T-cell metabolism in autoimmune disease

To protect the host from infections and malignancies, immune cells need to respond
promptly to antigens and danger signals, including massive expansion of T cells and
B cells, migration of cells to relevant tissue sites, and synthesis of cytokines and
effector molecules. Accordingly, immune stimulation imposes considerable demands for
energy and biosynthetic precursors. Lymphocytes fulfill these demands through swift
metabolic changes and rapidly generate energy and building blocks 4],5] (Figure 1). During their life cycle, lymphocytes transition between periods of rest and activity,
enforcing great flexibility in metabolic adaptations. Naïve and effector T cells differ
greatly in their energy needs and in the means to generate energy 6] (Table 1). Distinct T-cell subsets display unique metabolic programs, and data from metabolomics
studies and real-time bioenergetics analyses support the concept that wide variations
exist between CD4 and CD8 T cells 7], and between naïve, memory and effector T-cell subpopulations 8],9]. Environmental conditions, such as transitioning from normoxia to hypoxia, may impose
additional needs to adapt metabolic programs 10],11]. In essence, each T-cell subset has its very own metabolic profile.

Figure 1. Metabolic pathways match T cells’ functional demands. Schematic diagrams of metabolic pathways employed by T cells at different stages
of activation and differentiation. Dominant pathways are indicated as red cascades.
Blue arrows show pathways that are used at a steady level, and dashed arrows indicate
pathways that might be utilized but are insufficiently investigated. (Left) Resting
lymphocytes generate energy from glucose, fatty acids and amino acids. Most ATP is
produced in mitochondria by fermentation of acetyl-coenzyme A (CoA) in the tricarboxylic
acid (TCA) cycle and oxidative phosphorylation (OXPHOS). (Middle) Effector lymphocytes
(activated lymphocytes) swiftly and massively upregulate glycolysis and glutaminolysis,
while keeping the TCA cycle low. These cells switch lipid metabolism from beta-oxidation
towards fatty acid synthesis (lipogenesis). (Right) Memory lymphocytes mainly use
beta-oxidation to support their energy needs. 3PG, 3-phosphoglycerate; FFA, free fatty
acid; G-6-P, glucose-6-phosphate; NADPH, nicotinamide adenine dinucleotide phosphate;
PPP, pentose phosphate pathway; R-5-P, ribose 5-phosphate.

Table 1. Dominant metabolic pathways in resting and activated T cells

Table 2. Disease-specific metabolic abnormalities in rheumatoid arthritis and systemic lupus
erythematosus

Pathogenic T-cell populations can be expected to display metabolic and energy signatures.
Human autoimmune diseases typically proceed over decades and involve robust memory
responses 12]. Disease-relevant T cells depend on long-lasting energy supply. Vice versa, the metabolic status of the cell affects its specification and lineage commitment
and thus greatly influences the representation of functional effector cells in the
host’s immune system.

As an overarching rule, activated effector T cells are anabolic, employing primarily
glucose as their carbon source and utilizing glycolysis for fast access to adenosine
triphosphate (ATP). Memory cells are catabolic, able to metabolize fatty and amino
acids in addition to glucose, and depend on oxidative phosphorylation (OXPHOS) to
generate ATP 9] (Table 1). T cells and B cells seem to have evolved distinct approaches to generate energy
and macromolecules 13]. Upon stimulation, B cells proportionally increase lactate production and oxygen
consumption, optimizing the use of cytoplasmic glycolysis and mitochondrial energy
generation. In contrast, T cells tune down their glycolytic flux when resting and
disproportionally increase this pathway when encountering antigen. B cells thus thrive
in different microenvironments than T cells. T cells effectively utilize glucose via
glycolysis, glutamine via glutaminolysis and fatty acid via beta-oxidation, to refill
the tricarboxylic acid (TCA) cycle and fuel OXPHOS. We will briefly review the major
metabolic pathways to provide the appropriate context to compare the metabolomics
of normal and dysfunctional immune responses.

Glucose and glycolysis

Glucose serves as the primary source for the generation of ATP in the immune system,
and is essential for both resting and activated lymphocytes 14] (Figure 1). Nonactivated T cells and B cells predominantly oxidize glucose-derived pyruvate
in the TCA cycle and access lipids and amino acids as needed. The TCA cycle generates
nicotinamide adenine dinucleotide and reduced flavin adenine dinucleotide used to
fuel OXPHOS, an oxygen-dependent process in mitochondria that is highly efficient
in producing ATP. The end product of glycolysis, pyruvate, is imported into the mitochondria,
decarboxylated to acetyl-coenzyme A (CoA), and then condensed with oxaloacetate to
form citrate. Citrate can be exported from the mitochondria via the malate–citrate
shuttle system and used as a substrate for ATP citrate lyase. ATP citrate lyase catalyzes
the formation of acetyl-CoA and oxaloacetate from cytosolic citrate and CoA in the
presence of ATP. Accordingly, ATP citrate lyase serves as a cross-link between glucose
and fatty acid metabolism.

Upon recognition of foreign antigen and receipt of appropriate stimulatory signals,
T cells become activated and profoundly shift their metabolic program towards aerobic
glycolysis for ATP generation, which is less efficient but fast in providing the needed
energy. During glycolysis, a molecule of glucose is broken down into two molecules
of pyruvate, while yielding two molecules of ATP. Activated T cells convert pyruvate
into lactate rather than acetyl-CoA, even in the presence of sufficient oxygen, a
process known as aerobic glycolysis or the Warburg effect.

Mechanistically, upregulation of the transcription factor c-Myc is critical in boosting
activation-induced glycolysis 15]. c-Myc-dependent transcription directly targets several glycolytic enzymes, but is
not essential for fatty acid oxidation and OXPHOS. c-Myc target genes include glucose
transporter 1 (Glut1), the main glucose transporter in lymphocytes. Glut1 is not expressed at significant
levels on the surface of resting T cells, but is rapidly translocated to the plasma
membrane through the Akt signaling pathway, which also increases glycolysis by promoting
the activities of the rate-limiting glycolytic enzymes hexokinase and phosphofructokinase.
Glut1 induction greatly eases the delivery of glucose to T cells, and is considered
an essential step in supporting T-cell responsiveness.

Macintyre and colleagues identified Glut1 as being selectively essential for T-cell activation 16]. Glut1 deficiency severely impaired T-cell glucose metabolism and decreased effector T-cell
differentiation. On the contrary, regulatory T cells were functionally unaffected
and able to suppress inflammation regardless of Glut1 expression. Glut1-dependent
glycolytic reprogramming has also been implicated in T-cell helper function in antibody
production 13].

Although glycolysis provides less ATP than OXPHOS, favoring glycolysis provides T
cells with a means of generating the biosynthetic precursors that are required for
the synthesis of amino acids, nucleic acids and lipids (Figure 1). Glucose is therefore the optimal energy carrier for T cells and their functionality
is closely connected to how they access and break down this carbohydrate. B cells
require glucose not only as a source of ATP, but rely on glucose for de novo lipogenesis 17]. The reliance on glucose as a supplier of biosynthetic precursors predicts that the
level of glycolytic activity might directly influence the ability of activated T cells
to become either effector or long-lived memory cells 18]. Memory CD8⁺ T cells possess a markedly increased mitochondrial respiratory capacity when compared
with effector T cells 19], implicating OXPHOS as their major energy source.

In essence, T cells depend on glycolysis to support their unique demands for rapid
expansion and differentiation into distinct effector populations and have remarkable
plasticity to match metabolic and functional activities.

Glutamine and glutaminolysis

Besides glucose, amino acids are key nutrients for T cells because they can serve
both as a fuel source and as a pool of biosynthetic precursors for protein and nucleic
acid biosynthesis (Figure 1). T-cell activation imposes acute and delayed demands for protein synthesis. Elegant
studies have implicated amino acid transporters as absolute requirements for T cells
to adequately respond to antigenic challenge and to undergo clonal expansion and effector
differentiation 5]. Specifically, loss of the System L transporter Slc7a5, which mediates uptake of
large neutral amino acids, prevents the proliferation and differentiation of CD4⁺ and CD8⁺ T cells, while leaving the ability of CD4⁺ T cells to differentiate into regulatory T cells unaffected. Slc7a5-null T cells
fail to increase glutamine and glucose uptake and do not switch to aerobic glycolysis
after T-cell receptor stimulation. Cutting the supply of amino acids results in insufficient
activation of the amino acid monitor mammalian target of rapamycin complex 1 (mTORC1),
which is required for the differentiation of CD4⁺ cells into T-helper (Th)1 and Th17 subsets, while suppressing the differentiation
of regulatory FoxP3⁺ T cells 20]. mTORC1 has also been implicated in regulating the differentiation and migratory
ability of CD8⁺ cytotoxic T cells 21].

Among the amino acids, glutamine appears to be particularly important. T-cell activation
induces a substantial increase in the import of glutamine, but not glutamate 22]. T cells consume glutamine at rates comparable with or even higher than glucose 23]. During glutaminolysis, the amino acid is diverted into metabolic intermediates,
such as pyruvate and glutamate. Scientists have long known about the absolute requirement
for glutamine in proliferating T cells and have supplemented tissue culture media
for T-cell cultures with glutamine.

Recent studies by Nakaya and colleagues have clarified some of the contributions that
glutamine makes to T-cell immunity 24]. CD4 T cells uptake glutamine through the ASC amino-acid transporter 2 (ASCT2) and
this process influences the development of proinflammatory Th1 and Th17 cells in vitro and in vivo. Th2 and regulatory T-cell-dependent immune responses are unaffected by the genetic
ablation of ASCT2. Activated ASCT2^?/? T cells also have reduced glucose uptake, lactate production and oxygen consumption,
suggesting that glutamine has a key regulatory role in how T cells respond to abrupt
changes in their metabolic needs.

In addition to serving as a basic building block for protein synthesis, glutamine
contributes to other processes important for proliferating T cells, including fatty
acid synthesis, nucleotide synthesis and redox control. In activated lymphocytes,
citrate derived from glycolytic pyruvate is exported out of the mitochondria and used
in lipid synthesis. Glutamine-derived ?-ketoglutarate contributes to the production
of citrate by forward flux through the TCA cycle and malic enzyme-dependent production
of pyruvate 25], thus replenishing TCA cycle intermediates that are otherwise extracted for biosynthesis
in a process named anapleurosis. Citrate can then be used for the production of acetyl
groups for fatty acid synthesis. This pathway allows T cells to utilize glucose-derived
citrate to leave the mitochondria. Also, ?-ketoglutarate can provide precursors for
polyamine synthesis, indispensable for nucleotide synthesis. Finally, glutamate, the
first product of glutamine oxidation, serves as a metabolic nexus for the synthesis
of glutathione, critically influencing the redox status of lymphocytes.

Lipid metabolism

The key role of glucose and glutamine in sustaining cell growth, proliferation and
effector function of T cells is undebated. Less is known about fatty acid metabolism
and how it regulates T-cell fate and function (Figure 1, Table 1). In this context, it is important to consider kinetics of cellular responses, in
that glucose and glutamine are rapidly available and are easy to metabolize. Fatty
acids may be more important for long-term energy storage. As signaling molecules and
membrane building blocks, they play a compulsory role in the cell’s life cycle. Like
few other cell types, T cells need to be able to abruptly transition from quiescence
to massive expansion. Accordingly, they switch their lipid metabolism from energy
generation through fatty acid oxidation to fatty acid biosynthesis for membranes and
signaling molecules 26] (Figure 1). During steady state, both naïve and memory T cells catabolize fatty acids through
beta-oxidation into acetyl-CoA, which fuels the TCA cycle to provide most of the metabolic
support for basic cellular functions 27]. After activation, beta-oxidation is minimized while other metabolic pathways, including
glycolysis and glutaminolysis, increase. Lipids, such as phospholipids, glycolipids
and cholesterol, are the most abundant molecular species within cell membranes. Lymphocytes
are equipped with the enzymatic machinery to utilize acetyl-CoA and build complex
fatty acids. Expression of enzymes needed for fatty acid metabolism is markedly upregulated
post-stimulation, including the two key rate-limiting enzymes fatty acid synthase
and stearoyl-CoA desaturase-1 26]. T-cell activation is also associated with prompt induction of long-chain acyl-CoA
synthetases and lysophosphatidylcholine acyltransferases, known to catalyze the formation
of fatty acyl-CoA. Notably, removal of the stimulus in proliferating T cells results
in reversal of the lipid metabolism to resting state conditions and the immediate
hold of proliferation 28]. Accordingly, CD8 T cells with a defect of de novo lipogenesis fail to undergo T-cell expansion, unless they are supplied with exogenous
fatty acids 29]. In essence, proliferating lymphocytes appear to draw on external and internal lipid
sources to satisfy their enormous need for membrane building blocks.

Lipids integrated into membranes have a major influence on how T cells function. Lipid
rafts (also called lipid microdomains), which act as platforms for propagation of
signal transduction cascades, are composed primarily of phospholipids, sphingolipids
and cholesterol. Phospholipids are rich in unsaturated acyl chains and tend to pack
loosely into a liquid-disordered phase. Such membrane domains are considerably more
fluid, allowing rapid lateral movement within the lipid bilayer. By contrast, sphingolipids
have long and largely saturated acyl chains and easily pack tightly into a bilayer.
Adding cholesterol to the acyl chains further stabilizes the membranes. Cholesterol-containing
sphingolipid microdomains therefore present as a liquid-ordered phase. Miguel and
colleagues have examined the membrane lipid order in T lymphocytes using a fluorescent
lipid probe to distinguish liquid-ordered (raft) and liquid-disordered (nonraft) membranes
30]. They found proliferative activity closely correlated with the degree of membrane
lipid order. High lipid-order CD4 T cells proliferate robustly to T-cell receptor
activation, while intermediate-order cells have moderate proliferative ability and
low-order T cells have literally no response. Remarkably, different cytokine-producing
cells fall into distinctive membrane lipid-order populations; for example, interferon
gamma-producing CD4 T cells accumulate among intermediate lipid-order populations,
whereas interleukin (IL)-4-producing CD4 T cells are localized within the high-order
populations. Pharmacologic manipulation of membrane order by adding 7-ketocholesterol
and cholesterol into the culture media, which has been shown to reduce lipid order,
inhibits CD4 T-cell proliferation and IL-2 production.

Lipid metabolism is thus critically important in determining access to stored energy,
but even more relevant by altering the composition of cellular membranes.