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Nat Rev Cancer. Author manuscript; available in PMC 2021 April 12.
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Published in final edited form as:

Nat Rev Cancer. 2020 September ; 20(9): 516–531. doi:10.1038/s41568-020-0273-y.

Metabolism of immune cells in cancer

Robert D. Leone, Jonathan D. Powell
Bloomberg~Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer
Research Center, Department of Oncology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA.

Abstract
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Through the successes of checkpoint blockade and adoptive cellular therapy, immunotherapy has
become an established treatment modality for cancer. Cellular metabolism has emerged as a
critical determinant of the viability and function of both cancer cells and immune cells. In order to
sustain prodigious anabolic needs, tumours employ a specialized metabolism that differs from
untransformed somatic cells. This metabolism leads to a tumour microenvironment that is
commonly acidic, hypoxic and/or depleted of critical nutrients required by immune cells. In this
context, tumour metabolism itself is a checkpoint that can limit immune-mediated tumour
destruction. Because our understanding of immune cell metabolism and cancer metabolism has
grown significantly in the past decade, we are on the cusp of being able to unravel the interaction
of cancer cell metabolism and immune metabolism in therapeutically meaningful ways. Although
there are metabolic processes that are seemingly fundamental to both cancer and responding
immune cells, metabolic heterogeneity and plasticity may serve to distinguish the two. As such,
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understanding the differential metabolic requirements of the diverse cells that comprise an immune
response to cancer offers an opportunity to selectively regulate immune cell function. Such a
nuanced evaluation of cancer and immune metabolism can uncover metabolic vulnerabilities and
therapeutic windows upon which to intervene for enhanced immunotherapy.

Work over the past several decades has shown that activated immune cells employ many
metabolic pathways attributed to cancer cells1-3 (Fig. 1). This convergence of metabolic
adaptations creates a fundamental competition for nutrients required by cancer cells and
immune cells within the tumour microenvironment (TME). However, we are coming to find

[email protected].
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Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
J.D.P. is a scientific founder, a paid consultant and has equity in Dracen Pharmaceuticals. Technology arising in part from the studies
described herein was patented by Johns Hopkins University and subsequently licensed to Dracen Pharmaceuticals (JHU083 is
currently labelled as DRP-083). R.D.L. and J.D.P. are inventors for pending patent application no. PCT/US16/44829 submitted by
Johns Hopkins University that covers the use of glutamine analogues, such as JHU083 (DRP-083), for cancer immunotherapy. J.D.P
has been a paid consultant for Corvus Pharmaceuticals and has equity in the company.
Peer review information
Nature Reviews Cancer thanks N. Chandel, J. Fan and the other, anonymous, reviewer(s) for their contribution to the peer review of
this work.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
 Leone and Powell Page 2

fundamental differences between the metabolic programmes of cancer cells and immune
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cells, as well as between different immune cells. Understanding these differences can reveal
specific metabolic vulnerabilities and, consequently, novel targets for therapeutic approaches
aimed at metabolic programming in order to enhance cancer immunotherapy.

Although the ability of cancer cells and tumour tissue to upregulate glycolytic catabolism of
glucose to form lactate, even in oxygen-replete conditions (aerobic glycolysis), a process
known as the ‘Warburg effect’, has been considered a hallmark of malignancy, it has become
increasingly clear that cancer metabolism is heterogeneous, and that cancer cells can engage
in a broad range of metabolic programmes to meet the demands of growth and proliferation,
and that in addition to aerobic glycolysis, mitochondrial respiration is fundamentally
important in this regard4-7. Predictably, highly metabolically active cancer cells (Fig. 1)
impart profound effects on the TME, leading to nutrient depletion, hypoxia, acidity and the
generation of metabolites that can be toxic at certain concentrations. A significant amount of
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glucose from the TME is metabolized through aerobic glycolysis, generating high amounts
of lactate and H+, thereby lowering the intratumoural pH. That said, it is likely that the
balance between lactate-generating glycolysis and oxidative phosphorylation (OXPHOS) is
dependent on the degree of hypoxia, which can be both heterogeneous and wide ranging
within the TME. It is instructive to note that in moderately hypoxic regions, CO2 derived
from mitochondrial respiration is hydrated by extracellular carbonic anhydrase enzymes,
forming HCO3− and H+. Thus, oxidative metabolism can be a significant and often
overlooked source of extracellular acidification within the TME.

Given the recent establishment of cancer immunotherapy, including the use of blocking
antibodies against immune checkpoint pathways and adoptive cell therapy with chimeric
antigen receptor T cells (CAR T cells), several recent studies have begun to establish the
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relationship of tumour-intrinsic metabolism to successful immunotherapy. For instance, it

has been reported that increased glycolytic metabolism in melanoma cells is associated with
resistance to adoptive T cell therapy and checkpoint blockade8,9. Other studies have shown
that signalling through immune checkpoint proteins on tumour cells, including PD1 and B7-
H3, was responsible for increased glucose depletion within the TME10-12. Interestingly,
some immunosuppressive checkpoint pathways are actually induced as a direct consequence
of tumour acidification13. Further, immune checkpoint blockade can dampen glycolysis of
tumour cells, restore glucose in the TME and permit T cell glycolysis and cytokine
production14. Several recent studies have demonstrated that targeting specific aspects of
tumour-intrinsic metabolism, such as the hexosamine biosynthesis pathway (HBP) or
glutamine metabolism, could foster an immune response and sensitize tumours to
checkpoint blockade15,16.
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Because of the emergence of immunotherapy as a pillar of oncologic therapy, it is

increasingly vital to understand as much as possible about the metabolic interdependence of
infiltrating immune cells and cancer. This Review aims to discuss the following fundamental
questions: which metabolic programmes are critical for the function of specific cell subsets
involved in the immune response to cancer; how these metabolic programmes might be
perturbed within the TME; the implications of metabolic derangements in the TME for

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current immunotherapeutic paradigms; and how metabolic interventions might be leveraged

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to enhance the antitumour immune response.

The TME and immune contexture

Highly active metabolic pathways that are characteristic of cancer cells (Fig. 1) can create
profound changes in the composition of nutrients and other small molecules within the
TME. This can have critical effects on the immune response. The high metabolic activity of
cancer cells and disorganized vasculature within the TME can contribute to a nutrient-
depleted and hypoxic microenvironment, establishing metabolic competition between cancer
cells and infiltrating immune cells14,17,18. Indeed, the glucose uptake and effector function
of antitumour CD4+ T cells has been shown to be inversely proportional to glycolytic
activity of cancer cells in mouse models18, and glucose availability in the TME allows for
improved cytokine expression from antitumour CD8+ T cells14. Furthermore, transcriptomic
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analyses of patients with melanoma from The Cancer Genome Atlas revealed that effector T
(Teff) cell genes, such as CD40lg and IFNG, are inversely correlated with HK2 expression,
which encodes the rate-limiting enzyme in the glycolytic pathway18. Metabolic programmes
active within cells of the TME can also lead to the generation of toxic concentrations of
certain metabolites. Elevated levels of adenosine, kynurenine, ornithine, reactive oxygen
species (ROS) and potassium, as well as increased acidosis, have all been reported in the
TME, and each can have profound effects in suppressing the antitumour immune response.

The immune contexture of the TME comprises a range of distinct cell types19 (TAbLe 1).
Effector cells perform functions aimed at cell killing and can arise from either the innate
(non-specific) or adaptive (antigen-specific) arms of the immune system. Antitumour
effector cells arising from the adaptive system include CD4+ and CD8+ Teff cells, which
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orchestrate and carry out antigen-specific killing of cancer cells, respectively. CD8+ Teff
cells are critically important in direct tumour cell killing through the induction of apoptosis
and cytokine secretion. CD4+ T cells comprise numerous subsets. Some of these subsets, the
most well studied of which is the T helper 1 (TH1) subset, can also provide significant
antitumour activity. These antitumour CD4+ T cells, collectively termed conventional CD4+
(CD4+con)v T cells, are distinct from immunosuppressive, pro-tumorigenic CD4+ T cells
known as regulatory T (Treg) cells. Although CD4+
conv cells may engage in direct tumour cell
killing, they primarily contribute to antitumour immunity through cytokine secretion and
assisting in CD8+ T cell activation. Antitumour CD4+ conv T cells share significant
metabolic characteristics with CD8+Teff cells. Although less well understood in terms of
antitumour immunity, B cells may also perform effector roles in the TME20. Importantly, as
part of the adaptive immune system, T cells and B cells can give rise to memory cell
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populations, which can persist long after the resolution of an infection or tumour response.
CD8+ memory T (Tmem) cells are a crucial aspect of long-term tumour control. Innate cells,
such as natural killer (NK) cells and inflammatory macrophages, perform critical antitumour
effector functions as well. There are also immunosuppressive cell populations within the
TME, including CD4+FOXP3+Treg cells, myeloid-derived suppressor cells (MDSCs), anti-
inflammatory macrophages and some B cell populations20. Through various mechanisms,
including cytokine secretion and metabolic derangements, these cells can dampen or

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eliminate the effectiveness of antitumour effector cell populations. Lastly, antigen-presenting

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cells, such as intratumoural dendritic cells (DCs), have been shown to perform essential
roles in maintaining active adaptive immune response within the TME21,22. Numerous
excellent reviews can be referred to for more detailed discussions of tumour immunology
and immunotherapy19,23-25.

The metabolism of the antitumour response

Glucose metabolism of antitumour effector T cells.
CD4+conv and CD8+ Teff cells form the critical effector compartment of the antitumour
response. When naive CD4+ and CD8+ T cells, which are non-proliferative, recognize their
cognate antigen in the context of co-stimulatory signalling, they become proliferative and
enact metabolic features to support immense growth26-28. Although many early
investigations highlighted the upregulation of aerobic glycolysis as a hallmark of T cell
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activation, it is now clear that upregulated tricarboxylic acid (TCA) cycle metabolism and
OXPHOS are also a critical aspect of CD4+conv and CD8+ T cell activation. Although TCA
cycle metabolism is upregulated within 24 h post activation, upregulated aerobic glycolysis
appears to be a more rapid event, occurring within 6 h after activation27-32.

The transcriptional activity of MYC and hypoxia inducible factor 1 (HIF-1) are both
upregulated in response to T cell activation and promote metabolic
reprogramming26,29,33,34. Notably, although HIF-1 is well known to regulate metabolism in
response to hypoxia, its activity is also induced in response to T cell activation in the
absence of hypoxia. MYC and HIF-1 transcriptional activity leads to upregulation of genes
encoding enzymes that promote glycolysis, such as pyruvate kinase (PKM1), hexokinase 2
(HK2) and GLUT1 (ReFS29,34,35). Pathways emanating from proximal metabolites in the
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glycolytic pathway are also integral components of T cell activation and function (Fig. 1).
The pentose phosphate pathway (PPP) metabolizes glucose-6-phosphate to generate
NADPH and ribose-5-phosphate36. Glucose shuttling into the PPP is significantly increased
upon CD4+ T cell activation29. The PPP is the primary cellular source for NADPH, which is
required for fatty acid and plasma membrane synthesis in newly activated CD8+ T cells37.
NADPH is also critical for REDOX homeostasis in proliferating manunalian cells38-40. ROS
levels in activated T cells need to be finely regulated. Although dysregulated ROS levels can
be toxic39,41,42, ROS play an important role in Teff cell activation, having been shown to
promote nuclear factor of activated T cells (NFAT)-dependent IL-2 expression in CD4+ and
CD8+ T cells. Another pathway originating from early glycolytic reactions, the HBP, is the
primary cellular source of glycosylation substrates, which mediate a variety of effects on a
broad range of proteins, including stability, trafficking and function. The HBP relies on
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metabolism of glucose and glutamine and is responsive to their availability. The main
substrate produced by the HBP, UDP-GlcNAc, is critical for effector CD4+ and CD8+ T cell
expansion and function43. Lastly, the serine–glycine–one-carbon pathway allows cells the
ability to generate serine, glycine, NADPH and one-carbon units for use in the folate cycle.
Teff cell proliferation and function were dependent on sufficient serine metabolism in vitro
and in vivo44 (Fig. 1).

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Glucose carbons that are not metabolized to lactate or by proximal glycolytic pathways
contribute significantly to the TCA cycle in Teff cells6 (Fig. 1). In highly proliferative cells,
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intermediates of the TCA cycle are rapidly consumed to serve as building blocks for a broad
range of biomolecular syntheses, a process called cataplerosis45. For example, citrate can be
exported to the cytoplasm to regenerate acetyl-CoA for use in lipid and cholesterol
synthesis, both of which are critical for producing membranes in proliferative Teff cells.
Other TCA cycle intermediates function as building blocks for biosynthesis of, for example,
nucleotides and amino acids, which are in high demand during proliferation. Like cancer
cells, Teff cells are highly proliferative and upregulate specific glycolytic programmes,
including aerobic glycolysis, PPP, HBP and TCA cycle support, to allow massive cell
division and effector functions.

T cells and glucose restriction in the TME.

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Glucose limitation within the TME can markedly affect the T cell response. For example,
low-glucose conditions (0.1 mM) suppressed the generation of the glycolytic intermediate
phosphoenolpyruvate (PEP) in T cells, which disrupted calcium-dependent NFAT signalling
in vitro18. Compared with control, decreasing the glucose concentration in growth media has
been shown to suppress the extracellular acidification rate (a measure of aerobic glycolysis),
augment the oxygen consumption rate (a measure of OXPHOS), attenuate mTOR signalling
and suppress the effector function of both CD4+ and CD8+ Teff cells46-48. Reduced mTOR
complex 1 (mTORC1) signalling interfered with Teff cell differentiation and, in the case of
CD4+ T cells, specifically favoured the development of immunosuppressive, pro-
tumorigenic Treg cells49. Interestingly, in CD8+ T cells, mTOR blockade with rapamycin
favoured differentiation of long-lived Tmem cells, which may play an important role in
sustaining antitumour responses49-51. Decreasing glucose availability in culture suppressed
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production of the critical effector molecules interferon-γ (INFγ), IL-17 and granzyme B in
Teff cells compared with control growth media47,48,52,53. In activated CD4+ T cells cultured
in glucose-free media containing the alternative sugar fuel galactose (which suppresses
aerobic glycolysis), the glycolytic enzyme GAPDH assumed a moonlighting role, binding
the 3′ untranslated region of Ifng mRNA and suppressing its translation and Teff cell
function31. Glucose restriction in media conditioned by primary ovarian cancer cells led to
microRNA-mediated suppression of the histone methylase EZH2 (enhancer of zeste
homologue 2), leading to decreased NOTCH signalling, suppressed cytokine production and
decreased viability of Teff cells54.

Increasing the glycolytic capacity of mouse sarcoma cells through either pharmacologic
treatment with the AKT activator 4-hydroxytamoxifen in co-culture experiments or
overexpression of key glycolytic enzymes (for example, Glut1, Hk2 and Pdk1) in tumour
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cells followed by injection into mice led to suppression of CD8+ T cell effector function
compared with vehicle-treated tumour cells or empty vector overexpression, respectively14.
Similarly, compared with wild-type tumours, implanted Hk2- overexpressing melanoma
cells suppressed CD4+ T cell antitumour effector function and in vivo responses in mouse
models18. Furthermore, expression of glycolysis-related genes in tumour samples from
patients with melanoma and non-small-cell lung cancer was inversely correlated to T cell
infiltration8. Tipping the metabolic balance can also be accomplished through directly

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manipulating T cell metabolism. For example, overexpression of the glycolytic enzyme PEP
carboxykinase in tumour-specific CD4+ T cells improved antitumour responses compared
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with control vector-transfected T cells in an adoptive T cell model using melanoma-specific

T cells18.

Mitochondrial respiration is also a critical aspect of Teff cell metabolism. Several recent
studies have reported that T cells in patients with cancer (compared with healthy controls)
and tumour-infiltrating CD8+ T cells in tumour-bearing mice (compared with non-
infiltrating CD8+ T cells) displayed decreased mitochondrial mass as well as indicators of
mitochondrial dysfunction55-57. Mitochondrial fitness of resting peripheral CD8+ T cells
was impaired in patients with chronic lymphocytic leukaemia compared with healthy
controls55. Furthermore, the degree of response in these patients to CAR T cell therapy was
negatively correlated to the degree of mitochondrial impairment of infused CAR T cells55.
Tumour-infiltrating CD8+ T cells from patients with renal cell carcinoma showed
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dysregulated mitochondrial dynamics and function, including elevated levels of

mitochondrial ROS and hyperpolarization, compared with CD8+ T cells from healthy
donors56. Normal ex vivo activation of these T cells could be rescued with mitochondrial
ROS scavengers or pyruvate supplementation. Mitochondrial biogenesis and function are
particularly deranged in a subset of dysfunctional tumour-infiltrating CD8+ T cells termed
exhausted T cells (box 1). As a whole, these studies demonstrate that cancer itself can lead
to derangements in the metabolism of Teff cells, including mitochondrial dynamics, and that
a reciprocal relationship exists between the degree of glycolytic activity of cancer cells and
the antitumour effector function of infiltrating T cells.

Amino acids and the antitumour T cell response.

Like cancer cells, highly proliferative immune cells, such as activated T cells, are reliant on
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amino acid metabolism to support protein and nucleotide synthesis. As such, amino acid
transporters, including SLC7A5 (also known as LAT1)58, SLC38A1 (also known as
SNAT1), SLC38A2 (also known as SNAT2)59 and SLC1A5 (also known as ASCT2)60, have
been found to be highly upregulated during T cell activation compared with naive cells in in
vitro human and mouse studies61. Essential amino acids must be obtained exogenously. For
example, leucine was required for mTORC1 signalling, effector function and proper
differentiation in effector CD8+ and CD4+ CD4+ T cells. Interestingly, deletion of the
conv
leucine transporter, Slc7a5, in mouse models caused metabolic failure during in vitro
activation and cytokine-directed differentiation of CD4+ (TH1, IL-17-producing TH17) and
CD8+ Teff cells, but had no adverse effect on the differentiation of Treg cells26,62. Activated
T cells also rapidly metabolize arginine, and exogenous arginine supplementation leads to
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improved T cell fitness and increased generation of central Tmem cells63. Serine, tryptophan
and cysteine are also vital nutrients for T cell responses and, as such, are important
mediators of antitumour immune responses44,64-66. Tryptophan is an essential amino acid
and its availability within the TME is an important factor in determining strength and quality
of the T cell response. Human T cell proliferation and activation were strongly suppressed in
tryptophan-free media compared with normal growth media66,67. Cancer cells, tumour-
associated macrophages (TAMs), MDSCs, suppressive DCs and cancer-associated

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fibroblasts can deplete tryptophan levels through enzymatic activity of indoleamine 2,3-
dioxygenase (IDO)68, which can be expressed at high levels in these cells within the TME.
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Underlining the importance of this metabolic pathway for tumour growth, IDO expression
has been correlated with poor outcomes in patients with several cancer types, including
gastric cancer, colorectal cancer, non-small-cell lung cancer and melanoma69-71.

In proliferating cells, glutamine provides nitrogen for amino acid and nucleic acid synthesis
and carbon to replenish TCA cycle intermediates that are syphoned off as building blocks
for biosynthesis — a process called anaplerosis (Fig. 1). Cancer cells and some activated
immune cells, such as T cells and macrophages, are generally highly glutamine avid59,72.
The expression of glutamine transporters SLC1A5 and SLC38A1 and/or SLC38A2 was
significantly upregulated during in vitro stimulation of murine CD4+conv T cells60. Driven
by MYC, glutamine is metabolized by glutaminase (GLS) to glutamate, which may enter the
TCA cycle after conversion by glutamate dehydrogenase (GLUD1) to α-ketoglutarate
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(αKG; also known as 2-oxoglutarate). αKG is subsequently metabolized to succinate and

fumarate in the TCA cycle29. Interestingly, in settings of glutamine restriction, some cancer
cell lines switch to glucose-fuelled anaplerosis, wherein pyruvate is converted by pyruvate
carboxylase to oxaloacetate, which enters the TCA cycle73. Our group has recently shown
that effector CD8+ T cells are also capable of upregulating pyruvate carboxylase activity
under conditions of glutamine blockade in vitro16.

Although effector function and proliferation in differentiated CD8+ Teff cells was suppressed
by limiting glutamine in media59, if glutamine availability was restricted during activation of
CD8+ T cells, it altered differentiation towards a long-lived, memory phenotype74. This
effect on differentiation was shown to be mediated by αKG. αKG and other TCA
metabolites, such as succinate and fumarate, can modulate the activity of a wide range of
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cellular processes, including epigenetic remodelling and the stability of critical transcription
factors, such as HIF-1α (ReFS45,75).

Lipid metabolism and T cells.

Activated T cells also reprogramme lipid metabolism, upregulating de novo lipid synthesis
and cholesterol uptake, which are critical for membrane synthesis and mediated by the
transcription factors sterol regulatory element-binding protein 1 (SREBP1) and SREBP2,
respectively37,76. Proliferation, metabolic reprogramming and antiviral activity were
dramatically suppressed in activated mouse CD8+ T cells lacking SREBP1 and SREBP2
functionality. In addition, the cholesterol content in membranes during CD8+ T cell
activation and expansion in vitro was, in part, regulated by cholesterol esterification enzyme
acetyl-CoA acetyltransferase (ACAT1). Acat1 knockout CD8+ T cells showed increased
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membrane cholesterol and improved T cell receptor clustering and signalling, leading to
enhanced proliferation, function and improved tumour killing in adoptive-transfer mouse
tumour models76. Pharmacologic inhibition of ACAT1 with avasimide improved the
antitumour effect in mice compared with vehicle-treated control animals. Cholesterol
metabolism and antitumour T cell function is an evolving story, however. A recent study by
Ma et al. demonstrated that a high cholesterol content in tumours can induce T cell
dysfunction by activating the endo-plasmic reticulum stress response77. As such, although

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cholesterol is important for Teff cell proliferation and metabolism, the benefit of targeting
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specific aspects of cholesterol metabolism to improve the antitumour immune response

needs further study.

The metabolism of immunologic memory.

Unlike Teff cells, CD8+ Tmem cells preferentially rely on OXPEIOS78-81. Compared with
CD8+ Teff cells, enhanced spare respiratory capacity, a parameter indicative of the ability of
cells to upregulate OXPEIOS, is also highly characteristic of Tmem cells78. Initial studies
using etomoxir as an inhibitor of carnitine palmitoyl transferase 1A (CPT1A), a
mitochondrial transporter responsible for the import of long-chain fatty acids destined for
fatty acid β-oxidation (FAO), implicated FAO as the primary fuel for OXPEIOS in Tmem
cells. However, more recent work using T cell-specific Cpt1a knockout models has called
this into question and demonstrated that off-target effects of high-dose etomoxir (200 μM)
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are likely responsible for the earlier findings82. This should not be taken to imply that Tmem
cells do not use FAO in support of OXPHOS and spare respiratory capacity but, rather, that
FAO is not the sole pathway responsible for this metabolic phenotype. Indeed, the
expression of CPT1A is consistently upregulated in CD8+ Tmem cells compared with Teff
cells. Furthermore, a CD8+T cell subset known as tissue-resident memory cells were
specifically dependent on fatty acid binding protein 4 (FABP4) and FABP5 to import
extracellular fatty acids for FAO and for maintenance of a long-term memory phenotype83.

Intermediates of the TCA cycle, such as αKG, succinate and fumarate, are particularly
important in adaptive memory. Inhibition of 2-oxoglutarate-dependent dioxygenases
(2OGDD) through alterations in these TCA metabolites has been shown to increase memory
cell differentiation in CD8+ T cells84,85. Although glucose, glutamine and fatty acids are the
primary nutrient sources fuelling the TCA cycle, a range of other nutrients, such as amino
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acids and acetate, can also enter the cycle. In particular, acetate metabolism is emerging as
an important source of acetyl-CoA in CD8+ T cells and some cancer types16,86-88. In the
mitochondria, acetate can enter the TCA cycle after it is metabolized by acyl-CoA
synthetase short chain family member 1 (ACSS1) to form acetyl-CoA. Alternatively, acetate
can be converted to acetyl-CoA by ACSS2 in the cytoplasm, where it can contribute to fatty
acid synthesis and acetylation reactions important in epigenetic reprogramming and post-
translational modifications. The metabolism of acetate is an important metabolic pathway
for promoting the function of memory CD8+T cells88. Interestingly, blockade of glutamine
metabolism during T cell activation increased Tmemcell differentiation and induced acetate
metabolism and associated enzymes, including ACSS1 and ACSS216,85. As quiescent cells,
Tmem cells preferentially rely on OXPHOS relative to aerobic glycolysis and have significant
mitochondrial reserve that is required to upregulate OXPHOS further upon antigen
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activation. Tmem cells can adapt several distinct nutrient sources to fuel this metabolic
programme.

Hypoxia and the antitmnour T cell response.

Although tumours are highly heterogeneous, high levels of metabolic activity and associated
oxygen consumption, as well as disorganized, poorly functioning vasculature, can generate
hypoxic regions with median oxygen saturation levels <2 % (compared with a median of

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about 5% in normal tissues)89,90. The effect of hypoxia on Teff cells is not straightforward.
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Complicating this area of study is the fact that HIF-1 transcriptional activity is upregulated
in response to T cell activation in normoxic conditions34, so it is challenging to understand
the effect of hypoxia on further augmenting HIF-1 activity while also evaluating HIF-1-
independent effects. Early in vitro studies of CD8+ Teff cell activation, differentiation and
function showed that whereas proliferation and the expression of some cytokines were
suppressed in hypoxia, the lytic capacity, activation markers and survival were improved91.
Subsequent in vivo studies showed that CD4+ and CD8+ splenic T cells were more poorly
activated after concanavalin A challenge in mice exposed to subatmospheric O2 tension
(8%) compared with mice exposed to ambient O2 tension (20%)92. Other studies showed
that in vitro hypoxic exposure causes intracellular accumulation of the metabolite (S)-2-
hydroxyglutarate (S-2-HG), which profoundly alters CD8+ T cell activation and
differentiation, suppressing cytokine secretion and cytolytic capacity, but, interestingly,
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augmenting proliferation, long-term survival and antitumour response after in vivo transfer
in mouse models84. Contrary to previous findings demonstrating the necessity of oxidative
metabolism and oxidative metabolic capacity in forming long-lived memory CD8+ T cells,
glycolytic activity enforced through constitutive HIF-1α activity (achieved through
conditional knockout of the HIF-1 regulator Vhl) actually favoured the formation of long-
lived effector memory cells in mouse vaccine models93. Other work has demonstrated that
hypoxia induced the expression of the ectonucleotidases CD39 and CD73 on various cells in
the TME94,95. These enzymes break down ATP in the TME to adenosine. Adenosine is a
ligand for the A2A and A2B purinergic receptors, which are expressed on a large range of
immune cells, and is broadly immunosuppressive, inhibiting effector cell function and
proliferation of Teff cells96-101. Interestingly, supplemental oxygen enhanced the antitumour
immune response of T cells in mice by downregulating the adenosine signalling pathway102.
The effect of hypoxia on antitumour T cells is an evolving area of study. Further research
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will clearly benefit the field of immunotherapy, given both the prevalence of hypoxic regions
in tumours as well as the profound effects hypoxia can have on the adaptive immune
response.

Toxic metabolites.
In addition to adenosine, many other products generated from cancer cell metabolism
influence infiltrating T cells (Figs 1 and 2). Elevated levels of extracellular lactate and H+ in
the TME can suppress T cell proliferation, survival, cytotoxicity and cytokine production in
in vitro studies of mouse and human CD8+ T cells103,104. The upregulation of the gene
encoding the key Teff cell transcription factor NFAT was impaired during in vitro activation
of mouse CD8+ T cells in the presence of high levels of lactate and H+ compared with
standard growth media104. In vivo mouse studies showed that mouse melanoma cells that
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have Ldha knocked down produced less lactate and were more responsive to immune-
mediated tumour rejection than empty vector-transfected control melanoma cells104. MAP
kinase signalling was also severely impaired in human effector CD8+ T cells activated in the
presence of elevated lactate and H+ compared with control media105.

The accumulation of specific amino acids within tumours can also suppress the Teff cell
response. Probably the most well studied in this regard are the effects of tryptophan

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metabolites, especially kynurenine, which is generated through the activity of IDO1 106.
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Functioning as an endogenous ligand for aryl hydrocarbon receptors on T cells, kynurenine

caused upregulation of the PD1 co-inhibitory pathway on activated CD8+ T cells in vitro
compared with vehicle-treated control107. This upregulation of PD1 was also observed on
tumour-infiltrating CD8+ T cells in mouse models treated with exogenous kynurenine
compared with vehicle-treated controls. Tumour-infiltrating CD8+ T cells from kynurenine
treated tumour-bearing mice produced less IFNγ and TNF.

Cancer cells have also been reported to suppress T cell activity through release of the
oncometabolite (R)-2-hydroxyglutarate (R-2-HG). This metabolite can inhibit epigenetic
dioxygenase enzymes, such as histone demethylases, leading to increased methylation and
modified transcription. R-2-HG produced by isocitrate dehydrogenase (IDH)-mutant human
glioma was taken up by T cells in in vitro studies. R-2-HG interfered with proliferation, T
cell receptor signalling, NFAT activity and polyamine biosynthesis in activated human CD4+
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and CD8+ T cells in vitro108. This was corroborated by the finding that R-2-HG released
into the TME in IDH-mutant glioma-bearing mice inhibited complement-mediated
antitumour response as well as T cell migration, proliferation and cytokine secretion109.
These studies highlight the intricate interplay of cancer metabolites and immune function
within the TME (Fig. 2).

High levels of necrosis lead to increased levels of potassium within the TME, which limits T
cell effector function110. Mediated by reduced cytoplasmic levels of acetyl-CoA, this state
induced epigenetic remodelling of activated T cells, inducing a dysfunctional state of Teff
cells within the TME111. However, this dysfunctional state was enriched with characteristics
of T cell stemness. In accord with the induction of a stem-like state, ex vivo stimulation and
expansion of Teff cells in high potassium produced T cells with improved in vivo
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persistence, multipotency and capacity for tumour clearance111. The generation of T cell-
suppressive metabolites through tumour necrosis and metabolic activity intrinsic to the TME
forms an important mechanism of tumour immune evasion.

Metabolism and the innate effector response.

Because NK cells are particularly adept at cell killing during major histocompatibility
complex class I (MHC-I) down-regulation, a common evasion strategy of cancer cells, they
form a critical effector component of tire innate response. Metabolically, aerobic glycolysis
and OXPHOS were upregulated after in vitro cytokine stimulation (IL-12 and IL-15) of NK
cells112. Interestingly, SREBP transcription factors were required for these cytokine-induced
metabolic changes during in vitro NK cell stimulation113. Pharmacologic inhibition of
SREBP activity suppressed metabolic reprogramming, cytokine production and cytotoxicity
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in vitro and curtailed anti-tumour response in an adoptive NK cell mouse model.

Interestingly, it has been reported that endogenous SREBP inhibitors, such as 27-
hydroxycholesterol, can be increased within the TME and thus may be a mechanism of NK
cell suppression112,114-118. Lung cancer progression in mice and tumour-associated
transforming growth factor-β (TGFβ) are correlated with increased fructose-1,6-
bisphosphatase (FBP1) expression in tumour-associated NK cells119. FBP1 is a key enzyme
in gluconeogenesis, which, when activated, strongly suppressed glycolysis in NK cells,

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leading to dysfunction and diminished viability. Interestingly, pharmacologic inhibition of

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FBP1 was sufficient to re-establish glycolytic metabolism, as well as cytokine production

and cytotoxicity in vitro, and improve antitumour response in adoptive cell therapy mouse
models119. These studies showed that rescuing NK function through FBP1 inhibition was
dependent on restoration of glucose metabolism, as blocking glucose metabolism with 2-
deoxyglucose (2-DG) prevented the rescue caused by FBP1 inhibition. 2-DG by itself also
led to NK cell dysfunction119, implying that inhibition of glucose metabolism could have
profound effects on NK cell antitumour response. Other metabolic derangements within the
TME are likely to affect NK cell function as well. For instance, low arginine levels can
impair NK cell proliferation and IFNγ production120,121, and hypoxia can suppress cytolytic
activity122-124. Human NK cell-activating receptors, such as NKp46 and NKp30, are
suppressed in response to hypoxia or low arginine in in vitro studies121,124. High lactate
levels and associated low pH, as in the TME, also suppressed NK cell cytotoxicity, cytokine
production and NFAT signalling in in vitro studies104,125,126. Lastly, elevated adenosine
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levels within the TME can strongly suppress NK cell effector function and proliferation127.

Other innate cells, macrophages and DCs also enact specific metabolic programmes upon
activation. Although early in vitro work using activation schemes with specific cytokines
classified macrophages into inflammatory (M1) or immunosuppressive (M2) phenotypes,
there is poor evidence that these polarized phenotypes play distinct roles in vivo128,129.
More recent work has uncovered a spectrum of macrophage phenotypes characterized by
distinct transcriptional states130. That said, macrophages with inflammatory characteristics
can play an important role in antitumour immunity19, and in this regard it is instructive to
examine what has been established regarding the metabolic programming of in vitro derived
‘M1’ macrophages. Glucose metabolism is a vital aspect of the inflammatory phenotype in
macrophages. Upon activation, for example by a Toll-like receptor agonist, these cells
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showed increased expression of glycolytic genes, high levels of glucose uptake, increased
lactate production and upregulated glutamine anaplerosis131. This metabolic reprogramming
led to increased succinate levels, which increased expression of the inflammatory cytokine
IL-1β by stabilizing HIF-1 (ReF.132). Inflammatory macrophages are also particularly reliant
on the PPP for the generation of NADPH, with 13C-glucose tracing studies confirming
increased routing of glucose though this pathway upon activating inflammatory phenotypes
in culture133,134. NADPH is necessary to produce high levels of ROS as part of an oxidative
burst, a key effector mechanism for these cells132,135,136. Arginine is also a critical nutrient
in the function of pro-inflammatory ‘M1’ macrophages as they express high levels of
inducible nitric oxide synthase (iNOS) compared with alternatively activated or ‘M2’
polarized macrophages in in vitro studies137. iNOS requires arginine to generate cytotoxic
nitric oxide, an important pro-inflammatory mediator of antitumour response138,139.
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Specific nutrient deficits within the TME, particularly glucose and arginine, can severely
limit the metabolism and related elaboration of effector programmes in these cells. Glucose
limitation not only suppresses glycolysis as a whole but can curtail PPP activity and TCA
cycle function, thus limiting the generation of NADPH, ROS and succinate, all of which can
severely limit M1 macrophage function. Supporting this idea, the secretion of pro-
inflammatory cytokines by macrophages was significantly reduced by glycolysis inhibition
with 2-DG140.

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DCs are an important class of antigen-presenting cells involved in the antitumour response.
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Intratumoural DCs that are capable of antigen cross-presentation have specifically emerged
as a vital component of this response22. Upon activation, DCs undergo maturation allowing
antigen processing and presentation to T cells. This response was coupled to a metabolic
switch from OXPHOS to aerobic glycolysis, mediated by HIF-1α in response to in vitro
LPS activation141, and by the PI3K–AKT pathway in response to Toll-like receptor
stimulation in vitro142. This switch to glycolysis and away from OXPHOS during DC
activation is critical for DC survival, production of stimulatory cytokines and activation of T
cells142. Interestingly, pharmacologic activation of AMPK, which promotes mitochondrial
biogenesis and oxidative respiration143, was sufficient to block DC maturation in vitro142.
Given this critical dependence on aerobic glycolysis, glucose competition in the TME may
significantly suppress DC activation and viability and thus limit the ability of DCs to foster
an effective and persistent T cell response.
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The metabolism of cancer immune evasion

Metabolism of adaptive immune suppression.
Immunosuppressive Treg cells preferentially rely on TCA cycle function and mitochondrial
respiration144,145. Although initial studies demonstrating the dependence of Treg cells on
FAO did not account for off-target effects of etomoxir, other studies have shown that FAO
does support OXPHOS in Treg cells, although not as the sole pathway82,146,147. In contrast
to Teff cells, Treg cells showed decreased glucose uptake and expressed lower levels of
GLUT1 in vitro144. Interestingly, although glycolysis did not appear to play a crucial role in
Treg cell differentiation or a long-lived phenotype, our laboratory has reported that a subset
of highly active Treg cells, termed effector Treg cells, relied on the upregulation of glycolysis
for optimal function148. As such, Treg cells appear to be metabolically flexible, which may
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allow them to thrive within relatively harsh and heterogeneous conditions, such as the TME.
To this end, it has been reported that the Treg cell-defining transcription factor, FOXP3,
reprogrammes cellular metabolism through suppression of MYC favouring OXPHOS and
NAD(H) oxidation149. In conditions of low glucose and high lactate, such as found in the
TME, these adaptations allow for a metabolic advantage of these immunosuppressive cells,
allowing Treg cells to resist lactate-induced functional and proliferative suppression (unlike
Teff cells) in vitro149. Glucose or glutamine deprivation (leading to reduced intracellular
αKG) in media during in vitro skewing experiments can alter CD4 differentiation and favour
the development of Treg cells150,151.

Similar to Teff cells, Treg cell response to hypoxia is not entirely clear. Hypoxia has been
shown to promote cytokine-mediated recruitment of Treg cells into the tumour
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environment152. Other work has demonstrated that FOXP3 transcript is actually increased in
response to HIF-1α induction153-155. Also, adoptively transferred Treg cell-specific Hif1
knockout cells failed to migrate into brain tumours in mouse models compared with wild-
type controls, an effect that was also observed in dichloroacetate-treated Treg cells, in which
glycolysis is inhibited compared with vehicle-treated control Treg cells156. Interestingly,
hypoxia-responsive adenosine signalling through the adenosine receptor A2A on Treg cells
induced proliferation and significantly stronger immunoregulatory activity in mixed

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lymphocyte culture experiments157,158. Conversely, several groups have reported that

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hypoxia-induced HIF-1α can destabilize Treg cells, with reports demonstrating that hypoxia
can promote TH17 CD4+ T cells through direct HIF-1α interactions with the cell subtype-
defining transcription factors FOXP3 and RORγt, respectively52,53,159.

The unique metabolism of amino acids within the TME can also have a profound effect on
Treg cells. IDO1 activity can strongly promote Tregcell differentiation in vitro, an effect that
appears to be secondary to both tryptophan deficiency as well as the generation of
downstream metabolites, such as kynurenine160,161. Kynurenine has been found to induce
the generation of FOXP3-expressing Treg cells by functioning as an endogenous ligand for
aryl hydrocarbon receptors on T cells162. Interestingly, many of the qualities of the TME
that make it inhospitable for Teff cells are either well tolerated by Treg cells (elevated lactate
and H+) or can induce Treg cell responses (for example, accumulation of adenosine,
kynurenine and hypoxia).
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Metabolism of innate immune suppression.

TAMs can adopt phenotypes that are highly immunosuppressive. Although there is a
spectrum of TAM phenotypes as mentioned above, it is useful to examine the metabolic
programming of what heretofore had been established as an ‘M2’ anti-inflammatory
macrophage subset, characteristics of which are clearly evident within immunosuppressive
TAMs. Like Treg cells, M2 macrophages upregulate FAO and mitochondrial
respiration134,163. Although early studies demonstrating that FAO is obligatory in M2
macrophages did not take into account the off-target effects of the CPT1A inhibitor
etomoxir164, forced induction of FAO and mitochondrial biogenesis by overexpression of
Pgc1α primes macrophages for an immunosuppressive phenotype and strongly suppresses
the production of pro-inflammatory cytokines163.
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M2 macrophages metabolize amino acids in a distinct manner from inflammatory

macrophages, expressing high levels of arginase 1 (ARG1), which depletes arginine and
generates polyamines that are important mediators of wound healing but also highly
immunosuppressive165-168.

Another group of tumour-associated immunosuppressive innate cells, MDSCs, appear to be

highly metabolically active. Both aerobic glycolysis and OXPHOS were upregulated in
tumour-associated MDSCs compared with MDSCs in the periphery169. In another study,
granulocytic MDSCs from the spleens of tumour-bearing mice also showed increases in both
aerobic glycolysis and OXPHOS compared with splenic neutrophils from the same mice.
Interestingly, MD SC expansion in vitro and accumulation in the TME in mouse breast
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cancer models could be attenuated through blockade of glycolysis with 2-DG, likely by
causing increased ROS levels in these cells170.

Hypoxic regions within tumours have been associated with the accumulation of
macrophages, where they aid tumour development through the production of angiogenesis
factors, mitogenic factors and cytokines associated with tumour metastasis171-173.
Furthermore, hypoxia can promote the generation of immunosuppressive macrophage
phenotypes174. Adenosine, which can be generated as a result of hypoxia, can trigger

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signalling through A2A and A2B receptors on macrophages, both of which augment the
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differentiation and functional capabilities of immunosuppressive macrophages as well as

attenuate the cytokine release of pro-inflammatory macrophages in vitro96,175. Elevated
lactic acid in culture has been shown to promote the M2 phenotype, increasing ARG1
expression and polyamine-dependent immunosuppression176. High glycolytic rates in triple-
negative breast cancer were shown to promote MDSCs, whereas restricting glycolysis in
these cancer cells inhibited granulocyte colony-stimulating factor and granulocyte–
macrophage colony-stimulating factor secretion from cancer cells and limited MDSC
development177. Interestingly, hypoxia skewed MDSCs towards an immunosuppressive,
M2-like TAM phenotype compared with normoxic MDSCs in vitro. This occurred via
HIF-1α mechanisms, as Hif1a knockout MDSCs displayed increased tumour growth
compared with wild-type MDSCs in mouse melanoma tumour models178.
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Exploiting differential metabolic plasticity

Whereas the activation, proliferation and function of Teff cells can be attenuated through
inhibiting numerous metabolic pathways, other attributes, such as long-term viability or
effector function upon restimulation, may be enhanced. Although inhibiting glycolytic
metabolism with 2-DG inhibited Teff cell generation, it also conditioned T cells towards a
long-lived, memory-like phenotype50. Interestingly, blocking glycolysis during ex vivo T
cell activation and expansion, before reinfusion for tumour treatment, not only allowed for
increased survival of antitumour T cells but also for improved cytokine production and
cytotoxicity. Similar phenomena have been reported in CD8+ Teff cells in response to AKT
inhibition, glutamine blockade, hypoxia, arginine supplementation and potassium
supplementation16,63,74,93, 111,179. It may be possible to differentially affect cancer and the
immune response. For example, acetate metabolism can rescue T cell function in glucose-
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restricted CD8+Teff cells87. In addition, our group recently demonstrated the importance of
this pathway in maintaining metabolic homeostasis in CD8+ T cells undergoing glutamine
blockade16. These findings could imply a generalizable therapeutic strategy, in that blocking
the use of typical metabolic fuels, such as glucose or glutamine, may render some cancers
metabolically compromised, but may leave antitumour T cells metabolically intact and
functional given their ability to use alternative sources, such as acetate. Although specific
metabolic interventions may be introduced pharmacologically as adjuncts to checkpoint
blockade (TAbLe 2), these targets may be particularly applicable to CAR T cell therapy,
wherein manipulation of metabolic pathways can be precisely defined through genetic
means (box 2). Future studies delineating the degree of metabolic flexibility possible within
a given immune cell subset and functional capacity are clearly warranted.
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Checkpoint blockade and immunometabolism.

It is of great interest to define both the metabolic consequences of checkpoint therapy and
the metabolic determinants of response. Checkpoint signalling has been shown to regulate
metabolism in several studies. For example, PDL1 expression on cancer cells can drive Akt–
mTOR activation and glycolysis in cancer cells, increasing glucose uptake and augmenting
competition with T cells for glucose14. CD155-TIGIT signalling in T cells from human
gastric cancer tissue dampened glucose uptake, lactate production and expression of

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glycolytic enzymes GLUT1 and HK2180. Conversely, agonism of the co-stimulatory

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pathway GITR broadly increased T cell metabolic activity and proliferation compared with
isotype-treated control T cells181. Lastly, in vitro PD1 and CTLA4 signalling on activated
human T cells suppressed metabolic pathways, such as aerobic glycolysis, that are
associated with T cell activation182. To this end, the prospect of combining metabolic
inhibitors (TAbLe 2) with checkpoint inhibitors holds promise to enhance the efficacy of
checkpoint blockade. Targeting tumour metabolism by inhibiting glutamine metabolism in
mouse models inhibited tumour growth and conditioned the TME to be more hospitable for
antitumour effector cells16. Also, metabolically reprogramming T cells to make them more
robust, long-lasting memory cells might improve their response to checkpoint inhibitors.
This has been heralded by recent clinical trials combining the anti-folate pemetrexed with
anti-PDL1 immune checkpoint blockade183. In addition to having direct antitumour effects,
pemetrexed treatment enhanced the metabolic fitness and effector function of antitumour
CD8+ T cells, as well as induced immunologic cell death of cancer cells to trigger the
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immune response.

Conclusion and perspective

Although much of the foundation of immunometabolism has been informed by observations
of cancer metabolism, it is clear that there are distinct differences between cancer and
immunologic metabolic reprogramming. These differences provide opportunities to target
metabolism as a means of enhancing the efficacy of immunotherapy (Fig. 3). Such an
approach can be achieved through numerous different strategies. These include targeting
tumourmetabolic programmes to inhibit growth and alter the TME, targeting the metabolism
of suppressive immune cells to inhibit their function and targeting effector cell metabolism
to enhance tumour killing. Likewise, ex vivo pharmacologic or genetic reprogramming of T
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cell metabolic pathways prior to adoptive cellular therapy offers an opportunity to

dramatically engineer enhanced features, which may include longevity or enhanced effector
function (bOX 2).

Future work should begin to focus on the metabolic interdependence of immune cells and
cancer cells within the TME. In addition to nutrient depletion and the generation of
metabolites that can suppress the immune response at certain concentrations, cancer cells
can engage in metabolic crosstalk with other cells within the TME, wherein metabolic
programmes can be induced and co-opted to benefit malignant progression. It has been
reported that pancreatic stellate cells can provide alanine to cancer cells and, thus, fuel
proliferation184, and bone marrow stromal cells have been reported to provide cysteine to
promote survival of chronic lymphocytic leukaemia cells185. In another report, ammonia
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from cancer cell glutamine metabolism diffused through the TME and triggered autophagy
in cancer-associated fibroblasts, which in turn provided protein breakdown products, such as
glutamine itself, to further support cancer cell metabolism186. It will be important to
understand whether and by what mechanism immune-evading cancers may be co-opting the
metabolic machinery of immune cells and benefitting from their remarkable metabolic
flexibility.

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(2016). [PubMed: 27496729] This paper reports the suppression of glycolysis, mitochondrial
dysfunction and mitochondrial biogenesis in CD8+ T cells through PD1 signalling leading to an
exhausted T cell phenotype.
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250. Finney HM, Akbar AN & Lawson AD Activation of resting human primary T cells with chimeric
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Oxidative phosphorylation (OXPHOS).

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A highly efficient form of cellular respiration synthesizing ATP from the phosphorylation
of ADP using electrochemical potential energy generated by the transfer of electrons
from NADH or FADH2 to oxygen through a series of mitochondrial electron carriers.
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Immune checkpoint pathways

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Pathways mediated by cell surface proteins on immune cells, such as PD1 or CTLA4,
that serve to suppress the immune response, which can be activated by ligands within the
tumour microenvironment or draining lymph nodes.
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Chimeric antigen receptor T cells (CAR T cells).

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T cells harvested from a patien’s blood and genetically modified to express a special
receptor that can recognize and respond to specific, predefined molecular targets on
tumour cells.
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Hexosamine biosynthesis pathway (HbP).

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A branch of glycolysis that generates building blocks used for glycosylation of proteins
and lipids.
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Pentose phosphate pathway (PPP).

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A metabolic branch of glycolysis generating NADPH, used for fatty acid synthesis and
redox homeostasis, and 5-carbon sugars used in nucleotide synthesis.
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Nuclear factor of activated T cells (NFAT).

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A calcium-dependent transcription factor activated in response to T cell receptor

stimulation. Cooperation with the AP-1 transcription factor results in a productive
immune response and transcription of pro-inflammatory cytokines, such as iL-2 and
interferon-γ.
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Cataplerosis
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The loss of metabolic intermediates in a metabolic pathway (particularly the tricarboxylic

acid cycle) owing to consumption or degradation.
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Anaplerosis
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The process of replenishing intermediates of the tricarboxylic acid cycle to support

biosynthesis.
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De novo lipid synthesis

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The cellular biosynthesis of fatty acids, triglycerides, cholesterol and other lipids from
carbohydrates or other non-lipid precursors.
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2-Oxoglutarate-dependent dioxygenases (2OgDD).

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A family of enzymes that catalyse the hydroxylation of macromolecules, often as a

prerequisite to demethylation, reliant on α-ketoglutarate, Fe2+, ascorbate and oxygen as
cofactors.
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Major histocompatibility complex (MHC).

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MHC class i (MHC-i) is expressed on all nucleated cells, a molecular complex presenting
intracellular peptide epitopes for CD8+ T cell receptor recognition. Also expressed on
antigen-presenting cells, allowing initial antigen-specific activation of cytotoxic CD8+ T
cells. MHC-ii is highly expressed on antigen-presenting cells for presenting antigenic
epitopes for CD4+ T cell receptor recognition and activation.
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Antigen cross-presentation
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The ability of antigen-presenting cells to process extracellular antigens and present them
to CD8+ T cells through major histocompatibility complex class 1 presentation.
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Box 1 ∣
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Metabolism of T cell exhaustion

CD8+ T cells in the tumour microenvironment (TME) can adopt a state of functional
exhaustion wherein they are poorly proliferative and unable to generate sufficient
cytotoxicity against target cancer cells. A similar cell subset exists during chronic viral
infections, such as lymphocytic choriomeningitis virus (LCMV) clone 13 in mice or
hepatitis C virus in humans243. There are numerous metabolic features that are emerging
as characteristic of this set of immune cells, termed CD8+ exhausted T cells. Some of the
metabolic characteristics appear to be a consequence of co-inhibitory signalling, such as
PD1, which is highly characteristic of CD8+ exhausted T cells. In a chronic LCMV
mouse model, PD1 signalling inhibited the expression of peroxisome proliferator-
activated receptor-γ co-activator 1α (PGC1α), which disrupted mitochondrial and
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effector function and led to significantly less oxidative capacity compared with T cells
responding to an acute LCMV infection244. Overexpression of PGC1α in adoptively
transferred T cells improved mitochondrial function and restored T cell function. T cells
infiltrating tumours showed similar mitochondrial dysfunction and loss of oxidative
capacity secondary to inhibited PGC1α activity57. Interestingly, antitumour T cells also
regained function through overexpression of PGC1α, implying that the activity of a
metabolic programme can, in and of itself, overcome functional T cell exhaustion. PD1
signalling also suppressed mTOR complex 1 (mTORC1) signalling and glycolytic
activity in infiltrating CD8+ T cells in a progressive mouse tumour model14. Given the
dependence of T cell function (and loss of function) on metabolic programming, more
studies are needed to assess the determinants of metabolic dysfunction and associated T
cell exhaustion within the TME.
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Box 2 ∣
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Adoptive T cell therapies enable highly flexible approaches to metabolic

therapy through ex vivo culture conditioning or genetic targeting of
metabolic processes
T cells in such regimens can be genetically modified to express chimeric antigen
receptors (CARs) that recognize known tumour surface antigens and trigger T cell
receptor signalling in the absence of major histocompatibility complex presentation. T
cells can be metabolically conditioned through the use of chemical inhibitors or genetic
editing during ex vivo expansion, through metabolically engineered media or through
pharmacologic treatment after T cell reinfusion. Many of the interventions discussed,
including inhibiting glycolysis, glutamine metabolism with 6-diazo-5-oxo-l-norleucine
(DON), AKT–mTOR signalling and potassium supplementation, have been used in ex
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vivo T cell expansion and led to improved T cell persistence and antitumour
response50,74,111,179 (see the figure). Recent studies demonstrated that forced expression
of peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α), which
promotes mitochondrial biogenesis, in adoptively transferred CD8+ T cells resulted in
superior intratumoural metabolic and effector function57. Several groups have also
reported the importance of the co-stimulatory receptor 4-1BB in positively conditioning
mitochondrial health and biogenesis for robust antitumour immunity245,246. These
findings have been validated in the CAR T cell field, wherein the addition of the 4-1BB
receptor module has enhanced T cell persistence and increased therapeutic
efficacy247-251. 2-DG, 2-deoxyglucose; TCA, tricarboxylic acid.
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Fig. 1 ∣. Cancer cell metabolism and derangements in the TME.

Mitochondrial oxidation of nutrients, including glucose, amino acids and fatty acids, through
the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) is a highly
efficient means of producing energy for quiescent, differentiated cells. However, during
periods of increased proliferation, such as after immune activation or malignant
transformation, cells upregulate an alternative pathway for glucose metabolism, called
aerobic glycolysis. Although less efficient in generating ATP, aerobic glycolysis allows for
more rapid metabolism of glucose, efficient disposal of excess carbon and regeneration of
NAD+ while preserving mitochondrial enzymatic activity for anabolic processes242.
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Glycolytic intermediates are channelled through other essential pathways, such as the
pentose phosphate pathway, the one-carbon pathway and the hexosamine biosynthesis
pathway. These pathways support cellular processes that are critical for highly proliferative
cells, such as synthesis of fatty acids and nucleic acids. Pathways for the metabolism of
glutamine are also upregulated in the setting of increased proliferation72. In addition to
supplying the TCA cycle with carbon skeletons that maintain intermediates for biosynthesis
of amino acids, nucleic acids and fatty acids (a process known as anaplerosis), glutamine is
the primary source of nitrogen used for amino acid and nucleic acid synthesis. These cells
also upregulate a broad range of amino acid transporters and maintain tightly controlled
redox balance, primarily through NADPH synthesis. Many cells within the tumour
microenvironment (TME) express ectoenzymes, such as indoleamine 2,3-dioxygenase
(IDO), arginase 1 (ARG1) and CD73, which deplete nutrients, as well as increase
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immunosuppressive metabolites, such as kynurenine and adenosine. Along with a deranged

microvasculature, these metabolic adaptations can have profound effects on the metabolic
make-up of the TME, leading to depletion of vital nutrients, hypoxia, acidosis and the
generation of immune-toxic metabolites as shown. MDSC, myeloid-derived suppressor cell;
R-2-HG, (R)-2-hydroxyglutarate; ROS, reactive oxygen species; Teff, effector T; Treg,
regulatory T.

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Fig. 2 ∣. Metabolic derangements in the TME inhibit T cell function.

The metabolic milieu of the tumour microenvironment (TME) is a reflection of cancer
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metabolic programmes. Nutrient deprivation, hypoxia and toxic metabolites are conditions
within the TME that confront and influence T cell metabolism and function. The
consequences of TME conditions on immune cell responses can be predicted based on a
growing literature of preclinical, translational and clinical studies. AMPK, AMP kinase;
EZH2, enhancer of zeste homologue 2; Granz B, granzyme B; IFNγ, interferon-γ; MDSC,
myeloid-derived suppressor cell; miRNA, microRNA; NFAT, nuclear factor of activated T
cells; PKA, protein kinase A; R-2-HG, (R)-2-hydroxyglutarate; TCR, T cell receptor; Teff,
effector T; TH1, T helper 1; Tmem, memory T;Treg, regulatory T;Tscm, stem cell memory T;
TNF, tumour necrosis factor.
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Fig. 3 ∣. Potential metabolic targets for enhancing immune response in cancer.

Using small molecules, monoclonal antibodies and genetic editing, metabolic processes can
be targeted to either disable cancer and suppressive immune cell metabolism or, conversely,
engage and support effector cell metabolism. Metabolic processes in suppressive immune
populations and cancer cells can be targeted to directly decrease viability, as well as to
disable metabolic pathways that deplete nutrients (for example, arginase 1 (ARG1) and
indoleamine 2,3-dioxygenase (IDO)), lead to toxic metabolites (for example, lactate and
CD73) or induce metabolic control of effector cell populations (for example, mutant IDH1
generation of the oncometabolite (R)-2-hydroxyglutarate (R-2-HG)). Metabolic
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interventions may also be able to induce beneficial changes in effector populations, such as
increasing longevity and antigen-specific immunologic memory. A2AR, adenosine receptor
subtype A2A; AOA, amino-oxyacetic acid; 2-DG, 2-deoxyglucose; DON, 6-diazo-5-oxo-l-
norleucine; ETC, electron transport chain; G6PD, glucose-6-phosphate dehydrogenase;
MDSC, myeloid-derived suppressor cell; PGM3, phosphoglucomutase; TCA, tricarboxylic
acid; Teff, effector T; Treg, regulatory T.
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Table 1 ∣

Functional and metabolic phenotypes of immune cells within the TME

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Cell type Function Metabolic phenotype

Immune activation or inflammatory
NK cell MHC-independent cytotoxicity: Glycolysis and OXPHOS
Perforin, granzymes
FASL, TRAIL
IFNγ, TNF

Inflammatory TAM MHC-independent cytotoxicity: Glycolysis and PPP

TNF, IL-1β
Oxidative burst
Antigen presentation

DC DAMP processing Glycolysis

Teff cell activation
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Antigen presentation

Teffcell Antigen-specific cytotoxicity: Highly glycolytic and OXPHOS

Perforin, granzymes Amino acid metabolism (arginine, tryptophan, serine, leucine, glutamine,
FASL cysteine)
IFNγ, TNF PPP

Tmem Cell Maintain long-lived response OXPHOS

Immunosuppression
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MDSC IL-10, TGFβ Glycolysis and OXPHOS

Amino acid depletion
Polyamines, kynurenine

Immunosuppressive TAM IL-10 OXPHOS, HBP

Amino acid depletion
Polyamines, kynurenine
VEGF

Tregcell IL-2 sequestration: OXPHOS

Dampen APC co-stimulation
IL-10, TGFβ
Adenosine
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APC, antigen-presenting cell; DAMP, damage-associated molecular pattern; DC, dendritic cell; FASL, fas ligand; HBP, hexosamine biosynthesis
pathway; IFNγ, interferon-γ; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; NK, natural killer; OXPHOS,
oxidative phosphorylation; PPP, pentose phosphate pathway; TAM, tumour-associated macrophage; TGFβ, transforming growth factor-β; Teff,
effector T; TME, tumour microenvironment; Tmem, memory T; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand;
Treg, regulatory T; VEGF, vascular endothelial growth factor.

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Table 2 ∣

Metabolic inhibitors with potential for clinical translation

Pathway Target Representative drugs Representative Refs

phase II/III
clinical trials
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Glycolysis GLUT1 WZB117 NA 187

HK 2-DG NA 50
3-Bromopyruvate NA 188
PFKFB3 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO), PFK158 NA 189
GAPDH Dimethyl fumarate (DMF) Phase IIa 190,191

Heptelidic acid NA 192

PDHK1 Dichloroacetate (DCA) Phase II 193,194
LDHA NCI-737, NCI-006 NA 195
FAO CPT1A Etomoxir NA 196
Perhexidine NA 197
CD36 ABT-511 Phase II 198,199
FAS ACC1 Firsocostat NA 200
Cholesterol esterification ACAT Pactimibe NA 201
Electron transport and/or mitochondrial function AMPK Metformin a 202-205
Phase II
b GLS BPTES NA 206
Glutaminolysis
CB-839 a 207,208
Phase II
Glutamine metabolism Glutamine requiring enzymes DON NA 16
JHU083 NA 209

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One-carbon metabolism SHMT2 RZ-2994 NA 44
One-carbon metabolism TS, DHFR, GARFT Pemetrexed a a 192,210,211
Phase II and phase III
Methotrexate a 212-214
Phase II
TS 5-Fluorouracil a 215-217
Phase III
Reduction of ROS levels Antioxidant NAC Phase II 218,219
Increase of ROS levels GSH Menadione NA 220
Pentose phosphate pathway G6PD Polydatin NA 221
Hexosamine biosynthetic pathway PGM3 FR054 NA 222
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Pathway Target Representative drugs Representative Refs

phase II/III
clinical trials
Arginine pathway ARG1 CB-1158 a 223-225
Phase II
Arginine pathway Pegylated arginine deiminase ADI-PEG20 NA 226
IDO inhibitors IDO Epacadostat a 227-229
Phase II
Leone and Powell

Indoximod a 227,230,231
Phase II
Navoximod NA 227
R-2-HG synthesis Mutant IDH1 FT-2102 a 232-234
Phase II
Adenosine pathway CD73 Oleclumab a 98,235-237
Phase II
BMS-986179 a 98,235,238
Phase II
NZV930, CPI-006 NA 98,235
Adenosine receptor A2A PBF-509 a 98,239
Phase II
CPI-444 a 98,240
Phase II
AZD4635 a 98,241
Phase II

ACAT, acetyl-CoA acetyltransferase; ACC1, acetyl-CoA carboxylase; AMPK, AMP kinase; ARG1, arginase; CPT1A, carnitine palmitoyl transferase 1; 2-DG, 2-deoxyglucose; DHFR, dihydrofolate
reductase; DON, 6-diazo-5-oxo-l-norleucine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GARFT, glycinamide ribonucleotide formyltransferase; GLS, glutaminase; GLUT1, glucose transporter,
type 1; G6PD, glucose-6-phosphate dehydrogenase; GSH, glutathione; HK, hexokinase; IDH1, isocitrate dehydrogenase; IDO, indoleamine 2,3-dioxygenase; LDHA, lactate dehydrogenase A; NA, not
applicable; NAC, N-acetyl cysteine; PDHK1, pyruvate dehydrogenase kinase; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase; PGM3, phosphoglucomutase; R-2-HG, (R)-2-
hydroxyglutarate; ROS, reactive oxygen species; SHMT2, serine hydroxymethyltransferase, mitochondrial; TS, thymidylate synthase.
a
Denotes clinical trial in combination with established immunotherapy agents.
b
The enzymatic catabolism of glutamine to generate tricarboxylic acid cycle intermediates.

Nat Rev Cancer. Author manuscript; available in PMC 2021 April 12.
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