Microenvironmental influences on T cell immunity in cancer and inflammation

Abstract

T cell metabolism is dynamic and highly regulated. While the intrinsic metabolic programs of T cell subsets are integral to their distinct differentiation and functional patterns, the ability of cells to acquire nutrients and cope with hostile microenvironments can limit these pathways. T cells must function in a wide variety of tissue settings, and how T cells interpret these signals to maintain an appropriate metabolic program for their demands or if metabolic mechanisms of immune suppression restrain immunity is an area of growing importance. Both in inflamed and cancer tissues, a wide range of changes in physical conditions and nutrient availability are now acknowledged to shape immunity. These include fever and increased temperatures, depletion of critical micro and macro-nutrients, and accumulation of inhibitory waste products. Here we review several of these factors and how the tissue microenvironment both shapes and constrains immunity.

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REVIEW ARTICLE OPEN
Microenvironmental influences on T cell immunity in cancer
and inflammation
Darren R. Heintzman
1,3
, Emilie L. Fisher
1,3
and Jeffrey C. Rathmell
1,2
✉
© The Author(s), under exclusive licence to CSI and USTC 2022
T cell metabolism is dynamic and highly regulated. While the intrinsic metabolic programs of T cell subsets are integral to their
distinct differentiation and functional patterns, the ability of cells to acquire nutrients and cope with hostile microenvironments can
limit these pathways. T cells must function in a wide variety of tissue settings, and how T cells interpret these signals to maintain an
appropriate metabolic program for their demands or if metabolic mechanisms of immune suppression restrain immunity is an area
of growing importance. Both in inflamed and cancer tissues, a wide range of changes in physical conditions and nutrient availability
are now acknowledged to shape immunity. These include fever and increased temperatures, depletion of critical micro and macro-
nutrients, and accumulation of inhibitory waste products. Here we review several of these factors and how the tissue
microenvironment both shapes and constrains immunity.
Keywords: immunometabolism; T cell; microenvironment; cancer; inflammation
Cellular & Molecular Immunology; https://doi.org/10.1038/s41423-021-00833-2
INTRODUCTION
The classic metabolic chart present in every biology classroom and
textbook is a staple of biochemical training and represents the
culmination of over a century of detailed and rigorous studies.
These seminal discoveries define the landscape of fundamental
chemical reactions that integrate environmental signals and
nutrients to support the viability, growth, and activities of every
living cell. When cells receive signals to perform specific functions
such as to grow and proliferate, they adjust their nutrient uptake
and shift metabolic programs to meet the new demands. If,
however, adequate levels of essential nutrients are not available or
if end products or waste products accumulate, basic chemistry and
chemical equilibriums may bring these pathways to a halt or shift
their outcomes to disrupt or alter cell fates. Thermodynamic effects
such as temperature changes may also influence T cells. Much the
same as cell cycle checkpoints can stop or delay cell division,
metabolic checkpoints thus interpret nutrients and cell energetics
to determine cell differentiation, function, and fate. Cell metabo-
lism is thus the biochemistry of how cells interpret and integrate
signals from their microenvironment.
T cells and macrophage metabolism have been studied to the
greatest detail in the field of immunometabolism. In addition to
many studies focused on how cell activation signals can reprogram
metabolism from catabolic programs designed to generate energy
to anabolic programs that efficiently provide biosynthetic pre-
cursors to support cell growth and proliferation [1], it is now
apparent that each immune cell type and subset has specific
metabolic requirements for activation and differentiation that
reflect their specific roles and demands. Importantly, the tissue
microenvironment exerts a profound influence on these processes.
Cell activating signals, microenvironmental nutrients, and other
conditions integrate through upregulation of nutrient transporters
and the subsequent changes in intracellular nutrients influence cell
bioenergetics, biosynthesis, and signaling. Metabolic signaling
through generation of co-factors or posttranslational modifications
can then serve to shape cell fate or activate metabolic checkpoints.
These include nutrient sensing pathways such as the AMPK or
mTORC1 and HIF signaling axis, stress response pathways
including reactive oxygen species (ROS) and ER stress, or changes
to epigenetic marks and histone modifications that regulate gene
expression. These changes are broadly relevant to immunity and
inflammation and may have a particularly important impact in
settings such as obesity or in tumor microenvironments (TME).
Here we review how key changes in systemic nutrient status as
well as microenvironmental metabolites and other conditions are
integrated to shape the fate of T cells.
Micronutrients and ions
While cell metabolism typically focuses on intermediary metabo-
lites and central carbon metabolism pathways, ions and other
elements play key roles (Fig. 1A). Micronutrients such as ions have
increasingly been shown to impact the adaptive immune system,
particularly T cells. For example, recent literature has highlighted
that potassium (K
+
) can directly influence T cells. Increased K
+
ion
concentration in the tumor microenvironment can acutely silence
T cell effector function [2]. In contrast, high concentrations of this
ion preserve T cell stemness through acetyl CoA metabolism
and by epigenetically regulating gene transcription, nutrient
Received: 15 December 2021 Accepted: 19 December 2021
1
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37205, USA.
2
Vanderbilt Center for Immunobiology, Vanderbilt
University Medical Center, Nashville, TN 37205, USA.
3
These authors contributed equally: Darren R. Heintzman, Emilie L. Fisher. ✉email: jeff.rathmell@vumc.org
www.nature.com/cmi
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processing, and metabolism [3]. K
+
promoted induction of
mitochondrial AcCoA synthetase 1, promoting mitochondrial
metabolism, a metabolic feature of memory T cells. While these
studies focused on K
+
in the tumor microenvironment, ionic
concentrations not limited to potassium have been shown to
produce interesting cellular phenotypes in T cells in various tissue
settings. Therefore, the ionic composition of tissues represents a
relatively unexplored determinant of T cell polarization and T cell
effector functions.
H
+
concentration is a measure of acidity in tissues and high
concentrations of H
+
ions may be due to hypoxic conditions and
an increased abundance of cells utilizing aerobic glycolysis and
secreting lactate [4]. Most studies on acidity and T cell function
have been focused on the TME and how increased lactate, and
presumably acidic conditions, in the extracellular environment
may inhibit effector T cell functions in solid tumors. A major
consequence of low acidity seems to be through negative effects
on effector cytokine production by T cells, which can be severely
reduced in acidic conditions [5–7]. Acidity is, however, not only
encountered in the TME and inflammatory tissues can experience
pH conditions as low as ~5.5 pH [8,9] (Fig. 1B). Interestingly, a
recently published study shows evidence that lymph nodes also
harbor highly acidic pH environments that suppress inflammatory
effector T cell functions in T cell rich zones. In this study, naïve T
cell priming was unaffected, and T cells were able to rapidly
recover from acid induced inhibition within hours after pH rescue
[7] such as would occur upon exit from the lymph node. This
provided evidence that acidic inhibition of effector functions is
related to changes in metabolic programming, as low pH shifted
T cells from glycolytic metabolism to oxidative phosphorylation
and mitochondrial metabolism. Future studies involving therapeu-
tic shifts in tissue pH in inflammation and disease may aid in
relieving chronic inflammation and other T cell mediated diseases.
Sodium ions (Na
+
) also influence T cell differentiation and
function. Peripheral tissues accumulate NaCl in high concentra-
tions in both diet-intake dependent, such as in high-salt western
style diets, and independent mechanisms [10]. While sodium is a
vital nutrient, excess salt is associated with higher incidence of
autoimmune diseases such as arthritis, multiple sclerosis, and in
mouse studies, colitis [11–13]. One explanation for the increased
incidence of disease associated with high salt intake seems to be a
shift in T cell differentiation toward the Th17 subset [12,14–16].
Th17 differentiation in high salt conditions occurs by influencing
transcriptional networks during differentiation. NFAT5 and its
downstream kinase SGK1 seem to be critical for an anti-
inflammatory switch in Th17 cells [17,18]. In contrast, high salt
has a more nuanced effect on regulatory T cells subsets. Studies
have suggested that while induced CD4( +) Foxp3(+) regulatory
T cells are not affected in development or function by high salt
[19], thymically derived regulatory T cells can become less
suppressive [20]. Recently, NaCl has also been suggested to be
an ionic checkpoint for human Th2 differentiation [21], further
supporting a diverse role in the differentiation and function of
T cells. While NaCl has not been well studied in terms of its effect
on T cell metabolic programs, one may speculate that metabolic
pathways are impacted in T cells by high salt. This is highlighted by
the similarities between SGK1 and its close relative, Akt, a key
driver of the mTORC1 pathway and T cell metabolism [22], and the
shared activation of these kinases by mTORC2 [23]. These findings,
as well as future studies involving functional and metabolic
consequences of NaCl, have implications for a wide range of
chronic inflammatory and autoimmune diseases.
Iron (Fe
+
) has received much attention in recent T cell literature.
Iron deposition is a hallmark of many autoimmune diseases
including lupus and multiple sclerosis and plays a key role as a co-
factor for many enzymes, including TCA cycle enzymes that drive
mitochondrial metabolism. Excess iron, however, can also lead to
generation of ROS through Fenton reactions and lead to the
process of ferroptosis, or iron-dependent cell death. Iron uptake
and handling, therefore, must be tightly regulated, and aberrant
iron metabolism and homeostasis have been recently shown to
directly influence T cell effector functions. A direct indication of the
role of iron is shown by genetic variants of the iron uptake protein
Transferrin Receptor, (CD71), which are associated with a common
variable immune deficiency [24]. CD71 is also a highly dynamic
marker of lymphocyte activation and plasmablasts. Iron depriva-
tion can reduce clinical scores in EAE, a T cell dependent disease in
mice [25]. Conversely, excess intracellular available iron in CD4
T cells was linked to the pathophysiology of SLE through a
mechanism of altered DNA methylation states favoring enhanced
Fig. 1 Chemical and physical components of the tissue microenvironment that can modulate immunity. AChanges to micronutrients,
including iron and potassium, regulate T cell function and survival from ferroptosis. BIncreased H +and decreased pH also play important
roles in T cell metabolism and fate. CFever and high temperatures directly impact T cells to promote inflammatory states
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immune-related gene expression [26] and by regulating the
stability of the RNA binding protein Pcbp1 [27]. Ferroptosis can
play a key role to shape T cell responses. This process is redox
mediated and largely identified through the functions of
Glutathione peroxidase 4 (Gpx4), a selenoenzyme that reduces
membrane phospholipid hydroperoxides to maintain cellular
redox homeostasis [28]. Inactivation or depletion of Gpx4 in a
variety of cell types can induce ferroptosis [29,30]. A recent study
has shown that Tregs require Gpx4 to neutralize lipid peroxides
and prevent ferroptosis to maintain Treg cell activation and inhibit
anti-tumor immunity [31]. In follicular helper T cells (Tfh), inhibition
of ferroptosis by GPX4 also protected cells from cell death in
germinal centers [32]. In tumors, CD36-mediated lipid uptake can
enhance ferroptosis to dampen anti-tumor immunity by promot-
ing lipid peroxidation [33].
Temperature
A physical feature of tissue microenvironments that can affect
thermodynamic regulation of enzymatic rates and cell physiology
is temperature (Fig. 1C). Temperatures fluctuate extensively in the
human body, hovering around 37 °C in the core and central
organs, such as the spleen, to as low as 28 °C in peripheral organs
such as the skin at thermoneutrality [34,35]. Temperatures also
vary widely in response to several physiological and pathophysio-
logical mechanisms. Systemically, fevers can arise in many diseases
and are a common side effect in bacterial and fungal infections,
blood borne cancers like lymphoma, and a variety of autoimmune
and inflammatory diseases, such as Adult-Onset Stills Disease [36].
While fevers are responsible for systemically increased tempera-
tures, localized temperature changes are also common in
damaged and inflamed tissue regions and have long been
recognized. As early as the first century B.C., observations were
made by Celsius that heat is a cardinal signs of inflammation [37].
Over 100 years ago, studies showed that after breaking the femur
of a hamster, local temperatures at the break site rose by as many
as 1–4 °C compared to non-injured limbs [38]. Interestingly, when
ischemia was induced at the site of the break, the local
temperature rose substantially, arguing that cellular activity at
the site of inflammation was responsible for increased temperature
and blood perfusion was necessary to dissipate this heat. These
studies have been supported by more modern technological
methodologies such as the use of temperature sensitive ratio-
metric dyes [39]. Human biology reflects these findings from mice
and guinea pigs and is substantiated by several publications
involving rheumatoid arthritis joints. Even in remission, rheuma-
toid arthritis patients can exhibit elevated temperatures in
previously affected joints compared to healthy controls. In fact,
the degree of temperature elevation in RA joints has been shown
to be a reliable predictive indicator of disease progression [40,41].
To date, the actual cause of locally increased temperatures at
sites of inflammation is not well understood. Interestingly,
mitochondrial metabolism has been suggested to generate large
amounts of heat through ATP hydrolysis, with mitochondria
reaching temperatures close to 50 °C within cells [42]. Notably,
UCP1 expression seems to correlate with mitochondrial heat
generation and is highly expressed in brown fat where non-
shivering thermogenesis regulates body temperature [43]. One
could speculate that mitochondrial metabolism conducted by
immune cell infiltrate in inflammation may be responsible for
locally increased temperatures, however this has not been tested.
All together, these data suggest that heat generation and thermal
characteristics within the tissue microenvironment are highly
diverse, and likely important during immune challenge.
Even with a wealth of data suggestive of frequent and variable
temperature change in the tissue microenvironment, the effects of
temperature on immune cell function have received relatively
modest attention (Fig. 1C). Elevated temperatures have been
shown to promote T lymphocyte trafficking during infection
through the increased expression of alpha4 integrins and Heat
Shock Protein 90 [44]. Other temperature-inducible proteins like
heat shock factor-1 (HSF1) are induced at a lower temperature in T
lymphocytes than B lymphocytes (39 °C vs. 42 °C), indicating
specialized functions of T cells at febrile temperatures [45,46].
Several studies suggest temperature changes have significant
impacts on T cell activation, proliferation, and differentiation. In
vitro studies suggest that T cell proliferation occurs more rapidly
and to a greater extent at febrile temperatures [47]. Elevated
temperatures have also been shown to reduce costimulatory
thresholds to T cell activation due to a more fluid lipid bilayer,
perhaps providing a mechanism to explain elevated proliferation
rates [48]. Recent work has shown that T cell differentiation is also
influenced by changes in temperature. In a report where naïve CD4
T cells were primed in vitro at moderate fever temperatures (39 °C),
cells underwent transcriptional reprogramming which enhanced
commitment toward a Th2 phenotype and away from an IFNy
producing Th1 phenotype [49]. Interestingly, coculture with
dendritic cells inhibited this transcriptional transition, suggesting
that cellular composition of the tissue microenvironment can
influence T cell responses at febrile temperatures. Cellular
signaling mechanisms have been shown to be altered by changing
temperatures in other cell types, such as recent work showing that
NF-kB signaling is exquisitely temperature dependent in mouse
adult fibroblasts and human neuroblastoma cells [50]. An
intriguing paper was recently published focusing on the effects
of febrile temperatures on Th17 cell differentiation [51]. These
studies provided evidence that elevated temperatures predomi-
nantly impact Th17 cell differentiation, causing this helper subset
to enhance IL17a production. Febrile temperatures enhanced the
pathogenic gene transcription signatures of Th17 cells and caused
higher neutrophil invasion in bronchoalveolar lavage fluid in a
mouse model of allergic airway inflammation. This study high-
lighted the potential that not only are T cells able to respond
biochemically to elevated temperatures, but that this biochemical
response could have T cell-subset specific effects on immunity and
inflammation.
A potential explanation for subset specific adaptation to
elevated temperature may be metabolic programming and
mitochondrial adaptation. Exposure of CD8( +) T cells to febrile
temperatures during activation caused significant enhancement of
mitochondrial respiration in addition to enhanced extracellular
acidification rates [52]. RNA sequencing (RNA-Seq) of CD8( +) cells
exposed to febrile temperatures revealed that many upregulated
gene pathways involved mitochondrial processes. Enzyme activity
increases dramatically as temperature increases, and may predict
increased metabolic rates in T cells at febrile temperatures due to
faster enzymatic reactions [53]. It has been established that Th17
cells can utilize glycolysis at a much higher rate than other T cell
subsets[54,55] and seem to be especially sensitive to temperature
change [51,55]. Perhaps enzymatic activity involved in glycolysis is
enhanced at febrile temperatures. This remains a poorly under-
stood yet fundamental feature of inflammation.
Obesity
Presently over 671 million adults are classified as obese (BMI > 30
kg/m2) and obesity contributing to a wide range of diseases with
underlying inflammatory components (Fig. 2), including diabetes,
cardiovascular disease, and cancer [56,57]. Research on obesity
has widely examined systemic effects of insulin resistance, but
adipose tissue of obese individuals was also found to directly
produce high levels of pro-inflammatory cytokines which influence
T cell and macrophage differentiation and pro-inflammatory
phenotypes, including leptin, TNFα, and IL-6 [58–61]. Cell type
composition in obese tissues can also be dysregulated to favor
inflammation. Obesity-associated chronic inflammation is asso-
ciated with an accumulation classically activated “M1”polarized
macrophages (ATMs) in adipose tissues [62,63], which are highly
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inflammatory and secrete pro-inflammatory cytokines like TNFα
[63]. These cells contribute to a potentially hostile environment
which can shift the balance of immune cells toward a pro-
inflammatory phenotype. Trem2(+) Lipid Associated Macrophages
[64] are also implicated and associated with adipocyte hypertro-
phy, inflammation, and systemic metabolic dysregulation. While
macrophages are prominent inflammatory sources in adipose
tissue, mediators of macrophage and T cell polarization in adipose
tissue can seemingly promote either pro- or anti-inflammatory
phenotypes and include adipokines, fatty acids, and cytokines in
the tissue microenvironment. The adipokine leptin, which is
secreted in proportion to adipocyte mass [65], upregulates the
expression of Glut1 in T cells to promote increased glucose uptake
and glycolysis in T cells, fueling the expansion of T effector cells
like Th1 and Th17 subsets [66]. Anti-inflammatory adipokines like
Adiponectin are also present in obese adipose tissue and have
been shown to limit these effector cell populations by restricting
cell intrinsic glycolysis [67]. A constant battle thus maintains
homeostasis within obese tissues, and dysregulation can cause
meaningful swings in the outcomes of T cell differentiation and
function.
A key determinant of pro- or anti-inflammatory adipose
microenvironments is the frequency of regulatory T cells. Obese
adipose tissue seems to become a progressively hostile environ-
ment for regulatory T cells, as they are highly present in lean
adipose, but numbers decrease in obese adipose tissue [68]. Other
studies have suggested that circulating regulatory T cells are also
reduced in obesity [69]. One explanation for this is that regulatory
T cells can be negatively influenced by adipokines like leptin,
which cause regulatory T cell numbers to be reduced due to the
upregulation of glycolysis [66,70,71]. However, dietary lipids have
been shown to alter Treg cell metabolism and migratory function,
promoting an effector-like migratory phenotype in Tregs, and
biased migration toward sites of inflammation through a mechan-
ism of decreased mTORC1 signaling and increased fatty acid
oxidation [72]. Recently, Tregs were shown to be critical to adipose
tissue homeostasis, inhibiting white adipose tissue beiging
through secretion of IL-10 regulated by Blimp1 expression [73].
Interestingly, Treg-specific loss of IL-10 resulted in increased insulin
sensitivity and reduced obesity in high-fat diet-fed male mice,
suggesting that Tregs may promote healthy obesity in some
settings. Together, these studies suggest that regulatory T cells
play a unique yet currently poorly understood role in obesity.
The immunological consequences of obesity have been well
studied in terms of response to viral infections. In the recent
COVID-19 pandemic, obese individuals have been noted to be
hospitalized with COVID-19 at a much higher rate than lean
individuals [74]. Similarly, obesity has been shown to impair the
adaptive immune system in response to Influenza virus [75].
Recent work has shown that T cells within obese tissue become
easily exhausted through upregulation of PD-1 [76], and that PD-1
blockade can reverses T cell priming impairments seen in obesity
[77]. While reversing PD-1 mediated exhaustion can be beneficial
in terms of rescuing the adaptive response to viral infections, this
could also increase the risk of autoimmune disorders in obese
individuals. Many of the inhibitory responses in T cells due to
obesity can be linked to T cell metabolism. Elevated saturated fatty
acids increase T cell antigen responses and signaling through the
PI3K/Akt axis to fuel fatty acid oxidation in metabolically stressed
environments [78,79]. Obesity can also result in increased T cell
oxygen consumption and less effective response to pathogens,
and that loss of weight and return to lean state does not rescue
Fig. 2 Obesity leads to both systemic and local changes to T cell microenvironments. Obesity or weight loss can have striking effects on T cell
metabolism in the obesity paradox in which tumors are promoted yet sensitized to anti-PD1
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this metabolic change in T cells [80]. Activated CD4 +T cells from
obese mice had increased glucose uptake and oxygen consump-
tion rate (OCR), compared to T cells from lean controls, indicating
increased mitochondrial oxidation of glucose [81]. Interestingly,
treatment of obese mice with metformin improved T cell
responses to influenza and led to increased rates of survival.
Weight loss did not, however, rescue the defects in influenza
response. Why weight loss does not result in the rescue of T cell
dysfunction in obesity is not well understood, but epigenetic
factors associated with obesity may alter T cell chromatin
dynamics, creating a seemingly irreversible metabolic shift toward
higher mitochondrial respiration and worse outcomes to influenza
infections. Future work in this area will be of keen interest and may
identify mechanisms involved in metabolic reprogramming of
T cells in obesity.
Obesity, cancer, and the immunotherapy obesity paradox
In addition to important considerations of diabetes and heart
disease, it is becoming increasingly clear that obesity is associated
with cancer incidence and mortality. In this section we will focus
on how these factors alter the local TME (Fig. 2). The pro-
tumorigenic mechanisms behind obesity are multifactorial and
include direct effects of systemic hormones and nutrients on the
cancer cells [82]. Chronic inflammation, long-chain fatty acid
metabolism, and consumption of high fructose corn syrup have all
been implicated to directly promote tumorigenesis, independent
of their alterations to the anti-tumor immune response [83–85].
This adds complexity in elucidating the mechanisms involved in
altering the tumor vs immune system balance and may help
explain differences observed by groups in the field. Single cell
RNA-Seq studies of changes to immune cell populations in tumors
of lean and obese mice have shown a variety of potentially
impactful changes [86,87]. Ringel et al. showed that obesity led to
increased tumor growth that was particularly apparent in
immunocompetent mice and that tumors from obese mice had
decreased abundance of tumor infiltrating activated CD8 T cells.
They showed this was not correlated with elevated fatty acid
oxidation of the T cells, but rather that T cells maintained a more
naïve phenotype [86]. In addition, macrophage and monocyte
populations were found to change in obesity and to have reduced
expression of MHC-class II that may contribute to lower T cell
activation in the TME [87].
An interesting feature of obesity-induced cancer is the duality of
increased chronic systemic inflammation but reduced local
inflammation and T cell exhaustion in the TME. This has been
proposed to lead to the “obesity paradox”, in which obesity is a risk
factor for cancer, yet has the surprising outcome of sensitizing to
immunotherapy and improving outcomes upon immune check-
point blockade (ICB) therapy [88]. This effect is seen in multiple
human cancers [89–91] and recapitulated in animal models where
tumors grow faster with obesity, but these same tumors can
respond more thoroughly to PD-1 blockade therapy [92]. T cell
dysfunction in tumors of obese animals was found to be in part
driven by high levels of leptin that promoted PD-1 expression [92].
Similarly, leptin and PD-1 ligation both increased STAT3 signaling
in cytotoxic T cells (CTLs) in the TME and resulted in an increase in
CTL fatty acid oxidation and decreased glycolysis [93]. This led to
pronounced CTL dysfunction as marked by a decrease in tumor
infiltration, cytokine and granzyme B production, and tumor
control. This study recapitulated previous findings suggesting
STAT3 ablation increases granzyme B and CTL proliferation [94], as
well as the observation that CTL dysfunction may be in part due to
leptin/STAT3 signaling [89]. Leptin has also been shown to drive
accumulation of myeloid-derived suppressor cells in the TME,
which limit CTL activation and result in increased tumor burden
[89,95,96]. In addition, obesity reprograms macrophages in the
TME to become pro-tumorigenic [97]. Conversely, leptin can have
directly pro-inflammatory roles in obesity and may sensitize
macrophages with increased potential to drive inflammation [98].
Consistent with a pro-inflammatory role for leptin, treatment of
lean mice with leptin was sufficient to increase anti-tumor
immunity to an extent similar to PD-1 blockade [87]. Clearly,
obesity and hormones such as leptin play complex roles in the
TME. Given the prevalence of obesity and the potential for new
insight from obesity that may improve immunotherapy in lean
individuals, however, makes this an exciting and important area for
further discovery.
The tumor microenvironment
TMEs are heterogeneous and composed of mixed cell types,
nutrients, and stroma that can present a metabolically hostile setting
for immunity through a metabolic immune suppression (Fig. 3).
Dysplasia caused by cancer cell growth and subsequent tissue
responses including recruitment of fibroblasts and immune cells
disrupts normal vascular function to restrain nutrient exchange and
replenishment. This is driven in part by altered metabolism caused
by oncogenic signaling in the cancer cells themselves [99], although
the metabolic demands of inflammation and immune cells also
contribute. The complexity and heterogeneity of the TME is just
beginning to be understood and several key components contribute
to the ability to mount anti-tumor immune responses.
Hypoxia. T cell stimulation leads to activation that results in a broad
increase in anabolic metabolism through aerobic glycolysis, TCA
cycle metabolism, oxidative phosphorylation, the pentose phosphate
pathway, and others [100]. This requires increased nutrient uptake,
including oxygen to support mitochondrial metabolism. Oxygen is
among the best understood nutrient sensing pathways and hypoxia
is a hallmark of large or rapidly growing solid tumors. This occurs due
to insufficient vascular exchange or ineffective angiogenesis and
vascular maturation that cannot match oxygen consumption within
the tumor. Tumor spaces often have regions with hypoxic oxygen
tension well below 2% in areas distant to mature blood vessels
whereas oxygen levels are near 5% in healthy tissues or adjacent to
vessels. Cellular responses to low oxygen tension include induction
of hypoxia-inducible factors (HIFs), most notably HIF-1αand HIF-2α,
that lead to transcription of hypoxia response genes and a hypoxic
stress response to increase glycolysis and anerobic metabolism [101].
In T cells, HIF-1αis also activated independent of oxygen sensing in
response to TCR stimulation via the PI3K/mTOR pathway, TGF-B
signaling, and IL-6 [102].
Hypoxia promotes an immunosuppressive environment through
multiple mechanisms. Hypoxia induces expression of the ecto-
Fig. 3 The tumor microenvironment and metabolic immune suppres-
sion. Obesity leads to chronic systemic and local inflammation.
Adipose-resident CD8 T cells can be more naïve but are also sensitized
to reactivate with PD-1 blockade. Many factors contribute to this
obesity paradox, including elevated lactate, decreased glucose, and
altered amino acids
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nucleotidases CD39 and CD73, increasing TME levels of the
immunosuppressive nucleotide adenosine [102]. Increased expres-
sion of immune checkpoint molecules, including V-domain immu-
noglobulin suppressor of T cell activation (VISTA) and cancer-
associated fibroblast expression/secretion of TGF-B, IL10, VEGF, and
PD-L1 also occur under hypoxic conditions [103,104]. Consistent
with an immune suppressive role for a tumor hypoxic response,
targeting HIF-1αon tumor cells increased CTL infiltration and
improved combination immunotherapy outcomes in a preclinical
mouse melanoma model [105]. Hypoxia-targeted treatment also
improved CTL infiltration and tumor control in the “immune cold”
prostate cancer treated with ICB [106].
The actual role of hypoxia on CTLs, however, is complex and prior
adaptation to hypoxic conditions can also increase T cell function in
tumors. Increased HIF activity has been shown to increase CTL
invasion and function, synergizing with ICB [107] and culturing CTLs
under hypoxic conditions increased their cytotoxicity in an adoptive
cell transfer model [108]. Further supporting a hypoxic response to
intrinsically promote T cell anti-tumor responses, T cells made
genetically deficient in VHL or the PHD proteins that lead to
proteolytic degradation of HIF-1αhad increased anti-tumor activity
[107,109]. A potential explanation for these different observations
involves the other signals received by CTLs in the TME vs cultured in
hypoxia conditions in vitro. Continuous TCR stimulation in the
presence of hypoxia was recently shown to cause a Blimp-1
mediated repression of PGC-1αmitochondrial reprogramming,
eventually leading to an increase in mitochondrial ROS and
dysfunctional CTLs [110]. Hypoxia, therefore, may be insufficient to
cause dysfunction in isolation, but remains an important factor in the
complex TME.
Glucose. Glucose is the most abundant and prototypical carbo-
hydrate fuel, but availability of this nutrient can be heterogeneous
in the TME. Because cancer cells themselves can use glucose at
high rates and vascular exchange can be poor, the overall
availability of glucose in the interstitial space may be limited for
cells to uptake in the TME. While glucose remains only modestly
reduced and is generally available in many settings [111–113],
glucose levels in some tumor regions may be as low as 0.1 mM
[114,115]. This heterogeneity may reflect regions with efficient
vascular exchange relative and other regions with necrosis and
high levels of death caused by insufficient nutrient access. Effector
T cells, but interestingly not Treg, require high concentrations of
glucose and efficient glucose uptake to elicit inflammation.
Genetic deletion of the glucose transporter Slc2a1 (Glut1) can
prevent a variety of in vivo inflammatory conditions [116,117].
Indeed, glucose restriction in CTLs leads to decreased levels of the
glycolytic intermediate phosphoenolpyruvate (PEP) and decreased
Ca2+activation of the nuclear factor for T cell activation (NFAT)
signaling pathway to impair cytokine production [118,119].
Mitochondrial dysfunction, noted by small, fragmented, hyperpo-
larized mitochondria with decreased mitochondrial superoxide
dismutase 2 (SOD2) and increased ROS, also occurs with glucose
restriction [112]. Highlighting the importance of these metabolic
disturbances, overexpressing PEP carboxykinase 1 to increase
intracellular PEP levels, disrupting the PD-1/PD-L1 and CTLA-4
signaling pathways, and supplementation with pyruvate can
increase CTL anti-tumor effector function, cytokine production,
and mitochondrial ROS neutralization, respectively [112,118,119].
Conversely, there may be some benefit to reduced glucose uptake
for anti-tumor responses. T cells that fail to differentiate to terminal
effectors, may instead favor memory and long-lived states. This is
potentially helpful in adoptive cell therapy or to select T cells to
function in low glucose environments. Consistent with this
opportunity, activation of T cells in the glycolytic inhibitor
2-deoxyglucose (2DG) delayed T cell activation and effector
function to shift nutrient use and allow greater in vivo viability
and persistence that increased anti-tumor immunity [120,121].
Conversely, Treg do not require high levels of glucose and can
function without Glut1 [116].
While assays to measure glucose uptake and accessibility in
tumors suggested models where glucose limitation restricts anti-
tumor immunity, these studies have typically been performed with
bulk tumor tissue, with non-specific dye indicators [122], or at low
levels of resolution that preclude insight to which cells can capture
available glucose in the TME. This has led to a nutrient competition
model in which cancer cells consume the bulk of the available
glucose and cause a metabolic immune suppression by limiting T
cell access to this important nutrient [114,123]. To directly test this
model, radiolabeled Positron Emission Tomography tracers were
given to tumor bearing mice and tumor infiltrating cells were
fractionated to determine which population internalized the
labeled nutrient [113]. Interestingly,
18
F-2DG was primarily taken
up by macrophages, followed by T cells and cancer cells while
18
F-glutamine was primarily taken up by cancer cells followed by
macrophages and T cells. Treatment with a glutamine uptake
inhibitor, however, increased glucose uptake in all cell populations.
These data show that in most settings, glucose is broadly available
in the TME and that macrophages rather than cancer cells are the
dominant glucose consumers. Importantly, the ability of cells to
increase glucose uptake upon inhibition of glutamine uptake
demonstrated that glucose uptake was not widely limited by
accessibility, but rather by cell intrinsic metabolic and signaling
programs.
Amino acids. Growing evidence has highlighted the importance of
amino acid to modulate anti-tumor responses. A wide range of
amino acids including arginine, and serine or glycine have been
shown to be critical for anti-tumor effector T cell function [124] and T
cell activation even in glucose replete conditions [125]. As the most
abundant amino acid, glutamine is utilized as an anabolic and
anaplerotic nutrient by cancerous cells and effector T cells [113,126].
Effector T cells require glutamine uptake as genetic deletion of the
transporter Asct2 or SNAT2 impair effector T cell responses [127–
129]. Following uptake, glutamine is used in nucleotide or
hexosamine synthesis, transported back out of cells in exchange
for other amino acids, or converted to glutamate via the enzyme
glutaminase (Gls). Gls generation of glutamate then supports
glutathione synthesis, methylation reactions, catabolism, and the
TCA cycle substrate via conversion to alpha-ketoglutarate. Despite
these widespread and important roles for glutamine, genetic or
pharmacologic disruption of Gls had subset-specific effects on T cells
that showed that excessive glutamine uptake can be immunesup-
pressive [130]. Th17 cells required Gls and Th17-mediated inflam-
matory disorders were prevented by Gls inhibition. In contrast,
Th1 cells and CTLs responded to Gls inhibition by compensating
through increased glucose uptake and glycolysis that increased
mTORC1 signaling, glycolysis, and production of IFNy, granzyme B,
and perforin [130]. This observed phenotype of increased activation
has since been explored by multiple groups, showing an increase in
anti-tumor killing of CTLs treated with GLS inhibitors (telanglenastat,
or CB-839, and BPTES) when coupled with checkpoint immunother-
apy [131–133]. In one study, glutaminolysis inhibition resulted in a
decrease in GSH, which in turn led to global reduction in
glutathionylation in the cell, including SERCA glutathionylation that
increased NF-kB signaling and PD-1 expression [131]. In addition to
specific targeting of Gls, glutamine metabolism can be broadly
inhibited using the glutamine analog 6-diazo-5-oxo-L-norleucine
(DON). DON, and a modified DON molecule to target the TME more
directly due to toxicity concerns, reduced tumor burden even when
used as a monotherapy [134]. This approach both targets a
metabolic pathway preferentially used by cancer cells [113]and
promotes metabolic pathways favored by anti-tumor T cells
[130,134]. While promising, much remains to ensure T cells do
not exhaust and that essential glutamine metabolic pathways are
not also suppressed to ultimately limit T cell persistence or function.
D.R. Heintzman et al.
6
Cellular & Molecular Immunology

Cancer cells can also be dependent on methionine, and limiting
methionine may bolster current therapies [135]. Indeed, methionine
restriction can lead to decreased tumor burden in immunocompro-
mised mice to support a non-immune role for methionine in cancer
growth [135,136]. However, activated T cells upregulate and sustain
methionine transporters, and methionine restriction can decrease
cytokine expression and increase apoptosis [137,138]. Specifically,
methionine depletion resulted in decreased intracellular SAM
concentrations, loss of demethylation at lysine 79 of histone H3
via leukemia associated methyltransferase disruptor of telomeric
silencing 1-like (DOT1L), and functionally impaired CTLs [138].
Conversely, methionine supplementation, rather than restriction,
may reduce tumor burden in immunocompetent systems [138]. De
novo methionine synthesis also appears essential for maximal T cell
proliferation in vivo, as a CRISPR screen of genes in the methionine
cycle of one-carbon metabolism showed a loss of T cell fitness if
Mat2a, Mtr, or Mtrr were disrupted [139]. While further research is
needed to establish mechanistic details, our growing understanding
of methionine in the TME highlights the importance of studying
metabolic alterations in immunocompetent systems, as nutrient
availability alters the survival and function of immune cells as well as
cancerous ones.
Metabolism of the essential amino acid tryptophan by the
enzyme indoleamine 2,3-dioxygenase 1 (IDO1) of suppressive DCs,
TAMs, and CAFs results in both the depletion of tryptophan and
accumulation of the immunosuppressive metabolite kynurenine. As
an essential amino acid, tryptophan must be obtained through the
diet, and it cannot be replaced if it becomes limiting in tissues. IDO1
is overexpressed in most cancers, and kynurenine levels in the TME
correlate with poor prognosis in cancers such as melanoma, colon
cancer, ovarian cancer, and AML [140–142]. Kynurenine binds to the
aryl hydrocarbon receptor in naïve CD4 +T cells, promoting Treg
differentiation [142]. In addition, the depletion of tryptophan in the
TME activates the stress-response kinase GCN2 in T cells, which both
inhibits proliferation and induces differentiation into Tregs. GCN2 in
DCs and TAMs also leads to expression of inhibitory cytokines such
as IL-10 and TGFB, leading to a suppressive milieu [143]. Given the
role of IDO1 creating this environment, it has been the target of
multiple preclinical and early clinical trials over the past few years,
particularly in combination with ICB [142,144]. While initial trial
results were not positive, there remains a high potential for effective
combination therapies or in specific patient subsets.
Metabolites that accumulate to suppress immunity. In contrast to
the depletion of anabolic nutrients in the TME, many metabolites
in addition to kynurenine can accumulate in local microenviron-
ments as waste or secreted products to inhibit T cells. This is
particularly the case in tumors or inflamed tissues where vascular
exchange is poor. Levels of adenosine can be elevated in the TME
due to the release of ATP upon cell lysis which is converted to ADP
and adenosine by the ectonucleotidases CD39 and CD73 on the
surface of tumor cells. Adenosine may then act on the A2A- and
A2B-receptors on T cells and APCs, respectively, to reduce CD8 T
cell activation, proliferation, and anti-tumor function [145]. Local
accumulation of this immunosuppressive molecule impairs the
effectiveness of therapeutic approaches that induce ATP release
via tumor cell lysis, prompting multiple groups efforts to disrupt
conversion and adenosine signaling [145,146]. For example,
utilizing a CRISPR/Cas9 approach to disrupt A2AR on CAR-T cells in
culture led to an increase in IFNγ, TNFα, JAK/STAT pathway genes,
CAR-T cell survival, and control of tumor burden in a murine breast
cancer model [147].
As a result of elevated glucose uptake and glycolysis in tumors
that lead to a demand to convert NADH back to NAD by Lactate
Dehydrogenase, large quantities of pyruvate are shunted to lactic
acid formation rather than entering the TCA cycle. This can lead to
an interstitial accumulation of lactate and decreased pH. This
acidification has been shown to reduce anti-tumor CTL activation,
glycolysis, and expression of functional markers such as IFNγvia
diminished NFAT signaling [148,149]. In addition, the checkpoint
molecule VISTA suppresses T cells selectively in acidic environ-
ments and is upregulated in the TME [150]. While lactate can
inhibit effector T cells [151], it is not just as a waste product and
can serve as a metabolic fuel and signaling molecule. Treg are
oxidative and can take up and consume lactate in the TME
[152,153]. Clinically, levels of lactate dehydrogenase A (LDH-A),
which converts pyruvate into lactate, correlate with worse clinical
outcomes and fewer infiltrating T cells in multiple cancer types
[148,154,155].
Lactate accumulation in the TME is a rationale target for anti-
tumor preclinical research, yet there are complexities to the
feasibility of improving therapies via targeting this mechanism.
While LDH-A silencing in tumor cells via shRNA failed to alter
lactate levels, pH, or cell survival in the TME [156], a subsequent
study utilizing nanoparticle delivery of shRNA did show a reduction
in lactate, pH neutralization, and increased CTL infiltration and
tumor control [157]. Complicating the picture, another study found
tumor specific LDH-A knockdown with shRNA enhanced CAR-T
treatment and reduced tumor growth, however lactate levels and
pH in the TME were unchanged [154]. LDH-A was also shown to
play a role to regulate epigenetic marks through histone
acetylation to promote IFN-γexpression [158]. In addition, TME
neutralization has occurred in studies utilizing bicarbonate delivery
and inhibition of the lactate exporter monocarboxylate transport 1
[149,159]. One potential explanation for these discrepancies is the
differential expression of LDH-A vs the LDH-B isoform in various
tumors [156]. In addition, local lactate levels are dependent on
both tumor cell apoptosis and regional vascularization, which vary
widely between treatment conditions and tumor type. Importantly,
when these lactate-targeting treatments did improve outcomes,
multiple studies showed this to be a CTL-dependent effect and
treatment with LDH shRNA synergized with PD-1 ICB yet failed to
improve tumor outcomes in immunodeficient systems [157].
Supporting this notion, LDH inhibition controlled tumor growth
in a preclinical humanized non-small cell lung cancer model in a
CD8 +T cell dependent manner [160]. It was recently shown that
preconditioning CTLs with LDH-A inhibition resulted in less
terminally differentiated CTLs upon IL-2 treatment, while treatment
with IL-21 did not alter cellular metabolism but did decrease
transcription of LAG3, PD-1, and TIM3, leading to an overall
increase in cell persistence, tumor control, and host survival [161].
Conclusions and perspective
In as much as it is now apparent that metabolic pathways are
intimately linked to T cell fate, it is also clear that local nutrients
and physical conditions influence these processes. While we
reviewed several factors here, our understanding of these
processes and nutrient availability remains poor. These questions
are challenged by the need to consider in vivo tissue hetero-
geneity and the complexity of different cell types and activation
states. Nevertheless, the widespread and necessary use of
biochemical and bulk tissue assays obscure cell heterogeneity
in many settings. The variety of important factors for immunity in
tissues is beyond the ability to accurately model or quantitate
using simplified in vitro. It is important, however, to focus studies
on in vivo systems and consider microenvironmental factors and
tissue heterogeneity where possible. It is likewise important to
consider how different cell types may interact through metabo-
lites and how they may respond differently to the same nutrient
pool. These processes may have important implications for
immune cell function in different tissues, such as tumors relative
to lymph nodes, gut, adipose, liver, skin, or other disparate organ
sites. They also, however, offer an exciting opportunity to
modulate immunity in tissue and cell type specific ways with
implications for a wide variety of inflammatory conditions or
cancer types.
D.R. Heintzman et al.
7
Cellular & Molecular Immunology

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ACKNOWLEDGEMENTS
We thank members of the Rathmell lab for their input and discussions. Figures were
created using BioRender. This work was supported by R01s DK105550, HL136664,
AI153167, and CA217987 (JCR) and T32 GM007347 (ELF).
AUTHOR CONTRIBUTIONS
All authors contributed equally to the research, drafting, and editing of this
manuscript.
COMPETING INTERESTS
JCR is a founder, scientific advisory board member, and stockholder of Sitryx
Therapeutics, a scientific advisory board member and stockholder of Caribou
Biosciences, a member of the scientific advisory board of Nirogy Therapeutics, has
consulted for Merck, Pfizer, and Mitobridge within the past 3 years, and has received
research support from Incyte Corp., Calithera Biosciences, and Tempest Therapeutics.
ADDITIONAL INFORMATION
Correspondence and requests for materials should be addressed to Jeffrey C.
Rathmell.
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