Amino acid metabolism in immune cells: essential regulators of the effector functions, and promising opportunities to enhance cancer immunotherapy

Yang, Luming; Chu, Zhaole; Liu, Meng; Zou, Qiang; Li, Jinyang; Liu, Qin; Wang, Yazhou; Wang, Tao; Xiang, Junyu; Wang, Bin

doi:10.1186/s13045-023-01453-1

Review
Open access
Published: 05 June 2023

Amino acid metabolism in immune cells: essential regulators of the effector functions, and promising opportunities to enhance cancer immunotherapy

Luming Yang^1,2^na1,
Zhaole Chu²^na1,
Meng Liu^1,2,
Qiang Zou^1,2,
Jinyang Li²,
Qin Liu²,
Yazhou Wang¹,
Tao Wang²,
Junyu Xiang² &
…
Bin Wang^2,3,4

Journal of Hematology & Oncology volume 16, Article number: 59 (2023) Cite this article

9084 Accesses
25 Citations
10 Altmetric
Metrics details

Abstract

Amino acids are basic nutrients for immune cells during organ development, tissue homeostasis, and the immune response. Regarding metabolic reprogramming in the tumor microenvironment, dysregulation of amino acid consumption in immune cells is an important underlying mechanism leading to impaired anti-tumor immunity. Emerging studies have revealed that altered amino acid metabolism is tightly linked to tumor outgrowth, metastasis, and therapeutic resistance through governing the fate of various immune cells. During these processes, the concentration of free amino acids, their membrane bound transporters, key metabolic enzymes, and sensors such as mTOR and GCN2 play critical roles in controlling immune cell differentiation and function. As such, anti-cancer immune responses could be enhanced by supplement of specific essential amino acids, or targeting the metabolic enzymes or their sensors, thereby developing novel adjuvant immune therapeutic modalities. To further dissect metabolic regulation of anti-tumor immunity, this review summarizes the regulatory mechanisms governing reprogramming of amino acid metabolism and their effects on the phenotypes and functions of tumor-infiltrating immune cells to propose novel approaches that could be exploited to rewire amino acid metabolism and enhance cancer immunotherapy.

Introduction

Studies over the past few decades have demonstrated that malignant tumors and the immune system are intimately connected. The composition of the tumor microenvironment (TME) is complex, comprising various cell types such as immune cells, fibroblasts, and endothelial cells which exhibit intrinsic regulatory effects on tumor cells [1–4]. Immune cells, including myeloid and lymphoid cells, play critical roles in promoting inflammation and immunosurveillance on one hand; and inhibiting inflammation and enabling immune escape on the other. Some immune cells, such as helper T (Th) cells are dichotomized into Th1/Th2 subtypes. Th1 represents cells with a pro-inflammatory role, whereas Th2 cells exhibit an anti-inflammatory role. Similarly, macrophages are also dichotomized to M1/M2 subtypes. M1 subtypes promote a Th1 response and an anti-tumor function, and M2 subtypes display a Th2-like response and a pro-tumor effect. Thus, the differentiation status of immune cells may dictate their anti-tumor functions. The differentiation process of immune cells from their progenitors is directed by many factors, such as selective gene expression, cytokines stimulation, antigen presentation, nutrient metabolism and so on [5–7]. Besides governing cell fate, these factors also directly influence immune cell function. Whereas many of these factors have been extensively studied, metabolism-mediated regulatory mechanisms are still poorly understood [1, 8].

Metabolic reprogramming is a hallmark of the TME and has recently received increased attention. Various nutrients including glucose, lipids, and amino acids, play essential roles in regulating tumorigenesis through acting on both tumor cells and immune cells [9, 10]. These nutrients, as well as their metabolites, are sensed primarily by the mTOR complex through a series of biochemical networks [11, 12]. Among these major nutrients, amino acids play a dominant role in regulating mTORC1 [13]. During tumorigenesis, mTORC1 signaling regulates lipid synthesis, protein synthesis, cell survival, proliferation, and angiogenesis through targeting various substrate such as S6K1, hypoxia inducible factor 1 subunit alpha (HIF1α), and 4E-BP [11]. Collectively, mTOR functions as a central hub of nutrient signaling and cell growth across species [14]. While the roles of lipid metabolism in tumor immunity have been recently discussed [15], the regulation of amino acids metabolism in tumor immunity has not been extensively summarized. Therefore, we provide a comprehensive analysis of the roles of amino acid metabolism in regulating immune cells and their dysfunction within the TME.

The story of amino acids began from experiments carried out as early as 1827 when asparagine, isolated from asparagus juice, was hydrolyzed by Auguste-Arthur Plisson and Étienne Ossian, leading to the discovery of aspartic acid. In the subsequent 200 years, amino acids have been characterized as one of the fundamental units of life. In addition to serving as the basic building blocks of proteins, amino acid metabolism is also involved in many other cellular processes to dictate cellular functions. Given the marked increase in growth kinetics of malignant tumors, one can speculate that amino acid metabolism in the TME is significantly altered. Rewiring of amino acid metabolism in infiltrating immune cells directly impact their biological functions [16]. In this regard, amino acid transporters, sensors, and the rate-limiting metabolic enzymes are key gatekeepers of the metabolic flow. Aberrancies or deficiencies of this flow may reprogram amino acid metabolism in the infiltrating immune cells and impair anti-tumor immunity. Thus, key regulators of this pathway could be regarded as potential therapeutic targets.

Here, we briefly introduce the physiologic functions of immune cells and amino acids. We summarize the immunomodulatory effects of amino acid metabolism on various immune cell lineages under homeostatic and inflammatory settings, as well as in the TME, with an emphasis on amino acid competition between the immune cells and tumor cells. From a clinical perspective, we also discuss potential therapeutic strategies targeting amino acids metabolism pathways for improving cancer immune therapy.

Amino acid metabolism in lymphoid cells

T lymphocytes

T cells, or T lymphocytes, play a key role in cellular immunity and are responsible for eliminating cells expressing “foreign” antigens. With respect to cancer, once tumor cells begin to express tumor-associated antigens, specific T cells may view these cells as foreign and target them for cell death. T cells could be classified into two clusters depending on their membrane markers. The CD4⁺ cluster includes follicular helper T (Tfh) cells, T helper (Th) cells, and regulatory T cells (Treg); and the CD8⁺ cluster includes cytotoxic T cells and memory T cells. CD8 is predominantly an α/β heterodimer, but α/α homodimers are also present and recognizes endogenous antigens presented on MHCI, whereas CD4 recognizes exogenous antigens presented on MHCII. However, T cells may not be precisely defined by their cellular functions. For instance, the effector T cells (Teff) definition refers to Th cells or cytotoxic CD4⁺ and CD8⁺ T cells, which are characterized as secreting inflammatory cytokines and are cytotoxic to foreign cells [17].

It is noteworthy that Tregs are CD4+ CD25+ T cells with immune suppressive functions that are necessary for maintaining self-tolerance [18]. Subsequent studies identified Tregs as expressing the transcriptional factor FOXP3 [19, 20]. Tregs play important roles in regulating immune surveillance and antitumor immune response through suppressing the activities of CD4+ helper and CD8+ cytotoxic T cells. Upregulation of immunosuppressive molecules including CTLA-4, PD-1, PD-L1 is essential for immune evasion during tumorigenesis. The relationships between Tregs and other T cells, as well as the roles of immune checkpoints in anti-cancer immune therapy, has been previously reviewed [21–23]. Prior studies have indicated that both CD4⁺ and CD8⁺ T cells are essential for a robust immune response and diverse amino acids play a critical role in regulating T cells (Fig. 1).

Branched-chain amino acids (BCAAs)

The three BCAAs are leucine, isoleucine, and valine, all of which are essential amino acids that cannot be de novo synthesized in humans. Leucine was isolated by Joseph Louis Proust in an impure form called caseous oxide in cheese in 1818 [24]. Felix Ehrlich first discovered isoleucine and isolated it from fibrin in 1904, and named it “iso-” because of its similar structure and function with leucine [25]. Emil Fischer, who was awarded the Nobel Prize in Chemistry in 1902, isolated valine from casein in 1902 [26]. In addition to serving as a basic building block of protein synthesis, under physiological conditions BCAAs are also important regulators of global cellular protein synthesis. In the normal tissue microenvironment, a study in cats found that high doses of leucine enhanced the secretion of IL-10 by T cells to inhibit the immune response [27]. Another study found reduced survival of influenza virus infected mice with high BCAA diets, which might be attributed to an increase of the number of CD8⁺ T cells accompanied with overexpression of cytotoxic cytokines resulting in tissue damage [28].

The uptake and distribution of leucine is carried out by L-type amino acid transporter (LAT), which directs amino acids across the plasma membrane in a Na+-independent manner [29]. The structure of LAT1, encoded by SLC7A5, revealed the mechanism of LAT1 amino acid selectivity [30, 31]. In T cells, antigen signaling mediated through the interaction of the T cell receptor (TCR) and LAT1 increased leucine uptake [32], which may be a self-protective mechanism for T cells to compete with cancer cells for the limited supply of amino acids within the TME. It is possible that coupling amino acid uptake with T cell signaling exists for other amino acids as well. Interestingly, LAT1 itself is a bidirectional transporter that regulate the influx of leucine and the efflux of glutamine [33], whereas another transporter, ASCT2 (encoded by SLC1A5), allows cellular uptake of glutamine [34]. However, the functions of ASCT2 and LAT1 do not appear to be coupled [35]. BCAAs are catabolized by Branched-Chain Aminotransferases (BCATs), including BCAT1 and BCAT2, also known as cytosolic branched-chain aminotransferase (BCATc) and mitochondrial branched-chain aminotransferase (BCATm), respectively. BCATs transfer amino groups from BCAA to α-ketoglutarate to produce their respective branched-chain ketoacids (BCKA) and glutamate [36, 37]. β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of BCAAs, which favors a switch of Th1-type to Th2-type cells [38]. The concentration of extracellular L-arginine controls the expression of the T cell antigen receptor ζ chain (CD3ζ), to influence the antigen recognition function of T cells [39, 40].

Besides its effects on the TCR, another well characterized function of leucine is regulating the mTOR pathway. mTOR is found in two complexes, mTORC1 and mTORC2, which have different sensitivities to rapamycin and are composed of distinct subunits. Several reviews have covered the functions of mTOR on downstream T cell activation, differentiation, and homeostasis [41–43]. Expression of BCATc is induced by TCR signaling in CD4+ T cells. BCATc^−/− mice have increased levels of leucine which enhances mTOR activity, leading to increased phosphorylation of downstream substrates including S6 and 4EBP-1 [44]. Sestrin2, SAR1B, and GATOR2 are all the upstream regulators of mTOR. Both Sestrin2 and SAR1B are bonded to GATOR2 to inhibit mTORC1 signaling, and their interactions with GATOR2 can be disrupted by leucine, indicating a role for Sestrin2 and SAR1B as a sensor of leucine to activate mTOR activity [45, 46]. However, the function of these two sensors has some differences. For instance, Sestrin2 may show increased sensitivity to high concentration of leucine, whereas SAR1B may sense lower levels of leucine to maintain basal mTOR activity [46]. Although Sestrin2 and SAR1B have been studied in other cell types, they likely carry out important functions in T cells as well. While leucine controls mTOR activity to regulate T cell proliferation and differentiation, it does not appear to be required for the maintenance of naïve T cells [32].

However, mTOR activity is also essential for the immune suppressive Tregs. Treg-specific mTOR knockout mice revealed mTOR was critical for Treg differentiation, activation, and migration into non-lymphoid tissues [47]. A role for amino acids in regulating Treg function and immune tolerance through mTOR was further demonstrated to be dependent on RagA/B and Rheb1/2 GTPases where arginine and leucine functioned to induce and sustain mTORC1 activity in Treg cells [48]. Results such as these suggest that factors such as RagA/B and Rheb1/2 may serve as potential therapeutic targets. In addition to mTORC1, the cooperation of mTORC1 and mTORC2 also contributes to the maintenance of Tregs suppressive activity, but mTORC2 was not necessary for Tregs function [49]. Similarly, systemic lupus erythematosus, an autoimmune disease, exhibited increased activity of mTORC1 and mTORC2, but diminished Treg suppressive activity [50]. Therefore, the role of amino acids in activating mTOR and its role in controlling Tregs needs further exploration.

BCAA metabolism is hyperactive in multiple malignancies including leukemia, lung cancer, bone sarcomas, hepatic carcinoma, pancreatic ductal adenocarcinoma, and colorectal cancer [51–57]. Although there is no direct evidence demonstrating a role of BCAA metabolism in anti-tumor immunity, these observations indicate that activation of BCAA metabolism might be linked to dysfunction of tumor-infiltrating T cells.

Glutamine

Glutamine was first discovered by Schulze in 1883, with the synthesis of glutamine further described by Hans Krebs in 1935, and its importance in organismal homeostasis was explored in animal experiments [58]. Subsequently, research on glutamine in the fate of cancer cells and cancer therapy has been extensive, but its role in tumor-associated immune cell function is just emerging [59].

Overexpression of SLC38A1, a glutamine transporter, markedly enhanced mitochondrial function in human CD4⁺ T cells exposed to ascites derived from patients with ovarian cancer, a process shown to be associated with glucose metabolism [60]. Another glutamine transporter, SLC38A2, was found to be critical for the generation and memory T cells in part through modulating mTORC1 activity [61]. Furthermore, the proliferation and function of Tregs is dependent on the expression of SLC7A11, which is controlled by the nuclear factor erythroid 2-related factor 2 (NRF2) [62]. However, this study did not explore the exact substrate effects on Tregs regulation. Although SLC7A11 is regarded as an antiporter of cysteine and glutamate transporter, this study did not explore the exact substrate effects on Tregs regulation. Moreover, SLC7A11 is highly expressed in tumor cells where it imports cystine for GSH biosynthesis and antioxidant defense. This effect is partly due to suppression of ferroptosis, another powerful tumor suppression mechanism that may eliminate precancerous cells exposed to metabolic stress and nutrients starvation [63]. Thus, these observations support the notion that SLC7A11 may serve as an additional mechanism specific in tumor or immunosuppressive cells to capture free cystine in the TME to promote tumor growth and attenuate the function of anti-tumor immune cells. However, the role of SLC7A11 in anti-tumor immune cells needs further investigated.

As the precursor of protein O-GlcNAcylation, glutamine is involved in regulating T cell self-renewal [64]. Glutamine maintains Teff cell ATP concentrations through glutamine-dependent mitochondrial metabolism [65]. The catabolism of glutamine promotes de novo synthesis of glutathione (GSH) to impact T cell differentiation [66], a process that also involves mTOR activity [67, 68].

Despite glutamine regulating T cell activation and function, blocking glutamine metabolism does not weaken T cell function as anticipated, but rather enhances T cell antitumor activity [69]. One possible reason behind this observation is that tumor cells are addicted to glutamine, and thus glutamine starvation may have more direct consequences on tumor cell survival than T cells.

Tryptophan

L-tryptophan was discovered in 1901 by Frederick Hopkins and Syndey Cole from the lysate of casein digested by insulin [70]. And it was subsequently identified as one of the essential amino acids in 1912 by Hopkins [71]. Although a small fraction of free tryptophan is used in protein synthesis and to produce neurotransmitters such as serotonin, over 95% of free tryptophan is degraded through the kynurenine pathway [72], which provides metabolites such as melatonin, quinolinic acid, kynurenine, tryptamine, vitamin B3, nicotinamide adenine dinucleotide (NAD+), and nicotinamide adenine dinucleotide phosphate (NADP+).

Under physiological conditions, enzymes in the tryptophan-kynurenine pathway are constitutively expressed, thus tryptophan metabolism has widespread effects, from central excitatory neurotransmission to peripheral immune response and inflammation [73]. The function of tryptophan in T cells mainly serves as an immune inhibitory factor. In general, tryptophan is catabolized to kynurenine by Indoleamine 2,3-dioxygenase (IDO), which is then further catabolized by other kynureninases [72]. Besides IDO, another tryptophan-degrading enzyme, tryptophan-2,3-dioxygenase (TDO2), is expressed in the liver and catalyzes tryptophan to kynurenine [74]. While tryptophan conversion to kynurenine has not been shown to take place in T cells, this pathway functions in other cells within the TME including macrophages and tumor cells, thus tryptophan metabolism may indirectly influence T cell function.

In normal tissue environments, IDO activity has been directly associated with suppressing the immune response. During pregnancy, IDO prevented the fetal alloantigen response by inhibiting T cell-driven local inflammation [75], possibly due to attenuation of central and effector memory CD8⁺ T cell generation, an effect that was lost once these T cells were present [76]. During human hematopoietic stem-cell transplantation, the upregulation of extracellular kynurenine effectively suppressed T cell responses and promoted apoptosis of Teff cells [77]. Moreover, the ratio of tryptophan and kynurenine may be used as a predictive marker of CD4⁺ T cell populations [78]. Besides kynurenine, other metabolites of tryptophan also function in suppressing immune function. In a rat cardiac allograft model, 3-hydroxyanthranilic acid markedly prolonged survival [79], which may serve as a model of allograft transplantation induced immune tolerance. Interestingly, both kynurenine and picolinic acid inhibited T cell proliferation, but had no effect on the activation of resting T cells [80]. Furthermore, while 3-hydroxykynurenine and 3-hydroxyanthranilic acid inhibited T cell responses, anthranilic and quinolinic acid had no effect [76].

System L, a heterodimer composed of a heavy chain of CD98 (encoded by SLC3A2) and one of two catalytic L chains LAT1 (encoded by SLC7A5) or LAT2 (encoded by SLC7A8), transports tryptophan [81]. With low tryptophan levels in the TME, tumor cells may use two mechanisms to enhance tryptophan uptake to outcompete Teff cells for tryptophan. One mechanism may be for tumor cells to secrete toxic metabolites such as kynurenine to attenuate neighboring Teff cells anti-tumor function [82]. Another mechanism could be that system L is not a unique transporter for tryptophan, and another specific tryptophan transporter is induced in IDO expressed tumors [83]. Moreover, general control nonderepressible 2 (GCN2) kinase could serve as an intracellular amino acid sensor capable of phosphorylating eukaryotic translation initiation factor 2-alpha (eIF2α) to lower global protein synthesis rates in response to amino acid starvation [84]. Tryptophan depletion leads to T cell anergy and proliferation arrest, and disruption of GCN2 might prevent T cells from IDO-mediated inhibition [85].

Prior studies have shown that activation of GCN2 could inhibit inflammatory Th17 and Th9 cell differentiation [86, 87], potentially through GCN2-mediated activation and downregulated of key enzymes involved in fatty acid synthesis, which is a prerequisite of CD4⁺ T cell proliferation and differentiation, suggesting that GCN2 activity may weaken the immune response [88]. However, other studies have observed that GCN2 is essential for T cell function. For instance, GCN2 deficient T cells fail to proliferate when amino acids are limiting [89], and knockout of GCN2 impaired many aspects of T cell immunity in brain tumors [90]. In addition, both IDO and TDO inhibitors prevent the accumulation of immunosuppressive tryptophan catabolites but do not enhance T cell responses via rescuing GCN2 from tryptophan starvation, thus GCN2 might not play an important role in the response to tryptophan starvation [91]. Therefore, these varied conclusions reveal that the relationship between tryptophan metabolism and T lymphocytes remain unclear and worthy of further investigation.

Tryptophan metabolism also leads to the production of GCN2-dependent Tregs [92]. Kynurenine activated aryl hydrocarbon receptor (AhR)-expressing T cells to differentiate into Tregs but not AhR-null T cells, and TGF-β upregulated AhR expression to potentially induce the generation of Tregs [93]. Kynurenine is transported across the plasma membrane of T cells by SLC7A5 or SLC7A8 and sensed by AhR in the cytoplasm [82, 94] to upregulate PD-1 expression in CD8⁺ T cell, driving the generation and differentiation of Tregs [95, 96]. Inhibition of AhR was shown to suppress IDO/TDO mediated tumor progression, which synergizes with PD-1 blockade [95]. These studies indicate that the immunosuppressive axis, try-kyn-AhR, may participate in tumor progression through regulating Tregs.

Methionine

Methionine, initially named sulfurous amino acid, was isolated by J. H. Muller in 1923 [97]. Unfortunately, he made an incorrect summation formula. Three years later, his colleague S. Odake corrected that formula and named this amino acid methionine [98]. In 1928, the structure of methionine was solved by G. Barger and F. P. Coyne [99]. Methionine is another essential amino acid, and as with other amino acids, its primary function is in protein synthesis to satisfy T cell proliferation. Methionine also serves as a critical source of methyl groups in cells through the methionine cycle, during which methionine is converted into S-adenosylmethionine (SAM) by methionine adenosyltransferase (MTA), to participate in many important biochemical processes, including float metabolism, redox maintenance, polyamine synthesis, and as the methyl donor molecule for all methylation reactions [100]. In addition, methionine is also crucial for the production of endogenous hydrogen sulfide [101] and plays a role in inhibiting autophagy [102]. A recent study discovered that methionine also provides methyl groups for DNA and RNA methylation, which further promote differentiation and proliferation of T cells [103].

The importance of methionine in T cells is not dependent on differences between intracellular and extracellular methionine concentration gradients, but rather T cells upregulate the expression of the methionine transporter SLC7A5 following stimulation of with antigen. Based on this, SLC7A5 is recognized as the rate-limiting factor for the generation of methyl groups during T cell activation [103]. However, methionine import varies in tumor cells. Besides SLC7A5, the transporter SLC43A2 is also involved in methionine uptake in tumor cells allow them to outcompete T cells for limited methionine availability [104]. Following import of methionine, intracellular methionine regulates cellular functions within the nucleus. Methionine restriction reduces histone H3K4 methylation (H3K4me3) on promoters of key genes involved in Th17 cell proliferation and cytokine production, and exposing mice to a diet deficient in methionine reduced the expansion of pathogenic Th17 cells, leading to a reduction of T cell-mediated neuroinflammation and disease [105, 106]. In addition, tumor cells outcompete T cells for methionine leading to a decrease of intracellular methionine and SAM levels in CD8+ T cells reducing H3K79me2 and STAT5 gene expression to attenuate immune function [104]. There is also direct evidence revealing a relationship between methionine levels and anti-tumor function of T cells. In this context, elevation of SAM and its downstream catabolites 5-methylthioadenosine reprogrammed the global chromatin accessibility of CD8⁺ T cells, which attenuated T cell anti-tumor function [107]. Moreover, the role of SLC43A2 was also shown to be critical for the survival of Tregs, as the downregulation of SLC43A2 increased the Treg apoptosis due to the decrease of methionine uptake [108].

Arginine

Since the discovery of arginine from lupin seedling extracts in 1886 by Schulze and Steiger [109], studies of arginine focusing on its basic functions and clinical relevance have been of high interests over the past century. Arginine is a nonessential amino acid, but the de novo synthesis of arginine does not satisfy the daily demand of the human body. Thus, arginine is defined as a semi-essential, or conditionally essential. Under physiological conditions. Arginine is mainly metabolized by arginase-1 (Arg1) and nitric oxide synthase (NOS) in myeloid cells, generating urea and L-ornithine, and NO and L-citrulline, respectively [110].

Studies have found that reduction of arginine attenuates T cell function and proliferation, which can be reversed by arginine supplementation [111–114]. Using a mouse model of sepsis, arginine supplementation contributed to the maintenance of CD4⁺ T cells in the blood and para-aortic lymph node, but this was abrogated by inhibition of NOS, indicating that production of NO through NOS was important for proliferation of T cells [115]. Under arginine restriction, T cells downregulate CD3ζ expression, preventing the expression of the TCR leading to reduced proliferation and cytokine secretion [116]. In neonates, arginine might regulate DNA methylation to modulate the maturation of the immune system [117]. The arginine transporter, cationic amino acid transporter-1 (CAT1), supports both naïve and memory CD4⁺ T cells as well as CD8⁺ T cells to maintain T cell proliferation and activation [118].

More specifically, the uptake of arginine is controlled by the cationic amino acid transport (CAT1, 2a, 2b and 3, encoded by SLC7A) [119, 120], which function as monomers on the plasma membrane [120]. Generation and persistence of memory T cells, but not effector T cells, can be reprogrammed by arginine transported by SLC7A1 in a mechanism partially dependent on mTORC1 [61]. SLC38A9, a lysosomal transmembrane protein, serves as an intracellular arginine sensor to interact with lysosome membrane localized Rag GTPase and Ragulator, which acts as a scaffold to tether Rag GTPase and mTORC1 to the lysosome [121, 122]. Another key factor, CASTOR1, also senses arginine. However, the mechanism by which CASTOR1 and SLC38A9 sense arginine appears to be different. Arginine stimulates SLC38A9 to efflux essential amino acids, including leucine, from lysosomes to the cytosol to be recycled, but SLC38A9 has low affinity for arginine transport and minor effects on arginine concentration [123]. On the contrary, CASTOR1 binds to cytoplasmic arginine with high affinity [124]. Thus, SLC38A9 transmits an arginine sufficiency signal to mTORC1, leading to mTORC1 activation, and the overexpression of SLC38A9 partially maintains mTORC1 activation upon amino acid deprivation [122, 125]. Whereas CASTOR1 serves to transmit an arginine deprivation signal. When arginine is deprived, CASTOR1 and GATOR2 bind together promoting the dissociation of the GATOR1/GATOR2 complex (an upstream regulator of mTORC1), leading to the inhibition of mTORC1 activity [124, 126]. Moreover, the function of CASTOR1 is similar to Sestrin2 and SAR1B described above, except that they sense different amino acids. Therefore, SLC38A9 and CASTOR1 play a counterbalancing role in arginine sensing to prevent abnormal activation of mTORC1, which may play a role in regulating proliferation and differentiation of tumor-associated immune cells. Supporting this notion, L-arginine depletion dampened proliferation of T cells through GCN2, which may arrest T cells in the G1 phase of the cell cycle through impairment of cyclin D3 and cyclin-dependent kinase 4 (cdk4) activity [127, 128]. Interestingly, other studies have revealed that arginine levels might influence the cell cycle through regulating the mTOR pathway. In this context, arginine deprivation suppresses the activity of mTORC1, whereas the activity of mTORC2 increases, leading to cell cycle arrest, which can be reversed by arginine supplementation [129]. As important amino acid sensors, both GCN2 and mTOR signaling pathways regulate cell cycle progression of T cells. Regarding therapeutic T cells, such as chimeric antigen receptor (CAR)-T cells, recent evidence suggests that low arginine levels also impair their proliferation [130].

As a semi-essential amino acid, the de novo synthesis of arginine also plays an important role in maintaining intracellular arginine concentrations. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are key enzyme in arginine synthesis. ASS catalyzes condensation of citrulline and aspartate to argininosuccinate which is then converted to arginine and fumarate by ASL [131]. Citrulline, which is transported by SLC7A5 (LAT1), could partially substitute for arginine deficiency to promote T cell proliferation via ASS and ASL in T cells, whereas rescue of proliferation was not observed in complete absence of arginine [132].

Besides reprogramming of amino acid metabolism, additional studies have found that other metabolic pathways such as glucose metabolism also regulates the activity of Tregs through the mTOR pathway [49, 133]. Tregs are characterized by oxidative and catabolic metabolism. However, Toll-like receptor (TLR) signals promote Treg proliferation and increased glycolysis, anabolic metabolism, and expression of glucose transporter Glut1. Glut1 reduced expression of FOXP3 in Tregs and hampered their immunosuppressive capacity [134]. Thus, mTOR plays a key role in regulating amino acid, lipid, and glucose metabolism in Tregs, with further studies likely to uncover regulation of other metabolic pathways by mTOR in Tregs.

In the TME, owing to a lack of Arg1 and NOS in T lymphocytes, arginine may be consumed by Arg1 and NOS in tumor cells or myeloid cells leading to excessive arginine consumption and a weakening of the Teff immune response. Besides the direct regulation on T cells, L-arginine depletion induced myeloid-derived suppressor cells (MDSCs) to blunt the anti-tumor response of Teff cells [135]. Therefore, arginine depletion caused by elevated Arg1 in the TME has been considered a hallmark immunosuppressive mechanism. However, recent studies revealed Arg2, an isoform of Arg1 which functions in the mitochondria, is expressed in T cells and serves as a regulator of CD8⁺ T cell activation, anti-tumor cytotoxicity, and memory formation, independently of extracellular arginine concentration [136]. L-arginine is rapidly converted into downstream metabolites, and the addition of the arginase inhibitor norNOHA reduces this conversion, indicating that arginine is catabolized by Arg2 in T cells [137]. When Arg2^−/− CD8⁺ T cells and wild-type CD8⁺ T cells were co-transferred into wild-type mice, Arg2^−/− CD8⁺ T cells outcompeted wild-type cells suggesting that Arg2 was detrimental to T cell survival [137]. This may partially explain why Arg2^−/− CD8+ T cells suppress tumor growth and synergize with PD-1 blockade [136]. Therefore, Arg1 or Arg2 are both negative regulators of T cell survival and function with Arg1 existing in cells other than T cells to consume free arginine in the TME, and Arg2 is found in T cells to consume intracellular arginine. Both intracellular and extracellular deprivation of arginine are essential means to regulate the proliferation of T lymphocytes and maintain tissue homeostasis.

Serine

In 1865, Emil Cramer extracted component from silk, and named it serine from the Latin of silk, sericum [138]. As a non-essential amino acid, serine is synthesized de novo from the glycolytic intermediate 3-phosphoglycerate or transported by several transporters such as SLC1A5 into cells that cannot synthesize serine like mature neurons [139, 140]. Serine supports metabolic processes including protein and glutathione synthesis and serves as a crucial one-carbon donor for the folate cycle to participate in nucleotide synthesis, methylation reactions, and generation of NADPH [141, 142]. Based on these roles of serine, targeting cancer-associated serine metabolism is viewed as a potential cancer therapy.

Generally, following antigenic stimulation, the rate of serine synthesis increases in activated T cells to provide intracellular glycine and one-carbon metabolites to support the proliferation of T cells, indicating that serine is essential to the T cell adaptive immune response, and extracellular serine is dispensable to support optimal T cell expansion even when glucose is sufficient for T cell activation and function [143]. Another study found that in order to adapt a state of rapid proliferation of T cells, naïve T cells induce the reprograming of mitochondrial biogenesis and modeling, with one-carbon metabolism being the most highly induced pathway, which is fed by serine, and disruption of the key mitochondrial serine hydroxymethyltransferase (SHMT2) inhibiting T cells proliferation [144].

In the TME, serine activates mTOR to inhibit FOXP3 expression and further hinder Tregs immunosuppressive capacity, but the transformation of serine to glutathione through glutamate cysteine ligase (Gclc) contributed to preserve Tregs immunosuppressive function, and Gclc ablation showed autoimmunity and enhanced anti-tumor responses [145].

Given serine is a non-essential amino acid, relatively fewer studies have focused on its role in immunity. However, with the studies described above highlighting a key role for serine in T cell function, a role for serine metabolism in the tumor-associated immune response should be assessed further.

B lymphocytes

B cells have been recognized as a core component of humoral immunity. The mechanism by which B cells serve to defend against foreign antigens occurs through three different pathways: antibody secretion, antigen presentation, and their direct killing capacity. Following stimulation, B cells differentiate into plasma cells, and the immunoglobulins secreted by plasma cells facilitate the immune responses by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Several studies have revealed that a subset of amino acids play a role in these important processes (Fig. 2).

Leucine, transported into B cells through SLC7A5, targets mTORC1 to promote B cell differentiation, as well as supports the production of IgG and cytokines [146]. Furthermore, inhibition of the glutamine transporter SLC1A5 or key enzymes of glutamine metabolism, led to reduced production of IgM in B cells [147]. Moreover, restriction of amino acids such as tryptophan led to a developmental arrest of B cells in the bone marrow [148]. In addition, histidine is transported by SLC15A4 from the lysosome to cytosol, and loss of SLC15A4 disturbed the Toll-like receptor 7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to auto-antibody production [149]. Given this importance of amino acids in B cell function, further studies are necessary to fully understand a role for amino acids in regulating B cell function.γ-Aminobutyric acid (GABA) originates from glutamic acid, one of the non-essential amino acids, through key enzymes including glutamate decarboxylase (GAD). In the past, GABA has been extensively studied in the context of neurobiology as a central suppressive neurotransmitter. Interestingly, GABA has also been shown to play an important role in B cells anti-tumor immunity. GABA is synthesized and secreted by activated B cells and plasma cells, and this GABA further promotes monocytes to differentiate into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8⁺ T cell tumor killing function [150]. In addition, tumor cells aberrantly expressing GAD1 could enhance the tumors immune suppressive function through autocrine action of GABA [151]. Thus, GABA secreted by B cells may directly target tumor cells to promote tumor growth, therefore GAD should be studied as a potential anti-cancer target.

Germinal centers (GCs) are components of secondary lymphoid organs (SLOs) within the spleen, lymph nodes and mucosa-associated lymphoid tissue (MALT). They have important sites of B cell clonal expansion, somatic hypermutation, and affinity-based selection, a process that leads to the production of high-affinity antibodies [152]. GCs are divided into a light zone (LZ) and dark zone (DZ) which harbor distinct functions. The DZ is the site of B cell clonal expansion and somatic hypermutation, whereas the LZ is the region where B cells undergo positively selection for affinity-based mutations with the help of T follicular helper (Tfh) cells [153]. Rapamycin, an mTOR inhibitor, has been found block the formation of GCs [154]. Deficiency of the mTORC1 subunit Raptor reduce the population of B cells in GCs [155], and Raptor has been shown to fuel B cells differentiation and proliferation early after positive selection in the LZ, whereas it only regulates proliferation in the DZ [156, 157]. B cell homeostasis and function also requires Rictor, a subunit of mTORC2 [158]. Both mTORC1 and mTORC2 guard Tfh phenotypic and functional maturation to support B lymphocytes development [159, 160]. In addition, glucose, lipid and hypoxia influences GCs B cells through the metabolic sense of mTOR [161–163], and some chemokines such as BCL-6 [164] and CCL2 [165] are important regulators of the mTOR pathway in GC B cells. In Peyer’s patches (PPs), another SLO, nutrient deficiency drastically reduces secretion of CXCL13 from stromal cells via PI3K-Akt-mTOR axis to decrease the number of B cells in SLOs, and further attenuate the digestive immune system [166]. In addition, the PI3K-Akt-mTOR axis is also be activated by CXCL13/CCR5 to promote proliferation and migration of clear cell renal cell carcinoma [167]. Although this study did not explore the functional consequences to B cells or nutrient deprivation, one may speculate that nutrients could affect tumor progress through the regulation of B cells. Because B cells are critical for tertiary lymphoid structures (TLS), a tumor infiltrated ectopic lymphoid organ similar to SLOs, multiple studies found the higher number of infiltrating B cells in TLS indicate a better patient survival with malignant tumors, however the mechanism remains unclear [168, 169].

In summary, although several amino acids such as leucine, glutamine and glutamic acid have been shown to regulate tumor-associated B cells, the importance of mTOR in the formation of TLS and a correlation with the presence of B cells implicated within TLS suggests a key role of amino acids in both general biology and anti-tumor roles of B cells that deserves further investigation.

Natural killer cells

In 1975, Rolf Keissling and colleagues identified a new immune cell type mediating direct cytotoxicity against alloantigen and tumor-associated antigens, different from T or B lymphocytes [170, 171]. Subsequent studies of this cell type led to its classification as an innate lymphoid cell (ILC) named natural killer (NK) cells. Using high-throughput single-cell RNA-seq, NK cells have been further broken down into two major subsets based on marker expression. The first being NK1, which are CD56^dim in humans and CD27⁻CD11b⁺ NK cells in mice. NK2 cells are CD56^bright in humans and CD27⁺CD11b⁻ NK cells in mice [172]. NK cells are widely distributed throughout the human body, including lymphoid tissues such as bone marrow, lymph nodes, and spleen, as well as peripheral tissues such as liver, skin, intestine, and uterine [173]. Thus, it is important to explore the regulating factors, including amino acids, involved in controlling NK cell functions (Fig. 3).

Glutamine has been shown to be important in supporting physiological functions of NK cells. As an important regulator of the intestinal mucosa, glutamine supplementation increases the number of infiltrating NK cells and improves barrier function in a rat model of colitis [174]. In humans, glutamine supplementation in athletes with heavy load training led to an increase in NK cells activity [175]. However, another study provided contrary results regarding glutamine in regulating NK cell activity and function, where glutamine supplementation prevented the decline in plasm glutamine concentration but had no effect on NK cell cytotoxic activity [176]. However, in tubulointerstitial fibrosis, activated transglutaminase 2 in NK cells played an exacerbating role [177].

NK cells execute anti-tumor functions in multiple ways. First, they can recognize and respond to cancer cells lacking MHCI expression [178]. ADCC is another important mechanism by which NK cells function to restrict tumor growth and survival, and thus several studies have sought out ways to enhance ADCC such as assessing synergistic effects with anti-PD-L1, cytokine stimulation, and intercellular interactions [179–181]. NK cells also produce pro-inflammatory cytokines such as IFNγ and TNFα to promote anti-tumor activity of other immune cells or to directly inhibit tumor cells. Given NK cell characteristic ex vivo activation, expansion, genetic modification, and tumor cell recognition, mechanisms to improve their tumor-killing capacity for therapeutic purposes have been studied [182], and the metabolism of amino acids has been shown to play a role.

Recent studies have shown that SLC7A5 was the predominant system L-amino acid transporter in activated NK cells, and glutamine transported by SLC7A5 increases the expression of the transcription factor c-Myc through mTORC1 activation [183]. IL-12 induces the expression of the high-affinity IL-2 receptor CD25, and several amino acid transporters including SLC7A5, SLC1A5, and SLC3A2 are upregulated in response to IL-2 stimulation [183, 184]. NKG2D-mediated IFN-γ production and degranulation were decreased when SLC1A5 and SLC3A2 where inhibited [184], but whether this is through reducing amino acid uptake remains unclear. Besides IL-2 and IL-12, other cytokines such as IL-18 were shown to facilitate SLC7A5/SLC3A2 expression to promote proliferation of NK cells through the mTORC1 pathway [185]. mTORC1 and mTORC2 are both essential for NK cell activity, but through different mechanisms. mTORC1 appears to function in early NK cell development, whereas mTORC2 may function in terminal maturation, and thus they may cooperate in a non-redundant manner [186, 187]. Specifically, mTORC1 sustains mTORC2 activity through CD122-mediated IL-15 stimulation; however, mTORC2 negatively regulates mTORC1 function through the inhibition of STAT5-mediated SLC7A5 expression [186]. Another membrane protein, metabotropic glutamate receptor 5 (mGluR5), was found to enhance cytotoxicity of NK cells following activation by glutamate, and pharmacological activation of mGluR5 accelerated the regression of liver fibrosis [188].

While studies to date have focused largely on glutamine, amino acid transporters, and mTOR signaling in NK cells, it is likely that other amino acids play a role in this axis as well, such as arginine and leucine as key mTOR regulators, which needs further investigation.

Amino acid metabolism in myeloid cells

Macrophages

Macrophages are important innate myeloid cells and a major cell type within the mononuclear phagocyte system (MPS) [189]. Tissue-resident macrophages have various names tied to their specific function including microglia, Kupffer cells, alveolar macrophages, and osteoclasts [190-193]. In physiological conditions, tissue-resident macrophages play an important role in tissue homeostasis and organ development. The role of macrophages in development is often overlooked, including branching morphogenesis, neuronal patterning, angiogenesis, bone morphogenesis, generation of adipose tissue, and testicular organogenesis [194, 195]. As for homeostasis, Kupffer cells efficiently phagocytize pathogens entering from the portal or arterial circulation [191], and are essential for cholesterol transport to link lipid metabolism in the liver [196]. Alveolar macrophages play an important role in inhibiting pro-inflammatory responses to tissue debris or to innocuous antigens [192]. Osteoclasts are the only cells known to resorb bone to refresh or repair the skeletal system [193]. Spleen macrophages localize to the marginal zone and red pulp, which promote T cell immune responses and recycle iron elements from defective blood cells, respectively [197]. Testicular macrophages are known to contribute to spermatogenesis and the production of male hormones outside of their classical immune function [195].

Macrophages are important innate immune cells for tissue homeostasis and organ development under physiological conditions. It is generally recognized that M1/M2 polarized macrophages are two extremes within the immune response and are induced by various internal or external signals. These polarized macrophages further coordinate with diverse factors to regulate the outcome of pathogen infection, local tumor immune responses, and tissue repair and remodeling. In fact, M1-like and M2-like tumor-associate macrophages (TAMs) exist in the same tumor, but during different stages. They play an anti-tumor role through recognizing and eliminating tumor cells; or a pro-tumor role by promoting tumor growth, invasion, and metastasis. Such phenomenon is a so-called double-edged sword, and the function or polarization of macrophages is affected by many factors including amino acids and amino acid metabolites (Fig. 3) [198].

Tryptophan

Tryptophan regulates macrophages in many diseases besides cancer. Low levels of free tryptophan in serum may be associated with decreased IDO activity in macrophage and mental health syndromes such as anxiety and depression in patients with chronic hepatitis C [199]. Tryptophan-containing hexapeptide-coated gold nanoparticle hybrid effectively reduced the inflammatory response [200]. Consistently, reduced kynurenine production was shown to be associated with an increased inflammatory response in the lungs of aged individuals during an influenza infection [201]. Another study found kynurenine metabolism pathways were upregulated in the inflammatory disease, aortic atherosclerotic aneurysm, and the metabolism of kynurenines help downregulate inflammation [202]. With progression of the kynurenine metabolism pathways, tryptophan is ultimately broken down into small molecules such as NAD⁺, picolinic acid (PA), ATP, and CO₂ [72], which each having different roles in the anti-inflammatory function of macrophages. NAD⁺ was found to limit inflammation and promote phagocytosis [203], whereas PA is an activator of macrophage pro-inflammatory function [204].

Tryptophan is transported by system L in macrophages [81]. M2-like macrophages expressing IDO suppress the immune response by promoting the function of Tregs, and inhibiting Teff cells via the KYN-AhR pathway [205, 206]. AhR activation in tumor-associated macrophages might not only depend on tryptophan metabolism within macrophages, but might also require Lactobacillus metabolized dietary tryptophan to indoles to drive TAMs to acquire an immunosuppressive phenotype [207]. This observation emphasizes the importance of amino acid metabolism of the microbiota in the tumor immune response. Furthermore, high activity of AhR in glioblastoma is associated with reduced overall survival, suggesting AhR might become a potential therapeutic target in some tumor settings [208].