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The Journal of Immunology, 2006, 177: 852-862.
Copyright © 2006 by The American Association of Immunologists

Electron Transport Complex I Is Required for CD8+ T Cell Function1

John S. Yi, Beth C. Holbrook, Ryan D. Michalek, Nathan G. Laniewski and Jason M. Grayson2

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
After Ag encounter, CD8+ T cells become activated and begin to proliferate. Early during infection, when Ag-specific effector CD8+ T cells are proliferating, producing cytokines, and lysing infected cells in vivo, their mitochondrial potential is increased. The purpose of the experiments presented here was to determine whether mitochondrial function was required for CD8+ T cell function. To block mitochondrial function, transgenic CD8+ T cells were incubated with increasing doses of rotenone, an inhibitor of electron transport complex I. Within minutes of T cell activation, rotenone incubation decreased the production of H2O2, calcium flux, and ERK1/2 phosphorylation. Failure to undergo signal transduction resulted in a decrease in T cell division initiated by peptide-coated cells, CD3/CD28 Abs, and PMA/ionomycin stimulation. Decreased function following rotenone incubation was not restricted to naive cells, as effector and memory CD8+ T cells isolated directly ex vivo from lymphocytic choriomeningitis virus-infected mice displayed decreased production of IFN-{gamma} and TNF-{alpha} production after peptide stimulation. Furthermore, incubation with rotenone decreased degranulation of effector and memory cells, a critical step in the cytolysis of infected cells. These data suggest that electron transport complex I is required for CD8+ T cell signal transduction, proliferation, cytokine production, and degranulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
For the clearance of intracellular pathogens such as viruses, certain bacteria, and tumors, CD8+ T cells are critical (1, 2, 3). Following activation by an APC in the lymph nodes or spleen, naive CD8+ T cells initiate a new program of gene expression and differentiate into effector cells (4, 5, 6). This program is accompanied by increases in protein synthesis, cell growth, and eventual cell division (7). After 24 h, CD8+ T cells undergo a rapid expansion, dividing every 4–6 h (8, 9). This is necessary to increase the precursor frequency of virus-specific cells from an estimated 1:100,000 CD8+ T cells in uninfected mice (10) to ~1:2 CD8+ T cells found in lymphocytic choriomeningitis virus (LCMV)3-infected mice (11, 12). Although these cells are increasing in numbers, they also elaborate effector functions such as cytokine production and cytolysis of infected cells. CD8+ T cell effector function is critical for the clearance of many pathogens, including LCMV, vaccinia virus (13, 14), and Listeria monocytogenes in mice (15, 16), and it is thought to be critical in controlling HIV infection in humans (17). After pathogen clearance, the vast majority of effector cells undergo apoptosis by a process termed contraction, with the remaining CD127 high effector cells differentiating into memory cells (18). These cells can provide lifelong immunity to disease upon reinfection and, in the absence of Ag, undergo a slow homeostatic proliferation that is controlled by the cytokines IL-7 and IL-15 to maintain constant numbers (19, 20, 21). It is the goal of vaccination to induce the generation of memory cells in the absence of infection. Thus, the biological function of CD8+ T cells has three key attributes: clonal expansion, cytokine production, and cytolysis.

Understanding how the expansion and death of CD8+ T cells are controlled is critical to optimize the development of new vaccines. The release of proteins such as cytochrome c (22, 23) and apoptosis-inducing factor (24) from the mitochondria has been shown to initiate cell death. For this reason the loss of mitochondrial membrane potential ({Delta}{Psi}M), indicative of changes in mitochondrial membrane permeability, has often been used to identify populations of cells that are preparing to undergo apoptosis (25, 26, 27). Treatment of CD4+CD8+ double thymocytes with dexamethasone results in almost complete apoptosis 8 h after culture, and this is accompanied by the dissipation of {Delta}{Psi}M (28). Irradiation of T cells is another potent apoptotic stimulus that results in {Delta}{Psi}M dissipation. Previously (29), we determined the mitochondrial membrane potential in antiviral CD8+ T cell responses by incubating Ag-specific CD8+ T cells from LCMV infection with DiOC6, a fluorescent dye that is taken up by mitochondria based on their potential. We found that as the virus was cleared {Delta}{Psi}M decreased in effector cells, and by the memory phase its level was actually lower than that found in naive cells. In chronic LCMV infection, Ag-specific CD8+ T cells maintain their transmembrane potential and do so in a manner proportional to the level of virus found in the spleen (29). Studies from Perl and colleagues (30, 31) have shown that these results are not restricted to viral infection, as T cells from systemic lupus erythematosus patients contain increased potential compared with those found in healthy volunteers. In all of the cases described above, the highest level of {Delta}{Psi}M was observed when cells should be undergoing TCR stimulation and elaborating effector functions. This raises an important question. Is mitochondrial function required for T cell function during infection or autoimmune disease?

To determine the role of mitochondrial function in T cell function, we incubated CD8+ T cells with rotenone, a compound isolated from plants of the Derris genus. Rotenone is a very potent inhibitor of electron transport complex I (32), the largest mitochondrial respiratory chain complex. Photolabeling studies have suggested that rotenone binds to the ND1 (33, 34) and ND4 (35, 36) subunits of electron transport complex I. Because it is the proximal component of the electron transport chain, it contributes to ATP synthesis and {Delta}{psi}M. Severe defects in its function should decrease both of these functions. Prior studies of lymphocytes treated with rotenone demonstrated that excess superoxide production in lymphocytes from HIV-infected individuals was dependent on electron transport complex I (37). Additionally, T cell apoptosis induced by excess H2O2 production was also shown to be inhibited by rotenone incubation (38). We found that the clonal expansion in vitro of Ag-specific CD8+ T cells was decreased in a dose-dependent manner by rotenone incubation regardless of whether cells were activated by peptide, anti-CD3 and anti-CD28 Abs, or direct stimulation of signal transduction components by PMA and ionomycin (ION). Incubation with rotenone leads to decreases in protein synthesis, calcium flux, H2O2 production, and ERK1/2 phosphorylation. Additionally, effector and memory CD8+ T cells isolated directly ex vivo from LCMV-infected mice were inhibited in their ability to produce cytokines such as IFN-{gamma} and TNF-{alpha} upon peptide stimulation in the presence of rotenone. The ability of these cells to degranulate, a critical precursor of cytolysis of infected cells, was also inhibited by rotenone incubation. These studies demonstrate that Ag-specific CD8+ T cells require electron transport complex I function to execute their biological functions such as clonal expansion, cytokine production, and degranulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus 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 on Vero cells as described previously (39).

Cell isolation

The spleen was removed from mice after cervical dislocation. Following mechanical disruption of splenocytes on a wire mesh screen, RBCs were removed by osmotic lysis in ACK buffer (NH4Cl, KHCO3, and EDTA). Splenocytes were then resuspended in RPMI 1640 supplemented with 10% FCS and L-glutamine, penicillin-streptomycin, and beta2-ME.

Surface and intracellular staining

All Abs were purchased from BD Pharmingen. In this study, the following Abs were used: rat anti-mouse CD8{alpha}-PE, rat anti-mouse CD90.1-FITC, rat anti-mouse IFN-{gamma}-allophycocyanin, rat anti-mouse TNF-{alpha}-PE, rat anti-mouse CD107a-FITC, rat-anti-mouse CD107b-FITC, rat anti-mouse CD69-FITC, and rat anti-mouse CD8{alpha}-PerCP. Surface staining was performed by incubation of Abs at a 1/100 dilution in FACS buffer (2% FCS, PBS) for 30 min at 4°C. CD90.1 staining was performed at a 1/500 dilution. After three washes, cells were intracellularly stained using the BD Biosciences Cytofix/Cytoperm kit according to the manufacturer’s instructions. For the degranulation assay (40), GolgiStop was used instead of GolgiPlug (BD Biosciences), and CD107a and CD107b Abs were included in the peptide stimulation at a 1/50 dilution. Phospho-ERK1/2 staining has been described previously (41). Samples were acquired on a FACSCalibur flow cytometer and analyzed with FlowJo software (Tree Star).

CFSE labeling

CFSE was purchased from Invitrogen Life Technologies and dissolved in DMSO as a 5 mM stock. After resuspension, splenocytes were washed three times in PBS and suspended in PBS at 2 x 107 cells/ml. These cells were then mixed with 10 µM CFSE in PBS so that the final concentration was 5 µM. After 3 min, cells 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 sample was then washed three times with complete medium and used in reactions.

CD8+ T cell purification

Naive CD8+ T cells were purified by magnetic bead enrichment using the Miltenyi MicroBead system. Purity was >95% as determined by flow cytometry.

In vitro stimulation

After CFSE labeling, 106 splenocytes were resuspended in a 1-ml volume that contained DMSO or rotenone and 0.1 µg/ml gp33–41 peptide. Purified CD8+ T cells (5 x 105) were used for other stimulations. 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. Cells were then incubated in 1-ml complete medium that contained DMSO or rotenone. PMA and ION (ION) were added at 2 ng/ml and 10 µg/ml, respectively, in complete medium that contained DMSO or rotenone. Rotenone, PMA, and ION were purchased from Sigma-Aldrich.

Cell viability assay

Purified CD8+ T cells were either used directly ex vivo or after 24 h of incubation with PMA/ION. Cells were removed from the dish by gentle pipetting and diluted in trypan blue. Cells were scored as viable if they were able to exclude trypan blue.

Calcium flux assay

Fluo-3 acetoxymethyl ester (Fluo-3-AM) was purchased from Invitrogen Life Technologies and dissolved in DMSO as 1.25 mM stock. Purified CD8+ T cells were incubated in 5 µM µM Fluo-3-AM in PBS for 30 min and then acquired for 60 s on a FACSCalibur instrument, after which PMA/ION were added to the sample and recording was resumed.

ATP determination

ATP concentrations were determined by preparing cell lysates from purified CD8+ T cells that had been stimulated for 24 h in the presence or absence of rotenone. These lysates were then incubated with the substrate from the Enlighten kit (Promega), and luminescence was recorded using a Berthold Technologies luminometer. The amount of ATP per cell was determined by comparing cell lysates with the standard curve of ATP samples. Fold induction was determined by dividing the values at a given treatment by the ex vivo value.

Protein concentrations

Protein concentrations were determined by preparing cell lysates from purified CD8+ T cells that been stimulated with PMA/ION for 24 h in the presence of absence of rotenone using the BCA kit from Pierce.

Adoptive transfer and effector and memory CD8+ T cell generation

Naive P14 Thy1aPL/1 mice were sacrificed, and the spleen was excised. After osmotic lysis, splenocytes were stained with Abs specific for CD8{alpha} and CD90.1 and the Dbgp33–41 MHC class I tetramer. Enough splenocytes were transferred to ensure that 105 naive P14 CD8+ T cells were injected, with an engraftment of 104 cells. Four hours after cell transfer, mice were infected i.p. with 2 x 105 PFU of LCMV-Armstrong. For effector cell studies, splenocytes were harvested on day 8, and memory cells were used from mice at least 90 days after infection.

Preparation of MHC class I tetramers

The construction and purification of Dbgp33–41 has been described previously (11).

Statistical analysis

Data from control and rotenone treated samples were analyzed by using two-tailed Student’s t test, and a p value of ≤0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Rotenone incubation decreases CD8+ T cell proliferation after incubation with peptide-coated splenocytes, anti-CD3 and CD28 Abs, or PMA and ION

To determine the requirement of electron transport complex I for T cell proliferation, we incubated naive P14 splenocytes with their cognate peptide, gp33–41 of LCMV, along with increasing concentrations of rotenone. Rotenone is compound isolated from the roots of Derris sp. and is a potent inhibitor of mitochondrial electron transport by inhibition of electron transport complex I. Proliferation of CD8+ T cells was assessed by the loss of CFSE fluorescence. Samples with no peptide stimulation serve as a marker for undivided cells. In the absence of rotenone (Fig. 1A), no division was observed after 24 h regardless of stimulation. At 48 h after stimulation some cells had divided up to three times, whereas others had only managed to progress through one to two divisions. As the concentration of rotenone was increased from 0.001 to 5 µM, the number of divisions cells underwent progressively decreased until at 1 µM rotenone there was no division. After 72 h had passed, almost all of the cells have divided in the control samples, but in the rotenone-treated samples a dose-specific decrease in division was observed. To quantitate the effects of rotenone, the division and proliferative indexes were determined on day 3 after stimulation (Fig. 1, B and C). 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 underwent by those cells that divided. For control-treated samples the division index was 3.6, whereas the proliferative index was 4.3. There was no significant difference in the indexes at 0.001 and 0.01 µM rotenone, but as the concentration was increased to 0.1 µM both indexes dropped to 2. Finally, at 1 and 5 µM rotenone the division index had dropped almost to 0. To determine whether rotenone was functioning in an autonomous manner, CD8+ T cells were purified by positive selection using magnetic microbeads and then activated by either anti-CD3 and anti-CD28 Abs or PMA and ION incubation in the presence of rotenone. When the division and proliferation indices were determined on day 3 after stimulation, similar results as peptide activation were observed (Fig. 1, B and C). Thus, treatment with rotenone, a potent electron transport complex I inhibitor, decreases CD8 T cell proliferation in a dose-dependent and autonomous manner.


Figure 1
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  		FIGURE 1. Rotenone decreases the proliferation of naive CD8+ T cells. Splenocytes were isolated from naive P14 TCR transgenic mice, labeled with CFSE, and incubated with 0.1 µg/ml gp33–41 peptide or purified and activated with 10 µg/ml anti-CD3 or CD28 Abs or PMA and ION and increasing concentrations of rotenone. 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. Division (B) and proliferative indexes (C) were calculated for samples on day 3 after stimulation (Stim). At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
Rotenone does not affect initial cell survival

To determine the mechanism by which rotenone blocks T cell division, it was imperative to determine whether cells were surviving during incubation. The cells were examined at 24 h, because at this time point the effects of differential division would not confound interpretation of the results. Naive P14 CD8+ T cells were purified by magnetic microbeads, incubated in PMA and ION with increasing concentrations of rotenone for 24 h, and viability was determined by trypan blue exclusion assay. Compared with direct ex vivo cells, there was a slight decrease (5 x 105 to 3.8 x 105; Fig. 2) in cell number after activation with PMA and ION; but as rotenone was increased there was no significant effect, demonstrating that initial cell survival was not affected.


Figure 2
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  		FIGURE 2. In vitro survival of CD8+ T cells is not altered by rotenone incubation. Naive P14 CD8+ T cells were purified by magnetic microbeads and activated with PMA and ION in the absence or presence of rotenone. At 24 h after stimulation (Stim), cultures were harvested and viability was determined by trypan blue exclusion assay. At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
Rotenone blocks mitogen-induced cell growth

To determine whether rotenone was blocking the mitogen-induced program of cell growth, we examined the size of purified P14 CD8+ T cells 24 h after activation with PMA and ION (Fig. 3A). In the absence of stimulation, T cells remained small regardless of the concentration of rotenone with which they were incubated (Fig. 3A, top row). After stimulation, control-treated samples exhibit a large increase in forward scatter, indicative of T cell blasting. As the concentration of rotenone increased, the size of cells decreased, until at 5 µM they were similar to unstimulated cells. To determine the mechanism responsible for decreased blasting, we measured protein levels in T cells following PMA and ION stimulation (Fig. 3B). After mitogen stimulation we observed that protein levels increased from 29 to 49 µg per 106 cells. In rotenone-treated cells the total amount of protein decreased as drug levels were increased until 5 µM, where no difference was observed between unstimulated and stimulated cells. Because electron transport complex I function is critical for oxidative metabolism and ATP production, we measured steady-state levels of ATP in the cell. This level will reflect both synthesis and catabolism of ATP. Levels of ATP were determined after 24 h of PMA and ION stimulation in the presence of rotenone. We found that after stimulation with PMA and ION the steady state levels of ATP had increased almost 2-fold (Fig. 3C). At concentrations of rotenone up to 0.1 µM, the steady-state levels of stimulated cells were increased compared with unstimulated cells. However, at 1 and 5 µM rotenone, the levels were decreased 55 and 70%, respectively. Thus, prolonged rotenone incubation decreases steady-state ATP levels in CD8+ T cells following PMA and ION stimulation.


Figure 3
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  		FIGURE 3. Rotenone incubation inhibits CD8+ T cell blasting and increases in steady-state protein levels. Naive P14 CD8+ T cells were purified by magnetic microbeads and activated with PMA and ION in the absence or presence of rotenone. At 24 h after stimulation (Stim), T cell blasting (A) was determined by staining with an anti-CD8{alpha} Ab and a Dbgp33–41 MHC class I tetramer, and forward scatter was determined and plotted as a histogram. Protein levels (B) were determined by BCA assay, and steady-state ATP levels (C) were determined by luminescence assay. At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
Rotenone incubation decreases CD69 expression

Because rotenone decreased T cell proliferation, blasting, and protein and steady-state ATP levels after 24 h of activation, we examined whether rotenone incubation affected early T cell activation events. We first examined CD69 expression, which is a marker of recent TCR stimulation (42, 43). Directly ex vivo, ~1.6% of CD8+ T cells expressed this molecule on the cell surface (Fig. 4A). After PMA and ION stimulation, levels increased such that 22.6% (Fig. 4B) of cells expressed CD69 at 1 h after stimulation, and by 3 h after simulation 95% of cells expressed very high levels. As the level of rotenone was increased, CD69 staining decreased so that only 29.1% of cells were positive at 3 h after stimulation. When the results from several mice were analyzed, both the percentage of CD8+ T cells (Fig. 4C) and the mean fluorescent intensity (MFI) of CD69 expression were decreased at the 3-h point as the concentration of rotenone was increased (Fig. 4D). Thus, rotenone incubation blocked the ability of cells to respond to TCR stimulation, even at early time points after activation.


Figure 4
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  		FIGURE 4. Rotenone incubation blocks increases in CD69 expression following CD8+ T cell activation. Naive P14 CD8+ T cells were purified by magnetic microbeads and activated with PMA and ION in the absence or presence of rotenone. Cells were harvested directly ex vivo (A) or 30, 60, 120, and 180 min after stimulation (Stim) (B) and stained with anti-CD8{alpha} and anti-CD69 Abs. The fluorescence of CD69 is plotted as a histogram, and the number in the upper right-hand corner indicates the percentage of CD8+ T cells that fall into the gated region. The results for several mice were combined, and the percentage of CD69+ cells (C) and the fold induction (MFI stimulated/MFI unstimulated) (D) were determined. At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
ERK1/2 phosphorylation is decreased following rotenone incubation

To determine whether MAPK signaling, a critical component of cell proliferation and differentiation, was affected by rotenone incubation, phosphorylation of ERK1/2 following PMA and ION stimulation was measured in the presence of rotenone. Before stimulation (Fig. 5A), isotype staining and phospho-ERK1/2 staining were overlapping. Fifteen minutes after stimulation (Fig. 5, B and C), the phospho-ERK1/2 staining increased to levels almost 3-fold higher than those found in unstimulated cells, similarly as in previous studies (41, 44). As the concentration of rotenone was increased, the level of ERK1/2 phosphorylation decreased until at 5 µM rotenone it was comparable to that found in unstimulated cells. When the MFI values from several mice were compared, decreases with rotenone incubation were observed at 15 min. Thus rotenone incubation decreases the ability of T cells to undergo ERK1/2 phosphorylation early after T cell activation.


Figure 5
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  		FIGURE 5. Rotenone incubation decreases ERK1/2 phosphorylation following T cell activation. Naive P14 CD8+ T cells were purified by magnetic microbeads and activated with PMA and ION in the absence or presence of rotenone. Cells were harvested directly ex vivo (A) or 15 min after stimulation (Stim) (B) and stained with anti-CD8{alpha}, anti-Thy1.1, and anti-phospho-ERK1/2 Abs. The fluorescence of phospho-ERK1/2 is plotted as a histogram, and the numbers in the upper left-hand corner indicate the MFI of unstimulated and stimulated samples. The results from several mice were analyzed, and the fold induction of phospho-ERK1/2 was determined (C). At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
Rotenone incubation decreases calcium flux and H2O2 production

Because incubation with rotenone decreased the phosphorylation of ERK1/2, we examined whether components of signal transduction, besides MAP kinases, were affected by rotenone incubation. Because calcium flux is critical for T cell activation, we examined how rotenone affected this signaling event. Purified CD8+ T cells were incubated with Fluo-3-AM to detect intracellular calcium (Fig. 6A). Unstimulated cells had a basal level of fluorescence that increased rapidly after the addition of PMA and ION and was maintained throughout the interval examined. In rotenone-treated samples the level of fluorescence initially increased, but these levels were not maintained and began to decline over the time period examined.


Figure 6
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  		FIGURE 6. Rotenone incubation decreases calcium influx and H2O2 production following CD8+ T cell activation. A, Naive P14 CD8+ T cells were purified by magnetic microbeads, incubated with Fluo-3-AM, and then activated with PMA and ION in the absence or presence of rotenone. The fluorescence of Flou-3-AM is plotted as a dot plot as a function of time. The arrow in the plot indicates the time of addition of PMA and ION. B, Purified CD8+ T cells were also activated in the absence or presence of rotenone and then incubated with DCF. The fluorescence of DCF is plotted as a histogram, and the value in the upper right hand quadrant indicates the MFI of the population. Dot plots are representative of four to six mice in two independent experiments. Stim, stimulation.

 
In addition to calcium flux, the production of H2O2 has been demonstrated to be important for the activation of T cells (45). To determine the effect of rotenone incubation on the production of this signaling molecule, purified CD8+ T cells were activated with PMA and ION and incubated with dichlorofluorescein (DCF), a dye that fluoresces in the presence of H2O2. Directly ex vivo, the levels in purified cells are low (MFI 109; Fig. 6B). Within 30 min of stimulation, levels increased to 224 and remained elevated until 3 h after stimulation. Cells that were incubated for 3 h in the absence of PMA and ION had a MFI of 128. In rotenone-treated samples the levels of DCF staining were decreased as the concentration of rotenone was increased. At the 5-µM dose, the levels of DCF staining did not increase even at 3 h after stimulation. Thus, rotenone incubation inhibited calcium flux and the production of hydrogen peroxide early after T cell activation.

Rotenone incubation differentially decreases the production of cytokines by effector CD8+ P14T cells

Vigorous CD8+ T cell responses are characterized by clonal expansion, cytokine production, and cytolysis of infected cells. From the experiments described above we determined that rotenone incubation blocked clonal expansion and signal transduction of naive CD8+ T cells. To determine the role of rotenone in cytokine production, we generated effector CD8+ T cells from naive P14 cells. This was accomplished by the adoptive transfer of naive P14 CD8+ T cells into naive C57BL/6 mice followed by infection with LCMV-Armstrong. Eight days after infection the spleen was harvested. At this time there was massive expansion of the P14 cells (Fig. 7A), such that 75.8% of the CD8+ T cells were Dbgp33–41+. Staining with CD90.1 Abs revealed that 98% of these cells were derived from transgenic precursors. Incubation with 0–500 µM rotenone did not decrease the number of transgenic cells during 5 h of stimulation with gp33–41 peptide (Fig. 7B). Additionally, total cell recoveries were not significantly different between vehicle and rotenone-treated samples (data not shown). In the absence of peptide stimulation, very few CD8+CD90.1+ T cells made either IFN-{gamma} or TNF-{alpha} (Fig. 7C, top row). After gp33–41 stimulation, 94.2% of these cells made IFN-{gamma}, whereas 62.5% made both IFN-{gamma} and TNF-{alpha}. As the concentration of rotenone was increased, the level of TNF-{alpha} staining decreased first, such that by 100 µM only 0.93% of cells made TNF-{alpha}, whereas 51% made IFN-{gamma}. Further increases in rotenone up to 500 µM resulted in a further drop in IFN-{gamma}, producing cells to 25.9%. Incubation with PMA and ION could not rescue the production of cytokines (data not shown).


Figure 7
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  		FIGURE 7. Incubation with rotenone decreases the production of cytokines by effector P14 CD8+ T cells. Effector 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 day 8 after infection. Splenocytes were isolated and stained with anti-CD8{alpha}, Dbgp33–41 MHC class I tetramer, and either anti-CD44 or anti-CD90.1 Abs (A). Splenocytes were then incubated with 0.1 µg/ml gp33–41 peptide, varying concentrations of rotenone, and, after 5 h, stained with anti-CD8{alpha} and anti-CD90.1 (B) and anti-IFN-{gamma} and anti-TNF-{alpha} (C) Abs. The number in the dot plots in B represents the percentage of total splenocytes that are CD8+CD90.1+. The number of CD8+CD90.1+IFN-{gamma}+TNF-{alpha}+ cells was determined, the average and SD were plotted (D), and the percentage of maximal MFI was plotted (E) in a similar manner. At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05. Stim, stimulation.

 
Although percentages are important, it is always critical to determine the absolute numbers of cells. In the spleen there were 4 x 107 CD8+CD90.1+IFN-{gamma}+ T cells after gp33–41 peptide stimulation (Fig. 7D). As rotenone increased, the number dropped to 1.5x107 cells at 500 µM. This result contrasts with that for TNF-{alpha}, which reached the same level at 1 µM rotenone, and by 500 µM had reached a nadir of 105 cells. Plotting the data as a percentage of the maximal MFI of cytokine production illustrates the difference between the two cytokines (Fig. 7E). IFN-{gamma} reached 50% inhibition around 50 µM rotenone, whereas TNF-{alpha} reaches this inhibition at 1 µM. Thus, rotenone incubation decreases both the number of cytokine-producing effector cells and demonstrates that the production of TNF-{alpha} in effector CD8+ T cells is more sensitive to inhibition than IFN-{gamma}.

Rotenone incubation decreases the production of cytokines by memory CD8+ P14T cells

Following acute viral infection, a small subset of effector cells survive the contraction phase and differentiate into memory cells. One of the cardinal properties of these cells is their ability to rapidly respond to Ag by rapidly producing high levels of both IFN-{gamma} and TNF-{alpha} (46). To determine whether the rapid production of cytokines by memory CD8+ T cells requires electron transport complex I, we incubated splenocytes from LCMV-immune mice that received P14 cells with the gp33–41 peptide and increasing concentrations of rotenone. As the concentration of rotenone was increased (Fig. 8A), the number of IFN-{gamma}-producing cells declined initially to 50% of control values, and at 500 µM the number was 25% of untreated samples. This result contrasts with that for TNF-{alpha}, for which the number of cells rapidly decreased to 1% of untreated samples at 1 µM rotenone and was below the limit of detection by 100 µM. When the maximal MFI of cytokine-producing cells was determined (Fig. 8B), the levels of both cytokines were exquisitely sensitive to rotenone incubation and underwent a 99% decrease even at 1 µM. Thus, rotenone incubation potently decreases cytokine production in memory CD8+ T cells.


Figure 8
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  		FIGURE 8. Rotenone incubation decreases the production of cytokines by memory CD8+ T cells. Memory P14 cells were generated by adoptive transfer and LCMV-Armstrong infection. Splenocytes were isolated from mice >90 days after infection and incubated with 0.1 µg/ml gp33–41 peptide and increasing concentrations of rotenone. The number of CD8+CD90.1+IFN-{gamma}+TNF-{alpha}+ cells was determined, the average and SD were plotted (A), and the percentage of maximal MFI was plotted (B) in a similar manner. At each time point, four to six mice were examined in at least two independent experiments. *, significant difference between rotenone- and control-treated samples; p ≤ 0.05.

 
Rotenone incubation decreases degranulation by effector and memory CD8+ T cells

To address the effect of rotenone incubation on cytolytic activity, we performed a degranulation assay that measures the display of CD107a and CD107b on the cell surface and has been used as a surrogate of cytolysis (40). After incubation with the gp33–41 peptide (Fig. 9A), ~95% of the CD8+CD90.1+ P14 transgenic effector T cells were CD107a+ and CD107b+. As the concentration of rotenone was increased to 100 then 500 µM, the number of effector CD8+ T cells that degranulated decreased to 25% of the total. When absolute numbers were calculated (Fig. 9B) for either effector or memory CD8+ T cells, similar trends were observed. Thus, incubation with rotenone decreases the ability of both effector and memory CD8+ T cells to degranulate.


Figure 9
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  		FIGURE 9. Incubation with rotenone decreases degranulation by effector and memory CD8+ T cells. Effector and memory P14 cells were generated by adoptive transfer and LCMV-Armstrong infection. Splenocytes were isolated on day 8 (effector) or >90 days (memory) after infection and incubated with 0.1 µg/ml gp33–41 peptide, anti-CD107a and anti-CD107b Abs, and 100 and 500 µM rotenone. The contour plots (A) are gated on CD8+CD90.1+ T cells. The number in each quadrant represents the percentage of cells that fall into each quadrant. At each time point, six mice were examined in two independent experiments, and the average and SD were plotted for both effector and memory CD8+ T cells (B). *, Significant difference between rotenone- and control-treated samples; p ≤ 0.05. Stim, Stimulation.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have examined the effects of inhibition of mitochondrial function on the ability of CD8+ T cells to execute three of their critical biological functions: clonal expansion after activation, cytokine production, and killing of infected cells. First, we found that incubation of naive CD8+ T cells with rotenone, a known electron transport complex I inhibitor, decreased proliferation in a dose-dependent manner regardless of whether T cells were activated by peptide-coated splenocytes, TCR ligation, or direct stimulation of signal transduction components. This decrease in proliferation was accompanied by a failure to increase cell size in response to mitogen stimulation. Early signaling events such as calcium flux, H2O2 production, and ERK1/2 phosphorylation were decreased in rotenone-treated cells. We next demonstrated that effector and memory CD8+ T cells generated by LCMV infection in vivo required electron transport complex I function to produce cytokines such as IFN-{gamma} and TNF-{alpha}. Interestingly, the production of TNF-{alpha} in effector cells was 50-fold more sensitive to rotenone dose than IFN-{gamma}. In memory CD8+ T cells, the production of both cytokines was rapidly inhibited by rotenone incubation. Finally, we demonstrated that the degranulation capacity (a precursor of cytolytic function) of effector and memory cells was reduced by incubation with rotenone.

Mitochondria perform three key functions in the cell: energy production (47), execution and amplification of cell death pathways (48), and modulation of signal transduction (49). In actively respiring tissues, such as contracting skeletal and cardiac muscle, there are large numbers of mitochondria and high levels of proteins involved in oxidative phosphorylation (50). By the coupling of electron transport through four protein complexes to the establishment of a proton gradient followed by controlled reduction of oxygen, energy is generated. In other cells, such as eosinophils, very little respiration occurs, as cell rely almost completely on glycolysis for energy production (51). Although ATP production is a critical function of mitochondria, recent evidence has demonstrated that this organelle is an important site of cell death initiation and execution. Over the last 10 years a large body of evidence has accumulated demonstrating that the release of any of several mitochondrial intermembrane proteins such as cytochrome c (22, 23), apoptosis-inducing factor (52), endonuclease G (53), and SMAC/Diablo (54) can either initiate or amplify cell death cascades. Besides containing factors that initiate or sustain cell death pathways, many members of the Bcl-2 superfamily of proteins that control a cell’s susceptibility to apoptosis reside in this organelle or localize to it following stresses such as growth factor withdrawal or irradiation (55). Recently, it was documented that the p53 protein, a transcription factor responsible for genome integrity, will also localize to the mitochondria when apoptosis is initiated by irradiation (56, 57, 58). Although energy production and apoptosis control are two well-documented mitochondrial functions, mitochondria also exert cellular control through signal transduction cascades. This function is executed by controlled sequestration and release of calcium and by modulation of the levels of reactive oxygen intermediates, such as superoxide, that are produced during electron transport (59).

Our findings demonstrate that all three aspects of CD8+ T cell function are dependent on electron transport complex I function. What aspects of mitochondrial function may be missing in the rotenone-treated cells? The observation that cell survival does not appear to be dramatically affected in the initial phase of the proliferation and peptide stimulation experiments argues against the proposition that all of the effects we observed in short-term assays are due to cell death. It is interesting to note that during apoptosis in HeLa cells initiated by staurosporine incubation, caspases cleave proteins in electron transport complex I to inhibit its function (60). Our findings that cell survival in the initial phases of culture does not seem to be affected in a major way suggest that defects in signal transduction or metabolism are responsible for decreases in division, cytokine production, and degranulation.

After T cell activation, CD8+ T cells undergo a switch from a resting to a proliferating metabolic state. Studies by Frauwirth et al. (61) demonstrated that after T cell activation there is a massive increase in glucose uptake and increases in rates of glycolysis. This increase in glycolysis is dependent upon CD28-mediated activation of Akt. Once lymphocytes are actively proliferating, they are thought to become dependent upon glycolysis for energy production even though oxygen is present. This phenomena has been termed aerobic glycolysis (62). It is not restricted to proliferating lymphocytes and has been observed in tumor cells as well (63). Within the first 15 min following CD8+ T cell activation, where we observed decreases in calcium flux and ERK1/2 phosphorylation, no increase in O2 consumption was observed regardless of the concentration of rotenone added (data not shown). At these early time points rotenone incubation decreased the magnitude of all of these signal transduction events. At 24 h, however, rotenone appeared to also be affecting metabolism, as the steady-state levels of ATP were decreased in cells activated in high concentrations of rotenone.

In nonmuscle cell types, which lack a sarcoplasmic reticulum, mitochondria are a major site of calcium storage and can rapidly take up or release this molecule to influence signal transduction. In Jurkat T cells, previous studies indicated that functional mitochondria are required for calcium uptake to maintain T cell activation and enable NFAT translocation to the nucleus (64, 65). Loss of mitochondrial function by incubation with a protonophore, which should dissipate the mitochondrial potential, inhibits the ability of mitochondria to uptake calcium and ultimately decreases activation. When early signaling events were examined, we found that calcium flux was not maintained in the presence of rotenone. Additionally, we found that the production of H2O2 was also inhibited by rotenone incubation. In recent years, the production of reactive oxygen intermediates has been implicated in TCR-mediated signaling. Studies by Devedas et al. (45) have demonstrated that, following TCR stimulation, H2O2 is produced in a controlled fashion by Nox enzymes (66). Both of these key signaling events were decreased with rotenone incubation. In addition, the failure to up-regulate CD69 and undergo ERK1/2 phosphorylation demonstrates that cells are not undergoing full TCR signaling.

In addition to determining a requirement for mitochondrial function in the clonal expansion of naive CD8+ T cells, we demonstrate that mitochondrial function is required for cytokine production and degranulation. Decreases in cytokine production were not due to death of functional cells, as there was no cell loss during the 5 h of peptide stimulation. When IFN-{gamma} and TNF-{alpha} were examined, the production of both was sensitive to rotenone incubation, but TNF-{alpha} was almost two orders of magnitude more sensitive than IFN-{gamma}. One explanation for the difference in sensitivities could be the methodology used for their detection. Because both cytokines are detected by intracellular Ab staining, if one cytokine was much larger than the other, failure to complete peptide synthesis due to general metabolic defects would be read out as a negative. This should not be the case, as the predicted sizes of murine IFN{gamma} and TNF-{alpha} are 20 and 15 kDa, respectively. Interestingly, the order of loss of cytokine production (TNF-{alpha} first, then IFN-{gamma}) is very similar to that observed during chronic LCMV infection (67, 68). When mice are infected with LCMV clone 13, a chronic viral infection is established that takes 40–60 days to be cleared from the spleen. The initial responding population of virus-specific CD8+ T cells make cytokines such as Il-2, IFN-{gamma}, and TNF-{alpha}, but over time they cease to produce cytokines upon peptide stimulation and, in some cases, undergo physical deletion. These anergic CD8+ T cells, similar to rotenone-treated cells, are refractory to cytokine production by PMA and ION (69). It will be of critical importance to determine whether, during chronic viral infection, CD8+ T cells undergo decreases in mitochondrial function and, thus, decreased proliferation, cytokine production, and cytolysis.

In this study, we have demonstrated that the inhibition of electron complex I function by rotenone incubation decreases CD8+ T cell clonal expansion, cytokine production, and degranulation. These experiments implicate mitochondrial function as being critical for T cells to carry out their biological functions during infection. These experiments suggest that manipulating mitochondrial function may be a point of intervention to selectively increase T cell proliferation during vaccination or to attenuate proliferation and cytokine production during autoimmune syndromes or organ rejection.


   Disclosures
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 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 research was supported by American Cancer Society Research Scholar Grant RSG-04-066-01-MBC (to J.M.G.). 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: LCMV, lymphocytic choriomeningitis virus; {Delta}{Psi}M, mitochondrial membrane potential; DCF, dichlorofluorescein; Fluo-3-AM, fluo-3 acetoxymethyl ester; ION, ionomycin; MFI, mean fluorescent intensity. Back

Received for publication January 6, 2006. Accepted for publication May 1, 2006.


   References
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 Abstract
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 Results
 Discussion
 Disclosures
 References
 

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