2.1 Immunometabolism modulation in T cells differentiation and function

T cells are derived from the bone marrow's pluripotent hematopoietic stem cells (HSCs) and their lymphoid progeny. Upon egress from the bone marrow, these lymphoid progenitor cells migrate to the thymus, where they undergo a critical phase of T cell development characterized by the rearrangement of T cell receptor (TCR) genes. Thymocytes that express both the TCR β chain and the CD4 and CD8 coreceptors are termed double-positive (DP) cells.¹³ Before the β selection, thymocytes remain relatively quiescent, relying on highly efficient and energy-saving oxidative metabolism. Following β selection, pre-TCR signaling, Notch, and IL-7 signals activate the PI3K signaling pathway. Subsequently, Notch signaling and the PI3K–Akt cascade further activate glucose transporter type 1 (Glut1), leading to glycolysis.^14-16 In cells that have undergone β-selection, the rearrangement of the TCRα gene segments is initiated, leading to the assembly of αβTCR heterodimers on the cell surface. This αβTCR complex is crucial for the provision of survival signals and for guiding the subsequent maturation and differentiation of the thymocytes into functional T cells.¹³ The protein kinase mammalian target of rapamycin (mTOR) primarily functions in regulating cellular growth and metabolism through two complexes, mTOR complex 1 (mTORC1) and mTORC2.¹⁵ The PI3K–Akt pathway activates the tuberous sclerosis complex, which activates Rheb within the lysosomes, ultimately leading to the activation of mTORC1.¹⁷ mTORC1 is particularly involved in the development of αβ T cells in the thymus. It integrates signals from the TCR and Notch, leading to the induction of the cellular myelocytomatosis oncogene (c-Myc), a cytokine that facilitates lipid synthesis and ROS production. Furthermore, mTORC1 activates hypoxia-inducible factor 1-alpha (HIF1α), thereby increasing glycolytic metabolism and the pentose phosphate pathway (PPP).¹⁵

In the thymic environment, thymocytes that fail to express TCR are subjected to an intrinsic apoptotic mechanism. This process, known as “death-by-neglect,” is governed by the BCL-2 family of proteins, which regulate cell survival and death. Thymocytes that do not successfully undergo TCR gene rearrangement and surface expression are consequently targeted for elimination, ensuring the maintenance of a functional and self-tolerant T cell repertoire.¹⁸ Subsequently, the cells undergo a process of positive and negative selection based on their ability to react with the major histocompatibility complex (MHC) and peptide presented in the thymus with low affinity. During positive selection, cells that can bind to MHC class I molecules retain the expression of CD8, while the expression of CD4 diminishes; conversely, cells that can bind to MHC class II molecules maintain the expression of CD4 and lose CD8 expression. This selective process results in differentiated CD8⁺ and CD4⁺ single-positive (SP) cells, which are pivotal for the adaptive immune response.^{13, 19, 20} During this process, regulatory factors c-Myc and HIF1α are downregulated, leading to a sharp decrease in glucose metabolism within cells and a return to mitochondrial oxidative metabolism, which promotes the substantial generation of ATP.¹⁵

Beyond the TCR signaling pathway, the interaction between CD28 on the surface of thymocytes and its ligand B7 can significantly amplify TCR signals.²¹ This engagement of the CD28 costimulatory molecule with B7 family members on antigen-presenting cells (APCs) provides a crucial second signal that enhances the survival and proliferation of T cells. This costimulation is essential for fully activating T cells, promoting their transition from a quiescent state to one of clonal expansion and differentiation, thereby bolstering the immune response to antigenic challenges. The CD28–B7 interaction is a key component of the intricate network of signals that drive T cell development and function within the thymic environment.²² CD28 costimulation also mediates the upregulation and facilitates the cellular surface transport of the glucose transporter Glut1 through Akt-dependent and Akt-independent pathways, enhancing glucose uptake following TCR stimulation1. This metabolic reprogramming is essential for the activation of T cells and the development of memory-phenotype T cells.²³ Glutamine, an indispensable amino acid for T cell activation, does not merely increase intake during T cell activation. Instead, it is through CD28 costimulation that the expression of glutamine transporters is enhanced, along with the activity of enzymes required to utilize glutamine as a substrate in the Krebs cycle within T cells. This intricate modulation ensures that the demand for glutamine is met during T cell activation.^{24, 25} Thymocyte differentiation and development, influenced by various signaling pathways, are accompanied by changes in cellular metabolic profiles. This metabolic shift allows these cells to meet the heightened energy requirements for their activation, proliferation, and differentiation into mature T cells, underscoring the importance of metabolic regulation in immune cell development and function.

After activation, CD4 T cells differentiate into multiple effector T cell subtypes, including regulatory T (Treg) cells, Th1, Th2, Th17, and so on. Once pathogens invade the body, the combination of TCR and the peptide–MHC II complex presented by APCs triggers the differentiation of Th1 cells. The IL-12 and interferon-γ (IFN-γ) produced by APCs facilitate the further differentiation of T cells and activate signal transducer and activator of transcription 4 (STAT4) and STAT1. Under the combined effect of the transcription factor T-bet induced by TCR stimulation, Th1 cells are prompted to produce IFN-γ. The nuclear factor-κB (NF-κB) members c-Rel and RelB also exert significant roles in the differentiation, development, and function of Th1. RelA is recruited to the IFN-γ enhancer region in a T-bet-dependent manner and cooperates synergistically with STAT4 to enhance IFN-γ expression. c-Rel may act downstream of T-bet, while RelB influences the expression of T-bet and IFN-γ via STAT4.²⁶ Mannose metabolism is essential for the expression of IFN-γ in Th1 cells.²⁷ An intriguing finding suggests that the differentiation and effector functions of Th1 cells are mutually exclusive in terms of metabolic pathways. Only when the substrates produced by the initial proliferative and epigenetic reprogramming metabolic pathways required for TH1 cell activation are consumed by a different metabolic pathway, that is, after cell differentiation is suppressed, can the terminal effector functions of Th1 cells be exerted.²⁸

Upon stimulation by IL-4, CD4 T cells activate the transcription factor STAT6, which sets in motion the process of Th2 differentiation. This activation triggers a cascade of events, including the activation of the nuclear factor of activated T cells, activator protein-1, and NF-κB, as well as the induction of the pivotal transcription factor GATA3. GATA3 is responsible for the regulation of c-Maf expression. STAT6, GATA3, and c-Maf work in concert to reinforce the Th2 phenotype, ensuring the expression of signature cytokines and other genes characteristic of Th2 cells. Additionally, the NF-κB family member p50 is a crucial component in the Th2 development pathway, playing an indispensable role in the differentiation and function of these cells. Th2 cells are critical for the immune response to parasites and allergens.^{26, 29} The differentiation of Th2 cells entails a metabolic adaptation, transitioning from energy acquisition through the tricarboxylic acid (TCA) cycle in CD4 T cells to a metabolic mode that relies heavily on fatty acid oxidation (FAO) for substantial energy production. mTORC1 facilitates the differentiation of Th2 cells. By activating the SREBP transcription factor, mTORC1 promotes the lipid synthesis pathway, meeting the lipid metabolic demands during Th2 cell activation. Among effector helper T cells, Th2 cells exhibit the highest expression of Glut1 and the most robust glycolytic effect. Peroxisome proliferator-activated receptor gamma (PPAR-γ) links Th2 cell function and cellular metabolism by regulating fatty acid (FA) metabolism.³⁰

Th17 cells, characterized by their secretion of the cytokines IL-17A and IL-17F, follow a unique pathway of differentiation and development. Under the influence of transforming growth factor-b (TGF-b) and IL-6, naïve CD4⁺ T cells commit to the initial stage of Th17 differentiation. Upon stimulation by endogenous or exogenous pathogen-associated signals, dendritic cells (DCs) and tissue-resident macrophages secrete IL-23. The binding of IL-23 to its receptor, IL-23R, activates the Janus kinase 2 (JAK2) and tyrosine (Tyr) kinase 2 (TYK2), activating STAT3. This activation leads to the upregulation of the retinoic acid receptor-related orphan receptor γ-t (RORγt) transcription factor, which acts as the “master regulator” of Th17 cell differentiation.³¹ RORγt is a nuclear receptor that requires interaction with lipid ligands to achieve transactivation.³² The differentiation of Th17 cells relies on the de novo synthesis of FA.³³ Culturing Th17 cells with IL-23 and IL-1β leads to increased inflammatory phenotypes and upregulation of genes related to FA biosynthesis. Acetyl-CoA carboxylase 1 (ACC1) can regulate the biosynthesis of FAs and thereby control the differentiation of Th17 cells. The nuclear receptor PPAR-γ can influence the lipid metabolism of cells, thereby affecting the differentiation and function of Th17 cells.³⁴

In response to TCR signaling, CD4⁺ SP thymocytes differentiate into CD25⁺Foxp3⁻ CD4SP T regulatory cell precursors. The engagement of TCR–CD28 can activate the NF-κB and PI3K–Akt signaling pathways. Among the NF-κB family members, c-Rel is essential in developing thymus-derived Foxp3⁺ Treg cells, also known as tTreg cells.³⁵ Glutamine deprivation has been demonstrated to enhance the expression of the transcription factor Foxp3 in Treg cells. Besides, the inhibition of glutamate conversion to α-ketoglutarate has been shown to promote the differentiation of Th17 cells towards Treg cells through methylation of the Foxp3 locus.³⁶ However, the activation of the PI3K–Akt signaling pathway has been demonstrated to inhibit the differentiation of tTreg cells. Thus, the differentiation and maturation of tTreg cells are subject to the precise regulation of NF-κB and PI3K–Akt signaling pathways triggered by antigenic stimulation. Following this, CD25⁺Foxp3⁻ CD4SP Treg precursors, upon stimulation by the common γ-chain cytokine IL-2 and other signals, initiate the transcriptional mechanisms that govern the expression of the Foxp3 gene, culminating in the formation of tTreg cells. Furthermore, transcription factors such as STAT5 and the orphan nuclear receptors of the nuclear receptor 4a (Nr4a) family—comprising Nr4a1, Nr4a2, and Nr4a3—also influence the differentiation of tTreg cells to vary extents.³⁵ The expression levels of Glut1 on the cell surface of Th1, Th2, and Th17 cells are higher than those on Treg, yet Glut1 is essential for the differentiation and development of CD4⁺ Treg cells. Treg cells exhibit lower glycolysis rates and higher FAO rates, consistent with their higher levels of phosphorylated serine/threonine kinase AMP-activated protein kinase (AMPK). AMPK contributes to the stability and function of Treg cells by suppressing Glut1 expression, promoting FAO, and increasing the expression of Foxp3.³³

Emerging research posits that CD8 T cells with autoreactivity in the thymus do not necessarily undergo direct apoptosis following positive and negative selection. Instead, signals mediated by TCR induce a decrease in the expression of the transcriptional repressor Gfi1. This downregulation of Gfi1 activates the sphingosine-1-phosphate receptor-1 (S1P1) on the surface of immature thymocytes. Consequently, these immature CD8 T cells are prematurely ejected from the thymus into the periphery. There, they are believed to undergo further maturation, eventually developing into mature CD8 T cells that have acquired self-tolerance.³⁷ This paradigm underscores the intricate regulatory mechanisms within the thymus, highlighting the dynamic interplay between TCR signaling, transcriptional regulation, and cellular egress. It provides a nuanced view of how the immune system refines its T cell repertoire, balancing eliminating potentially self-reactive cells with maintaining a diverse and functional immune response. Naive CD8 T cells reside in a metabolically quiescent state, primarily fueled by FAO. Upon antigenic stimulation of CD8 T cells, the costimulatory molecule CD28 becomes activated, leading to increased expression of Glut1, lactate dehydrogenase A, and hexokinase, which enhances glycolysis and directs metabolized glucose towards the synthesis of nucleotides and serine. The activation of CD28 also results in decreased expression of carnitine palmitoyltransferase 1A, coupled with the mTOR-regulated sterol regulatory element-binding proteins, which leads to increased FA synthesis.³⁸

2.2 Function regulation of DC by immunometabolism pathway

DCs, in their specialized role as APCs, connect the innate and adaptive arms of immunity.^{39, 40} As sentinels of the immune response, they are adept at capturing, processing, and presenting antigens to T cells, thus initiating and shaping adaptive immune reactions. DCs originate from HSC, and their differentiation process is modulated by the hematopoietic growth factor Fms-like TYK3 ligand (FLT3L).⁴¹ FLT3L is pivotal in regulating HSC status by binding to its cognate receptor FLT3. This interaction induces receptor homodimerization and subsequent autophosphorylation, activating downstream signaling cascades. FLT3L engagement activates the PI3K–Akt pathway, which is instrumental in promoting cell survival, proliferation, and metabolism. Additionally, FLT3L signaling intersects with the Ras/MAPK pathway, known for its role in cell growth and differentiation. By modulating these critical pathways, FLT3L influences the maintenance, self-renewal, and differentiation potential of HSCs, thereby shaping the hematopoietic landscape.^42-45 FLT3L stimulates the expression of nutrient transport proteins CD71, CD98, and Glut1, enhancing dextran uptake by conventional type 1 DCs (cDC1).⁴⁶ When aerobic glycolysis, which is associated with anabolic metabolism, is inhibited by the inhibitor 2-DG, the cell proliferation induced by Flt3L is blocked by 2-DG in a dose-dependent manner, indicating that the development of DCs is dependent on aerobic glycolysis.⁴⁷ When bone marrow-derived DCs are cultured in the presence of FLT3L (FL-DCs), there is an upregulation of the expression of key surface molecules such as CD80, CD86, and MHC II, which are critical for effective T cell activation. Upon encountering an infection, FL-DCs exhibit an enhanced capacity to produce many proinflammatory cytokines, including IL-6, IL-12, IL-23, IFN-γ, and tumor necrosis factor-α (TNF-α).⁴⁸ Research has demonstrated that FL-DCs exhibit superior migratory capacity when administered via subcutaneous injection, effectively trafficking to the draining lymph nodes. This enhanced migration is a critical aspect of their function in initiating immune responses at peripheral sites. From a cellular perspective, FL-DCs possess characteristics that resemble those of steady-state resident DCs more closely.⁴⁹

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is recognized as a pivotal regulatory cytokine for DC development under both homeostatic and inflammatory conditions, serving as a central mediator of tissue inflammation.⁵⁰ Under homeostatic conditions, the expression of GM-CSF is typically maintained at low levels, ensuring a baseline level of DC function. However, upon encountering infection and inflammation, the expression of GM-CSF escalates, endowing DCs with enhanced capabilities for rapid antigen uptake and cross-presentation.⁵¹ Upon binding to its receptor, GM-CSF activates the cytoplasmic Tyr kinase JAK2, triggering the JAK/STAT pathway, MAPK pathway, and PI3K pathway was also activated. These constitute key signaling modules that regulate diverse cellular processes such as proliferation, survival, and differentiation. Furthermore, GM-CSF activation leads to the canonical NF-κB signaling cascade, a central component in immune and inflammatory responses. The activation of these pathways by GM-CSF is essential for the maturation and functional specialization of various DC subsets, thereby facilitating their role in antigen presentation and the modulation of adaptive immunity.⁵⁰ DCs cultured with GM-CSF exhibit a distinct cytokine profile when stimulated with Toll-like receptor (TLR) agonists. Compared with FL-DCs, GM-CSF-cultured DCs produce a unique array of cytokines, including TNF-α, IL-10, and the chemokine CCL2.⁴⁹ This differential cytokine production is reflective of the cells' activation state and their role in shaping the immune response. The secretion of TNF-α and IL-10 suggests a role in both proinflammatory and regulatory processes, while CCL2 contributes to leukocyte chemotaxis and inflammation. GM-CSF can also augment dextran uptake by DCs and effectively sustain the vitality of cDCs.^{46, 52} Under the stimulation of GM-CSF and IL-4 in vitro, PPAR-γ, which plays a crucial role in FAO, is significantly upregulated in DCs. The mRNA levels of PPAR-γ target genes, including fatty-acid-binding protein-4, adipose differentiation-related protein, and apolipoprotein E, are increased, modulating the differentiation of DC subpopulations.⁴⁷

In plasmacytoid DCs (pDCs), the induction of the JAK2–STAT5 signaling pathway by IL-3 culminates in generating transport proteins SLC7A5 and SLC3A2. These transport proteins, involved in the uptake of the essential amino acid leucine, work in concert with TLR activation to engage mTORC1. The engagement of transport proteins and TLR signaling leads to the activation of mTORC1, a key instigator of anabolic metabolism, which can promote a suite of synthetic metabolic activities associated with increased glycolysis, thereby facilitating the production of cytokines and chemokines by this specific subset of DCs.^53-55

2.3 Immunometabolism modulation in macrophage polarization and function

Macrophages are a class of immunoeffector cells with remarkable plasticity, ubiquitously present in all tissues of living organisms. They possess the innate ability to rapidly alter their functional characteristics in response to various stimuli, thereby playing pivotal roles in both innate and adaptive immune responses.⁵⁶ Cytokines such as IFN-γ, TNF-α, IL-1, IL-4, and IL-10 are crucial in orchestrating the polarization and metabolic reprogramming of macrophages. IFN-γ promotes the polarization of M1-type macrophages by activating signaling pathways such as JAK/STAT and NF-κB.^{57, 58} Additionally, lipopolysaccharide (LPS) can also induce M1 polarization in macrophages, a process that is typically accompanied by an increase in aerobic glycolysis. M1 macrophages, aerobic glycolysis is coupled to the oxidative PPP to generate NADPH. This NADPH is subsequently utilized by NADPH oxidases to produce reactive oxygen species (ROS). The generation of ROS is pivotal for activating the NLRP3 inflammasome, which is crucial for the phagocytic activity of M1 macrophages.^{58, 59} The interplay between aerobic glycolysis, NADPH production, and ROS generation is integral to the proinflammatory functions of M1 macrophages, highlighting the critical role of metabolic pathways in shaping immune responses.

IL-4 and IL-13 are pivotal cytokines that promote the polarization of M2 macrophages through the activation of the JAK/STAT signaling pathway.⁶⁰ The polarization of M2 macrophages is also contingent upon FAO. Research has demonstrated that M2 macrophages utilize the uptake and oxidation of FAs to fuel the TCA cycle, subsequently providing energy for oxidative phosphorylation (OxPhos). The inhibition of FAO results in the suppression of M2 macrophage activation induced by IL-4, underscoring the critical role of FAO in their metabolic programming and functional activation. In addition to FAO, glycolysis is also essential for the biosynthesis of cytokines in M2 macrophages.^{61, 62} This metabolic pathway not only generates ATP but also serves as a source of precursors for anabolic processes, highlighting the dual importance of glycolysis in both energy production and the inflammatory response of M2 macrophages.

2.4 Immunometabolism regulation in natural killer cell development and cytotoxicity

Natural killer (NK) cells originate from HSC and are integral components of the innate immune system, characterized by their ability to exert nonspecific cytotoxic effects against target cells. Resting NK cells maintain a low level of glycolysis and OxPhos, a metabolic rate sufficient to produce IFN-γ, thereby sustaining acute NK cell responses. Notably, inhibiting either OxPhos or glycolysis can impact the generation of IFN-γ.⁶³ IL-15 plays a pivotal role in the differentiation and development of NK cells, serving as a critical cytokine in their maturation process. Upon binding to the IL-15Rβ/γ complex, IL-15 activates JAK1 and JAK3, promoting the dimerization or tetramerization of STAT5. This signaling cascade leads to the transcriptional activation of the target gene Mcl1, resulting in the production of the antiapoptotic protein MCL1. MCL1 functions to sequester proapoptotic proteins such as BIM and NOXA, thereby sustaining the continuous survival of NK cells. Conversely, the withdrawal of IL-15 signaling can precipitate apoptosis in NK cells. Furthermore, the enhancement of IL-15 signaling augments the expression of key effector molecules within NK cells, including IFN-γ, perforin, and granzymes. This upregulation not only bolsters the cytotoxic potential of NK cells but also stimulates their proliferation and differentiation.⁶⁴ Activated NK cells under the stimulation of cytokines, such as IL-2, experience an increase in mTORC1 activity, which leads to elevated expression of nutrient transport proteins and glycolytic enzymes. When the activity of mTORC1 is inhibited, the glycolysis in NK cells decreases. It is noteworthy that the increase in glycolysis in NK cells stimulated by IL-15 does not require the active involvement of mTORC1. Furthermore, mTORC2 restricts the activity of mTORC1 by inhibiting the expression of the amino acid transporter SLC7A5, which is mediated by STAT5.⁶³

IL-2, a cytokine belonging to the same 4-α-helix bundle family as IL-15, rapidly enhances NK cells’ ability to bind to their target cells. This effect is particularly notable in augmenting NK cells’ reactivity against weak target cells, thereby strengthening their overall cytotoxic function. However, IL-2 does not exert an influence on the formation and cytolytic activity of NK cells against strong target ligands, which suggests a nuanced role in modulating the responsiveness of NK cells based on the strength of target cell recognition.⁶⁵ NK cells activated by IL-2 primarily derive their energy from glucose metabolism, and they rely on the activity of mTORC1 to enhance glycolysis levels.⁶³

Traditionally, NK cells have been considered to lack memory capabilities. However, recent findings challenge this notion. Under the combined stimulation of cytokines IL-12, IL-15, and IL-18, NK cells exhibit heightened metabolic activity and induce a “memory-like” phenotype. This subset of NK cells, after brief activation, can mount a more robust response upon subsequent cytokine re-stimulation, characterized by the production of increased levels of IFN-γ.⁶⁶

3.1 Maintenance of metabolic balance in immune cells

Metabolic pathways are not only crucial for energy production but also serve as a signaling hub that integrates extracellular signals with cellular responses. From monitoring and defending against pathogens to resolving inflammation and tissue repair, immune cells require the delicate regulation of metabolic activities to perform their diverse functions and maintain systemic homeostasis.

Within the immune cell repertoire, the mTOR serves as a central modulator of cellular growth and metabolic pathways. Upon activation, the mTOR pathway orchestrates enhancing anabolic processes, which are crucial for facilitating T lymphocytes’ clonal expansion and developmental trajectory.⁶⁷ Autophagy is a critical degradation and recycling process within immune cells, essential for maintaining their homeostasis and functionality. In T cell-mediated immune responses, autophagy exerts its effects through various mechanisms. In conditions of nutrient abundance, the activation of mTORC1 typically inhibits autophagy.⁶⁸ However, under various environmental stresses, such as nutrient deprivation, absence of growth factors, and hypoxia, mTORC1 is either not activated or its activity is subdued. This leads to the induction of autophagy through multiple pathways, which helps cells maintain sufficient nutrition and energy supply to ensure their survival.⁶⁹ Glucose starvation can activate AMPK by increasing the relative levels of AMP or ADP to ATP, thereby triggering autophagy, or it can initiate autophagy by enhancing amino acid catabolism, leading to an accumulation of ammonia.⁷⁰ Autophagy is a crucial mechanism for promoting cellular homeostasis, adapting to metabolic demands within the body, and maintaining metabolic balance within cells. This intricate process is vital for the overall health and proper functioning of cells, ensuring they can meet the energetic and material needs of the organism.

SREBP plays a crucial role in the metabolic reprogramming induced by the activation of CD8⁺ T cells. The absence of SREBP not only suppresses lipid synthesis but also markedly inhibits the proliferation of T cells.³⁴ SREBP-1a and -2 are the principal isoforms of SREBP, which activate the synthesis of FAs and cholesterol, respectively. The activation of SREBP by mTORC1 is mediated through regulating cholesterol transport. When mTORC1 is highly active, it suppresses autophagy and inhibits the transport of cholesterol from lysosomes to the endoplasmic reticulum, leading to reduced cholesterol levels in the endoplasmic reticulum and consequently promoting the activation of SREBP-2 and the expression of genes involved in cholesterol synthesis. In contrast, when mTORC1 activity is low, autophagy is induced, and cholesterol is transported to lysosomes through two membrane transport pathways, resulting in increased endoplasmic reticulum cholesterol levels, which suppresses the activation of SREBP-2 and inhibits cholesterol synthesis.⁷¹

Liver kinase B1 (LKB1) plays a crucial role in maintaining T cell homeostasis by regulating mTORC1 through the phosphorylation of phosphatase and tensin homolog (PTEN). In the absence of LKB1, T cells exhibit a metabolic shift characterized by increased glycolysis mediated by mTORC1–HIF1α, which subsequently influences the differentiation of Th1 and Th17 cells, thereby contributing to the development of inflammation.⁷²

3.2 Influence of the microenvironment on immune cell metabolism

The metabolic activities of immune cells are fundamental to their functional execution,⁶ with these activities being significantly influenced by the microenvironment in which they reside. Nutrients, oxygen levels, and cytokines within the microenvironment are pivotal in sculpting the metabolic pathways of immune cells.⁷³ These factors subsequently dictate the survival, proliferation, and functional performance of immune cells.

Under homeostatic conditions, immune cells meticulously regulate their metabolic pathways to maintain their functionality and self-renewal capabilities. Quiescent T cells predominantly rely on the breakdown of glucose, and amino acids to actively inhibit the aberrant activation of mTORC1. Following activation, T cells escalate their glucose metabolism and enhance the catabolism of glutamine to foster cellular growth, division, and differentiation.⁷⁴ However, this delicate metabolic balance is often disrupted in the tumor microenvironment. Tumor cells, through enhanced aerobic glycolysis, consume a significant amount of glucose, leading to reduced glucose levels in the tumor interstitial fluid, which in turn affects the survival and functionality of T cells.⁷⁵ In the hypoxic or glucose-deprived cancer microenvironments, CD8⁺ tumor-infiltrating T lymphocytes (TILs) can augment their uptake and catabolism of FAs to generate more energy and restore their antitumor capabilities.⁷⁶ Because Treg cells are less dependent on glucose metabolism, they are more capable of adapting to the tumor microenvironment, characterized by high lactate and low glucose levels. In highly glycolytic tumors, Treg cells absorb lactate through the monocarboxylate transporter 1 (MCT1), which increases the expression of PD-1 on Treg cells, thereby affecting the curative effect.³³ Tumor cells and tumor-associated macrophages (TAMs) that highly express PD-L1 can impair the PI3K/AKT/mTOR pathway, directly affecting glycolysis. Additionally, PD-L1 can suppress glutaminolysis, blocking energy supply to CD8⁺ T cells derived from glutamate metabolism. Furthermore, PD-L1 may also activate STAT3, promoting FAO in CD8⁺ T cells, thereby harming these cells and inhibiting antitumor immunity mediated by CD8⁺ T cells.⁷⁷ In tumors with high glycolysis, cancer cells produce a large amount of lactate through an enhanced glycolytic process. This lactate can affect the function of pyruvate carboxylase, thereby impacting the TCA cycle, and ultimately influencing the cytotoxic killing function of CD8⁺ T cells, leading to tumor immune evasion. However, pyruvate dehydrogenase can inhibit this process, allowing CD8⁺ T cells to maintain their cytotoxicity and overcome the lactate-rich TME.⁷⁸ An increase in the extracellular asparagine level facilitates the uptake of Asn by T cells through the SLC1A5-mediated antiporter mechanism. This process leads to the direct binding of Asn to lymphocyte-specific protein Tyr kinase (LCK), inducing Tyr394 phosphorylation and subsequently activating LCK signaling. As a result, the activation of CD8⁺ T cells is enhanced, bolstering their antitumor activity in vivo.⁷⁹

In mice infected with various pathogens such as vaccinia virus, Listeria monocytogenes, and lymphocytic choriomeningitis virus, researchers have observed that IL-7 and IL-15 can promote the expression of the antiapoptotic protein Bcl-2, thereby facilitating the homeostatic turnover of memory T cells. Furthermore, IL-7 and IL-15 can also induce the expression of the glycerol channel protein aquaporin-9 in CD8⁺ T cells, promoting the esterification of FAs and the formation of triglycerides, allowing memory T cells to maintain a stable and long-term energy supply, thus extending the lifespan of CD8⁺ T cells.³³

Primary splenic DCs or GM-CSF BMDCs that ingest excessive FAs may compromise their ability to stimulate T cell proliferation, resulting in DC dysfunction.⁸⁰ In TME, DCs acquire exogenous lipids, which diminish their immunogenicity, thereby facilitating the immune evasion of tumor cells.⁸¹ In contrast, liver DCs with higher lipid content can more effectively activate proinflammatory T cells, NK T cells, and NK cells. It is the distinct microenvironments that shape the varying lipid requirements of DCs.⁸⁰

4.1 Immunometabolic checkpoints on T cells

4.1.2 IDO1/2 and TDO

IDO1 and IDO2, along with TDO, act as the primary rate-limiting enzymes in the catabolism of tryptophan (Trp) via the kynurenine (Kyn) pathway. IDO1 and IDO2 are two distinct isoforms, with IDO1 characterized by a heme monomer nestled between a pair of α-helical domains,¹¹³ and IDO2 exhibiting greater substrate specificity for Trp with comparatively lower catalytic activity.¹¹⁴ TDO, a tetrameric enzyme, is versatile in its substrate specificity, catalyzing not only Trp but also its fluorinated analogs.¹¹⁵ IDO1 is predominantly found in glial cells, neurons, microglia, DCs, monocytes, and macrophages,¹¹⁶ whereas IDO2 is primarily expressed in certain tumor cells.^{114, 116} Additionally, IDO2 is mainly detected in certain tumor cells and is constitutively present in the liver, placenta, and central nervous system.¹¹⁷ TDO's primary site of expression is the liver.¹¹⁸ The expression of IDO1 is primarily triggered by IFNs, TNF-α, LPS, and IL-10,^{119, 120} whereas IDO2 expression is induced by the aryl hydrocarbon receptor (AHR).¹²¹ TDO expression can be stimulated by glucocorticoids.¹²²

Functionally, both IDO and TDO cleave the C2-C3 bond of indole ring of Trp introducing an oxygen molecule to form N-formyl-Kyn, which rapidly and spontaneously transforms into Kyn.¹²³ Kyn is further processed by a series of enzymes into immunosuppressive metabolites, including kynurenic acid (KYNA), xanthurenic acid (XA), 3-hydroxyanthranilate (3-HAA), and quinolinic acid (QA), ultimately yielding NAD, a vital coenzyme within the body.^{118, 124-127}

IDO1's immunosuppressive effects are exerted through three principal mechanisms: “Trp depletion,” “accumulation of Trp metabolites,¹²⁸” and “cell metabolism involvement (Figure 2).”¹²⁹ Initially, cells expressing IDO1 consume Trp aggressively. In Trp-sufficient conditions, the activated mTOR phosphorylates ribosomal S6 kinase, which in turn phosphorylates ribosomal protein S6, promoting cellular growth, proliferation, and protein synthesis. Conversely, Trp deficiency triggers the storage of uncharged Trp-tRNA, activating the Trp-sensing kinase general control nonderepressible 2 (GCN2) and leading to the phosphorylation of eukaryotic initiation factor 2 alpha. This phosphorylation event disrupts mRNA translation and curbs protein synthesis, resulting in T cell cycle arrest and a consequential impact on T cell proliferation. Additionally, Trp scarcity impairs mTOR pathway activity and induces mTOR-dependent autophagy.^130-135 Second, IDO1 enzymatically degrades Trp into Kyn, which activates AHR and fosters the expression of Foxp3 in T cells, thus driving the differentiation of CD4⁺ T cells into Tregs.^136-138 This process also attracts TAMs,¹³⁹ modulates DC maturation,¹⁴⁰ and creates an immunosuppressive tumor microenvironment conducive to cancer cell evasion, metastasis, and drug resistance development. Kyn has been shown to induce NK cell apoptosis via a ROS-mediated pathway and impairs NK cell function by dampening the surface expression of activating receptors NKp46 and NK group 2D (NKG2D).¹⁴¹ AHR also regulates IDO1 expression.¹⁴² Kyn is further metabolized into KYNA, 3-HAA, QA, and other catabolic products that bind to immune cell receptors, promoting immune tolerance and dampening T cell responses, leading to T cell anergy, apoptosis, Treg and Th17 cell proliferation, and alterations in Th1/Th2 balance.¹¹⁷

Lastly, IDO1 diminishes glucose uptake in activated lymphocytes, enhances mitochondrial function, curbs aerobic glycolysis, and stimulates the production of the immunosuppressive cytokine IL-10 by lymphocytes. This metabolic interference by IDO1 further exerts regulatory effects on the immune response. Studies have revealed a synergistic interaction between “Trp depletion” and “accumulation of Trp metabolites” within the IDO1 regulatory network, culminating in an amplified immunosuppressive effect that significantly influences the body's immune response.¹²⁹

The expression of IDO2 curtails the proliferation of CD4⁺ and CD8⁺T cells,¹⁴³ and impedes the infiltration of CD4⁺ T cells into tumor sites.¹⁴⁴ IDO2, triggered by LPS, directly stimulates cytokine production by modulating the IL-6/stat3 signaling pathway, thereby influencing T cell function.¹⁴⁵ Studies have further revealed that the differentiation of Tregs induced by IDO1 significantly depends on IDO2.¹²⁷ Additionally, TDO suppresses T cell responses by generating the tryptophan metabolite Kyn.¹⁴⁶

4.2 Immunometabolic checkpoints on DCs

4.2.1 Indoleamine 2,3-dioxygenase 1

The upregulated expression of IDO in DCs is particularly significant, as it wields immunosuppressive effects that directly target APCs, fostering the emergence of regulatory DCs and indirectly modulating local T cells.^{128, 174-176} IDO's role is multifaceted; it curbs the secretion of the proinflammatory cytokine IL-12 by DCs while simultaneously promoting the release of the anti-inflammatory cytokine IL-10. This duality induces T cell tolerance by suppressing Th1 cell differentiation and encouraging the differentiation of Tregs.¹⁷⁷ IDO1's influence extends to pDCs, where it mitigates IL-6 production through a GCN2-dependent mechanism, thus preserving the suppressive phenotype of Tregs.¹⁷⁸

IDO-expressing DCs are capable of effectively curbing the proliferation of CD4⁺CD25⁻ T cells in vitro and expanding the population of CD4⁺CD25⁺Foxp3⁺ Tregs via the IDO–Kyn–AHR axis.^{179, 180} The metabolite Kyn, produced by IDO, and its derivatives have been recognized as AHR ligands capable of activating T cell AHR signaling, further bolstering Treg differentiation.¹⁸¹ Moreover, IDO-expressing DCs can enhance the expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) and PD-1 on Tregs.¹⁸² The interaction between CTLA-4-expressing Tregs and the B7 molecule on DCs induces IDO expression, suggesting a self-regulatory network where CTLA-4⁺ Tregs harness IDO to dampen T cell responses and instill tolerance.^183-186

IDO⁺ DC can induce apoptosis in both CD4⁺ T cells and CD8⁺ T cells, as well as significantly inhibit the activity of CD8⁺ T cells.¹⁸⁷ In addition to T cells, IDO⁺ DC can also reduce B cell activation factor expression on mononuclear cells, thereby affecting B cell maturation and survival and resulting in a decrease in the number of plasma cells in the spleen and local lymph nodes.¹⁸⁸

IDO's enzymatic function is complemented by its nonenzymatic roles. TGF-β may activate IDO1 in DCs to mediate nonenzymatic signals through the phosphorylation and activation of the immune receptor Tyr-based inhibitory motif (ITIM). The ITIM on IDO serves as a docking site for Tyr phosphatases SHP-1 and SHP-2, initiating the noncanonical NF-κB pathway. This pathway inhibits IL-1 receptor-associated kinase 1, averting the production of the proinflammatory cytokine IL-6. Furthermore, IDO1 harbors a YENM motif that can directly interact with p85, activating the class IA PI3Ks p110 subunit, which anchors IDO1 to the early endosome and subsequently activates the immune regulation pathway in pDCs. The results emphasize the crucial role of IDO1 in the long-term regulation of immune responses within DCs (Figure 3).¹⁸⁹

4.3 Immunometabolic checkpoints on Macrophage

4.4 Immunometabolic checkpoints on NK cells

Beyond its influence on T cells, CD38 is a pivotal regulator of immune function, particularly modulating the activity of NK cells. Constitutive expression of CD38 in NK cells is crucial, as the interaction between CD38 and CD16 equips CD16-expressing NK cells with an effector cytotoxicity phenotype. This interaction bolsters NK cell activation and the release of cytokines, such as granzyme and IFN-γ.^{222, 223} Elevated CD38 expression is observed in rheumatoid arthritis (RA), where higher levels of CD38⁺ cells correlate with the presence of rheumatoid factors. CD38's regulatory role extends to dampening TNF-α levels and enhancing IFN-γ secretion in NK cells by suppressing Sirtuin 6 expression, inhibiting the differentiation of monocytes into Treg cells, leading to immune imbalance. CD38⁺ NK cells also activate mTOR signaling in cocultured CD4⁺ T cells, significantly altering the Th1/Th2 and Th17/Treg ratios and fostering inflammation.²²⁴

However, the role of CD38 in NK cells varies among different subsets. The expression level of CD38 in NK cells can be increased by IL-12, IL-15, and IL-18.²²⁵ In secondary lymphoid organs, the CD16⁻CD56^bright NK cell subset, which is less mature, releases anti-inflammatory cytokines upon stimulation and suppresses CD4⁺ T cell proliferation through a G protein-coupled receptor and CD38-dependent pathway.²²⁶ In HIV patients, the proportion of CD38⁺CD39⁺ NK cells is closely tied to disease progression, with these cells expressing high levels of inhibitory receptors LAG-3 and TIM-3, leading to increased TGF-β and IL-10 expression and a regulatory phenotype.²²⁵ In systemic lupus erythematosus patients, overexpression of CD38 in NK cells impairs the response of surface receptors SLAMF1 and SLAMF7 to stimulation, compromising NK cell function and contributing to disease pathogenesis.²²⁷

5.1 Targeting CD38

The landscape of CD38-targeted therapeutics has seen remarkable progress, with the development of anti-CD38 antibodies, small molecule inhibitors, and CD38-directed CAR-T/CAR-NK therapies taking center stage.

Biologically inspired therapies, such as anti-CD38 monoclonal antibodies (mAbs), have shown considerable promise in clinical trials. A plethora of CD38 antibodies is under global development, with daratumumab and isatuximab leading the way, having received market authorization. Both mAbs are approved for treating MM.²²⁸ Daratumumab, a human IgG1κ mAb, targets CD38's two β chains with high specificity for malignant tumor cells. Its antitumor effects are multifaceted, encompassing antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), macrophage activation via FcγR for antibody-dependent cell phagocytosis (ADCP), direct induction of apoptosis, and reduction of immunosuppressive adenosine secretion. Daratumumab also depletes MDSCs, Tregs, Bregs, and other immune subsets, while promoting CD4⁺ and CD8⁺ T cell expansion.^229-234 Daratumumab is approved for MM treatment as a monotherapy or combined with melphalan, immunomodulatory drugs, and proteasome inhibitors.²²⁸ In phase I/II GEN501 and phase 2 SIRIUS trials for relapsed or refractory multiple myeloma (RRMM), daratumumab (16 mg/kg) achieved overall response rates (ORRs) of 36 and 29%, respectively. The combined clinical benefit rate (ORR+MR) was 37.2%, with 83.1% of patients attaining stable disease (SD) or better, showcasing its efficacy.²³⁵ The phase III clinical trial of carfilzomib, dexamethasone, and daratumumab for RRMM revealed significantly improved median progression-free survival (PFS) and median overall survival (mOS) for the carfilzomib, daratumumab, and dexamethasone (KdD) group.^{236, 237}

Although daratumumab has shown encouraging results in the treatment of MM, it has been found in studies that some patients fail to benefit from it, and eventually, all patients face the dilemma of disease progression. In addition to inherent resistance to daratumumab due to interindividual variability in CD38 expression levels, acquired resistance can also develop in patients receiving this treatment. Studies have demonstrated that daratumumab treatment leads to the depletion of CD38 cells. However, given the expression of CD38 in MM cells and its widespread distribution in NK, T, and B cells, such depletion will inevitably impact the immune status of the organism. Furthermore, the CD38–dara complex formed by CD38 and daratumumab is phagocytosed by monocytes and granulocytes, leading to MM cells displaying altered surface antigen profiles and thus promoting immune evasion. Additionally, daratumumab induces self-destruction of CD38-expressing NK cells through ADCC, and depletion of NK cells consequently impacts the NK-mediated ADCC activity of daratumumab. MM cells also exhibit overexpression of CD47, which binds to signal-regulated protein α on TAM, leading to the inhibition of ADCP and promotion of immune escape in tumor cells. Additionally, homeostasis mechanisms such as humoral regulators or complement inhibitors hinder daratumumab-mediated CDC, thereby facilitating tumor immune evasion and compromising the efficacy of daratumumab treatment.^{234, 238} Ongoing research is being conducted to explore new combination therapies to further broaden the scope of daratumumab, with multiple studies initiating large-scale phase III clinical trials (Table 1).

Isatuximab, an IgG-κ chimeric anti-CD38 mAb,²³² isatuximab can induce apoptosis through both Fc-dependent and Fc-independent activities by inhibiting CD38 enzyme activity. The Fc-dependent mechanisms include CDC, ADCC, and ADCP, while the Fc-independent mechanisms involve caspase-dependent and lysosome-associated pathways. The fragment of daratumumab that binds to antigens and its Fc fragments are sufficient to activate NK cells, whereas the activation of NK cells by isatuximab depends on the cross-linking of CD16 and CD38 with full-length isatuximab.^{239, 240} Isatuximab can inhibit Treg proliferation and migration, suppress IL-10 secretion, and enhance the cytotoxic capabilities of CD8⁺ T cells and NK cells against tumor cells.²⁴¹ Isatuximab's combination with immunomodulators shows synergistic potential in preclinical studies.²⁴² Isatuximab can bind to CD38 molecules previously bound by HB-7 or AT13/5 antibodies, making it primarily suitable for adult patients with RRMM who have received prior treatment. It can be administered in combination with dexamethasone, pomalidomide, or carfilzomib.²²⁸ In phase 3 trials, this combination demonstrated significant improvements in PFS, with promising results anticipated in upcoming trials.^{242, 243} The ongoing phase 3 trials of isatuximab are anticipated to yield positive results soon, and we eagerly await the outcome (Table 1).

In addition to the approved CD38 antibodies, a variety of other CD38-targeting antibodies are currently in clinical trials, showcasing the diversity of therapeutic strategies under investigation. MOR202 is a fully human CD38 antibody that primarily induces tumor cell lysis through ADCC and ADCP mechanisms, demonstrating promising therapeutic efficacy in clinical trials for RRMM at stage I/IIa.²⁴⁴ Lenalidomide has been found to potentially enhance the macrophage-mediated cytotoxicity dependent on MOR202 via the vitamin D pathway.²⁴⁵ TAK-079 is an all-human IgG1λ mAb that specifically targets CD38, inhibits its enzymatic activity, and induces apoptosis and cytolysis through ADCC and CDC mechanisms.²⁴⁶ In arthritis studies, TAK-079 has shown potential for prophylactic administration, inhibiting the progression of arthritis and joint damage by reducing various immune cells, including lymphocytes, NK cells, B cells, and T cells.²⁴⁷ TNB-738 is a human anti-CD38 biparatopic antibody with high affinity for two distinct epitopes on CD38, synergistically achieving maximal CD38 inhibition without inducing cytokine production in CD38⁺ T cells.²⁴⁸ ISB 1342 is a bispecific antibody targeting both CD38 and CD3, potentially allowing for combination or sequential use with daratumumab.²⁴⁹ SAR442257 is a trispecific T cell-engaging antibody that targets CD38 on MM cells, CD3 on T cells, and CD28 on both cell types, offering extensive potential for treating patients resistant to both CD38 monoclonal antibodies and BCMA-targeted therapy.²⁵⁰

The expression of CD38 has been linked to increased PD-1 expression, contributing to resistance against PD-1/PD-L1 blockade in some studies. Tumor cells may inhibit ADCC mediated by anti-CD38 monoclonal antibodies through the PD-L1/PD-1 pathway, and the subsequent upregulation of CD38 expression can inhibit CD8 T cells. Studies combining CD38 antibodies with PD-L1 monoclonal antibodies have shown promise in preclinical models,^{87, 96, 240} but clinical trials combining durvalumab with daratumumab and atezolizumab with isatuximab have not met expectations,^{251, 252} indicating a need for further investigation.

Small molecule inhibitors targeting CD38 have emerged as a novel class of drugs with significant potential. Thiazoloquin(az)olin(on)es, such as compound 78c, are highly compatible with CD38 and exhibit excellent cell permeability, high steady-state distribution volume, and long half-life.²⁵³ Compound 78c is a potent reversible inhibitor of CD38, specifically suppressing its NADase activity without affecting other isoenzymes or homologous enzymes.²⁵⁴ In vivo, 78c elevates NAD levels, potentially improving age-related conditions and extending the lifespan and health span of mice without significant antitumor effects, suggesting its primary therapeutic effect is through metabolic regulation.^254-256

Apigenin (4′,5,7-trihydroxyflavone), a natural flavonoid, has shown promise in decreasing CD38 expression and inhibiting its activity, with potential applications in a variety of diseases, including cancer and central nervous system inflammation.^{88, 255, 257-259}

MK-0159 is another compound that effectively inhibits CD38 enzymatic activity and restores NAD and NADP levels in cells, presenting a promising avenue for CD38 inhibitor research.²⁶⁰

In addition to antibodies and small molecule inhibitors targeting CD38, numerous studies have focused on developing anti-CD38 CAR cells, including the engineering of anti-CD38 CAR-T cells and CAR-NK cells through genetic technology. These can be utilized for effective tumor treatment by transfusing specific T or NK cells capable of recognizing and attacking CD38⁺ tumor cells. To prevent the autolysis reaction caused by the binding of anti-CD38 CAR-T to CD38 on the cell surface, some researchers have attempted to combine CD38 antibody with anti-CD38 CAR-T, resulting in significant and specific tumor cell killing in both in vitro and mouse experiments.¹⁰² CD38 CAR-T cells have been developed for phase 1/2 clinical trials, and a double-targeted DCAR-T cell, which targets CD38 and SLAMF7, has also been constructed for the treatment of MM. This approach can rapidly induce an immune response without causing toxicity.²⁶¹ CD38-LMP1 tandem CAR-T cells were engineered for the treatment of NK/T cell lymphoma (NKTCL). Tandem CAR-T cells exhibited superior cytolytic activity against tumor cells compared to single-target CAR-T cells in both in vitro and in vivo experiments. Furthermore, even if one target molecule was lost during treatment, the tandem CAR-T cells still demonstrated synergistic enhancement by recognizing the target and exerting cytolytic activity against NKTCL cells.²⁶² Furthermore, the dual split CAR strategy is employed to simultaneously target CD38 and CD138 for the construction of CAR-T cells. Interestingly, this dual split CAR-T cell therapy may exhibit potential therapeutic benefits for patients who are refractory to daratumumab.²⁶³ The CD38-targeted CAR was engineered utilizing the NK cell line NK92. The CAR-NK cells demonstrated enhanced expression of IFN-γ, perforin, and granzyme, and exhibited CD38-dependent lysis of Daudi and Jurkat cells.²⁶⁴ Another form of CAR-NK involves the use of CRISPR–Cas9 technology to knock out CD38, followed by mRNA electroporation to introduce an enhanced CAR targeting CD38. This CD38-knockout NK cell with anti-CD38 CAR can evade NK cell-mediated autophagy and enhance cytotoxic activity against CD38⁻ expressing MM cells.²⁶⁵

5.2 Targeting IDO/TDO

IDO, an immune checkpoint enzyme with well-defined biochemical properties, is an attractive target for small-molecule inhibitors. Currently, 11 IDO inhibitors are in clinical investigation,²⁶⁶ including two in phase III and two in phase II trials. IDO inhibitors as monotherapy have generally shown limited efficacy in tumor treatment, and research has indicated a complex interplay between IDO1 expression, PD-1 expression, and immune therapy resistance.^267-270 The combination of IDO inhibitors with PD-1/PD-L1 monoclonal antibodies is a strategy being explored to modulate the tumor immunosuppressive microenvironment and enhance the efficacy of cancer therapy. Clinical trials of IDO inhibitors have yielded mixed results, and ongoing trials are being closely monitored for further insights (Tables 2 and 3).

5.3 Targeting GAPDH

4-Octyl itaconate, a cell-permeable itaconate derivative, reduces GAPDH activity by directly alkylating cysteine residues at position 22 on the enzyme.²⁹² Heptelidic acid (koningic acid) binds to the cysteine-149 residue at the active site of GAPDH enzyme in a dosage-dependent manner, leading to irreversible inhibition of its activity.²⁹³ TCH-346 is a pharmacological agent with antiapoptotic properties that targets GAPDH, inhibiting cell apoptosis by preventing GAPDH nitrosylation and nuclear accumulation. It is primarily used for managing Parkinson's disease and motor neuron disease.^294-296

5.4 Targeting ACOD1

Citraconate, an endogenous competitive inhibitor of ACOD1 and a natural isomer of itaconate, serves as a substrate analogue due to its structural similarity to cis-aconitate. This allows citraconate to specifically bind to the active site of ACOD1, significantly inhibiting the production and accumulation of itaconate.²⁹⁷ Higher concentrations of citraconate have been detected in various disease states, including colorectal cancer and type II diabetes, highlighting its potential for inhibiting ACOD1-mediated antitumor activity and its demonstrated anti-inflammatory, antioxidant, and antiviral properties.^{297, 298}

5.5 Targeting arginase

2-(S)-amino-6-boronohexanoic acid (ABH) marks a new era as the first arginase inhibitor to incorporate boron. Its analog, S-(2-boronoethyl)-l-cysteine (BEC), is crafted by substituting sulfur for carbon in ABH's main chain. Both ABH and BEC interact with arginase through a mechanism similar to the hydrolysis of l-arginine.²⁹⁹ Nω-hydroxy-nor-arginine (nor-NOHA) emerges from the endogenous compound N(ω)-hydroxy-l-arginine (NOHA) as an arginase inhibitor. Despite NOHA's potent arginase inhibition, its role as a transient intermediate in NOS-mediated NO production restricts its widespread application.³⁰⁰ As nonboron analogues of ABH, both NOHA and nor-NOHA have been shown to effectively neutralize the immunosuppressive effects of MDSCs by inhibiting arginase1, thus alleviating MDSC-mediated T cell proliferation.^{301, 302} In the context of Candida infection, nor-NOHA has demonstrated a multifaceted approach to enhancing immune responses by inhibiting arginase, regulating eosinophil infiltration, and promoting a protective type 1 immune response. This leads to an upregulation of IFN-γ, IL-6, TNF-α, and other cytokines and chemokines, ultimately improving infection control and survival rates.³⁰³ In a model of cutaneous squamous cell carcinomas, nor-NOHA has been shown to modulate tumor progression by enhancing T cell and DC infiltration and PD-1 expression, with a synergistic effect observed when combined with the anti-PD-1 monoclonal antibody nivolumab.³⁰³ Currently, nor-NOHA is being explored in clinical trials for coronary artery disease and diabetes management. The development of N-substituted 3-amino-4-(3-boronopropyl) pyrrolidine-3-carboxylic acids, a novel ABH derivative with enhanced inhibitory activity against both arginase1 and arginase2, promises a more potent therapeutic option,³⁰⁴ although further research is warranted.

5.6 Targeting HDAC6

Despite discovering several HDAC6 inhibitors, including Nexturastat A (NextA),³⁰⁵ Tubacin,³⁰⁶ and TSA,³⁰⁷ their clinical application has been limited due to insufficient anticancer efficacy and the requirement for high drug concentrations. The development of dual-target HDAC6 inhibitors for cancer therapy is an area of active research, with promising candidates such as dual HDAC6/PI3K inhibitors, dual HDAC6/mTOR inhibitors, dual HDAC6/BRD4 inhibitors, and dual HDAC6/HSP90 inhibitors in various stages of development. While these inhibitors are still largely experimental, they hold great promise for the future of targeted cancer therapies.¹⁶¹