Increased Mitochondrial Biogenesis and

Reactive Oxygen Species Production

This information is current as
of October 29, 2018.
Accompany Prolonged CD4+ T Cell
Activation
Billur Akkaya, Alexander S. Roesler, Pietro Miozzo,
Brandon P. Theall, Jafar Al Souz, Margery G. Smelkinson,
Juraj Kabat, Javier Traba, Michael N. Sack, Joseph A.
Brzostowski, Mirna Pena, David W. Dorward, Susan K.
Pierce and Munir Akkaya

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J Immunol published online 29 October 2018
http://www.jimmunol.org/content/early/2018/10/28/jimmun
ol.1800753

Supplementary http://www.jimmunol.org/content/suppl/2018/10/29/jimmunol.180075
Material 3.DCSupplemental

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Published October 29, 2018, doi:10.4049/jimmunol.1800753
The Journal of Immunology

Increased Mitochondrial Biogenesis and Reactive Oxygen

Species Production Accompany Prolonged CD4+ T
Cell Activation

Billur Akkaya,*,1 Alexander S. Roesler,†,1,2 Pietro Miozzo,†,3 Brandon P. Theall,†

Jafar Al Souz,* Margery G. Smelkinson,‡ Juraj Kabat,‡ Javier Traba,x Michael N. Sack,x
Joseph A. Brzostowski,† Mirna Pena,† David W. Dorward,{ Susan K. Pierce,† and
Munir Akkaya†
Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria
primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen
species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes

Downloaded from http://www.jimmunol.org/ by guest on October 29, 2018

in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling
using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following
activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased
expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed
increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial
content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation,
CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling,
resulting in increased mitochondrial activity. The Journal of Immunology, 2018, 201: 000–000.

A
ctivation in response to foreign molecules results in a
cascade of changes in immune cells. These changes
include not only rapid proliferation and differentiation
but also the extensive cellular remodeling that accompanies them.
The cellular changes that immune cells go through to meet the
needs of activated cells have gained considerable interest in recent
years (1, 2). Most of the advances in the area focus on how im-
mune activation is coupled to changes in cellular metabolism and
how these metabolic changes are maintained. Although cellular
activation and differentiation is a process that demands high en-
ergy production, the way cells meet this demand is not uniform.
For instance, recent studies showed that both B and T lymphocytes
rely on aerobic respiration and fatty acid oxidation for their qui-
escent state energy needs, and upon activation, they shift toward
glucose as the main energy source (3–5). However, in contrast to
T cells, which, upon activation, remodel their energy production
machinery predominantly toward glycolysis with limited increase
in oxidative phosphorylation (OXPHOS) (6), activated B cells
show a more proportional increase in both glycolysis and
OXPHOS, and they maintain part of their OXPHOS capacity by
diverting a portion of glucose toward oxidation in mitochondria
through increased pyruvate dehydrogenase activity (7). So, as
compared with activated T cells, mitochondria in activated B cells
contribute more to overall energy production (3).
The process through which activated T cells use their mito-
chondria for generation of macromolecular intermediates, such as
lipid biosynthesis from citrate and nucleic acids through 1-carbon
metabolism, rather than energy production, resembles a similar
choice that exists in rapidly proliferating tumor cells (8–11). This
phenomenon, termed the Warburg Effect, represents a shift in

*Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, This work was supported by the Intramural Research Programs of the National
National Institutes of Health, Bethesda, MD 20892; †Laboratory of Immunogenetics, Institute of Allergy and Infectious Diseases and the National Heart, Lung, and Blood
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Institute, National Institutes of Health.
Rockville, MD 20852; ‡Research Technologies Branch, National Institute of Allergy
M.A. conceived and supervised the project; M.A., B.A., A.S.R., P.M., B.P.T., and J.T.
and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
x designed experiments; M.A., B.A., A.S.R., P.M., B.P.T., J.A.S., M.G.S., M.P., J.T., and
Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Insti-
D.W.D. carried out experiments; M.A., B.A., B.P.T., J.K., and J.T. analyzed data; M.A.
tutes of Health, Bethesda, MD 20892; and {Research Technologies Branch, National
and B.A. wrote the manuscript; S.K.P., M.N.S., and J.A.B. edited the manuscript.
Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
MT 59840 Address correspondence and reprint requests to Dr. Munir Akkaya, Laboratory of
1 Immunogenetics, 5625 Fishers Lane, Room 4S04B, Rockville, MD 20852. E-mail
B.A. and A.S.R. contributed equally to this work.
address: munir.akkaya@nih.gov
2
Current address: Duke University School of Medicine, Durham, NC.
The online version of this article contains supplemental material.
3
Current address: University of Massachusetts Medical School, Worcester, MA.
Abbreviations used in this article: A/R, antimycin/rotenone; COXIV, cytochrome c
ORCIDs: 0000-0002-6808-3776 (B.A.); 0000-0001-5768-1589 (A.S.R.); 0000-0003- oxidase subunit IV; DC, dendritic cell; 2-DG, 2-deoxy-D-glucose; DNP, dinitrophe-
3486-4061 (P.M.); 0000-0003-3852-5139 (B.P.T.); 0000-0003-2345-2803 (J.A.S.); nol; ECAR, extracellular acidification rate; MFI, mean fluorescence intensity;
0000-0001-7777-5574 (M.G.S.); 0000-0002-3411-0000 (M.N.S.); 0000-0003-0257- mTORC1, mTOR complex 1; 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
3905 (J.A.B.); 0000-0002-1710-731X (D.W.D.); 0000-0001-7261-3437 (S.K.P.); amino)-2-deoxyglucose; OCR, oxygen consumption rate; OXPHOS, oxidative phos-
0000-0002-9949-9424 (M.A.). phorylation; qPCR, quantitative PCR; ROS, reactive oxygen species; RT, room tem-
perature; STED, stimulated emission depletion; TMRM, tetramethylrhodamine,
Received for publication May 29, 2018. Accepted for publication September 25,
methyl ester, perchlorate; TOM20, translocase of the outer membrane 20; VDAC1,
2018.
voltage-dependent anion channel 1; WT, wild-type.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800753
2 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING
rapidly proliferating cells toward glycolysis and lactate production
even in the presence of oxygen (12). Despite the inefficiency of
glycolysis as a source of energy compared with OXPHOS, this
commitment allows for the use of the mitochondrial TCA cycle
for macromolecular synthesis in proliferating T cells (9, 10,
13–15). Furthermore, recent studies showed that mitochondrial
reactive oxygen species (ROS) production increases upon T cell
activation, and this acts as a second signal in regulating multiple
downstream elements (15, 16).
However, despite the advances in our understanding of T cell
immunometabolism, key questions remain unanswered. Because
most studies focus on early time points after T cell activation, we do
not clearly know whether the shift toward glycolysis is sustained
after prolonged activation. In addition, we do not know what type of
structural mitochondrial remodeling, if any, accompanies sustained
T cell activation.
In this study, we addressed these key questions by applying a
range of metabolic and cellular analyses to naive and CD4+ T cells
activated both in vitro and in vivo in a comparative fashion. Our
data showed a continued dominance of glycolysis over OXPHOS
as the source of energy in activated T cells even 4 d after acti-
vation. This was accompanied by a gradual increase in the ex-
pression of GLUT transporters, which allowed for increased
glucose uptake. Using a novel mitochondrial imaging strategy
optimized for lymphocytes, we showed that mitochondrial
remodeling goes in favor of increasing mitochondrial size, vol-
ume, and number in activated cells, which highlights the impor-
tance of mitochondria in rapidly proliferating activated T cells
despite their limited role in energy production.
Materials and Methods
Animals, cells, and reagents
Eight- to twelve-week-old C57BL/6 female mice purchased from
The Jackson Laboratory (Bar Harbor, ME) were used for isolation
of lymphocyte subsets. CD45.1+ CD45.2+ mice were generated by
breeding C57BL/6 mice with B6.SJL-Ptprca Pepcb/BoyJ mice (purchased
from The Jackson Laboratory). OT-II TCR transgenic mice were obtained
from Taconic Farms (Hudson, NY). Mice were maintained at National
Institute of Allergy and Infectious Diseases animal facilities, according to
Animal Care and Use Committee standards.
For lymphocyte isolation, mice were euthanized by CO2 asphyxiation
followed by cervical dislocation, and spleens were harvested. Naive CD4+
T cells were isolated using an isolation strategy described elsewhere (17).
Cell purities were checked by staining with phenotyping Abs and found to
be over 95% as measured by flow cytometry. For the isolation of dendritic
cells (DCs), spleens were removed and flushed by complete RPMI 1640
containing Liberase Blendzyme II and 2 mg/ml DNase, both purchased
from Roche (Indianapolis, IN). Spleens were then fragmented and incu-
bated at 37˚C for 30 min. After incubation, RBCs were lysed with
ammonium-chloride-potassium lysing buffer. DCs were isolated using
CD11c Microbeads (Miltenyi Biotec, Auburn, CA) and autoMACS
(Miltenyi Biotec) according to the manufacturer’s protocol. For
experiments involving cell culture, cells were maintained in complete
media (RPMI 1640 media containing 50 U/ml penicillin, 50 mM 2-ME,
50 mM streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino
acids, 10% FCS, 1 mM sodium pyruvate, and10 mM HEPES.)
To test the involvement of NO and mTOR in the regulation of
metabolic remodeling, NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl-3-oxide potassium (Carboxy-PTIO) and
mTOR inhibitor rapamycin (both purchased from Sigma-Aldrich,
St. Louis, MO) were added to cultures at 500 mM and 200 nM final
concentrations, respectively.
In vitro CD4+ T cell activation
For nonpolarizing CD4+ T cell (Th0) activation, a protocol previously
described elsewhere was used (18). Briefly, aseptically purified naive CD4+
T cells were resuspended in complete media supplemented with 100 U/ml
IL-2 (Peprotech, Rocky Hill, NJ) and 10 mg/ml anti-TGF-b (Bioxcell,
West Lebanon, NH) and plated on sterile 24-well tissue culture plates
(Corning/Life Sciences, Tewksbury, MA) previously coated with 4 mg/ml
anti-mouse CD3ε (Clone 145-2C11; Biolegend, San Diego, CA) and
4 mg/ml anti-mouse CD28 (Clone 37.12; Biolegend). Cells were incubated
for up to 96 h in a 5% CO2 humidified tissue culture incubator at 37˚C.
Adoptive transfer and in vivo CD4+ T cell activation
For adoptive transfer experiments, naive CD4+ T cells purified from spleens
of CD45.1+CD45.2+ wild-type (WT) mice and CD45.2+ OT-II transgenic
mice were mixed at a 1:1 ratio, stained with e450 cell proliferation dye
(Thermo Fisher, Waltham, MA) according to the manufacturer’s recom-
mendations, and resuspended in PBS. Two hundred microliters of PBS
containing 2 3 106 e450-stained CD4+ T cells were transferred into a
CD45.2 WT mouse i.v. via tail vein injection. Twenty-four hours after
T cell transfer, mice were injected i.v. with 100 ml of PBS containing 7.5 3
105 DCs previously loaded with either OVA(323–339) or lymphocytic cho-
riomeningitis virus gp(61–80) peptides. Four days after DC transfer, mice
were euthanized, and spleens were harvested.
Preparation of samples of light microscopy
Eighteen-millimeter no. 1.5 circular glass coverslips (catalog no. 64-0714;
Warner Instruments, Hamden, CT) were placed inside wells of 12-well
tissue culture plates (Corning), and 600 ml of 0.01% poly-L-lysine solution
(Sigma-Aldrich) was carefully applied on top of the coverslip, avoiding
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spillover to the underside. After 5 min of incubation at room temperature
(RT), liquid was aspired, and coverslip was dried for 2 h to overnight in a
ventilating tissue culture hood.
Cells harvested upon activation or purification were stained with RPMI
1640 without phenol red supplemented with 2% FCS, 1% HEPES, and 1/250
dilution of LIVE/DEAD Fixable Green Dead Cell Stain (Thermo Fisher
Scientific) for 20 min at 4˚C protected from light. Cells were then washed
and resuspended with prewarmed RPMI 1640 without phenol red supple-
mented with 2% FCS, 1% HEPES and 150 nM MitoTracker Red CMXROS
(Thermo Fisher Scientific). Cells were incubated at 37˚C for 30 min, fol-
lowed by two washes in a protein-free buffer (RPMI 1640 without phenol
red, PBS, or HBSS). After the final wash, cells were resuspended in protein-
free buffer at ∼3 3 106 cells/ml, and 500 ml of the cell suspension was
transferred onto the poly-L-lysine–coated coverslip (note that although these
numbers yielded optimal coating density for lymphocytes tested here, ad-
justments might be required if dramatic alterations in the size of cells are
anticipated because of specific experimental conditions). After 10 min of
incubation at RT, suspension was aspirated and washed using FACS buffer
(HBSS containing 2% FCS and sodium azide). This was followed by a
10-min incubation with anti-CD16/32 Ab (Biolegend). Cells were then
washed with FACS buffer, fixed, and permeabilized using the Cytofix/
Cytoperm kit (BD Biosciences, San Jose, CA). Rabbit anti-mouse translo-
case of the outer membrane 20 (TOM20) (2 mg/ml) (Abcam, Cambridge,
MA) followed by ATTO-647N–conjugated anti-rabbit secondary (2 mg/ml)
(Sigma-Aldrich) was used to stain TOM20 intracellularly. Fixation, per-
meabilization, and intracellular staining steps were carried out by following
the BD Cytofix/Cytoperm protocol. DAPI staining of nuclei was carried out
next by incubating the cells with PBS supplemented with 300 nM DAPI
for 5 min at RT. Coverslips were washed once and mounted on slides by
using ∼10–15 ml of mounting media prepared by mixing dissolved
Mowiol 4-88 (Polysciences, Warrington, PA) with 0.1% aqueous solution
of p-phenylenediamine at a 9:1 ratio, according to the manufacturer’s guide-
lines. Slides were left at RT in the dark overnight to ensure proper hard-
ening before imaging. For long-term storage, samples were kept at 220˚C.
Light microscopy and image analysis
Images were collected on a Leica TCS SP8 STED 33 system equipped with
white light and UV excitation lasers, a pulsed 775-nm depletion laser, an
HC PL APO 1003/1.40 oil STED White objective, and gated HyD de-
tectors. To exclude dead cells from images, an initial single-slide image
was taken from the area of interest using all four lasers, and upon con-
firmation of cell viability, a 488-nm laser used to detect the LIVE/DEAD
stain was switched off to decrease sampling time and photobleaching.
Image analysis was carried out using Imaris software version 9.1
(Bitplane, Zurich, Switzerland). Channel arithmetic extension in Imaris
was used to sum the TOM20 and MitoTracker fluorescence intensities
together, and the resulting channel was selected to create a three-
dimensional surface rendering of the mitochondrion via surface crea-
tion and watershed splitting algorithm. The seed points were determined
with a region growing estimated diameter of 0.5 mm. Other parameters
used in the three-dimensional surface creation, such as surface grain size
and diameter of the largest sphere, were adjusted individually, according
to the fluorescence intensities.
The Journal of Immunology
Transmission electron microscopy
For transmission electron microscope imaging, activated and naive CD4+
T cells were harvested and fixed by directly mixing the cell culture sus-
pensions with excess amounts of the modified Karnovsky fixative (0.1 M
sodium phosphate buffer containing 4% paraformaldehyde and 2.5%
glutheraldehyde) (Electron Microscopy Sciences, Hatfield, PA). Samples
were processed following a previously published microwave irradiation
strategy (19) with the following modifications: centrifugations were carried
out at 800 3 g, cells were infiltrated in Araldite resin (SPI, West Chester,
PA), sections were cut at 200-nm thickness, and no additional staining was
performed on sections. UltraScan 4000 camera (Gatan, Pleasanton, CA)
was used for image collection.
Extracellular flux assay
Oxygen consumption rate (OCR) and extracellular acidification rate
(ECAR) measurements were performed using Seahorse XF96 analyzer
(Agilent Technologies, Santa Clara, CA) following a previously published
protocol (20). For each experiment, activated and naive CD4+ T cells were
FACS sorted prior to assay to exclude nonviable cells. Chemicals used for
glycolysis and mitochondrial stress tests were administered at the fol-
lowing final concentrations: oligomycin (1 mM), 2,4 dinitrophenol (DNP)
(0.1 mM), antimycin A (1 mM), rotenone (1 mM), glucose (10 mM), and
2-deoxy-D-glucose (2-DG) (50 mM).
Flow cytometry
For measurement of mitochondrial membrane potential, a final concen-
tration of 5 mM FCCP (depolarizes the membrane to show the back-
ground staining), 6 mM oligomycin (hyperpolarizes the membrane to
show maximum achievable potential), or plain media (to show current
potential) were added directly to the cell cultures. After 10 min incu-
bation at 37˚C, staining mixture containing tetramethylrhodamine,
methyl ester, perchlorate (TMRM) (Thermo Fisher Scientific) (30 nM
final concentration) and SYTOX Blue Dead Cell Stain (Thermo Fisher
Scientific) (1 mM final concentration) were added to the cells. Cultures
were incubated for an additional 30 min and then analyzed in flow
cytometry. The formula 100 3 (mean fluorescence intensity [MFI](TMRM alone) 2
MFI(TMRM + FCCP))/(MFI(TMRM + oligomycin) 2 MFI(TMRM + FCCP)) was used to
standardize the measured TMRM MFI levels in the percentage of the maximum
potential format.
For flow cytometric detection of ROS production, 5 mM MitoSOX Red
(Thermo Fisher Scientific) or 500 nM CellROX (Thermo Fisher Scientific)
were used together with 1 mM SYTOX Blue Dead Cell Stain.
For intracellular staining of GLUT transporters and mitochondrial
markers, harvested cells were resuspended in FACS buffer supplemented
with 1/250 dilution of LIVE/DEAD Near-IR Dead Cell Stain (Thermo
Fisher Scientific) and any relevant surface-staining Abs. Upon 30 min
incubation at 4˚C, cells were washed, fixed, and permeabilized by either
BD Biosciences CytoFix/Cytoperm kit (for GLUT stains) or Biolegend
Foxp3 stain kit (for mitochondrial stains). Fluorochrome-conjugated mAbs
against GLUT-1, GLUT-3, voltage-dependent anion channel 1 (VDAC1),
TOM 20, and cytochrome c oxidase subunit IV (COXIV) used in these
experiments were purchased from Abcam.
For identification of the origin of adoptively transferred cells, Abs against
mouse CD4, CD45.1, and CD45.2 were used. Activation states of cells were
detected by Abs against CD44. mTOR complex 1 (mTORC1) activity was
evaluated by measuring surface expressions of mTOR-dependent markers
CD71 and CD98 (21–23), using fluorescently labeled mAbs. These Abs
were purchased from Biolegend.
Real-time glucose uptake of the cells was monitored by adding 2-(N-
(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (Thermo
Fisher Scientific) to cultured cells. For this purpose, activated and naive
T cells were stained with a viability marker and then incubated with 2-
NBDG for various durations. Cells remained in 37˚C culture until being
harvested at various time points and were then rapidly analyzed in a flow
cytometer.
Data acquisition was done using BD LSR II or BD 320 cytometers,
and data were analyzed using FlowJo software (version 10) (FlowJo,
Ashland, OR).
Western blot
For mitochondrial content detection by Western blot, freshly isolated naive
CD4+ T cells and in vitro–activated CD4+ T cells were stained with a
viability dye, and live cells were FACS sorted into protein-free buffer.
Equal numbers of viable cells from both conditions were lysed using
radioimmunoprecipitation assay buffer. Lysates were run on SDS-PAGE
and transferred to nitrocellulose membranes. The following Abs were
3
used: Histon 3 (9715S; Cell Signaling Technology), HSP60 (4870S; Cell
Signaling Technology), SIRT3 (5490S; Cell Signaling Technology), and
TOM20 (sc-11415; Santa Cruz Biotechnology). Images were captured
using the Odyssey system (LI-COR).
Quantitative PCR
The ratio of mitochondrial to genomic DNA was used to assess the mi-
tochondrial biogenesis potential of T cells. For this purpose, viable cells
were FACS sorted, and DNA was isolated using DNeasy Blood and Tissue
Kit (Qiagen, Hilden, Germany) and quantitative PCR (qPCR) was carried
out with different dilutions of DNA using iQ SYBR Green Supermix (Bio-
Rad, Hercules, CA). To represent genomic DNA, Mouse 18S ribosomal
DNA was amplified using 59-TAGAGGGACAAGTGGCGTTC-39 and 59-
CGCTGAGCCAGTCAGTGT-39. To represent mitochondrial DNA, mouse
cytochrome oxidase subunit I was amplified using 59-GCCCCAGATA-
TAGCATTCCC-39 and 59-GTTCATCCTGTTCCTGCTCC-39, and 12S
ribosomal DNA was amplified using 59-ACCGCGGTCATACGATTAAC-
39 and 59-CCCAGTTTGGGTCTTAGCTG-39. The transcriptional activity
of various genes encoding antioxidant enzymes was measured using RNA
isolated from freshly isolated naive CD4+ T cells and viable FACS-sorted
4-d activated CD4+ T cells following a protocol and primer sets published
elsewhere (7).
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Statistical significance analysis
Methods used to analyze statistical significance between experimental
groups and the p value ranges are described in respective figure
legends. Statistical analysis was calculated using Graph Pad Prism
Version 7.
Results
Prolonged activation of CD4+T cells results in cellular
changes to facilitate glycolysis
Naive CD4+ T cells harvested from mouse spleens were acti-
vated in vitro using plate-bound Abs specific for CD3 and CD28
in media containing soluble IL-2 and anti–TGF-b Ab for 4 d to
promote nonpolarized activation and proliferation. As expected,
T cells proliferated and upregulated the activation marker CD44
as assessed by flow cytometry (Supplemental Fig. 1A). We
initially compared freshly isolated naive and activated CD4+
T cells in a Seahorse analyzer using a glucose stress test as
described previously (20). For this assay, both cell types were
immobilized in chambers and starved of glucose for 30 min.
Following three baseline recordings of ECAR, which correlates
to lactate production by glycolysis, glucose was added to the
wells to stimulate glycolysis. Next antimycin/rotenone (A/R)
was added to block complex I and III of the respiratory chain,
which is expected to increase glycolytic capacity to its maxi-
mum (24). Finally, 2-DG was added to the wells to stop gly-
colysis to demonstrate the direct link between the ECAR and the
glycolytic performance. We observed that activated T cells
rapidly responded to glucose by increasing their ECAR val-
ues, which was further enhanced by adding A/R. In contrast,
naive T cells did not show any significant response to stimula-
tion, showing the relative insignificance of glycolysis in resting
T cell energy production (Fig. 1A, 1B). These observations were
in line with the findings of a previous study that showed gradual
increases in ECAR values and lactate production for both hu-
man CD4+ and CD8+ T cells during the course of a 3-d in vitro
activation (25).
Next, we ruled out the possibility that confounding effects
resulting from glucose starvation might be accounting for these
findings by repeating the experiment in the absence of glucose
deprivation, which showed similar results (Supplemental Fig.
1B). Consistent with these observations, as compared with
naive T cells, activated T cells increased GLUT-1 and GLUT-3
expressions (Fig. 1C–F) that corresponded to their increased
ability to take up glucose as measured by 2-NBDG (Fig. 1G).
4 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING

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FIGURE 1. Activated CD4+ T cells remodel their glucose uptake machinery to meet increased energy demand. For all experiments, purified naive
and 96-h ex vivo–activated CD4+ T cells were used. (A and B) Cells were FACS sorted for viability, and 5 3 105 viable cells were immobilized onto
each well of a 96-well Seahorse analyzer in which they were starved of glucose for 30 min, and basal ECAR levels were measured. Glucose, A/R, and
2-DG were consequently added after this period, and changes in ECAR values were recorded. (A) Arrowheads indicate the time points treatments were
added. Symbols and error bars refer to mean and SD of triplicates. (B) ECAR values pooled from four independent glycolysis stress tests (glycolysis =
ECARpostglucose 2 ECARbasal; glycolytic capacity = ECARpost–A/R 2 ECARpost–2-DG). Symbols demonstrate the means of individual experiments, and
lines mark the mean of the pooled data. (C–F) The expression levels of GLUT-1 (C and D) and GLUT-3 (E and F) as measured by flow cytometry.
Representative histograms (C and E) and bar graphs (D and F) demonstrating the MFI values are shown. Bars and error bars represent mean and SD of
triplicates. (G) Cells were stained with LIVE/DEAD and incubated in the presence of 10 mM 2-NBDG for up to 90 min. At each time point, aliquots
were harvested and run on a flow cytometer. MFI values were then normalized by subtracting the background MFI. Symbols and error bars represent
mean and SD of quadruplicates, respectively. (H) Expressions of Slc2a1 and Slc2a3 genes encoding GLUT-1 and GLUT-3, respectively, are quantified
using RNA isolated from naive and activated T cells. Bars and error bars represent mean and SEM of five independent experiments, respectively. Data
in (A) and (C)–(G) represent four independent experiments. Statistical significance was measured with Welch t test (B, D, F, and H) or two-way ANOVA
with Sidak multiple comparisons analysis (G). *0.01 , p # 0.05, **0.001 , p # 0.01, ****p # 0.0001.
The Journal of Immunology 5

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FIGURE 2. T cells continuously activated in vitro also increase their mitochondrial respiration despite greatly accelerated glycolysis. For all
experiments, purified naive and 96-h ex vivo–activated CD4+ T cells were used. (A–C) Cells were FACS sorted for viability, and 5 3 105 viable cells
were immobilized onto each well of a 96-well Seahorse analyzer. Upon basal OCR measurements, cells were sequentially treated with oligomycin;
2,4 DNP; and A/R on time points indicated with arrowheads. (A) Graph represents the changes in OCR values. Symbols and error bars refer to mean
and SD of each recording. (B) OCR values pooled from four independent mitochondrial stress tests (basal: OCRinitial 2 OCRpost–A/R; maximal
respiration = OCRpost-DNP 2 OCRpost–A/R; ATP-coupled respiration = OCRbasal 2 OCR postoligomycin). Symbols demonstrate means of individual
experiments, and lines mark the mean of the pooled data. (C) Ratio of the basal OCR to basal ECAR values, pooled from the mitochondrial stress
tests above. (D–F) Mitochondrial membrane potentials were measured using TMRM either alone or in combination with oligomycin or FCCP.
Representative histograms (D), MFI bar graphs (E), and percentage of the maximum potential graphs (F) are shown. Data represent four independent
experiments each carried out with triplicates. Statistical significance was calculated using Welch t test. p . 0.05 = ns; *0.01 , p # 0.05, ***0.0001
, p # 0.001. ns, not significant.

Activated T cells were actively transcribing Slc2a1 and Slc2a3, Thus, upon prolonged activation, T cells sustained increased
the genes encoding GLUT1 and GLUT3, respectively. These glycolytic activity, and the high expression levels of GLUT1
genes were up to 40-fold more transcriptionally active in and GLUT3 facilitated glucose uptake to fuel the glycolytic
activated T cells compared with naive T cells (Fig. 1H). activity.
6 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING

FIGURE 3. Increased ROS production following

T cell stimulation persists during prolonged acti-
vation without causing oxidative stress-induced
mitochondrial dysfunction. For all experiments,
purified naive and 96-h ex vivo–activated CD4+
T cells were used. (A and B) Cells were stained

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with SYTOX Blue viability dye together with
CellROX (A) or MitoSOX (B). Representative his-
togram overlays (top panels) and graphs of MFI
values (bottom panels) are shown. Bars and error
bars represent mean and SD, respectively. Data
represent three independent experiments, each
carried out with quadruplicates. (C) Transcription
of genes coding for antioxidant enzymes gluta-
thione reductase (Gsr), glutathione peroxidase 1
(Gpx1), superoxide dismutase (Sod1 and Sod2), and
catalase (Cat), as measured by qPCR, are shown for
both activated and naive T cells. Bars and error bars
refer to the mean of individual samples obtained
from five independent experiments and SEM, re-
spectively. (D) Transmission electron microscopy
electron micrographs of naive and activated T cells.
Images represent sections obtained from 10 indi-
vidual cells. N refers to nucleus (scale bar, 400
nm). Statistical significance was calculated using
Welch t test. p . 0.05 = ns, **0.001 , p # 0.01,
***0.0001 , p # 0.001. ns, not significant.

T cells preserve their mitochondrial reserves upon
prolonged activation
We performed a mitochondrial stress test to determine the con-
tribution of mitochondria to cellular energy production in ac-
tivated and naive CD4+ T cells using a Seahorse extracellular
flux analyzer. Cells were immobilized, and their basal oxygen
consumption was recorded. This was followed by oligomycin
treatment to inhibit ATP production, which leads to a decrease
in oxygen consumption. 2,4 DNP was added to uncouple
OXPHOS from electron transport, which increases oxygen
consumption to its maximum possible level. Finally, the
electron transport chain through complex I and III was blocked
using A/R, which dropped the oxygen consumption to the basal
level, confirming the link between the oxygen consumption
measurements and mitochondrial activity. We showed that,
compared with naive T cells, activated T cells used their mi-
tochondria more actively for energy production as shown by
significantly higher oxygen consumption levels (Fig. 2A, 2B).
However, the OCR/ECAR ratio was significantly lower in ac-
tivated cells, indicating that the prolonged activation-induced
increase in glycolysis surpasses the increase in mitochondrial
OXPHOS (Fig. 2C).
The Journal of Immunology 7

FIGURE 4. Prolonged T cell ac-

tivation leads to an increase in total
mitochondrial content. For all ex-
periments, purified naive and 96-h
ex vivo–activated CD4+ T cells were
used. (A and B) Cells were stained
with a viability dye together with
various mitochondria-specific markers
and analyzed in flow cytometry. Rep-
resentative histogram overlays (A) and
MFI graphs (B) are shown. Bars and
error bars represent mean of triplicates
and SD, respectively. (C) Live cells
were FACS sorted, and equal num-
bers of naive and activated cells
were lysed, separated in protein gel,
and immunoblotted for mitochon-
drial markers as well as H3 for load-
ing control. Representative Western

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blot image is shown. Data represent
two independent experiments. (D)
Total DNA was isolated from FACS-
sorted live naive and activated CD4+
T cells. Graphs show mitochondrial
DNA copy numbers relative to ge-
nomic DNA that were measured
using qPCR. Bars and error bars
refer to the mean of individual
samples obtained from five inde-
pendent experiments and SEM, re-
spectively. Statistical significance
was calculated using Welch t test.
*0.01 , p # 0.05; ***0.0001 ,
p # 0.001; ****p # 0.0001.

Next, we compared the mitochondrial performance between
naive and activated CD4+ T cells by measuring their relative
mitochondrial membrane potential using TMRM either alone or in
combination with oligomycin or FCCP. We showed that activated
T cells have higher total membrane potential (Fig. 2D, 2E) but use
a similar percentage of their maximum potential compared with
naive T cells (Fig. 2F). These findings suggest that the increase in
TMRM staining may either represent a continuation of mito-
chondrial membrane hyperpolarization that was shown to occur
shortly after activation (26, 27) or merely a consequence of a
greater total mitochondrial content in activated T cells. Because
both activated and resting T cells use ,30% of their mitochondrial
membrane potential, both can be considered efficient in terms of
mitochondrial ATP production with adequate reserves.
Activated T cells maintain their mitochondrial activity despite
prolonged ROS production
It was shown previously that T cell activation leads to rapid in-
creases in mitochondrial ROS production, which acts as a second
messenger to induce downstream cellular changes (16). We asked
whether this early increase in ROS production is sustained upon
prolonged activation. We observed higher ROS production in ac-
tivated T cells, as measured by both CellROX (Fig. 3A), a marker
for total intracellular ROS production, and MitoSOX (Fig. 3B), a
specific dye indicating mitochondrial ROS production. The in-
creases in intracellular ROS often trigger transcriptional activation
of antioxidant enzymes such as SOD1 and SOD2 to prevent
pathologic consequences of oxidative stress, such as induction of
mitochondrial swelling, by increasing mitochondrial membrane
permeability (28). However, we observed no change in tran-
scriptional activity of the genes encoding antioxidant enzymes in
activated T cells (Fig. 3C), suggesting either ROS levels were
insufficient to induce transcription or there are other mechanisms
that prevent upregulation of antioxidant genes to preserve
beneficial increase in ROS levels. Consistent with a beneficial role
of increased ROS production, we observed no increase in per-
centage of maximum mitochondrial membrane potential (Fig. 2),
which would rise during mitochondrial dysfunction. Moreover,
electron micrographs of both naive and activated T cells showed
healthy cristae structures and absence of swollen or vesicular-
swollen mitochondrial matrix areas, similar to those exemplified
in Refs. 7, 29, and 30, all of which rule out any ROS-induced
gross pathology in mitochondrial structures (Fig. 3D). Therefore,
sustained increase in ROS can be considered as a regulatory
component of activation rather than a signature of pathology.
Prolonged T cell activation leads to increases in
mitochondrial content
We explored whether during prolonged activation, T cells increase
their mitochondrial content. To do so, we measured the expression
levels of various proteins specifically localized in mitochondria and
widely used to assess mitochondrial content (7, 31–33). T cells
activated for 4 d in vitro as compared with naive T cells showed
increased levels of mitofilin and COXIV, as measured by flow
cytometry; sirtuin 3 (Sirt3) and heat shock protein 60 (Hsp60), as
measured by western blot; and VDAC1 and TOM20 as measured
by both (Fig. 4A–C). Upregulation was observed for all these
markers, demonstrating an increase in the total mitochondrial
8 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING

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FIGURE 5. Metabolic remodeling due to activation can be prevented by the inhibition of NO or mTOR pathways. Purified naive CD4+ T cells were
activated up to 96 h ex vivo. (A and B) Time-dependent changes in activation status (CD44), GLUT1 expression, and mitochondrial content (TOM20 and
COXIV). Representative histogram overlays (A) and MFI graphs (B) are shown. Symbols and error bars represent the mean of triplicates and SD, re-
spectively. Time points were compared against 0 h. (C–E) Naive CD4+ T cells were activated for 24 h ex vivo in the presence of rapamycin or Carboxy-PTIO where
indicated. Representative histogram overlays and MFI graphs for NO levels (C), mTOR-dependent surface markers (D), (Figure legend continues)
The Journal of Immunology
content in T cells upon prolonged activation, consistent with the
increased TMRM staining shown in Fig. 2D, 2E. To assess
the mitochondrial biogenic potential of the cells, we compared the
ratio of COI or 12S ribosomal DNA (genes encoded in the mi-
tochondrial genome) to 18S ribosomal DNA (a gene encoded in
the nuclear genome). This analysis revealed that 4 d poststimu-
lation in vitro, T cells retain an increased mitochondrial DNA
mass, which further supports increased mitochondrial biogenesis
capacity (Fig. 4D).
Metabolic remodeling following T cell activation is linked to
NO-mediated increases in mTOR activity
Our data, so far, showed that prolonged T cell activation leads to a
remodeling that involves both glucose metabolism and mito-
chondria. To confirm that these changes directly stem from TCR
stimulus, we repeated our 4-d culture experiments using different
combinations of activation conditions. This comparative analysis
revealed that the observed increases in GLUT and mitochondrial
markers can be recapitulated almost perfectly by plate-bound anti-
CD3 stimulation alone and that addition of IL-2 in culture or
costimulation with CD28 have no effects in the absence of TCR
stimulation (Supplemental Fig. 2A, 2B).
Next, to have a more in-depth understanding of the dynamics of
metabolic and cellular remodeling resulting from prolonged T cell
activation, we measured the changes in the expression of CD44,
GLUT1, and mitochondrial markers on a daily basis during the course
of 4-d CD4+ T cell activation in vitro (Fig. 5A, 5B). This analysis
showed gradual increases in the expression levels of the activation
marker CD44 and glucose transporter GLUT1. In contrast, markers of
mitochondrial content increased within the first 24 h and plateaued
after that, showing that the need for increased glucose uptake is
corelated with the duration of activation, whereas the majority of
mitochondrial remodeling occurs within the first day of activation.
These data also complemented the sustained high transcriptional ac-
tivity we showed in Slc2a genes in Fig. 1H.
Earlier studies on human samples showed a direct link between
T cell stimulation-induced NO production and the increase in
mitochondrial calcium levels and mitochondrial membrane hy-
perpolarization (34, 35). NO, through induction of mTORC1 ac-
tivity, was shown to be responsible for prevention of mitophagy by
Rab4A-mediated depletion of Drp1 (36–38). Furthermore, a re-
cent clinical trial in systemic lupus erythematosus patients showed
that mTOR blockade leads to a reduction in mitochondrial mass
(39). To address whether a similar link between T cell activation,
NO production, and mTOR activity might be responsible for the
changes we observed in the expression of GLUT transporters and/
or mitochondrial markers, we activated CD4+ T cells in vitro in
the presence or absence of NO scavenger (Carboxy-PTIO) or
mTOR inhibitor (rapamycin). We first showed that T cell
activation-induced NO can be efficiently scavenged in the pres-
ence of Carboxy-PTIO (Fig. 5C) and that the blockage of NO
results in a level of reduction in mTORC1-dependent cell surface
markers CD71 and CD98 similar to those observed in the presence
of rapamycin (Fig. 5D). Upon confirming the link between T cell
activation-mediated NO production and the induction of mTOR
activity, we showed that both rapamycin and Carboxy-PTIO
prevented the upregulation of GLUT1, VDAC1, and TOM20 in
and metabolic markers (E) are shown. Bars and error bars represent the mean of triplicates and SD, respectively. Data are representative of two independent
experiments. Freshly isolated naive CD4+ T cells were used as control. Statistical significance was measured using one-way ANOVA with Dunnett (B)
or Tukey (C–E) multiple comparisons analysis. p . 0.05 = ns, *0.01 , p # 0.05, **0.001 , p # 0.01, ***0.0001 , p # 0.001, ****p # 0.0001.
ns, not significant.
9
activated T cells. Carboxy-PTIO–mediated inhibition of the in-
crease in mitochondrial content and glucose transporters was more
pronounced compared with the level of inhibition, observed in the
presence of rapamycin (Fig. 5E). This suggests that NO may be
using both mTOR-dependent and -independent pathways during
T cell activation-induced metabolic remodeling.
Changes observed in in vitro–activated T cells can be
recapitulated in vivo
Thus far, we have shown that prolonged in vitro activation of
isolated T cells results in increases in glucose transporters,
total mitochondrial content, and mitochondrial ROS production
(Figs. 1–4). To test whether these changes also occur upon
in vivo activation of the cells, we carried out an adoptive transfer
experiment (Fig. 6A). For this purpose, naive TCR transgenic
CD4+ T cells isolated from CD45.1- CD45.2+ OT-II mice were
mixed with naive polyclonal CD4+ T cells isolated from WT
CD45.1+ CD45.2+ congenic mice at a 1:1 ratio. Cells were la-
beled with e-450 cell proliferation dye and adoptively transferred
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to WT CD45.2+ animal. Twenty-four hours posttransfer, recipi-
ent mice were injected with DCs pulsed with either OVA(323–339)
or control peptide, namely lymphocytic choriomeningitis virus
gp(61–80). Four days poststimulation, spleens were harvested and
analyzed in a flow cytometer using a gating strategy outlined in
Fig. 6B. As expected, OT-II transgenic T cells that were acti-
vated by OVA(323–339)-pulsed DCs in vivo had gone through a
few rounds of proliferation and induced the expression of acti-
vation marker CD44 (Fig. 6C). In vivo activation of OT-II T cells
also increased the fluorescent intensities of GLUT-1, COXIV,
TOM20, and MitoSOX as compared with control groups, indi-
cating that changes accompanying in vitro activation can be
recapitulated in vivo (Fig. 6D–G).
Multicolor imaging strategy offers a comprehensive analysis of
activation-induced mitochondrial changes in T cells
Having observed that both mitochondrial content and biogenesis
increase during prolonged activation, we wanted to visualize the
morphology of the resulting mitochondria. Visualization of mi-
tochondria in live T cells is challenging given the small cytoplasmic
volume and the relative fragility of T cells ex vivo.
To visualize T cell mitochondria, we developed a strategy using a
combination of a MitoTracker dye (MitoTracker Red CMXROS)
that accumulates in the mitochondrial matrix (40) and a fluo-
rescently labeled Ab specific for a subunit of TOM20 (41). The
nucleus was stained with DAPI, and dead cells were marked by
a viability dye (Fig. 7A). We used high-resolution stimulated
emission depletion (STED) microscopy to distinguish individual
mitochondria inside the small cytoplasmic volume of T cells. To
reduce excessive photobleaching due to high laser power with
STED imaging, we used photostable ATTO-647 fluorochrome to
label TOM20-specific Ab. This dual mitochondrial staining ap-
proach both eliminated staining artifacts and also provided a more
precise image of mitochondrial morphology. We then applied
computer algorithms to sum the fluorescence intensities of TOM20
and MitoTracker stains and to create artificial surfaces based on the
combined fluorescence intensity to predict the gross mitochon-
drial morphology and measure total mitochondrial volume. The
10 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING

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FIGURE 6. In vivo activation of CD4+ T cells leads to changes that are similar to those observed in vitro. (A) Schematic outline of the adoptive transfer
experiment. (B) Gating strategy used to discriminate adoptively transferred OT-II and polyclonal T cells in recipient mouse spleens. (C) Histogram overlays
showing the levels of CD44 expression (left) and the extent of proliferation (right) in adoptively transferred polyclonal and OT-II T cells 4 d poststimulation
with DCs pulsed with control glycoprotein peptide (top) or OVA peptide (bottom). (D–G) T cells were activated in vivo for (Figure legend continues)
The Journal of Immunology 11

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FIGURE 7. High-resolution multicolor imaging of lymphocytes reveals mitochondrial changes in CD4+ T cells induced upon prolonged activation. Cells
activated for 4 d in vitro or freshly isolated from spleens of C57BL/6 mice were stained with LIVE/DEAD and MitoTracker CMXROS red, then mounted
on poly-L-lysine–coated coverslips. Cells were then fixed, permeabilized, and stained for TOM20 and DAPI as described in Materials and Methods. (A)
Microscope image demonstrating viable (white arrowheads) and nonviable (yellow arrowheads) cells in the same area of interest (scale bar, 4 mm). (B)
Outline of the sequential image processing strategy used for distinguishing mitochondrial boundaries in lymphocytes. Naive CD4+ T cells, stained as
outlined above, were imaged for TOM20, MitoTracker, and DAPI in STED system. Images left to right show microscope images of TOM20, MitoTracker,
their merged view with DAPI, combined channel showing the sum of both mitochondrial markers in one channel, computer-generated surface of the
combined channel, and individual mitochondria estimates based on the watershed splitting algorithm applied to the combined surface (scale bar, 1 mm).
(C–F) Naive and activated T cells were imaged and analyzed in groups of five cells per area of interest as described above. Representative STED microscope
images (C) and data derived from image analysis showing average total mitochondrial volumes per cell (D), average number of mitochondria per cell (E),
and volume distribution of individual mitochondria (F) are given. Lines represent mean values, and symbols represent averages of five cells (D and E) or
individual mitochondrion (F). Data represent two independent experiments, each with at least 25 cells analyzed per condition. Statistical significance was
calculated using Welch t test. Scale bars, 1 mm. ****p # 0.0001.

surfaces, consisting of both matrix and membrane compartments of
the mitochondria, were also split via watershed splitting algorithm
to estimate the size and number of individual mitochondria in each
cell (Fig. 7B) (Supplemental Video 1).
We used this imaging strategy to compare freshly isolated naive
CD4+ T cells with T cells that were activated for 4 d in culture.
The images showed fewer, sparsely distributed mitochondria in
naive T cells as compared with activated T cells (Fig. 7C). Image
analysis showed that the average total mitochondrial volume per
cell is almost 3-fold higher in activated cells (Fig. 7D).
Furthermore, compared with naive cells, activated T cells had at
least a 2-fold increase in the estimated number of mitochondria

4 d as illustrated in (A). Histogram overlays (left) and MFI graphs (right) show the levels of GLUT1 (D), TOM20 (E), COXIV (F), and MitoSOX (G). Each
circle is an individual mouse. Data represent two independent experiments. Statistical significance was calculated using two-way ANOVA with Sidak multiple
comparisons test. p . 0.05 = ns, ****p # 0.0001. ns, not significant.
12 T CELL ACTIVATION INDUCES MITOCHONDRIAL REMODELING
(Fig. 7E), and the average volume of individual mitochondria in
these cells was larger (Fig. 7F). These results not only comple-
ment our observations on total mitochondrial content (Figs. 4–6)
but also show that the increased total mitochondrial content in
activated T cells is a direct consequence of both individual mi-
tochondrial enlargement and increased mitochondria numbers.
Altogether, our data suggest that activation-induced mitochondrial
biogenesis serves the purpose of increasing mitochondrial content
in multiple ways.
Discussion
In this study, we have addressed key questions related to metabolic
and cellular changes that accompany prolonged T cell activation
and proliferation. Our data showed that the early increases in
glycolysis and OXPHOS in activated T cells are maintained during
prolonged activation. We have shown that after 4 d of both in vitro
and in vivo activation, rapidly proliferating CD4+ T cells have
already increased their GLUT transporters and are more able to
import glucose into the cell and yet are still transcriptionally ac-
tive to induce further GLUT expression.
Our findings mainly focused on the changes in mitochondrial
dynamics. This was an unexplored area because of the lack of high-
resolution imaging strategies optimized for lymphocytes. These
cells are not only difficult to handle and culture ex vivo (20), but
also their massive nucleus to cytoplasm ratio poses a great diffi-
culty for any researcher aiming to visualize cytoplasmic com-
partments. We addressed these shortcomings by introducing
viability staining to exclude nonviable cells as well as target the
mitochondria with two independent markers for added specificity.
By taking advantage of this novel multicolor mitochondria stain-
ing protocol in a high-resolution STED microscope setting and
processing the raw data further by using an optimized computer
algorithm, we have been able to visualize individual mitochondria
and estimate their boundaries even at close proximity areas.
These analyses showed that upon continuous activation, CD4+
T cells upregulate their total mitochondrial volume and number as
well as the size of individual mitochondria. Our comparative anal-
ysis on the ratio of mitochondrial DNA to genomic DNA revealed
that activated T cells have increased mitochondrial biogenesis. In
addition to the structural mitochondrial remodeling, we showed that
increases in ROS production is sustained through long-term acti-
vation, which is likely maintained at least in part by lack of increase
in antioxidant enzyme synthesis. Nevertheless, despite sustained
increases in cellular ROS levels, our data demonstrated that the
functional and structural integrity of mitochondria is preserved,
which is in line with the previous observations suggesting a
physiological role for the ROS production.
For many cell types, the function of mitochondria goes far beyond
its role in energy production. Recent studies showed that there is a
clear correlation between the intracellular organization of mito-
chondria and the stress response of cells (42, 43). Mitochondria are
also known to actively participate in programmed cell death (44).
Furthermore, recently, we have identified a novel contribution of
mitochondria in modulating activation-induced cell death in B
lymphocytes. Our assays showed that Ag stimulation of B cells
requires a spatiotemporally distinct second stimulus in the form of
either cognate B–T interaction or TLR activation. Lack of this
second signal triggered a cascade of events, including mitochon-
drial swelling, inefficient mitochondrial respiration, and increased
production of ROS, all of which eventually led to the death of the
cell (7). All of these examples show the importance of accurately
assessing mitochondrial changes.
Altogether, our findings provide the most comprehensive
mitochondria-centric approach to decipher changes that accompany
T cell activation and highlight the functional importance of mito-
chondria in activated T cells despite its limited role in energy pro-
duction. The data presented in this article not only increase our
understanding of mitochondrial remodeling following prolonged
T cell activation but also offer novel methodological approaches that
can be applied to decipher changes in the mitochondrial dynamics in
other cell types under numerous experimental settings.
Disclosures
The authors have no financial conflicts of interest.
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