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Immunity

Review

Metabolic and Epigenetic Coordination


of T Cell and Macrophage Immunity
Anthony T. Phan,2 Ananda W. Goldrath,2,* and Christopher K. Glass1,*
1Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA
2Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA
*Correspondence: agoldrath@ucsd.edu (A.W.G.), cglass@ucsd.edu (C.K.G.)
http://dx.doi.org/10.1016/j.immuni.2017.04.016

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular
processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosyn-
thetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule produc-
tion. In parallel, activation initiates context-specific gene-expression programs that drive effector functions
and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying en-
zymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing poten-
tial cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss
recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environ-
mental signals with activation-induced gene-expression programs through modulation of the epigenome
and speculate as to how this may influence context-specific macrophage and T cell responses to infection.

Introduction lation of individual epigenetic modifications, such as histone


Activation of immune cells in response to infection is the result of methylation and acetylation, as well as DNA methylation, are a
the summation of antigen-induced gene-expression programs recent topic of interest. Progress toward characterization of
integrated with environmental signals. Both innate and adaptive the global epigenetic landscapes of many immune cell popula-
immune cells increase their metabolic throughput upon stimula- tions in distinct differentiation states allows the linkage of epige-
tion, promoting energy generation and biosynthesis, while shift- netic modifications to alterations in differentiation and function.
ing the relative usage of metabolic pathways to support prolifer- Further, numerous signals (i.e., T cell receptor [TCR], Toll-like
ation, effector molecule production, and differentiation (O’Neill receptors [TLRs], inhibitory receptors, and cytokines) drive
and Pearce, 2016; MacIver et al., 2013; Pollizzi and Powell, changes in the epigenome that result in downstream modulation
2014; Buck et al., 2015). While the rapid transitions in cellular of immune responses.
metabolism have long been described, few studies have focused As studies of immunometabolism and epigenetics continue to
on determining the intrinsic cellular impacts metabolic activity reveal mechanisms that regulate immune cell differentiation and
can have on gene expression, effector molecule production, function, the exciting possibility has emerged that alterations in
and, ultimately, the fate and function of a given cell. Recent inter- availability of metabolic intermediates may directly impact the
est in understanding the impact of these fundamental metabolic epigenome of immune cells as well. This fits well with studies
changes on immune cell differentiation and function has yielded in cancer cells and stem cells suggesting a link between cellular
numerous studies examining the pathways and metabolites metabolism and alterations in epigenetic landscapes (Kinnaird
involved in driving protective immune responses. A key question et al., 2016; Etchegaray and Mostoslavsky, 2016). The enzymes
that has emerged is whether differential metabolic activity (i.e., required for epigenetic modifications, for example DNA deme-
use of glycolysis versus mitochondrial respiration) is coincident thylases (Ten-eleven Translocation [Tet] enzymes) and histone
with differentiation, a consequence of changes in phenotype or demethylases (HMTs), often require cofactors and substrates
localization, or a direct driver of alterations in immune cell differ- that are also critical intermediate metabolites. For example,
entiation and function. The complex interplay metabolism can 2-oxoglutarate is a cofactor for Tet enzymes, suggesting a link
have on nearly all cellular processes and the link between metab- between cellular metabolism and regulation of gene expression
olites and epigenetic modifiers have been extensively reviewed through alterations in the epigenetic landscape of immune cells.
(Kinnaird et al., 2016; Kaelin and McKnight, 2013; Etchegaray While this link has been hypothesized and explored in non-im-
and Mostoslavsky, 2016). Here we focus on studies that lay mune cell types (Kinnaird et al., 2016; Kaelin and McKnight,
the groundwork for a direct link between metabolic intermedi- 2013; Etchegaray and Mostoslavsky, 2016), it remains a nascent
ates and regulation of epigenetic landscapes of two key immune area of study in the context of immunity. In this review, we high-
cells: T cells and macrophages. light recent studies that emphasize the impact stimulation-
The discovery of numerous epigenetic modifications and the induced metabolism has on macrophage and T cell phenotype
enzymes responsible for them have revealed another extensive and effector function. Further, we discuss recent discoveries in
network of potential regulators of immune cell function. Tradi- the unique epigenetic profiles of macrophage and T cell subsets
tionally, attempts to identify factors critical for the differentiation as differentiation is induced after activation. Finally, we address
and function of immune cell populations have relied on compar- the exciting possibility that alterations in cellular metabolism may
isons of global gene-expression changes after stimuli. The regu- provide an additional mechanistic link between environmental

714 Immunity 46, May 16, 2017 ª 2017 Elsevier Inc.


Immunity

Review

signals and downstream gene-expression changes by immune


A
cells critical for the specification of immune cell function due to
recent studies that suggest coordination of epigenetic modifica-
tions by metabolic activity.

Epigenetic Landscapes
The program of gene expression within each cell type is
controlled by interactions of sequence-specific transcription
factors (TFs) with promoters and distal enhancers. While pro-
moters represent the obligatory sites of initiation of mRNAs, en-
hancers are major determinants of cell-specific patterns of
expression and physiologic responses to internal and external
signals (Heinz et al., 2015; Levine, 2010). Promoters and en-
hancers exhibit distinct chromatin signatures defined by modifi-
cations of both DNA and histones. This topic has recently been
reviewed (Tessarz and Kouzarides, 2014; Allis and Jenuwein,
2016) and for the purposes of this discussion we consider pro-
moters to exhibit a histone modification signature of trimethyla-
B tion of histone H3 lysine 4 (H3K4me3), whereas enhancers are
typically characterized by monomethylation of histone H3 lysine
4 (H3K4me1) (Figure 1A; Heintzman et al., 2007, 2009). Active
enhancers and promoters are further distinguished by histone
acetylation, for example at histone H3 lysine 27 (H3K27ac)
(Creyghton et al., 2010). Intriguingly, recent studies of macro-
phages indicate that the histone acetyltransferase p300 can
use Crotonyl-CoA as an alternative substrate to Acetyl-CoA for
histone tail modification as an activating mark (Sabari et al.,
2015). These modifications are recognized by ‘‘reader’’ proteins
that play various roles in transcriptional activation or repression.
For example, histone acetylation is recognized by proteins that
contain bromo domains, such as Brd4, which interacts with the
positive transcription elongation factor (pTEFb) and stimulates
the transition of RNA Pol II from a paused to elongating form
C (Brès et al., 2008). Many other DNA and histone modifications

H3K27ac and H3K4me1). Diversity in epigenetic landscapes correlates with


diversity in responses and plays a role in specifying differentiation.
(B) Classical activation of macrophages elicits increased glycolytic meta-
bolism and reduced reliance on mitochondrial respiration. These gross alter-
ations in metabolic pathway usage underlie specific changes in the abundance
of several metabolites. In particular, increased glycolytic metabolism drives
production of lactate and supports a pool of acetyl-CoA by slowing the TCA
cycle, resulting in a buildup of citrate that can be converted to acetyl-CoA as
well. Reduced flux through the TCA cycle also reduces the production of
2-oxoglutarate and increases the production of itaconate. Alternatively, TLR
stimulation of macrophages along with IL-4 reduces glycolytic metabolism and
drives increased mitochondrial respiration and oxidative phosphorylation.
(C) TCR and co-receptor stimulation of Th1, Th2, Th17, Tr1, and CD8+ T cells
drives a general increase in both glycolytic metabolism and mitochondrial
metabolism as measured by extracellular flux analysis. Glucose transport is
increased substantially to facilitate glycolytic metabolism and mitochondrial
mass increases as well. Treg cells, on the other hand, do not exhibit as dra-
matic an increase in glucose uptake and glycolytic metabolism, but do show
increased reliance on oxidative phosphorylation and FAO. Biosynthetic
pathways are engaged to fuel proliferation, in particular serine-driven folate
metabolism, a branch of one-carbon metabolism, that promotes nucleotide
synthesis. Extensive characterization of metabolic changes that occur during
memory CD8+ T cell differentiation show that in general memory CD8+ T cells
Figure 1. Regulation of Epigenetic State and Metabolic Pathway exhibit reduced metabolic activity, glycolytic and mitochondrial, with meta-
Usage in Macrophages and T Cells after Activation bolic heterogeneity exhibited within the memory pool. Tcm cells maintain high
(A) Activation of immune cells results in substantial remodeling of epigenetic mitochondrial mass, substantially increased SRC fueled by increased FAO.
landscapes. Genes associated with the naive state are repressed and effector Tem cells maintain lower levels of SRC and can be supported by high levels of
genes become active through deposition of repressive marks (i.e., H3K9me3 glycolytic metabolism. Examination of Trm cell metabolism show that skin Trm
and H3K27me3) and activating marks at promoters and enhancers (i.e., cells uniquely rely on high levels of fatty acid import to fuel FAO and SRC.

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Immunity

Review

are associated with distinct chromatin functions. For example, and DNA (DNMTs and Tet) (Allis and Jenuwein, 2016). As our
DNA methylation of cytosine at CpG and histone methylation mechanistic understanding of these writer and eraser proteins
of H3K9 or H3K27 are associated with active repression (Blattler grows, an appreciation for the importance of cofactors that
and Farnham, 2013; Wiles and Selker, 2016; Wang et al., 2016), take part in the establishment of epigenetic marks as well as
whereas CpG methylation within gene bodies prevents aberrant those necessary for their removal has emerged. While there is
intragenic transcriptional initiation (Neri et al., 2017). an extensive array of cofactors and substrates necessary for
The recent application of assay for transposase-accessible the epigenetic modification of the genome (reviewed extensively
chromatin with high-throughput sequencing (ATAC-seq) has in Kinnaird et al., 2016; Kaelin and McKnight, 2013; Etchegaray
facilitated the global identification of open chromatin corre- and Mostoslavsky, 2016), we will focus on several key metabo-
sponding to sites of TF binding with high genomic resolution lites that have demonstrated roles in deposition of epigenetic
and minimal cellular input, which allows for feasible ex vivo ana- marks that have been implicated in regulation of T cell and
lyses (Buenrostro et al., 2013; Amit et al., 2016). TF binding mo- macrophage activation.
tifs within accessible chromatin regions can be used to infer the
binding of known TFs and identify potential gene targets of these Acetyl-CoA
TFs. When combined with chromatin immunoprecipitation with Histone subunits contain multiple positively charged lysine resi-
high-throughput sequencing (ChIP-seq) that identifies active dues that attract negatively charged DNA. Acetylation of these
promoters and enhancers, ATAC-seq proves especially power- residues not only creates docking sites for Bromodomain-con-
ful to pinpoint TF binding sites inside the regulatory elements. taining readers, but also neutralizes the positive charge of the
For instance, studies examining open chromatin identified by lysine residue, resulting in loosening of the interaction between
ATAC-seq with corresponding chromatin modifications have DNA and histone (Huang et al., 2015). This results in a more
successfully revealed tissue-specific TF and enhancers respon- accessible chromatin structure permissive to transcriptional ma-
sible for the specific identity of tissue-resident macrophages, chinery. Histone acetylation is performed by HATs and requires
lineage-determining TF (LDTF) in hematopoiesis (Lavin et al., the availability of acetyl-CoA, which is the sole source of acetyl
2014; Lara-Astiaso et al., 2014), and more recently TFs regu- groups in eukaryotic cells (Choudhary et al., 2009). Thus, regula-
lating T cell differentiation to infection (Sen et al., 2016; Yu tion of the production of acetyl-CoA, which can be produced
et al., 2017). from cytoplasmic acetate, citrate, or pyruvate by distinct en-
zymes, may serve as a critical sensing step for HAT activity
Cofactor Requirements for Epigenetic Modifications within cells (Kinnaird et al., 2016). This sensing mechanism is
Gene expression within cell types is tightly regulated by TFs and supported in studies of Saccharomyces cerevisiae where reduc-
their interactions with promoters and distal enhancers. There- tion of cytosolic acetyl-CoA production was sufficient to drive a
fore, regulation of TF binding and activity by the epigenome is rapid reduction in histone acetylation within the nucleus (Takaha-
a crucial step in modulating differentiation programs and in im- shi et al., 2006). A seminal study in colon cell carcinoma cells also
mune cells can be essential for permitting or inhibiting the execu- showed that modulation of acetyl-CoA levels produced by ATP
tion of gene expression downstream of immune stimuli (Kanno citrate lyase (ACLY) was sufficient to regulate HAT activity
et al., 2012). For example, Ezh2, the catalytic subunit of (Wellen et al., 2009). Building on those findings, the culture of
polycomb repressive complex 2 (PRC2), is a histone methyl- mammalian cells in nutrient-limiting conditions initiates protein
transferase (HMT) that mediates gene repression by facilitating kinase B (also known as AKT)-driven uptake and metabolism
deposition of H3K27 trimethylation (H3K27me3) and has been of glucose and phosphorylation of ACLY, that sustains high
implicated in repressing cytokine production and limiting CD4+ ACLY activity despite reduced citrate levels, increasing the
T helper (Th) cell subset plasticity (Tumes et al., 2013). Specif- pool of acetyl-CoA, and promoting HAT activity (Lee et al.,
ically, analysis of Ezh2 binding and H3K27me3 of in vitro differ- 2014). Notably, a recent study of CD4+ T cells draws a link be-
entiated Th1 and Th2 cells demonstrated that Ezh2 activity tween glycolytic metabolism and acetyl-CoA levels; deletion of
downstream of polarizing cytokine conditions was necessary the glycolytic enzyme lactate dehydrogenase A (LDHA) inhibited
to repress expression of alternative Th lineage TFs and cyto- glycolytic metabolism, reduced acetyl-CoA levels, and lowered
kines, maintaining the differentiation state of CD4+ Th cell sub- H3K9ac of the Ifng enhancer (Peng et al., 2016). This study ar-
sets (Tumes et al., 2013). Thus, understanding the regulation of gues that the reduction in H3K9ac is a direct result of reduced
enzymes that add chemical groups to histones or DNA acetyl-CoA levels in LDHA-deficient CD4+ T cells. However, it
(‘‘writers,’’ histone acetyltransferases [HATs], HMTs, and DNA is unclear how LDHA deficiency directly modulates acetyl-CoA
methyltransferases [DNMTs]) and enzymes that remove those levels, whether by reducing levels of citrate available for conver-
same modifications (‘‘erasers,’’ histone deacetylases [HDACs] sion to acetyl-CoA or through inhibition of acetyl-CoA synthetase
and Tet enzymes) is essential for building a model of how im- (Peng et al., 2016). Furthermore, manipulation of any single
mune cells modulate chromatin to regulate function and cell fate. metabolic pathway may result in disruption of other metabolic
Several families of writer and eraser enzymes that shape the and signaling pathways as energy or biosynthetic processes
epigenetic landscape have been identified, of which several are perturbed, requiring significant care in interpreting data in
have been shown to be critical regulators of immune cell activity complex scenarios such as immune cell activation and the
(Chang et al., 2014; Wherry and Kurachi, 2015; Link et al., 2015). downstream impacts on epigenetic enzymes.
These enzymes are often grouped based on the substrate In contrast to promotion of acetylation by increasing acetyl-
and the mark they regulate: acetylation of histones (HATs and CoA levels, CoA-SH, a product of HAT-catalyzed histone acety-
HDACs) and methylation of histones (HMTs and HDMs) lation, can serve as a negative regulator of HAT activity. This

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Immunity

Review

negative feedback regulates HAT activity in tumor cells and may (Ma et al., 2017). Multiple metabolic inputs affect one-carbon
serve as a critical upper limit for histone acetylation in non-trans- metabolism and the regulation of this pathway is complex, mak-
formed cells and suggest that clearance of CoA-SH is an equally ing it difficult to predict outcomes given changes in any branch.
important regulator of HAT activity as acetyl-CoA levels (Lee In mouse embryonic stem cells, catabolism of threonine (one
et al., 2014). Given the central role acetyl-CoA plays in energy branch of one-carbon metabolism) was important for maintain-
production, many other pathways may indirectly regulate ing high levels SAM as removal of threonine from culture media
acetyl-CoA levels and subsequently histone acetylation as resulted in reduced SAM levels and diminished H3K4me3
well. Whether regulation of histone acetylation on such a global (Shyh-Chang et al., 2013). At the simplest level, high levels of
level can drive cell fate decisions remains an open question. SAH can be equated with low nutrient conditions and high
SAM concentrations with nutrient-rich conditions, highlighting
Nicotinamide Adenine Dinucleotide a role for the overall nutritional state of a cell as a modulator of
NAD+ and NADH are central coenzymes necessary to accept or DNMT and HMT activity.
carry electrons in numerous cellular processes. Reflective of
their importance, numerous processes regulate their abun- 2-oxoglutarate
dance. The discovery of SIR2, the first identified Sirtuin (SIRT) A citric acid cycle intermediate, 2-oxoglutarate, also called
NAD-dependent deacetylase, as the enzyme responsible for a-ketoglutarate, is typically generated in the mitochondria
mediating the effects of caloric restriction on yeast lifespan from isocitrate by isocitrate dehydrogenase (IDH). 2-oxogluta-
linked energy balance to histone acetylation through levels of rate functions as a critical cofactor for the function of 2-oxoglu-
NAD+ (Imai et al., 2000). Specifically, in yeast and mice, SIR2 tarate-dependent dioxygenases that hydroxylate proteins
required cleavage of NAD+ to deacetylate histones, and yeast (prolylhydroxylases), de-methylate histones (JmjC domain-con-
mutants with defects in NAD+ metabolism showed reduced taining histone demethylases [JHDMs]), or de-methylate DNA
SIR2-dependent repression of gene expression (Imai et al., (Tet enzymes). In the case of JHDMs, 2-oxoglutarate is decar-
2000; Tanner et al., 2000; Smith et al., 2000). Expanding on boxylated to succinate during demethylation of the substrate
these findings, nutrient-deficient conditions activated AMPK, (Chervona and Costa, 2012). The importance of 2-oxoglutarate
increased NAD+ levels, and supported SIRT activity in skeletal on JHDM and Tet function has primarily been examined in
muscle cells, resulting in deacetylation of peroxisome prolifera- cancer cell lines where tumor-associated mutations in IDH
tor-activated receptor g coactivator 1a (PGC1a) (Cantó et al., result in the production of the R-enantiomer of 2-hydroxygluta-
2009, 2010). These studies detail how cellular energy balance, rate (2-HG), which along with succinate and fumarate can
read out by NAD+ and NADH levels, can regulate protein acety- inhibit 2-oxoglutarate-dependent dioxygenases (Kaelin and
lation including histone acetylation. McKnight, 2013). This raises the possibility that regulation of
The complex regulation of NAD+ availability and the impact on 2-oxoglutarate or competitors (2-HG) can regulate the epige-
SIRTs have been reviewed previously, highlighting that regula- nome via modulation of 2-oxoglutarate-dependent dioxyge-
tion of NAD+ levels are dependent on synthesis, compartmental- nase activity. Little is currently known about how physiologic
ization, and usage by metabolic and non-metabolic pathways fluctuations in 2-oxoglutarate levels, compartmentalization, or
(Houtkooper et al., 2012). For example, poly ADP-ribose poly- competition between 2-oxoglutarate-consuming enzymes
merases (PARPs) have been shown to be NAD+-consuming en- play into the regulation of JHDM or Tet enzyme activity.
zymes implicated in the regulation of NAD+ levels and Sirt activity The metabolites discussed above by no means constitute the
(Schreiber et al., 2006). Specifically, deletion of Parp1 in mice only metabolic intermediates that exhibit rapid alterations in con-
increased intracellular NAD+ levels in brown adipose tissue centration or use following macrophage and T cell stimulation
and muscle, resulting in increased SIRT1 activity (Bai et al., and can modulate writer and eraser activity. For example,
2011). Particularly relevant to immune cell responses, the lactate, which can play a role in modulating T cell function in
compound resveratrol indirectly activates SIRTs, driving histone the tumor microenvironment, was demonstrated to be a weak in-
deacetylation, by increasing the availability of NAD+ in an AMPK- hibitor of HDACs and may impact immune cell epigenomes
dependent fashion (Um et al., 2010). (Latham et al., 2012; Brand et al., 2016). Furthermore, many
epigenetic modifying enzymes (i.e., HATs and SIRTs) have sub-
S-adenosylmethionine strates beyond histones and DNA, which are beyond the scope
DNMTs and HMTs transfer methyl groups to DNA and histones of this review, but must be considered when evaluating the activ-
via the same molecular mechanism, utilizing a methyl group ity of these enzymes. In the following sections, we focus the dis-
from S-adenosylmethionine (SAM) to generate methylated cussion on central metabolic molecules that are dynamically
DNA/histones and a molecule of S-adenosyl homocysteine regulated or have been shown to influence epigenetic enzymes
(SAH). SAM is produced via one-carbon metabolism from methi- in T cells or macrophages directly.
onine by the enzyme methionine adenosyltransferase (Sakata
et al., 1993). One-carbon metabolism is a critical component of Epigenetic Landscapes in Macrophages
cellular biosynthesis in immune cells and can regulate prolifera- Genome-wide analyses of open chromatin and histone modifi-
tion, as evidenced by a recent study that restricted serine, a cations associated with active enhancers in many cell types
methyl donor for the conversion of tetrahydrofolate (THF) to and tissues indicate that mammalian genomes contain on the
5,10-metheylene-THF in the folate cycle of one-carbon meta- order of a million potential regulatory elements (Kundaje et al.,
bolism, suppressing T cell proliferation by abrogating purine 2015). Each cell type typically selects 30,000 such elements
nucleotide biosynthesis without affecting energy production that act as primary drivers of that cell’s program of gene

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Review

expression and specific pattern of responses to internal and The transcriptional response to IL-4 in macrophages is driven
external signals. How each cell acquires its unique set of en- by a STAT-6-PPARg-PGC1a signaling axis (Vats et al., 2006),
hancers remains a major question. As in other cell types, pro- which plays key roles in regulating mitochondrial function and
moters and enhancers in macrophages are selected from the oxidative metabolism. In addition, metabolic reprogramming
genome by specific combinations of lineage-determining tran- has also recently been proposed to involve a mammalian target
scription factors (LDTFs) and more broadly expressed TFs of rapamycin complex 2 (mTORC2)-IRF4 signaling axis (Huang
(Link et al., 2015). Cell type-specific transcriptional responses et al., 2016). As with LPS treatment, responses to IL-4 are asso-
are primarily driven by enhancers, which are the most numerous ciated with extensive alterations of the epigenetic landscapes
sites of DNA binding of LDTFs. In peritoneal and bone marrow- (Piccolo et al., 2017). Intriguingly, IFN-g and IL-4 were found to
derived macrophages, PU.1, AP-1, CCAAT/Enhancer binding mutually inhibit the epigenomic and transcriptional changes
protein (C/EBP), and interferon regulatory factors (IRF) are major induced by each cytokine alone (Piccolo et al., 2017).
LDTFs and ChIP-seq experiments demonstrate that they are Metabolic reprogramming is also a notable aspect of the phe-
found alone or in combination at most macrophage enhancers nomenon of trained immunity, in which an initial response of an
(Ostuni et al., 2013; Ghisletti et al., 2010; Heinz et al., 2010; Kaik- innate immune cell to a particular stimulus results in altered re-
konen et al., 2013). These factors play important pioneering roles sponses to a subsequent stimulus by mechanisms that are
in establishing open regions of chromatin that become sites of thought to be epigenetic in nature (Netea et al., 2016; Arts
action for signal-dependent TFs such as NF-kB and nuclear hor- et al., 2016). Immune tolerance of macrophages is a well-studied
mone receptors (Glass and Natoli, 2016). Specific combinations example in which treatment with an initial dose of LPS renders
of TFs recruit co-activator and/or corepressor complexes that in them less responsive to a second dose for a subset of inducible
turn control the transcriptional functions of the corresponding genes (distinct from those associated with latent enhancers
regulatory elements. Many of these complexes contain enzy- noted above). This phenomenon may contribute to the immune
matic activities that modify chromatin through effects on DNA deficiency of sepsis (Netea et al., 2016). Treatment of monocytes
methylation, histone tail methylation, acetylation, etc. Studies with the Candida albicans cell wall constituent b-glucan provides
of nuclear hormone receptors established a paradigm by which an additional model of trained immunity, in which trained (i.e.,
signaling could induce a switch in the activity of a regulatory b-glucan-treated) monocytes exhibit an increased response to
element by inducing the exchange of co-repressor complexes LPS treatment 7 days later relative to naive monocytes (Cheng
for co-activator complexes (Rosenfeld et al., 2006). For example, et al., 2014). In this model, b-glucan treatment is associated
unliganded retinoic acid receptors bind NCoR and SMRT co- with immediate changes in expression of genes associated
repressor complexes that contain HDACs and repressive with immune responses and metabolism and corresponding
HMTs that exert active repression functions. Upon binding reti- changes in the epigenetic landscape. While the initial transcrip-
noic acid, NCoR/SMRT complexes are exchanged for SRC co- tional response resolves, epigenetic changes persist (Cheng
activator complexes that contain HATs such as CBP and p300 et al., 2014), suggesting a mechanistic basis for training, analo-
and activating HMTs such as CARM1, leading to gene activation. gous to findings made in LPS-treated macrophages. Intriguingly,
Thus, enhancers and promoters occupied by LDTFs and other b-glucan treatment has recently been found to partially reverse
sequence-specific TFs can range from being strongly repressed LPS tolerance in vitro through mechanisms suggested to involve
to maximally activated depending on the specific combinations reprogramming the epigenetic landscape (Novakovic et al.,
of factors and their activity states (Figure 1A). 2016). The two most significant functional annotations associ-
Treatment of macrophages with lipopolysaccharides (LPS) in ated with b-glucan-induced genes were oxidative reduction
the presence or absence of interferon-g (IFN-g) to induce a and metabolism, suggesting roles of these processes in training.
‘‘classically’’ activated phenotype or with interleukin-4 (IL-4) to
induce an ‘‘alternatively’’ activated phenotype have been exten- Metabolic Reprogramming in Macrophages
sively used as in vitro paradigms for investigation of signal- A conventional view of signal-dependent gene expression pla-
dependent gene expression and metabolic reprogramming ces metabolic intermediates downstream of TFs and their tar-
(O’Neill and Pearce, 2016; Baardman et al., 2015; Pearce and gets that play direct roles in regulation of metabolic processes
Pearce, 2013; Glass and Natoli, 2016; Wynn et al., 2013). The (e.g., PPARg and enzymes involved in fatty acid oxidation).
LPS response in macrophages is initially driven by latent TFs This view is challenged, however, by numerous recent findings
that include NF-kB, AP-1, and IRFs that subsequently result in indicating that metabolic intermediates are required for aspects
secondary transcriptional responses to type I interferons and of gene expression that are specific for distinct polarization
other cytokines. Hundreds of genes are activated or repressed states. There are potentially a broad range of mechanisms that
and are associated with corresponding changes in the surround- could account for these findings, acting at both epigenetic and
ing epigenetic landscapes (Kaikkonen et al., 2013; Ostuni et al., non-epigenetic levels.
2013). Although most changes in epigenetic features occur at LPS ligation of TLR4, while exerting direct effects on gene
pre-existing regulatory regions, there are also inactive genomic expression by activating signal-dependent TFs such as NF-kB,
loci that acquire features of active enhancers (Kaikkonen et al., AP-1, and IRFs, also results in a shift to glycolytic metabolism
2013; Ostuni et al., 2013), so-called ‘‘latent’’ or ‘‘de novo’’ en- and impaired mitochondrial respiration (Rodrı́guez-Prados
hancers. Some of these features are retained after resolution of et al., 2010). The shift to glycolysis in response to LPS is thought
the initial LPS response and are associated with more rapid re- to be important in the context of acute bacterial infection
sponses of higher magnitude to subsequent stimuli, suggesting because glycolysis, although less efficient in generating adeno-
a form of epigenetic ‘‘memory’’ (Ostuni et al., 2013). sine triphosphate (ATP), can be upregulated many fold and

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Review

therefore results in a faster production of ATP compared to and cytokine milieu. Naive CD4+ T cells give rise to effector pop-
oxidative phosphorylation (Arts et al., 2016). Glycolysis results ulations with helper and regulatory functions distinguished on
in an increase in lactate, which is a weak inhibitor of class II his- the basis of cytokine production and expression of ‘‘lineage-
tone de-acetylases (HDACs) (Latham et al., 2012). TLR signaling defining’’ TF, i.e., Th1 (IFN-g and T-bet), Th2 (IL-4 and GATA3),
also results in marked shifts in NAD+-NADH ratios, which influ- Th17 (IL-17 and RORgt), follicular helper T (Tfh) (IL-21 and
ence the activities of the class III HDACs SIRT1 and SIRT6, Bcl-6), and regulatory T (Treg) (IL-10 and FoxP3) cells (Bonelli
potentially altering de-acetylation of histone and non-histone et al., 2014). Notably, significant complexity within each Th cell
substrates (Figure 1B; Liu et al., 2012). population including overlapping TF expression and cytokine
The response to LPS plus IFN-g also results in alterations in production as well as plasticity among fates are observed (Bone-
the tricarboxylic acid (TCA) cycle (Lampropoulou et al., 2016; lli et al., 2014), particularly when studied ex vivo. In contrast, acti-
Jha et al., 2015). Downregulation of isocitrate dehydrogenase, vation of naive CD8+ T cells leads to an effector population that
which converts isocitrate to 2-oxoglutarate, leads to accumula- broadly produces IFN-g and TNFa and possesses cytotoxic ac-
tion of citrate, required for fatty acid biosynthesis and itaconic tivity but with phenotypic complexity and TF dependence that
acid formation. Citrate can be converted to acetyl-CoA by can indicate potential for differentiation to long-lived memory
ATP-citrate lyase, which is used as a substrate for histone cells, e.g., IL-7R is expressed by memory-precursor cells that
acetylation in cancer cells (Wellen et al., 2009). Thus, one conse- depend on TCF-1, Eomes, Id3, and FOXO1, while KLRG1 is ex-
quence of the metabolic switch of classical macrophage activa- pressed by shorter-lived effector and effector-memory cells that
tion could be generation of acetyl-CoA for histone acetylation depend on T-bet, Blimp-1, Id2, IRF4, and Zeb2 (Chang et al.,
and reduced levels of 2-oxoglutarate, needed for JHDMs and 2014). In contexts where antigen exposure is sustained beyond
Tet proteins (Figure 1B). These possibilities have yet to be exam- the initial expansion and effector phases, such as in chronic in-
ined directly. The break in the TCA cycle induced by LPS and fections or tumors, responding T cells often exhibit a progressive
IFN-g is compensated for by a variant of the aspartate-argino- loss of function or ‘‘exhaustion’’ that can modulate immunopa-
succinate shunt. Notably, inhibition of aspartate-aminotrans- thology as well as lead to a permissive dysfunctional state and
ferase, a key enzyme of the shunt, inhibited nitric oxide and escape of virus or tumors. Exhaustion of CD8+ T cells is regu-
IL-6 production in M1 macrophages, indicating a role of shunt in- lated by expression of inhibitory receptors such as PD-1 and
termediates in establishing the classically activated phenotype the TFs T-bet and Eomes and is accompanied by metabolic
(Jha et al., 2015). The underlying mechanisms have not been and epigenetic states distinct from both effector and memory
established. T cell subsets (Wherry and Kurachi, 2015). Recent studies
Succinate represents another TCA cycle intermediate with make clear that LDTFs provide only a portion of the information
regulatory functions. Succinate has been shown to activate needed to establish stable regulatory networks that drive T cell
HIF-1a and promote inflammatory gene expression (Kelly and effector function and memory cell fates; the epigenetic land-
O’Neill, 2015). Succinate levels were recently shown to be modu- scapes accompanying T cell activation and effector cell polariza-
lated by itaconate, which is one of the most highly induced tion show dynamic remodeling of DNA and histone modifications
metabolites in classically activated macrophages (Figure 1B; that mediate access of key TFs to their targets and provide the
Lampropoulou et al., 2016). Itaconate modulates macrophage framework for the relative stability or flexibility between cell fates.
metabolism and effector functions by inhibiting succinate Comparisons of global DNA methylation among naive and
dehydrogenase-mediated oxidation of succinate. Bone marrow effector CD8+ T cell populations in the response to acute viral
macrophages from mice lacking Irg1, required for production infection reveal differentially methylated regions at promoters
of itaconate, exhibit marked reductions in succinate levels and and enhancers consistent with the dynamic changes in gene
reductions in levels of some inflammatory cytokines after treat- expression after T cell activation and emphasize that gains in
ment with LPS (Lampropoulou et al., 2016). methylation in effector cells compared to naive T cells are
In contrast to classical activation of macrophages, treatment consistent with repression of the naive state in addition to deme-
of macrophages with IL-4 in addition to TLR signaling results thylation of necessary effector loci to drive differentiation and
in a shift to oxidative metabolism (Vats et al., 2006; Jha et al., effector function (Scharer et al., 2013). More recent studies using
2015). Overall, the program of macrophage alternative activation ATAC-seq to capture dynamic changes in chromatin accessi-
is coupled to changes in polyamine synthesis and fatty acid bility as antigen-specific polyclonal or TCR transgenic CD8+
oxidation (Figure 1B). In addition, enhanced use of glucose T cells differentiated in the response to acute or chronic LCMV
for UDP-GlcNAc synthesis has been suggested as a meta- infection allowed the identification of stable changes in chro-
bolic signature of alternatively activated macrophages (Jha matin accessibility by effector, memory, and exhausted popula-
et al., 2015). tions (Scott-Browne et al., 2016; Scharer et al., 2017). More than
70,000 regions that were ‘‘open’’ in at least one differentiation
Epigenetic Landscapes in T Cell Activation state could be discerned and approximately half of those were
Activation of T cells initiates dramatic changes in gene expres- stable in all cell types examined, indicating a core pattern of
sion within hours, and over the course of days the T cell response chromatin organization common to the CD8+ lineage regardless
evolves with acquisition of heterogeneous effector fates distin- of activation state (Scott-Browne et al., 2016). Integration of
guished by phenotype, effector capacity, and potential for es- chromatin accessibility with known TF binding motifs allow the
tablishing long-lived protective immunity. Many conditions influ- prediction of TFs that promote gene expression at each differen-
ence the composition and outcome of T cell responses including tiation state (Scott-Browne et al., 2016; Scharer et al., 2017).
the duration of antigen exposure, availability of costimulation, Memory CD8+ T cells maintained an open configuration at

Immunity 46, May 16, 2017 719


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Review

many regulatory sites, providing evidence for ‘‘memory primed’’ emphasize a role for epigenetic regulation at each stage of the
genes that can be more rapidly induced compared to naive pop- CD8+ T cell response to infection and show that accessibility
ulations. The ATAC-seq analysis of CD8+ T cells in chronic LCMV of specific regulatory regions by differentiation intermediates
infection of mouse or HIV-specific cells in humans revealed a provide the basis for TFs that are ubiquitously expressed to drive
unique chromatin state associated with exhaustion, where one cell fate over another.
many accessible sites were shared with effector populations in Similar studies providing a comparative epigenetic land-
acute infection, but additional regions were uniquely lost or scape for CD4+ Th cell subsets, generated by in vitro polariza-
gained that could mediate expression of genes that drive tion and directly ex vivo from a range of disease states, reveal
exhaustion such as PD-1 (Scott-Browne et al., 2016; Pauken dynamic changes in DNA and histone modifications as Th cells
et al., 2016; Sen et al., 2016). Notably, blockade of PD-1 initiated differentiate and have recently been reviewed (Tripathi and
the re-engagement of genes associated with effector function Lahesmaa 2014; Bonelli et al., 2014; Vahedi et al., 2013).
but could not reverse the exhausted configuration of chromatin, Notably, the presence of H3K4me3 and H3K27me3 modifica-
further emphasizing the central role that epigenetic landscapes tions that indicate a potential for both activation and repression
play in differentiation and functional state of T cells during at key regulatory sites of LDTF in differentiated Th cells pro-
infection. vides insight into the substantial plasticity observed between
Genome-wide analysis of H3K4me3 and H3K27me3 histone distinct Th cell subsets (Figure 1A). Observations that many
methylation for polyclonal naive and endogenous memory sub- of the Th cell subset-distinct enhancer landscapes established
populations at the steady state and antigen-specific naive, in response to external stimuli such as cytokines are regulated
effector, and memory CD8+ T cells responding to influenza A by STAT family TFs and not LDTFs emphasize that differentia-
virus revealed dynamic changes in promoter methylation corre- tion is regulated not only by expression of key TFs but also by
lating with gene expression (with H3K4me3 associated with accessibility of regulatory elements for which the landscape
expressed genes and H3K27me3 associated with reduced may be remodeled in response to triggering of environmental
expression) (Araki et al., 2009a; Russ et al., 2014). Notably, naive sensors (Bonelli et al., 2014; Tripathi and Lahesmaa, 2014;
T cells showed co-deposition of H3K4me3 and H3K27me3 at Vahedi et al., 2013).
promoter regions of genes important for cellular differentiation
whereas genes associated with immune effector function lacked Metabolic Reprogramming in T Cells
the permissive H3K4me3 modification, suggesting epigenetic A number of reviews have detailed the changes in T cell meta-
mechanisms underlying CD8+ T cell differentiation and acquisi- bolism that occur after activation (MacIver et al., 2013; Pollizzi
tion of effector function. Interestingly, a subset of effector-asso- and Powell, 2014) and here we will focus on highlighting studies
ciated genes are rapidly, and in some cases transiently, induced that suggest metabolic links between T cell receptor stimulation
after T cell activation (Best et al., 2013; Kakaradov et al., 2017) and writers and erasers of the epigenetic code discussed above.
and may be marked by H3K4me2 modification in naive T cells Activation of T cells initiates a molecular program that rapidly
(Russ et al., 2014). Treatment of effector or memory T cells sub- alters gene expression and metabolic activity in T cells.
sets with histone acetylase or deacetylase inhibitors modulates Numerous signaling pathways induce increases in metabolic ca-
expression of effector-associated molecules (Araki et al., pacity and the engagement of biosynthetic pathways after TCR
2009a) and affects memory function and potential (Northrop engagement and costimulation (reviewed extensively by Ma-
et al., 2008), further emphasizing a role for epigenetic changes cIver et al., 2013; Pollizzi and Powell, 2014), promoting the
in regulating gene expression at different stages of the T cell activity of TFs such as c-MYC, mTOR, PI3K, and AKT, Hypoxia
response (Figure 1A). Recent studies of antigen-specific CD8+ inducible factor 1 alpha (HIF1a), and AMP-activated protein ki-
T cells expand these analyses, including genome-wide assess- nase (AMPK).
ment of histone modification of H3K4me1 and H3K27ac marks Initial reports suggested that both CD4+ and CD8+ T cells
(He et al., 2016) as well as ATAC-seq (Yu et al., 2017), to show engaged the same metabolic program upon activation, empha-
extensive remodeling of enhancers as the effector and memory sizing the use of glycolytic metabolism to fuel energy production
populations differentiate and, along with improved computa- and preserve non-glucose carbon sources for biosynthesis
tional approaches, predict TF binding activity at each differenti- (Wang et al., 2011; Bental and Deutsch, 1993). However, recent
ation state with greater resolution. Of more than 50,000 en- studies have revealed complexity in both the usage of TFs as well
hancers identified, approximately 50% were unchanged over as differential reliance on glycolysis versus mitochondrial respi-
the course of the immune response. However, of enhancers ration after activation reflective of the differences between
that showed dynamic regulation, as many or more were lost as CD4+ Th cell subsets and CD8+ T cells (Wang et al., 2011; Cao
gained at each stage, in agreement with the idea that repression et al., 2014). For example, studies of mTORC1 versus mTORC2
of alternative fates is key to differentiation (Yu et al., 2017). usage revealed distinct roles for each pathway in Th cell subsets
Furthermore, single-cell gene expression analysis at the first and CD8+ T cell differentiation. Specifically, loss of mTORC1 or
division after infection reveals an early burst of transcriptional mTORC2 signaling in CD4+ T cells resulted in failure of CD4+
activity by daughter cells destined for terminal differentiation T cells to differentiate into Th1 and Th17 or Th2 cells, respec-
compared to memory-precursor cells at the same stage (Kakar- tively (Figure 1C; Delgoffe et al., 2011). Notably, mTORC1 and
adov et al., 2017). This burst was followed by epigenetic mTORC2 signaling in CD4+ T cells contributed to Tfh cell differ-
silencing of genes associated with long-lived memory formation entiation, while conversely loss of mTORC signaling in CD4+
with deposition of H3K27me3 and a requirement of Ezh2 (Kakar- T cells promoted Foxp3 expression but reduced suppressive
adov et al., 2017; Gray et al., 2017). These recent studies further ability (Delgoffe et al., 2011; Zeng et al., 2013, 2016). In contrast

720 Immunity 46, May 16, 2017


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Review

for CD8+ T cells, loss of mTORC1 significantly perturbed the gen- Impact of Metabolic Switching on T Cell Function and
eration of effector responses, while mTORC2 loss did not impact Differentiation
effector responses but appeared to promote memory CD8+ A strategy by which T cells integrate the substantial array of
T cell generation (Pollizzi et al., 2015). In each of these scenarios, in situ signals that initiate, sustain, and mediate effector
mTORC1 and mTORC2 signaling is thought to promote glyco- responses may be reflected in the marked shifts in energy pro-
lytic metabolism, but critically, it remains to be determined duction and biosynthetic pathways after activation (Pollizzi and
whether the alterations in differentiation after their loss is primar- Powell, 2014; Chang et al., 2014). At present, the fundamental
ily a function of changes in energy production or biogenesis due question of whether metabolic reprogramming or switching dur-
to reduced glycolytic metabolism or loss of regulation of non- ing the course of T cell responses drives fate decisions remains
metabolic target genes. These questions (and many others unanswered primarily due to the difficulty in distinguishing
discussed further in Jones and Pearce, 2017) are areas of active defects in survival versus alterations in differentiation. However,
investigation and a number of studies that propose some there is strong data for both CD4+ and CD8+ T cells demon-
direct effects on differentiation and gene expression are dis- strating that usage of glycolysis, mitochondrial respiration,
cussed below. biosynthetic pathways, and the factors that regulate them facil-
Recent studies examining the energetic profile of CD4+ and itate distinct aspects of effector functionality (Bengsch et al.,
CD8+ T cells after activation supported glycolytic metabolism 2016; Chang et al., 2013; Doedens et al., 2013; Scharping
as a crucial contributor to fueling effector responses and prolifer- et al., 2016; Peng et al., 2016; Ma et al., 2017; Ho et al., 2015;
ation (Peng et al., 2016; Doedens et al., 2013; Chang et al., 2013). Shi et al., 2011; Zeng et al., 2013; Gerriets et al., 2015). Moreover,
It is also clear that mitochondrial metabolism contributes to recent studies support the hypothesis that microenvironmental
effector CD8+ T cell metabolism beyond the established para- nutrient availability (discussed below) can directly impact T cell
digm of supporting memory T cells (Figure 1C; Cao et al., metabolism and result in modulation of gene expression, func-
2014; Scharping et al., 2016; Bengsch et al., 2016). After tion, and T cell fate (Ho et al., 2015; Ananieva et al., 2014).
in vitro activation, CD4+ and CD8+ T cell proliferation was Studies examining the level of metabolic heterogeneity in
inhibited by pharmacological blockade of either glycolysis, via CD4+ T cells and Th cell subsets show that Th1, Th2, Th17,
2-deoxyglucose treatment, or mitochondrial respiration, via oli- and Tfh cell subsets exhibit increased expression of Glut1 and
gomycin treatment (Cao et al., 2014; Phan et al., 2016). Examina- glycolytic rate in comparison to Treg cells after activation
tion of exhausted antigen-specific CD8+ T cells responding in (Figure 1C; Zeng et al., 2016; Shi et al., 2011; Michalek et al.,
chronic viral infection or tumors treated with anti-PD-1 sug- 2011). In particular, HIF1a-driven glycolytic metabolism can sup-
gested that in some inflammatory contexts mitochondrial func- port differentiation of Th17 cells, and pharmacologic inhibition of
tion is critical for reinvigorating T cell responses (Bengsch glycolytic metabolism in activated CD4+ T cells skewed them to-
et al., 2016; Scharping et al., 2016). ward iTreg cell differentiation and reduced disease severity in a
Given the substantial increase in the use of glycolytic meta- mouse model of multiple sclerosis (Shi et al., 2011). In line with
bolism by T cells after activation, most studies have focused the initial observations that Th1, Th2, and Th17 cells relied on
on elucidating the impact of energy-producing pathways on glycolytic metabolism, abrogated mTOR signaling in CD4+
T cell biology (Blagih et al., 2015; O’Sullivan et al., 2014; Chang T cells increased Foxp3 expression (Zeng et al., 2013). As
et al., 2013; van der Windt et al., 2012, 2013; Phan et al., 2016; mentioned above, a nuance to the increased generation of
Wang et al., 2011; Pearce et al., 2009; Sukumar et al., 2013). Foxp3+ cells is the reduced capacity of those cells to suppress.
This focus has yielded a number of critical insights, including A number of hypotheses could explain this loss of function,
how energy-producing pathways support production of cyto- including a requirement for mTOR in effector Treg cell differenti-
kines by effector CD8+ T cells and CD4+ Th cell subsets (Chang ation, but perhaps the more intriguing question is whether glyco-
et al., 2013; Peng et al., 2016; Ho et al., 2015). Specifically, the lytic metabolism serves to facilitate cytokine production by sup-
regulation of IFN-g production by glycolytic metabolism has re- pressive Treg cells. The hypothesis that glycolysis also maintains
vealed two potential mechanisms of metabolism-dependent suppressive cytokine production in Treg cells is supported by the
cytokine production (Chang et al., 2013; Peng et al., 2016). observation that type 1 regulatory (Tr1) cells, an IL-10-producing
One centers on sustained glycolytic throughput occupying CD4+ T cell subset that does not express Foxp3, exhibited a
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) thereby metabolite profile associated with glycolytic metabolism similar
preventing inhibition of IFN-g mRNA translation, while the other to Th17 cells (Mascanfroni et al., 2015).
links glycolytic metabolism to deposition of epigenetic marks In contrast to the effect metabolic activity has on effector func-
that drive IFN-g expression (Chang et al., 2013; Peng et al., tion and proliferation of T cells, the causative impacts on differen-
2016). While the exact mechanism by which glycolytic meta- tiation of CD4+ and CD8+ T cell subsets remains unclear. Two key
bolism facilitates production of IFN-g remains controversial, studies renewed interest in T cell metabolism as a modulator of
the correlative changes in T cell metabolism after activation cell-fate decisions (Araki et al., 2009b; Pearce et al., 2009). Inhi-
clearly have functional ramifications and support survival of bition of mTOR by treatment with rapamycin or activation of
long-lived memory T cells as well as set the stage for rapid AMPK by treatment with metformin potentiated the formation of
secondary responses by memory T cells (Chang et al., 2014; memory CD8+ T cells after infection by inhibiting glycolytic meta-
Pollizzi and Powell, 2014). Importantly, this encompasses bolism and supporting fatty acid oxidation (FAO) (Araki et al.,
biosynthetic pathways as well, such as serine-fueled one car- 2009b; Pearce et al., 2009). These studies suggested that a crit-
bon metabolism, essential for fueling efficient proliferation (Ma ical step in the specification of the memory T cell fate is a transi-
et al., 2017). tion away from ‘‘effector’’ metabolism (i.e., reliance on glycolysis)

Immunity 46, May 16, 2017 721


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Review

to ‘‘memory’’ metabolism (i.e., reliance on fatty acid oxidation T cells. These data strongly argue that the memory CD8+ T cell
and mitochondrial respiration). Memory CD8+ T cells exhibited fate in general is independent of the usage of either glycolytic
greater oxygen consumption, a surrogate for mitochondria- or mitochondrial metabolism and that the specification of
driven energy production, than that of naive and effector CD8+ memory relies on the provision of adequate cellular energy for
T cells and most importantly, maintained a greater maximal survival, allowing other mechanisms (i.e., LDTFs) to delineate
capacity for oxygen consumption dubbed mitochondrial spare T cell fate (Phan et al., 2016). Furthermore, examination of meta-
respiratory capacity (SRC) (van der Windt et al., 2012). Further- bolic activity of memory CD8+ T cell subsets, central memory T
more, this increased SRC was shown to be a product of (Tcm) and effector memory T (Tem) cells, revealed a previously
increased mitochondrial mass and fueled by T cell-intrinsic lipol- unappreciated level of metabolic heterogeneity within the mem-
ysis that supplied fatty acids for use within the mitochondria ory CD8+ T cell pool that may reflect the distinct functional and
(O’Sullivan et al., 2014; van der Windt et al., 2012). migratory characteristics of each subset (Figure 1C; Phan
These studies established a paradigm in which T cells require et al., 2016). Whether this metabolic heterogeneity drives the dif-
the transition from glycolytic metabolism to mitochondria- ferentiation of Tcm or Tem cells is unknown, but a recent study of
dependent oxidative phosphorylation (OxPhos) for the genera- tissue-resident memory CD8+ T (Trm) cell metabolism showed
tion of memory and it has been argued that the shift in T cell that skin Trm cells utilize distinct strategies relative to circulating
metabolism itself drives the formation of memory CD8+ T cells memory cells for acquiring fatty acids for energy production and
(O’Sullivan et al., 2014; van der Windt et al., 2012). Deletion or in- survival, potentially reflective of their localization, which supports
hibition of molecules critical for various metabolic processes the premise that T cells exhibit substantial metabolic flexibility
provide complementary evidence showing that metabolic transi- (Figure 1C; Pan et al., 2017).
tions and differential usage of glycolysis and OxPhos occur dur- Similarly, metabolic heterogeneity has been observed in Th
ing T cell responses and can impact the generation of effector cell subsets that express LDTFs, as discussed above, and
and memory cell function and survival (Pollizzi et al., 2015; although differential metabolic pathway usage affects function-
Chaoul et al., 2015; Rolf et al., 2013; Sena et al., 2013; Rao ality it remains to be determined whether the metabolic hetero-
et al., 2010; Shrestha et al., 2014; Balmer et al., 2016). For geneity observed contributes mechanistically to Th cell subset
example, inhibition of glycolysis with 2-deoxyglucose during differentiation (Michalek et al., 2011; Shi et al., 2011; Mascan-
activation can promote a memory-like gene expression program froni et al., 2015; Zeng et al., 2016). These studies raise a number
and longer-term survival of CD8+ T cells responsive to infection of questions about when, where, and how fluctuations in T cell
or tumors, supporting the necessity of a metabolic transition in metabolism impact T cell function and support a model in which
the specification of a memory cell fate (Sukumar et al., 2013). cellular metabolism serves as a context-specific integrator of
Additionally, IL-7-driven expression of glycerol channel aqua- extracellular cues such as cytokines and nutrients (discussed
porin 9 (AQP9) was essential for the production of triacylglcer- more thoroughly in this issue by Olenchock et al., 2017).
ides, necessary for FAO-driven ATP production and survival of
memory T cells (Cui et al., 2015). Similarly, increased intracellular Impacts of Metabolism on Gene Expression and Links to
levels of L-arginine improved survival of human and mouse Epigenetics
T cells (Geiger et al., 2016). While these studies certainly demon- One of the difficulties in studying the impact of metabolism on
strate that T cell metabolism supports the differentiation of T cell function and differentiation lies in the limited ability to
memory T cells and that the shift between reliance on glycolytic manipulate metabolic pathways in vivo where the full comple-
metabolism and FAO-driven mitochondrial respiration correlate ment of signals governing the immune response are present.
with the memory T cell fate, it remains to be determined whether Many studies have relied on in vitro models that at times trans-
metabolic pathways function as a driving force behind cell-fate late well to T cell responses in situ, but some conclusions drawn
decisions such as memory differentiation. More specifically, directly about T cell metabolism require further examination in
parsing out the difference between inducing differentiation and physiological contexts as the availability of nutrients, cytokines,
enhancing the likelihood of survival by improving energy produc- and cellular interactions are critical and support a model where
tion will need to be considered in future studies. shifts in cellular metabolism are a likely means for integration of
The importance of discriminating improvements in survival multiple exogenous and endogenous signals downstream of
versus driving memory differentiation is shown in studies of pathogen recognition. In particular, two recent studies demon-
von Hippel Lindau tumor suppressor (VHL)-deficient CD8+ strate in CD4+ T cells that levels of phosphoenolpyruvate (PEP)
T cells that sustain high levels of glycolytic metabolism while and leucine feed back into well-studied signaling pathways,
actively suppressing mitochondrial respiration due to constitu- altering T cell gene expression (Ho et al., 2015; Ananieva
tive stabilization of HIFa subunits (Phan et al., 2016). After acute et al., 2014). PEP, a glycolytic intermediate, inhibited SERCA-
viral infection, VHL-deficient CD8+ T cells form protective long- mediated calcium uptake and thus supported a model where
lived memory T cell populations despite sustained reliance on PEP accumulation is critical for sustaining Ca2+-NFAT signaling,
glycolytic metabolism, reduced SRC, and downregulated linking the upregulation of glycolytic metabolism in T cells
mitochondrial respiration deemed essential for memory differen- to maintenance of TCR signaling (Ho et al., 2015). Functionally,
tiation by previous studies (Phan et al., 2016). Intriguingly, the this is particularly relevant in the context of tumors where
sustained usage of glycolytic metabolism by VHL-deficient glucose can be limiting due to competition for uptake by
CD8+ T cells did not result in a bioenergetic disadvantage as tumor cells as overexpression of phosphoenolypyruvate
cellular ATP levels during the contraction or memory phases carboxykinase 1, which converts oxaloacetate into PEP,
of the response to infection were similar to wild-type CD8+ improved Ca2+-NFAT signaling in tumor-infiltrating CD4+

722 Immunity 46, May 16, 2017


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Review

activation, measured by phosphorylation of S6 and 4EBP-1,


further increasing glycolytic metabolism and demonstrating
Pathogen
another metabolic layer of regulation of TCR-driven signaling
(Ananieva et al., 2014). These data highlight the direct impact al-
terations in metabolite levels can have on gene expression in
Activation signal
T cells and support the possible regulation of T cell function
via epigenetic enzymes.
Glycolysis Two recent studies directly raised the possibility that metabo-
Glucose
lites may modulate the epigenome of T cells after activation. As
discussed above, glycolytic metabolism supports effector func-
3-phosphoglycerate
tion and, in the case of IFN-g production, one proposed mecha-
Phosphoenolpyruvate nism is that engagement of glycolytic metabolism by T cells can
Lactate Butyrate
facilitate the production of IFN-g through maintenance of high
Pyruvate Nucleotide
acetyl-CoA levels that facilitated deposition of H3K9Ac at the
HDAC NAD+ synthesis
Lactate NADH IFN-g promoter driving IFN-g expression (Figure 2; Peng et al.,
2016). It remains unclear what other loci exhibit increased acet-
Acetyl-CoA HAT CoA-SH ylation after LDHA deletion, and whether this can be modulated
5,10-methylene-THF
on a gene-specific level remains unexplored. The second study
Acyl-CoA 5-methyl-THF Glycine that shows direct modulation of the epigenome in T cells centers
Citrate Folate cycle around the oncometabolite 2-hydroxyglutarate (Tyrakis et al.,
Oxaloacetate 2016). In vitro activated CD8+ T cells treated with S-enantiomer
Hemocysteine
Isocitrate
THF
of 2-hydroxyglutarate (S-2-HG) acquired a Tcm cell-like pheno-
Malate
TCA cycle Methionine cycle Methionine type that was sustained after transfer into wild-type host mice.
2-HG HMT Furthermore, upon stimulation in vivo, S-2-HG-treated CD8+
2-oxoglutarate SAH DMNT SAM Serine T cells exhibited enhanced proliferation, persistence, and anti-
Fumarate
tumor capacity after adoptive transfer. These changes coincided
Succinate Succinyl-CoA
2-oxoglutarate with alterations in global DNA and histone methylation, that may
JHDM be a direct effect of S-2-HG inhibition of Tet2 and Utx3 (Figure 2;
TET
Tyrakis et al., 2016). Critically, although it was observed that
Electron transport chain
Succinate 2-HG T cells do produce S-2-HG at physiologically significant levels
Fumarate after in vitro activation and culture, it remains to be determined
whether S-2-HG is produced in situ at levels necessary to have
ATP
similar impacts (Tyrakis et al., 2016). Furthermore, whether
global DNA and histone methylation changes occur and at
Figure 2. Pathogen Recognition Initiates Induction of Biosynthetic what level of specificity in physiological conditions remain to
and Energetic Pathways, Impacting Writers and Erasers of the be defined. Clearly the wealth of data regarding the impact of
Epigenetic Code metabolic pathways on T cells supports modulation of enzymes
Recognition of pathogen by immune cells initiates a genetic program that
that modify epigenetic landscapes, but the small number of
promotes use of numerous metabolic pathways for biogenesis (i.e., nucleotide
synthesis) and energy production (i.e., glycolysis, TCA cycle). Metabolites studies directly examining this connection highlight that further
involved in glycolysis (teal), TCA cycle (orange), and one-carbon metabolism exploration of molecular mechanisms is needed.
(light green) can play critical roles as substrates such as acetyl-CoA and SAM,
for histone acetylation and methylation, respectively, or cofactors such as
2-oxoglutarate and NAD+, for demethylation of DNA and histone deacetyla- Influence of Tissue Environment on Gene Expression
tion, respectively. Epigenetic enzymes (blue) catalyze the addition (HAT, HMT, and Epigenetic Landscapes
or DNMT) and removal (HDAC, JHDM, or TET) of epigenetic marks and can be These studies raise a number of exciting questions about how
impacted by multiple metabolic pathways. Metabolites shown to be controlled
by pathways relevant in T cells or macrophages (bold) are critical for energy
metabolites regulate macrophage and T cell biology and
production (i.e., lactate, acetyl-CoA) and proliferation (i.e., serine). Flexibility in whether physiological conditions such as diverse tissue
metabolic pathway usage is reflected by the small network depicted here; for microenvironments, inflammation, or diet may significantly
example, 3-phosphoglcerate can be converted to serine for use in one-carbon
modulate their function and differentiation. Nearly all studies
metabolism. Clear links between activity of epigenome-modifying enzymes
and metabolites relevant in T cells and macrophages that will impact the connecting signal-dependent gene expression, metabolism,
epigenetic landscape of activated cells can be drawn. and epigenetic consequences have been performed in vitro.
However, in vivo, these interactions occur in the context of
T cells, bolstering their tumoricidal ability. Similar effects were specific tissue environments, in which infection and disease
also seen for CD8+ T cells activated in vitro and cultured in can radically alter both the populations of immune cells within
glucose-poor conditions (Ho et al., 2015). Cytosolic branched tissues as well as gene-expression patterns with a given
chain aminotransferase (BCATc) is induced upon T cell activa- cell type.
tion and catalyzes the transamination of cytosolic leucine (Ana- In the case of macrophages, the steady-state populations
nieva et al., 2014). Loss of BCATc in CD4+ T cells resulted in are derived from primitive erythro-myeloid progenitor cells
an accumulation of cytosolic leucine, a known activator of during fetal development and/or monocyte-derived macro-
mTORC1, after activation and promoted increased mTORC1 phages that originate from hematopoietic stem cells (Ginhoux

Immunity 46, May 16, 2017 723


Immunity

Review

eases, including cardiovascular, metabolic, neurodegenera-


tive, and neoplastic diseases. Therefore, dissecting the full
complement of signals that modulate and maintain tissue-resi-
dent macrophage gene expression may yield insight into novel
stimuli that drive human diseases.
Similar to tissue-resident macrophages, recent studies have
identified Trm cells that reside in peripheral tissues without recir-
culation that are critical contributors to host defense upon rein-
fection (Jiang et al., 2012; Schenkel et al., 2013, 2014; Iijima
and Iwasaki 2014; Ariotti et al., 2014). Both CD8+ and CD4+
Trm cells have been identified and exhibit important sentinel
functions that drive recruitment of circulating adaptive immune
cells as well as unique niche-driven roles in the case of CD4+
Trm cells in the female reproductive tract (FRT) (Schenkel
et al., 2014; Iijima and Iwasaki 2014; Ariotti et al., 2014). Due to
the recent identification of Trm cells, only a small number of
Trm cell populations have been profiled for gene expression
from different tissues. However similar to tissue-resident macro-
phages, these studies show tissue-specific gene expression in
addition to the core Trm cell gene signature necessary for
long-term survival and function in non-lymphoid tissues (Mackay
et al., 2013). In the case of T cells, it remains to be seen whether
Figure 3. Effect of Extrinsic Metabolism/Metabolites on Epigenome there are essential tissue-specific gene programs beyond the
of Macrophages and T Cells core Trm cell signature currently identified. Further studies char-
Activated immune cells initiate rapid metabolic and epigenetic changes that
are responsive to additional stimuli encountered in peripheral tissues. Migra-
acterizing populations from other tissues where Trm cells have
tion to peripheral tissues submit immune cells to additional challenges such as been identified, coupled with analysis of enhancer landscapes
changes in nutrient availability and tissue-specific signals, as well as alter the and chromatin dynamics, will provide clarity as to whether there
metabolic pathways that are utilized by the cell in situ. Cells that enter the gut
are additional tissue-specific functions of Trm cells similar to the
microenvironment must contend with microbiome-derived metabolites, such
as butyrate, that has been shown to inhibit HDACs and modulate macrophage CD4+ Trm cells in the FRT and hint at tissue-specific signals that
and T cell differentiation and function. Similarly, immune cells that infiltrate may regulate these programs. Furthermore, clarifying our under-
tumors encounter a challenging environment with increased competition for standing of Trm cell biology may contribute to our understanding
glucose, higher concentrations of lactate, and reduced oxygen levels.
Reduced glucose availability could impair glycolytic metabolism, reduce of a diverse subset of human diseases in which memory T cells
acetyl-CoA levels, and inhibit the acetylation of histones necessary for gene are thought to participate.
expression. Increased lactate concentrations may inhibit HDAC activity, Tissue environment has emerged as an important determi-
further dysregulating the epigenetic landscapes of tumor-infiltrating immune
nant of tissue-specific macrophage gene expression pro-
cells. These examples highlight the possible effects of extrinsic signals on
epigenetic enzymes and support a role for metabolites as integrators of cell- grams. Primitive macrophages acquire tissue-specific gene
intrinsic and -extrinsic signals for the modulation of gene expression. signatures soon after entering developing organs in the mouse
embryo (Matcovitch-Natan et al., 2016; Mass et al., 2016).
et al., 2016; Schulz et al., 2012). In addition to playing general Plasticity in the tissue-specific program of adult macrophage
roles in innate immunity, each tissue-resident macrophage is gene expression was demonstrated by transfer of peritoneal
specialized to support tissue-specific homeostatic functions macrophages to the alveolar air spaces of the lung, which
(Wynn et al., 2013). For example, microglia, the main resident resulted in substantial reprogramming of gene expression to
macrophage population of the central nervous system, secrete an alveolar macrophage-like pattern (Lavin et al., 2014).
factors that modulate neurogenesis and participate in the Conversely, transfer of microglia or peritoneal macrophages
refinement and elimination of synapses, while resident perito- to an in vitro environment resulted in loss of expression of a
neal macrophages play roles in gut immunity. Consistent with large fraction of the genes that are specific for each cell type
this functional specialization, different tissue-resident macro- (Gosselin et al., 2014). Collectively, these observations imply
phages express tissue-specific genes in addition to genes that each tissue environment provides signals that instruct
that enable core macrophage functions, such as phagocytosis entering macrophages to acquire tissue-specific programs of
and pathogen surveillance (Gautier et al., 2012). Comparing gene expression (Figure 3). However, the identities of the
microglia to peritoneal macrophages, for example, nearly signaling molecules that play these instructive roles are for
2,000 mRNAs are more than 16-fold differentially expressed the most part unknown.
that are enriched for functional annotations related to brain ho- In the case of T cells, it remains to be determined whether
meostasis and gut immunity, respectively (Gosselin et al., tissue environment directly defines the residency characteris-
2014). These differences in gene expression are associated tics of each Trm cell population and whether any level of plas-
with corresponding differences in enhancer landscapes (Gos- ticity exists in Trm cell populations. However, a number of
selin et al., 2014; Lavin et al., 2014). While tissue macrophages studies provide a hint that local tissue environment provides
normally serve adaptive functions, dysregulation of macro- essential cues that regulate T cell responses. For example,
phage activities contributes to a diverse range of human dis- T cell-intrinsic expression of prolyl hydroxylases (PHD),

724 Immunity 46, May 16, 2017


Immunity

Review

oxygen-sensing negative regulators of HIF TFs, limited the in- macrophages and T cells. Many questions remain to be
duction of Th1 cell responses, promoted Treg cell induction, explored. Studies to date have focused analyses on particular
and suppressed CD8+ effector function specifically in the loci of interest, but have yet to define the broad effects metabo-
lung, suggesting that oxygen levels regulate T cell responses lite levels have on global epigenetic landscapes. Further, the
(Clever et al., 2016). Furthermore, PHD deletion in T cells abro- extent to which global changes in metabolic intermediates
gated the number of lung tumors in mice but did not alter the impact the epigenome in a gene-specific manner remains unde-
size of the subcutaneous tumor in a B16 model of secondary termined. It is likely that modulation of global metabolite levels
tumor colonization, indicating that oxygen sensing specifically drive broad effects that require additional mechanisms to target
restrained pulmonary T cell responses (Clever et al., 2016). In epigenetic modifications to specific loci. Moreover, defining the
line with these observations, the gut microenvironment, which compartmentalization of particular metabolites in relation to
contains diet-derived antigens, commensal microbes and their epigenetic ‘‘writers’’ and ‘‘erasers’’ may clarify the impact of
metabolic byproducts, and local inflammatory signals, induces macrophage and T cell metabolism on epigenetic landscapes
Treg cell differentiation through several mechanisms and mod- after activation. In sum, there is an enticing link between cell-
ulates immune responses (Mucida et al., 2005; Sun et al., 2007; intrinsic and -extrinsic metabolites and gene expression with
Arpaia et al., 2013; Atarashi et al., 2011). As the field moves to sparse experimental evidence of molecular mechanisms result-
interrogate signals critical for T cell responses to understand ing in functional consequences in immune cells that warrant
Trm cell ontogeny and function, it is likely that microenviron- further exploration.
mental cues may regulate or play a role in T cell metabolism
and epigenetics (Figure 3). ACKNOWLEDGMENTS
A reasonable assumption is that many of the factors that regu-
late tissue-specific macrophage phenotypes are classical Funding of this work provided by the NIH (AI072117 and AI067545 for A.W.G.
signaling molecules that bind to specific receptors that in turn and DK091183 and NS096170 for C.K.G.). C.K.G. was also supported by a Le-
ducq Transatlantic Network Grant and the Ben and Wanda Hildyard Chair in
regulate gene expression. For example, local retinoic acid has Hereditary Diseases. We thank Nathan Spann and Kyla Omilusik for critical
been suggested to be a key regulator of peritoneal macrophage reading of the manuscript.
phenotypes by inducing expression of GATA6 in a retinoic acid-
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