Download and Function Activation Sulfenic Acid Formation for T Cell The

The Requirement of Reversible Cysteine
Sulfenic Acid Formation for T Cell Activation
and Function
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of July 31, 2017.
Ryan D. Michalek, Kimberly J. Nelson, Beth C. Holbrook,
John S. Yi, Daya Stridiron, Larry W. Daniel, Jacquelyn S.
Fetrow, S. Bruce King, Leslie B. Poole and Jason M.
Grayson
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:6456-6467; ;
doi: 10.4049/jimmunol.179.10.6456
http://www.jimmunol.org/content/179/10/6456
The Journal of Immunology
The Requirement of Reversible Cysteine Sulfenic Acid
Formation for T Cell Activation and Function1
Ryan D. Michalek,* Kimberly J. Nelson,† Beth C. Holbrook,* John S. Yi,* Daya Stridiron,*
Larry W. Daniel,† Jacquelyn S. Fetrow,§ S. Bruce King,‡ Leslie B. Poole,†
and Jason M. Grayson2*
he CD8⫹ T cell is a critical component of the immune
system’s response to infectious agents and tumors (1–3).
Activation of naive T cells begins when the TCR encounters cognate Ag presented by MHC proteins on the surface of
APCs. This recognition event orchestrates a signaling cascade
marked by protein tyrosine phosphorylation and a rise in intracellular
calcium (4 – 6). These signals initiate a new program of gene expression and differentiation into effector cells that is accompanied by increased protein synthesis, cell growth, and effector functions such as
cytokine production and cytolytic activity (7, 8). Activated T cells
also undergo massive clonal expansion beginning 24 h after stimulation and continuing with one division every 6 to 8 h (9). Understanding the mechanisms regulating these events is essential for designing
new vaccine and immunotherapy strategies.
The critical role of reactive oxygen intermediates (ROI)3 in innate immune responses has been well documented. Upon activa-
T
*Department of Microbiology and Immunology and †Department of Biochemistry,
Wake Forest University School of Medicine, Winston-Salem, NC 27157; and ‡Department of Chemistry and §Department of Computer Science and Department of
Physics, Wake Forest University, Winston-Salem, NC 27109
Received for publication January 9, 2007. Accepted for publication August
30, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by American Cancer Society Research Scholar Grant No.
RSG-04-066-01-MBC (to J.M.G.). Additional support was provided by the Wake
Forest University Cross Campus Collaborative Fund (to J.M.G. and J.S.F.).
2
Address correspondence and reprint requests to Dr. Jason M. Grayson, 5100A Gray
Building, Department of Microbiology and Immunology, Wake Forest University
School of Medicine, Winston-Salem, NC 27157. E-mail address: jgrayson@
wfubmc.edu
3
Abbreviations used in this paper: ROI, reactive oxygen intermediate; 7-AAD,
7-amino-actinomycin D; DCFDA, 5-(and-6)-chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein diacetate acetyl ester; dimedone, 5,5-dimethyl-1,3-cyclohexanedione;
Fluo-3-AM, Fluo-3 acetoxymethyl ester; GP, glycoprotein; ION, ionomycin; LCMV,
lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; PTEN, phos-
www.jimmunol.org
tion, phagocytic cells such as neutrophils and macrophages increase the production of O2. , H2O2, and NO (NO䡠) in preparation
for the respiratory burst. This process is vital to innate immunity
because individuals who suffer from chronic granulomatous disease, in which the O⫺
2 -producing NADPH oxidase enzyme complex is defective, are vulnerable to severe recurrent bacterial and
fungal infections (10). More recently, studies have demonstrated
ROI production in adaptive immune responses. Shortly following
Ab-mediated TCR cross-linking, Devadas et al. (11) documented
that T cell blasts increase ROI levels. This result was consistent
with an earlier report that PHA and PMA induce oxidative product
formation in the Jurkat malignant T cell line (12). Although the
intracellular source of ROI production in T cells is still being investigated, studies have implicated the mitochondria as well as a
phagocyte-type NADPH oxidase as contributors (13–16). An essential role for ROI in T cell function was initially demonstrated
by reports indicating that antioxidants inhibit proliferation and
IL-2 production when administered during the early stages of T
cell activation (17, 18). Subsequent in vivo studies found that antioxidant treatment of mice decreased the proliferation and cytokine production of Ag-specific T cells in both autoimmune and
infectious models (19, 20). These findings suggest that ROI generated in response to receptor stimulation act as positive mediators
involved in lymphocyte activation.
Although ROI possess the ability to modify all biological macromolecules, reversible oxidation of cysteine is an important
mechanism by which signaling proteins can be regulated. Phosphatases, such as phosphatase and tensin homologue deleted on
chromosome 10 (PTEN) and Src homology 2 domain-containing
phosphatase (SHP)-2, as well as transcription factors, such as
NF-␬B and AP-1, use reversible cysteine oxidation to modulate
phatase and tension homology deleted on chromosome 10; PTP, protein tyrosine
phosphatase; SHP, Src homology 2 domain-containing phosphatase.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8ⴙ T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8ⴙ T cells increased ROI levels in a manner dependent upon
Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins
revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification
following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures.
Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation,
calcium flux, cell growth, and proliferation of naive CD8ⴙ and CD4ⴙ T cells. We also found that TNF-␣ production by effector and
memory CD8ⴙ T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-␥. Together, these
results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8ⴙ T cells are
able to modulate signaling, proliferation, and function. The Journal of Immunology, 2007, 179: 6456 – 6467.
The Journal of Immunology
Materials and Methods
Adoptive transfer and effector and memory CD8⫹
T cell generation
A naive P14 Thy1aPL/1 mouse was sacrificed and the spleen was excised.
After osmotic lysis, splenocytes were stained with Abs specific for CD8␣,
CD90.1, and the Db glycoprotein (GP) peptide 33– 41 (DbGP33– 41) MHC
class I tetramer. Splenocytes containing 105 naive P14 CD8⫹ T cells were
transferred into naive C57BL/6 mice with an engraftment of 104 cells.
Mice were then infected with 2 ⫻ 105 PFU of lymphocytic choriomeningitis virus (LCMV) strain Armstrong by i.p. injection and were sacrificed
on day 8 for effector cell isolation or ⬎60 days after infection for memory
cell isolation.
CD8⫹ and CD4⫹ T cell purification
Naive CD8⫹ or CD4⫹ T cells were negatively selected by magnetic bead
enrichment from the spleens of naive C57BL/6 mice using the Miltenyi
MicroBead system according to the manufacturer’s protocol. Purity was
⬎95% as determined by flow cytometry.
CFSE labeling
CFSE was purchased from Invitrogen Life Technologies and dissolved in
DMSO as a 5 mM stock. After purification, cells were washed three times
in PBS and suspended at a concentration of 2 ⫻ 107 cells/ml in PBS. The
CFSE stock was diluted to 10 ␮M in PBS and mixed with cells 1:1 (v/v),
resulting in a final concentration of 5 ␮M CFSE. After 3 min, samples were
vortexed and then continued incubating for an additional 2 min. After this
time, 1/10 volume of FCS was added for 1 min followed by vortexing. The
cells were then washed three times with complete medium and used in
experiments.
Calcium flux assay
Fluo-3 acetoxymethyl ester (Fluo-3-AM) was purchased from Invitrogen
Life Technologies and dissolved in DMSO as a 1.25 mM stock. Purified
CD8⫹ T cells were incubated in 5 ␮M Fluo-3-AM in PBS with 5% FCS
and dimedone for 60 min. Samples were washed two times and resuspended in the same medium containing dimedone. Cells were acquired for
60 s on a FACSCalibur instrument, after which PMA and ionomycin (ION)
were added to the sample and recording was resumed.
Cell isolation and medium
The spleen was removed from mice after cervical dislocation. Following
mechanical disruption of splenocytes on a wire mesh screen, RBC were
removed by osmotic lysis in ACK buffer (NH4Cl, KHCO3, and EDTA).
Splenocytes were then resuspended in RPMI 1640 supplemented with 10%
FCS, L-glutamine, penicillin-streptomycin, and 2-ME (complete medium).
Cell viability assay
Purified CD8⫹ T cells were used either directly ex vivo or after 24 h of
incubation in the presence or absence of dimedone. Cells were removed
from the dish by gentle pipetting and diluted in trypan blue. If cells were
able to exclude trypan blue they were scored as viable.
5-(and-6)-chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein
diacetate-acetyl ester (DCFDA) oxidation
DCFDA was purchased from Invitrogen Life Technologies and resuspended in DMSO as a 2 mM stock. Cells were activated in a 96-well
flat-bottom plate, transferred to a 96-well round-bottom plate after stimulation, and loaded with 5 ␮M DCFDA in complete medium. Cells were
incubated for 30 min at 37°C before being washed in FACS buffer (2%
FCS and PBS), stained with an anti-CD8␣ Ab and DbGP33– 41 tetramer,
and acquired immediately on a FACSCalibur instrument. Data are presented as change in mean fluorescent intensities compared with unstimulated cells.
In vitro stimulation and dimedone pretreatment
For all stimulations, cells were pretreated with DMSO or dimedone. The
highest concentration of DMSO used in any experimental condition was
1% (v/v). For peptide stimulation of naive CD8⫹ T cells, 106 P14 splenocytes were resuspended in complete medium that contained DMSO or
dimedone for 1 h before stimulation with 10⫺7 M GP33– 41 peptide. To
activate OT-2 transgenic CD4⫹ T cells, 10⫺6 M OVA323–339 peptide was
used. Purified T cells (2.5 ⫻ 105) were used for the other stimulations.
Cells were incubated in DMSO or dimedone and complete medium for one
hour before PMA and ION were added at 2 ng/ml and 10 ␮g/ml, respectively. For CD3/CD28 stimulation experiments, 96-well flat-bottom plates
were coated with 10 ␮g/ml anti-CD3 and anti-CD28 or 20 ␮g/ml control
IgG in PBS overnight at 4°C. Purified T cells were incubated in complete
medium that contained DMSO or dimedone for 1 h before being transferred to the Ab-coated plate. For ZAP70 stimulation, white aldehyde/
sulfate latex beads (Interfacial Dynamics) were coated with 10 ␮g/ml antiCD3 and anti-CD28 Abs at 37°C for 1 h before being used to stimulate T
cells. Dimedone, PMA, and ION were purchased from Sigma-Aldrich.
Anti-CD3, anti-CD28, and hamster IgG were purchased from BD
Pharmingen.
Effector and memory CD8⫹ T cells were harvested from LCMVArmstrong-infected mice on days 8 and ⬎60, respectively. Intracellular
cytokine staining was performed as described below. For restimulation,
1 ⫻ 106 splenocytes were pretreated in complete medium containing dimedone or DMSO for 1 h before 5 h of peptide stimulation.
Dimedone removal experiment
Purified CD8⫹ T cells were prepared as described above and incubated
with dimedone or DMSO for 1 h before stimulation with PMA and ION.
A duplicate set of samples was prepared and washed with complete medium three times before stimulation with PMA and ION.
Preparation of MHC class I tetramers
The construction and purification of DbGP33– 41 has been described previously (27).
Protein assay
Steady-state protein levels were measured by preparing lysates from 106
purified CD8⫹ T cells. Cells were stimulated with PMA/ION for 24 h in the
presence or absence of dimedone. Protein concentration was determined
using the Micro BCA protein assay kit from Pierce.
Sulfenic acid labeling
Purified CD8⫹ T cells (1 ⫻ 106) were stimulated with PMA and ION or
anti-CD3 and anti-CD28 Abs. Additionally, one set of samples was pretreated with 10 mM N-acetyl cysteine or 20 ␮M ebselen for 1 h before
stimulation. At each time point, cells were lysed in the presence of 5 mM
biotin-linked dimedone derivative (L. B. Poole et al., submitted for publication), 50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 20 mM ␤-glycerophosphate, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal, 0.5%
Triton X-100, 1 mM Na3VO4, 20 mM NaF, 1 mM PMSF, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin (pH 8.0). Lysates were immediately sonicated and incubated at room temperature for 10 min. N-ethyl maleimide at
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
protein function (21–24). In the presence of ROI, cysteine thiolates
can be transiently oxidized to cysteine sulfenic acid (-SOH). This
species can be stabilized, reduced, or further irreversibly oxidized
to sulfinic acid (-SO2H) and sulfonic acid (-SO3H) (25). Cysteine
sulfenic acid also acts as an intermediate in the formation of disulfide bonds and glutathione conjugation (26). Because of its transitory nature and intermediate role in multiple reactions, the reversible formation of cysteine sulfenic acid is a mechanism by
which ROI can modulate signaling.
In this study, we have demonstrated that ROI are generated during peptide-, Ab-, and mitogen-induced activation of naive CD8⫹
T cells. Using a biotin-linked derivative of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently binds to
cysteine sulfenic acid, we show that sulfenic acid formation increases during naive CD8⫹ T cell activation. Activating cells in
the presence of dimedone diminished calcium flux and ERK1/2
phosphorylation. This decrease in cellular signaling resulted from
reversible cysteine sulfenic acid events occurring after activation
and led to a reduction in naive T cell growth, S phase entry, and
proliferation. In the case of T cell function, we demonstrated that
production of the cytokine TNF-␣ by effector and memory CD8⫹
T cells isolated directly ex vivo was more sensitive to a blockade
of reversible cysteine sulfenic acid formation than IFN-␥. These
studies illustrate the important role of reversible cysteine sulfenic
acid formation in regulating the activation, proliferation, and function of CD8⫹ T cells.
6457
6458
ROLE OF CYSTEINE SULFENIC ACID IN T CELL ACTIVATION
10 mM was then added to block free thiols, and samples were incubated an
additional 20 min at room temperature before being frozen at ⫺20°C.
Conjugated dimedone derivatives have been shown in previous reports to
retain their specificity and reactivity with cysteine sulfenic acid (28, 54).
For immunoprecipitation experiments, 2– 4 ⫻ 106 naive CD8⫹ T cells
were stimulated with anti-CD3 and anti-CD28 Abs. Cell lysates were prepared as described above and cleared with protein G magnetic beads (Dynal) for 1 h at 4°C. The magnetic beads were removed and 2.5 ␮g/ml
anti-SHP-2 (BD Pharmingen), anti-SHP-1, or anti-actin (Santa Cruz Biotechnology) Ab was added and incubated overnight at 4°C. The following
day, protein G magnetic beads were added to the extracts, and lysates
continued to incubate for 3 h at 4°C. Samples were then placed on a magnetic column, washed, and resuspended in lysis buffer. Protein was eluted
from beads by boiling in reducing sample buffer from Pierce.
Protein precipitation
Sulfenic acid detection
For sulfenic acid detection, samples were separated on a 7.5, 10, or 12%
SDS-denaturing gel, transferred to a nitrocellulose membrane, and blocked
overnight in 5% FCS. Sulfenic acid containing proteins were detected by
a 1/50,000 dilution of streptavidin-HRP at room temperature for 1 h and
visualized using the SuperSignal West Pico Chemiluminescent Substrate
from Pierce according to the manufacturer’s protocol. The blot was then
stripped with Restore Western blot stripping buffer (Pierce) for 10 min at
room temperature, blocked, and probed with a 1/1000 dilution of either
anti-actin, anti-SHP-1, or anti-SHP-2 Ab for 2 h at room temperature. After
three washes, the blot was incubated in either rabbit anti-goat, goat antirabbit (Southern Biotechnology), or goat anti-mouse (Pierce) HRP-conjugated secondary Ab (1/10,000 dilution) for 1 h and developed as described
above. For quantitation of sulfenic acid, actin and biotin levels were normalized between samples using a Kodak Image Station 2000RT and Kodak
Molecular Imaging software. To determine sulfenic acid levels, the entire
length of the gel lane was scanned, whereas only the protein band was
quantitated for actin. The fold increase in cysteine sulfenic acid was then
calculated by multiplying the fold difference in the normalized actin value
by the biotin signal.
Statistical analysis
Data from control and dimedone-treated samples were analyzed using twotailed Student’s t test, and p ⱕ 0.05 was considered significant.
Surface and intracellular staining
In this study the following Abs were used: rat anti-mouse CD8␣-PE; rat
anti-mouse CD8␣-PerCP; rat anti-mouse CD90.1-FITC; rat anti-mouse
IFN-␥-allophycocyanin; rat anti-mouse TNF-␣-PE; rabbit anti-mouse
phospho-ZAP70 (Tyr319), rabbit anti-mouse phospho-p44/42 MAPK
(Tyr202/Tyr204); mouse anti-mouse phospho-p38 MAPK (Thr180/Tyr182),
and mouse anti-mouse phospho-JNK (Thr183/Thr185). Phospho-ZAP70,
phospho-ERK1/2, phospho-JNK, and phospho-p38 Abs were purchased
from Cell Signaling. All other Abs were purchased from BD Pharmingen.
Surface staining was performed by the incubation of Abs at a 1/100 dilution in FACS buffer for 30 min at 4°C. CD90.1 staining was performed at
a 1/750 dilution. To measure intracellular cytokine levels, cells were
treated with BD Biosciences Cytofix/Cytoperm kit according to the manufacturer’s instructions.
For phospho-ZAP70, phospho-ERK1/2, phospho-JNK, and phosphop38 staining, purified CD8⫹ T cells were fixed in 2% paraformaldehyde at
37°C for 10 min. Samples were then permeabilized with 90% methanol
before Ab staining according to the manufacturer’s protocol (Cell Signaling). A FITC-conjugated goat anti-rabbit secondary was used for visualization of phospho-ZAP70 and phospho-ERK1/2 (Caltag Laboratories).
Samples were acquired on a FACSCalibur instrument and analyzed using
FlowJo software.
Six- to 8-wk-old C57BL/6 mice were purchased from the National Cancer
Institute (Frederick, MD). LCMV-Armstrong stocks were propagated on
BHK-21 cells and quantitated as described previously (29).
Cell cycle analysis
BrdU (Sigma-Aldrich) labeling was performed as described previously by
Tebo et al. (30). Briefly, purified CD8⫹ T cells were stimulated with PMA
and ION. At 23 h, samples were pulsed with 10 ␮M BrdU for 45 min,
resuspended in 1% paraformaldehyde with 0.05% Igepal (Sigma-Aldrich),
shaken, and incubated overnight at 4°C. Cells were then washed two times
in room temperature PBS at 290 ⫻ g for 6 min, resuspended in 1 ml of PBS
and 4.2 mM MgCl2 containing 50 Kunitz U/ml DNase I (Sigma-Aldrich),
and incubated for 30 min at 37°C. After two washes with wash buffer (5%
FCS with 0.5% Igepal in PBS) at 290 ⫻ g and 4°C for 6 min, cells were
resuspended in the same buffer containing 2% mouse serum, a 1/5 dilution
of anti-BrdU-FITC (BD Pharmingen), and incubated on ice for 45 min.
Samples were washed two times in wash buffer at 290 g and 4°C for 6 min.
For 7-amino-actinomycin D (7-AAD) staining, cells were resuspended
in 20 ␮l of 7-AAD (Pharmingen) plus FACS buffer for 10 min on ice.
Samples were acquired immediately using a FACSCalibur instrument.
Results
Naive CD8⫹ T cells increase DCFDA oxidation after incubation
with peptide-coated splenocytes, anti-CD3 and anti-CD28 Abs,
or PMA and ION
Previously, it is has been demonstrated that 9C127 murine T cell
hybridomas increase ROI levels upon anti-CD3 stimulation and
that this increase is required for cellular signaling (11). To determine ROI production after the activation of naive CD8⫹ T cells by
their cognate Ag, the kinetics of GP33– 41-stimulated ROI production were measured in naive P14 transgenic T cells by using the
oxidation-sensitive dye DCFDA. DCFDA is a cell-permeant
dye that is nonfluorescent until it is oxidized by peroxides, peroxynitrite, and/or hydroxyl radicals. Oxidation increases the
fluorescence of the dye, which can be recorded by flow cytometry. Fig. 1A demonstrates an increase in CD8⫹GP33– 41⫹ T
cell DCFDA oxidation within 15 min of 10⫺7 M peptide stimulation. This increased level of oxidation remained elevated for
up to 6 h (Fig. 1B).
The production of ROI was dependent on peptide concentration.
At 10⫺9 M, the lowest concentration of GP33– 41 in which full
proliferation is observed, there was a delay in maximal ROI production during the initial hour. However, by 3 h after stimulation
the levels of DCFDA oxidation were comparable to those of T
cells stimulated with 10⫺7 M peptide. At a peptide concentration
(10⫺10 M) in which minimal recruitment and very limited proliferation were observed, ROI production during the first 6 h of activation did not increase significantly compared with the
OVA257–264 noncognate peptide control.
To determine whether DCFDA oxidation was due to autonomous ROI production, CD8⫹ T cells were magnetically purified
from the spleens of naive C57BL/6 mice by negative selection.
Cells were then activated with plate bound anti-CD3 and antiCD28 Abs. Within 15 min of Ab stimulation there was an increase
in DCFDA oxidation (Fig. 1C) that remained elevated for at least
6 h. A similar observation was made when cells were stimulated
through just the TCR alone with an anti-CD3 Ab. However, when
the CD28 costimulatory molecule was cross-linked in the absence
of TCR stimulation, there was an initial increase in ROI production
that was not maintained and had declined to ex vivo levels by 3 h
after stimulation.
Purified CD8⫹ T cells were also stimulated with PMA and ION,
PMA alone, or ION alone to determine whether ROI production
was exclusive to receptor proximal stimulation events. PMA is a
mitogen that stimulates cells by activating protein kinase C,
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Soluble protein was extracted after sulfenic acid labeling and precipitated
by adding 5:1 (v/v) cold acetone to the sample for 10 min at ⫺20°C.
Protein was then pelleted at max speed in an Eppendorf 5415 C centrifuge
for 5 min, resuspended in 10% TCA for 15 min at ⫺20°C, and pelleted
again at maximum speed for 5 min. Samples were washed with 1:1 (v/v)
ethanol:ether and then pelleted for 5 min at maximum speed. After discarding the supernatant, the pellet was rehydrated in 8 M urea lysis buffer.
Protein concentrations were determined using the DC protein assay from
Bio-Rad according to manufacturer’s protocol.
Viral infection and mice
The Journal of Immunology
6459
whereas ION is a calcium ionophore that increases the cytosolic
calcium concentration. Fifteen minutes after PMA and ION stimulation there was a 2-fold increase in DCFDA oxidation that was
similar to the increase observed with peptide and anti-CD3 and
anti-CD28 stimulation (Fig. 1D). When cells were stimulated with
PMA alone there was also a comparable increase in DCFDA oxidation. However, ION stimulation failed to sustain DCFDA oxidation although ROI levels were still increased compared with the
DMSO solvent control sample. Thus, ROI are generated by naive
CD8⫹ T cells in response to Ag, TCR cross-linking, and mitogen
stimulation.
Cysteine sulfenic acid levels increase during activation of naive
CD8⫹ T cells
Increased ROI production following receptor stimulation has been
associated with cellular signaling in response to insulin, plateletderived growth factor (PGDF), basic fibroblast growth factor
(bFGF), and TNF-␣ (31–34). To determine whether increased ROI
levels following T cell stimulation lead to the formation of cysteine sulfenic acid, a biotin-linked derivative of dimedone was
used to specifically alkylate sulfenic acid-modified proteins. Dimedone is a synthetic compound used in mass spectrometry for the
detection of sulfenic acid-containing proteins. It is a highly specific
alkylator that attacks cysteine sulfenic acid in a nucleophilic fashion and forms a covalent bond (35). Within 5 min of TCR stimulation, total cysteine sulfenic acid levels increased (Fig. 2A).
These levels continued to rise for 120 min, which was the latest
time point recorded. The actin in the lower panel of Fig. 2A serves
as a loading control. To verify that this increase in sulfenic acid
formation resulted from ROI production, naive CD8⫹ T cells were
treated with either N-acetyl cysteine or ebselen (Fig. 2B). At 120
min after receptor stimulation both of these antioxidants decreased
ROI (data not shown) and sulfenic acid to levels similar to those of
ex vivo samples. In addition to receptor stimulation, we measured
sulfenic acid levels following mitogen stimulation of protein kinase C and calcium flux. PMA and ION activation resulted in
elevated levels of cysteine sulfenic acid by 5 min (Fig. 2C). In
contrast to receptor activation, these levels reached a peak at 60
min before declining slightly by 120 min. The total sulfenic acid
signal in each lane was quantitated and normalized to actin intensity (Fig. 2D). Both stimulations resulted in an ⬃2-fold increase in
sulfenic acid levels at the maximum time point.
In addition to total proteome changes, we identified specific proteins that underwent cysteine sulfenic acid formation. Multiple
studies have demonstrated that protein tyrosine phosphatases
(PTPs) undergo oxidation following activation (21, 22). To better
understand this process in naive T cells, we chose to measure the
kinetics of sulfenic acid formation in two PTPs important for T cell
activation: SHP-2 and SHP-1. After immunoprecipitation of
SHP-2 we observed an increase in cysteine sulfenic acid levels
within 5 min of TCR stimulation. These levels quickly declined by
15 min and were ROI dependent as shown by N-acetyl cysteine
treatment (Fig. 2E). A similar peak at 5 min was observed for
SHP-1, although labeling appeared to occur within 1 min of activation and could still be visualized at low levels by 15 min (Fig.
2F). We also measured the oxidation of actin, because glutathionylation is important for cell spreading and cytoskeleton reorganization (36). In contrast to the PTPs, sulfenic acid levels in actin
did not peak until 120 min after activation (Fig. 2G). Together,
these data establish that T cell activation is accompanied by increases in both ROI production and cysteine oxidation.
Dimedone incubation decreases CD8⫹ T cell proliferation after
incubation with peptide-coated splenocytes, anti-CD3 and
anti-CD28 Abs, or PMA and ION
To determine the requirement for reversible cysteine sulfenic acid
formation in naive CD8⫹ T cell proliferation, naive P14 splenocytes were incubated with their cognate peptide, GP33– 41 of
LCMV, in the presence of increasing concentrations of dimedone.
Dimedone prevents the further oxidation or reduction of cysteine
sulfenic acid-modified proteins through a covalent interaction. Unlike antioxidants, treatment of cells with dimedone did not decrease DCFDA oxidation following activation (data not shown).
Proliferation of CD8⫹ T cells was assessed by the loss of CFSE
fluorescence in comparison to undivided cells. In the absence of
dimedone pretreatment, no division was observed at 24 h regardless of stimulation (Fig. 3A). By 48 h after stimulation, control
cells had proliferated up to four divisions. At 0.5 and 1.0 mM
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FIGURE 1. Naive CD8⫹ T cells increase DCFDA oxidation after activation. Naive P14 splenocytes were stimulated with the indicated concentrations
of GP33– 41 peptide followed by incubation with DCFDA. CD8⫹ T cells were specifically analyzed by staining with anti-CD8␣ and DbGP33– 41. A,
DCFDA fluorescence at 15 min after stimulation with 10⫺7 M GP33– 41 peptide is plotted as a histogram compared with unstimulated cells. B, Data are
presented as the percentage increase in DCFDA MFI compared to unstimulated cells, with the average and SD shown. OVA257–264 is used as a noncognate
peptide control. C and D, CD8⫹ T cells were purified by magnetic microbeads from naive C57BL/6 mice. Cells were then stimulated with Ab or mitogen
before incubation with DCFDA. ␣CD3, Anti-CD3; ␣CD28, anti-CD28. Results are plotted as in B. At each time point, 5–9 mice were examined in a
minimum of two independent experiments. ⴱ, p ⱕ 0.05; significant difference between ex vivo and stimulated samples.
6460
ROLE OF CYSTEINE SULFENIC ACID IN T CELL ACTIVATION
dimedone there were minimal effects on proliferation. However, increasing the concentration from 2.5 to 10 mM decreased proliferation
of the cells. After 72 h of stimulation, ⬎90% of cells in the control
sample had divided. However, T cells incubated with dimedone exhibited a concentration-dependent inhibition of proliferation.
FIGURE 3. Dimedone incubation
decreases naive CD8⫹ T cell proliferation. Splenocytes were isolated
from naive P14 TCR transgenic mice,
labeled with CFSE, and incubated
with 10⫺7 M GP33– 41 peptide or purified and activated with anti-CD3
(␣CD3) and anti-CD28 (␣CD28) Abs
or PMA and ION and increasing concentrations of dimedone. Proliferation
(A) was assessed by loss of CFSE fluorescence after activation. Histograms were gated on CD8⫹DbGP33–
41⫹ cells, and a representative plot is
shown. The division (B) and proliferation (C) indices were calculated for
samples on day 3 after stimulation. At
each time point, 5– 8 mice were examined in a minimum of two independent experiments. The average
and SD are plotted. ⴱ, p ⱕ 0.05; significant difference between dimedone- and vehicle-treated samples.
Analysis of the division and proliferation indices on day 3 after
stimulation demonstrated a significant decrease in T cell proliferation at dimedone concentrations ⱖ2.5 mM. The division index is
defined as the average number of divisions that a cell has undergone, whereas the proliferation index is the average number of
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FIGURE 2. Cysteine sulfenic acid levels increase following naive CD8⫹ T cell activation. Naive CD8⫹ T cells were purified by magnetic microbeads
from C57BL/6 mice. Cells were then stimulated with anti-CD3 (␣CD3) and anti-CD28 (␣CD28) Abs (A, B, and D–G) or PMA and ION (C and D) for
the indicated time before lysis in the presence of biotin-linked dimedone derivative. A–C, Representative blots indicating total cysteine sulfenic acid (top
panel) and actin (bottom panel). B, Samples were treated with 10 mM N-acetyl cysteine (NAC) or 20 ␮M ebselen (EBS) before stimulation. D, Data are
depicted as the fold increase in cysteine sulfenic acid formation relative to unstimulated cells after normalization of the actin signal. Results are representative of three independent experiments. The average and SD are shown. ⴱ, p ⱕ 0.05; significant difference between stimulated and ex vivo samples.
E–G, SHP-2, SHP-1, or actin were immunoprecipitated (IP) following stimulation and biotin labeling. Samples were probed for sulfenic acid as described
above. The blot was then stripped and total protein is shown as a loading control.
The Journal of Immunology
6461
This indicates that the cysteine sulfenic acid formation that occurs
following naive CD8⫹ T cell activation plays a key role in regulating proliferation.
Dimedone incubation decreases CD4⫹ T cell proliferation after
incubation with peptide-coated splenocytes, anti-CD3 and
anti-CD28 Abs, or PMA and ION
Considering that cysteine sulfenic acid formation plays a key role
in naive CD8⫹ T cell proliferation, it was important to determine
whether the division of naive CD4⫹ T cells was also susceptible to
dimedone inhibition. The representative histograms in Fig. 5A
demonstrate that naive CD4⫹ T cell proliferation was decreased in
a concentration-dependent manner by dimedone. Similarly as for
CD8⫹ T cells, this inhibition was observed in response to antiCD3 and anti-CD28 or PMA and ION stimulation (Fig. 5, B and
C). However, proliferation due to peptide stimulation was slightly
increased at low concentrations of dimedone. Thus, reversible cysteine sulfenic acid formation plays a key role in the proliferation of
both naive CD8⫹ and CD4⫹ T cells.
divisions that those cells that divided underwent. Fig. 3, B and C
demonstrate that control-treated cells possess a division index of
3.0 and a proliferation index of 4.2 when stimulated with 10⫺7 M
GP33– 41 peptide. At 0.5 and 1.0 mM dimedone the indices were
not affected, but as the concentration increased the division and
proliferation indices decreased, indicating that both the initial and
subsequent divisions of the cells were inhibited.
To determine whether dimedone was functioning in an autonomous manner, purified CD8⫹ T cells were activated with anti-CD3
and anti-CD28 Abs or PMA and ION. By day 3, the proliferation
and division indices of the control samples were comparable to
those of the peptide stimulation (Fig. 3, B and C). In addition, a
similar concentration-dependent inhibition of proliferation was observed when dimedone was present during either stimulation.
Thus, the treatment of cells with dimedone, a compound that binds
irreversibly to cysteine sulfenic acid, decreases CD8⫹ T cell proliferation in a concentration-dependent and autonomous manner.
Inhibition of naive CD8⫹ T cell proliferation by dimedone
occurs at an early stage following T cell activation
Even though DCFDA oxidation and sulfenic acid levels increase
upon naive T cell activation, it is possible that the inhibition of
proliferation is due to the reaction of dimedone with the cysteine
sulfenic acid-modified proteins present in unactivated T cells. To
address this, purified naive CD8⫹ T cells were pretreated with
dimedone for 1 h as described above. Afterward, one set of samples was stimulated with PMA and ION in the continuous presence
of dimedone (Fig. 4, A and B). These samples displayed a similar
proliferative profile as those in Fig. 3. A duplicate set of pretreated
samples were washed three times in complete medium to remove
the dimedone before stimulation with PMA and ION. Analysis of
the division and proliferation indices showed that there was no
significant difference between the washed and the control samples.
Dimedone decreases PMA and ION-stimulated S phase entry in
naive CD8⫹ T cells
To determine the mechanism of dimedone inhibition of CD8⫹ T
cell proliferation, cell cycle progression was analyzed by measuring BrdU and 7-AAD incorporation. BrdU is an analog of the
DNA precursor thymidine and is incorporated into newly synthesized DNA in cells progressing through S phase. 7-AAD is a fluorescent dye that binds to nucleic acid.
Purified naive CD8⫹ T cells were stimulated for 24 h with PMA
and ION in the presence or absence of dimedone. During the last
45 min of stimulation the cells were pulsed with BrdU to measure
DNA synthesis. When the cells were activated in the absence of
dimedone, 27% of the cells were progressing through S phase
compared with only 0.3% of the unstimulated cells (Fig. 6, A and
B). At 0.5–2.5 mM dimedone there were minimal effects on the
percentage of cells in S phase. However, at 5 and 10 mM there was
a decrease in the percent of cells in S phase to 15.7 and 3% respectively. Thus, reversible cysteine sulfenic acid plays a role in
regulating S phase entry.
Dimedone does not alter the survival of PMA and
ION-stimulated naive CD8⫹ T cells
To determine whether reduced cell division and S phase entry were
due to cell death, naive CD8⫹ T cells were purified from C57BL/6
mice and stimulated for 24 h with PMA and ION. Cells were
incubated with increasing concentrations of dimedone, and viability at 24 h was determined by trypan blue exclusion. Directly ex
vivo there was a decrease in cell number (5 ⫻ 105 to 3.1 ⫻ 105)
after PMA and ION activation (Fig. 7A) that was similar to previously published results (16). As the concentration of dimedone
increased there was no significant decrease in the number of viable
cells, indicating that dimedone does not alter the initial survival of
activated CD8⫹ T cells.
Dimedone decreases mitogen-induced naive CD8⫹ T cell growth
When naive CD8⫹ T cells become activated they undergo an increase in cell size termed blasting. Because the proliferation of T
cells was inhibited by dimedone, the role of cysteine sulfenic acid
in the mitogen-induced program of cell growth was examined in
purified CD8⫹ T cells 24 h after activation with PMA and ION. In
the absence of stimulation, T cells remained small as indicated by
small forward scatter (Fig. 7B). Twenty-four hours after stimulation, the control samples exhibited a large increase in forward scatter that was reflective of T cell blasting. As the concentration of
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FIGURE 4. Inhibition of naive CD8⫹ T cell proliferation by dimedone
is reversible. CD8⫹ T cells were purified by magnetic microbeads and
stimulated with PMA and ION in the absence or presence of dimedone
(continuous). A duplicate set was incubated with dimedone for 1 h and then
washed three times before stimulation (removed at t ⫽ 0). The division (A)
and proliferation (B) indices at day 3 are shown. At each time point, five
mice were examined in two independent experiments. The average and SD
are shown. ⴱ, p ⱕ 0.05; significant difference between dimedone- and
vehicle-treated samples.
6462
ROLE OF CYSTEINE SULFENIC ACID IN T CELL ACTIVATION
dimedone increased, the size of cells decreased, indicating that
reversible cysteine sulfenic acid plays a role in T cell blasting.
To determine the mechanism responsible for decreased blasting,
we measured protein levels in T cells following PMA and ION stimulation. Twenty-four hours after activation we observed that the protein levels increased from 22 to 40 ␮g per 106 cells between the ex
vivo and control samples (Fig. 7C). In dimedone-treated cells the total
amount of protein was similar to that of the control at the 0.5 and 1.0
mM concentration. As the concentration increased, there was a de-
crease in total protein levels so that at 10 mM there was no significant
difference between the unstimulated and stimulated cells. Thus, reversible cysteine sulfenic acid formation is critical to T cell blasting
and increases in steady-state protein levels.
Dimedone incubation does not decrease ZAP70 phosphorylation
following TCR stimulation
To determine the signaling processes that require reversible
sulfenic acid formation, we examined ZAP70 phosphorylation in
FIGURE 6. Dimedone decreases naive CD8⫹ T cell S phase entry. CD8⫹ T cells were purified by magnetic microbeads from naive mice and were
stimulated (Stim) for 24 h with PMA and ION in the presence or absence of dimedone. During the last 45 min of stimulation, BrdU was added to each
culture. Cells were stained with anti-BrdU Ab and 7-AAD. A, Representative dot plot of BrdU and 7-AAD staining is shown. The top gate indicates the
percentage of cells in S phase. B, Data are depicted as the percentage of cells that are in S phase and are representative of five mice in two independent
experiments. The average and SD are shown. ⴱ, p ⱕ 0.05; significant difference between dimedone- and vehicle-treated samples.
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FIGURE 5. Dimedone incubation
decreases naive CD4⫹ T cell proliferation. Splenocytes were isolated
from naive OT-2 TCR transgenic
mice, labeled with CFSE, and incubated with 10⫺6 M OVA323–339
peptide or purified and activated with
anti-CD3 (␣CD3) and anti-CD28
(␣CD28) Abs or PMA and ION and
increasing concentrations of dimedone. Proliferation (A) was assessed
by loss of CFSE fluorescence after activation. Histograms were gated on
CD4⫹ cells and a representative plot
is shown. The division (B) and proliferation (C) indices were calculated
for samples on day 3 after stimulation. At each time point, five mice
were examined in two independent
experiments. The average and SD are
shown. ⴱ, p ⱕ 0.05; significant difference between dimedone- and vehicletreated samples.
The Journal of Immunology
6463
the presence of dimedone. Phosphorylation of ZAP70 is a key
early step in T cell activation that increases the kinase activity of
the protein. Intracellular staining of phospho-ZAP70 revealed that
there was an ⬃1.7-fold increase in phosphorylation within 2 min
of TCR stimulation (Fig. 8A). Treatment of cells with 10 mM
dimedone did not decrease the phosphorylation of ZAP70. H2O2
was used as positive control for ZAP70 phosphorylation because it
decreases total phosphatase activity. Examining phosphorylation
kinetics demonstrated that except for a slight delay at 30 s, phospho-ZAP70 was similar between vehicle- and dimedone-treated
samples for the duration of the experiment (Fig. 8B). Thus, reversible cysteine sulfenic acid formation is not required for ZAP70
phosphorylation.
ERK1/2 phosphorylation is decreased following
dimedone incubation
To determine whether MAPK signaling, a critical component of T
cell proliferation and differentiation, was affected by reversible
cysteine sulfenic acid formation, we performed intracellular staining to detect the phosphorylation of ERK1/2, JNK, and p38 following PMA and ION stimulation. Before activation, isotype and
phospho-ERK1/2 staining were overlapping (Fig. 8C). Fifteen
minutes after stimulation, phospho-ERK1/2 staining increased to
levels at least 3-fold higher than those found in unstimulated cells
(Fig. 8D). This fold change was similar to that in previously published studies (37, 38). As the concentration of dimedone increased, the level of ERK1/2 phosphorylation decreased until at 10
mM it was comparable to the levels found in unstimulated cells.
The decrease in ERK1/2 phosphorylation was still present at 60
min after stimulation. Thus, reversible cysteine sulfenic acid formation plays a role in the ERK1/2 phosphorylation during T cell
activation.
Phosphorylation of JNK and p38 were also measured in the
presence of dimedone. Fig. 8E demonstrates within 15 min after
PMA and ION stimulation there was an ⬃1.6 and 1.8-fold increase
in the phosphorylation of p38 and JNK, respectively, which was
similar to that in previous reports (38). As the concentration of
dimedone increased, there was no significant decrease in JNK or
p38 phosphorylation. These results indicate that reversible cysteine
sulfenic acid formation plays a key role in the phosphorylation of
ERK1/2, whereas the phosphorylations of p38 and JNK are less
sensitive.
Dimedone treatment decreases calcium flux
Because reversible cysteine sulfenic acid formation plays a role in
the phosphorylation of ERK1/2, we examined whether other components of signal transduction were affected by dimedone treatment. Because calcium flux is critical for naive T cell activation,
we examined how dimedone affected this signaling event. To measure calcium flux, purified CD8⫹ T cells were incubated with
Fluo-3-AM to detect intracellular calcium levels. Fluo-3-AM is a
membrane-permeable dye that is hydrolyzed by cellular esterases
to release the calcium sensitive form, Fluo-3. Unstimulated cells
had a basal level of fluorescence that increased rapidly after the
addition of PMA and ION and was sustained throughout the experiment (Fig. 8F). In contrast, cells in the presence of 5 mM
dimedone only exhibited a small increase in intracellular calcium.
As the concentration of dimedone was increased to 10 mM, calcium levels did not increase. Thus, reversible cysteine sulfenic
acid formation plays a role in calcium flux following initial T cell
stimulation.
Dimedone incubation differentially alters the production of
cytokines by effector and memory CD8⫹ P14 T cells
Vigorous CD8⫹ T cell responses are characterized by clonal expansion and cytokine production. From the experiments described
above, we determined that dimedone incubation blocked clonal
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FIGURE 7. Dimedone does not alter in vitro survival of CD8⫹ T cells but inhibits T cell blasting and increases in steady-state protein levels. CD8⫹ T
cells were purified by magnetic microbeads and activated with PMA and ION in the presence or absence of dimedone for 24 h. A, At 24 h, cultures were
harvested and viability was determined by a trypan blue exclusion assay. B, T cell blasting was determined by staining with anti-CD8␣ Ab and forward
scatter was determined and plotted as a histogram. C, Protein levels were determined by bicinchoninic acid (BCA) assay. At each time point 5 or 6 mice
were examined in a minimum of two independent experiments. The average and SD are shown. ⴱ, p ⱕ 0.05; significant difference between ex vivo and
stimulated samples. Stim, Stimulation.
6464
ROLE OF CYSTEINE SULFENIC ACID IN T CELL ACTIVATION
expansion and signal transduction of naive CD8⫹ T cells, implicating reversible cysteine sulfenic acid formation in these processes. To determine whether reversible cysteine sulfenic acid formation is important for effector and memory cell function, we
generated these cells in vivo from naive P14 mice. This was accomplished by the adoptive transfer of naive P14 CD8⫹CD90.1⫹
T cells into naive CD90.2⫹ C57BL/6 mice followed by acute infection with LCMV-Armstrong. Eight days after infection the
spleen was harvested to isolate effector cells. At this time there was
massive expansion of the P14 cells such that 54% of the CD8⫹ T
cells were DbGP33– 41⫹CD44⫹ (Fig. 9A). Staining with CD90.1
Abs revealed that 98% of these cells were derived from transgenic
precursors.
To measure effector cell function, IFN-␥ and TNF-␣ were measured in transgenic CD8⫹ T cells following 5 h of restimulation
with 10⫺7 M GP33– 41 in the presence or absence of dimedone.
The top panel in Fig. 9B demonstrates that in the absence of peptide stimulation few CD8⫹ CD90.1⫹ T cells made either IFN-␥ or
TNF-␣. Following GP33– 41 stimulation, vehicle-treated samples
were 96% positive for IFN-␥ and 75% of the cells were producing
both IFN-␥ and TNF-␣. 〈s dimedone was increased, the percentage of TNF-␣⫹ cells decreased by 32% compared with the vehicle.
Additionally, there was no difference in the number of transgenic
or total cells recovered after GP33– 41 stimulation regardless of
dimedone concentration (data not shown).
To determine whether cytokine production was different on a
per cell basis, the mean fluorescence intensity (MFI) of IFN-␥ and
TNF-␣ were quantitated. Plotting the data as a percentage of the
vehicle MFI, the IFN-␥ MFI at 10 mM dimedone was 70% of the
vehicle (Fig. 9C). Although there was a small decrease in IFN-␥
production on a per cell basis, there was no difference in the total
number of CD8⫹ CD90.1⫹ T cells producing IFN-␥ (data not
shown). In contrast, the TNF-␣ MFI exhibited a slight increase at
0.5–2.5 mM dimedone in comparison to the control. By 5 mM
dimedone, the intensity of TNF-␣ staining decreased to vehicle
levels and continued declining until the MFI was only 42% of the
control at 10 mM. These results demonstrate that in effector CD8⫹
T cells the production of TNF-␣ is more sensitive to reversible
cysteine sulfenic acid formation than IFN-␥.
Following acute viral infection, T cells undergo a period of massive contraction, with only a small subset of effector cells surviving and differentiating into memory cells. These cells posses the
ability to rapidly respond to Ag by producing high levels of both
IFN-␥ and TNF-␣. To determine whether the rapid production of
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FIGURE 8. Dimedone incubation decreases ERK1/2 phosphorylation and calcium flux. CD8⫹ T cells were purified by magnetic microbeads and
activated with anti-CD3 (␣CD3) and anti-CD28 (␣CD28) Abs (A and B) or PMA and ION (C–F) in the presence or absence of dimedone. A, Cells were
harvested directly ex vivo or after 2 min of stimulation and stained for phospho-ZAP70. Incubation with 200 ␮M H2O2 for 10 min was used as a positive
control. Numbers in the upper left-hand corners indicate the MFI of unstimulated and stimulated samples. B, Data are presented as the fold increase in
phospho-protein induction compared with unstimulated cells. For MAPK measurement, cells were harvested after 15 (C–E) or 60 min (D) of stimulation
and stained with anti-phopho-ERK1/2 (C and D), anti-phospho JNK (E), or anti-phospho p38 (E). Graphs are representative of 4 – 6 mice in a minimum
of two independent experiments. The average and SD are shown. ⴱ, p ⱕ 0.05; significant difference between dimedone- and vehicle-treated samples. F,
Purified CD8⫹ T cells were incubated with Fluo-3-AM (Fluo-3) and activated with PMA and ION in the presence or absence of dimedone. The fluorescence
of Fluo-3-AM is plotted as a function of time. The arrow indicates the time of addition of PMA and ION. The histogram is representative of four mice
in two independent experiments.
The Journal of Immunology
6465
cytokines by memory CD8⫹ T cells requires reversible cysteine
sulfenic acid formation, we incubated splenocytes from LCMVimmune mice with GP33– 41 peptide in the presence or absence of
dimedone. When splenocytes were harvested beyond day 60 after
infection, ⬃6% of cells were CD8⫹ DbGP33– 41⫹. Staining for
the congenic marker CD90.1 showed that 97% of this population
was derived from the initially transferred P14 cells.
Following peptide restimulation, here was a slight decrease in
the MFI of IFN-␥ in the dimedone-treated samples compared with
that of the vehicle (Fig. 9D). When TNF-␣ production was examined in memory cells, a trend similar to that of the effector cells
was observed. At 0.5–5 mM dimedone cells exhibited a small increase in TNF-␣ production. However, the TNF-␣ MFI at 10 mM
was only 68% of the vehicle. Additional analysis found that the
decrease in TNF-␣ production at 10 mM dimedone was significantly greater in effector cells compared with memory CD8⫹ T
cells. Thus, TNF-␣ production is more sensitive to reversible cysteine sulfenic acid formation than IFN-␥ in both Ag-specific effector and memory CD8⫹ T cells.
Discussion
In this study we have examined the role of reversible cysteine
sulfenic acid formation in the activation, proliferation, and function of CD8⫹ T cells. We demonstrate that ROI generated during
Ag and mitogen-induced stimulation are associated with increased
cysteine sulfenic acid levels during naive CD8⫹ T cell activation.
Using dimedone to covalently bind cysteine sulfenic acid showed
that reversible formation was important for naive T cell division
and S phase entry. In addition to proliferative events, we observed
that reversible cysteine sulfenic acid formation played a role in
several early aspects of T cell activation, including ERK1/2 phosphorylation and calcium flux. Effector and memory cell cytokine production were also affected by blocking the reversible formation of
cysteine sulfenic acid, demonstrating that TNF-␣ production was
more sensitive to inhibition than that of IFN-␥ in both cell types.
Interaction of the TCR and its cognate Ag initiates a complex
signal transduction cascade inside the naive T cell. Over the last 20
years a large amount of data has emerged documenting the extensive changes in protein phosphorylation, localization, and interaction that occur following activation (39, 40). Only recently has the
role of ROI and their effects on T cell biology begun to be identified. Previous studies have found that, following TCR or mitogen-induced stimulation, human T cell blasts and T cell hybridomas increase their ROI levels (11). In an earlier report we
extended this finding by showing that Ag-specific CD8⫹ T cells
isolated directly ex vivo from LCMV-infected mice had increased
levels of superoxide (41). Increased levels of ROI have also been
observed in lymphocytes from systemic lupus erythematosus patients compared with those found in healthy controls (42). In this
study we expand upon these findings by demonstrating that the
generation of ROI in naive CD8⫹ T cells is strongly related to the
magnitude of the stimulus. For the first time to our knowledge, we
document that the production of ROI in naive CD8⫹ T cells is
proportional to Ag concentration. When naive P14 cells were stimulated with 10⫺7 M GP33– 41 there was a rapid and sustained
increase in DCFDA oxidation, whereas cells incubated with 10⫺9
M peptide took longer to reach maximal levels of oxidation. This
contrasts with cells incubated with 10⫺10 M peptide, where no
increase in ROI was detected. The decreased production of ROI
could be due to decreased TCR engagement or increased time for
naive T cells to encounter their cognate Ag. Matsue et al. (43)
demonstrated that naive CD4⫹ T cells activated with high concentrations of peptide had increased DCFDA oxidation at 6 h compared with cells activated with lower doses. Differences in our
observations compared with theirs could be due to inherent differences between naive CD8⫹ and CD4⫹ T cells, as the time it takes
to reach maximal production of ROI in naive CD4⫹ T cells following PMA and ION activation is greater than that of CD8⫹ T
cells (R. Michalek, J. Yi, and J. Grayson, unpublished observations). In addition to ROI production being controlled through the
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FIGURE 9. Dimedone incubation
differentially alters the production of
cytokines by effector and memory
CD8⫹ P14 T cells. Effector and memory cells were generated by adoptive
transfer of P14 splenocytes, such that
104 naive P14 CD8⫹ T cells became
engrafted. Four hours after transfer,
mice were infected with LCMV-Armstrong and sacrificed on either day 8 for
effector or beyond day 60 for memory
CD8⫹ T cells. A, Day 8 splenocytes
were isolated and stained with antiCD8␣, DbGP33– 41 MHC class I tetramer, and either anti-CD44 or antiCD90.1 Abs. B, Splenocytes were
incubated with 10⫺7 M GP33– 41 peptide and varying concentrations of
dimedone for 5 h, stained with antiCD8␣,anti-CD90.1,anti-IFN-␥,andantiTNF-␣ Abs. C and D, Samples were
gated on CD8␣⫹ Thy1.1⫹ cells and the
percentage of the vehicle cytokine MFI
was plotted for effector and memory
cells, respectively. The average and SD
are shown for six mice in two independent experiments. ⴱ, p ⱕ 0.05; significant difference between dimedone- and
vehicle-treated samples.
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ROLE OF CYSTEINE SULFENIC ACID IN T CELL ACTIVATION
Our studies suggest a differential requirement for ROI production and cysteine sulfenic acid formation depending on the specific
stage of T cell development. In naive CD8⫹ T cells, reversible
cysteine sulfenic acid formation plays a key role in activation and
proliferation. Some early events, such as ZAP70, JNK, and p38
phosphorylation, are not sensitive to reversible sulfenic acid formation, whereas ERK1/2 phosphorylation and Ca2⫹ flux require it.
Effector CD8⫹ T cells did not exhibit a strong decrease in cytokine
production until the highest concentration of dimedone. Memory
CD8⫹ T cells were even less sensitive to cytokine inhibition by
dimedone than effector cells. In both effector and memory cells,
low concentrations of dimedone had a positive effect by slightly
increasing TNF-␣ production. This differential sensitivity to reversible cysteine sulfenic acid formation is similar to that reported
in earlier studies, which implied that differentiated T cells may be
less dependent on ROI. Laniewski and Grayson (20) found that
day 8 effector CD8⫹ T cells produced similar levels of cytokines
when cultured in the presence or absence of the antioxidant manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP).
They also demonstrated that although antioxidant treatment could
reduce T cell expansion when present during a primary CD8⫹ T
cell response, proliferation and cytokine production during the secondary CD8⫹ T cell response were not affected. These results,
combined with our findings, suggest that effector and memory
CD8⫹ T cells have a smaller dependence on ROI production and
reversible cysteine sulfenic acid formation than naive CD8⫹ T
cells.
Because dimedone forms an adduct upon binding to cysteine
sulfenic acid, the possibility that it provides a neo-function to the
protein cannot be excluded. However, in previous studies dimedone binding to cysteine sulfenic acid has promoted the inhibition
of specific enzymes. Benitez and Allison (52) showed that treatment of the sulfenic acid form of GAPDH with dimedone completely inactivated the acyl phosphate reaction catalyzed by the
oxidized enzyme. In the case of papain, Allison (53) demonstrated
that sulfenic acid formation and the subsequent dimedone binding
inhibited the protein’s active site. In addition to these specific examples, it is important to reiterate that dimedone can only bind to
a protein that has already formed cysteine sulfenic acid. Our removal experiment demonstrates that the cysteine sulfenic acid formation events that occur following T cell activation are critical for
proliferation.
In conclusion, our studies demonstrate that reversible cysteine
sulfenic acid formation is an important process during naive T cell
activation. Preventing further oxidation or reduction of cysteine
sulfenic acid inhibits cellular signaling and proliferation of naive
CD8⫹ and CD4⫹ T cells. In addition, TNF-␣ production in effector and memory CD8⫹ T cells is more dependent on reversible
cysteine sulfenic acid formation than IFN-␥. Understanding that
cysteine sulfenic acid plays a role in these cellular processes allows a focus on identifying proteins in these pathways that are
modulated by oxidation. Further studies will provide insight into
the regulation of T cell activation and may ultimately be applied to
improved vaccine and autoimmunity therapy.
Disclosures
The authors have no financial conflict of interest.
References
1. Alexander-Miller, M. A. 2005. High-avidity CD8⫹ T cells: optimal soldiers in
the war against viruses and tumors. Immunol. Res. 31: 13–24.
2. Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell
differentiation: implications for vaccine development. Nat. Rev. Immunol. 2:
251–262.
3. Wherry, E. J., and R. Ahmed. 2004. Memory CD8 T-cell differentiation during
viral infection. J. Virol. 78: 5535–5545.
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TCR, we also document that signaling through CD28 induces transient ROI production. These results reinforce the idea that while
some aspects of T cell activation such as up-regulation of glycolysis, Bcl-xL, and IL-4 (44 – 46) are more directly regulated by
costimulatory signaling, other events such as IL-2 and IFN-␥ production use costimulation to amplify signals from the TCR (47).
In recent years a critical role for ROI in regulating cellular signaling has emerged. Although multiple mechanisms may control
signaling, reversible oxidation of cysteine would allow cells to
modulate protein activity. For example, multiple studies have
shown that PTPs such as PTEN, SHP-2, and Cdc25C can be inactivated by oxidation (21, 22, 48). In many of these enzymes,
oxidation of the active site cysteine decreases enzymatic activity.
Additionally, some transcription factors are regulated by cysteine
oxidation. Abate et al. (24) demonstrated that the DNA binding
activity of c-Fos and c-Jun is sensitive to reversible cysteine oxidation at amino acids 154 and 252, respectively. NF-␬B is also
subject to redox regulation on cysteine 62 on the p50 subunit (49).
The DNA binding activity of the transcription factor is inhibited by
oxidation (23). Although cysteine oxidation inhibits the function of
some proteins, in others it promotes activation. In the case of protein kinase C, oxidation by a superoxide stimulates enzymatic activity by thiol oxidation and the subsequent release of zinc from a
cysteine-rich region in the amino terminus (50). Thus by modifying one amino acid in a reversible manner, cells are able to modulate signaling.
Few studies have focused on determining the oxidative modifications that are essential for naive T cell activation. Most reports
have used exogenously added high concentrations of H2O2, antioxidants, or overexpressed enzymes to alter ROI levels and to
determine the effects on T cell activation. These methods make it
difficult to focus on the contribution of one specific oxidative modification. Using these techniques also alters the normal levels of
ROI during activation. By using dimedone, we have selectively
focused on the contribution of reversible cysteine sulfenic acid
formation during T cell activation without altering ROI production.
Unlike previously published sulfenic acid detection techniques requiring complex masking and reduction reactions, we were able to assess
cysteine sulfenic acid levels in one direct step using a biotin-linked
derivative of dimedone (54). Our results are the first to show that the
total levels of cysteine sulfenic acid rise during naive CD8⫹ T cell
activation. The 2-fold increase we observed in the total levels of
sulfenic acid was comparable to an earlier report where rat hearts were
perfused with 10 mM H2O2 before sulfenic acid detection (51).
Aside from the increase in the total proteome, we observed that
sulfenic acid levels increased in the PTPs SHP-1 and SHP-2 following activation. A previous study (22) using transformed Jurkat
T cells reported that SHP-2 cysteine oxidation occurs within 5 min
of TCR stimulation. Our finding is the first evidence that naive
CD8⫹ T cells also oxidize SHP-2 following T cell activation. The
authors also examined SHP-1, but were only able to detect oxidation following 1 mM H2O2 treatment. In contrast, we observed
sulfenic acid formation in SHP-1 following receptor stimulation. It
is possible that our labeling method and cell choice may account
for the difference between the two studies. We also observed that
actin oxidation occurred with differential kinetics compared with
the PTPs. It has been previously shown in mouse fibroblasts that
cysteine 374 in actin is sensitive to oxidation and that this modification plays a role in leading to glutathionylation of the protein
(36). Glutathionylation of actin is required for spreading and cytoskeleton organization. Dimedone binding to cysteine sulfenic
acid in actin could therefore inhibit this modification. Taken together, these results suggest that cells tightly regulate sulfenic acid
levels during signal transduction.
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