Targeting Metabolic Reprogramming of T-Cells for Enhanced Anti-Tumor Response.

Cancer immunotherapy is an effective treatment option against cancer. One of the approaches of cancer immunotherapy is the modification of T cell-based anti-tumor immune responses. T-cells, a type of adaptive immune response cells responsible for cell-mediated immunity, have long been recognized as key regulators of immune-mediated anti-tumor immunity. T-cell activities have been reported to be suppressed or enhanced by changes in cell metabolism. Moreover, metabolic reprogramming during activation of T cells is required for the development of distinct differentiation profiles of these cells, which may allow the development of long-term cell-mediated anti-tumor immunity. However, T cells have been shown to undergo metabolic exhaustion in tumor microenvironment (TME) as it poses several obstacles to their function. Applications of several mechanistic solutions to improve the efficacy of T cell-based therapies including chimeric antigen receptor (CAR) T cell therapy are yet to be determined. Modifying the metabolic properties of these cells and employing them in cancer immunotherapy is a potential strategy for improving their anti-tumor activity and therapeutic efficacy. To give an insight, in this review paper, we endeavoured to cover metabolic reprogramming in cancer and T cells, signalling mechanisms involved in immuno-metabolic regulation, the effects of the TME on T cell metabolic fitness, and targeting metabolic reprogramming of T cells for an enhanced anti-tumor response.

Introduction

Cancer immunotherapy is defined as therapeutic technique that use the immune system in defense against cancers.1 Modification of T cell-based anti-tumor immune responses is one of the approaches of cancer immunotherapy,2 because due to the presence of unique mutations or protein expression patterns, tumor cells may be recognized and eliminated by T cells with a high degree of specificity.1,3,4

T cells, a type of adaptive immune response cells responsible for cell-mediated immunity, have long been recognized as key regulators of immune-mediated anti-tumor immunity. Changes in cell metabolism have been shown to enhance or suppress diverse T cell activities.5 Naive T cells have low metabolic requirements and oxidative phosphorylation (OXPHOS) is their main source of energy.6 However, upon antigen encounter, T cell receptor (TCR)-mediated signalling will be activated and this, in turn, induces changes in T cells’ metabolism that causes an increase in proliferation and differentiation of these cells into effector T cells (Teff).7 Activation of naive T cells enhances the up-regulation of glucose and amino acid transporters at their surface and leads to metabolic reprogramming of these cells from OXPHOS to glycolysis.8,9

T cells have been shown to undergo metabolic exhaustion in TME.10 Thus, mechanistic understanding of tumor-specific T lymphocytes metabolic reprogramming may provide an important therapeutic approach along with immunotherapy methods.11 It has been demonstrated that modulation of T cell metabolism can be a potential therapeutic target to inhibit or enhance immune responses including anti-tumor responses.12 This review paper focuses on targeting metabolic reprogramming of T cells for enhanced anti-tumor response in cancer immunotherapy.

Metabolic Reprogramming in Cancer and T Cells

Metabolic Reprogramming in Cancer Cells

Because of metabolic reprogramming, cancer cells’ metabolic properties, as well as the pathways by which they acquire and refill their metabolic demands, differ from those of normal cells.13 Under normoxic conditions, non-malignant (quiescent) cells rely on OXPHOS as their primary source of energy.6 Unlike normal cells, cancer cells generate energy primarily through increased glycolysis in the cytosol, even under aerobic conditions. This metabolic shift of cancer cells to aerobic glycolysis is often known as the Warburg effect in recognition of Otto Warburg who originally discovered it in 1926.14

The difference in cancer cell metabolism from normal cells is a result of disruption of intracellular signalling pathways caused by mutated oncogenes and tumor-suppressor genes.15 Among oncogenic mutations, alterations in the phosphoinositide 3-kinase (PI3K) pathway has repeatedly been demonstrated to be altered, and consequently plays a significant role in tumor proliferation and survival in a wide range of human malignancies.16 When activated, the PI3K pathway induces a glycolytic phenotype in tumors and increases ATP generation via its downstream effector, Protein kinase B also known as Akt1, ensuring that cells have the bioenergetics capacity to respond to growth signals.17,18 The PI3K enzyme itself inhibits the tumor suppressor PTEN and its loss enhances glycolysis via Akt and hypoxia-inducible factor 1-alpha (HIF-1) activation.17 Akt promotes glycolysis by boosting the expression and membrane translocation of glucose transporters, as well as by phosphorylating glycolytic enzymes including hexokinase (HK) and phosphofructokinase (PFK).17,19

Furthermore, even under normoxic settings, Akt1 substantially promotes the mammalian target of rapamycin (mTOR) signalling pathway by causing inhibitory phosphorylation of tuberous sclerosis 2 (TSC2), a negative regulator of mTOR,16,19 which indirectly influences other metabolic pathways by activating HIF-1.19

In addition to its role in cell cycle control and cell death,20 p53 inhibits glycolysis21 by increasing the production of tumor protein 53 (TP53)-induced glycolysis and apoptosis regulator (TIGAR), an enzyme that reduces levels of the glycolytic activator fructose-2,6-bisphosphate.22 Furthermore, p53 promotes the expression of PTEN, which inhibits the PI3K pathway and hence suppress glycolysis.23 Moreover, p53 enhances OXPHOS by increasing the expression of cytochrome C oxidase assembly protein (SCO2), which is essential for the assembly of the electron transport chain’s cytochrome C oxidase complex.21 Hence, loss of p53 shifts metabolism from OXPHOS towards glycolysis.

c-Myc is a transcription factor that promotes the expression of genes that encode glucose transporters and enzymes involved in glycolysis that includes: HK, phosphoglucose isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase.24 Tumor cells that overexpress c-Myc have enhanced metabolic flux,25 which compensates for the low efficiency of ATP synthesis via glycolysis. This occurs because increased expression of glucose transporters, such as Glucose transporter (GLUT1) 126 and GLUT4,27 leads to higher glucose uptake in cancer cells.

In addition to glucose, cancer cells also rely on glutamine to fuel their metabolic need. Through glutaminolysis, glutamine is catabolized to glutamate, α-ketoglutarate which further fuels the TCA cycle of tumor cells. Furthermore, the intermediates of TCA cycles could be used for the synthesis of lipid, cholesterol, amino acids, and other essential metabolites.28–30 Supporting their high need for nucleotides and other materials for biosynthesis due to increased proliferation of cancer cells demand for Pentose Phosphate Pathway (PPP) is important and frequently upregulated in many types of tumors.29,31

Aside from the classic Warburg effect in cancer cells, evidence of mutations in genes encoding for Krebs cycle enzymes, as well as the emerging paradigm of Oncometabolites-Driven Tumorigenesis, bolstered the role of metabolic change in cancer cell development.32–35 The presence of oncogenic transformation in metabolic enzymes, as well as the metabolic difference between tumor and normal cells, suggests that a novel anticancer strategy targeting cancer cell metabolism could be developed.

Metabolic Reprogramming in T Cells

In a naive state, T cells rely primarily on the use of small amounts of nutrients such as glucose, fatty acids, and amino acids, as well as the oxidation of pyruvate and glutamine via the tri-carboxylic acid (TCA) cycle. TCR-mediated activation and T helper (Th) lineage differentiation are intricately linked to metabolic reprogramming, a shift in cellular metabolic processes.36

Different T cell subsets have distinct metabolic properties. Basically, compared to activated T cells, the circulating naive T cells are quiescent and their metabolic demand is primarily mediated by OXPHOS.6 Naive T cells require cell extrinsic signals such as Interleukin (IL)-7 to maintain their basal energy-generating metabolism and to support their continued migration through secondary lymphoid tissues and immune surveillance.9 Antigen recognition in the context of the major histocompatibility complex, together with appropriate co-stimulation, causes naive T cells to escape quiescence, become highly proliferative and differentiate into Teff that exerts their function as a cluster of differentiation (CD)4+Th cells, such as Th1, Th2 or Th17 or as CD8+ cytotoxic T lymphocyte (CTL). T cells expand in size and undergo a metabolic shift from OXPHOS to glycolysis when activated.37,38

In addition to the transition to glycolysis, T-cell activation alters several metabolic processes including reduced fatty acid oxidation, decreased pyruvate flux into the TCA cycle, increased glucose flux into the PPP, and enhanced glutamine metabolism.39–41 It has been previously evidenced that glutamine uptake and metabolism is indispensable for the functional aspect of T cells.42

After the antigen is cleared through several T cell responses, most Teff cells die and a small population of antigen-specific T cells that survive become T memory cells (Tm). Tm, unlike Teff, does not grow fast and as a result, they do not require a high rate of anabolic metabolism. Instead, they produce energy to aid in self-renewal.8,12

Tm cells have an increased mitochondrial mass and consequently a higher mitochondrial spare respiratory capacity (SRC), which is the maximum mitochondrial respiratory capacity available to a cell to produce energy under conditions of increased effort or stress.9,43,44 Tm cells rely on β-oxidation of de novo generated fatty acids that have been synthesized from glucose during the effector phase by fatty acid synthesis and stored intracellularly for energy generation, rather than uptake and use of extracellular lipids.45 On the other hand, FAO and other ATP-generating catabolic pathways are actively suppressed in Teff cells. In general, the metabolism-cell fate connection has been demonstrated with the switch to glycolysis that occurs with Teff differentiation and the switch to FAO that occurs with Teff to Tm conversion.46 Moreover, conversion toward a T regulatory (Treg) phenotype is also favored in conditions of increased OXPHOS and decreased glycolysis.47

Signalling Pathways Involved in Immunometabolic Regulation

The activation of various metabolic pathways has a significant impact on cell differentiation and function. Metabolic reprogramming is influenced by key receptor signalling events, growth factor cytokines, and nutrient availability.8 Understanding how specific cellular signalling pathways involved in immunometabolic regulation may uncover therapeutic targets to modulate metabolic programming and T cell responses that can lead to new cancer therapies.

PI3K Signalling Pathway

The PI3K signalling system regulates cell survival, growth, metabolism, and glucose homeostasis.16 The PI3K family consists of three classes including class I, class II, and class III.48 Class 1 PI3K, lipid kinases that phosphorylate phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] to generate the lipid signalling molecule phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P3], play a crucial role in many aspects of T cell function.49

TCR, CD 28, and Interleukin 2 receptor (IL-2R) activation phosphorylate and activates PI3K while inactivating PI3K-suppressing molecules including PTEN and PIK3IP1. PI3K activity converts PIP2 to PIP3, and PIP3 assists in the recruitment and activation of downstream signalling molecules such as pyruvate dehydrogenase kinase 1(PDK1) and Akt. mTORC2 further activates Akt, enabling increased metabolism and T cell effector activity.50

mTOR Signaling Pathway

Signals from the TCR, costimulatory molecules, and growth factor cytokines activate signaling pathways that enhance transcriptional programs required for effector actions. These signals also activate the kinase mTOR, which causes glycolysis induction across numerous routes to support cell growth, proliferation, and function.51

mTOR, a serine/threonine-protein kinase, plays an important role in metabolism control by recognizing and integrating signals in response to nutrients, growth factors, energy, and stress.52 It exists in two different complexes: mTOR complex 1(mTORC 1) and mTOR complex 2 (mTORC2).53 T cell fate decisions are determined by the interaction of mTOR and metabolism.54 mTOR signaling is required for the generation of CD4+ effector T cells, as mTOR-deficient T cells fail to differentiate into Th1, Th2, and Th17 cells both in vitro and in vivo. T cells lacking in mTOR, on the other hand, evolve into Treg cells.55 mTORC1 signals govern Th1 and Th17 lineage differentiation, whereas mTORC2 signals promote Th2 development.56

The mTORC1 signaling pathway promotes metabolic reprogramming toward enhanced aerobic glycolysis, glutaminolysis, and mitochondrial metabolism remodeling.57 mTORC2 activity is also linked to metabolic reprogramming by regulating the activation of AGC kinases, however, its function appears to be less critical for early metabolic programming (ie, that occurs during first quiescence exit) that promotes T cell activation.58

mTOR is a crucial regulator of translation59 and cell growth60 that drives glycolysis and cellular metabolism61 by raising glycolytic enzyme activity and increasing the expression of nutrient transporters. Interestingly, activated Akt stimulates the mTOR pathway, allowing for greater consumption of glucose and amino acids.17,30,62 mTOR activation increases the expression of GLUT1163 and transgenic expression of GLUT1 increases T cell proliferation and cytokine production.64 Increased glycolysis and glucose absorption are linked to the enhanced effector actions that occur upon T cell activation.65

mTOR activation and glycolysis activation result in the production of downstream transcriptional regulators such as HIF-1α, c-Myc, and estrogen-related receptor alpha (ERRα), which regulate metabolism in T cells and activate pathways involved in rapid cell proliferation and effector function.66 The HIF-1α-dependent transcriptional pathway promotes glycolysis in T cells and promotes the development of the TH17 fraction while suppressing regulatory T cells (Treg).47 When c-Myc is activated, enzymes involved in glycolysis and glutaminolysis are expressed, the results of which contribute to the production of lipids, amino acids, and nucleic acids for cellular expansion.41 ERRα functions as a metabolic regulator of effector CD4+ T-cell homeostasis and function by influencing metabolic gene expression and glucose metabolism in a wide range of ways.67

In addition to transcriptional regulation, post-translational modulation of glucose uptake and glycolysis is essential for T cell effector function development. The PI3K/mTOR pathway, in particular, plays an important role in promoting the glucose metabolism required for effector T cell differentiation while inhibiting Treg generation.52,68 mTOR signaling also has pleiotropic effects on mitochondrial metabolism. In naive T cells, mitochondrial metabolism is catabolic, which supports cellular homeostasis, and mTOR signaling is actively retained at lower levels, establishing these cells’ quiescence.69 The up-regulation of mitochondrial metabolism by mTORC1 enhances efficient OXPHOS as well as the production of many epigenetic-regulating metabolites to modulate T cell functional programming.70–72

LKB1–AMPK Signaling Pathway

Liver kinase B1 (LKB1) and Adenosine monophosphate(AMP) activated protein kinase (AMPK) contribute to T cell development and function through regulating metabolic reprogramming.50 The LKB1-AMPK signaling pathway regulates cellular metabolism, growth, and survival in response to changes in nutrient and energy needs. It also stimulates catabolic pathways that generate ATP and allow metabolic reprogramming in T cells. AMPK promotes T cell survival by enhancing glutaminolysis and mitochondrial OXPHOS in maintaining intracellular ATP levels in the absence of glucose through promoting the expression of glutamine uptake and metabolism genes.73

Increased intracellular AMP-to-ATP concentrations activate the energy stress sensor AMPK, which promotes FAO.62 AMPK is required for the production of Tm; permits effector T cells to physiologically adjust to nutritional stress; and controls T cell effector function via mTOR inhibition.73,74

Activated T cells were discovered to have a glucose-sensitive metabolic checkpoint regulated by the energy sensor AMPK that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability, implying that AMPK-dependent regulation of metabolic homeostasis is a key regulator of T cell-mediated adaptive immunity.

Effects of Tumor Microenvironment on T Cell Metabolic Fitness

Cancer cells, stromal tissue and the extracellular matrix (ECM) that surrounds it all make up a complex milieu called TME.75 The TME is regarded as a critical component of cancer initiation and spread. The TME’s intricacy is considered to be related to uncontrolled cancer cell growth and faulty blood vessel formation.76 According to studies, the TME is distinguished by acidic pH conditions, hypoxia, endogenous H2O2, and changes in the expression of ECM proteins, all of which play important roles in tumor development and cancer metabolism.77,78

The acidic pH is produced by membrane proteins such as ATPase, monocarboxylate transporter 1 (MCT1) and MCT4 excreting protons (H+) and lactate during anaerobic glycolysis.79 Acidic pH promotes cancer cell migration and invasion by increasing the production of angiogenic molecules such as vascular endothelial growth factor A (VEGFA) and IL-8.79 Hypoxia (partial oxygen pressure of 10 mmHg) has been observed in a number of solid tumors.80,81

Cancer cells’ high metabolic activity, along with a weak vascular blood supply in the TME, might cause nutritional deficiency.82 These TME circumstances can affect TCR signaling, glycolytic metabolism, amino acid absorption, and metabolism, all of which are characteristics of Teff, leading to decreased anti-tumor effector activities of tumor-specific T cells. Treg cells, on the other hand, which rely mostly on FAO,9,63,81 may survive in these circumstances and exert inhibitory effects on tumor-specific Teff. The activation of AMPK, is also connected to the expansion of Treg cells in the TME.73

Waste produced by hypermetabolic cancer cells, such as lactate and amino acid metabolic products such as kynurenine, can limit T cell activation and cytolytic activity while supporting Treg differentiation.6,83 HIF1, which is activated by TME hypoxia, can also enhance the formation and maintenance of Treg cells.84 Hypoxia-induced (HIF1) promotes programmed death-ligand 1 (PD-L1) expression in myeloid-derived suppressor cells (MDSC), resulting in strong immunosuppressive effects in tumor-specific Teff cells.85

T cell “metabolic fitness” is critical for efficient antitumor immunity, but it is hampered by the tumor nutritional microenvironment’s specific circumstances of restricted food supply and the impact of immunological checkpoints.86 T cells develop an “exhausted” phenotype inside the immune-suppressive tumor milieu, which is characterized by gradual loss of effector activities, changes in the expression and function of critical transcription factors.10

Furthermore, “exhausted” T cells exhibit a persistent overexpression and co-expression of numerous inhibitory receptors, which can have significant effects on T cell activity.87,88 T cell exhaustion is characterized by reduced glycolysis and OXPHOS and indications of mitochondrial dysfunction.88–90 Lower levels of glycolysis have been linked to decreased GLUT1 expression, lower phosphoenolpyruvate (PEP) levels in T cells and glucose concentrations in the TME.91–94

Signaling through the TCR and the costimulatory protein CD28 activates the PI3K-AKT-mTOR pathway, resulting in enhanced aerobic glycolysis and OXPHOS during a normal immunological response that culminates in antigen clearance.95 CD28 costimulation during activation is also crucial in boosting mitochondrial biogenesis and, as a result, sparing respiratory capacity in Teff cells transitioning to the Tm state.96 However, the inhibitory molecule PD-1 is constitutively high in exhausted T cells, inhibiting CD28 signaling and CD28-dependent metabolic activities.97,98 Thus, identifying different bioenergetics patterns in exhausted T cell subsets might give new methods for determining the amount of T cell exhaustion as well as uncover novel targets for reversing depletion.

Modulation of T Cells Metabolism for Enhanced Anti-Tumor Response

Recent advances in cancer biology shed more definitive light on the therapeutic use of immune cells for efficient antitumor immune response. This includes tumor-infiltrating lymphocytes (TILs) and the latest CAR T cell therapy. Although immunotherapy has been increasingly shown wonderful clinical outcomes in patients with Leukemia, and other cancer types, there are still challenges related to specificity, and side effects possibly of life-threatening immune-related toxicities.99,100 This could be associated with TME that creates many impediments to immune cell activity, including a metabolically demanding and immunosuppressive milieu.75,78

Cellular metabolic pathways have been demonstrated to play critical roles in controlling T cell fate, function, and longevity.101 And regulation of T cell metabolism has been investigated as a possible therapeutic target for enhancing or suppressing immunological responses in a variety of situations, including anti-tumor immunity.102 Modifying the metabolic characteristics of T cells used in cancer immunotherapy is therefore a viable method for enhancing anti-tumor activity and therapeutic effectiveness.12

CRISPR/Cas9 genome editing technology has been applied to enhance T cell effector function for therapeutic applications.103 Regulating cell metabolism to improve CAR T cell activity is an essential modulation method for better immunotherapy against cancer.104 Because T cells are controlled by metabolic molecules such as Diacylglycerol Kinases (DGKs), a class of enzymes that catabolize diacylglycerols (DAGs),105 using CRISPR/Cas9 genome editing to knock out DGK isoforms increases TCR signalling in CAR T cells.106 Furthermore, Kawalekar et al show that CAR T cells engineered to express 4-1BB signaling domains have increased in vitro persistence, central memory differentiation, and mitochondrial biogenesis,107 all of which play a role in enhancing the anti-tumor response of metabolically reprogrammed T cells.

The production of tumor-specific Tm cells in conjunction with the generation of Teff cells is a primary objective of new immunomodulatory methods. Instead of a transitory anti-tumor impact, this will allow for persistent immune-mediated anti-tumor action. One approach is drug repositioning (DR), which involves searching for anti-cancer therapeutic effects in commonly prescribed drugs for non-malignant diseases because the safety and frequency of adverse effects of these treatments have previously been established.108

Metformin is a commonly used and well-tolerated medication for the treatment of type 2 diabetes mellitus (T2DM),109 and it has been demonstrated to have anti-tumor properties through a variety of mechanisms.46,110–112 Metformin has been shown in studies to influence Teff cells and increase the formation of Tm cells via AMPK activation.46,63 Eikawa et al discovered that metformin can protect CD8+ T cells from eventual functional depletion and death, as well as enhance T cell functioning in the TME.113 This may also assist exhausted T cells in regaining function. In addition, mTOR inhibitors such as rapamycin can have metabolism-targeting effects on T cells, through increasing memory CD8+ T cell production.114,115

The longevity and durability of T cells used in immunotherapy are likely to be key factors in determining treatment effectiveness. Geiger et al demonstrated that higher L-arginine levels can have a pleiotropic influence on T cell activation, differentiation, and function, ranging from enhanced bioenergetics and survival to anti-tumor efficacy in vivo.5 Increased L-arginine levels may upregulate the serine biosynthesis pathway, which has been demonstrated to feed the TCA cycle and, as a result, OXPHOS.116

Another study conducted by Jaccard et al indicates that pharmacological suppression of the metabolic enzyme isocitrate dehydrogenase 2 (IDH2) during CD8+ T cell priming resulted in greater memory formation and tumor growth inhibition upon adoptive cellular therapy (ACT) into melanoma tumor-bearing mice.117

In addition, manipulation of cellular fatty acid metabolism may potentially be of therapeutic relevance, since changes in basic cellular lipid metabolism can have a major impact on T cell destiny and function.118 Fatty acid synthesis (FAS) promotes the proliferation and differentiation of Teff cells in response to stimulation, whereas FAO is required for the formation of CD8+ T cell memory cells.45 Kim et al were able to provide evidence on activation of anticancer effector functions of T cells through nanoparticle‑induced lipid metabolic reprogramming.119

Metabolic pathways or enzymes that specifically inhibit cancer cell growth while enhancing anti-tumor T cell function can be targeted. Leone et al show that inhibiting glutamine metabolism in tumor-bearing mice suppresses cancer cell oxidative and glycolytic metabolism, resulting in decreased hypoxia, acidosis, and nutrient depletion, whereas Teff cells responded to glutamine antagonism by significantly up-regulating oxidative metabolism and adopting a long-lived, highly activated phenotype, allowing restoration of antitumor immunity.120 It has been demonstrated that inhibiting cancer cell glycolysis preserves antitumor T-cell function and improves response to checkpoint immunotherapy.121

Conclusion and Future Perspectives

To adapt to changing extracellular and intracellular circumstances, T cells undergo metabolic reprogramming. The presence of intense nutritional competition between cancer and T cells inside the tumor microenvironment causes T cells to exhaust, resulting in diminished antitumor responses. Several studies have shown that metabolic reprogramming plays an important role in supporting the transition from a resting to an active state, as well as how numerous signalling pathways are involved in immunometabolic regulation and therefore T cell functions.

As a result, mechanistic knowledge of such immunometabolic alterations allows for the identification of novel therapeutic targets to enhance T cell immunological activity. The synergistic effects of repurposed medications that target metabolic pathways, such as metformin, with established anti-cancer immunotherapies should be studied in clinical trials to aid in the development of novel treatments. Future research should focus on developing a strategy that can halt cancer cell growth while improving anti-tumor T cell function by targeting metabolic enzymes. Furthermore, metabolic programs that can boost anti-tumor activity should be included in CAR T cell design.