Abstract
Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetracholordibenzo- p-dioxin (TCDD) during an acute graft-versus-host response induces a population of alloreactive donor CD4+CD25+ regulatory T (Treg)-like cells that have potent suppressive activity in vitro. In the present studies, we show that TCDD induced a similar population of donor CD8+CD25+ T-cells with suppressive activity in vitro. Like the CD4+ Treg cells, donor CD8+CD25+ T-cells also expressed higher levels of CD28, glucocorticoid-induced TNFR (GITR) and CTLA-4 along with low levels of CD62L. These TCDD-induced phenotypic changes were not observed if donor T-cells were obtained from AhR-KO mice. When CD4+ and CD8+ donor T-cells from AhR-WT and AhR-KO mice were injected in various combinations into F1 mice, the enhanced expression of CD25 on CD8+ T-cells required AhR in donor CD4+ T-cells, while down-regulation of CD62L required AhR in the donor CD8+ T-cells themselves. Changes in GITR and CTLA-4 on donor CD8+ T-cells were partially mediated by AhR in both T-cells subsets. In contrast, all phenotypic changes in donor CD4+ T-cells were dependent on the presence of AhR in the CD4+ T-cells themselves. These findings suggest that the direct effects of AhR-mediated signaling in CD8+ T-cells are more limited than the direct effects in CD4+ T-cells, and that AhR signaling in CD4+ T-cells may be a unique pathway for the induction of both CD4+ and CD8+ adaptive Treg.
INTRODUCTION
AhR is a ligand-activated transcription factor and a member of the basic helix-loop-helix PER-ARNT-SIM (PAS) family of proteins. PAS proteins function as environmental sensors for light intensity, oxygen tension, redox potential, and xenobiotic chemicals (Gu et al., Citation2000). Upon binding ligand, AhR translocates to the nucleus where dimerization with ARNT occurs, forming a complex that is capable of binding to the consensus DNA sequence known as a dioxin response element (DRE) (Schmidt and Bradfield, Citation1996; Mimura and Fujii-Kuriyama, Citation2003). AhR can be activated by a number of different ligands including products of cellular metabolism, dietary components, and a large class of chemicals known as halogenated aromatic hydrocarbons (HAH) (Ciolino et al., Citation1999; Denison and Nagy, Citation2003). The prototypic HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been studied extensively due to its high binding affinity for AhR (Kd = 10− 10 to 10− 9 M) and its potency to induce AhR-dependent effects in animals (Ema et al., Citation1994).
TCDD has long been recognized for its immunosuppressive effects, which are mediated by AhR (Kerkvliet, Citation2002; Kerkvliet et al., Citation2002; Lawrence and Kerkvliet, Citation2007). Expression of AhR in both CD4+ and CD8+ T-cells is essential for complete suppression of allograft responses generated in an acute parent-into-F1 GVH model (Kerkvliet et al., Citation2002). Additionally, AhR-KO mice are completely resistant to the immunosuppressive effects of TCDD, even when treated with higher doses of TCDD (Vorderstrasse et al., Citation2001), further indicating that AhR is necessary for mediating the immunosuppressive effects of TCDD. Recently, studies to identify the changes induced by TCDD in CD4+ T-cells that may account for its potent immune suppression were carried out in our laboratory using the acute parent-into-F1 GVH model. We used flow cytometry to monitor the alloresponsive donor T-cells at various times after injection into F1 hosts and showed that TCDD increased the frequency of donor CD4+ T-cells that expressed high levels of CD25, GITR, and CTLA-4, as well as low levels of CD62L (Funatake et al., Citation2005). This CD4+CD25+ subpopulation was not induced in TCDD-treated host mice if the donor T-cells did not express AhR, indicating that AhR-mediated signaling in the donor T-cells was required for these TCDD-induced changes. Furthermore, donor CD4+CD25+ T-cells isolated on Day 2 from TCDD-treated F1 mice exhibited potent suppressive activity in vitro (Funatake et al., Citation2005), suggesting that activation of AhR induces a subpopulation of CD4+CD25+ donor T-cells that are similar to adaptive Treg.
Consistent with our hypothesis that TCDD-induced adaptive Treg mediate suppression of the GVH response (Funatake et al., Citation2005), it has been observed that alloantigen-induced CD4+CD25+ Treg can mediate tolerance to allografts (Walsh et al., Citation2004). These antigen-induced, adaptive Treg, like natural Treg, are potent suppressors of immune responses. While natural Treg are derived from the thymus and are noted for their expression of the transcription factor Foxp3, a variety of adaptive CD4+ Treg which may or may not express Foxp3 have been described following antigen exposure (Bluestone and Abbas, Citation2003; Shevach, Citation2006). The fact that adaptive Treg do not necessarily express Foxp3 suggests that other signaling pathways mediate induction and expansion of adaptive Treg.
Although less studied than CD4+ Treg, CD8+ Treg have also been described. Both natural and adaptive CD8+ Treg are phenotypically diverse and can be induced by a number of different stimuli (Tang et al., Citation2005; reviewed in Shevach, Citation2006). Naturally occurring CD8+ Treg that express CD25 and Foxp3 have been identified in both humans (Cosmi et al., Citation2003) and mice (Sharma et al., Citation2006). Like CD4+ Treg, adaptive CD8+ Treg expressing CD25, but not necessarily Foxp3, have been induced in several model systems (Zheng et al., Citation2004b; Bisikirska et al., Citation2005; Noble et al., Citation2006). For example, different combinations of cytokines such as interleukin (IL)-2 and transforming growth factor (TGF)-β (Zheng et al., Citation2004b) or IL-4 and IL-12 (Noble et al., Citation2006) have been shown to induce CD8+CD25+ Treg that suppress the proliferation of both CD4+ and CD8+ T-cells. These cytokine-induced CD8+ Treg have also been shown to impair the development of chronic GVH disease (Zheng et al., Citation2004b; Noble et al., Citation2006), though their role in acute GVH is less clear.
In the present study, we identified a subpopulation of the alloresponsive donor CD8+ T-cells in TCDD-treated mice that also resemble Treg. They potently suppress the proliferation of naïve CD4+ T-cells in vitro and express a Treg-like phenotype including increased expression of CD25, GITR and CTLA-4, and low levels of CD62L. However, whereas similar phenotypic changes induced by TCDD in donor CD4+ cells were strictly dependent on activation of AhR within the CD4+ T-cells themselves, most of the effects of TCDD on donor CD8+ cells were driven by activation of AhR in the donor CD4+ cells. These data suggest that activation of AhR triggers signaling pathways involved in the development of adaptive Treg and confers in donor CD4+ cells an ability to induce Treg-like characteristics in donor CD8+ T-cells. The mechanisms by which AhR-induced Treg-like cells mediate suppression are currently under investigation.
METHODS
Mice and Treatment with TCDD
C57Bl/6J (B6) mice (H-2b, Thy1.2) and B6D2F1/J (F1) mice (H-2b/d, Thy1.2) were purchased from The Jackson Laboratory. B6.PL-Thy1a/CyJ (Thy1.1, H-2b) mice and B6.129-AhRtm1Bra/J (AhR-KO; H-2b) mice (originally purchased from The Jackson Laboratory, Bar Harbor, ME) were bred and maintained in our specific pathogen-free animal facility at Oregon State University. B6 mice purchased from The Jackson Laboratory were used as AhR-WT controls for the AhR-KO mice. In later studies, AhR-KO mice were backcrossed onto the Thy1.1 background, and Thy1.1 mice were used as AhR-WT controls. The Institutional Animal Care and Use Committee approved all animal procedures. F1 mice were dosed orally with vehicle or 15 μg TCDD/kg body weight one day before the injection of donor B6 T-cells as previously described (Kerkvliet et al., Citation2002).
Preparation of Donor T-cells
T-Cells were purified from pooled spleens from B6 mice by magnetic bead sorting (Pan T isolation kit, Miltenyi Biotec, Auburn, CA). The purity of the T-cells was > 90% and viability was > 95%. F1 host mice were injected intravenously with 2 × 107 donor T-cells. In some experiments, cell division was assessed by labeling the donor T-cells with 1 μ M CFSE (Invitrogen, Carlsbad, CA) prior to injection into F1 hosts.
In some experiments, individual donor T-cell subsets (CD4+ or CD8+) were purified from the pooled spleens from AhR-WT or AhR-KO mice using sequential positive-selection, first for CD4+ cells, then for CD8+ cells (CD4 or CD8 MicroBeads, Miltenyi Biotec). Donor CD4+ cells were > 95% pure, and donor CD8+ cells were > 90% pure and the contaminating cells were not from the other T-cell subset (< 1%). After purification, the T-cells were recombined giving rise to four possible combinations: CD4-WT CD8-WT, CD4-WT CD8-KO, CD4-KO CD8-WT, or CD4-KO CD8-KO, where WT and KO refer to the AhR-status of the donor mice. The cells were recombined so that the ratio of CD4:CD8 T-cells was the same as it was in the donor mice before separation (between 1.4:1 and 1.7:1).
Flow Cytometry
Spleen cells were stained with anti-H-2Dd and anti-CD8 mAb to identify the donor CD8+ T-cells along with mAb to the following markers: CD62L, CD25, CD28, (BD Pharmingen, San Diego, CA) and GITR (R&D Systems, Minneapolis, MN). In some experiments, Thy1.1 mice were used as donors and identified using anti-Thy1.1 and anti-CD8 mAb. Following surface staining, the cells were fixed and permeabilized (Cytofix/Cytoperm Plus Kit™; BD Pharmingen) and stained with anti-CTLA-4 (BD Pharmingen). Isotype-matched fluorochrome-conjugated Ab were used as controls for nonspecific fluorescence. After gating on live spleen cells, list-mode data on 3000–7000 donor CD8+ T-cells were collected using either a Coulter XL or FC500 flow cytometer (Beckman Coulter, Hialeah, FL). All data analyses, including software compensation, were performed using WinList software (Verity Software House, Topsham, ME).
In Vitro Suppression Assay
The ability of GVH-derived donor CD8+ T-cells or naïve CD4+CD25+ cells to suppress the proliferation of CFSE-labeled naïve CD4+CD25− cells was assessed as described (Kruisbeek et al., Citation2004). Because the frequency of donor CD8+CD25+ cells in F1 mice on Day 2 represented less than 0.05% of the total spleen cells, sorting for these cells specifically was not technically feasible. In an initial experiment, we purified the entire donor CD8+ population as follows. Donor Thy1.1+ T-cells were enriched from pooled spleens of 6-7 vehicle- or TCDD-treated F1 mice on Day 2 by panning to remove B-cells (anti-IgG) and host T-cells (anti-Thy1.2); the CD8+ fraction was purified in two steps (an enrichment sort followed by a purity sort) using a MoFlo® high speed cell sorter (Dako, Fort Collins, CO). The resulting CD8+ population was > 95% pure. In a second experiment, we enriched for the donor CD8+ cells that expressed the highest levels of CD25 by sorting for those cells that had divided two or more times on Day 2. In this experiment, the donor T-cells were labeled with CFSE before injection into F1 mice. After enriching for the donor Thy1.1+ cells by panning as described above, the cells were enriched for CFSE+ cells followed by high purity sorting for the donor CD8+ cells in divisions 2–5 using a MoFlo® high speed cell sorter. The resulting donor CD8+ cells that had divided 2-5 times were > 95% pure, and the percentage of cells that were CD25+ was 23–29% for vehicle and 54–55% for TCDD, which was the same both before and after sorting, indicating that there was not a selective loss of any subset during the sorting process. Similar results were seen in both experiments.
Pooled spleen cells from three naïve B6 mice were sorted into CD4−, CD4 +CD25−, and CD4+CD25+ fractions using a CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec). The CD4− cells were irradiated (3000 rad) and used as accessory cells. The naïve CD4+CD25− cells were labeled with 1 μ M CFSE prior to culturing with anti-CD3 (BD Pharmingen) and accessory cells; donor-derived CD8+ T-cells or CD4+CD25+ cells were added in increasing numbers to the wells. After 72 hr, dilution of CFSE in the naïve CD4+ cells was measured by flow cytometry. Interleukin (IL)-2 and interferon (IFN)-γ were measured by flow cytometry using FlowCytomix mouse simplex kits according to the manufacturer's instructions (Bender MedSystems, Vienna, Austria).
Statistical Analysis
All statistical analyses were performed using SAS statistical software (SAS Institute, Inc., Cary, North Carolina). Comparisons between means were made using the least significance difference multiple comparison (Tukey-Kramer) t-test, with p < 0.05 considered statistically significant.
RESULTS
Early Expansion of the Donor CD8+ T-Cell Population in F1 Mice is not Impaired by TCDD
The ability of donor CD8+ cells to seed to and proliferate in the spleen of F1 mice was determined by tracking the donor CD8+ cells over time using flow cytometry. After gating on live splenocytes (), two strategies for tracking the donor CD8+ cells were used based on staining for H-2Dd ( or Thy1.1 (). As shown in the number of donor CD8+ cells in the spleen of vehicle-treated F1 mice increased steadily through Day 5, with most of the expansion occurring between Days 5 and 10. In TCDD-treated host mice, the number of donor CD8+ cells in the F1 spleen also increased through Day 3 ( inset). However, by Day 5, the number of donor CD8+ cells was significantly less compared to vehicle-treated mice, and by Day 10 there were nearly 5-fold fewer donor CD8+ cells present in the spleen of TCDD-treated mice (). Analysis of CFSE-labeled donor T-cells showed that donor CD8+ cells in the spleen underwent early, robust proliferation beginning on Day 2 after injection (). On Day 2, 58.9 ± 1.8% of the donor CD8+ cells had undergone at least one and as many as five divisions in vehicle-treated mice. In TCDD-treated mice, there was a small but significant increase in the frequency of donor CD8+ cells that had divided three or more times, contributing to a significant increase in the percentage of donor CD8+ cells in cell divisions 1–5 (73.3 ± 0.8%, p < 0.0001). The same pattern of increased cell division in TCDD-treated mice was observed on Day 3 for cells that had divided seven or more times (vehicle = 50.2 ± 0.8%, TCDD = 62.2 ± 1.3%, p = 0.0002). The increase in cell division sometimes correlated with a small increase in the number of donor CD8+ cells in the spleen of TCDD-treated mice. The inconsistency of this finding likely reflects variability in both the cell division and migration of donor CD8+ cells in and out of the spleen at any given point in time. The significance, if any, of this small but reproducible increase in division of donor CD8+ cells to the subsequent suppression of the acute GVH response is not known.
FIG. 1 Exposure to TCDD promotes early cell cycling and inhibits the continued expansion of donor CD8+ cells. F1 mice were dosed with vehicle or TCDD one day before the injection of B6 donor T-cells. After gating on live spleen cells (A), donor CD8+ cells were identified as H2Dd − CD8+ (B) or Thy1.1+CD8+ (C) on Days 1–5. On Day 10, the alloreactive donor CD8+ cells were identified as blasting CD8+ cells. (D) The number of donor CD8+ cells in the spleen was determined on Days 1–5 (see also inset) and on Day 10 in vehicle- (open symbol) and TCDD-treated (filled symbol) mice. The data are combined from several independent experiments, with n = 10–20 mice per group per day. (E) Donor T-cells were labeled with CFSE before injection into F1 hosts and 2 d later the donor CD8+ cells were analyzed for cell division by dilution of CFSE. Five independent experiments were conducted with n = 4–5 mice per group in each experiment. Representative histograms from vehicle- and TCDD-treated mice are shown. *p < 0.05, compared to vehicle.
Exposure to TCDD Promotes an Activated Phenotype in Alloresponsive CD8+ T-Cells
To further characterize the effects of TCDD on donor CD8+ cells, changes in the expression of two well-known activation markers, CD25 and CD62L were assessed over the first five days of the GVH response. As shown in the frequency of CD25+ donor CD8+ cells increased within the first day in vehicle-treated mice when compared to syngeneic controls, with the highest frequency observed on Day 3. Treatment with TCDD resulted in a much greater increase in the percentage of CD25+ donor CD8+ cells with the highest frequency occurring on Day 2 (). After Day 3, the percentage of CD25+ donor CD8+ cells decreased and remained low thereafter in both vehicle- and TCDD-treated mice. The down-regulation of CD62L occurred incrementally on Days 1-5 in vehicle-treated mice (). Exposure to TCDD significantly increased the percentage of CD62Llow donor CD8+ cells on Days 2–4 (). The frequency of CD62Llow donor CD8+ cells in vehicle-treated mice did not reach similar levels until Day 5. These TCDD-induced changes in expression of CD25 and CD62L on donor CD8+ cells are similar to the changes induced by TCDD on donor CD4+ cells (Funatake et al., Citation2005), suggesting that TCDD could be promoting the development of alloresponsive CD8+ Treg.
FIG. 2 Up-regulation of CD25 and down-regulation of CD62L are enhanced following exposure to TCDD. F1 mice were dosed with vehicle or TCDD one day before the injection of donor T-cells. On Days 1–5, the expression of CD25 (A) and CD62L (B) was measured on donor CD8+ cells in the spleen. The data are combined from several independent experiments, with n = 6–20 mice per group per day. *p < 0.05, compared to vehicle.
To further characterize the phenotype of the alloresponsive donor CD8+ T-cells, the co-expression of CD25 with CD62L, CD28, GITR or CTLA-4 was analyzed on Day 2, at the peak of the expression of CD25. As shown in , most of the CD25+ cells in TCDD-treated mice were CD62Llow and co-expressed CD28, GITR and CTLA-4. Far fewer donor CD8+ cells from vehicle-treated mice expressed this phenotype, due largely to the low frequency of CD25+ donor CD8+ cells (). A similar phenotype is expressed by the CD4+CD25+ donor T-cells in TCDD-treated mice previously shown to possess potent suppressive activity in vitro (Funatake et al., Citation2005).
FIG. 3 Donor CD8+CD25+cells in TCDD-treated mice co-express CD62Llow, CD28, GITR, and CTLA-4. F1 mice were dosed with vehicle or TCDD one day before the injection of donor T-cells. On Day 2, donor CD8+ cells were analyzed for the expression of CD25 together with CD62L (A), CD28 (B), GITR (C), and CTLA-4 (D). Representative contour histograms gated on donor CD8+ cells are shown and quadrants were set based on isotype controls. Data are representative of two independent experiments; n = 4–5 mice per group in each experiment. Numbers on the histograms indicate the mean percentage of cells within each quadrant for n = 4–5 mice per group. *Indicates a statistically significant difference (p < 0.05) for the Treg phenotype as compared to vehicle; SEM is not shown for clarity.
Alloresponsive Donor CD8+ Cells from TCDD-treated F1 Mice Potently Inhibit the Proliferation of Naïve CD4+ T-Cells In Vitro
Given the similarity in phenotype between donor CD4+ and CD8+ cells in TCDD-treated mice, it was important to know if the donor CD8+CD25+ T-cells also possessed similar suppressive activity in vitro. Using a sorting regimen designed to isolate the alloresponsive donor CD8+ cells that expressed the highest levels of CD25 (see Methods), the activity of the sorted cells from vehicle- and TCDD-treated mice was tested using a standard in vitro suppression assay (Kruisbeek et al., Citation2004). For comparative purposes, the suppressive activity of natural CD4+CD25+ Treg obtained from naïve, untreated mice was also tested. As shown in , stimulation of naïve CFSE-labeled CD4+ T-cells with anti-CD3 for 72 hr resulted in 4–5 rounds of cell division, with the dividing cells representing 56.3 ± 1.1% of the naïve CD4+ T-cell population. Addition of increasing numbers of donor CD8+ cells from vehicle-treated mice significantly decreased the division of naïve CD4+ cells in a cell concentration-dependent manner. The degree of suppression was greater than that produced by a similar number of natural Treg. In contrast, the addition of donor CD8+ cells from TCDD-treated mice almost completely inhibited the cell division of naïve CD4+ cells at all of the cell concentrations tested ( and ). This suppression of cell division was not due to increased death of naïve CD4+ cells, which was 27–30% for all samples, regardless of the source of regulatory cells added to the cultures (data not shown). Nor did suppression of division appear to be due to infectious anergy in which Treg confer their own non-responsiveness to conventional T-cells (Qin et al., Citation1993; Jonuleit et al., Citation2002; Stassen et al., Citation2004; Waldmann et al., Citation2006), because donor CD8+ cells from both vehicle- and TCDD-treated mice continued to divide to a similar extent ex vivo (data not shown).
FIG. 4 Exposure to TCDD enhances the in vitro suppressive activity of alloresponsive donor CD8+ cells. F1 mice were dosed with vehicle or TCDD one day before injection of CFSE-labeled donor T-cells. On Day 2, the donor CD8+ cells that had divided two or more times were sorted as described in the Methods. Donor CD8+ cells from vehicle- or TCDD-treated mice were added to wells at 1:8, 1:4, or 1:2 per CFSE-labeled naïve CD4+ cell. Three days later, the cultures were harvested and the dilution of CFSE in the naïve CD4+ cells was measured. (A) Representative histograms for each ratio are shown as overlays, with 10,000 events per histogram. (B) The percentage of CFSE-labeled naïve CD4+ cells that had divided after three days in culture was determined for each culture condition. (C) IL-2 and (D) IFNγ were measured in the supernatants after three days of culture using cytokine bead arrays. Each data point represents the average of triplicate wells. Data shown are representative of two independent experiments.
We also measured the levels of IL-2 and IFNγ present in the supernatant of the suppression assay cultures ( and ). Stimulation of naïve CD4+ T-cells in the absence of any Treg induced production of IL-2 but not IFNγ compared to unstimulated cells ( and ). Addition of natural CD4+ Treg as well as donor CD8+ cells from both vehicle- and TCDD-treated mice dramatically decreased the amount of IL-2 present in the cultures. The decrease in IL-2 was nearly complete when the CD8+ cells were from TCDD-treated mice, while CD8+ cells from vehicle-treated mice appeared to mediate their effects in a cell-number dependent manner, similar to natural CD4+ Treg (). Cultures containing donor CD8+ cells from vehicle-treated mice had very high levels of IFNγ that progressively decreased upon addition of greater numbers of the same donor CD8+ cells, possibly due to autocrine and/or paracrine consumption (). In contrast, very low levels of IFNγ were detected in wells containing donor CD8+ cells from TCDD-treated mice and the amount of IFNγ was higher than background only when the fewest number of donor CD8+ cells were added to the wells. When cultured alone (no naïve CD4+ cells were added to the wells), donor CD8+ cells from vehicle- or TCDD-treated mice did not produce IL-2 or IFNγ in response to stimulation with anti-CD3 (data not shown).
Expression of AhR in the Donor T-Cells is Required for TCDD-Induced Phenotypic Changes on Donor CD8+ Cells
AhR has been shown to mediate the majority of the biological effects of TCDD, including the induction of CD4+ Treg (Funatake et al., Citation2005). To determine if AhR was required for the changes induced by TCDD in CD8+ cells, vehicle- or TCDD-treated F1 mice were injected with CFSE-labeled donor T-cells obtained from AhR-WT or AhR-KO mice, and phenotypic analysis of donor CD8+ cells was carried out two days later. As shown in and , the enhanced up-regulation of CD25 and down-regulation of CD62L on donor CD8+ cells occurred only when the donor T-cells came from AhR-WT mice. Likewise, TCDD-mediated up-regulation of GITR and CTLA-4 on donor CD8+ cells was observed only when the donor T-cells came from AhR-WT mice ( and ). Thus, the phenotypic changes induced by TCDD on donor CD8+ cells are dependent on the expression of AhR in the donor T-cells.
FIG. 5 TCDD-mediated changes in the phenotype of donor CD8+ cells are dependent on AhR in the donor T-cells. F1 mice were dosed with vehicle or TCDD one day before the injection of CFSE-labeled AhR-WT or AhR-KO donor T-cells. On Day 2, donor CD8+ cells were analyzed for expression of CD25 (A), CD62L (B), GITR (C), and CTLA-4 (D). Data shown are representative of 2–4 independent experiments; n = 4 mice per group. *p < 0.02, compared to vehicle.
While the phenotype of the donor CD8+ cells was altered in an AhR-dependent manner, the same was not true when the effects of TCDD on cell division were examined. As shown in and , donor CD8+ cells from all treatment groups divided up to five times by Day 2 after injection, and, as indicated previously (see ), treatment with TCDD led to a small but significant increase in cell division. Surprisingly, increased cell division was observed independently of AhR expression in the donor T-cells (), suggesting that enhanced division of donor CD8+ cells following exposure to TCDD is not mediated by direct, AhR-dependent signaling in the donor T-cells. This could result from an AhR-independent effect of TCDD on the donor T-cells, or more likely, from an AhR-dependent effect of TCDD in the F1 host cells thereby indirectly influencing the proliferation of donor CD8+ T-cells.
FIG. 6 Exposure to TCDD enhances cell division of donor CD8+ cells independent of AhR. F1 mice were dosed with vehicle or TCDD one day before the injection of CFSE-labeled AhR-WT (A) or AhR-KO (B) donor T-cells. On Day 2, the cell division of donor CD8+ cells in the spleen was measured by flow cytometry. The data shown are representative of five independent experiments; n = 4 mice per group. *p < 0.02, compared to vehicle.
The down-regulation of CD62L and the up-regulation of CD25 have been shown to be linked to cell division (Oehen and Brduscha-Riem, Citation1998; Gudmundsdottir et al., Citation1999). However, based on the above data, it appears that exposure to TCDD dissociates cell division from altered expression of CD62L and CD25. To determine this directly, we looked at the changes in expression of CD25 and CD62L as a function of cell division number. As shown in , enhanced up-regulation of CD25 as well as down-regulation of CD62L occurred only on AhR-WT donor CD8+ cells beginning as early as the second round of division; no changes were seen on AhR-KO donor CD8+ T-cells. Thus, the phenotypic changes, although occurring on the cells that were dividing, were not strictly linked to enhanced cell division but were mediated by activation of AhR in the donor T-cells.
FIG. 7 Enhanced up-regulation of CD25 and down-regulation of CD62L is correlated with cell division and is dependent on activation of AhR within the donor T-cells. F1 mice were dosed with vehicle or TCDD one day before the injection of CFSE-labeled AhR-WT or AhR-KO donor T-cells. On Day 2, donor CD8+ cells were analyzed for cell division and the mean channel fluorescence (MCF) of CD25 (A) and CD62Llow (B) was examined in each cell division. Data shown are representative of 4 independent experiments; n = 4 mice per group. *p < 0.05, compared to VEH WT.
AhR-Mediated Signaling in Donor CD4+ and CD8+ Cells Elicits Different Phenotypic Changes in Each Subset
Previous studies have shown that expression of AhR in both the CD4+ and the CD8+ donor T-cells is required for TCDD to fully suppress the anti-host CTL response on Day 10, and only partial suppression of CTL activity is seen if only one of the T-cell subsets expressed AhR (Kerkvliet et al., Citation2002). To determine if the phenotypic changes induced by TCDD in the CD4+ and CD8+ donor T-cells were dependent on selective expression of the AhR in either T-cell subset, CD4+ and CD8+ T-cells were separately purified from AhR-WT or AhR-KO mice and then recombined before injection into F1 hosts (see Methods). On Day 2 after injection, the expression of CD25, CD62L, GITR, and CTLA-4 were analyzed on both donor CD4+ and CD8+ cells. As shown in (left two bar pairs), increased expression of CD25, CD62Llow, GITR and CTLA-4 occurred on donor CD4+ cells only when the donor CD4+ cells themselves expressed AhR. If the donor CD4+ cells did not express AhR, TCDD had no effect on the phenotype of donor CD4+ cells, regardless of the AhR status of the CD8+ T-cells (, right two bar pairs).
FIG. 8 TCDD-mediated phenotypic changes on donor CD4+ and CD8+ cells display a differential requirement for AhR within each donor T-cell subpopulation. F1 mice were dosed with vehicle (white bars) or TCDD (black bars) one day before the injection of CFSE-labeled donor T-cells that had been purified and recombined as described in the Methods. Naïve B6 mice were injected with the same donor T-cell inoculum as F1 mice (one for each combination) and served as syngeneic controls (SYN, grey bar). The donor T-cells in B6 mice were identified based on CFSE. On Day 2, donor CD4+ cells (A) and donor CD8+ cells (B) were analyzed for the expression of CD25, CD62L, GITR, and CTLA-4. Data shown are representative of 2-3 independent experiments; n = 3-4 mice per group. *p < 0.05, compared to vehicle-control with the same donor T-cell inoculum.
In contrast to donor CD4+ T-cells, AhR-driven phenotypic changes in donor CD8+ cells were more complicated. Most surprisingly, the enhanced up-regulation of CD25 on donor CD8+ cells was dependent on the presence of AhR in the donor CD4+ cells and not on the AhR in the donor CD8+ cells (, left two bar pairs). Up-regulation of GITR and CTLA-4 on donor CD8+ cells was partially mediated by AhR in CD4+ cells and also by the AhR in the CD8+ cells. The increased frequency of CD62Llow donor CD8+ cells was the only phenotypic change induced by TCDD that was strictly dependent on the expression of AhR within the donor CD8+ cells themselves. TCDD had no effect on either subset of T-cells when both donor CD4+ and CD8+ cells lacked AhR. In addition, in no instance was expression of any of the markers on the donor T-cells different between the vehicle-treated groups, indicating that the lack of AhR in both or either of the donor T-cell subsets had no effect on the phenotypic response. This is consistent with earlier studies that showed AhR-KO mice as well as donor T-cells from AhR-KO mice generate a normal CTL response to alloantigen stimulation (Vorderstrasse et al., Citation2001; Kerkvliet et al., Citation2002). Taken together, the data suggest that activation of AhR in CD4+ T-cells is the primary determinant for the induction of the CD4+CD25+ and CD8+CD25+ Treg-like phenotype.
DISCUSSION
Adaptive CD4+ and CD8+ Treg have been described in many different systems (reviewed in Shevach, Citation2006). Strategies to induce adaptive Treg are important not only for understanding the ontogeny of Treg but also for their therapeutic use in the treatment of autoimmune diseases as well as transplant recipients. Previous studies from our laboratory found that activation of the transcription factor AhR by its most potent ligand TCDD during an acute GVH response leads to the generation of donor CD4+CD25+ T-cells with potent Treg-like activity in vitro (Funatake et al., Citation2005). In those and additional experiments, we examined the phenotypic and functional characteristics of the donor CD8+ cells in F1 host mice as influenced by exposure to TCDD. As reported here, activation of AhR by TCDD induced a Treg-like phenotype in donor CD8+ cells on Day 2 of the GVH response along with suppressive activity in vitro similar to that seen for the donor CD4+ cells. Surprisingly, the activation of AhR in donor CD4+ cells alone was sufficient to enhance the up-regulation of CD25 on both donor CD4+ and CD8+ cells, and fully to partially induced the up-regulation of GITR and CTLA-4 on the donor CD4+ and CD8+ cells, respectively. These findings suggest that activation of AhR in donor CD4+ cells facilitates the induction of both CD4+ and CD8+ Treg-like cells by TCDD. Thus, AhR represents a potentially important therapeutic target for the induction of adaptive Treg.
CD8+ Treg have been described in many different models. For each model, the CD8+ Treg are unique, not only by phenotype but also in the manner in which they are derived (Jiang et al., Citation1998; Cortesini et al., Citation2001; Cosmi et al., Citation2003; Filaci et al., Citation2004; Hu et al., Citation2004; Rifa'i et al., Citation2004; Zheng et al., Citation2004b; Bienvenu et al., Citation2005; Bisikirska et al., Citation2005; Endharti et al., Citation2005; Wei et al., Citation2005; Maile et al., Citation2006; Noble et al., Citation2006; Shevach, Citation2006). In our model, activation of AhR during the GVH response induced a profound enhancement of the expression of CD25 on the CD8+ donor T-cells. In association with the up-regulation of CD25, activation of AhR also enhanced the up-regulation of CD28, GITR, and CTLA-4, while at the same time enhancing the down-regulation of CD62L. Interestingly, this phenotype is similar to the phenotype of other adaptive CD8+ Treg induced by modified anti-CD3 antibody that is used clinically to treat certain conditions such as allograft rejection and Type I diabetes mellitus (Woodle et al., Citation1999; Herold et al., Citation2002; Bisikirska et al., Citation2005). We have also looked at other markers associated with activated or regulatory CD8+ T-cells (CD44, CD69, CD30, 41BB, CD103, CD71) and found no TCDD-mediated changes in expression on the donor CD8+ T-cells on Day 2 (data not shown). Cytokine analysis of the supernatants from the suppression assay revealed that donor CD8+ cells from vehicle-treated mice were capable of producing large amounts of IFNγ when naïve CD4+ cells were present in cultures. In contrast, donor CD8+ cells from TCDD-treated mice did not produce any IFNγ. These data suggest that enhanced or premature T-cell activation per se is not the underlying mechanism of TCDD's immunosuppressive effects.
The induction of allospecific CD4+ and CD8+ Treg has been described using various combinations of IL-10, TGF-β and IL-2 (Chen et al., Citation2003; Horwitz et al., Citation2003; Zheng et al., Citation2004a and b). We have found that TCDD significantly increases the frequency of IL-2+ donor CD4+ T-cells 24 hr after injection into F1 hosts (Funatake et al., manuscript in preparation), which is consistent with earlier findings showing that the IL-2 gene possesses three DRE in its promoter/enhancer region (Lai et al., Citation1997; Jean and Esser, Citation2000). However, excess IL-2 alone, given during the first three days of the GVH response, did not recapitulate the effects of TCDD on the phenotype of donor T-cells (Funatake et al., manuscript in preparation). Gene array analysis of purified donor T-cells (containing a mixture of both CD4+ and CD8+ cells) isolated from F1 mice on Day 2 showed that exposure to TCDD increased transcription of IL-10 and TGF-β 3 genes (unpublished observation), and putative DRE have been identified in the promoter regions of both of these genes (Sun et al., Citation2004). Although we detected an increase in IL-10 mRNA, no IL-10 was detected in the supernatants of the suppression assay cultures (data not shown). This suggests that donor CD8+ cells are not the source of increased IL-10 mRNA detected in the gene array analysis. The increase in transcription of TGF-β 3 has not been confirmed at the protein level, and any role it plays in AhR-mediated induction of adaptive Treg remains to be determined.
The signaling pathways required for the induction of adaptive CD8+ Treg are not well understood. Recently, Maile et al. (Citation2006) have reported that low avidity stimulation of CD8+ T-cells can induce CD8+ Treg. This finding may be relevant to AhR-induced Treg in that activation of AhR induces down-regulation of CD62L earlier and more profoundly on donor T-cells from TCDD-treated mice as compared to donor cells from vehicle-treated mice. Since CD62L has been shown to play a role in T-cell activation by enhancing the adhesion between the antigen presenting cell (APC) and the T-cell through activation of CD11a (Giblin et al., Citation1997), the low expression of CD62L on donor T-cells in TCDD-treated mice may result in a reduced ability to maintain prolonged contact with the APC. Interestingly, the level of expression of CD11a on donor CD8+ cells was reduced by TCDD on Day 3 of the GVH response (unpublished data). Induction of CD8+ Treg by low-avidity stimulation is consistent with a previously proposed model of AhR-mediated immune suppression in which exposure to TCDD caused reduced expression of CD62L and CD11a on DO11.10 transgenic T-cells (Shephard et al., 2000; Funatake et al., Citation2004).
Although induction of CD25+CD62Llow Treg-like cells depends on expression of AhR in the donor T-cells, prior studies had only shown this dependence using whole donor T-cells prepared from AhR-WT or AhR-KO mice. Using separately purified CD4+ and CD8+ T-cells from AhR-WT and AhR-KO mice that were recombined before injection into F1 mice, the present studies showed that activation of AhR within the donor CD4+ T-cells was responsible for all of the phenotypic changes induced in the donor CD4+ cells by TCDD (i.e., autoregulated effects) as well as the increased expression of CD25 on donor CD8+ T-cells when the CD8+ T-cells did not express AhR themselves. Activation of AhR in donor CD4+ cells also played a significant role in the up-regulation of CTLA-4 and GITR on the donor CD8+ cells. On the other hand, AhR in the donor CD8+ cells drove the down-regulation of CD62L and partially mediated the up-regulation of CTLA-4 and GITR. If CD25 is the discriminating Treg marker, these results indicate that activation of AhR in the donor CD4+ cells mediates the induction of adaptive CD4+ and CD8+ Treg. However, the degree to which each of the other phenotypic changes (CD62Llow, GITR+, CTLA-4+) contribute to the suppressive activity of the CD25+ donor T-cells remains to be determined. Recently, we found that a constitutively active form of AhR expressed exclusively in donor T-cells caused the same level of down-regulation of CD62L as exposure to TCDD, but did not up-regulate CD25 nor did it result in suppression of the CTL response (Funatake et al., manuscript in preparation), supporting the likelihood that early up-regulation of CD25 is a critical phenotypic change associated with the immunosuppressive effects of TCDD.
Natural Treg are characterized by expression of Foxp3, and natural CD8+CD25+ Foxp3+ Treg have been described in normal mice (Sharma et al., Citation2006). However, depletion of CD25+ cells from the donor cell inoculum prior to injection into F1 hosts did not impair the induction of CD8+CD25+ or CD4+CD25+ cells following exposure to TCDD (unpublished observations and Funatake et al., Citation2005), indicating that these subpopulations induced by TCDD were not derived from naturally occurring Treg. In further support of this, neither the CD4+ nor CD8+ Treg-like cells express Foxp3 on Day 2 (Marshall et al., manuscript in preparation and data not shown). These results are consistent with other studies showing that the induction of adaptive CD4+ Treg is not dependent on expression of Foxp3 (Vieira et al., Citation2004). In addition to expression of Foxp3, natural Treg are also characterized by a state of anergy. We found that although donor CD8+ cells from TCDD-treated mice do not make cytokines in response to ex vivo stimulation with anti-CD3 they do continue to proliferate, indicating that the donor CD8+ cells are not anergic. Taken together, our findings suggest that exposure to TCDD does not simply expand the existing pool of natural Treg. Thus, AhR may be a transcription factor important for the induction of some adaptive CD4+ and CD8+ Treg, just as Foxp3 is important for the development of natural Treg.
The dissimilarities between natural Treg and AhR-induced Treg suggest that the mechanism by which they mediate suppression is also unique. We observed that donor CD8+ cells from both vehicle- and TCDD-treated mice suppressed the proliferation of naïve CD4+ cells, but donor CD8+ cells from TCDD-treated mice were more potent in this regard. One explanation for this is the high expression of CD25 and the consumption of IL-2. A much greater percentage of donor CD8+ cells from TCDD treated mice expressed CD25 compared to cells from vehicle-treated mice. However, the natural CD4+ Treg all expressed high levels of CD25 and they were the least suppressive. Thus, expression of high levels of CD25 alone does not explain the differences in suppressive potency observed in vitro. Natural Treg have been shown to mediate suppression through a process termed “infectious anergy” (Qin et al., Citation1993; Jonuleit et al., Citation2002; Stassen et al., Citation2004; Waldmann et al., Citation2006). This does not appear to be involved in the suppressive mechanism of donor CD8+ cells because, as mentioned above, the donor CD8+ cells continue to divide and do not appear to be anergic. As a negative co-stimulatory molecule, CTLA-4 has been suggested to mediate suppression by Treg (Takahashi et al., Citation2000; Tang et al., Citation2004). A greater proportion of donor CD8+ cells from TCDD-treated mice are CTLA-4+ as compared to donor CD8+ cells from vehicle-treated mice and could help explain the difference in potency between the two sources of donor CD8+ cells. However, the specific role of CTLA-4 in mediating suppression by the donor CD8+ cells has not yet been determined. Current studies in the laboratory are aimed at elucidating the mechanism(s) by which AhR-induced Treg-like cells suppress the proliferation of naïve T-cells.
ACKNOWLEDGMENTS
We would like to thank Linda Steppan for her outstanding technical assistance and Julie Oughton for her exceptional assistance with cell sorting and data analysis. We would also like to thank the Cell and Tissue Analysis Facilities and Services Core of the Environmental Health Sciences Center at Oregon State University for the use of the flow cytometers. This work was supported by National Institutes of Health Grants P01ES0040, P30ES0210, and T32ES07060.
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