoxicity, whereas excessive or prolonged oxidative stress drives exhaustion and apoptosis. GCN2 links amino acid availability, particularly methionine and cysteine, to adaptive transcriptional and metabolic programs that regulate glutathione synthesis and redox homeostasis. We highlight how therapeutic manipulation of methionine availability, GCN2 signaling and ROS produces highly context-dependent outcomes across immune checkpoint blockade and adoptive cell therapy settings in solid tumors. Finally, we discuss emerging strategies to interrogate and modulate this circuit using integrated omics, CRISPR-based screening, and pharmacological approaches, emphasizing the need for context-aware and temporally controlled metabolic interventions to enhance T cell–based immunotherapies in solid tumors.
Introduction
Metabolism has emerged as a central driver of T cell anti-tumor immunity. It is well established that T cells shift their metabolism based on their differentiation state and functional capacity: namely, T naïve (Tn) and T memory (Tm) cells mainly rely on oxidative phosphorylation (OXPHOS) for their bioenergetic needs, whereas T effector (Te) cells, which are dominantly responsible for tumor killing, are fueled by glycolysis (–). Similarly, exhausted T cells display reduced glycolytic flux and spare respiratory capacity (). This knowledge has laid a foundation for modulating T cell metabolism as a strategy to augment therapeutic need, such as strengthening immunotherapies to treat solid tumors. For example, T cell-based cancer immunotherapy is now being looked at through the lens of metabolic engineering, conditioning and pharmacological intervention (–).
Redox metabolism, methionine metabolism, and the general control nonderepressible 2 (GCN2) pathway have all garnered increasing attention in the cancer immunotherapy field as potential therapeutic targets. This review will highlight the underappreciated connection between GCN2, methionine metabolism, and the transsulfuration pathway, and how these pathways collectively tune the redox state of T cells. We will discuss these pathways as components of an integrated metabolic circuit that is essential for T cell function and that can be manipulated to enhance cancer immunotherapy.
Sources of ROS in T cells
Redox metabolism encompasses the cellular oxidation–reduction reactions that occur within cells, often mediated by redox cofactors such as NAD+/NADH, as well as the production of reactive oxygen species (ROS) and the antioxidant systems that regulate and eliminate ROS (). The primary focus of this review will be on the production and downstream functions of ROS, however, other components of redox metabolism will be briefly discussed.
ROS are a byproduct of metabolic pathways in T cells, while also playing an indispensable role in T cell biology. The primary contributors to ROS production reside in the mitochondria, which supply over 90% of cellular ROS (). The electron transport chain (ETC), a component of OXPHOS and the dominant producer of adenosine triphosphate (ATP), is located in the inner mitochondrial membrane. During the electron transport, superoxide (O2-) is generated through reactions catalyzed by complex I, II and III (). Most superoxide is rapidly converted into hydrogen peroxide (H2O2), which can freely diffuse into the cytosol (, ).
Another important source of ROS in T cells is the family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, including NOX2 and DUOX2. These enzymes play a role in maintaining redox cycling during the many metabolic reactions activated upon T cell receptor (TCR) stimulation (). NOX2 produces superoxide as a byproduct of NADPH oxidation to NADP+ at the plasma membrane ().
The endoplasmic reticulum (ER) also contributes to ROS production in T cells (). Proteins that contain a disulfide bond, such as the TCR, produce hydrogen peroxide as a byproduct of disulfide bond formation. This process occurs through the oxidation of protein disulfide isomerase (PDI), a molecular chaperone critical for disulfide bond formation and rearrangement, which transfers electrons to molecular oxygen during catalysis (). Furthermore, the ER indirectly influences ROS through calcium (Ca2+) signalling upon TCR stimulation (). Calcium influx into mitochondria enhances ETC activity, thereby increasing mitochondrial ROS (mtROS) generation (). Another indirect contribution of the ER to ROS arises during protein overload, when the ER reaches its folding capacity and activates the unfolded protein response (UPR). Although the UPR ultimately induces antioxidant programs, it initially increases ROS levels through the activation of stress response pathways (, ).
Finally, a recurring theme in this review is amino acid deprivation as a stimulator of ROS accumulation. Amino acid limitation can indirectly increase ROS through dysregulated mitochondrial dynamics and ER stress. This review, however, will primarily focus on glutathione (GSH) depletion as a central mechanism of ROS dysregulation. GSH is the primary intracellular redox buffer in T cells and plays an essential role in supporting anti-tumor responses (). Deprivation of amino acids required for GSH synthesis, particularly methionine, cysteine, and glycine limits GSH production, leaving T cells unable to adequately buffer ROS ().
Overall, multiple pathways in T cells indirectly or directly contribute to ROS production, including ones not mentioned here (e.g. ROS produced in the peroxisome), with the mitochondrial ETC being the dominant means of ROS production.
Measuring cellular ROS
The simplest and most widely used methods to detect ROS rely on fluorescent dyes that can be quantified by flow cytometry or microscopy. Common examples include 2’,7’-dichlorodihydrofluorescein diacetate (DCF), MitoSOX Red, and dihydroethidium (DHE) (). Additionally, ROS can also be assessed using genetically encoded sensors, such as roGFP2-based probes or members of the HyPer family, which allow for more targeted and dynamic measurements (). In addition to these direct approaches, ROS can be measured indirectly through downstream readouts, including the GSH/GSSG and NADPH/NAD+ ratios, often quantified using metabolomics-based techniques (). Each of these methods has distinct advantages and limitations and is best suited to specific experimental contexts, as reviewed extensively by Murphy et al. (). Refer to Table 1 for a summary of methods to measure ROS.
Table 1
| Category | Method / probe | What it measures | Key advantages | Key limitations / notes |
|---|---|---|---|---|
| Fluorescent dyes (general ROS) | DCF (H2DCFDA) | Broad intracellular ROS (mainly H2O2-derived oxidation) | Simple, widely used, flow cytometry compatible | Non-specific; oxidation-dependent signal; prone to artifacts |
| CellROX dyes | General oxidative stress | More stable than DCF; good for imaging | Still relatively non-specific | |
| Superoxide-specific dyes | DHE | Cytosolic superoxide (O2•−) | Common, relatively sensitive | Can form multiple oxidation products; requires careful interpretation |
| MitoSOX Red | Mitochondrial superoxide | Mitochondria-targeted; useful for metabolic studies | Can be oxidized non-specifically at high ROS | |
| Genetically encoded sensors | roGFP2 (± Grx1, Orp1) | Redox potential / H2O2 (depending on fusion) | Ratiometric, reversible, quantitative | Requires transduction; lower throughput |
| HyPer family | H2O2 specifically | High specificity, real-time dynamics | pH sensitivity; needs controls | |
| Indirect metabolic readouts | GSH/GSSG ratio | Cellular redox buffering capacity | Quantitative, biologically meaningful | Indirect; does not measure ROS species directly |
| NADPH/NADP+ ratio | Redox metabolism / antioxidant capacity | Links to metabolic state | Indirect; influenced by many pathways | |
| Mass spectrometry / metabolomics | Untargeted or targeted metabolomics | Redox metabolites (e.g., glutathione, cysteine) | High depth, systems-level insight | Expensive; indirect |
| Protein oxidation markers | Protein carbonylation, sulfenylation (e.g., dimedone-based probes) | Oxidative damage to proteins | Biologically relevant readout | Endpoint measurement; not dynamic |
| Lipid peroxidation assays | MDA, 4-HNE | Oxidative damage to lipids | Clinically relevant biomarkers | Indirect; reflects accumulated damage |
| DNA oxidation | 8-oxo-dG | Oxidative DNA damage | Useful for stress/damage studies | Not a real-time ROS measure |
| Electron spin resonance (ESR/EPR) | Spin trapping of free radicals | Direct detection of specific ROS species | Gold standard, highly specific | Technically demanding, low throughput |
| Mitochondrial function assays | Seahorse (OCR/ECAR) | Indirect ROS-linked metabolic stress | Functional metabolic context | Does not measure ROS directly |
Methods of ROS detection.
Antioxidant pathways in T cells
Given the multitude of metabolic pathways that generate ROS in T cells, robust antioxidant systems are required to prevent ROS accumulation beyond levels compatible with optimal T cell function. As noted above, the primary antioxidant hub in T cells is the GSH pathway, which is readily available in the cytosol during periods of elevated ROS production, such as TCR stimulation (). GSH is highly nucleophilic, making it an effective scavenger of ROS. With catalytic assistance from glutathione peroxidases (GPXs), GSH reduces a wide range of ROS to water (, ).
Another important antioxidant system in T cells is the peroxiredoxin (PRDX) family. PRDX enzymes primarily regulate ROS generated during TCR signalling, rather than ROS produced during overt stress responses. There are six PRDX isoforms, each of which plays distinct roles in T cells (–). PRDX1 and PRDX2 are cytosolic and contribute to ROS regulation. However, PRDX1 additionally participates in cellular signaling, whereas PRDX2 functions predominantly as an antioxidant (). PRDX3 and PRDX4 scavenge ROS within the mitochondria and ER, respectively, while PRDX5 and PRDX6 localize to multiple cellular compartments ().
Additional detoxification systems include the thioredoxin (TRX1/2) pathway, which is particularly important for neutralizing ROS generated during disulfide bond formation in the ER, and superoxide dismutase 2 (SOD2), which catalyzes the conversion of superoxide into hydrogen peroxide (, ). Beyond enzymes that directly detoxify ROS, several metabolic pathways indirectly support antioxidant capacity. For example, the pentose phosphate pathway (PPP) regenerates NADPH, which is required to maintain GSH in its reduced state (). Similarly, the transcription factor NRF2 is activated under oxidative stress and drives the expression of numerous antioxidant genes, including those encoding PRDXs and enzymes involved in GSH metabolism ().
ROS: a careful balance
Maintenance of ROS homeostasis is critical across all cell types, and T cells are no exception. ROS levels increase dramatically upon TCR stimulation, yet ROS are not merely metabolic byproducts. Instead, ROS produced by T cells and surrounding immune cells are essential for TCR signaling and sustained effector function. Conversely, excessive ROS accumulation leads to DNA damage, induction of p53 and Fas ligand (FasL), and ultimately T cell apoptosis (Figure 1), which can be advantageous in terms of controlling autoimmunity (). However, in environments such as the tumor microenvironment [e.g. pancreatic ductal adenocarcinoma (PDAC), lung cancer, melanoma, etc.], excessive ROS can cause high T cell dysfunction and death (–). Many reviews have covered the topic of ROS balance in T cells extensively (–).
Figure 1
Given this finite balance, it is not surprising that ROS are generated by multiple pathways following TCR activation and, in parallel, eliminated by multiple antioxidant systems. This redundancy is essential to maintain ROS at concentrations that support, rather than impair, T cell function.
What is the GCN2 pathway?
General Control Non-derepressible-2 (GCN2) is a translational and metabolic regulator that coordinates cellular processes in response to stress, including amino acid deprivation (). Upon depletion of amino acids, GCN2 binds to uncharged tRNAs – tRNAs not attached to amino acids – and becomes activated through autophosphorylation (). Activated GCN2 phosphorylates Eukaryotic Translation Initiation Factor 2α (eIF2α), resulting in a global reduction in translation while selectively enhancing the translation of mRNAs containing upstream open reading frames (uORFs), including Activating Transcription Factor 4 (ATF4) mRNA (–). The GCN2-ATF4 pathway subsequently induces the expression of genes involved in amino acid transportation and metabolism (, ). In addition, this pathway also promotes survival through AKT activation, suppression of mTORC1 and induction of autophagy (–), the latter includes mitophagy (57), which plays a key role in limiting excess ROS production by removing damaged mitochondria (58). The GCN2-ATF4 pathway also modulates redox homeostasis by inducing the expression of Glutathione peroxidase 1 (GPX1), a key antioxidant enzyme (59). Moreover, the pathway has also been shown to activate the NRF2 signaling pathway in macrophages in response to leucine depletion (60).
The GCN2-ATF4 axis further regulates redox balance by upregulating amino acid transporters, including SLC1A4, SLC7A5/SLC3A2 (LAT1), SLC7A11 (xCT), and SLC1A5 (ASCT2), thereby increasing uptake of methionine and cysteine, among other amino acids (, ). Both methionine and cysteine contribute to GSH biosynthesis. In addition, in response to cysteine depletion, the GCN2-ATF4 pathway maintains redox homeostasis by inducing enzymes of the transsulfuration pathway, which synthesizes cysteine from methionine in a methionine cycle-dependent manner (61). Thus, methionine and cysteine connect nutrient sensing to ROS regulation through GCN2 signaling and GSH synthesis.
Methionine and cysteine fuel GSH synthesis through the methionine cycle and the transsulfuration pathway
GSH is synthesized through two energy-dependent steps. The first, rate-limiting step involves the ligation of cysteine to glutamate to form γ-glutamylcysteine, catalyzed by γ-glutamylcysteine ligase (GCL). The second step is mediated by glutathione synthase (GS), which conjugates γ-glutamylcysteine with glutamine to generate GSH (). This process is primarily limited by the availability of cysteine (62).
T cells acquire cysteine through the transporters SLC1A4 and SLC1A5, as well as the cystine (oxidized cysteine) transporter, xCT (63). Alternatively, T cells can acquire cysteine de novo synthesis by converting methionine to cysteine through the methionine cycle and the transsulfuration pathway, a process that is crucial for promoting T cell survival under oxidative stress (64). In the methionine cycle, methionine is first converted to homocysteine, losing a methyl group in the process (65). Homocysteine is then ligated with serine by cystathionine β-synthase (CBS) to form cystathionine, which is subsequently cleaved by cystathionine γ-lyase (CTH) to generate cysteine (61).
Beyond its role in fueling GSH synthesis, the methionine cycle also produces metabolites essential for multiple crucial cellular functions. One of such metabolites, S-adenosylmethionine (SAM), serves as a universal methyl group donor required for proteins, RNAs, and DNA methylation. SAM shapes T cell activation, differentiation, and antitumor function through epigenetic and posttranscriptional modifications (66–70). The role of SAM in epigenetic regulation of T cell activation and effector function has been reviewed previously (65). Additionally, SAM is required for de novo polyamine synthesis, which governs the fidelity of T cell differentiation and can suppress T cell anti-tumor function (71–74). Besides SAM, other metabolites derived from the methionine cycle also contribute to the regulation of T cell responses, as reviewed previously (65).
Section 1: importance of ROS, GCN2, and methionine pathways in regulating T cell anti-tumor immunity
Importance of ROS in T cells
Reactive oxygen species (ROS) play an essential role in T cell activation, subset formation, and effector function. The most direct role that ROS play in T cell function occurs during activation. Once the T cell receptor (TCR) is stimulated along with a costimulatory signal from an antigen presenting cell (APC), mitochondrial electron flux is increased. This results in a substantial buildup of hydrogen peroxide in the cytosol, which works to reversibly oxidize catalytic cysteine residues in phosphatases, ultimately preventing them from dephosphorylating key downstream TCR signaling proteins, including ZAP-70, PTEN, and LAT (75, 76). Importantly, ROS-mediated signaling during T cell activation occurs within a tightly regulated spatial and temporal window, as excessive diffusion of hydrogen peroxide would otherwise oxidize iron–sulfur cluster–containing electron transport chain (ETC) components and impair mitochondrial function. Activation of TCR signalling proteins leads to robust signal amplification that drives the imminent effector phenotype of the T cell. Similarly, ROS enhance calcium signaling by sustaining store-operated calcium entry (SOCE) activity, thereby prolonging calcium flux into the cytosol (77). Calcium signaling plays many important roles in T cell activation; notably, it is essential for the translocation of Nuclear Factor of Activated T cells (NFAT) into the nucleus (Figure 1) (78). Furthermore, ROS play a multitude of additional roles in initiating the T cell response, such as activating key transcription factors (e.g., NF-κB, AP-1) and the mechanistic target of rapamycin complex 1 (mTORC1), the master regulator of cellular metabolism (79).
ROS also play a critical role in delineating T cell subset formation and, reciprocally, cytokines, the messengers of the immune system, greatly influence ROS. As a byproduct of amplified TCR activation, T cells shift toward an effector phenotype, the key subset responsible for tumor killing (80, 81). In CD4 T cells, this ROS-induced effector phenotype is characterized by a bias toward T helper 1 (Th1) cells, the dominant anti-tumor CD4 subset, as well as Th17 cells (, 82). Conversely, this phenotype is reversed if high levels of ROS linger after activation, in which case a Th2 phenotype is promoted (83). Likewise, low ROS levels during initial activation can favor regulatory T cell (Treg) formation by promoting a shift toward oxidative phosphorylation (OXPHOS) and stabilizing FOXP3 (82, 84). Furthermore, antioxidant defenses are essential for maintaining Treg suppressive capacity, and treatment with N-acetyl cysteine (NAC), an ROS scavenger, can reverse Treg dysfunction induced by elevated ROS levels (84, 85). When T cell activation is sustained for prolonged periods, such as within the tumor microenvironment, elevated cellular ROS can push T cells toward an exhausted phenotype due to damage to DNA and mitochondria (86, 87). Following the initial ROS spike after activation, intracellular ROS levels decline. Memory T cell formation depends on moderate levels of mitochondrial ROS generation, originating from complex III (88). Together, these findings highlight the “Goldilocks” role of ROS: too little fails to induce effector T cell formation and instead promotes Tregs, whereas excessive or prolonged ROS drives exhaustion and apoptosis.
T cell–mediated tumor killing relies on cells that adopt a Warburg-like metabolic phenotype characterized by high glycolytic flux (89–91). Strong evidence suggests that ROS play an important role in amplifying and maintaining a T cell glycolytic phenotype through multiple mechanisms. Primarily, ROS sustain TCR signaling via PTEN inactivation, activating mTORC1 and AKT to promote the translation and stabilization of glycolytic enzymes (92–95). In addition, ROS stabilize and enhance hypoxia-inducible factor-1α (HIF-1α), the oxygen-sensitive subunit of the HIF-1 transcription factor, by inhibiting prolyl hydroxylase–mediated degradation under both hypoxic and inflammatory conditions. Stabilized HIF-1α translocate to the nucleus, where it dimerizes with HIF-1β and drives the transcription of genes that promote glycolytic metabolism, including glucose transporters and key glycolytic enzymes such as GLUT1 and lactate dehydrogenase A (LDHA), thereby reinforcing a metabolic shift toward aerobic glycolysis (94–96). Similarly, ROS play a critical role in NFAT activation, which promotes the transcription of genes that support glycolysis. NFAT induces expression of genes such as MYC, a key regulator of glycolytic metabolism, and IL-2, which signals through the PI3K–AKT–mTOR pathway to enhance glycolytic activity (, 97). In addition, NFAT can increase chromatin accessibility by recruiting the histone acetyltransferases CBP/p300, leading to H3K27 acetylation and facilitating the binding of glycolysis-promoting transcription factors, including HIF-1α (98, 99). Collectively, ROS-supported signaling and transcription factor driven glycolytic programming promote the secretion of effector molecules, including IFN-γ and perforin/granzyme, that are essential for T cell function.
Taken together, ROS play an indispensable role in promoting anti-tumor immunity by sustaining T cell activation, biasing differentiation away from a Treg phenotype, and promoting the glycolytic metabolism required for T cell cytotoxicity. Importantly, ROS enhance T cell–mediated anti-tumor immunity only within a low to moderate range, whereas excessive ROS drive T cell exhaustion and apoptosis.
GCN2 regulates T cell anti-tumor function by sensing amino acid depletion
Not only are amino acids building blocks for protein synthesis, but they also fuel metabolic pathways to support T cell proliferation and function. For example, methionine supports epigenetic reprogramming during T cell differentiation by generating the methyl group donor SAM (66, 67). Cystine, cysteine and methionine are essential for maintaining redox balance during T cell activation, as they are required for GSH synthesis (64, 100). Serine and glycine serve as key precursors for purine synthesis in T cells, enabling proliferation upon activation, while glutamine fuels the tricarboxylic acid (TCA) cycle to support ATP and pyrimidine synthesis during T cell activation (101, 102). In addition to these metabolic roles, several amino acids directly regulate T cell anti-tumor immunity. Arginine promotes oxidative phosphorylation and drives T cell memory formation, thereby enhancing anti-tumor function (103). The branched-chain amino acids, leucine, isoleucine, and valine, enhance glucose metabolism through the activation of mTOR, leading to improved anti-tumor function of T cells (100, 101). In addition, tryptophan depletion arrests T proliferation and sensitizes T cells to apoptosis (104). Moreover, the catabolism of tryptophan into kynurenine by indoleamine 2,3-dioxygenase (IDO) downregulates TCR ζ-chain expression and promotes the differentiation of regulatory T cells, contributing to immunosuppression (105).
However, the tumor microenvironment is often nutrient-poor, including depletion of specific amino acids (106). For example, arginine, cystine, and tryptophan are depleted in the interstitial fluid of pancreatic ductal adenocarcinoma (PDAC) (107). Similarly, glutamine, glutamate, and arginine levels were downregulated in melanoma interstitial fluid (108). As such, a mechanism to sense and respond to amino acid depletion may be crucial for T cell anti-tumor response in solid tumors.
One such mechanism is the GCN2 pathway. Early studies on the role of GCN2 in T cells have primarily focused on its role in IDO-mediated immune suppression. IDO converts tryptophan into the immunosuppressive metabolites kynurenine, resulting in tryptophan depletion and kynurenine accumulation – an immune tolerance mechanism often hijacked by tumors. These studies demonstrated that GCN2-deficient T cells are resistant to IDO-mediated proliferation arrest and TCR ζ-chain downregulation, suggesting that GCN2 is crucial for IDO-mediated immune suppression (105, 109). However, GCN2 is not required for T cell proliferation arrest induced by tryptophan depletion alone. In contrast, subsequent studies showed that GCN2 upregulates stress response genes during T cell activation under amino acid-restricted conditions and is required for optimal T cell proliferation upon antigen stimulation, suggesting a supportive role for GCN2 in T cell activation in nutrient-restricted environments (110). Interestingly, one study reported that GCN2 is dispensable for T cell anti-tumor activities in murine melanoma models, as T cell-specific GCN2 knockout neither impairs T cell responses against tumors, nor does it significantly affect FoxP3 expression in CD4+ T cells or IFN-γ expression in both CD4+ and CD8+ T cells in the tumor. Notably, GCN2 was not activated in T cells in these tumors, likely due to sufficient intratumoral tryptophan levels (111). By contrast, a more recent study demonstrated that GCN2 is crucial for CD8+ T cell anti-tumor function in murine glioma models exhibiting IDO activities. In this setting, knocking out GCN2 reduced CD8+ T cell survival and protein kinase C θ (PKCθ) phosphorylation under low-tryptophan conditions in vitro, and GCN2 knockout leads to decreased CD8+ T cell numbers, and reduced CD44 and granzyme B expression in CD8+ T cells within the tumor, resulting in reduced survival (112). Consistent with these findings, treatment of activated CD8+ T cells ex vivo with a GCN2 agonist halofuginone (halo) increased IFN-γ and Granzyme B expression. In addition, halo-treated OTI T cells showed enhanced anti-tumor function in murine lymphoma models, leading to reduced tumor growth and improved survival. Mechanistically, halo augments T cell effector function by increasing oxidative phosphorylation and activating the CD98 (SLC7A5/SLC3A2)-mTOR axis. Interestingly, halo-treated CD8+ T cells also showed elevated intracellular methionine and glutathione (GSH) levels, suggesting enhanced antioxidant response induced by GCN2 (113). Whether this increased antioxidative response directly contributes to improved effector function remains to be determined.
Role of methionine in T cell anti-tumor function
Studies examining the role of methionine in regulating T cell anti-tumor function have yielded conflicting findings. In cases where tumor cells outcompete T cells for methionine, methionine depletion reduces levels of the histone methylation mark H3K79me2, which decreases the expression of signal transducer and activator of transcription 5 (STAT5) in endogenous CD8+ T cells. Consequently, expression of IL-2, TNF-α, and IFN-γ was reduced, leading to impaired anti-tumor function (68). Additionally, reduced H3K79me2 levels caused by tumor-driven methionine depletion also increases the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) in endogenous CD4+ T cells, further suppressing T cell function (69). Restoring methionine availability in the tumor microenvironment rescues T cell function and improves tumor burden control, suggesting that methionine supports the anti-tumor function of endogenous T cells (69). Consistent with these findings, a recent study demonstrated that methionine restriction during early T cell activation induces T cell exhaustion by inhibiting methylation of the potassium channel KCa3.1, resulting in increased Ca2+ influx and excessive activation of NFAT (70).
Conversely, studies examining the effects of methionine in the context of immune checkpoint blockade (ICB) therapies indicate a more suppressive role. For example, dietary methionine restriction (MR) in combination with PD-1 blockade increases the abundance of tumor-infiltrating CD8+ T cells and enhances granzyme B expression. Crucially, the increase in CD8+ T cell effector function is associated with reduced presence of the immunosuppressive M2 macrophages in the tumor, which may result from suppressed M2 polarization driven by increased ROS production and nuclear factor of activated T cells 5 (NFAT5) expression under MR (114). These findings suggest that methionine indirectly suppress anti-tumor T cell function by promoting immunosuppressive macrophages. Consistent with this, Li et al. reported that dietary MR synergizes with PD-1 inhibition by reducing methylation of immune checkpoint mRNAs, including programmed death ligand 1 (PD-L1) and v-domain Ig suppressor of T cell activation (VISTA), in tumor cells, thereby enhancing CD8+ T cell infiltration and increasing granzyme B and IFN-γ expression (115). Similarly, a recent study showed that dietary MR enhances the efficacy of PD-1 blockade by increasing T cell number and granzyme B expression within tumors. Mechanistically, dietary MR was found to downregulate proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of MHC-I expression and TCR signaling, through inhibition of DNA methylation (116). In line with these findings, two additional studies suggest a suppressive role for methionine in anti-tumor immunity in the context of ICB, but these studies did not examine T cells (117, 118). In contrast, one study reported that methionine supports T cell activation and anti-tumor immunity during ICB by supporting hydrogen sulfide production via the gut microbiota, indicating that methionine promotes T cell anti-tumor function in the presence of ICB (119). Consistently, supplementation of methionine through peritumoral injection reduces the expression of inhibitory histone methylation marks in CD8+ T cells and enhances the efficacy of ICB (70).
Beyond these conflicting observations, recent work has shown that intermittent dietary MR, rather than sustained dietary MR, accelerates tumor ferroptosis and enhances T cell infiltration and effector function. These findings suggest that the role of methionine in regulating T cell anti-tumor function is not only context-dependent but also time-dependent (120).
Lastly, although many studies have focused on methionine’s role in ICB, its impact on adoptive T cell therapies (ACT) has received considerably less attention. To address this gap, a recent study examined the effect of MR specifically on activated T cells, as T cells are activated and expanded ex vivo prior to infusion in ACT. Methionine was shown to play a time-dependent roles in regulating the effector function of activated T cells. Culturing activated T cells transiently in MR media inhibits synthesis of the immune suppressive polyamines, spermidine and spermine, leading to enhanced IFN-γ production and cytotoxicity against lymphoma cells in vitro. In contrast, prolonged exposure to MR in vitro induces expression of inhibitory receptors PD-1 and TIM-3. However, transient MR conditioning of CD8+ T cells prior to adoptive transfer into tumor-bearing mice does not improve anti-tumor efficacy, possibly because their function is reversed upon entering a methionine-replete environment. In contrast, sustained dietary MR reduces tumor control and overall survival, potentially due to increased inhibitory receptor expression, suggesting that continuous methionine availability is crucial for ACT efficacy (74). Consistent with this, Sharma et al. reported that daily peritumoral injection of methionine improves ACT efficacy in murine tumor models (70). Together, these findings suggest that methionine is required to support the anti-tumor function of adoptively transferred T cells.
GCN2-Methionine-ROS metabolic circuit: role in tuning T cell function
GCN2, methionine, and ROS each play independent yet integrated roles in tuning T cell responses to tumors, such that modulation of one pathway inevitably influences the others. Here, we outline how these pathways from a crucial metabolic circuit that is a key determinant of T cell function (Figure 2).
Figure 2
As mentioned, the primary trigger for GCN2 activation is amino acid deprivation, which results in the accumulation of uncharged tRNAs. High ROS environments can trigger amino acid deprivation in multiple ways, including oxidizing amino acids, such as methionine. MetO, the oxidized form of methionine, is a poor substrate for MetRS, the methionine-specific tRNA (, 59, 121). GCN2 activation leads to downstream activation of ATF4, a central mediator of the integrated stress response (ISR) (). ATF4 regulates a broad transcriptional program, including rerouting methionine into the methionine salvage and transsulfuration pathways. The transsulfuration pathway supports the synthesis of GSH, a critical antioxidant in T cells (61). In parallel, the ISR promotes expansion of intracellular amino acid pools, largely through the upregulation of amino acid transporters. Thus, two major outcomes of GCN2 activation are increased amino acid availability and reduced cellular ROS levels ().
The GCN2–methionine–ROS circuit becomes particularly important when considering T cells within the tumor microenvironment (TME). In this context, GCN2 activity is elevated due to sustained environmental stress, including limited amino acid availability and high oxidative burden (109). This milieu can drive constitutive GCN2 activation, ultimately leading to reduced cellular ROS levels and the potential for impaired effector function (109). This phenomenon may play an integral role in dampening T cell activity within the TME. Therefore, modulation of the GCN2–methionine–ROS circuit may represent a promising strategy to enhance T cell–based immunotherapies for solid tumors.
Section 2: therapeutic modulation
Manipulating ROS to enhance T cell anti-tumor immunity
Given that reactive oxygen species (ROS) play multiple roles in priming T cells for a potent anti-tumor response, it is reasonable to suggest that modulating ROS may improve T-cell immunotherapies against solid tumors. Rather than operating as two mutually exclusive strategies, redox modulation could be designed to do both: engineering T cells to withstand chronic, damaging oxidative stress within the tumor microenvironment (TME) while preserving, or enhancing, transient, signaling-competent ROS required for effector function. This strategy becomes particularly important in solid tumors, where immunotherapy has shown limited success.
Several studies have demonstrated that disruption of the NRF2 gene, which encodes a transcription factor responsible for inducing many antioxidant proteins, enhances T cell anti-tumor immunity. One study showed that NRF2 expression was increased in tumor-infiltrating lymphocytes (TILs) isolated from a hypoxic TME compared to control T cells (122). Likewise, IFN-γ production was dramatically increased in NRF2-deficient cells (122). Another study reported similar findings, showing that deletion of Nrf2 in a murine adoptive T cell therapy (ACT) model enhanced tumor-killing capacity across several solid tumor models, including melanoma, lymphoma, colon carcinoma, and lung carcinoma (123). A separate group demonstrated that pre-treating T cells with auranofin prior to adoptive transfer, an Nrf2-activating compound, increased T cell effector function and tumor killing in a blood cancer model (124). These seemingly contradictory results suggest that NRF2 may play a context-dependent role in modulating T cell function, such as differences between solid and liquid tumors or between high- and low-oxidative-stress environments.
Similarly, one study showed that Tmed4(-/-) mice had endogenous regulatory T (Treg) cells that shifted toward a more inflammatory phenotype with enhanced anti-tumor activity in a colorectal cancer model (84). TMED4 is a potent activator of NRF2, and loss of TMED4 prevents NRF2 from inducing an antioxidant state within the cell. Indeed, the authors demonstrated that the increase in anti-tumor function resulting from Tmed4 deletion was due to elevated ROS levels, which contributed to inhibition of FOXP3 (84). This work further supports the conclusion that abrogating NRF2 signaling in T cells can increase anti-tumor capacity against solid tumors.
Likewise, multiple groups have shown that increasing hydrogen peroxide levels in T cells can have beneficial effects on T cell function. One study demonstrated that Prdx2-/- mice, where PRDX2 is responsible for cytosolic hydrolysis of hydrogen peroxide, enhanced T cell function (125). In an lymphocytic choriomeningitis (LCMV) murine model, effector CD8+ T cells lacking Prdx2 exhibited increased persistence and initial effector function, although this phenotype was reversed during chronic infection (125). While this effect was observed in a viral model, it suggests that loss of antioxidant capacity may confer an advantage in tissue environments, with implications for solid tumors. Another group showed that intratumoral injection of hydrogen peroxide alongside radiotherapy significantly reduced tumor growth in colon cancer and melanoma murine models (126). Importantly, this effect was partially T cell-dependent, as increased IFN-γ production and T cell persistence were observed in hydrogen peroxide-treated tumors (126). Together, these studies indicate that increasing cellular or environmental hydrogen peroxide can enhance T cell effector function.
It has been shown that conditioning T cells prior to ACT in a low-glucose environment primes them for increased effector function and persistence in vivo, leading to complete tumor clearance in a lymphoma murine model (). One study sought to uncover the mechanism underlying this effect and found that glucose restriction alters the cellular redox state, ultimately increasing ROS production from mitochondrial complex III (127). Importantly, blunting mitochondrial ROS reversed the benefits conferred by glucose restriction prior to ACT (127). These findings lend further support to the idea that increasing ROS in specific contexts can enhance T cell anti-tumor immunity.
In the context of chimeric antigen receptor (CAR) T cell therapy, an adoptive cell ACT approach that engineers T cells to recognize tumour antigens, both enhancing and limiting antioxidant capacity have shown therapeutic promise. Specifically, overexpression of catalase in CAR-T cells improved tumour killing of an ovarian cancer cell line under oxidative stress, while TRX1 overexpression enhanced pro-inflammatory cytokine production, proliferation, and cytotoxicity against a breast cancer cell line in oxidative conditions (, 128). On the other hand, another study showed that NRF2 knockdown in human CAR-T cells enhanced anti-tumor immunity in a xenograft murine model implanted with a B-lymphoblastoid cell line, showing that eliminating the antioxidant effect may be beneficial in a lymphoma setting (124).
The seemingly contradictory findings regarding whether increasing or decreasing antioxidant capacity in T cells is beneficial warrant closer examination of the TME itself. It has long been known that the TME contains high levels of ROS, leading to the hypothesis that enhancing antioxidant capacity may help T cells retain function within this environment (129). However, multiple studies have demonstrated that oxidative stress within the TME is required for the efficacy of adoptive T cell therapy. One study showed that ROS accumulate in tumors following ACT and actively contribute to treatment efficacy (130). Furthermore, another study found that macrophage-dependent accumulation of peroxynitrite, a byproduct of superoxide, is triggered by T cell recognition of tumors and contributes to tumor cell cytotoxicity (131). Together, these findings suggest that ROS within the TME both directly enhance T cell anti-tumor immunity and cooperate with intratumoral immune cells to drive tumor cell killing.
In terms of clinical translation, several ROS-modulating agents already used in clinical settings warrant further investigation for their effects on T cell anti-tumor immunity. For example, N-acetyl cysteine (NAC), an ROS scavenger commonly taken as an over-the-counter supplement, has been shown in preclinical studies to enhance adoptive T cell therapy in melanoma (132, 133). Similarly, as discussed previously, auranofin has demonstrated the ability to enhance T cell anti-tumor immunity in preclinical models and holds strong potential for repurposing as a cancer therapeutic, given its established use in rheumatoid arthritis (134). Given that both compounds have well-characterized and tolerable safety profiles, it may be valuable to evaluate their effects on T cell function and anti-cancer efficacy in clinical studies (135, 136).
Manipulating methionine to enhance T cell anti-tumor immunity
To date, many studies have sought to enhance anti-tumor responses in solid tumors by modulating methionine metabolism (68–70, 74, 114–118, 120). Collectively, these studies have suggested that methionine can exert both beneficial and suppressive effects on T cell responses in solid tumors. In the context of ACT or spontaneous anti-tumor response, methionine restriction (MR) has been shown to induce immune checkpoint receptor expression and T cell exhaustion, resulting in impaired anti-tumor T cell responses (69, 70, 74). In contrast, most studies examining methionine metabolism in the settings of immune checkpoint blockade (ICB) suggest that MR can enhance anti-tumor immune response by suppressing immune evasion mechanisms in tumor cells (114, 115).
Together, these findings indicate that MR may have dual functions: suppressing T cell response while spontaneously reducing tumor-mediated suppression of anti-tumor immunity. The net effect of MR on anti-tumor T cell response is therefore likely to be determined by the balance between these two opposing effects. Moreover, the observed synergy between MR and ICB suggests that ICB may shift this balance toward favoring anti-tumor immunity. If so, combining MR and ICB may present a promising strategy in solid tumors.
Conversely, in the absence of ICB, such as in ACT and spontaneous immune response, supplementation with methionine, either intratumorally or systematically, enhances T cell anti-tumor immunity, suggesting that methionine supplementation may improve therapeutic efficacy in these settings (68–70). Methionine supplementation attenuates T cell exhaustion marker expression, including TOX, PD-1 and TIM-3 (70). In the context of chimeric antigen receptor (CAR)-T cell therapy, peritumoral injection of methionine improves efficacy in a syngeneic mouse osteosarcoma model, leading to enhanced tumor control and improved overall survival (70). Consistently, methionine restriction in CAR-T cell-tumor cell cocultures reduces central memory T cell frequency and promotes PD-1 and TIM-3 expression (137). Moreover, dietary methionine restriction impairs human CAR-T cell efficacy against lung squamous cell carcinoma in mice (137). Notably, Bian et al. reported that methionine supplementation enhances T cell response in a small cohort of colorectal cancer patients – oral supplement at 1000 mg/day for two weeks significantly improved CD8+ T cell cytokine production and reduced T cell apoptosis, highlighting the potential for clinical translation (68).
Manipulating GCN2 to enhance T cell anti-tumor immunity
GCN2 plays a crucial role in regulating T cell immunity, particularly in amino acid-limited conditions and indoleamine 2,3-dioxygenase (IDO)-mediated immune suppression (105, 109, 110, 112). Despite this, relatively few studies have explored the potential of strengthening T cell anti-tumor response through the modulation of GCN2 signaling. A study by St. Paul et al. demonstrated that activation of GCN2 in activated T cells using halo prior to adoptive transfer enhances their anti-tumor activity in murine lymphoma models (113). Importantly, halo treatment induces a similar phenotype in human T cells in vitro, suggesting translational potential (113). Consistently, a recent study found that GCN2 activation by dabrafenib, an anticancer reagent, blocks the generation of myeloid-derived suppressor cells (MDSCs) in murine melanoma, indicating that systemic activation of GCN2 may enhance anti-tumor immunity (138). In contrast, another study showed that GCN2 deletion in myeloid cells induces inflammatory macrophages and attenuates MDSC function, leading to enhanced anti-tumor T cell response in murine melanoma models (139). Furthermore, treatment of melanoma-bearing mice with nanoparticles delivering ATF4 siRNA markedly inhibited tumor growth, suggesting that suppression of the GCN2-ATF4 axis may represent a viable therapeutic strategy (139). Together, these conflicting findings underscore the need for future studies to further investigate strategies for modulating GCN2 signaling to strengthen T cell responses in solid tumors.
Temporary conditioning human CAR-T cells in leucine-deprived medium increased the transcription of genes involved in IFN-α response and NF-κB-mediated TNF-α signaling and IFN-γ response. This effect is reversed by GCN2 inhibition, suggesting that GCN2 activation during CAR-T cell manufacturing enhances effector function (140). However, whether this benefit can be translated into improved efficacy against solid tumors remain to be examined.
Clinically, \an GCN2 inhibitor, APL-4098, has been studied in a phase 1 trial for acute myeloid leukemia (AML), where it was well tolerated and reduced AML blast counts in 5 out of 9 of the patients (141). In addition, a phase 1 trial is currently underway to test an GCN2 activator, HC-7366, in AML (142). However, both studies primarily focus on cancer cell–intrinsic effects, it remains unclear how modulating GCN2 signaling influences antitumor T cell responses. Nonetheless, blood samples collected from these clinical trials, if available, would be highly valuable for addressing this question.
Table 2 provides a summary of the preclinical interventions targeting the GCN2–methionine–ROS pathways.
Table 2
| Target axis | Tumor type / model | Intervention strategy | Key outcomes | Ref(s) |
|---|---|---|---|---|
| GCN2 | Melanoma (murine) | GCN2 knockout (T cells) | No impairment of T cell anti-tumor function | (111) |
| Glioma (murine) | GCN2 knockout (T cells) | ↓ CD8+ T cell survival, activation, effector function → ↓ tumor control | (112) | |
| Lymphoma (murine) | Halofuginone (GCN2 agonist) + ACT | ↑ OXPHOS, ↑ effector function, ↑ tumor control | (113) | |
| Melanoma (murine) | Dabrafenib (GCN2 activation) | ↓ MDSCs → enhanced anti-tumor immunity | (138) | |
| Melanoma (murine) | GCN2 deletion (myeloid) or ATF4 siRNA | ↓ MDSCs, ↑ inflammatory macrophages, ↓ tumor growth | (139) | |
| ROS | Melanoma, lymphoma, colon carcinoma, and lung carcinoma | Nrf2 knockout in ACT | ↑ tumor killing, ↑ T cell function | (123) |
| Colorectal cancer (murine) | Tmed4 knockout | ↑ Treg inflammatory phenotype, ↓ FOXP3, ↑ anti-tumor activity | (84) | |
| Colon cancer & melanoma | Intratumoral H2O2 + radiotherapy | ↓ tumor growth, ↑ IFN-γ, ↑ T cell persistence | (126) | |
| Viral (LCMV model) | Prdx2 knockout | ↑ CD8+ T cell effector function and persistence (context-dependent) | (125) | |
| Lymphoma (ACT) | Glucose restriction (↑ mito ROS) | ↑ persistence, complete tumor clearance; ROS-dependent | (, 127) | |
| Melanoma (ACT) | NAC treatment | ↑ ACT efficacy | (132, 133) | |
| Blood cancer (murine) | Auranofin | ↑ T cell effector function and tumor killing | (124) | |
| Ovarian cancer (in vitro) | Catalase overexpression (CAR-T) | ↑ tumor killing under oxidative stress | (128) | |
| Breast cancer (in vitro) | TRX1 overexpression (CAR-T) | ↑ cytokines, proliferation, cytotoxicity | () | |
| Lymphoma (xenograft) | NRF2 knockdown (CAR-T) | ↑ anti-tumor immunity | (124) | |
| Methionine | Colorectal cancer (human/murine) | Methionine supplementation | ↑ CD8+ T cell function, ↓ apoptosis | (68) |
| Prostate cancer (murine) Renal cancer (murine) | Methionine restriction + PD-1 blockade | ↑ CD8+ T cells, ↑ GZMB/IFNγ, ↓ M2 macrophages | (114) | |
| Colon cancer (murine) | MR + PD-1 blockade | ↑ T cell infiltration, ↑ effector function and cytotoxicity | (115) | |
| Colorectal cancer (murine) | MR + PD-1 blockade | ↑ tumor control | (116) | |
| Lymphoma (murine) | Transient MR and sustained MR + ACT | ↑ IFN-γ, ↑ cytotoxicity (in vitro only) ↓ tumor control and survival (in vivo) | (74) | |
| Osteosarcoma (murine) | Methionine supplementation + ACT | ↑ ACT efficacy | (70) | |
| Melanoma (murine) | Intermittent MR + ICB | ↑ ferroptosis, ↑ T cell infiltration/cytotoxicity | (120) |
Preclinical studies targeting the GCN2–methionine–ROS circuit.
↓ = decreased; → = leads to; ↑ = increased.
Section 3: perspectives
It is clear from the existing literature that individually modulating methionine, GCN2, or reactive oxygen species (ROS) does not yield predictable outcomes in the context of T cell immunotherapies. This likely reflects a combination of factors. First, the tumor microenvironment (TME) differs substantially across tumor types [e.g. lymphoma versus pancreatic ductal adenocarcinoma (PDAC)], exerting distinct pressures on T cell metabolism and function and precluding a blanket therapeutic approach (143). For example, a therapeutic approach of activating the GCN2 pathway and increasing antioxidant capacity of T cells may be prudent in the PDAC environment, where oxidative and metabolic stress is high, but may not work for other tumor microenvironments that don’t share the same stress, such as lymphoma and melanoma. Second, the type of T cell–based therapy employed [e.g. immune checkpoint blockade (ICB) versus adoptive T cell therapy (ACT)] is likely to influence how these pathways are engaged. Finally, whether modulation is transient or permanent may play a dominant role in determining therapeutic value. For example, inducing a naïve- or memory-like phenotype during ex vivo expansion of ACT products has been shown to be beneficial; however, once transferred into the tumor, these cells must transition to an effector phenotype to mediate tumor killing (144). Here, we will (a) examine approaches to elucidate the GCN2–methionine–ROS circuit to determine what forms of therapeutic modulation for T cell immunotherapies may be beneficial in a given context, and (b) explore different nodes within the GCN2-methionine-ROS circuit where intervention may be advantageous.
Evaluating the impact of GCN2–methionine–ROS circuit modulation
Given the contradictory literature surrounding interventions in GCN2, methionine, and ROS, it is critical to determine whether targeting these pathways will ultimately enhance T cell function. There are many approaches to address this question. In this review, we focus on multi-omics strategies, CRISPR–Cas9 screening, and drug screening. To extract meaningful insight from these tools, it is essential to develop experimental models that accurately capture relevant environmental contexts. Specifically, the therapeutic setting should closely mirror the therapy of interest [e.g. tumor infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR)-T cell therapy, PD-1 or CTLA-4 checkpoint blockade]. Likewise, the tumor model, and critically, the TME, must reflect the cancer under study, particularly ones that are less immunogenic or hard to treat. This can be achieved using appropriate syngeneic or xenograft mouse models, as well as in vitro systems incorporating patient-derived samples or organoids. Importantly, metabolic interventions are often beneficial in a time-dependent manner (e.g. pre– versus post–adoptive transfer), and this temporal dimension should be explicitly considered when evaluating therapeutic strategies.
Transcriptomics, proteomics, and metabolomics have evolved substantially over the past decades, providing increasingly comprehensive readouts of T cell states. Transcriptomics offers a high-level overview of gene expression and is useful for broadly characterizing cellular phenotypes, such as exhaustion (145). However, it does not capture cellular activity at a functional level. Metabolomics, and to a lesser extent proteomics, can provide a more direct and integrated picture of cellular phenotype (146, 147). Metabolites act as key cellular messengers, conveying signals that tune T cell states and functions (148, 149). As such, profiling cellular metabolite composition can be particularly powerful for understanding T cell phenotypes within specific environments. Together, these omics approaches enable assessment of which pathways are activated or suppressed in T cells under defined conditions, providing insight into whether targeted modulation of a given pathway may be beneficial.
Genome-wide screening offers a complementary and highly scalable approach to interrogate cellular pathways in a systematic manner (150, 151). While CRISPR-Cas9 screens are commonly used to probe a broad range of biological processes, smaller, pathway-focused libraries are increasingly accessible and can be designed to specifically target pathways such as the methionine cycle or GCN2 signaling (152, 153). Using this approach, individual components of a pathway can be perturbed, and the functional consequences of each edit assessed within the same experimental framework. Because CRISPR–Cas9 knockout screens result in permanent gene deletion, this strategy is particularly well suited for evaluating the effects of sustained pathway modulation.
Drug screening provides another powerful, high-throughput method to assess whether modulation of specific genes or pathways enhances T cell function (154, 155). In contrast to CRISPR-based approaches, pharmacological interventions can be inherently transient. This distinction may be especially relevant in ACT settings, where T cells are expanded ex vivo prior to infusion and where transient induction of a stem-like or memory-like phenotype has been shown to improve therapeutic outcomes. Drug screens may also be applied after initial tumor–T cell interactions (e.g. in serial killing assays) to identify interventions capable of reinvigorating exhausted T cells.
Furthermore, leveraging clinically available biomarkers may help determine whether modulation of the GCN2–methionine–ROS circuit is beneficial in a given context. For example, several peripheral biomarkers can indirectly reflect ROS levels, such as malondialdehyde (MDA) (156). Similarly, circulating methionine levels can be measured and may serve as an indicator of whether methionine restriction or supplementation would be advantageous within a specific cancer treatment regimen (157).
Engineering the GCN2–methionine–ROS circuit is likely to produce T cell products with context-dependent and potentially unpredictable outcomes. As such, it is essential to employ screening strategies within appropriately defined experimental settings that can capture this complexity.
Tuning the GCN2–methionine–ROS circuit
There are multiple intervention points within the GCN2–methionine–ROS circuit that may prove useful for enhancing T cell–based immunotherapies, including therapies that act on endogenous T cells (e.g. ICB), against solid tumors. Here, we outline three multiplex strategies that warrant further investigation.
Within ACT paradigms where T cells are expanded ex vivo prior to reinfusion, evidence suggests that methionine restriction can impair T cell function, whereas methionine supplementation may be beneficial (70, 74). Similarly, a study has demonstrated that inhibiting the GCN2 stress response in T cells can enhance adoptively transferred T cell proliferation (109). Mechanistically, these interventions are also likely to reshape intracellular redox balance. Methionine supplementation enhances antioxidant capacity through glutathione biosynthesis and methionine-dependent redox buffering, thereby limiting excessive oxidative stress during rapid ex vivo proliferation. Concurrent inhibition or deletion of GCN2 may permit sustained mitochondrial and anabolic activity, which can increase ROS generation, while preventing activation of stress-response pathways that otherwise restrain T cell proliferation and effector differentiation. However, it is worth noting that sustained GCN2 inhibition (e.g. by gene deletion) could potentially lead to T cell exhaustion. Together, these manipulations are predicted to maintain ROS at levels that are permissive for TCR signaling, metabolic activation, and effector programming, without triggering oxidative dysfunction. Thus, combining GCN2 inhibition or deletion with methionine supplementation may improve T cell–based therapies for solid tumors in the ACT setting (Figure 3A).
Figure 3
Regulatory T cells (Tregs) have been shown in multiple models to rely on a high antioxidant capacity, characterized by low ROS levels and elevated glutathione (GSH), as well as active GCN2 signaling (158, 159). In cancers where poor prognosis is associated with high endogenous Treg infiltration, a multiplex intervention combining inhibition of both GCN2 (e.g. clinical-grade inhibitors APL-4098 and HC-7366) and glutamate–cysteine ligase catalytic subunit (GCLC) [e.g. clinical-grade inhibitor buthionine sulfoximine (BSO)], the enzyme that synthesizes GSH, may synergistically destabilize Treg phenotypes and promote a more inflammatory state (Figure 3B) (160, 161). Mechanistically, this combination is also expected to reduce intracellular methionine availability by increasing anabolic demand (via GCN2 inhibition) while imposing oxidative and metabolic stress (via GCLC inhibition), creating a state of methionine limitation without the normal GCN2-mediated adaptive response.
In the context of ICB, therapeutic efficacy relies on reinvigorating endogenous T cells by blocking inhibitory receptors at the cell surface. Multiple studies have shown that dietary MR can be beneficial in this setting (114, 115). Mechanistically, MR would be expected to indirectly activate GCN2. In this context, combining MR and ICB with an additional intervention, such as a pharmacological GCN2 activator (e.g. halo), may synergistically enhance T cell reinvigoration (Figure 3C). MR would limit GSH synthesis, whereas GCN2 signaling would enhance GSH production, potentially maintaining ROS at an optimal, intermediate level. This multiplex strategy may be particularly valuable for solid tumors that have historically been resistant to immunotherapy.
Although tuning the GCN2–methionine–ROS circuit is not straightforward, it represents a potentially powerful strategy when applied in the appropriate context. Moreover, targeting this circuit from multiple angles using multiplex approaches may ultimately prove more effective than single-pathway interventions.
Conclusions
This review highlights the GCN2–methionine–reactive oxygen species (ROS) axis as an integrated redox-metabolic circuit that plays a central and context-dependent role in shaping T cell anti-tumor immunity. Rather than acting in isolation, GCN2 signaling, methionine metabolism, and reactive oxygen species dynamically intersect to regulate T cell activation, differentiation, metabolic state, and effector function, particularly within the nutrient-poor and oxidative tumor microenvironment. Physiological levels of ROS are essential for sustaining T cell receptor (TCR) signaling, glycolytic reprogramming, and cytotoxic activity, while excessive or prolonged oxidative stress drives exhaustion and apoptosis; GCN2 functions as a key sensor that links amino acid availability and redox stress to adaptive transcriptional and metabolic programs, in part by sustaining glutathione (GSH) synthesis through methionine and cysteine metabolism. Importantly, therapeutic manipulation of any single node in this circuit yields highly variable outcomes that depend on tumor type, microenvironmental pressures, therapy modality (ICB versus ACT), and timing of intervention. As such, blanket metabolic interventions need to be approached with caution and instead emphasizes the need for context-aware, temporally controlled, and multiplex strategies to tune the GCN2–methionine–ROS circuit. Leveraging integrated omics, CRISPR-based perturbation screens, and drug discovery platforms in physiologically relevant models will be essential for identifying when and how modulation of this circuit can be harnessed to enhance T cell–based immunotherapies for solid tumors.
Statements
Author contributions
SM: Writing – review & editing, Writing – original draft. TZ: Writing – original draft, Writing – review & editing. JL: Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. S.M. is supported by a Doctoral Research Award (Cancer Research Society) and BC Cancer Rising Stars Award (BC Cancer Foundation). This manuscript was supported by grants from the Canadian Institutes of Health Research (PJT192015, JJL) and the Terry Fox Research Institute New Frontiers Program Project (1125, JJL).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
GCN2, immunometabolism, immunotherapy, methionine, redox, T cells
Citation
McPhedran S, Zhao T and Lum JJ (2026) Redox-metabolic circuits as a central regulator of T cell-based immunotherapy. Front. Immunol. 17:1807087. doi: 10.3389/fimmu.2026.1807087
Received
09 February 2026
Revised
18 April 2026
Accepted
27 April 2026
Published
14 May 2026
Volume
17 - 2026
Edited by
Marit van Buuren, BioNTech US, United States
Reviewed by
David O’Sullivan, Victoria University of Wellington, New Zealand
Yanying Yang, Fudan University, China
Updates
Copyright
© 2026 McPhedran, Zhao and Lum.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Julian J. Lum, jlum@bccancer.bc.ca
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.