Download during T Lymphocyte Activation Coordinately Regulated by ERK

Glutamine Uptake and Metabolism Are
Coordinately Regulated by ERK/MAPK
during T Lymphocyte Activation
This information is current as
of May 8, 2017.
Erikka L. Carr, Alina Kelman, Glendon S. Wu, Ravindra
Gopaul, Emilee Senkevitch, Anahit Aghvanyan, Achmed M.
Turay and Kenneth A. Frauwirth
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 185:1037-1044; Prepublished online 16
June 2010;
doi: 10.4049/jimmunol.0903586
http://www.jimmunol.org/content/185/2/1037
The Journal of Immunology
Glutamine Uptake and Metabolism Are Coordinately
Regulated by ERK/MAPK during T Lymphocyte Activation
Erikka L. Carr, Alina Kelman, Glendon S. Wu, Ravindra Gopaul, Emilee Senkevitch,
Anahit Aghvanyan, Achmed M. Turay, and Kenneth A. Frauwirth
A
ctivation of a T lymphocyte induces cell growth, proliferation, and cytokine production, placing significant
energetic and biosynthetic demands on the cell. In order
for the cell to meet these demands, increased uptake and metabolism of nutrients must occur. This includes large changes in amino
acid metabolism (1–6). In addition to serving as the basic building
blocks of protein synthesis, amino acids contribute to many processes critical for growing and dividing cells, including nucleotide
synthesis, energy metabolism, and redox control. Several genes
associated with amino acid transport and amino acid biosynthesis
are induced under starvation conditions in various cell types, including T cells (7–13). However, although amino acids are the
basic building blocks of protein synthesis and serve as substrates
for many other metabolic processes, the regulation of amino acid
use during T cell activation is poorly understood.
One potentially key amino acid for T cells is glutamine. Glutamine is the most abundant amino acid in serum, making it a
readily available resource, and is involved in numerous processes
important for lymphocyte activation (14, 15). Glutamine serves as
an amine group donor for nucleotide synthesis, and glutamate (the
first product of glutamine metabolism) is a metabolic nexus, playing
direct roles in amino acid and glutathione synthesis. Glutamate can
also be converted into the Krebs cycle intermediate a-ketoglutarate,
Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research
Institute, University of Maryland, College Park, MD 20742
Received for publication November 5, 2009. Accepted for publication May 13, 2010.
This work was supported by funds from the National Institutes of Health (Grant K01
CA092156) and the Leukemia Research Foundation (to K.A.F.).
Address correspondence and reprint requests to Dr. Kenneth Frauwirth, University of
Maryland, 2113 Microbiology Building, College Park, MD 20742. E-mail address:
[email protected]
Abbreviations used in this paper: E.C., Enzyme Commission; GCN2, general control
2; GDH, glutamate dehydrogenase; GOT, glutamic-oxaloacetic transaminase; GPT,
glutamic-pyruvic transaminase; SNAT, sodium-dependent neutral amino acid transporter; TOR, target of rapamycin.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903586
providing a two-step pathway for glutamine to enter energy metabolism. Lymphocytes and thymocytes consume glutamine at rates
comparable to, or even higher than, glucose consumption (1–3),
and mitogen-induced T cell proliferation and cytokine production
in culture require high levels of glutamine (16–19). Thus, pathways
of glutamine use may serve as novel targets for immune modulation.
To investigate the role of glutamine in T cell function, we examined the changes in glutamine use during activation of purified
T cells. We found that T cells are highly sensitive to glutamine
levels, and this sensitivity is specific in that glutamine cannot be
replaced by metabolic precursors or products. T cell activation
leads to a selective increase in glutamine import, and this is
reflected by increased expression of glutamine transporters. Activities of enzymes involved in glutamine metabolism are also
increased during T cell activation, likely allowing enhanced use of
glutamine as a substrate for Krebs cycle metabolism.
Materials and Methods
Abs and reagents
Anti-CD3 (mAb 145-2C11) and anti-CD28 (mAb 37.51) Abs, control hamster IgG, and PE-labeled anti-Thy1.2, anti-CD69, anti-CD25, and anti-CD98
Abs were purchased from eBioscience (San Diego, CA). HRP-conjugated
anti-mouse IgG and anti-rabbit IgG were from Jackson ImmunoResearch
Laboratories (West Grove, PA). The MEK inhibitor PD98059 was purchased from BIOMOL (Plymouth Meeting, PA) and used at a concentration
of 40 mM. ADP, lactate dehydrogenase (Enzyme Commission [E.C.]
1.1.1.27), and malate dehydrogenase (E.C. 1.1.1.37) were purchased from
Calbiochem (San Diego, CA). 1-Bromododecane, pyridoxal phosphate,
NADH, triethanolamine-HCl, hydrazine dihydrochloride, a-ketoglutarate,
glutaminase (E.C. 3.5.1.2), glutamate dehydrogenase (GDH; E.C. 1.4.1.3),
glutamic-oxaloacetic transaminase (GOT; E.C. 2.6.1.1), and glutamicpyruvic transaminase (GPT; E.C. 2.6.1.2) were purchased from SigmaAldrich (St. Louis, MO). L-2,3,4-[3H]glutamine and L-2,3,4-[3H]glutamic
acid were from American Radiolabeled Chemicals (St. Louis, MO).
Animals
C57BL/6J mice (6-wk-old) were purchased from The Jackson Laboratory
(Bar Harbor, ME). All of the mice were maintained in ventilated microisolator cages (Animal Care Systems, Centennial, CO) in the University of
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Activation of a naive T cell is a highly energetic event, which requires a substantial increase in nutrient metabolism. Upon stimulation, T cells increase in size, rapidly proliferate, and differentiate, all of which lead to a high demand for energetic and biosynthetic precursors. Although amino acids are the basic building blocks of protein biosynthesis and contribute to many other
metabolic processes, the role of amino acid metabolism in T cell activation has not been well characterized. We have found that
glutamine in particular is required for T cell function. Depletion of glutamine blocks proliferation and cytokine production, and this
cannot be rescued by supplying biosynthetic precursors of glutamine. Correlating with the absolute requirement for glutamine,
T cell activation induces a large increase in glutamine import, but not glutamate import, and this increase is CD28-dependent.
Activation coordinately enhances expression of glutamine transporters and activities of enzymes required to allow the use of glutamine as a Krebs cycle substrate in T cells. The induction of glutamine uptake and metabolism requires ERK function, providing
a link to TCR signaling. Together, these data indicate that regulation of glutamine use is an important component of T cell activation.
Thus, a better understanding of glutamine sensing and use in T cells may reveal novel targets for immunomodulation. The Journal
of Immunology, 2010, 185: 1037–1044.
1038
Maryland animal facility. Animals received humane care in compliance
with the Guide for the Care and Use of Laboratory Animals (Institute of
Laboratory Animal Resources). All of the mice were euthanized by carbon
dioxide inhalation, as recommended by the American Veterinary Medical
Association Panel on Euthanasia.
T cell purification
Murine T cells were purified from spleens using the EasySep negativeselection system (Stem Cell Technologies, Vancouver, British Columbia,
Canada) according to the manufacturer’s protocol. Purified T cells were
generally .95% Thy1-positive, as determined by flow cytometry.
Cell lines and culture
The murine EL-4 thymoma cell line was purchased from American Type
Tissue Collection (Manassas, VA). All of the cells were maintained in
RPMI 1640 medium (Mediatech, Manassas, VA) supplemented with
10% FBS (Hyclone, Logan, UT), penicillin/streptomycin, 10 mM HEPES
buffer, and 55 mM 2-ME, with or without 2 mM glutamine, at 37˚C in
a 5% CO2 atmosphere. For glutamine withdrawal experiments, the FBS
was replaced with 10% dialyzed FBS (J R Scientific, Woodland, CA). For
amino acid uptake experiments, glutamine-deficient and glutamatedeficient RPMI 1640 media were produced in-house, using the standard
RPMI 1640 formulation but omitting either glutamine or glutamic acid.
Anti-CD3 and anti-CD28 Abs were covalently attached to tosyl-activated
Dynabeads (Invitrogen, Carlsbad, CA) following the manufacturer’s
instructions, using 75 mg/ml of each Ab. Beads were mixed with T cells
at a 3:1 ratio of beads/cells and incubated at 37˚C for the desired time. For
unstimulated samples, T cells were mixed with control hamster IgG-linked
Dynabeads and were then either used immediately or cultured in parallel
with stimulated samples.
Proliferation and cytokine assays
Unfractionated C57BL/6J splenocytes were stimulated with soluble antiCD3 Ab. Proliferation after 3 d of stimulation in vitro was determined by
[3H]thymidine incorporation. Cells were pulsed for 6–8 h with 1 mCi per
well of [3H]thymidine (MP Biomedicals, Irvine, CA), transferred to glass
fiber filters with a 96-well cell harvester (Tomtec, Hamden, CT), and
analyzed by liquid scintillation using a 1450 MicroBeta TriLux scintillation counter (Wallac, Turku, Finland). IL-2 and IFN-g levels in 36 h
stimulation supernatants were determined using a sandwich ELISA.
Primary and biotinylated secondary anti-cytokine Abs and recombinant
cytokine standards were purchased from eBioscience and used at the concentrations recommended by the manufacturer. Alkaline phosphataseconjugated avidin was purchased from Jackson ImmunoResearch Laboratories and used at a 1:3000 dilution. Colorimetric alkaline phosphatase substrate was purchased from Sigma-Aldrich and used at a concentration of 1
mg/ml in 10% diethanolamine buffer, and quantification was performed on
a VersaMax spectrophotometer (Molecular Devices, Sunnyvale, CA). Data
were analyzed using SoftMax Pro software (Molecular Devices). All of the
data are presented as the mean of triplicate samples 6 SD.
Amino acid uptake
Glutamine and glutamate uptake were measured using a modification of the
amino acid uptake protocol of Edinger and Thompson (20). Briefly,
unstimulated or stimulated T cells were resuspended at a concentration of
2.5 3 107cells per milliliter in serum-free RPMI 1640 lacking the amino
acid being assayed (“uptake medium”). An aliquot of 2.5 3 106 cells was
added to the top layer of a 0.7-ml microfuge tube containing 25 ml 8%
sucrose/20% perchloric acid (bottom layer), 200 ml 1-bromododecane
(middle layer), and 50 ml uptake medium containing 45 nmol L-2,3,4[3H]glutamine or L-2,3,4-[3H]glutamic acid. Cells were allowed to take up
radiolabeled amino acid for 10 min at room temperature and then spun
through the bromododecane into the acid/sucrose, stopping the reaction
and separating the cells from unincorporated 3H-labeled amino acid. The
perchloric acid/sucrose/T cell layer was removed and analyzed by liquid
scintillation using a 1450 MicroBeta TriLux scintillation counter, and cellfree controls were subtracted. In comparisons of glutamine and glutamate
uptake, final counts were adjusted to account for the higher sp. act. of [3H]
glutamic acid (50 versus 44 Ci/mol for [3H]glutamine). Data points for all of
the analyses are presented as the mean of triplicate samples 6 SD.
Flow cytometry
Surface levels of activation markers were determined on unstimulated and
stimulated purified T cells by flow cytometry. After harvest, cells were
washed once in PBS and resuspended in staining buffer (1% BSA and 0.01%
sodium azide in PBS) containing PE-conjugated anti-CD69, anti-CD25, or
anti-CD98 Abs for 30 min at 4˚C. Fluorescence values were then read on
a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Forward
and side scatter gates were used to exclude dead cells. Data from ∼104 live
cells were analyzed using CellQuest software (BD Biosciences).
Analysis of cell-surface sodium-dependent neutral amino acid
transporter 2
Unstimulated or stimulated T cells were surface-biotinylated with EZ-Link
Sulfo-NHS-Biotin (Pierce, Rockford, IL) according to the manufacturer’s
instructions. Cells were lysed in immunoprecipitation lysis buffer (0.5%
Nonidet P-40, 10 mM Tris-HCl, and 140 mM NaCl [pH 8.0]) supplemented
with 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF, and Complete Mini
protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland) and
precleared overnight at 4˚C with protein G-agarose (Sigma-Aldrich). Precleared lysates were incubated with anti–sodium-dependent neutral amino
acid transporter (SNAT2) Ab (Santa Cruz Biotechnology, Santa Cruz, CA)
and protein G-agarose (Sigma-Aldrich) for 5 h at 4˚C, and immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. Blots
were probed with avidin–HRP (Pierce) and visualized using SuperSignal
West Pico chemiluminescence reagents (Pierce).
Real-time PCR
Total RNA was extracted from unstimulated or stimulated T cells using the
Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany). cDNA was
generated using the iScript reverse transcriptase kit (Bio-Rad, Hercules,
CA). Primers for real-time PCR were designed using Beacon Designer
software (Premier Biosoft International, Palo Alto, CA), except for glutaminase (21), and were as follows: SNAT1, forward 59-TTACCAACCATCGCCTTC-39, reverse 59-ATGAGAATGTCGCCTGTG-39; SNAT2, forward
59-GGTATCTGAACGGTGACTATCTG-39, reverse 59-TCTGCGGTGCTATTGAATGC-39; glutaminase, forward 59-GCACTACACTTTGGACACCA39, reverse 59-TAGCAACCCGTCGAGATT-39; 18S, forward 59-ATGCGGCGGCGTTATTCC-39; reverse 59-GCTATCAATCGTTCAATCCTGTCC-39.
Real-time PCR was performed in an iCycler iQ system (Bio-Rad) using iQ
Supermix reagents (Bio-Rad) and analyzed with MyiQ software (Bio-Rad).
Melt curve analysis was performed to confirm the presence of a single PCR
product for each reaction, and agarose gel electrophoresis was used to confirm
that PCR products were the expected sizes. Fold induction was calculated by
the DDCt method, using 18S rRNA as the reference. Data are presented as the
means of triplicate samples 6 SD.
Enzyme activity assays
The assay for glutaminase activity was adapted from Graham and Aprison
(22) and Ardawi and Newsholme (23). Resting or stimulated T cells were
lysed by sonication in 150 mM potassium phosphate buffer (an equimolar
mixture of K2HPO4 and KH2PO4), 50 mM Tris-HCl (pH 8.6), 1 mM
EDTA, and 1% Triton X-100 and assayed at a concentration of 5 3 106
cell equivalents per milliliter. The reaction was initiated by adding 15 ml
lysate to 85 ml reaction buffer (180 mM Tris-HCl [pH 9.4], 80 mM glutamine, 70 mM potassium phosphate buffer [an equimolar mixture of
K2HPO4 and KH2PO4], 28 mM NAD, 20 mM hydrazine dihydrochloride,
2 mM ADP, 0.05% Triton X-100, and 20 U/ml GDH), and the reaction was
carried out at 37˚C. GDH, GOT, and GPT activities were determined as
described by Ardawi and Newsholme (23), with slight modifications.
T cells were lysed by sonication in 50 mM triethanolamine-HCl, 1 mM
EDTA, 5 mM MgCl2, and 30 mM 2-ME, adjusted to pH 7.5 with KOH,
and assayed at a concentration of 5 3 106 cell equivalents per milliliter.
Assay conditions for GDH were: 70 mM Tris-HCl (pH 7.6), 100 mM
ammonium acetate, 0.2 mM NADH, 2 mM ADP, and 8 mM a-ketoglutarate. Assay conditions for GOT were: 75 mM potassium phosphate (equimolar mixture of K2HPO4 and KH2PO4), 0.2 mM pyridoxal phosphate,
0.2 mM NADH, 40 mM a-ketoglutarate, 12.5 U/ml malate dehydrogenase,
and 438 mM aspartate. Assay conditions for GPT were: 75 mM potassium
phosphate (equimolar mixture of K2HPO4 and KH2PO4), 0.2 mM pyridoxal phosphate, 0.2 mM NADH, 10 mM a-ketoglutarate, 3 U/ml lactate
dehydrogenase, and 1 M alanine. Reactions were initiated by addition of
a-ketoglutarate and were carried out at room temperature. Negative controls lacking a-ketoglutarate or cell lysate were run in parallel. For all
of the enzymes, activity was determined by the change in absorbance at
340 nm, using a VersaMax spectrophotometer and SoftMax Pro software
(Molecular Devices). OD readings were taken every 8–10 s for at least
20 min, and rate was calculated from the linear portion of the curve. A340
values were converted into substrate concentrations using Beer’s Law (A =
εLc, where A is absorption, ε is the extinction coefficient, L is the path
length, and c is concentration) and the extinction coefficient of NADH
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T cell stimulation
GLUTAMINE USE IN ACTIVATED T CELLS
The Journal of Immunology
(6.22 mmol21 cm21 l). The reported rates are representative of three independent experiments, and are averages of triplicate samples (6SD).
Statistical analysis
All of the statistical analyses were performed using Prism software, version
5 (GraphPad, San Diego, CA). The minimal level of confidence at which
experimental results were considered significant was p , 0.05. Statistical
significance between samples in proliferation assays and between glutamine and glutamate uptake was determined by two-way ANOVA with
Bonferroni posttest analysis. Statistical significance of glutamine uptake
was determined by one-way ANOVA with Bonferroni posttest analysis or
by two-tailed t test. Statistical significance of enzyme activity assays in
resting versus stimulated T cells was determined by two-tailed t test.
Results
T cell activation is absolutely dependent on extracellular
glutamine
FIGURE 1. T cell activation
requires high levels of glutamine. A,
C57BL/6J splenocytes were stimulated with titrated amounts of antiCD3 Ab in medium containing the
indicated concentration of glutamine. The standard glutamine concentration in RPMI 1640 medium
is 2 mM. Proliferation was measured
after 3 d of stimulation. p , 0.05,
means at a given Ab concentration
without a common letter differ from
each other and from 0 mM glutamine. Means with no letter do not
differ significantly from 0 mM glutamine. B and C, C57BL/6J splenocytes were stimulated for 36 h with
1 mg/ml anti-CD3 Ab in complete
(2 mM glutamine) or glutaminefree RPMI 1640 medium, and IL-2
(B) and IFN-g (C) levels were
measured in culture supernatants.
For resting samples, cells were
cultured in complete medium in the
absence of anti-CD3 Ab. All of
the results are representative of at
least three independent experiments.
pp , 0.01; pppp , 0.0001
possible that glutamine depletion has such a dramatic effect because there are insufficient levels of other amino acids to compensate. We therefore tested whether supplementing the medium
with glutamic acid, which is readily converted into glutamine,
could restore T cell proliferation in glutamine-free medium. As
shown in Fig. 2A, even medium supplemented with an extra 2 mM
glutamic acid was unable to support T cell proliferation. Similarly,
supplementing the medium with either 2 mM proline or 2 mM
asparagine, each of which can serve as a precursor to synthesize
glutamine, was unable to restore T cell proliferation in glutaminefree medium (Fig. 2B). Glutamate, asparagine, and proline were
also unable to substitute for glutamine in supporting cytokine production (data not shown). Thus, T cells specifically require glutamine for activation, consistent with previous studies using
mitogenic lectins to activate lymphocytes (1).
Proliferation and cytokine secretion are relatively late events in
T cell activation. To determine whether glutamine depletion also
inhibits earlier events, we examined the expression of T cell surface
markers after 24 h of stimulation (Fig. 3A–C). T cells stimulated
in complete or glutamine-free medium induced expression of
CD69, CD25, and CD98/4F2hc comparably, indicating that cells
are able to initiate activation events in the absence of glutamine.
However, cell growth was severely impaired in glutamine-free
medium, as seen by the reduced forward scatter values (Fig.
3D). Thus, T cells require glutamine for growth, cytokine secretion, and proliferation but not for expression of activation markers,
largely consistent with the findings of Hörig et al. (17).
T cell activation selectively increases glutamine uptake
The requirement for glutamine during T cell activation and the
inability of metabolic precursors to serve as substitutes suggested
that activated T cells would consume glutamine at a substantially
higher rate than resting cells. To test this, glutamine uptake rates in
unstimulated and 24 h-stimulated T cells were compared. We consistently saw a 5- to 10-fold induction of glutamine uptake in activated T cells relative to that of unstimulated controls (Fig. 4A).
Similar to previous findings with glucose uptake and metabolism
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To define the amino acid requirements for T cell activation, splenic
T cells were stimulated in tissue culture medium depleted of individual amino acids, and proliferation in culture was measured.
Not surprisingly, removal of any of the essential amino acids
(valine, leucine, isoleucine, phenylalanine, tryptophan, lysine,
threonine, and methionine), which cannot be synthesized by
mammalian cells, blocked T cell proliferation (data not shown).
However, we also found that several nonessential amino acids were
required for T cell proliferation and the removal of glutamine
consistently gave the strongest inhibition of any amino acid. Reducing glutamine levels by as little as 50% produced a detectable
impairment in T cell proliferation, and reduction below 10% of
normal culture levels led to an absolute proliferation block (Fig.
1A). Similarly, removal of glutamine blocked secretion of IL-2
(Fig. 1B) and IFN-g (Fig. 1C) by activated T cells. Depletion of
other amino acids had variable effects on cytokine production, but
glutamine was unique in being absolutely required for both IL-2
and IFN-g. Notably, no other single depletion inhibited IL-2 production by .50%, and the essential amino acid leucine was not
required for either IL-2 or IFN- g secretion (data not shown).
The concentration of glutamine in RPMI 1640 culture medium is
2 mM, which is .30% of the total amino acid pool. Thus, it is
1039
1040
GLUTAMINE USE IN ACTIVATED T CELLS
To distinguish these possibilities, we compared the changes in
glutamine and glutamate uptake during T cell activation. As seen in
Fig. 4C, import of glutamine was induced by T cell activation, but
glutamate uptake in activated T cells was comparable to uptake in
resting cells. Thus, activated T cells selectively enhance glutamine
uptake. Strikingly, the T lymphoma cell line EL-4 constitutively
imported glutamine at an even higher rate than activated T cells
(Fig. 4D) and had a similarly strong preference for glutamine over
glutamate (Fig. 4E).
T cell activation induces expression of glutamine transporters
(24), the enhanced glutamine uptake required CD28 costimulation
(Fig. 4B), implicating CD28 more broadly in metabolic control
of T cells.
The increased uptake by activated T cells could be specific to
glutamine or might be part of a general increase in amino acid use.
Glutamine uptake in T cells is regulated by ERK activity
FIGURE 3. Glutamine is not required for induction of early T cell activation markers. Purified C57BL/6J T cells were stimulated for 24 h with
immobilized anti-CD3 and anti-CD28 Abs in complete or glutamine-free
medium. A–C, Cells were stained with PE-conjugated anti-CD69 (A), antiCD25 (B), or anti-CD98 (C) Abs and analyzed by flow cytometry. For resting
samples, cells were cultured in complete medium in the presence of immobilized control hamster Abs. D, Forward scatter plots of the T cells analyzed
in A–C. Results are representative of three independent experiments.
The importance of glutamine for T cell function and the large
increase in glutamine uptake during activation suggested that
glutamine use might be regulated by TCR-initiated signals. TCR
ligation results in activation of multiple signal transduction pathways, including members of the MAPK family (26). The MAPK
family member ERK has been implicated in the control of SNAT
expression and transport activity in response to amino acid starvation (27, 28). We therefore tested whether ERK activity was
required for the increase in glutamine uptake during T cell activation. T cells were stimulated in the presence or absence of the
MEK inhibitor PD98059, which blocks ERK activation, and
glutamine uptake was determined. As shown in Fig. 6, 40 mM
PD98059 [a concentration that inhibits MEK1 and MEK2 but not
other kinases (29)] completely blocked the increased glutamine
uptake. This was not due to a global block in activation, because
PD98059 had little or no effect on the induction of several activation markers, including CD69 and CD98 (data not shown).
Thus, ERK acts downstream of TCR/CD28 signaling to positively
regulate glutamine uptake.
T cell activation induces enzymes involved in glutamine
metabolism
One possible explanation for the dependence of T cells on glutamine is that T cells use glutamine as a primary energy source.
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FIGURE 2. Biosynthetic precursors cannot substitute for glutamine in
T cell activation. C57BL/6J splenocytes were stimulated as in Fig. 1A, in
complete medium or glutamine-free medium, or in glutamine-free medium
supplemented with 2 mM glutamate (A), asparagine (B), or proline (B).
Proliferation was measured after 3 d of stimulation. All of the results are
representative of at least three independent experiments. pp , 0.05, different from glutamine-free; pppp , 0.001, different from glutamine-free.
The increase in glutamine uptake during T cell activation suggested
that expression of glutamine transporters was also induced. Although cells can import glutamine via multiple systems, the major
glutamine transporters are members of the SNAT family (25).
We therefore used quantitative real-time PCR to measure SNAT
mRNA expression levels during T cell activation. As shown in
Fig. 5A, stimulation of T cells through CD3 and CD28 induced
expression of SNAT1 and SNAT2, with SNAT1 being induced
more rapidly and to a higher level. mRNA expression peaked at
8 h and had begun to fall by 24 h. Thus, induction of glutamine
transporter gene expression is consistent with the increased glutamine uptake after T cell activation.
In addition to overall transporter levels, nutrient transport can
be regulated by cellular localization of the transporter proteins.
For SNAT1 and SNAT2, it has been found that the bulk of the
protein is located in intracellular vesicles (25), suggesting that
increased transport may be due to a combination of increased
protein expression and relocation of transporters from intracellular
stores to the cell surface. We examined surface levels of SNAT2
during T cell activation by biotinylation of total surface protein,
followed by immunoprecipitation of SNAT2 and detection by
avidin–HRP. Immediately after stimulation, there was a moderate
increase in surface SNAT2 levels, followed by a large increase
after 15–18 h (Fig. 5B). Stimulation also induced changes in the
pattern of surface proteins that associated with SNAT2 (seen as
coprecipitating bands), suggesting that glutamine transport may
be further regulated by proteins that interact with the transporters.
The Journal of Immunology
1041
Although T cells are highly glycolytic, most pyruvate is converted
to lactate rather than oxidized in the Krebs cycle (24, 30, 31). It has
been suggested that glutamine serves as an alternate Krebs cycle
substrate for lymphocytes (1, 3). We therefore examined the expression and activity of key enzymes in this metabolic pathway.
A major route of glutamine metabolism is initiated by the enzyme glutaminase, which hydrolyzes glutamine to produce glutamate. T cell activation induced glutaminase activity, consistent
with an important role for glutamine in T cell function (Fig. 7A). As
with glutamine uptake, glutaminase activity in EL-4 cells was
constitutively high (Fig. 7A). Glutaminase mRNA was also induced
early after T cell stimulation, peaking at 3 h (Fig. 7B). We have
found that IL-2 mRNA also becomes readily detectable at 3 h
poststimulation (data not shown), indicating that changes in glutamine metabolism occur with similar kinetics to other early T cell
activation events.
The entry point into the Krebs cycle for glutamine is aketoglutarate. We therefore examined the enzymes GDH, GOT,
and GPT, each of which converts glutamate to a-ketoglutarate.
GDH catalyzes the oxidative deamination of glutamate, producing
ammonia and reducing NAD+ to NADH. GOT and GPT transfer
the amine group from glutamate to oxaloacetate and pyruvate,
producing aspartate and alanine, respectively. The activities of
all three enzymes were induced by T cell activation, but GOT
activity was greater than the activities of GDH and GPT combined
(Fig. 7C). This is consistent with analysis of glutamine metabolism in lymphocytes, because ammonia generation appears limited
mainly to the initial step (deamidation to glutamate) and aspartate
is a major product (1, 3). Notably, the activity of glutaminase was
consistently lower than the activity of GDH or GOT, for both
resting and activated cells, indicating that glutaminase activity is
likely limiting for glutamine metabolic flux. EL-4 lymphoma
cells demonstrated constitutively high enzyme activities, with
GOT and GPT activities nearly 10 times the level found in activated T cells, but GOT remained the dominant enzyme (Fig. 7D).
As with glutamine uptake, induction of all four enzymes during
T cell activation was blocked by ERK inhibition (Fig. 7A–C).
Thus, ERK signaling serves as a common regulatory pathway
for both uptake and metabolism of glutamine in T cells.
Discussion
The role of metabolism as a regulator of cellular function has
recently attracted increasing attention. There is a growing sentiment that metabolic pathways do not merely respond to changing
conditions to maintain homeostasis but also are important participants in defining the activation state of a cell and can respond to
signal transduction events. Much of this renewed attention has
come from studies demonstrating that altered glucose and glutamine metabolism is important for tumor cell growth and survival
(32–35). However, rather than being limited to transformed cells,
similar changes in cellular metabolism are likely to be important
for any rapidly dividing cells, included activated T cells (36, 37).
To investigate the regulation of amino acid use by T cells, we
began by testing the importance of individual amino acids for T cell
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FIGURE 4. T cells selectively enhance
glutamine uptake during activation. A, Purified T cells were cultured for 24 h with either
control hamster IgG (rest) or anti-CD3 plus
anti-CD28 Abs (24 hr stim), and glutamine
uptake was measured. pppp , 0.001, different from resting. B, Purified T cells were
stimulated with either control hamster IgG
(rest), anti-CD3 Abs alone (CD3), or antiCD3 plus anti-CD28 Abs (CD3/28), and glutamine uptake was measured. C, Purified
T cells were stimulated as in A, and rates of
uptake of glutamine and glutamate were measured. pppp , 0.001, different. D, Constitutive glutamine uptake in EL-4 lymphoma
cells was compared with uptake in resting
and stimulated T cells. p , 0.01, means without a common letter differ. E, Rates of constitutive glutamine and glutamate uptake
were measured in EL-4 lymphoma cells.
ppp , 0.01, different from glutamate. All
of the results are representative of at least
three independent experiments, except for
B, which is representative of two independent
experiments.
1042
FIGURE 5. T cell activation induces expression of SNAT transporters.
A, Purified C57BL/6J T cells were stimulated with immobilized anti-CD3
and anti-CD28 Abs for the indicated times. SNAT1 and SNAT2 mRNA
levels were determined by quantitative real-time PCR. B, Purified T cells
were stimulated for the indicated times, and surface proteins were biotinylated prior to lysis. SNAT2 protein was precipitated from lysates with antiSNAT2 Ab, resolved by SDS-PAGE, and detected with avidin–HRP. The
predicted mobility of SNAT2 is indicated to the left. Results are representative of three (A) or two (B) independent experiments.
FIGURE 6. Glutamine uptake in activated T cells is regulated by ERK
signaling. Purified T cells were stimulated in the presence or absence of
the ERK inhibitor PD98059 (40 mM), and glutamine uptake was measured
as in Fig. 4. Results are representative of three independent experiments.
ppp , 0.01, different.
FIGURE 7. T cell activation induces enzymes involved in glutamine
metabolism. A, Purified T cells were cultured for 24 h with immobilized
control (rest) or anti-CD3 plus anti-CD28 (stim) Abs, in the presence or
absence of 40 mM PD98059 (stim + PD), and enzymatic activity of glutaminase was measured. Glutaminase activity in continuously growing EL-4
cells was also measured. B, T cells were cultured with control (rest) or
anti-CD3 plus anti-CD28 Abs for the indicated times, in the absence (stim)
or presence of 40 mM PD98059 (stim + PD), and glutaminase mRNA
levels were determined by quantitative real-time PCR. C, Purified T cells
were cultured as in A, and enzymatic activities of GDH, GOT, and GPT
were measured. D, Activities of GDH, GOT, and GPT in continuously
growing EL-4 cells were determined. p , 0.05, means without a common
letter differ. All of the results are representative of at least three independent
experiments.
bolism more generally, possibly serving as a point of coordination
for many metabolic pathways.
Glutamine is nonessential in the diet and is readily synthesized
by mammalian cells (and is used by many as a mechanism of
nitrogen excretion). Lymphocytes have been found to express
glutamine synthetase activity (38), suggesting that they are also
able to synthesize glutamine from glutamate. It is therefore unclear why T cells are unable to substitute other amino acids for
glutamine. One possibility is that transport of other amino acids is
insufficient to compensate for a lack of glutamine. We found that
T cells increase glutamine uptake 5- to 10-fold upon activation but
fail to increase glutamate import. This is consistent with studies of
the anionic amino acid transport system xc2, which appears to
have low activity in lymphocytes (39–41). Thus, even in the presence of high glutamate levels, T cells would be unable to import
enough to replace glutamine. In addition, import of a given amino
acid is frequently influenced by the intracellular concentration of
other amino acids (42). It is therefore possible that other amino
acids are unable to replace glutamine because T cells cannot import them efficiently in the absence of a glutamine supply.
An alternative (and complementary) possibility is that glutamine
is directly sensed in T cells, and so other amino acids cannot
substitute for it. Amino acid sensing by mammalian cells is still
poorly understood, and sensor pathways for glutamine have not
been well characterized. Two major signaling kinases involved in
amino acid sensing are the general control 2 (GCN2) kinase (43)
and the target of rapamycin (TOR, known as mTOR in mammalian cells) kinase (44). Although sensing by GCN2 has been
established for several amino acids in mammalian cells (45–48),
studies examining GCN2 responsiveness to glutamine have not
been reported. Investigation of glutamine sensing by mTOR has
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activation. We found that T cells have an absolute requirement for
a large external supply of glutamine and that even moderate
reductions within the normal physiological range impair T cell
function. Other amino acids, including direct biosynthetic precursors, cannot substitute for glutamine, indicating that the need for
glutamine is not simply due to insufficient levels of other nutrients
in tissue culture medium.
This dependence on glutamine is similar to results found for
unfractionated human and rodent lymphocytes stimulated with the
T cell mitogens Con A and PHA (16–19). However, in these
systems, T cells represent #50% of the total cell population. This
makes it difficult to assign metabolic effects directly to T cells,
because activated T cells may in turn regulate the metabolism of
other cells (e.g., via cytokines and/or direct cell–cell contact or by
changing the metabolite composition of culture medium). In addition, Con A and PHA likely stimulate cells by clustering large
numbers of surface receptors, including some that might not
normally be involved in T cell activation. Our results confirm the
importance of glutamine in a more defined system, using purified
T cells and specific stimulation via CD3 and CD28. Notably, we
were able to show a specific requirement for CD28 costimulation,
similar to previous studies of glucose metabolism (24). It thus
appears that CD28 is important for regulation of T cell meta-
GLUTAMINE USE IN ACTIVATED T CELLS
The Journal of Immunology
that the ERK signaling pathway is another important regulator of
cellular metabolism. ERK signaling is required for enhanced glutamine uptake and increased activity of key metabolic enzymes.
ERK activation has also been found to be involved in the induction
of system A transporter activity (mediated by expression of SNAT
transporter proteins) in response to amino acid starvation in human fibroblasts (27) and Chinese hamster ovary cells (28). We
have further found that ERK activity is required for TCR/CD28induced changes in glucose metabolism (A. Marko, R. Miller, and
K. Frauwirth, manuscript in preparation). Together, these results
implicate ERK as another central player in cellular metabolic
control.
Although ERK regulates glutamine uptake and metabolism in
a coordinated fashion, it appears that multiple pathways are likely
to be involved in these processes. Induction of glutaminase
mRNA requires ERK activity, indicating that ERK regulates
either transcription or stability of glutaminase mRNA. This differs
from the induction of glutaminase via c-Myc, which blocks expression of regulatory microRNAs but does not appear to increase
glutaminase mRNA itself (60). Thus, glutaminase expression is
regulated by at least two distinct mechanisms.
The reliance of activated T cells on glutamine metabolism is
strikingly similar to that of tumor cells and may represent a
common feature of all rapidly dividing cells (37, 61). However, the
pattern of enzyme activity that we observed (high GOT, low GPT)
is distinct from the pattern recently reported for tumor (glioma)
cells, in which GPT appeared to be the dominant activity (62). We
found that the EL-4 lymphoma line showed a pattern of activity
similar to that of activated T cells, albeit with higher levels for
both enzymes, suggesting that GPT activity does not necessarily
predominate in tumor cells. It remains unclear whether the differences between our findings and those of the Thompson group
represent a divergence between tissue types or between the methods used to measure enzyme function, and analysis of a wider
range of normal and tumor cells will be required to draw conclusions. However, this difference suggests the possibility that different aspects of glutamine metabolism may provide specificity for
targeting therapeutics toward tumor cells versus normal cells or
toward specific cell types (e.g., lymphocytes).
Acknowledgments
We thank Dr. Brooke D. Humphrey for technical assistance with real-time
PCR primer design and for many helpful discussions and Dr. Jeffrey
C. Rathmell and Dr. Brian J. Bequette for critical reading of the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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