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The Journal of Immunology, 2006, 177: 5819-5828.
Copyright © 2006 by The American Association of Immunologists, Inc.

Aryl Hydrocarbon Receptor Activation Impairs the Priming but Not the Recall of Influenza Virus-Specific CD8+ T Cells in the Lung1

B. Paige Lawrence2,*, Alan D. Roberts{dagger}, Joshua J. Neumiller*, Jennifer A. Cundiff* and David L. Woodland{dagger}

* Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164; and {dagger} Trudeau Institute, Saranac Lake, NY 12983


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR+/+ recipients, and the generation of CD45.2AhR–/–->CD45.1AhR+/+ chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The aryl hydrocarbon receptor (AhR)3 is a ligand-activated transcription factor that is expressed in cells of the immune system (1, 2, 3, 4, 5). It is a member of the basic helix-loop-helix Per-Arnt-Sim family of transcriptional regulators, which play roles in toxin metabolism, development, circadian rhythm, response to hypoxia, and hormone signaling (6, 7, 8). The AhR is often considered an orphan receptor, because it has been difficult to identify an endogenous ligand; however, it binds many different types of exogenous compounds, including many environmental contaminants. Polychlorinated dibenzo-p-dioxins, polychlorinated biphenyls, and polyaromatic hydrocarbons, such as benzo(a)pyrene, which are found in diesel exhaust and cigarette smoke, are well-known AhR agonists. Additional AhR ligands include compounds derived from numerous plant species, such as catechins found in green tea, indole-containing compounds and their metabolites, and bioflavanoids such as genistein and resveratrol (6, 9, 10). In summary, AhR ligands are a combination of widely dispersed and persistent environmental contaminants and common phytochemicals found in regularly consumed foods. Consequently, humans are exposed to AhR ligands daily through ingestion and inhalation.

The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is used as a model AhR agonist because of all the AhR agonists identified to date, it has the highest binding affinity, and has been used for the majority of studies characterizing the consequences of AhR activation (6, 11, 12, 13, 14). Although very little is known about the endogenous function of the AhR in the immune system, TCDD has a very profound effect on immune function. In fact, T cell-dependent responses and host resistance to infection are extremely sensitive targets for modulation by AhR agonists (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Although the molecular mechanisms that underlie these effects are not entirely clear, epidemiological data suggest that exposure to pollutants that contain AhR agonists correlates with diminished host resistance, altered immune function, and an increased incidence of influenza and other respiratory infections (27, 28, 29), suggesting a possible cause and effect relationship between exposure to AhR ligands and altered host resistance to infection.

We have investigated the mechanisms underlying the immunosuppressive effects of TCDD, and have shown previously that exposure of mice to TCDD impairs multiple facets of the immune response to influenza A virus, resulting in suppressed virus-specific IgG levels, enhanced pulmonary inflammation, and altered cytokine production in the lung and draining lymph nodes (25, 30, 31). Importantly, we have found that during primary infection, exposure to a single dose of TCDD suppresses the proliferation of CD8+ T cells in the mediastinal lymph nodes (MLN), reduces their production of IFN-{gamma}, and impairs the generation of CTL (25, 30). In addition to suppressive effects on the response of endogenous CD8+ T cells, exposure to TCDD impairs expansion, differentiation, and IFN-{gamma} production by influenza virus-specific TCR transgenic CD8+ T cells that have been adoptively transferred into wild-type recipients (32).

In a recent study, we analyzed the impact of TCDD on the primary and recall CD8+ T cell response to an immunodominant epitope of influenza A virus (nucleoprotein (NP)366–374/Db) in C57BL/6 mice. These studies demonstrated that a single dose of TCDD resulted in 50% fewer NP366–374/Db-specific memory CD8+ T cells in the MLN 60 days after infection. Furthermore, upon recall challenge, the expansion of NP366–374/Db-specific memory CD8+ T cells was delayed by several days compared with the response in infected mice that were not treated with TCDD (33). These observations suggest that AhR activation by TCDD not only impairs the primary CD8+ T cell response, but may also adversely affect the recall response of Ag-specific CD8+ memory T cells. However, it is not clear whether the decreased frequency of NP366–374/Db-specific memory CD8+ T cells in the MLN reflects an altered distribution of memory cells or a direct impairment in their generation. Likewise, the delayed expansion of memory CD8+ T cells upon reinfection could result from either a reduced functional capacity or the smaller number of memory cells available at the time of recall challenge. The present study dissects the effects of AhR activation on the in vivo distribution and frequency of CD8+ T cells specific for different viral epitopes during and after the resolution of a primary infection, and the effects of TCDD on virus-specific memory CD8+ T cells during recall challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, TCDD treatment, viruses, and infection

Female C57BL/6 (B6) mice (age 5–8 wk) were obtained from The Jackson Laboratory or National Cancer Institute. Female B6.Pl-Thy-1a/Cy (CD90.1) and B6.SJLptprcapep3b/BoyJ (CD45.1) mice were purchased from The Jackson Laboratory. Female B6.AhRtm1Bra (AhR–/–) mice were obtained by breeding AhR–/+ females with AhR–/– males. AhR–/– mice were bred at Washington State University and genotyped by PCR, as described previously (31). All mice were housed in microisolator cages in a specified pathogen-free facility at either Washington State University or the Trudeau Institute, and were provided food and water ad libitum. All animal treatments were conducted with approval of Institutional Animal Care and Use Committees at Washington State University and the Trudeau Institute.

TCDD (≥98% purity; Cambridge Isotope Laboratories) was dissolved in anisole and diluted in peanut oil to 1 µg/ml. Mice were given a single oral dose of 10 µg of TCDD/kg body weight by gavage. Except where indicated, TCDD was administered only once, 1 day before primary infection. Control mice received the peanut oil-anisole vehicle in the same manner as described above.

Influenza A virus strains HK-x31 (x31; H3N2) and PR8/34 (PR8; H1N1) were prepared, titered, and stored, as described previously (34, 35). For primary infection, mice were anesthetized by i.p. injection of 2,2,2-tribromoethanol and intranasally (i.n.) infected with either 120 hemagglutinating units or 300 egg ID50 (EID50) of x31. In some experiments, mice were i.n. challenged 30–40 days after x31 infection with 3000 EID50 PR8. In the adoptive transfer experiments, donor mice and naive recipient mice were i.n. infected with x31.

Collection and preparation of cells

Leukocytes were obtained from lung airways by bronchoalveolar lavage (BAL) into cold RPMI 1640 containing 1% BSA, and 10 mM HEPES or HBSS. Single-cell suspensions from lavaged lungs, spleen, and MLN were prepared by passing tissues through cell strainers. Erythrocytes were removed by treatment with ammonium chloride. Cells obtained from the lung were resuspended in 80% isotonic Percoll, overlaid with 40% isotonic Percoll, and centrifuged (400 x g for 25 min). Cells were enumerated using either a hemacytometer or a Coulter Counter (Beckman Coulter). For some experiments, MLN and spleen cells were prepared under aseptic conditions by pressing MLN from a single animal between the frosted ends of two sterile microscope slides. Cellular debris was removed by sedimentation, and the cells were enumerated using a Coulter Counter (Beckman Coulter). For in vitro restimulation, MLN cells were resuspended and transferred to cultures containing x31-infected, irradiated dendritic cell (DC) 2.4 cells, which serve as APCs, as described previously (30). MLN cells from naive mice and wells with only DC2.4 cells do not contain detectable IFN-{gamma} (data not shown).

Immunophenotypic analyses

MHC class I tetramers and peptides corresponding to the major C57BL/6 epitopes in x31 (NP366–374/Db; acid polymerase (PA)224–233/Db; polymerase B1 (PB-1)703–711/Kb) were prepared by the Molecular Biology Core Facility at the Trudeau Institute. Cells were stained with allophycocyanin- or PE-labeled tetrameric reagents for 1 h at room temperature and then incubated with previously determined optimal concentrations of fluorochrome-conjugated Abs against the following cell surface Ags: CD8a, CD44, CD45.1, CD45.2, CD62L, CD69, CD90.1, CD90.2, CD122, and Ly-6C. All Abs were purchased from BD Pharmingen, e-Biosciences, or Caltag Laboratories. Nonspecific staining was blocked by incubating single-cell suspensions with anti-mouse CD16/CD32. Appropriately labeled, isotype-matched Igs were used as controls for nonspecific fluorescence. Data were collected from 50,000 to 200,000 CD8+ cells by listmode acquisition using FACSCalibur and FACSort flow cytometers (BD Biosciences). Data were analyzed using WinList (Verity Software) and FlowJo (Tree Star) software programs.

Bone marrow chimeric mice were generated, as described by Staples et al. (36). Briefly, 5-wk-old female B6.Ly-5.1 congenic mice (CD45.1+AhR+/+) were lethally irradiated with two doses of 600 rad, spaced 3.5 h apart. One hour after the second irradiation, mice were given 1.5 x 106 bone marrow cells (i.v.) from either C57BL/6 (CD45.2+AhR+/+) or B6.AhR-deficient (CD45.2+AhR–/–) mice. Five weeks later, bone marrow chimeric mice were gavaged with TCDD (10 µg/kg) or peanut oil vehicle and infected, as described above. Irradiated CD45.1+AhR+/+ mice that did not receive bone marrow cells did not survive. Chimerization was further validated using flow cytometry, which showed that for recipients of either CD45.2+AhR+/+or CD45.2+AhR–/– cells, >90% of the bone marrow in the irradiated Ly-5.1 congenic mice were CD45.2+ and <3% of the bone marrow cells were CD45.1+ (data not shown).

Statistical analyses

Statistical analyses were performed using Statview (SAS Institute). Using a one-way ANOVA, followed by post hoc tests (Fisher least significant difference test), differences between independent variables were compared over time and between each treatment group. Differences between two groups at a single point in time were evaluated using Student’s t test. Differences were considered significant when p values were <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCDD-mediated suppression of CD8+ T cell expansion during primary influenza infection is independent of Ag specificity

Previous reports have shown that exposure to the potent AhR agonist TCDD suppresses the expansion and differentiation of CD8+ T cells (17, 25, 30). However, it is not known whether this effect is limited to specific CD8+ T cell epitopes or whether it alters the anatomical distribution of virus-specific CD8+ T cells during the response. This is an important consideration because it has been shown that CD8+ T cells that recognize different peptide epitopes of influenza A virus may be differentially activated by different types of APCs and undergo activation and clonal expansion in a hierarchical manner (37, 38, 39, 40). Therefore, we determined the frequency and distribution of CD8+ T cells specific for three immunodominant epitopes of influenza A virus (NP366–374/Db, PA224–233/Db, PB-1703–711/Kb) in the MLN, spleen, lung airways, and lung interstitium during the primary response to i.n. infection with x31 influenza virus. As shown in Fig. 1, mice that were given a single dose of TCDD immediately before infection had up to 30 times fewer NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells in all anatomical sites examined on day 8 postinfection. In general, the number of NP366–374/Db- and PA224–233/Db-specific CD8+ T cells in the MLN and spleen were more profoundly suppressed than in lung airways (BAL) and lung interstitium (lung). Although there were ~10 times fewer PB-1703–711/Kb-specific CD8+ T cells detected in MLN from TCDD-treated mice, the number of these cells in the BAL and lung was not suppressed. Similar effects of TCDD on the number and distribution of virus-specific CD8+ T cells were observed 11 days after infection, indicating that the decrease in cell number observed 8 days after infection is due to suppression rather than a delay of the response (see Fig. 3). These observations extend earlier findings and demonstrate that exposure to TCDD suppresses the clonal expansion of CD8+ T cells specific for multiple epitopes of influenza virus.


Figure 1
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  		FIGURE 1. The expansion of virus-specific CD8+ cells during a primary response is diminished by exposure to TCDD. Naive 6- to 8-wk-old female C57BL/6 mice were administered (p.o.) either peanut oil vehicle control (Veh) or 10 µg/kg TCDD 1 day before x31 infection. Animals were sacrificed 8 days after infection, and isolated cells were stained with MHC class I-restricted tetramers specific for the indicated viral peptides and anti-CD8 Abs, as described in Materials and Methods. Representative dot plots depict the frequency of NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells in the MLN, spleen, lung airways (BAL), and lung parenchyma (lung). The number in the upper right quadrant indicates the average number of epitope-specific CD8+ T cells. *, Significant difference in cell number compared with vehicle-treated mice (n = 5/group; p ≤ 0.05).

 

Figure 3
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  		FIGURE 3. Exposure to TCDD during primary infection has similar effects on the distribution of memory CD8+ T cells and the kinetics of the recall response regardless of peptide specificity. The average number of NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells in the MLN, spleen, BAL, and lung during primary, memory, and recall phases of infection is depicted. Mice were treated, as described in Figs. 1 and 2. V, Cells from peanut oil vehicle control-treated mice; T, cells from TCDD-treated mice. At each point in time, the data are derived from 5 (primary and recall) or 15 (memory) mice in each treatment group. *, Significant difference in cell number compared with vehicle-treated mice (p ≤ 0.05).

 
The recall response to secondary virus challenge is delayed in TCDD-treated mice

We next examined the effects of TCDD on the distribution of virus-specific memory cells, and their responsiveness during a recall response to heterosubtypic influenza virus infection. Five weeks after primary infection, the percentage and number of NP366–374/Db-specific memory T cells in the MLN, spleen, BAL, and lungs from mice treated with TCDD were less than in the vehicle control group (Figs. 2 and 3). These mice were then i.n. challenged with PR8. Three days after challenge, mice that had been given TCDD before primary infection had ~50% fewer NP366–374/Db-specific CD8+ T cells in the spleen and MLN compared with mice in the vehicle control group (Fig. 2); however, by day 10, there was no difference in the frequency of NP-specific CD8+ T cells. Importantly, the t1/2 of TCDD in C57BL/6 mice is 7–10 days (41); therefore, the amount of TCDD remaining 35 days later is <1.25 µg/kg, a level that has been shown previously to be insufficient to suppress a primary CD8+ T cell response to x31 (42). We extended this assessment to include CD8+ T cells specific for multiple influenza epitopes, and converted these data into the absolute number of each population in each anatomical site (Fig. 3). Similar to effects on NP366–374/Db-specific CD8+ T cells, mice that had been given TCDD before x31 infection had half as many PA224–233/Db- and PB-1703–711/Kb-specific CD8+ T cells as the vehicle control group in all tissues tested on day 3 postinfection, but equivalent numbers to the vehicle control group on day 10 postinfection.


Figure 2
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  		FIGURE 2. AhR activation during primary infection reduces the frequency of NP366–374/Db-specific memory CD8+ T cells and delays their expansion during recall challenge. C57BL/6 mice treated were with peanut oil vehicle (Veh) or TCDD and infected with x31, as described in Fig. 1. Five weeks after primary infection, mice were either sacrificed to examine memory CD8+ T cells, or infected with 3000 EID50 PR8 to assess the recall response. Representative dot plots depict the frequency of NP366–374/Db-specific CD8+ T cells in the MLN, spleen, BAL, and lung. The number in the upper right quadrant on each dot plot indicates the average percentage of NP366–374/Db+CD8+ T cells in each treatment group. *, Significant difference compared with vehicle-treated mice (n = 5/group; p ≤ 0.05).

 
The virus-specific memory CD8+ T cells generated in absence and presence of TCDD are phenotypically similar and expand similarly when adoptively transferred to the same host

Possible explanations for the delayed kinetics of the recall response in mice treated with TCDD are that fewer memory cells are generated in the primary response, different lymphocyte subsets are activated, or there is an overall diminution in the responsive capacity of Ag-specific CD8+ T cells in TCDD-treated mice. To further characterize the population of memory CD8+ T cells generated in the absence and presence of TCDD, we examined the expression of several activation markers on virus-specific CD8+ T cells in lungs and lymphoid organs in mice that had recovered from a prior x31 infection. Memory CD8+ T cells from vehicle- and TCDD-treated mice had similar phenotypic profiles. For example, all of the NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells expressed similar levels of CD44 and CD122 in both TCDD- and vehicle-treated mice (data not shown). Furthermore, in response to infection, CD69 expression levels increased on virus-specific CD8+ T cells in airways, lung, and MLN (data not shown). In the airways, increased Ly-6C expression on virus-specific CD8+ cells correlates with the arrival of activated memory cells (43). BAL cells from vehicle- and TCDD-treated mice demonstrated a similar switch in the relative ratio of Ly-6Clow and Ly-6Chigh CD8+ cells (data not shown). Thus, based upon the representative activation molecules examined, it appears that although fewer memory CD8+ T cells are generated, exposure to TCDD before primary infection does not have a marked impact on the phenotype of the virus-specific memory CD8+ T cells.

Therefore, we sought to determine whether exposure to TCDD during primary infection creates a population of memory CD8+ T cells that are inherently less able to respond to recall challenge. To accomplish this, we used a dual adoptive transfer approach. CD44highCD8+ memory T cells were isolated from mice that had recovered from a prior x31 infection in the presence or absence of either TCDD or the peanut oil vehicle. The cells were then mixed such that the NP366–374/Db-specific CD8+ T cells from each group were present in a 1:1 ratio, and adoptively transferred into naive recipients. The two sets of donor cells were from congenic mice expressing either CD90.1 or CD45.1, allowing us to distinguish between vehicle- and TCDD-treated donors and the recipient (Fig. 4A).


Figure 4
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  		FIGURE 4. Influenza virus-specific memory CD8+ T cells from TCDD- and vehicle-treated mice expand similarly. A, Experimental design: C57BL/6 (CD45.2/CD90.2), B6.CD45.1/CD90.2, and B6.CD45.2/CD90.1 congenic mice were treated with TCDD (10 µg/kg) or peanut oil vehicle control (VC) 1 day before i.n. x31 infection. One month later, spleen cells were recovered and CD8+ T cells were enriched by negative selection, and then sorted by FACS to obtain CD44high/CD8+ (memory) cells. The frequency of NP366–374/Db-specific T cells within the CD44high/CD8+ population was determined by flow cytometry, and cells derived from vehicle- and TCDD-treated donors were combined such that the number of NP366–374/Db-specific CD8+ T cells from each donor was equivalent. Between 2600 and 6000 NP366–374/Db-specific CD44high/CD8+ (1300–3000 from each donor) donor T cells were injected into congenic recipients that expressed a distinct pattern of phenotypic markers, and the recipient mice were then infected with x31 1 day later. Eleven days postinfection, tissues were isolated and stained with fluorochrome-conjugated anti-CD8, NP366–374/Db, and PA224–233/Db tetramers. Representative dot plots show the NP366–374/Db-specific CD8+ T cells identified in the MLN, spleen, BAL, and lung 11 days postinfection. Dot plots on the top row show NP366–374/Db-specific CD8+ T cells in each anatomical site. Dot plots on the bottom row depict the frequency of NP366–374/Db-specific CD8+ T cells derived from the vehicle- and TCDD-treated donors. B, The relative frequencies of NP366–374/Db- and PA224–233/Db-specific CD8+ cells are expressed as the ratio of cells (vehicle control:TCDD) in 12 adoptive transfer recipients. The circles and triangles indicate whether the data are derived from experiments using B6.CD45.1/CD90.2 or B6.CD45.2/CD90.1 congenic mice, respectively. Similar results were obtained regardless of whether we used either B6.CD45.1/CD90.2 or B6.CD45.2/CD90.1 congenic mice as the recipients, indicating that allelic variation in CD45 and CD90 did not influence the experimental outcome.

 
There was a strong expansion of donor cells derived from both vehicle- and TCDD-treated mice, such that 11 days after infection a population of NP366–374/Db-specific CD8+ T cells derived from the two different donors was clearly observed in the spleen, MLN, lung, airways, and pleural cavity (Fig. 4). In fact, regardless of the treatment group from which the cells were derived, 11 days after infection there were generally between 50 and 150 times the number of cells that were adoptively transferred (data not shown). By comparing the expansion of memory CD8+ T cells in these studies, it was apparent that memory T cells that had been generated in the presence of TCDD were mildly impaired compared with controls (Fig. 4B). This experiment demonstrates that although they are slightly less responsive than the cells derived from vehicle-treated donors, memory CD8+ T cells from TCDD-treated donors nevertheless exhibited a strong recall response. Therefore, the delay in clonal expansion upon recall challenge observed in Fig. 2 is due to the diminished number of memory cells generated during the primary phase of the response and a mild impairment of proliferative capacity of these cells. In addition, the fact that we observed no signs of morbidity and no mortality in the TCDD-treated memory mice upon infection with PR8 indicates that although the memory pool is smaller, the CD8+ T cells are fully functional.

Memory CD8+ cells are much less susceptible to suppression by TCDD

We next examined whether treatment with TCDD just before secondary infection affects the expansion of memory cells that were previously generated in the absence of TCDD. For this study, TCDD (or peanut oil vehicle) was administered to mice 1 mo after x31 infection, and the following day the mice were challenged with PR8 (Fig. 5). In both treatment groups, virus-specific CD8+ T cells expanded rapidly upon recall challenge. Interestingly, in the TCDD-treated mice, there were fewer virus-specific CD8+ cells in the spleen and MLN, but the number of cells in the airways and lung interstitium was unaffected by TCDD. However, it is important to note that the reduced frequency of virus-specific cells in the lymphoid organs was of a much smaller magnitude (<2-fold) than the suppression of the primary response (5- to 30-fold), suggesting that once they are formed, memory CD8+ T cells are much less susceptible to suppression by TCDD. In fact, given that during anamnestic responses a subpopulation of responding cells in the lymphoid organs is naive, the decreased number of NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells in the spleen and MLN may simply reflect suppressive effects of TCDD on the clonal expansion of naive CD8+ T cells.


Figure 5
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  		FIGURE 5. Existing CD8 memory cells are much less susceptible to suppression by TCDD. One month after infection with x31, mice were given either peanut oil vehicle control (V) or TCDD (T, 10 µg/kg) by gavage. The following day, all mice were infected (i.n.) with 3000 EID50 PR8. Mice were sacrificed 3 or 10 days later, and isolated cells were stained with MHC class I-restricted tetramers specific for the indicated viral peptides and anti-CD8 Abs, as described in Materials and Methods. Graphs show the average number of NP366–374/Db-, PA224–233/Db-, and PB-1703–711/Kb-specific CD8+ T cells in the MLN, spleen, BAL, and lung. *, Indicates a significant difference in cell number compared with vehicle-treated mice (n = 5/group at each point in time; p ≤ 0.05).

 
Events during the activation of naive CD8+ T cells are critical targets of the AhR-mediated immune suppression

Our finding that the responsiveness of memory CD8+ T cells is much less sensitive to perturbation by TCDD than the primary response provides a very important new piece of information regarding the mechanism by which TCDD suppresses CD8+ T cell responses. Specifically, it indicates that events during the activation of naive CD8+ T cells are the targets of AhR-mediated suppression. However, the mechanism that underlies the suppressive effects of TCDD on naive CD8+ T cells has remained elusive. In particular, it is not clear whether AhR ligands, such as TCDD, affect CD8+ T cells directly or whether their impaired function occurs indirectly, as a consequence of direct action on accessory cells, such as DC. Therefore, we first determined whether AhR-deficient mice mount a CD8+ T cell response to influenza virus infection that is equivalent to age-matched wild-type mice. Upon infection with x31, AhR–/– mice generated Ab and T cell responses that were not different from age-matched AhR+/+ mice (data not shown). Furthermore, in contrast to AhR+/+ mice, treatment of AhR–/– mice with TCDD did not suppress the clonal expansion of NP366–374/Db-specific T cells in the MLN (Fig. 6A), nor did it impair IFN-{gamma} production (Fig. 6B), differentiation into CTL, or the production of virus-specific IgG (data not shown). This is consistent with a previous report that TCDD does not suppress other types of immune responses in AhR–/– mice (24).


Figure 6
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  		FIGURE 6. AhR-mediated events within the hemopoietic system underlie the suppressed clonal expansion and differentiation of naive, virus-specific CD8+ T cells in TCDD-treated mice. Top row, Age-matched C57BL/6 (AhR+/+) and B6.AhR-deficient (AhR–/–) mice were treated with vehicle (Veh) or 10 µg/kg TCDD 1 day before infection with x31. Mice were sacrificed 7 days after infection, and the MLN were removed. A, The average number of NP366–374/Db-specific CD8+ T cells was determined using flow cytometry, as described in Materials and Methods. B, MLN cells were restimulated for 24 h with x31-pulsed, irradiated DC2.4 cells, which served as APCs, and IFN-{gamma} levels in culture supernatants were determined using an ELISA. Results are representative of three separate experiments. In each experiment, there were three to six mice in each group. Bottom row, MLN cells from vehicle- or TCDD-treated bone marrow chimeric mice were obtained 7 days after infection. The average number of C, Ly-5.2+CTLe (i.e., CD8+ cells bearing a CD44highCD62Llow phenotype), and D, Ly-5.2+NP366–374/Db-specific CD8+ T cells was determined using flow cytometry. Results are representative of two separate experiments. Error bars, SEM. *, Significant difference between the vehicle and TCDD treatment groups (n = 6–8 mice/group; p ≤ 0.05).

 
To determine whether the AhR-driven events that suppress the response of virus-specific CD8+ T cells are within or extrinsic to the immune system, we generated chimeric mice in which the hemopoietic system lacks the AhR, but all other tissues express it. To accomplish this, we reconstituted lethally irradiated B6.CD45.1 congenic recipient mice (AhR+/+) with bone marrow cells from either C57BL/6 (AhR+/+CD45.2+) or B6.AhR-deficient (AhR–/–CD45.2+) donor mice, generating CD45.2AhR+/+->CD45.1AhR+/+ and CD45.2AhR–/–->CD45.1AhR+/+ chimeric mice. As expected, the expansion of NP366–374/Db-specific T cells and generation of effector cells were suppressed in TCDD-exposed, infected mice in which immune system expresses a functional AhR. In contrast, when the immune system lacked the AhR, exposure to TCDD did not suppress this response (Fig. 6, C and D), indicating events within the hemopoietic system underlie the suppressed clonal expansion and differentiation of virus-specific CD8+ T cells in TCDD-treated mice.

To determine whether TCDD suppresses the response of CD8+ T cells directly or indirectly, we adoptively transferred isolated CD8+ cells derived from either C57BL/6 (AhR+/+CD45.2+) or B6.AhR-deficient (AhR–/–CD45.2+) mice into naive B6.CD45.1 (AhR+/+) recipients. Using congenic markers to distinguish the adoptively transferred and endogenous CD8+ T cells, we found that regardless of whether the CD8+ T cells express the AhR, their response to infection was suppressed by TCDD treatment (Fig. 7), indicating that the target of AhR-mediated suppression of the CD8+ response is within the immune system, but not within CD8+ T cells. Thus, we show in this study that the impact of TCDD on CD8+ T cell responsiveness during viral infection does not depend on AhR expression within the CD8+ T cells. This finding indicates that TCDD acts, via the AhR, on other cells of the immune system that are involved in the priming of CD8+ T cells.


Figure 7
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  		FIGURE 7. CD8+ T cells are not directly affected by TCDD. Isolated CD8+ cells from C57BL/6 (B6, CD45.2+AhR+/+) and B6.AhR-deficient (CD45.2+AhR–/–) mice were injected (i.v.) into naive B6.CD45.1 (AhR+/+) congenic recipients. Two days later, recipient mice were treated with peanut oil vehicle (VEH) or TCDD (10 µg/kg) 1 day before infection with x31. Mice were sacrificed 9 days after infection. The average number of CD44highCD62LlowCD8+ T cells (CTLe) in the MLN (A and B), and NP366–374/Db-specific CD8+ T cells in the MLN (C and D) and lung airways (E and F) was determined using flow cytometry. Exposure to TCDD suppressed the expansion and differentiation of endogenous (CD45.1+) CD8+ T cells to infection (A, C, and E). TCDD treatment similarly decreased the number of CD45.2+CTLe in the MLN (B), and the number of CD45.2+ DbNP366-specific CD8+ T cells in the MLN (D) and lung airways (F) regardless of the AhR status of the adoptively transferred CD8+ cells. This experiment was repeated three times with similar results. In each experiment, there were five to six mice in each treatment group. *, Statistical significance at p ≤ 0.05 when compared with the vehicle treatment group that received cells of the same genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
AhR activation by TCDD causes profound suppression of immune responses to a variety of Ags, and epidemiological data show that exposure to dioxins, polychlorinated biphenyls, and PAHs correlates with diminished vaccine efficacy and an increased incidence of influenza and other respiratory infections (27, 28, 29), suggesting that environmental exposure to AhR agonists affects antiviral immunity. However, the underlying mechanism for AhR-mediated immune suppression is not entirely clear. In the present study, we determined the effects of AhR activation on the in vivo distribution of virus-specific CD8+ T cells during primary and recall responses to influenza virus infection. Using a dual adoptive transfer system, we directly compared the expansion of memory CD8+ T cells created in the absence or presence of TCDD in the same host. Given that the magnitude of a memory response is thought to be influenced by the primary CD8+ T cell response (38, 40, 44, 45), it is logical to hypothesize that impairment of the primary response by an immunosuppressive compound will lead to an insufficient recall response. However, we show in this study that this is not necessarily the case. Despite profound suppression in the primary response to influenza virus, the recall response of virus-specific CD8+ T cells in TCDD-treated mice is only mildly impaired. Moreover, we show that although the number of virus-specific memory CD8+ T cells formed is greatly reduced, the reduction in frequency does not correlate with diminished host resistance.

These findings suggest that, contrary to our expectations, suppression of the primary response does not result in an inherent defect in the response of memory CD8+ T cells. Immunodominance hierarchies for the activation and expansion of CD8+ T cells that recognize different epitopes of influenza A virus have been established (37, 38, 39, 40). Although the underlying cause is not clear, the type of APC, affinity of the peptide-MHC interaction, and relative levels of viral proteins contribute to the dominance of certain viral epitopes over others (46). If we had examined only NP366–374/Db-specific CD8+ cells, one possible explanation for our observations could be that AhR activation preferentially impairs the activation of NP366–374/Db-specific CD8+ T cells, whereas the activation of CD8+ T cells specific for other viral epitopes is unaffected. However, by carefully examining effects of TCDD on the distribution and frequency of CD8+ cells specific for different viral peptides presented by different MHC class I molecules, we show that this is probably not the case.

The discovery that the memory CD8+ T cell response is not particularly sensitive to TCDD provides a very important piece of new information regarding how AhR activation suppresses CD8+ T cell responses. Specifically, it clearly demonstrates that the critical target of the AhR affects the activation of naive CD8+ T cells, but is not essential for the activation of memory cells. The idea that TCDD preferentially disrupts the function of naive T cells is supported by previous work in an allogeneic tumor model, in which the ability of TCDD to suppress the CTL response was limited to the first 4 days during alloantigen exposure, but once the CTL response passed this window it was resistant to suppression by TCDD (17). The idea that naive CD8+ T cells are more sensitive to AhR-mediated immune suppression than effector or memory cells is also consistent with the sensitivity Ab responses to suppression by TCDD. Primary and secondary Ab responses to a variety of Ags are suppressed in rodents exposed to a single dose of TCDD (33, 47, 48). However, in mice infected with influenza virus, exposure to TCDD immediately before recall challenge (i.e., after virus-specific memory B cells had been generated) did not affect the Ab response (33). This suggests a more general mechanism that, once activated, TCDD targets events necessary for the activation of naive, but not previously activated lymphocytes.

Therefore, understanding how AhR activation by TCDD disrupts the activation and clonal expansion of naive CD8+ T cells is most likely the critical step in determining the mechanism of AhR-mediated suppression of CD8-mediated responses to viral infection. However, despite considerable effort, the mechanism by which TCDD suppresses the expansion and differentiation of CD8+ T cells is not known, and progress has been hampered by the fact that direct effects of TCDD on the function of purified T cells in vitro have been very difficult to demonstrate (3, 20, 49, 50, 51). In fact, the inability of many laboratories to reproduce the suppressive effects of TCDD on CD8+ T cell cytokine production, proliferation, and differentiation that are observed in vivo, when they are exposed to TCDD in vitro, provides circumstantial evidence that the function of CD8+ T cells may not be directly affected by TCDD.

To resolve this issue directly, we generated bone marrow chimeric mice to determine whether AhR-mediated events within the immune system underlie suppression of the primary CD8+ cell response to influenza virus, and monitored the expansion of adoptively transferred AhR–/–CD8+ cells in AhR+/+ congenic mice to determine whether TCDD affects CD8+ T cells directly. Using these approaches, we found that although AhR-dependent events within hemopoietic cells underlie the suppressive effects of TCDD, CD8+ T cells do not appear to be directly affected. At first glance, this latter finding is in contrast to a study using CD4+ and CD8+ T cells isolated from AhR+/+ and AhR–/– mice in the context of a graft-vs-host (GVH) model (52). In this study, AhR expression in both CD4+ and CD8+ T cells was required for full suppression of the CTL response. However, when only the CD8+ T cells lacked the AhR (i.e., CD4+ cells were AhR+/+), the CTL response was only partially suppressed by TCDD, indicating that AhR-mediated events extrinsic to CD8+ T cells contribute to suppression of the CTL response in this model. Further evidence that CD8+ T cells are not the direct targets of TCDD comes from studies using a P815 tumor model, wherein the provision of CD8+ T cells with enhanced costimulation or exogenous IL-2 renders them insensitive to suppression by TCDD (22, 53). Thus, the mechanism by which AhR activation suppresses CD8+ T cell responses most likely depends on the conditions under which the CD8+ T cells are activated.

Activation of naive CD8+ T cells during a primary immune response to viral infection requires interaction with and costimulation by APCs, in particular DC (38, 54). Interestingly, we observed that the TCDD-induced suppression was more pronounced in lymphoid organs than in the lung, which is consistent with the idea that TCDD, via the AhR, disrupts a function of accessory cells that is necessary for the proper activation of naive CD8+ T cells in the MLN. The difference in the level of suppression in lymphoid organs and lung also suggests that regardless of the magnitude of the CD8 response in lymphoid organs, the immune system has a mechanism for insuring that the site of infection acquires the CD8+ T cells that it needs to successfully eliminate the virus. In fact, we and others (55, 56, 57) have shown previously that there is no difference in the amount of virus in lungs of vehicle control and TCDD-treated mice.

Previous reports have shown that exposure to TCDD suppresses IL-12 production (25, 58), and affects the expression of costimulatory and adhesion molecules on DC, macrophages, and B cells (23, 53, 59, 60). Suppressed IL-12 production and changes in DC number and phenotype were not observed in TCDD-treated AhR–/– mice (60) (our unpublished observations), indicating that that these changes are AhR dependent. Moreover, TCDD affects DC maturation and survival both in vivo and in vitro (59, 60, 61, 62), suggesting that DC are affected by TCDD directly. Therefore, deregulated DC function provides an indirect mechanism by which AhR activation could lead to a suppressed CD8+ T cell response to influenza virus. In the P815 model, DC are involved in activating CD4+ T cells, which in turn provide help to CD8+ T cells. This is an important mechanistic consideration because, in contrast to the primary CD8 T cell response to influenza virus, the CTL response to P815 mastocytoma cells is CD4 dependent (53). Although their precise role is unclear, costimulatory signals from DC may be less important in the GVH model because CD4+ and CD8+ T cells can be activated directly by MHC class I and class II alloantigens. Therefore, in the P815 tumor and GVH models, AhR-mediated events in DC and/or CD4+ cells may contribute to suppressed activation of CD8+ T cells.

In addition to direct effects of TCDD on DC, AhR activation could suppress the expansion of virus-specific CD8+ T cells by enhancing the function of immunosuppressive CD4+CD25+ regulatory T cells (Treg). Treg play an important role in the prevention of autoimmune disease and the regulation of immune responses to bacteria, tumors, and in GVH responses (63, 64); however, the role of Treg in CD8+ T cell responses during viral infection is less clear (65). Nevertheless, it was shown recently that in the context of a GVH response, AhR activation by TCDD stimulated a CD4+CD62LlowCD25+ subpopulation that expresses glucocorticoid-induced TNF receptor and CTLA-4, suggesting a role for Treg in TCDD-mediated suppression of CD8+ T cell responses (66).

Although these studies provide evidence that DC and Treg may play a role in AhR-mediated disruption of CD8+ T cell function, the specific molecular events within these cells that follow AhR activation are not clear. The ligand-activated AhR binds to a well-defined response element in the genome (Ah response element (AhRE), 5'-GCGTG-3'), and this core pentameric sequence has been identified in the promoter region of many rodent and human cytokine genes (67). Genomic analyses have also revealed that exposure to TCDD altered the expression of numerous other immunoregulatory genes in the bone marrow-derived DC, spleen, thymus, and liver (61, 68, 69). Whether these putative AhRE are functional and the consequences of these reported changes in gene expression are only now being evaluated. AhR activation may also affect the level of expression of immunoregulatory proteins whose genes do not contain AhRE. For example, the AhR has been reported to interact with the p65 subunit of NF-{kappa}B (62, 70, 71) and immunophilin-like proteins (72, 73, 74). Therefore, it is possible that AhR-dependent alterations in DC or Treg involve modulation of NF-{kappa}B and immunophilin function as well as AhRE-mediated changes in gene expression.

In conclusion, this study provides strong evidence that AhR activation by TCDD disrupts events within bone marrow-derived cells that are important for the activation of naive CD8+ T cells. These critical AhR-regulated processes are extrinsic to virus-specific CD8+ T cells, a finding that is consistent with difficulty demonstrating direct effects of TCDD on the function of cultured T cells. When put into context with our observations that the recall response of memory CD8+ T cells created in TCDD-treated mice is not inherently defective and that, once established, memory CD8+ T cells are not particularly sensitive to TCDD, the novel findings reported in this work suggest that identifying defects in DC and other factors essential for generating a primary response is the critical next step in unraveling the immunomodulatory mechanisms regulated by the AhR.


   Acknowledgments
 
We thank the Molecular Biology Core at the Trudeau Institute for synthesis of tetrameric reagents; B. Sells for assistance with the cell sorting; and Drs. Marcia Blackman and Nancy Kerkvliet for thoughtful discussion of our data and helpful comments during the preparation of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the following research and training grants from National Institutes of Health: R01ES10619 and K02ES012409 (awarded to B.P.L.), HL63925 and AI055500 (awarded to D.L.W.), and T32AI07025 (awarded to J.J.N.). J.J.N. is the recipient of a Merck Research Scholar Award from the American Association of Colleges of Pharmacy and the Merck Company Foundation, a Seattle Chapter Achievement Rewards for College Scientists Scholarship, and a Rho Chi, Schering-Plough, American Foundation for Pharmaceutical Education First Year Graduate Fellowship. Back

2 Address correspondence and reprint requests to Dr. B. Paige Lawrence, Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642. E-mail address: paige_lawrence{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: AhR, aryl hydrocarbon receptor; AhRE, Ah response element; BAL, bronchoalveolar lavage; DC, dendritic cell; EID50, egg ID50; GVH, graft-vs-host; i.n., intranasal; MLN, mediastinal lymph node; NP, nucleoprotein; PA, acid polymerase; PB-1, polymerase B1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Treg, regulatory T cell. Back

Received for publication May 31, 2006. Accepted for publication August 14, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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