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. 2014 Dec 9;143(2):418–429. doi: 10.1093/toxsci/kfu242

In vitro Exposure to the Herbicide Atrazine Inhibits T Cell Activation, Proliferation, and Cytokine Production and Significantly Increases the Frequency of Foxp3+ Regulatory T Cells

Lindsay E Thueson *, Tiffany R Emmons *, Dianna L Browning †, Joanna M Kreitinger *, David M Shepherd ‡, Scott A Wetzel *,†,1
PMCID: PMC4306722  PMID: 25433234

Abstract

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4+ T lymphocytes. We found that atrazine exposure significantly inhibited CD4+ T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3+ CD4+ T cells.

Keywords: atrazine, Foxp3, CD4+ T cell, cAMP, regulatory T cells

Atrazine (ATR), a chlorotriazine herbicide (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) used to control annual broadleaf and grassy weeds, is one of the most widely applied herbicides in the United States with more than 80 million pounds applied annually (Grube et al., 2011). It is found in ∼70% of all surface and fresh ground water in the United States, making it the most common water contaminant (Bexfield, 2008; Solomon et al., 1999). Surveys conducted by the US Environmental Protection Agency (2003) have reported >88% of well water in agricultural areas is contaminated with ATR at levels above the maximum containment level.

ATR is not acutely toxic and, owing to its relatively short half-life in the body, it does not bioaccumulate (Mcmullin et al., 2003; Ross et al., 2009). However, its prolonged persistence in the environment results in chronic exposure to tens of millions of people. Adverse effects on fetal growth and development (Ochoa-Acuna et al., 2009; Waller et al., 2010; Winchester et al., 2009), male fertility (Swan, 2006), and the nervous (Hossain and Filipov, 2008) and immune systems (Karrow et al., 2005; Rowe et al., 2006) have been described.

At the biochemical level, ATR is a potent phosphodiesterase (PDE) inhibitor, leading to elevated levels of cAMP (Roberge et al., 2004). ATR is also classified as an Endocrine-Disrupting Compound (EDC), which at low concentrations increases levels of estrogen via induction of CYP19 (aromatase) transcription (Laville et al., 2006). Intriguingly, both cAMP (Averill et al., 1988; Tasken and Stokka, 2006) and estrogen (Tai et al., 2008) are implicated in control of T lymphocyte activation and effector functions, suggesting that ATR may be capable of modulating immune function.

Previous studies have shown that ATR affects components of the immune system. Immunotoxic effects such as inhibition of natural killer cell degranulation (Rowe et al., 2007) and dendritic cell maturation (Pinchuk et al., 2007) have been associated with in vitro ATR exposure. ATR exposure also reduces pro-inflammatory cytokine secretion by mitogen-activated human peripheral blood mononuclear cells (PBMC) (Devos et al., 2003; Hooghe et al., 2000), induces mast cell degranulation (Mizota and Ueda, 2006) and modulates macrophage functions (Karrow et al., 2005). In vivo, ATR exposure has been shown to decrease tumor resistance (Karrow et al., 2005). Furthermore, in utero exposure to ATR has been shown to result in immune dysfunction of adult male offspring (Rooney et al., 2003; Rowe et al., 2006). Although these previous studies have provided important clues as to the effects of ATR on the immune system, much remains unknown.

The previously published studies have predominantly focused on the interaction between ATR and innate immune cells. The focus of this study is to characterize how ATR exposure in vitro modulates adaptive immunity, in particular the activation and effector functions CD4+ T lymphocytes. To better understand how ATR may modulate CD4+ helper T cell activity we have exposed primary murine T cells to ATR during activation in vitro. We observed that ATR exposure significantly inhibited CD4+ T cell activation, proliferation, and pro-inflammatory effector cytokine production. We also found that ATR exposure caused a significant increase in the frequency of CD4+ Foxp3+ regulatory T cells (Tregs). ATR exposure significantly increased cAMP levels in CD4+ T cells and the ATR phenotype was partially mimicked by compounds that elevate cAMP, consistent with the ability of ATR to act as a PDE inhibitor. Interestingly, the effect was more pronounced in cells derived from male animals, suggesting that ATR-induced elevated estrogen levels may also play an important role in the observed immune modulation. Taken together, our results show that in vitro, ATR exposure leads to a significant increase in the frequency of Foxp3+ regulatory T cells (Treg), which suppress the activation and effector functions of conventional CD4+ T cells (Tconv). This may have important implications for the generation of protective immune responses by chronically exposed individuals.

MATERIALS AND METHODS

Animals

Spleens were harvested from several mouse strains to provide primary CD4+ T lymphocytes in this study. Heterozygous AD10 T cell receptor (TCR) transgenic mice (Vβ3+) specific for pigeon cytochrome c fragment 88-104 (Kaye et al., 1992) and reactive against moth cytochrome c (MCC) fragment 88-103 in the context of I-EK were provided by David Parker (Oregon Health and Science University, Portland, OR). Balb/c mice carrying the Foxp3gfp knock-in allele (Fontenot et al., 2005) were provided by Alexander Rudensky (University of Washington, Seattle, WA) by way of Kevan Roberts (University of Montana, Missoula, MT).

Mice were housed under specific pathogen-free conditions in the University of Montana Laboratory Animal Resources facilities and were allowed food and water ad libitum. All procedures were supervised and in accordance with the University of Montana Institutional Animal Care and Use Committee.

Staining reagents

The following unconjugated or fluorescent-conjugated antibodies were purchased from BioLegend (San Diego, CA): CD4 (RM4-5), CD25 (3C7 and PC61), CD62L (MEL14), Fc Block (Anti-CD16/CD32; clone 93), and CD69 (H1.2F3). Anti TCR Vβ3 (KJ25) was purchased from BD Biosciences (San Jose, CA). Intracellular staining for Foxp3 was done using the Alexa-Fluor 647 anti-mouse/rat/human FoxP3 (150D) Flow Kit (BioLegend).

Peptides

MCC88-103 was purchased from New England Peptide (Gardiner, MA) and resuspended at 500 µM in PBS. It was diluted in complete RPMI for use.

Preparation of splenic single-cell suspensions

Mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Spleens were harvested and single-cell suspensions were prepared by gentle grinding between sterile ground glass slides. Single-cell suspensions of splenocytes from 6- to 12-week-old mice were depleted of erythrocytes by hypotonic lysis. After hypotonic lysis of red blood cells, lymphocytes were enriched by density centrifugation using Lympholyte M (Cedarlane, Burlington, NC). The lymphocyte-enriched cell suspension was resuspended at 6 × 106/ml in “complete RPMI” containing RPMI (Life Technologies) supplemented with 10% FBS (Atlanta Biologicals, Atlanta, GA), 1 mM L-glutamine, 100 mg/ml sodium pyruvate, 50 µM 2-ME, essential and nonessential amino acids (Life Technologies), 100 U/ml penicillin G, 100 U/ml streptomycin, and 50 µg/ml gentamicin (Sigma, St. Louis, MO.).

Atrazine solution

A 60 mM stock Atrazine (Supleco PS-380, Chem Service, West Chester, PA) suspension was prepared in 100% ethanol (EtOH) and stored at −80°C. For experimental use, the stock solution was vortexed to resuspend the ATR before preparation of a 1000x working solution in absolute EtOH. These 1000x working solutions were then further diluted 1:1000 in culture media before addition of cells. The final EtOH concentration was 0.1% in all cultures, including the vehicle only controls.

Standard ATR exposure assay

Splenic single-cell suspensions at 6 × 106/ml were established from the TCR transgenic or Balb/c Foxp3gfp mouse strains, as described above. To activate the AD10 TCR transgenic T cells, antigenic peptide (MCC88-103) was added to cultures at a final concentration of 2.5 µM. In experiments with Balb/c Foxp3gfp knock-in mice, cells were activated with plate bound anti-CD3 (145-2C11; BD Bioscience) and anti-CD28 (37.51; eBioscience), both at 1 mg/ml. The splenic cell cultures were treated with 3, 15, or 30 µM Atrazine in 0.1% EtOH or the vehicle control, 0.1% EtOH, on day 0. To evaluate the effects of ATR on cell proliferation, cells were stained with 5-(and 6-)-carboxy-2′, 7′-dichlorofluorscein diacetate, succinmidyl ester (CFSE) or Cell Trace Violet (Life Technologies, Eugene, OR) on day 0, according to the manufacturer’s protocol. To allow for cell growth without media exhaustion and acidification, the media volume was doubled on day 2 and ATR or EtOH were added to maintain the treatment concentration. On day 4 cells were recovered from single-cell suspensions, counted, and prepared for flow cytometry analysis.

Flow cytometry

Cells were recovered from the cultures and resuspended at 106 cells/ml in flow cytometry buffer (FACS) (phosphate buffered saline (PBS) containing 2% BSA fraction V [Sigma] and 0.1% Sodium Azide). After treatment with 10 µg/ml Fc Block for 15 min to prevent nonspecific binding of antibodies to Fc receptors, primary antibodies were added and the cell suspension was incubated for 30 min in the dark at 4°C. After 3 washes in FACS buffer, cells were stained for 30 min with secondary reagents in FACS buffer, when necessary. Following a final set of 3 washes in FACS buffer, cells were resuspended in 500 µl of FACS buffer and stored on ice until analyzed using a FACSAria II (BD Biosciences) in the UM Fluorescence Cytometry Core. Alternatively, after the final wash cells were fixed by addition of ice-cold fixative (4% paraformaldehyde and 0.5% glutaraldehyde) followed by a 45-min incubation at room temperature. Following an additional set of washes in FACS buffer, cells were resuspended in 500 µl FACS buffer and were stored at 4°C in the dark for up to 3 days until analysis. Unstained, isotype stained, and single color controls were collected and used in data analysis. Data were analyzed using FlowJo Software (version 8.8.7; Treestar, Ashland, OR).

Identification of Foxp3+ Treg

Cells from day 0 and day 4 cultures were stained for Foxp3 expression using the BioLegend Foxp3 kit, according to the manufacturer’s directions. Briefly, cells were stained for extracellular markers before fixation and detergent permeabilization. The cells were then stained for intracellular anti-Foxp3. With the Foxp3gfp knockin mice, Foxp3 expression was monitored directly by flow cytometry analysis of GFP in conjunction with the surface markers analyzed in the standard exposure assay.

Evaluating apoptosis and necrosis

To examine apoptosis and/or necrosis in ATR-treated and EtOH control cultures, cells were stained with Annexin V-PE and 7-aminoactinomycin D (7-AAD) using the BD Annexin V Staining Kit (BD Bioscience, San Jose, CA) according to the manufacturer’s instructions. Annexin V+ 7-AAD− cells were classified as apoptotic, whereas Annexin V− 7-AAD+ cells and Annexin V+ 7-AAD+ cells were considered necrotic.

Cytokine production

IFNγ production was determined by enzyme-linked immunosorbent assay (ELISA). AD10 splenocytes were harvested from culture after the standard 4-day incubation period. Viable lymphoid cells were enriched by differential density centrifugation, using Lympholyte M (Cedarlane Labs, Canada). These cells were re-cultured in triplicate at 2 × 106/ml and were stimulated with 1 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μM Ionomycin for 24 h. Supernatants were collected after 24 h for ELISA. Purified anti-IFNγ (Clone R4-6A2; BioLegend) was used to capture the cytokine. To detect the cytokine in the supernatants, biotin-conjugated anti-IFNγ (clone XMG 1.2; BioLegend) followed by Horse Radish Peroxidase conjugated-streptavidin and high sensitivity TMB substrate (BioLegend) were used. The samples were run in triplicate and a standard curve was generated using purified IFNγ (R&D Systems). The optical density of each well was determined using a Molecular Devices microplate reader and SoftMax Pro software.

Regulatory T cell suppression assay

To confirm the suppressive ability of the Treg associated with ATR exposure, we used a flow cytometry-based T cell suppression assay. Splenocytes from male AD10 TCR transgenic mice were collected and stimulated in the standard ATR exposure assay. As a positive control, male AD10 splenocytes were stimulated in the presence of 5 ng/ml TGFβ (R&D Systems, Minneapolis, MN.) to induce Treg expansion/conversion. On day 4, an aliquot was stained and the frequency of CD4+CD25+ Foxp3+ cells (Treg) was determined for each culture.

To serve as responders in the suppression assay, fresh naive AD10 TCR transgenic spleen cells were collected. The frequency of CD4+CD25− cells in the spleen cell preparation was determined by flow cytometry. Whole spleen cell suspensions were added to individual wells in a 6-well plate such that 106 CFSE-labeled, naïve CD4+CD25− responder cells were placed to each well. These cells were stimulated with 2.5 µM MCC88–103. To these naïve CFSE+ responder CD4+ cells, 106, 2.5 × 106, or 4 × 106 putative Treg cells from the EtOH and 30 µM ATR cultures were added. After 3 days, the cells were harvested and stained for CD4, CD69, and CD25. CFSE dilution was assessed for the various Treg:Tconv ratio treatment groups. Statistically significant inhibition of naïve T cell proliferation and activation in the presence of the CD4+CD25+ cells was taken to indicate Treg-mediated T cell suppression.

cAMP modulation experiments

To assess the potential role of increased cAMP in the ATR-associated phenotype, the pharmacological PDE inhibitor Pentoxifylline (3. 7-Dimethyl-1- (5-oxohexyl) xanthine; Tocris Biologicals) or the cell-permeant, non-cleavable cAMP analog dibutyryl cAMP (N6, O2′-dibutyryl adenosine 3′, 5′ cyclic monophosphate; Tocris Biologicals) were added to parallel cultures on day 0. Both compounds function to increase intracellular cAMP levels. After 4 days of incubation, the cells were recovered and examined by flow cytometry, as described above.

Measurement of cAMP in CD4+ T cells by ELISA

The concentration of intracelluar cAMP in the 30 µM ATR-treated and EtOH vehicle control cultures was determined using the R&D Systems Parameter murine cAMP ELISA kit (Minneapolis, MN.). Briefly, CD4+ T cells were FACS sorted from the 30 µM ATR and EtOH cultures at the end of the 4-day incubation period using the FACSAria IIu in the University of Montana Fluorescence Cytometry Core. The purity of CD4+ cells was >93% in each experimental group. Cells were resuspended at 107/ml in lysis buffer and the cAMP levels were determined by ELISA following the manufacturer’s directions.

Statistical analysis

To compare the different treatment groups, data were analyzed by student’s t-test or an analysis of variance (ANOVA) using GraphPad Prism 4.0 software. P values of ≤.05 are considered statistically significant.

RESULTS

Several previous studies have shown that ATR exposure modulates the activity of innate immune cells (Karrow et al., 2005; Mizota and Ueda, 2006; Pinchuk et al., 2007; Rowe et al., 2007). To better understand the immunotoxic potential of ATR on adaptive immunity, in this study we have examined the impact of in vitro ATR exposure on the activation, expansion, and effector functions of primary murine CD4+ T cells.

In the studies presented here, cells were exposed to 30 µM ATR unless otherwise noted. The 30 µM ATR dose was chosen based upon preliminary data (data not shown), which found that this concentration resulted in maximal biological effects (reduced cell number, reduced cell size, and reduced expression of activation markers in various cell types in the whole splenocyte culture) with no observed increase in cytotoxicity. As seen in Figure 1A, ATR exposure resulted in a dose-dependent reduction in CD62L down-modulation on CD4+ T cells. CD62L is expressed at high levels on naïve T cells and is reduced upon T cell activation. In the EtOH-only vehicle controls, 77.2% of the cells are CD62L−. In comparison, the frequency of CD62L− cells was reduced to 61.1% in 10 µM ATR cultures, with 46.7% in 30 µM cultures and 33.5% CD62L− in 50 µM cultures. In addition, cell size data (Forward Scatter) showed that T cell blastogenesis was consistently inhibited at 30 µM, with no apparent decrease in cell viability (data not shown). The use of the 30 µM concentration is consistent with the National Toxicology Program guidelines and is similar to, or significantly lower than the concentration used in several previous studies that examined aspects of ATR immunotoxicology (Devos et al., 2003; Hooghe et al., 2000; Pinchuk et al., 2007). This concentration is significantly higher than the MCL and the level from ingesting contaminated drinking water, but comparable levels of ATR metabolites have been found in the urine of farmers and non-farmers in the Midwestern United States (Perry et al., 2001).

FIG. 1.

FIG. 1.

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In vitro Atrazine (ATR) exposure significantly reduces CD4+ T cell proliferation and accumulation, but does not increase apoptosis. A, Male AD10 spleen cells were peptide-stimulated for 4 days in the presence of 0.1% EtOH (vehicle control) or 10 µM, 30 µM, or 50 µM ATR in 0.1% EtOH. ATR exposure results in a dose-dependent reduction of CD62L down-modulation on CD4+ cells. The marker in the graph indicates the CD62L+ population and the frequency of CD62L+ and CD62L− cells for each dose is shown in the associated table. This data is representative of 3 separate experiments. B, The percent reduction in the absolute number of CD4+ T cells in the 30 µM ATR-treated cultures compared with the EtOH-only control cultures is shown for 6 separate experiments. The ATR-associated mean reduction in CD4+ T cells for these 6 experiments was statistically significant; **P = .0058. C, T cells were stained with Annexin V to monitor apoptosis induction and with 7-AAD to monitor necrosis. Apoptotic cells are in the Annexin V+ 7-AAD− lower right quadrant, whereas necrotic cells are found in the upper left Annexin V− 7-AAD+ (upper left) and Annexin V+ 7-AAD+ (upper right) quadrants. Data are representative of 3 separate experiments. D, Male TCR transgenic splenic T cells were stimulated for 4 days in the presence of 30 µM ATR (center) or EtOH vehicle control (left). Proliferation (CFSE dilution) vesus size (forward scatter) of CD4+Vβ3+ gated cells is shown. An overlay of CFSE (right panel) is also shown. These results are representative of 11 separate experiments. E, T cells from AD10 male (gray line) and female (black line) mice were CFSE-labeled before being peptide-stimulated in the presence of EtOH (gray histogram) or 30 μM ATR for 4 days. CFSE levels on CD4+Vβ3+ gated T cells are shown. The data are representative of 6 separate experiments.

In vitro Atrazine Exposure Significantly Reduces Antigen-Driven CD4+ T Cell Accumulation

Fewer CD4+ T cells were consistently recovered from the ATR-treated cultures compared with from the EtOH-only vehicle controls. To determine the impact of ATR exposure on CD4+ T cells, primary male TCR transgenic T cells were activated for 4 days in the presence of 30 µM ATR or the EtOH vehicle control. The number of CD4+ T cells in each culture was calculated and the number of cells in the ATR-treated cultures was compared with the EtOH-only vehicle control cultures. The reduction in the number of CD4+ T cells from ATR cultures compared with the EtOH-only cultures is shown in Figure 1B. In each of 6 separate experiments, there were significantly fewer CD4+ T cells in the ATR-exposed cultures compared with the EtOH vehicle control (Figure 1B). The mean reduction of CD4+ T cell numbers in the ATR-exposed cultures compared with the EtOH control was 70.6%, which was statistically significant (P = .0058). There were also small, but not significant reductions in the number of CD19+ B cells and F4/80+ macrophages in the ATR cultures compared with the EtOH controls (data not shown). Thus, antigen-dependent accumulation of CD4+ T cells was significantly reduced in the presence of 30 µM ATR.

Atrazine Inhibits T Cell Proliferation but does not Increase Apoptotic Cell Death

It was unclear how ATR exposure significantly reduced the antigen-driven accumulation of CD4+ T cells in vitro as seen in Figure 1B. Conflicting reports using transformed cell lines have suggested that in vitro ATR exposure can either inhibit (Kmetic et al., 2008) or induce (Greenman et al., 1997) cellular proliferation. Thus, it was possible that ATR was directly inhibiting the proliferation of the T cells. Alternatively, the cells may proliferate normally in response to antigen, but the presence of ATR could lead to a significant increase in cell death via the induction of apoptosis and/or necrosis.

To test the hypothesis that ATR exposure mediates increased CD4+ T cell death, on day 4 CD4+ T cells were stained with Annexin V and 7-AAD to monitor apoptosis and necrosis, respectively. The results in Figure 1C show that there was not a significant difference in the frequency of apoptotic cells (Annexin V+ 7-AAD− population) between untreated, EtOH-only vehicle controls, and 30 µM ATR-exposed cultures. Similarly, the frequency of necrotic cells (Annexin V− 7-AAD+ and Annexin V+ 7-AAD+) was not significantly different between the treatment groups. From this data, we concluded that ATR-exposure was not reducing the accumulation of CD4+ T cells by increasing cell death in the in vitro cultures.

To examine the alternate hypothesis that ATR exposure inhibits CD4+ T cell proliferation, cells were stained with CFSE or CellTrace Violet at the initiation of the experiments. After 4 days proliferation was evaluated by CFSE or Cell Trace Violet dilution. The results in Figure 1D show that CD4+ T cell proliferation was significantly inhibited by the presence of 30 µM ATR. In addition, the ATR-treated cells have minimal increases in cell size (forward scatter) suggesting that ATR exposure also inhibits their activation. Similar results were seen with antigen-stimulated AND, OT-II, and 3.L2 TCR transgenic CD4+ T cells as well as anti-CD3 + anti-CD28 stimulated non-transgenic CD4+ T cells (data not shown). Thus, the significant reduction in CD4+ T cell accumulation observed in the ATR-exposed cultures was due to inhibition of proliferation and not increased cell death.

Atrazine Effects are More Pronounced in Cells from Male Mice

Previous in vivo studies have shown that in utero ATR exposure leads to immune dysfunction, predominantly in male offspring (Rooney et al., 2003; Rowe et al., 2006). ATR has been classified as an endocrine disrupting compound because it induces the enzyme aromatase, which converts androgens to estrogen causing an imbalance in steroid hormones (Laville et al., 2006). These findings suggest that CD4+ T cells from male animals might be more sensitive to ATR exposure than cells from female mice potentially due to elevated estrogen levels. To examine this possibility, male and female-derived spleen cells were stimulated in the presence of ATR or EtOH and their proliferation was compared. As seen in Figure 1E, the effects of ATR exposure on T cells were more pronounced in cells derived from male mice compared with cells from female mice. The EtOH control (shaded histogram) has gone through multiple divisions, whereas the ATR-treated female cells (black line) show a decrease in proliferation. By comparison, the ATR-treated cells from male mice (gray line) were much more severely inhibited by ATR exposure with >90% remaining undivided. Due to the apparent increased sensitivity to ATR exposure, the subsequent experiments described in this study were performed with cells from male mice.

Atrazine Exposure Inhibits CD4+ T Cell Activation and Effector Cytokine Production

The results from Figure 1 show that in vitro ATR exposure reduced CD4+ T cell accumulation by inhibiting T cell proliferation rather than by enhancing T cell apoptosis. The inhibition of blastogenesis also suggested the possibility that ATR exposure inhibited T cell activation. To further characterize the effects of ATR exposure, the expression of the surface membrane activation markers CD69 and CD25 was monitored. The upper right quadrant of each graph represents CD4+CD25+CD69+ activated T cell population. In Figure 2A, the left panel shows the expression of CD69 and CD25 on CD4+Vβ3+ gated transgenic T cells on day 0. These naïve cells were generally quiescent with only 1.6% in the CD25+CD69+ quadrant. The center and right panels in Figure 2A display CD69 and CD25 expression on cells after 4 days in culture in the presence of EtOH (center) or 30 µM ATR (right). More than 77% of the EtOH control cells were CD25+CD69+, indicating that they had been activated. In contrast, only a minor population (13.2%) of cells in the ATR-treated cultures was CD25+CD69+. The majority (70.5%) remained CD25–CD69–, suggesting they had not been activated. These results are consistent with the inhibition of T cell proliferation and the reduced cell size seen in Figure 1D. In the context of Figure 1, the results in Figure 2A show that both CD4+ T cell activation and proliferation were inhibited by ATR exposure.

FIG. 2.

FIG. 2.

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In vitro Atrazine exposure inhibits the CD4+ T cell activation and effector cytokine production. A, Male AD10 TCR transgenic splenocytes were antigen stimulated for 4 days in the presence of EtOH (center) or 30 µM Atrazine (ATR; right). Cells were stained with anti-CD25 and anti-CD69 to assess activation status. Day 0 cells are shown for comparison (left). The data are representative of 8 separate experiments. B, On day 4 the cells from cultures stimulated in the presence of EtOH, 30 µM ATR or media-only cultures were recovered and stimulated with PMA + ionomycin for 24 h. The concentration of IFNγ in culture supernatants was quantified by ELISA. Values are shown as mean ± SEM. The difference between the EtOH and Atrazine cultures is significant (P = .011). Data are representative of 3 separate experiments.

Having shown that ATR exposure inhibits CD4+ T cell activation, we went on to examine the effects of ATR on CD4+ T cell effector functions. To determine whether ATR exposure alters CD4+ T cell effector function, we quantitated IFNγ production by ELISA. As shown in Figure 2B, ATR exposure during in vitro antigen-driven activation resulted in a statistically significant 28% reduction of IFNγ production. The mean reduction in IFNγ production in the ATR cultures in 3 separate experiments was 36%. These results are consistent with a previous report using non-physiologic activation of human PBMC (Devos et al., 2003; Hooghe et al., 2000). The data in Figure 2B show that in addition to inhibiting proliferation and activation of the T cells, ATR exposure during antigen stimulation resulted in a significant reduction of effector cytokine production.

ATR-Mediated Inhibition of CD4+ T Cell Activation and Proliferation is Dose-Dependent

The results in Figures 1 and 2 show that in vitro exposure to 30 µM ATR inhibits T cell activation, proliferation, and effector cytokine production. To determine whether the observed ATR effects were dose-dependent, male AD10 splenocytes were exposed to 3, 15, or 30 µM ATR during the 4-day, in vitro culture. The results in Figure 3 show that the ATR-mediated inhibition of CD4+ T cell proliferation and activation were indeed dose-dependent. In each graph, the shaded histogram represents the EtOH-only control group. For the EtOH-only control, 85% of the cells divided and the mean fluorescence intensity (MFI) for CD69 and CD25 are indicated in the panel insets. The 3 µM ATR group (thin black line) closely mimics the control group with minimal differences in proliferation (79.5% had divided), CD69 (6% reduction in MFI) and the CD25 MFI (16.2% MFI reduction). In contrast, 30 µM ATR exposure (thick black line) almost completely inhibited T cell proliferation and significantly reduced both CD69 (by 50%) and CD25 (by 94.4%) expression levels. The cells treated with 15 µM ATR (thick gray line) show an intermediate effect for all three parameters tested. In the 15 µM ATR culture, 52.5% have divided, the CD25 MFI was reduced by 55.6% and the CD69 MFI was reduced by 26.3%. Interestingly, for both CFSE and CD25 the 15 µM sample shows a bimodal population where some cells have been activated and were proliferating, whereas the majority of cells have not. These results show that the effects of ATR on T cell activation and proliferation are dose-dependent.

FIG. 3.

FIG. 3.

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Atrazine (ATR)-mediated inhibition of CD4+ T cell activation and proliferation is dose dependent. Male AD10 TCR transgenic spleen cells were antigen-stimulated for 4 days in the presence of 0.1% EtOH only (filled histogram) or 3 μM (thin black line), 15 μM (gray line), or 30 µM ATR (thick black line) in 0.1% EtOH. CFSE dilution (left) and expression of CD69 (center) and CD25 (right) on CD4+Vβ3+ cells are shown. The region marker in the left panel was used to determine the undivided frequency. The frequency of dividing cells (left panel) or the mean fluorescent intensity values (center and right panels) is found in the insets for each panel. Results are representative of 4 separate experiments.

Pharmacologically Increased Intracellular cAMP Partially Mimics the ATR Phenotype

The mechanism of the ATR-associated suppression of T cell activation, proliferation, and effector cytokine production was unclear. ATR is a potent phosphodiesterase inhibitor that causes an accumulation of intracellular cAMP (Roberge et al., 2004). Elevated intracellular concentrations of cAMP have long been implicated in immune modulation (Tasken and Stokka, 2006). The effects of cAMP include inhibition of TCR proximal signaling (Vang et al., 2001), inhibition of IL-2 synthesis and proliferation (Averill et al., 1988; Tasken and Stokka, 2006), and stabilization of Foxp3 expression via control of DNA methylation (Kim and Leonard, 2007). In addition, elevated cAMP is an effector mechanism used by regulatory T cells (Treg) to suppress conventional T cells (Bopp et al., 2007).

Based upon these previous studies, we examined the possibility that elevated cAMP was involved in the observed suppression of the T cells. If ATR’s effects were due to elevated cAMP levels in the T cells, pharmacological reagents that elevate cAMP would mimic the effects of ATR. To test this hypothesis, AD10 T cells were antigen stimulated for 4 days in the presence of the PDE inhibitor Pentoxifylline (PTX) at 250 and 500 µM or the cell-permeable and non-cleavable cAMP analog dibutyryl cAMP (dbcAMP) at 50 and 100 µM. The proliferation and activation of cells from these treatment groups were compared with the effects of ATR exposure. As seen in the top row of figure 4A and enumerated in the table, 500 µM PTX, 100 µM dcAMP and 30 µM ATR all significantly inhibited CD4+ T cell proliferation. When these treatments were compared, 30 µM ATR, which reduced proliferation by 97.1% compared with the EtOH vehicle control, was the most potent of the three followed by 500 µM PTX (which reduced proliferation by 86.9%) and dbcAMP (which reduced proliferation by 72.3%). As shown in Figures 3 and 4A, treatment with ATR resulted in a dose-dependent inhibition of CD4+ T cell proliferation. Similar to the ATR treatments, PTX or dbcAMP treatment significantly reduced proliferation in a dose-dependent manner cells. CD4+ T cell proliferation was inhibited by each of these compounds in a dose-dependent manner, but as seen in Figure 4A, the effects were more severe in the ATR-treated cells.

FIG. 4.

FIG. 4.

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Pharmacological reagents that elevate intracellular cAMP partially mimic Atrazine (ATR) phenotype in a dose-dependent manner. Male AD10 spleen cells were peptide-stimulated for 4 days in the presence of 0.1% EtOH vehicle control or increasing concentrations of ATR (30 and 15 µM; left column), PTX (PDE inhibitor at 500 and 250 nM; center column) and dbcAMP (cAMP analog at 100 and 50 µM; right column). A, Proliferation of CD4+Vβ3+ AD10 cells, as monitored by CFSE dilution, was measured for each treatment group. The region marker in the CFSE plots indicates the position of the non-dividing cells. B, Activation of CD4+Vβ3+ T cells was monitored by examining CD25 expression and C, CD69 expression. The region markers in the CD25 plots indicate the position of the CD25+ population. D, A summary table of numerical data from the plots in figures, A–C is shown. E, The expression levels of CD25 and CD69 on cells from the EtOH, 30 µM ATR, 500 nM PTX, and 100 µM dbcAMP-treated cultures are shown. The table indicates the frequency of CD25+ and CD69+ cells for each treatment. Results are representative of 5 separate experiments.

To examine the potential dose-dependence of elevated cAMP and ATR on T cell activation, the expression of CD25 and CD69 were also assessed. In contrast to the CD25+ population in the EtOH vehicle control, expression of CD25 in the ATR, PTX and dbcAMP-treated cultures was bimodal with both CD25+ and CD25− populations clearly present. As with the proliferation results (Figure 4A), the CD25 data in Figure 4B show that as the concentration of each of the compounds increased, the frequency of CD25+ cells and the overall CD25 MFI decreased. The data, summarized in the table in Figure 4D, showed that compared with the EtOH controls, treatment with 30 µM ATR reduced the frequency of CD25+ cells by 86.4% and the MFI by 94%, whereas treatment with 15 µM ATR reduced CD25+ cells by 64.3% and the MFI by 56%. Similarly, both PTX and dbcAMP reduced the frequency of CD25+ cells and the CD25 MFI in a dose-dependent manner.

We also examined CD4+ T cell activation by assessing the expression of the early activation marker, CD69 (Figure 4C). Compared with the EtOH control, 30 µM ATR reduced the CD69 MFI by 50% and 15 µM ATR reduced it 26.3%. As with the proliferation and CD25 expression data, treatment with PTX or dbcAMP also reduced CD69 MFI in a dose-dependent manner. Thus, for T cell proliferation and each of the parameters of T cell activation tested (CD25 and CD69 expression), ATR and pharmacological reagents that elevate intracellular cAMP inhibit T cell activation in a dose-dependent manner.

To more clearly visualize the effects of these compounds on T cell activation, the plots in Figure 4E show the expression of both CD25 and CD69 on individual cells. Only the highest concentration of each compound (30 µM ATR, 500 µM PTX, and 100 µM dbcAMP) are shown because they had the greatest effect on the CD4+ T cell activation. As can be seen, the frequency of highly activated CD25+CD69+ cells was significantly reduced by each of the treatments compared with the EtOH vehicle control. Consistent with the results in Figures 4 A–D, the effect of ATR exposure was slightly more severe than exposure with either PTX or dbcAMP.

Overall, the results in Figure 4 are consistent with the hypothesis that ATR functions, in part, by elevating intracellular cAMP levels, which contributes to the observed inhibition of T cell activation, proliferation, and effector cytokine production associated with in vitro ATR exposure. This supported by the observation that the concentration of cAMP, as measured by ELISA, was significantly higher in FACS-purified CD4+ T cells from 30 µM ATR-treated cultures (12.7 pMol/106 cells) than in FACS-purified CD4+ T cells from EtOH vehicle control cultures (6.1 pMol/106 cells).

Exogenous IL-2 Augments Rather Than Prevents ATR-Mediated T Cell Suppression

Since the 1980’s it has been known that elevated cAMP levels inhibit T cell IL-2 production and the proliferation of conventional T cells (Averill et al., 1988; Bodor et al., 2007; Cone et al., 1996; Tasken and Stokka, 2006). As ATR is a PDE inhibitor and reagents that elevated intracellular cAMP mimicked the observed ATR effects, ATR-mediated PDE inhibition could account for the loss of proliferation in ATR-treated cultures due to the associated loss of IL-2 production. If insufficient IL-2 in the cultures was responsible for the ATR-associated effects, the addition of recombinant IL-2 should overcome this effect, restoring activation and proliferation of the ATR-treated T cells. To examine this possibility, 50 U/ml recombinant murine IL-2 (rmIL-2) was added to an ATR-containing culture on day 2 of the 4-day incubation period. Unexpectedly, the addition of rmIL-2 increased the potency of ATR’s effect. The data in Figure 5A show that 39.7% of the cells had divided in the ATR culture compared with 89.9% in the EtOH control. In contrast, proliferation was almost completely inhibited in the ATR cultures that had been supplemented with 50 U/ml rmIL-2. The effects on CD25 expression were similar. In Figure 5B, CD25 expression on the ATR-treated cells was bi-modal in the absence of exogenous IL-2, with 53.2% being CD25+. The higher frequency of CD25+ cells compared with Figure 4 is due to experiment-to-experiment variation. Addition of IL-2 to the ATR-treated cultures reduced the number of CD25+ cells to 31.4% and cut the MFI by more than 2-fold. These data suggest that rather than countering the effects of ATR on the T cells, exogenous IL-2 augmented the reduction in T cell activation and proliferation observed in the ATR-treated cultures. Thus, the defective activation and proliferation associated with ATR exposure was not due to insufficient IL-2 production, suggesting another mechanism was responsible for the ATR-mediated inhibition of CD4+ T cells.

FIG. 5.

FIG. 5.

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Exogenous IL-2 does not reverse Atrazine (ATR)-mediated T cell suppression. Male AD10 TCR transgenic T cells were stimulated in the presence of 30 µM ATR (gray line) or EtOH (shaded histogram) for 4 days. On day 2, 50 U/ml rmIL-2 was added to some cultures (black line). A, CFSE dilution and B, CD25 expression on the CD4+Vβ3+ gated cells for each treatment condition is shown. The region marker in the CD25 plot indicates the position of the CD25+ population. The table indicates the CFSE and CD25 mean fluorescent intensity values as well as the frequency of dividing and non-dividing cells. These data are representative of 4 separate experiments.

In vitro Atrazine Exposure Significantly Increases the Frequency of Foxp3+ Regulatory T Cells

The data in Figure 5 showed that exogenous IL-2 potentiated the inhibition of proliferation and activation in ATR-exposed cultures rather than reversing the ATR phenotype. Treg have constitutively high concentrations of cytoplasmic cAMP (Bopp et al., 2007), which stabilizes expression of the master transcriptional regulator for Treg cells, Foxp3 (Yagi et al., 2004). Foxp3+ Tregs also require elevated IL-2 concentrations for expansion and effector functions (Zeiser and Negrin, 2008). Our findings suggest the possibility that ATR exposure might be increasing the frequency of regulatory T cells and/or augment their suppressive activity. To test this hypothesis, we determined the frequency of Foxp3+ Treg in the ATR and EtOH-treated cultures.

To examine the effects of ATR exposure on the frequency of Foxp3+ Treg, peptide-stimulated AD10 T cells were cultured in EtOH or 30 µM ATR for 4 days before fixation and antibody staining for Foxp3. Figure 6A shows that the frequency of Foxp3+ cells in the CD4+CD25+ population increased more than 5-fold in the ATR-treated cultures compared with the EtOH controls. In 7 additional experiments the frequency of Foxp3+ CD4+ T cells was consistently increased by 2- to 5-fold compared with the EtOH controls.

FIG. 6.

FIG. 6.

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Atrazine (ATR) treatment significantly increases the frequency of Foxp3+ regulatory T cells. A, Male AD10 TCR transgenic T cells were antigen-stimulated in the presence of 30 µM ATR (right) or EtOH vehicle control (left) and on day 4 cells were fixed and permeabilized for intracellular Foxp3 antibody staining. The frequency of CD25+Foxp3+ cells in the CD4+Vβ3+ transgenic population is shown. Data are representative of 11 separate experiments. B, Male splenocytes from Balb/c Foxp3gfp+ mice were stimulated for 4 days by immobilized 1 mg/ml anti-CD3 and 1 mg/ml anti-CD28 in the presence of EtOH (left) or 30 µM ATR (right). The frequency of GFP+ Foxp3+ Tregs in CD4+CD25+ gated cells is shown. At the initiation of the experiment 1.52% of the CD4+CD25+ cells were Foxp3gfp+. Data are representative of 5 separate experiments. C, On day 4, splenocytes from EtOH- or ATR-exposed AD10 TCR transgenic male mice were recovered and the frequency of CD4+CD25+ cells was determined by flow cytometry. The CD4+CD25+ putative Tregs from the EtOH or 30 µM ATR cultures were incubated with peptide-stimulated, CFSE-labeled naïve AD10 responder cells at a ratio of 2.5 Treg: 1 responder T cell. CFSE dilution by the responder cells in the presence of the EtOH (thick black line) or 30 µM ATR (thin gray line) CD4+CD25+ cells is shown. Proliferation of the responders in the absence of Tregs (gray histogram) is shown for comparison. Data are representative of 3 separate experiments.

These experiments were repeated using male Balb/c Foxp3gfp knock-in mice (Fontenot et al., 2005) stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of EtOH or 30 µM ATR. Similar to the antibody staining results seen with AD10 cells in Figure 6A, Figure 6B shows that ATR exposure significantly increased the frequency of Foxp3gfp+ cells compared with the EtOH controls. As with antigen stimulation of TCR transgenic cells, in 3 separate experiments, there was a 2- to 5-fold increase in the frequency of Foxp3+ Treg in the ATR cultures compared with the EtOH-only controls. The antibody-stimulation results are consistent with the possibility that ATR is working directly on the CD4+ T cells, but they could also be interpreted as ATR working indirectly via the antigen presenting cells present in the cultures. The significant increase in Foxp3+ CD4+ T cells in the ATR-treated cultures is a novel and very significant finding and is consistent with the effects of ATR exposure on CD4+ T cells.

To confirm that these cells were functional Tregs contributing to the ATR-associated inhibition of proliferation, activation and effector function observed in the ATR-treated cultures, their ability to inhibit naïve AD10 T cell proliferation in vitro was examined using a flow cytometry Treg suppression assay. Naïve AD10 T cells were labeled with CFSE and incubated with the putative Tregs at varying Treg:Tconv ratios. The results in Figure 6C show the CFSE dilution in the CD4+ gated responder population from the 2.5 Treg: 1 Tconv culture. The tinted gray histogram represents proliferation of the naïve AD10 T responder cells in the absence of Treg. When the Tregs from the EtOH culture (black line) were added, the proliferation of the naïve T cells was significantly inhibited. Similarly, when Treg from the ATR culture were added (gray line), responder T cell proliferation was also significantly inhibited. Interestingly, there was not a significant difference in the Treg suppressive potency between the treatment groups. The data in Figure 6C confirm that the CD4+CD25+Foxp3+ cells associated with ATR exposure seen in Figures 6A and B are, indeed, functional Treg.

DISCUSSION

In this study, we have examined the impact of in vitro ATR exposure on the activation, proliferation, and effector functions of CD4+ T cells. Cells were activated in the presence of 30 µM ATR for 4 days before they were examined. This concentration is significantly higher than the level in people exposed through drinking water, but is lower than urinary levels in Midwestern farmers (Perry et al., 2001) and is similar or lower than previous in vitro studies examining the immunotoxicity of ATR (Devos et al., 2003; Hooghe et al., 2000; Pinchuk et al., 2007). We initially observed that ATR exposure significantly reduced the antigen-driven accumulation of CD4+ cells. Seemingly at odds with these results, previous reports have demonstrated that the total CD4+ T cell number is not altered by Atrazine exposure (Filipov et al., 2005; National Institute of Environmental Health Sciences, 1994). This discrepancy is likely due to differences in the experimental design as in those previous reports the in vivo ATR exposure was not associated with lymphocyte activation (Filipov et al., 2005). In our experiments, TCR triggering must occur to induce the ATR-associated phenotype. In the absence of TCR triggering, we observed minimal effects of ATR exposure on CD4+ T cell numbers (data not shown). The requirement for T cell activation in the induction of the ATR-associated phenotype is consistent with our results that elevated intracellular cAMP is mediating, in part, the observed ATR affects on CD4+ T cells (Figure 4). It is well established that antigen recognition leads to adenylyl cyclase activation and the production of cAMP within T cells.

We reasoned that the significant reduction in the accumulation of CD4+ T cells observed in presence of ATR could be due to inhibition of proliferation or to increased cell death. To test whether in vitro 30 µM ATR exposure was cytotoxic to the CD4+ T cells, we monitored apoptosis and necrosis by staining with Annexin V and 7-AAD, respectively. As shown in Figure 1C, ATR exposure did not increase the frequency of apoptotic or necrotic cells. As enhanced cell death was not responsible for the ATR-associated significant reduction in CD4+ T cell accumulation, we tested whether ATR exposure reduced T cell proliferation. As seen in Figure 1D, in vitro ATR exposure inhibited blastogenesis and almost completely blocked CD4+ T cell proliferation. The lack of proliferation seen in Figure 1D was dose-dependent (Figure 3) and correlated with a dose-dependent inhibition of T cell activation (Figures 1A, 2A, 3, and 4). Exposure to ATR also significantly reduced T cell effector function as measured by reduced IFNγ secretion (Figure 2B). These results demonstrated that ATR suppressed T cell activation, effector cytokine production and proliferation in a dose-dependent manner.

The mechanism of this ATR-associated suppression was unclear. However, based upon its known biochemical functions it is it unlikely that ATR exposure directly inhibited signaling from the TCR. This is supported by the data in Figure 4, which show a small percentage of cells still upregulated CD69 and proliferated in the presence of ATR and also down-modulated the TCR (data not shown). Biochemically, ATR is a potent PDE inhibitor that leads to elevated cAMP (Roberge et al., 2004). Elevated cAMP levels are known to inhibit IL-2 production and proliferation of conventional T cells (Averill et al., 1988; Bodor et al., 2007). We reasoned that if ATR exposure significantly increased cAMP within helper T cells, in turn, causing a suppressive effect, then this ATR effect would be comparable with the addition of Pentoxifylline (PTX), a synthetic PDE inhibitor, or dbcAMP, a cell-permeable and non-cleavable cAMP analog. The results showed in Figure 4 showed that PTX and dbcAMP closely resemble the effects of ATR exposure in blocking proliferation and interrupting activation, although the effects of ATR exposure were consistently more severe. The effects of these compounds were dose-dependent (Figure 4). Our ELISA data confirms that the ATR-treated cells contain significantly higher levels of cAMP than the EtOH vehicle controls. These data strongly suggest that cAMP is an important component driving the ATR-associated T cell suppression. These results are consistent with the previously reported inhibition of NK secretion (Rowe et al., 2007) and induction of mast cell degranulation (Mizota and Ueda, 2006), which are both mediated by elevated cAMP levels.

As IL-2 production is reduced by elevated cAMP (Averill et al., 1988; Bodor et al., 2007), the ATR-mediated PDE inhibition could account for the loss of proliferation in ATR-treated cultures as a result of the associated loss of IL-2 production. If insufficient IL-2 was responsible for the ATR-associated effects, the addition of recombinant IL-2 should overcome this effect, restoring activation and proliferation of the ATR-treated T cells. Somewhat unexpectedly, the results in Figure 5 show that, rather than enhancing proliferation, the exogenous IL-2 significantly increased the ATR-associated inhibition of proliferation and activation. Thus, insufficient IL-2 production was not the reason for the inhibition of activation and proliferation in the ATR-treated cultures.

The effects of elevated cAMP differ between conventional and regulatory T cells (Bazhin et al., 2010). In conventional T cells, cAMP inhibits IL-2 secretion, whereas elevated cAMP stabilizes Foxp3 expression, leading to increases in the number or effector potency of Foxp3+ Tregs (Kim and Leonard, 2007). Tregs contain constitutively high concentrations of cAMP and the contact-dependent transfer of cAMP from Treg to Tconv is one effector mechanism they use to attenuate an immune response (Bopp et al., 2007). It is possible then, that ATR exposure elevates intracellular cAMP levels, which could result in Treg conversion and/or expansion and increased Treg effector functions. The fact that supplemental IL-2 potentiated the inhibition of proliferation in ATR-exposed cultures (Figure 5) and Tregs require elevated IL-2 concentrations for expansion and effector function (Zeiser and Negrin, 2008) also supports the possibility that ATR exposure is increasing the frequency of Tregs.

To assess the possibility that ATR was altering the frequency of Foxp3+ Treg, we examined changes in the number of Foxp3+ cells after 4 days in the presence of EtOH or ATR. Our results showed that the frequency of Foxp3+ CD4+ cells consistently increase 2- to 5-fold in ATR-treated cultures compared with the EtOH vehicle controls (Figure 6). This was observed in 2 different models of T cell activation including antigen-stimulated AD10 transgenic T cells stained with anti-Foxp3 antibodies (Figure 6A) and antibody-stimulated Balb/c Foxp3gfp mice (Figure 6B). The fact that the phenotype was similar among the different experimental systems supports the conclusion that the effect is universal. The link between ATR and increased frequency of Foxp3+ Treg has not previously been reported. The Foxp3+ cells from the ATR cultures were functional Tregs, that displayed similar potency to EtOH vehicle control Tregs (Figure 6C). At present, it is not clear whether the increase in Treg frequency is due to expansion of pre-existing “natural” Treg (nTreg) or conversion of conventional T cells (Tconv) to induced Treg (iTreg).

It is interesting to note that the ATR phenotype was slightly more severe than either PTX or dbcAMP treatment (Figure 4). This raises the possibility that elevated cAMP does not account for all of ATR’s effects. We observed a significant difference in the sensitivity to ATR-mediated suppression between cells derived from male and female mice (Figure 1E). These differences in sex-associated sensitivity to ATR are supported by previous studies that have shown that in utero exposure to ATR has a more severe impact on the male offspring than the females (Rooney et al., 2003; Rowe et al., 2006). In addition to being a PDE inhibitor, ATR is classified as an endocrine disrupting compound, because it activates CYP19 (aromatase), which converts androgens to estrogen (Laville et al., 2006). It is possible that the differential sensitivity of male and female cells, as well as the differences between ATR and PTX, could be due to ATR’s modulation of estrogen levels via aromatase II induction (Laville et al., 2006). At physiological levels, estrogen promotes the expansion of CD4+CD25+ Foxp3+ Treg cells (Tai et al., 2008) and engagement of the estrogen receptor (ER) and G protein coupled estrogen receptor (GPER1) can lead to an increase in Treg-like cells (Yates et al., 2010). We are currently pursuing the hypothesis that ATR is inducing the production of elevated estrogen in serum-containing media, which is playing a role in the ATR-mediated CD4+ T cell inhibition and the observed increased frequency of Foxp3+ Tregs.

The results from this study demonstrate that in vitro ATR exposure increases the frequency of functional Foxp3+ Treg. This increase is consistent with the ATR-associated inhibition of conventional CD4+ T cell activation, proliferation and effector cytokine production that was observed. Such a finding suggests that the immune status of people chronically exposed to ATR occupationally or in their drinking water may be altered. The results of the antibody-stimulated cultures (Figure 6B) are consistent with the possibility that ATR is working directly on the CD4+ T cells, however, because whole splenic cultures were used rather than purified T cells, the results are also consistent with the possibility that ATR might be working indirectly via the antigen presenting cells (macrophages and dendritic cells) present in the cultures. Studies are currently underway to determine whether ATR is acting directly on CD4+ T cells or indirectly via antigen presenting cells in the culture. Further work is also required to determine whether the elevated frequency of Foxp3+ Tregs and suppression of conventional T cell activation, proliferation and effector cytokine production observed here results in alterations in the ability of exposed individuals to mount protective adaptive immune responses.

FUNDING

The National Institute of Environmental Health Sciences [grant R03 ES022463] (to SAW) and The University of Montana Small Grant Program [MRA801] (to SAW).

ACKNOWLEDGMENTS

The authors thank Dr Stephanie Lathrop for critical review of manuscript. We also thank Pam Shaw and the University of Montana Fluorescence Cytometry core facility (supported by the National Institute of General Medicine Sciences [NIGMS] P20RR017670) for technical assistance with flow cytometry.

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