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Infection and Immunity, January 2007, p. 260-269, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.01358-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Long-Term Staphylococcal Enterotoxin C1 Exposure Induces Soluble Factor-Mediated Immunosuppression by Bovine CD4+ and CD8+ T Cells{triangledown}

Keun Seok Seo,1 Sang Un Lee,1 Yong Ho Park,2 William C. Davis,3 Lawrence K. Fox,4 and Gregory A. Bohach1*

Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83844,1 Department of Microbiology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea,2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164,3 Department of Veterinary Medicine, Washington State University, Pullman, Washington 991644

Received 23 August 2006/ Returned for modification 25 September 2006/ Accepted 27 September 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulatory T cells (Tregs) help control the development and maintenance of protective immunity and can lead to aberrant immune responses to some pathogens. Several lines of evidence suggest that Tregs are induced by exposure to superantigens (SAgs) in vitro or in vivo. In this study, bovine peripheral blood mononuclear cells (PBMC) were exposed in vitro to a relatively low dose (5 ng/ml) of staphylococcal enterotoxin C1 (SEC1) for up to 10 days. Upon stimulation, CD4+ and CD8+ T cells initially proliferated at similar rates. Subsequently, from days 6 through 10, most CD4+ and CD8+ T cells proliferated regardless of Vß specificity, but the proliferation of CD8+ T cells occurred more vigorously. The transcription of CD25 and CD152 genes increased, whereas that of interleukin-2 (IL-2) decreased. {gamma}{delta} T cells appeared to be unresponsive. An increase in the transcription of IL-10 and transforming growth factor ß (TGF-ß) genes in SEC1-stimulated cultures was attributed to the CD4+ CD25+ T-cell subpopulation. The expression of Foxp3 mRNA also increased and was accompanied by the upregulation of CD152 and the downregulation of IL-2 transcription, suggesting that cells in this subpopulation are Tregs. Functionally, SEC1-stimulated CD4+ T cells suppressed the proliferation of naive PBMC in response to heat-killed-fixed Staphylococcus aureus. The suppression was partially mediated by IL-10 and TGF-ß, another characteristic of certain types of Tregs. The CD8+ T-cell population also suppressed naive PBMC through another mechanism not mediated by IL-10 or TGF-ß. These results provide further insight into the potential mechanisms by which SAgs could contribute to evasion of the immune response, affecting the outcome of infection or colonization.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Staphylococcal enterotoxins and other pyrogenic toxins produced by Staphylococcus aureus and Streptococcus pyogenes are prototype microbial superantigens (SAgs). Conventional antigens induce T-cell activation by antigen-specific signaling through the major histocompatibility complex-peptide-T-cell receptor (TCR) complex and costimulatory signals through CD28/CTLA-4 (CD152) on T cells and B7 (CD80/86) on antigen-presenting cells (APCs). The interaction of CD28 with CD80/CD86 leads to T-cell proliferation and the production of cytokines (1, 5, 15), while the interaction of CD152 with CD80/CD86 leads to a downregulation in the production of cytokines (23, 43). Unlike conventional antigens, most T-cell SAgs bind to major histocompatibility complex class II molecules outside of the peptide binding groove and to TCR-bearing specific Vß sequences (25). The binding triggers extensive proliferation of T cells and uncontrolled release of proinflammatory cytokines such as interleukin-1 (IL-1), IL-2, gamma interferon (IFN-{gamma}), and tumor necrosis factor alpha (TNF-{alpha}). These factors increase sensitivity to lipopolysaccharide and induce the toxic shock syndrome (28). The initial expansion of T cells is followed by activation-induced cell death, or apoptosis, leading to the clonal deletion of T cells bearing SAg-reactive Vß TCR sequences (26). SAg-reactive T cells which escape, however, fail to proliferate and secrete IL-2 in response to subsequent exposure to SAg. This is often referred to as anergy (26). The immune dysfunction caused by SAgs is associated with multiple diseases, including the toxic shock syndrome and autoimmune diseases in humans (3, 45) and has been proposed to lead to long-term chronic infections in animals such as bovine mastitis (8).

Peripheral T-cell tolerance is required for immune system homeostasis. In addition to clonal deletion of self-reactive T cells (27, 31) and functional inactivation of antigen-specific T cells (17, 21), suppression involving T-cell-derived soluble factors and cell-to-cell contact also help maintain tolerance (16, 39). Regulatory T cells (Tregs), able to control immune responses to self and foreign antigens, have been identified in humans and mice (38). Their absence is associated with autoimmune and inflammatory bowl diseases (35, 38). Evidence suggests that SAgs induce the development of Tregs, which are capable of suppressing the primary immune response in humans and in the mouse model (11, 42, 44). Low-dose stimulation of human CD4+ CD25– T cells with staphylococcal enterotoxin B (SEB) in the presence of transforming growth factor ß (TGF-ß) induces CD4+ CD25+ Tregs that express high levels of CD25 and CD152, with potent TGF-ß-dependent suppressive activity (47). Repeated low-dose stimulation with staphylococcal enterotoxin A (SEA) in mice induces downregulation in the cytotoxic activity of SEA-reactive IFN-{gamma}-producing CD8+ CD25+ CD152+ T cells (42).

Tregs from bovine or other ruminants have not been studied despite the fact that these animals are frequently exposed SAgs (41). The finding that Tregs are induced with low doses of SAgs in other animals led us to postulate that analogous cells are induced when bovine cells are exposed to staphylococcal enterotoxin C1 (SEC1), a class of SAg often produced by staphylococcal bovine mastitis isolates (10, 46). Previously, we demonstrated that toxins in the SEC group stimulate the Vß-dependent proliferation of bovine {alpha}ß T cells similar to the response observed in humans and mice (6). We also partially characterized cell phenotypes and cytokine profiles resulting from the exposure of peripheral blood mononuclear cells (PBMC) to a relatively high dose (1 µg/ml) of SEC1 in vitro (6, 10). The present study was undertaken to extend those earlier reports and to determine whether low doses of this SAg could induce bovine Tregs under certain conditions. We determined that SEC1 exposure results in the development of CD4+ Tregs with immunosuppressive activity mediated in part by IL-10 and TGF-ß. An immunosuppressive CD8+ T-cell population, not requiring IL-10 or TGF-ß, was also induced.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of SEC1. SEC1 was purified from cultures of S. aureus RN4220(pMIN121), harboring the recombinant sec structural gene cloned from S. aureus MNDON (19). Cultures were grown in medium containing dialyzable beef heart broth extract and erythromycin (5 µg/ml). SEC1 was purified by ethanol precipitation of the bacterial cultures in the cold, followed by resolubilization and preparative isoelectric focusing with broad isoelectric point ( 3 to 10) and narrow isoelectric point (6 to 8) ranges of ampholytes in succession as described previously (6).

Preparation of heat-killed fixed S. aureus. Overnight cultures of S. aureus Novel strain (29) were harvested, washed in phosphate-buffered saline (PBS), incubated at 80°C for 15 min, fixed in 2% paraformaldehyde, and then resuspended in culture medium as previously described (22).

Animals and purification of PBMC. Blood was obtained from purebred adult healthy Holstein-Frisian steers (10 to 18 months old) via venipuncture of the jugular vein. The animals were maintained according to Association for the Assessment and Accreditation of Laboratory Animal Care, International, guidelines and regulations established by the Animal Care and Use Committee at the University of Idaho. PBMC were isolated from blood by density gradient centrifugation using Ficoll-Hypaque (Pharmacia Biotechnology) according to standard techniques (6). The RPMI 1640 culture medium used in the present study contained 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 2 µM L-glutamine, 100 U of penicillin-streptomycin/ml, and 5 x 10–5 M 2-mercaptoethanol.

Cell stimulation. To determine the level of lymphocyte proliferation induced by SEC1, PBMC were processed as described above, adjusted to a concentration of 106 cells/ml in RPMI 1640 culture medium with various concentrations of SEC1 (0.05 ng/ml to 10 µg/ml) added to PBMC cultures (200 µl of PBMC suspension per well in 96-well culture plates), and incubated at 37°C in 5% CO2 for 72 h. Incorporation of [3H]thymidine into cellular DNA in a standard 4-day assay (34) was used to measure the level of lymphocyte proliferation. Cultures were pulsed (18 h) with 1 µCi of [3H]thymidine. Cellular DNA was harvested on glass fiber filters, and [3H]thymidine incorporation was quantified by liquid scintillation counting.

For phenotypic and gene expression analyses, 12 ml of the PBMC suspension (concentration of 106 cells per ml in RPMI 1640 culture medium) was incubated in tissue culture dish (Falcon, Lincoln Park, NJ) for 2, 4, 6, 8, or 10 days at 37°C in 5% CO2. SEC1 was added to some cultures to obtain a concentration of 5 ng/ml. In some experiments, PBMC were stained with 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) at 2 µM for 15 min at 37°C as described previously (36). CFSE-stained PBMC were stimulated with SEC1 for up to 10 days and immunolabeled by using antibodies specific for a variety of cell surface markers as described below.

MAbs. Monoclonal antibodies (MAbs) specific for bovine CD4, CD8, CD62 ligand (CD62L) (BAQ92A, immunoglobulin G1 [IgG1]), CD45R (GS5A, IgG1), CD25 (CACT116A, IgG1), CD26 (CACT114A, IgG2b), and CD45R0 (GC44A, IgG3) were purchased from the Washington State University Monoclonal Antibody Center. Anti-IL-10 and IgG1 isotype control MAbs were purchased from Serotec Immunological (Oxford, United Kingdom); anti-TGF-ß MAb was purchased from R&D Systems (Minneapolis, MN).

FC analysis of bovine PBMC. Some properties of SEC1-stimulated bovine PBMC were analyzed by indirect immunofluorescence staining and flow cytometry (FC). After stimulation with SEC1 in culture, PBMC were harvested and incubated with the appropriate MAb at 4°C for 30 min. After being washed with PBS, the cells were incubated at 4°C for 30 min in the dark with isotype-specific anti-mouse immunoglobulins conjugated to fluorescein isothiocyanate or phycoerythrin (PE; Caltag Laboratories, Burlingame, CA). After further washing, PBMC were analyzed with FACSAria or FACScalibur flow cytometers equipped with FACSdiva and CellQuest software, respectively (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Cell purification. Bovine CD4+, CD4–, CD8+, and CD8– T-cell populations were purified from PBMC prior to or after stimulation with SEC1 by positive and negative selection. Briefly, cell suspensions were labeled with an anti-CD4 or anti-CD8 MAb in PBS for 15 min at 4°C. The cells were washed and then incubated with PE-conjugated isotype-specific antibodies in PBS for 15 min at 4°C. After washing, the cells were incubated with anti-PE-coated microbeads (Miltenyi Biotech, Auburn, CA) for 15 min at 4°C. Cell separation was performed with an LS column (Miltenyi Biotech) according to the manufacturer's instructions. In some experiments, CD4+ and CD8+ T-cell populations were further separated into CD25+ and CD25– subpopulations. Each cell population was incubated with the anti-CD25 MAb at 4°C for 30 min. After a washing step, the cells were incubated with isotype-specific anti-mouse immunoglobulins conjugated with fluorescein isothiocyanate (Caltag Laboratories). The cells were washed again, and the various subpopulations were sorted by using the FACSAria cytometer. The purities of sorted subpopulations were determined to be 98.5 ± 0.5% in three separate experiments, as assessed by FC (data not shown).

Real-time PCR. RNA was extracted from approximately 5 x 106 SEC1-stimulated PBMC or purified T cells by using TRIzol reagent (Life Technologies, Gaithersburg, MD). First-strand cDNA was generated from 1 µg of RNA by using Superscript II reverse transcriptase and oligo(dT) primers (both from Life Technologies). The reverse transcription reaction was performed in a 20-µl volume according to the manufacturer's specifications.

Primers for PCR amplification were designed by Primer Express (version 2.0; PE Applied Biosystems, Foster City, CA) using GenBank sequences (Table 1) . The real-time PCRs were performed by using the SYBR green I dye master mix and ABI Prism 7500 real-time PCR system (PE Applied Biosystems) according to the manufacturer's instructions. After 40 cycles of amplification, a melting curve was generated by slowly increasing the temperature of the reaction mixture at a rate of 0.1°C/s from 60 to 95°C. During this time, the fluorescence was measured continuously to verify the amplification specificity.


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  		TABLE 1. Primers for real-time PCR used in this study

 
Real-time PCR data were analyzed by using sequence detector systems software (version 1.2.2; PE Applied Biosystems). Briefly, the threshold cycle (CT) was calculated by the sequence detection software as the cycle number at which the {Delta}Rn crossed the baseline. The data were normalized by calculating the {Delta}CT (CT of target – CT of the internal control [ß-actin gene]). Normalized {Delta}CT data were used for the comparative CT method ({Delta}{Delta}CT) for relative quantification. Normalized {Delta}CT data from each time point of stimulation were compared to data from unstimulated cells (day 0).

Identification of the bovine foxp3 gene. A partial sequence of the bovine foxp3 gene was obtained from the bovine genome database (24) based on its high similarity to human foxp3 and used to generate the following PCR primers: forward, 5'-CACTGGTTTACACGCATGTTTG-3', and reverse, 5'-TGACTGAGGCAGGCTGTGTGT-3'. PCRs, using these primers with cDNA produced from SEC1-stimulated PBMC, generated the expected size amplicons (330 bp). Based on the sequence of this product, a reverse primer (5'-GTGCACACCTTACTTCTTGGT-3') was designed and used with a RACE (rapid amplification of cDNA ends) adaptor primer to obtain the complete cDNA sequence of bovine foxp3, using the FirstChoice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer's instructions.

Coculture experiments. Coculture experiments were conducted in Transwell (Corning Life Sciences, Nagog Park Acton, MA) culture plates to determine the ability of soluble factors from SEC1-stimulated PBMC or purified subpopulations to suppress naive PBMC proliferation in response to heat-killed fixed S. aureus. Naive PBMC (105 or 106) were placed in 24-well culture plates (Corning Life Sciences) in the presence or absence of heat-killed fixed S. aureus. Various numbers of SEC1-stimulated PBMC (from 0 to 106) or purified SEC1-stimulated CD4+ or CD8+ T cells (105) were seeded into the upper chamber of Transwell plates, which were then inserted into 24-well culture plates. After 72 h of culture, [3H]thymidine (1 µCi/well) was added. The PBMC from 24-well plates were harvested 18 h later, and the [3H]thymidine incorporation was quantified as described above. In some experiments, anti-IL-10 and/or anti-TGF-ß MAb (1 or 10 µg/ml) or isotype control MAbs were added to 24-well plates to determine whether the suppression was mediated by IL-10 and TGF-ß.

Statistical analysis. Statistical significance was analyzed with the Student t test using OriginPro software (OriginLab, Northampton, MA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential activation of bovine T-cell populations by SEC1. Bovine PBMC were stimulated for 4 days with various concentrations of SEC1 (0.5 to 10 µg/ml). [3H]thymidine incorporation was measured as an indicator of cell proliferation (Fig. 1A). A near-linear dose response was observed between 50 and 500 ng/ml. Subsequent experiments were conducted using the 5-ng/ml concentration of SEC1, which was near the midpoint of the dose-response curve and generated a response that was ca. 50% of the maximum observed.


Figure 1
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  		FIG. 1. Activation of bovine T-cell populations by SEC1. (A) PBMC proliferation after 4 days of SEC1 exposure as indicated by the incorporation of [3H]thymidine. The data are means of triplicate measurements ± the standard error of the mean (SEM). (B) Percentages of major T-cell populations after various periods of exposure to SEC1. (C and D) Phenotypic characteristics of SEC1-stimulated bovine CD4+ and CD8+ T cells were analyzed by FC using MAbs specific for naive T-cell markers (CD62L and CD45R) or T-cell activation markers (CD25 and CD26) and CD45R0. The results using antigen-specific MAbs are indicated as solid lines, and dotted lines represent isotype controls. The data shown in panels B, C, and D are from a single representative experiment that was conducted three times. "Unstimulated" refers to data acquired from cultures without added toxin and prior to incubation.

 
The phenotypes of SEC1-stimulated PBMC were analyzed by FC. Before stimulation, the CD4/CD8 ratio, normally 1.18 to 1.82 (32), was initially 1.35 but decreased to 0.91 by day 10 (Fig. 1B). The proportion of {gamma}{delta}+ T cells present in the culture dramatically decreased from ~39 to 7% between days 4 and 10 of exposure to SEC1 (Fig. 1B).

The percentages of CD4+ and CD8+ T cells expressing naive T-cell markers (CD62L and CD45R) dramatically decreased after 4 days of exposure to SEC1 in culture. This observation was accompanied by a corresponding, but more gradual, increase in cells expressing T-cell activation markers (CD25 and CD26) and CD45R0 (Fig. 1C and D). These data indicated that both CD4+ and CD8+ T cells were activated by SEC1.

To determine whether the decrease in the percentages of {gamma}{delta}+ T cells was attributable to a lack of stimulation by SEC1 or to a difference in the relative rate of proliferation compared to that of CD4+ and CD8+ T cells, PBMC were labeled with CFSE prior to incubation to monitor the rate of proliferation of each cell population. As expected, unstimulated cells were represented as a single peak of fluorescence (Fig. 2A). Upon stimulation, additional peaks representing PBMC fractions with progressively lower concentrations of CFSE appeared as a result of CFSE dilution during successive rounds of cell division at 4 and 10 days. The rates of cell division by the CD4+ and CD8+ populations were similar at 4 days based on the distribution of the peaks. At 10 days, very few cells remained among the CD4+ and CD8+ populations represented by the high fluorescence levels in the initial peaks, indicating that most of CD4+ and CD8+ T cells had proliferated. In contrast, the majority of {gamma}{delta} T cells contained maximal CSFE levels throughout the entire 10-day period of culture, indicating that few of these cells had proliferated.


Figure 2
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  		FIG. 2. Assessment of cell proliferation by CFSE labeling. (A) Bovine PBMC were labeled with CFSE prior to culture and stimulated with SEC1 (5 ng/ml) for 10 days. Cells were harvested during stimulation (middle panel [day 4] and right panel [day 10]) or before stimulation (left panel), and stained for CD4+, CD8+, and {gamma}{delta}+ T cells, and CFSE fluorescence was analyzed before (Whole PBMCs) or after gating on CD4+, CD8+, and {gamma}{delta}+ T cells. The dotted lines represent the superimposed profile of the unstimulated cells. (B) The cell fraction containing the lowest CSFE concentrations from CFSE-labeled bovine PBMC stimulated with SEC1 for 10 days was gated and further analyzed for CD4+, CD8+, and {gamma}{delta}+ T-cell content. The data shown are from a single representative experiment that was performed twice.

 
The distribution of CD4+, CD8+, and {gamma}{delta}+ T cells in the CFSE-stained fraction representing the most highly divided (lowest CSFE content) cells after 10 days was determined (Fig. 2B). CD4+, CD8+, and {gamma}{delta}+ T cells represented 25, 43.8, and 9.2% of the PBMC, respectively (Fig. 2B). The results indicated that CD4+ and CD8+ T cells proliferated in response to stimulation with SEC1, with the proliferation of CD8+ T cells being more extensive than that of CD4+ T cells. This observation could explain, at least in part, the reversal of the CD4/CD8 ratio after 10 days of culture and also indicates that the relative decrease in {gamma}{delta}+ T cells after 10 days was due to their failure to proliferate.

Functional characterization of bovine T-cell subpopulations induced by SEC1. The transcriptional levels of several key activation marker and cytokine genes were analyzed by real-time PCR. The data from SEC1-stimulated and unstimulated PBMC were compared. The relative transcriptional level resulting from this comparison is expressed on a log2 scale in Fig. 3 and a natural scale in the text. Consistent with the FC results, transcription of CD25 increased (~10-fold at 10 days) rapidly and dramatically during exposure to the toxin (Fig. 3A). The transcription of CD152 increased (~10-fold at 10 days), and the pattern of expression was similar to that for CD25. The transcription of CD28 also increased but only modestly (~1.8-fold) compared to CD25 and CD152 (Fig. 3A). The transcription of IFN-{gamma} and granulocyte-macrophage colony-stimulating factor (GM-CSF) also increased (~60-fold at 10 days). After induction to maximal levels on 4 days, mRNA levels for both genes were sustained throughout the remainder of the culture period (Fig. 3B). Transcription of IL-2 increased early, peaked at 4 days (~13-fold), and then gradually decreased to the baseline level by 10 days. Based on these data, one may conclude that CD4+ and CD8+ T-cell proliferation at later time points (after 4 days) was accompanied by increased transcription of CD25 but not of IL-2. The transcription of IL-12 initially declined (at 2 days) and then increased slightly at later time points before returning to baseline levels on 10 days. A small but persistent increase (~1.6-fold) in the transcription of IL-4 was observed beginning in 4-day-old cultures. In contrast, the increase in the transcription of IL-5 and IL-13 after 4 days of culture was much more dramatic (ca. 20- to 160-fold) (Fig. 3C). The transcription of TGF-ß gradually increased and peaked on 8 days (~2.8-fold), whereas that of IL-6 and IL-10 decreased during the entire culture period (~25-fold) (Fig. 3C and D).


Figure 3
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  		FIG. 3. Transcription of T-cell activation molecules and cytokines in SEC1-stimulated bovine PBMC at various time points measured by real-time PCR. The results shown represent the means ± the SEM of data combined from three separate experiments (n = 9).

 
The CD4+ CD25+ and CD8+ CD25+ T-cell subpopulations were isolated from 10-day-old SEC1-stimulated PBMC cultures (CD4+ CD25– and CD8+ CD25– T-cell subpopulations were isolated from unstimulated control cultures) and subjected to gene expression studies by using real-time PCR. Compared to unstimulated controls, SEC1 exposure increased the transcription of CD25 and CD152 in both CD4+ CD25+ and CD8+ CD25+ T-cell subpopulations (Fig. 4A). A smaller but consistent increase in CD28 transcription (~1.4-fold) was measured in CD4+CD25+ T cells, whereas a corresponding relative decline in expression occurred in the CD8+ CD25+ subpopulation (~1.2-fold) (Fig. 4A). The transcription of IL-2 increased ~7-fold in CD8+ CD25+ T cells; a smaller but consistent increase was observed in CD4+ CD25+ T cells (~2-fold) (Fig. 4B). Consistent with data acquired from analysis of SEC1-stimiulated PBMC cultures, the transcription of IFN-{gamma} and GM-CSF was highly increased within both purified T-cell subpopulations (ca. 20- to 40-fold) (Fig. 4B). The transcription of IL-12 was increased in both purified T-cell subpopulations (approximately four- to eightfold) (Fig. 4B). This level of increase was not observed in PBMC exposed for 10 days, suggesting that IL-12 expression among other T-cell subpopulations was dramatically reduced. The transcription of IL-5 and IL-13 increased in both subpopulations, whereas IL-4 transcription decreased in CD4+ CD25+ T cells and slightly increased in CD8+ CD25+ T cells (Fig. 4C). The transcription of IL-6 was consistently decreased in both subpopulations (ca. 50- to 200-fold) (Fig. 4C). The levels of TGF-ß transcription in either subpopulation stimulated with SEC1 did not change substantially compared to baseline levels in unstimulated controls (Fig. 4D). However, it is noteworthy that baseline TGF-ß transcription was inherently high and was comparable to that of ß-actin prior to SEC1 exposure (data not shown). These data suggest that, even though the transcription of TGF-ß did not change, it remained at a high level of transcription during SEC1 stimulation. The transcription of IL-10 increased in CD4+ CD25+ T cells (~10-fold) and decreased in CD8+ CD25+ subpopulation (~30-fold) (Fig. 4D). This result is consistent with the overall decline in IL-10 expression in PBMC as demonstrated in Fig. 3D.


Figure 4
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  		FIG. 4. T-cell activation molecules and cytokine expression in CD4+ CD25+ and CD8+ CD25+ subpopulations purified from bovine PBMC cultures stimulated with SEC1 for 10 days and analyzed by real-time PCR. The relative transcription was calculated by subtracting the {Delta}CT of SEC1-stimulated CD4+ CD25+ or CD8+ CD25+ subpopulations from that of unstimulated CD4+CD25– or CD8+ CD25– subpopulations, respectively. The results shown represent the means of triplicate measurements ± the SEM of data combined from two separate experiments (n = 6).

 
Bovine foxp3 identification and expression characterization. The demonstration that SEC1 exposure upregulates the transcription of CD25, CD152, and IL-10 and maintains constitutive transcription levels of TGF-ß in CD4+ CD25+ T cells suggested that this subpopulation was analogous to Tregs in other species. In T cells from other species, Foxp3 (forkhead/winged helix transcription factor 3) is a key marker for Tregs (38). Since the sequence of the bovine foxp3 gene had not been determined at the time of the study, we cloned and sequenced the bovine orthologue of foxp3. The identified foxp3 sequence was deposited in GenBank (GenBank accession no. DQ322170). The translated protein sequence of bovine Foxp3 was compared to other known orthologues of Foxp3 by using the multisequence alignment program CLUSTALX (20) and showed a high similarity with other orthologues of this protein (human, 90%; monkey, 89%; mouse, 86%; rat, 85%) (4, 14).

Real-time PCR primers for bovine foxp3 were designed (Table 1) and used to determine the level of transcription in SEC1-stimulated PBMC cultures. A gradual increase in the transcription of foxp3 was clearly evident by 6 days of culture, and mRNA levels reached a peak by between 8 and 10 days (ca. four- to fivefold) (Fig. 5A). In other species, Foxp3 is a transcriptional repressor and activator of the IL-2 and CD152 genes, respectively (18). The transcription patterns of IL-2 and CD152 after SEC1 exposure (Fig. 3B and A, respectively) were consistent with the pattern of expression of foxp3 (Fig. 5A). After a rapid induction and peak on 4 days, IL-2 mRNA gradually decreased thereafter, when foxp3 transcription increased dramatically. In contrast, expression of CD152 closely paralleled that of foxp3. Real-time PCR analysis of various purified T-cell subpopulations after exposure to SEC1 indicated that the transcription of foxp3 occurred in CD4+ T cells. No transcription increase could be detected in CD8+ T cells, regardless of whether or not they expressed CD25 (Fig. 5B).


Figure 5
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  		FIG. 5. Transcription patterns of bovine foxp3. (A) Transcription of foxp3 in SEC1-stimulated bovine PBMC was measured at various time points by real-time PCR. (B) Real-time PCR analysis of foxp3 in various purified T-cell populations after 10 days of exposure to SEC1. The relative transcription was calculated by subtracting the {Delta}CT of SEC1-stimulated CD4+, CD4+ CD25+ CD8–, CD4–, CD8+, or CD8+ CD25+ cells from that of unstimulated CD4+, CD4+ CD25–, CD8–, CD4–, CD8+, or CD8+ CD25– cells, respectively. The results shown represent the means of triplicate measurements ± the SEM of data combined from two separate experiments (n = 6).

 
SEC1-stimulated PBMC inhibit the proliferation of naive PBMC in response to heat-killed fixed S. aureus via soluble factors. In other species, some Tregs are immunosuppressive through a mechanism involving the secretion of IL-10 and TGF-ß. To assess whether SAgs could induce this type of bovine Treg cell, we conducted experiments to determine whether the exposure of bovine PBMC to SEC1 induces T cells with this property. Naive PBMC, with heat-killed and fixed S. aureus cells, were cocultured with SEC1-stimulated PBMC. Cell-to-cell contact between naive and SEC1-stimulated PBMC was prevented by using a Transwell apparatus. In control cultures without SEC1-stimulated PBMC, naive PBMC proliferated well in response to heat-killed fixed S. aureus (Fig. 6A). The addition of SEC1-stimulated PBMC to the cultures resulted in a dose-dependent suppression of proliferation to levels of ca. 22.51% compared to the control when both cell types were cocultured in a 1:1 ratio (Fig. 6A). IL-10-specific or TGF-ß-specific MAbs added to the cultures resulted in partial restoration of the naive PBMC proliferative response to heat-killed S. aureus. Both MAbs used in combination resulted in an additive effect (Fig. 6B). However, the suppressive activity of SEC1-stimulated PBMC was not fully abrogated, suggesting the MAbs only partially neutralized their respective cytokines or the existence of additional mechanisms of suppression induced by SEC1.


Figure 6
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  		FIG. 6. Suppressive activity induced by the stimulation of bovine PBMC with SEC1 for 10 days. (A) SEC1-stimulated bovine PBMC (from 0 to 106/ml) cocultured with naive PBMC (106/ml) and heat-killed and/or -fixed S. aureus cells. After 96 h, the proliferation of the naive PBMC was assessed by measuring [3H]thymidine incorporation. The results shown are the means of triplicate measurements ± the SEM of data combined from two separate experiments. (B, C, and D) Cocultures incubated in a 1:1 ratio (105 each) (naive PBMC cocultured with the same number of SEC1-stimulated PBMC or purified stimulated T-cell subpopulations) were either not supplemented or supplemented with MAbs specific for IL-10 and/or TGF-ß or with an irrelevant isotype control MAb. (B to D) The proliferation of the naive PBMC in response to heat-killed and/or -fixed S. aureus cells was quantified after cocultures with SEC1-stimulated PBMC (B), CD4+ T cells (C), or CD8+ T cells (D) purified from SEC1-stimulated PBMC cultures. In panel B, "Naive PBMC" indicates data from control wells lacking SEC1-stimlated cells. The results shown are the means of triplicate measurements ± the SEM of data combined from experiments that were performed two times. Statistical analysis was performed to assess differences between results obtained with the isotype control antibody and cytokine-specific MAbs (P < 0.01 indicated by and asterisk).

 
To address which T-cell population was responsible for the suppression, CD4+ and CD8+ T cells were purified from SEC1-stimulated PBMC and used in coculture experiments. Either CD4+ or CD8+ T cells generated from SEC1 stimulation strongly suppressed naive PBMC proliferation in response to heat-killed S. aureus (Fig. 6C and D). The CD4+ suppressive effect was partially abrogated by adding anti-IL-10-specific MAb, anti-TGF-ß-specific MAb, or a combination of both MAbs. The IL-10-specific MAb had a small effect on the CD8+ T-cell suppression. Used alone, only the higher concentration of this MAb induced a statistically significant reversal (Fig. 6D), which was much less dramatic than that observed with CD4+ T cells or with PBMC. The TGF-ß-specific MAb did not have a significant effect at either concentration tested. These results indicate CD4+ and CD8+ T cells mediate their suppression by different mechanisms.


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Staphylococcal diseases, including those associated with SAgs, are a significant cause of morbidity in many animals. In one prevalent disease of dairy cows, staphylococcal mastitis, more than 50% of the S. aureus isolates from infected cows produced one or more staphylococcal SAgs (41). It has been postulated that chronic staphylococcal infection in humans and animals could be exacerbated by SAgs, which induced the aberrant activation of T cells and the accompanying production of cytokines and altered patterns of cell signaling to dysregulate the response to S. aureus (28, 40, 45). Recent studies in humans and mice demonstrate that SAgs induce the development of Tregs, capable of suppressing the primary immune response (11, 42, 44). Despite this, limited information has been obtained on the effects of SAgs on cows other than primates or rodents. In the present study, we established the bovine Treg cell model system, wherein bovine Tregs could be induced by long-term, low-dose exposure to the SAg.

Studies in humans and mice showed that SAg-induced immunomodulation is dose and time dependent (31, 42, 47). Most of the previous studies on the effects of SAgs on the bovine immune system have been performed with a relatively high dose of toxin (1 µg/ml) despite the fact that we have observed a much lower concentration (5 ng/ml) of SEC1 when S. aureus is grown in whole milk (unpublished data). Therefore, the present study was conducted with a much lower and more physiologically relevant dose (5 ng/ml).

Long-term exposure of bovine PBMC cultures to SEC1 resulted in the initial proliferation of CD4+ and CD8+ T cells at similar rates. The more rapid proliferation of CD8+ T cells later resulted in a CD4/CD8 ratio reversal at 10 days. This is consistent with our previous finding (9), except that the reversal of the CD4/CD8 ratio occurred later in the present study, presumably due to our use of a lower dose of SEC1.

Early (prior to 4 days) T-cell proliferation, assessed by CFSE labeling, likely represents the T-cell response due to the direct molecular interaction between the toxin and compatible Vß sequences. Similarly, Renno et al. (36) found that CFSE-stained murine Vß8+ T cells, reactive to SEB, proliferated in response to SEB at 3 days after the administration of SEB, whereas Vß8– T cells did not proliferate during this time period. Unfortunately, molecular or immunological reagents to differentiate among the various bovine TCR families are not currently available. It is noteworthy that few cells remained in the undivided cell fraction at 10 days. This observation suggests that most of the CD4+ and CD8+ T cells eventually proliferated, regardless of their TCR Vß compatibilities with SEC1. We suspect that nonspecific stimulation occurred later in the culture and included the proliferation of T cells bearing Vß without specificity for SEC1. It is likely that the proliferation of these nonreactive cells resulted from the influence of signals from SEC1-specific reactive T cells.

Fikri et al. reported that bovine {gamma}{delta}+ T cells proliferated in response to SEA, SEB, and toxic shock syndrome toxin 1 in the presence of exogenous IL-2 and costimulatory signals from APCs (12). Although SEB and SEC1 are closely related phylogenetically, structurally, and in Vß specificity (33), we did not observe {gamma}{delta}+ T-cell proliferation after SEC1 exposure. This is despite the presence of APCs in the cultures and the expression of IL-2 up to at least 4 days. In the present study, the proportion of {gamma}{delta}+ T cells gradually decreased and constituted only ca. 8% of the T cells at 10 days.

Although Tregs have not been confirmed in bovines, several key findings in the present study suggest strongly that the exposure of bovine T cells to SAgs induces the development of a functionally analogous cell population. SEC1 exposure induced the upregulation of T-cell activation markers (CD25 and CD26) and CD45R0 on CD4+ and CD8+ T cells. Cells expressing naive T-cell markers (CD62L and CD45R) decreased in SEC1-stimulated cultures. The cytokine profiles of SEC1-stimulated PBMC and CD4+ CD25+ T cells showed that the transcription of IL-10 and TGF-ß was increased, whereas the IL-2 levels were decreased. Foxp3 is a key regulatory transcription factor involved in the development of Tregs (18). We identified the bovine foxp3 gene and showed there is a high similarity with other orthologues of foxp3. Consistent with the findings of Brunkow et al. (4), who showed the highest levels of foxp3 expression in the murine CD4+ T-cell population, the transcription of foxp3 was only elevated in SEC1-stimulated CD4+ T cells. Importantly, the transcription of foxp3 peaked at 8 days, indicating that Tregs develop after long-term exposure to low doses of SAg. Whether they are derived from cells with compatible Vßs by direct stimulation with SEC1, or secondarily, is under investigation.

The role of Tregs as effectors of immune responses has been extensively studied in mice and humans for approximately one decade (38). To our knowledge, this is the first report of Tregs in cattle. Several types of Tregs have been documented (38). Among them, Th3 cells, which secrete IL-10 and TGF-ß (7), appear to be most similar to the CD4+ CD25+ subpopulation characterized here. In addition to the cell surface markers typical of Th3 cells, functional assays also yielded results consistent with this conclusion. Coculture experiments using neutralizing MAbs demonstrated that the suppression of the response to heat-killed and fixed S aureus by SEC1-stimulated PBMC was at least partially mediated by IL-10 and TGF-ß. TGF-ß appeared to be the predominant cytokine mediating suppression observed with separated CD4+ T cells. However, the suppression was not fully inhibited by IL-10-specific or TGF-ß-specific MAbs, individually or in combination, suggesting that other mechanisms could contribute to the suppression also.

Similarly, Miller et al. (30), using SEA in a murine model demonstrated that IL-10 and TGF-ß mediated suppression by CD4+ T cells was only partly reversed by treatment with anti-IL-10 and anti-TGF-ß-specific MAbs.

Cell phenotype and cytokine profiles of SEC1-stimulated CD8+ CD25+ T cells showed there was a decrease in the transcription of CD28 and a large increase in the transcription of IFN-{gamma}, findings similar to those recently reported for human CD8+ CD28– suppressor T cells (13). The development of suppressor T cells in a human model required the incubation of CD8+ T cells with alloantigenic monocytes and an exogenous source of IL-2 and GM-CSF (2). In the present study, the stimulation of bovine PBMC with SEC1 induced a dramatic increase in IL-2 at an early time point and a sustained increase in GM-CSF, which might fulfill the requirement for the development of CD8+ CD28– suppressor T cells. Human CD8+ CD28– suppressor T-cell-mediated suppression requires IFN-{gamma} and IL-6. An anti-IFN-{gamma} MAb or antisense oligonucleotide for IL-6 abrogates this suppression (13). In the present study, the transcription of IL-6 was highly decreased, whereas transcription of IFN-{gamma} was highly increased in SEC1-stimulated CD8+ CD25+ T cells. In addition, Tr1 Tregs produce high levels of IL-10 and TGF-ß with moderate levels of IFN-{gamma} and IL-5 (37). Oral tolerance mediated by Th3 Tregs is preceded by the priming of IFN-{gamma}-producing cells. Th3 cells also produce variable amounts of IFN-{gamma} (7). These data strongly suggest that IFN-{gamma} might also be involved in the suppressive activity, together with IL-10 and TGF-ß. Clearly, the role of IFN-{gamma} in Tregs requires further investigation.

These results provide evidence that long-term, low-dose exposure to SEC1 results in at least two immunosuppressive bovine T-cell subpopulations. One of these is phenotypically and functionally characteristic of one type of Treg cell in other animals. The established model in the present study offers an approach to further analyze the immunopathological roles of SAgs in bovine diseases. We are currently extending the present study to determine whether SAg exposure induces the Tregs described in the present study in vivo and predisposes animals to long-term chronic infections or other adverse effects.


   ACKNOWLEDGMENTS
 
This study was supported by the National Institutes of Health grants P20 RR15587, P20 RR016454, and U54AI57141 and the Idaho Agricultural Experimental Station.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, South Line Street, Agricultural Biotechnology Building 222, Moscow, ID 83844. Phone: (208) 885-6666. Fax: (208) 885-6518. E-mail: gbohach{at}uidaho.edu. Back

{triangledown} Published ahead of print on 9 October 2006. Back

Editor: R. P. Morrison


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Infection and Immunity, January 2007, p. 260-269, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.01358-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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