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The Journal of Immunology, 2007, 179, 6456 -6467
Copyright © 2007 by The American Association of Immunologists, Inc.
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*CYSTEINE

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function1

Ryan D. Michalek*, Kimberly J. Nelson{dagger}, Beth C. Holbrook*, John S. Yi*, Daya Stridiron*, Larry W. Daniel{dagger}, Jacquelyn S. Fetrow§, S. Bruce King{ddagger}, Leslie B. Poole{dagger} and Jason M. Grayson2,*

* Department of Microbiology and Immunology and {dagger} Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157; and {ddagger} Department of Chemistry and § Department of Computer Science and Department of Physics, Wake Forest University, Winston-Salem, NC 27109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 CD8+ T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8+ 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 CD8+ and CD4+ T cells. We also found that TNF-{alpha} production by effector and memory CD8+ T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-{gamma}. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8+ T cells are able to modulate signaling, proliferation, and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T cell is a critical component of the immune system’s response to infectious agents and tumors (1, 2, 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, 5, 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 activation, phagocytic cells such as neutrophils and macrophages increase the production of OFormula 2, H2O2, and NO (NO·) in preparation for the respiratory burst. This process is vital to innate immunity because individuals who suffer from chronic granulomatous disease, in which the O2–-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, 14, 15, 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-{kappa}B and AP-1, use reversible cysteine oxidation to modulate protein function (21, 22, 23, 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-{alpha} 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-{gamma}. These studies illustrate the important role of reversible cysteine sulfenic acid formation in regulating the activation, proliferation, and function of CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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{alpha}, 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 x 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 x 107 cells/ml in PBS. The CFSE stock was diluted to 10 µM in PBS and mixed with cells 1:1 (v/v), resulting in a final concentration of 5 µM 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 µM 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 µM 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{alpha} 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 x 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 µg/ml, respectively. For CD3/CD28 stimulation experiments, 96-well flat-bottom plates were coated with 10 µg/ml anti-CD3 and anti-CD28 or 20 µg/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 µg/ml anti-CD3 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 LCMV-Armstrong-infected mice on days 8 and >60, respectively. Intracellular cytokine staining was performed as described below. For restimulation, 1 x 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 x 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 µM 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 β-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 µg/ml aprotinin, and 10 µg/ml leupeptin (pH 8.0). Lysates were immediately sonicated and incubated at room temperature for 10 min. N-ethyl maleimide at 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 x 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 µg/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

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.

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 anti-rabbit (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 two-tailed Student’s t test, and p ≤ 0.05 was considered significant.

Surface and intracellular staining

In this study the following Abs were used: rat anti-mouse CD8{alpha}-PE; rat anti-mouse CD8{alpha}-PerCP; rat anti-mouse CD90.1-FITC; rat anti-mouse IFN-{gamma}-allophycocyanin; rat anti-mouse TNF-{alpha}-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 phospho-p38 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.

Viral infection and mice

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 µM 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 x 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 x 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 µl of 7-AAD (Pharmingen) plus FACS buffer for 10 min on ice. Samples were acquired immediately using a FACSCalibur instrument.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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).


Figure 1
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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{alpha} 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. {alpha}CD3, Anti-CD3; {alpha}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. *, p ≤ 0.05; significant difference between ex vivo and stimulated samples.

 
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 anti-CD28 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, 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, platelet-derived growth factor (PGDF), basic fibroblast growth factor (bFGF), and TNF-{alpha} (31, 32, 33, 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.


Figure 2
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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 ({alpha}CD3) and anti-CD28 ({alpha}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 µM 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. *, p ≤ 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.

 
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 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
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  		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 ({alpha}CD3) and anti-CD28 ({alpha}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. *, p ≤ 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 ≥2.5 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 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. This indicates that the cysteine sulfenic acid formation that occurs following naive CD8+ T cell activation plays a key role in regulating proliferation.


Figure 4
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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. *, p ≤ 0.05; significant difference between dimedone- and vehicle-treated samples.

 
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 anti-CD3 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.


Figure 5
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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 ({alpha}CD3) and anti-CD28 ({alpha}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. *, p ≤ 0.05; significant difference between dimedone- and vehicle-treated 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.


Figure 6
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  		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. *, p ≤ 0.05; significant difference between dimedone- and vehicle-treated samples.

 
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 x 105 to 3.1 x 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.


Figure 7
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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{alpha} 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. *, p ≤ 0.05; significant difference between ex vivo and stimulated samples. Stim, Stimulation.

 
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 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 µg 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 decrease 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 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.


Figure 8
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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 ({alpha}CD3) and anti-CD28 ({alpha}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 µM 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. *, p ≤ 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.

 
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 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.


Figure 9
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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 anti-CD8{alpha}, DbGP33–41 MHC class I tetramer, and either anti-CD44 or anti-CD90.1 Abs. B, Splenocytes were incubated with 10–7 M GP33–41 peptide and varying concentrations of dimedone for 5 h, stained with anti-CD8{alpha}, anti-CD90.1, anti-IFN-{gamma}, and anti-TNF-{alpha} Abs. C and D, Samples were gated on CD8{alpha}+ 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. *, p ≤ 0.05; significant difference between dimedone- and vehicle-treated samples.

 
To measure effector cell function, IFN-{gamma} and TNF-{alpha} 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-{gamma} or TNF-{alpha}. Following GP33–41 stimulation, vehicle-treated samples were 96% positive for IFN-{gamma} and 75% of the cells were producing both IFN-{gamma} and TNF-{alpha}. As dimedone was increased, the percentage of TNF-{alpha}+ 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-{gamma} and TNF-{alpha} were quantitated. Plotting the data as a percentage of the vehicle MFI, the IFN-{gamma} MFI at 10 mM dimedone was 70% of the vehicle (Fig. 9C). Although there was a small decrease in IFN-{gamma} production on a per cell basis, there was no difference in the total number of CD8+ CD90.1+ T cells producing IFN-{gamma} (data not shown). In contrast, the TNF-{alpha} 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-{alpha} 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-{alpha} is more sensitive to reversible cysteine sulfenic acid formation than IFN-{gamma}.

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-{gamma} and TNF-{alpha}. To determine whether the rapid production of cytokines by memory CD8+ T cells requires reversible cysteine sulfenic acid formation, we incubated splenocytes from LCMV-immune 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-{gamma} in the dimedone-treated samples compared with that of the vehicle (Fig. 9D). When TNF-{alpha} 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-{alpha} production. However, the TNF-{alpha} MFI at 10 mM was only 68% of the vehicle. Additional analysis found that the decrease in TNF-{alpha} production at 10 mM dimedone was significantly greater in effector cells compared with memory CD8+ T cells. Thus, TNF-{alpha} production is more sensitive to reversible cysteine sulfenic acid formation than IFN-{gamma} in both Ag-specific effector and memory CD8+ T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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-{alpha} production was more sensitive to inhibition than that of IFN-{gamma} 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 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, 45, 46) are more directly regulated by costimulatory signaling, other events such as IL-2 and IFN-{gamma} 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-{kappa}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.

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-{alpha} 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-{alpha} production in effector and memory CD8+ T cells is more dependent on reversible cysteine sulfenic acid formation than IFN-{gamma}. 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.). Back

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{at}wfubmc.edu Back

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, phosphatase and tension homology deleted on chromosome 10; PTP, protein tyrosine phosphatase; SHP, Src homology 2 domain-containing phosphatase. Back

Received for publication January 9, 2007. Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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