Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Renders Influenza Virus-Specific CD8+ T Cells Hyporesponsive to Antigen

Kristen A. Mitchell and B. Paige Lawrence1

Graduate Program in Pharmacology/Toxicology, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164

Received February 20, 2003; accepted April 3, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While considerable evidence indicates that exposure to the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs T cell function, the precise mechanism underlying this effect is not well understood. Furthermore, relatively little is known about the effects of TCDD on the fate of activated, antigen-specific T cells in vivo. In the present study, we took advantage of major histocompatibility complex (MHC) class I-restricted tetramers and clonotypic anti-T cell receptor (TCR) antibodies to follow the fate of influenza virus-specific CD8+ T cells in mice treated with TCDD. Exposure to TCDD suppressed the clonal expansion of influenza virus-specific CD8+ T cells, resulting in a three- to five-fold reduction in the number of cytotoxic T lymphocytes (CTL) in the lymph node, as compared to vehicle-treated mice. Studies to address possible mechanisms for the diminished CTL response failed to show evidence for increased apoptosis in virus-specific CD8+ T cells from TCDD-exposed mice. However, treatment with TCDD reduced the number of proliferating virus-specific CD8+ T cells by as much as 70% on day 7 post infection. Moreover, ex vivo restimulation of lymph node cells with influenza virus nucleoprotein (NP366–374) peptide and exogenous interleukin-2 (IL-2) only partially restored the proliferation of influenza virus-specific CD8+ T cells from TCDD-exposed mice and failed to stimulate interferon-gamma (IFN{gamma}) production by these cells. The observation that neither proliferation nor IFN{gamma} production by CD8+ T cells could be completely restored, even when cells were provided with optimal stimulation, suggests that exposure to TCDD drives antigen-specific CD8+ T cells into a state of unresponsiveness similar to anergy.

Key Words: TCDD (dioxin); immunosuppression; CD8+ T lymphocytes; antigen-specific; tetramer, influenza virus; anergy; mouse; lymph node; in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies conducted over the past 25 years have clearly established that the immune system is one of the most sensitive targets for the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Produced through a variety of manufacturing and incineration processes, TCDD and structurally related chemicals are widely distributed and persistent environmental contaminants that elicit their toxic and biological effects by binding to a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Studies with AhR-null mice indicate that most, if not all, of the immunotoxic effects of TCDD are mediated through an AhR-dependent pathway (Vorderstrasse et al., 2001Go). Despite these findings, the precise mechanisms by which TCDD impairs the immune system are not known, nor have the immune cells directly targeted by TCDD been identified.

Although the mechanisms of TCDD immunotoxicity are not well understood, one adverse effect of TCDD that is consistently reported under a variety of exposure paradigms is suppression of T cell-mediated immune responses. In vivo exposure to TCDD impairs T cell functions such as proliferation, differentiation, cytokine production, and antibody responses to T-dependent antigens (reviewed in Kerkvliet, 2002Go). Suppression of the cytotoxic T lymphocyte (CTL) response has also been reported in mice exposed to TCDD (Clark et al., 1983Go; Kerkvliet et al., 1996Go; Warren et al., 2000Go). For example, using a P815 tumor allograft model, Kerkvliet et al.(1996)Go showed that exposure to TCDD dose-responsively suppressed splenic cytolytic activity, decreased the production of interleukin-2 (IL-2) and interferon-gamma (IFN{gamma}) by CD8+ T cells, and reduced the number of splenocytes bearing an effector CTL (CTLe) phenotype, defined as CD8+CD44hiCD62Ll°(Hou and Doherty, 1993Go; Mobley and Dailey, 1992Go). Likewise, we have shown that exposure to TCDD suppresses the CTL response in mice infected with influenza virus, resulting in fewer CTL in the lung of TCDD-treated mice (Warren et al., 2000Go). We attribute this to impaired CTL generation in the regional lymph node, as exposure to TCDD reduced the number of CD4+ and CD8+ T cells in the lymph node, and diminished IL-2 and IFN{gamma} production and cytolytic activity in restimulated lymph node cells (Warren et al., 2000Go).

Although the CTL response is clearly impaired in TCDD-treated mice, the basis for this suppression is unclear. Specific unanswered questions include: Why do CD8+ T cells in TCDD-treated mice fail to respond adequately to antigen? How does exposure to TCDD affect the fate of stimulated antigen-specific CD8+ T cells in vivo? Answering these questions has been hindered by several factors. First of all, the finding that T cell functions are not altered by in vitro exposure to TCDD precludes the use of T cell clones or other simple in vitro approaches for studying TCDD immunotoxicity (De Krey and Kerkvliet 1995Go; Lang et al., 1994Go; Lawrence et al., 1996Go; Lundberg et al., 1992Go). Second, whereas the function of antigen-stimulated T cells is clearly suppressed by in vivo exposure to TCDD, there have been no documented effects of TCDD on naive T cells (Kerkvliet et al., 1996Go; Lundberg et al., 1992Go; Prell et al., 1995Go; Pryputniewicz et al., 1998Go; Rhile et al., 1996Go). Consequently, determining how exposure to TCDD impairs T cell function requires the assessment of antigen-specific T cells activated in vivo. However, until recently, such mechanistic studies were limited by the technical difficulties inherent in identifying and monitoring the fate of activated antigen-specific T cells in vivo.

To address mechanisms by which exposure to TCDD causes T cell dysfunction, we characterized the effects of TCDD on the fate of antigen-specific CD8+ T cells following infection with influenza virus. Infection with influenza virus is a suitable model for studying how exposure to TCDD impairs antigen-specific T cells for several reasons. First, anti-influenza immunity is exquisitely sensitive to TCDD. In fact, decreased host resistance to influenza virus has been observed in mice exposed to as little as 10 ng TCDD/kg, representing the most sensitive immunotoxic effect of TCDD reported to date (Burleson et al., 1996Go). Second, exposure to TCDD clearly impairs T cell-mediated immune responses during infection with influenza virus, as evidenced by reduced CTL in the lung and suppressed virus-specific cytolytic activity in the lymph node of TCDD-treated mice (Warren et al., 2000Go). Additionally, several tools are available to identify influenza virus-specific CD8+ T cells in vivo. For example, because the T cell receptor (TCR) usage of influenza virus-specific CTL has been well characterized, the majority of influenza virus-specific CD8+ T cells can be identified with clonotypic antibodies specific for a predominant Vß chain of the TCR (Deckhut et al., 1993Go; Palmer et al., 1989Go; Tomonari and Lovering 1988Go; Townsend et al., 1983Go). Furthermore, CD8+ T cells specific for the immunodominant epitope of influenza virus nucleoprotein (NP366–374) can be visualized using soluble tetrameric complexes consisting of H-2Db+NP366 (DbNP366). In the present study, we used clonotypic antibodies and DbNP366 tetramers to address why antigen-specific CD8+ T cells fail to generate an adequate CTL response in TCDD-exposed mice. Specifically, we determined how TCDD affects the fate of antigen-specific CD8+ T cells upon antigen stimulation, using clonal expansion, apoptosis, and anergy as endpoints.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Virus strains and infection.
Influenza virus strains A/HKx31 (gift from Dr. Michael Coppola, Argonex, Charlottesville, VA) or A/Memphis/102/72 (gift from Dr. Demetrius Moskophidis, Medical College of Georgia, Augusta, GA) were used in all experiments. A/HKx31 and A/Memphis/102/72 are both H3N2 strains of influenza virus with immunodominant nucleoprotein (NP) epitopes that differ at one amino acid residue. Virus strains were propagated in 10-day-old fertilized chicken eggs as previously described (Barrett and Inglis, 1985Go). A/HKx31 titer was determined based on hemagglutination assay with chicken red blood cells. A/Memphis/102/72 titer was quantified using a plaque assay with Madin-Darby canine kidney cells (Barrett and Inglis, 1985Go). Mice were anesthetized with avertin (2,2,2-tribromoethanol; Aldrich, Milwaukee, WI) and inoculated intranasally with 20 µl sterile PBS containing either 120 hemagglutinating units (HAU) of A/HKx31 or 107 plaque forming units of A/Memphis/102/72 (equivalent to 125 HAU).

Mice.
C57Bl/6 (B6) and C57Bl/10 (B10) female mice (6–8 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in microisolator units on a 12/12 h light cycle under pathogen-free conditions. Two separate strains of Ah-responsive mice were used for these studies to take advantage of two different tools for detecting influenza virus-specific CD8+ T cells in vivo. In B6 mice infected with HKx31, virus-specific CD8+ T cells can be detected using class I-major histocompatibility complex (MHC) tetramers (Belz et al., 2000Go; Flynn et al., 1998Go). In B10 mice infected with Memphis102/72, virus-specific CD8+ T cells can be identified using a monoclonal antibody against the Vß11 chain of the TCR (Tomonari and Lovering, 1988Go).

TCDD.
TCDD (≥99% pure, Cambridge Isotope Laboratories, Inc., Woburn, MA) was dissolved in anisole and diluted in peanut oil to yield a working stock of 1 µg/ml. Mice were gavaged with TCDD (10 µg/kg body weight) or peanut oil vehicle one day prior to infection with influenza virus. Although clearly immunosuppressive (Kerkvliet and Burleson, 1994Go), this dose of TCDD is not overtly toxic and is well below the LD50 in C57Bl mice (>120 µg/kg; Birnbaum, 1986Go).

Flow cytometric analysis.
Freshly isolated mediastinal lymph node (MLN) cells were stained with combinations of the following fluorochrome-conjugated antibodies purchased from BD Pharmingen (San Diego, CA) or Caltag Laboratories (Burlingame, CA): FITC-labeled anti-CD44, PE-labeled anti-CD62L, Tricolor- or APC-labeled anti-CD8, and APC-labeled anti-CD4. Appropriately labeled, isotype-matched antibodies were used to determine nonspecific fluorescence. Cells from B10 mice infected with A/Memphis/102/72 were stained with a biotinylated or PE-labeled clonotypic antibody specific for the Vß11 chain of the TCR to detect a majority of influenza virus-specific CD8+ T cells. To identify influenza virus-specific CD8+ T cells in B6 mice infected with A/HKx31, cells were incubated for 1 h at room temperature with PE-labeled DbNP366 tetrameric complexes (generously provided by Dr. Peter Doherty, University of Melbourne, Australia). In order to evaluate apoptosis, cells were stained with FITC-labeled annexin V and 7-amino-actinomycin D (7-AAD; BD Pharmingen). Apoptotic cells were identified as annexin V-positive, 7-AAD-negative (van Engeland et al., 1998Go). For all experiments, data were collected from 25,000 to 50,000 cells using a FACScan or FACSort flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using WinList software (Verity Software, Topsham, ME).

Ex vivo restimulation of lymphoid cells.
MLN cells were harvested, processed, and counted as previously described (Warren et al., 2000Go). Cells were resuspended in cRPMI (RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, and 50 µM 2-ME) and plated at 4 x 105 cells/well in 96-well flat-bottom tissue culture plates. Exogenous murine IL-2 (R&D Systems, Minneapolis, MN) was added to wells at 12.5 U/ml. NP366–374 peptides from strain A/HKx31 (ASNENMETM) or strain A/Memphis/102/72 (ASNENMDTM) were added to wells at a final concentration of 1 µM. Both peptides were synthesized at the Molecular Biology Core Facility at Washington State University. Plates were incubated at 37°C and 5% CO2 for 24 to 72 h.

In some experiments, MLN cells were depleted of CD4+ or CD8+ T cells prior to restimulation. Briefly, anti-CD4- or anti-CD8-conjugated magnetic beads (Dynal ASA, Oslo, Norway) were added to 5–10 x 106 MLN cells at a ratio of four beads per target cell, incubated 20 min at 4°C with continuous end-over-end mixing, and removed by magnetic separation. The remaining cells were washed three times and resuspended in cRPMI. Depletion efficacy was assessed by flow cytometry and was found to be ≥ 82%. For preparation of antigen presenting cells, DC2.4 cells (provided by Dr. Ken Rock, Dana Farber Cancer Institute, Boston, MA) were resuspended at 2 x 106 cells/ml in RPMI 1640, containing 10 mM HEPES and 1% BSA (Sigma Chemical Co., St. Louis, MO) and incubated with influenza A virus at 300 HAU/ml for 3 h at 37°C. Infected DC2.4 cells were subsequently irradiated at 3000 rad, washed twice and resuspended in cRPMI. For restimulation, 2 x 106 MLN cells were cultured with 1 x 106 DC2.4 cells in 24-well, flat-bottom plates and incubated at 37°C. After 24 h, supernatants were analyzed for IFN{gamma} by ELISA. Wells containing MLN cells from naive mice and wells with only DC2.4 cells were included as negative controls.

Measurement of IFN{gamma} production.
Supernatants from ex vivo restimulated MLN cells were collected after 24 h. IFN{gamma} levels were measured using a sandwich enzyme-linked immunosorbant assay (ELISA) with reagents purchased from BD Pharmingen. The ELISA was performed according to the manufacturer’s protocol. Briefly, 96-well plates (Nunc Maxisorp Immunoplates) were coated with 2µg/ml rat anti-mouse IFN{gamma} antibody (R4-6A2) diluted in 0.1 M NaHCO3 (pH 8.3). Samples or serially diluted recombinant murine IFN{gamma} standards were added to the plate in duplicate. IFN{gamma} was detected using 1 µg/ml biotinylated rat anti-mouse IFN{gamma} antibody (XMG.1) followed by HRP-conjugated streptavidin. Plates were developed using 2,2'-azinobis(3-ethylebenzthiazoline)-6-sulfonic acid (ABTS). The lower limit of detection for this assay is 125 pg/ml.

Analysis of proliferation.
To measure T cell proliferation in vivo, 5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co.) was added at 0.8 mg/ml to the drinking water three days before sacrificing mice (Flynn et al., 1999Go; Wynford-Thomas and Williams, 1986Go). Water bottles that contained BrdU were protected from light and changed daily. Freshly isolated MLN cells were stained with anti-CD8 and either anti-Vß11 or DbNP366 tetramers. To measure BrdU incorporation, cells were fixed, permeabilized (Tough and Sprent, 1994Go), and stained with 20 µl of FITC-labeled anti-BrdU or a matched isotypic control antibody (BD Pharmingen). To measure in vitro proliferation, ex vivo restimulated lymph node cells were cultured for a total of 72 h; cells were pulsed with 3H-thymidine (2 µCi/well; Amersham Pharmacia Biotech, Piscataway, NJ) for the last 24 h. Contents of wells were harvested onto glass fiber filter strips using a cell harvester (PHD model, Cambridge Technology, Watertown, MA), and 3H-thymidine incorporation was measured using a liquid scintillation counter.

Statistical analyses.
Statistical analyses were performed using Statview (version 4.01, Abacus Concepts, Berkeley, CA). Data were evaluated by ANOVA, followed by a Fisher’s least significant difference multigroup comparison. Data were considered significantly different at p ≤ 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Exposure to TCDD Suppresses the Expansion of Influenza Virus-Specific CD8+ T Cells
The generation of CTL during infection with influenza virus is accompanied by an increase in the number of influenza virus-specific CD8+ T cells in the regional lymph node (Allan et al., 1990Go). To investigate how exposure to TCDD suppresses the development of an effective CTL response, clonotypic antibodies (anti-Vß11) were used to identify a population of influenza virus-specific CD8+ T cells in B10 mice and characterize the effects of TCDD on the expansion of these cells. As shown in Figure 1AGo, infection induced a three- to fivefold increase in the number of Vß11+CD8+ T cells in the MLN of vehicle-treated mice, with maximum cell number observed on day 7. In contrast, the expansion of Vß11+CD8+ T cells during infection with influenza virus was reduced 40–50% in mice exposed to TCDD. Suppressed cell recovery in TCDD-exposed mice was not observed before day 5, as the numbers of Vß11+CD8+ T cells in vehicle- and TCDD-treated mice were similar on day 3. Expansion of antigen-specific cells was also suppressed in TCDD-treated mice when DbNP366 tetramers were used to identify influenza virus-specific CD8+ T cells in B6 mice infected with A/HKx31 (Fig. 1BGo). Exposure to TCDD did not alter the percentage or number of Vß11+CD8+ or DbNP366+CD8+ T cells in uninfected mice (data not shown).



View larger version (30K):
[in this window]
[in a new window]
 
FIG. 1. Exposure to TCDD suppresses the expansion of influenza virus-specific CD8+ T cells in the regional lymph node. (A) MLN cells were isolated from vehicle- or TCDD-treated B10 mice 3 to 12 days after infection with influenza virus strain A/Memphis/102/72. Cells were stained with fluorochrome-conjugated antibodies specific for Vß11 and CD8 and analyzed by flow cytometry. Data represent mean number of Vß11+CD8+ T cells in the MLN. Error bars represent SEM. For each day, five to six mice were used per treatment group. (B) MLN cells were isolated from vehicle- or TCDD-treated B6 mice infected with influenza virus strain A/HKx31. Cells were stained with Tricolor-labeled anti-CD8 and PE-labeled DbNP366 tetramer and analyzed by flow cytometry. Numbers in upper right quadrants indicate the average number of DbNP366+CD8+ T cells in samples from five vehicle- and five TCDD-treated mice on day 8 post infection. The SEM for the vehicle group was 5.7 x 104 and the SEM for the TCDD group was 2.1 x 104. Results are representative of five separate experiments. Asterisk depicts a significant difference when compared to the vehicle group (p ≤ 0.05).

 
Differentiation of Naive Influenza Virus-Specific CD8+ T Cells into Functional Effector Cells Is Reduced in TCDD-Treated Mice
Reduced expansion of influenza virus-specific CD8+ T cells in the lymph nodes of TCDD-treated mice suggested that TCDD impaired the ability of these cells to differentiate into CTL. Therefore, we examined the effects of TCDD on the development of virus-specific effector CTL (CTLe), which were identified as Vß11+CD8+ or DbNP366+CD8+ T cells expressing a CD44hiCD62Ll°phenotype. As shown in Table 1Go, exposure to TCDD reduced the percent and number of Vß11+ CTLe in the lymph nodes of B10 mice infected with influenza virus. Suppression began on day 7 post infection and peaked on day 9, which is consistent with the time at which influenza virus-specific CTLe are typically detected in the lung (Flynn et al., 1998Go). Similarly, when tetramers were used to identify influenza virus-specific CTLe on day 9 post infection, exposure to TCDD reduced the number of DbNP366+ CTLe by 80% (Table 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 1 Exposure to TCDD Reduces the Percent and Number of Influenza Virus-Specific Effector Cytotoxic T Lymphocytes (CTLe) in the Mediastinal Lymph Nodes of Mice
 
Another characteristic of influenza virus-specific CTL is the production of IFN{gamma} (Belz et al., 2000Go; Flynn et al., 1998Go; Price et al., 2000Go; Taylor et al., 1989Go). We have previously shown that IFN{gamma} levels are diminished 60% in restimulated bulk MLN cultures from TCDD-treated mice (Warren et al., 2000Go). To determine the cellular source of IFN{gamma} in the MLN cultures, and to quantify the effects of TCDD on IFN{gamma} produced by CD8+ T cells in particular, magnetic beads were used to deplete CD8+ or CD4+ T cells prior to restimulation. As shown in Figure 2Go, removal of CD4+ T cells did not alter IFN{gamma} levels in restimulated MLN cells from vehicle- or TCDD-treated mice on day 9 post infection. However, depletion of CD8+ T cells ablated IFN{gamma} production in restimulated cells from either treatment group, indicating that CD8+ T cells were the primary source of IFN{gamma} in the MLN cultures. Similar results were obtained on days 5 and 8 post infection (data not shown). It is important to note that diminished levels of IFN{gamma} in intact or CD4-depleted cultures from TCDD-exposed mice were not simply due to fewer CD8+ T cells in the wells containing cells from TCDD-treated mice. The percentage and number of CD4+ and CD8+ T cells in these cultures were equivalent among treatment groups (data not shown), therefore, decreased levels of IFN{gamma} are not simply due to putting fewer T cells into the well. Taken together, these functional and phenotypic analyses demonstrate that the development of influenza virus-specific CTL is suppressed in mice exposed to TCDD. Thus, additional studies were conducted to address possible mechanisms by which virus-specific CD8+ T cells failed to develop into functional CTL in TCDD-exposed mice.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 2. Treatment with TCDD reduces IFN{gamma} production by CD8+ T cells. B6 mice were treated with vehicle or TCDD one day prior to infection with influenza virus strain A/HKx31. On day 9, MLN cells were isolated and depleted of T cell subsets using anti-CD4 or anti-CD8-conjugated magnetic beads, as described in the Materials and Methods. Remaining MLN cells were plated at 2 x 106 cells/well and restimulated with 1 x 106 influenza virus-infected, irradiated DC2.4 cells, which served as antigen presenting cells. Supernatants were collected after 24 h and IFN{gamma} levels were measured by ELISA. Data represent mean (± SEM) from three samples per treatment group. Results are representative of two separate experiments in which mice were sacrificed on days 5, 8, and 9 post infection. Asterisk indicates a significant difference when compared to vehicle group under same depletion conditions (p ≤ 0.05).

 
Treatment with TCDD Does Not Increase Apoptosis in Antigen-Specific CD8+ T Cells
The clonal expansion and differentiation of activated CD8+ T cells is counterbalanced by mechanisms to prevent or terminate T cell responses (reviewed in Van Parijs and Abbas, 1998Go). One mechanism involved in T cell homeostasis is the deletion of activated T cells via apoptosis (Jones et al., 1990Go; Kyburz et al., 1993Go; Webb et al., 1990Go). Based on the observation that there were fewer CTLe and less IFN{gamma}-producing CD8+ T cells in TCDD-treated mice, we addressed the possibility that TCDD impaired the development of CTL by increasing apoptosis in virus-specific CD8+ T cells. To test this, virus-specific CD8+ T cells from the MLN were stained immediately with annexin V and 7-AAD to assess apoptosis. The percentage of virus-specific CD8+ T cells expressing an apoptotic phenotype (annexin V-positive, 7-AAD-negative) was determined by flow cytometry. Figure 3AGo shows representative histograms for apoptosis staining on Vß11+CD8+ T cells from vehicle- or TCDD-treated B10 mice on day 7 post infection with influenza virus. Exposure to TCDD did not alter the percentage of Vß11+CD8+ T cells expressing an apoptotic phenotype, nor did it affect the percentage of cells bearing a late apoptotic/necrotic (7-AAD-positive) phenotype. Furthermore, no evidence of increased apoptosis was observed in influenza virus-specific CD8+ T cells from TCDD-exposed mice sacrificed throughout the course of infection (Fig. 3BGo). Although increases in the number of annexin V+ cells were observed in samples from TCDD-treated mice, these changes were not statistically significant. Furthermore, we saw no consistent change (increase or decrease) in the percentage of apoptotic influenza virus-specific CD8+ T cells in repetitions of this experiment. Likewise, no alterations in the percentage of annexin V-positive, 7-AAD-negative cells were observed when DbNP366 tetramers were used to identify virus-specific CD8+ T cells on days 8 and 9 (Fig. 3BGo). Together, these results suggest that enhanced apoptosis is not a likely mechanism for the diminished generation of CTL in TCDD-treated mice.



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 3. Apoptosis is not enhanced in influenza virus-specific CD8+ T cells from TCDD-exposed mice. B6 and B10 mice were exposed to vehicle or TCDD one day prior to infection with influenza virus. Freshly isolated MLN cells were stained with FITC-labeled annexin V, 7-AAD, and APC-labeled anti-CD8. Additionally, PE-labeled DbNP366 tetramers were used to identify influenza virus-specific CD8+ T cells from B6 mice infected with A/HKx31, whereas PE-labeled anti-Vß11 was used to detect influenza virus-specific CD8+ T cells from B10 mice infected with A/Mempis/102/72. (A) Representative histograms depict annexin V and 7-AAD staining in Vß11+CD8+ T cells from vehicle- or TCDD-treated B6 mice 7 days after infection with influenza virus. Numbers refer to the percentage of Vß11+CD8+ T cells in each quadrant. (B) Percentage of Vß11+CD8+ or DbNP366+ CD8+ T cells that stain annexin V-positive, 7-AAD-negative, based on flow cytometric analysis. On every day tested, each treatment group consisted of 6–8 B10 mice or 3–7 B6 mice. Results are representative of two separate experiments for each strain of mouse.

 
In Vivo Proliferation of Influenza Virus-Specific CD8+ T Cells Is Impaired by Exposure to TCDD
Another mechanism that may account for the decreased CTL response in TCDD-exposed mice is the induction of T cell unresponsiveness, or anergy. This was initially addressed using in vivo BrdU incorporation to determine whether exposure to TCDD impaired the proliferation of influenza virus-specific CD8+ T cells in the MLN. Consistent with a report by Flynn et al.(1999)Go, we found that infection with influenza virus induced the proliferation of Vß11+CD8+ and DbNP366+CD8+ T cells in vehicle-treated mice (Fig. 4Go). In contrast, the proliferation of Vß11+CD8+ T cells in TCDD-treated mice infected with influenza virus was reduced by as much as 66% on day 7 (Fig.4AGo). Exposure to TCDD did not appear to suppress proliferation before day 5, as BrdU staining on day 3 was comparable in Vß11+CD8+ T cells from vehicle- and TCDD-treated mice (Fig. 4AGo). Reduced proliferation in TCDD-treated mice was also observed when DbNP366 tetramers were used to identify influenza virus-specific CD8+ T cells on days 5 to 9 post infection (Fig. 4BGo). These data clearly demonstrate that exposure to TCDD impairs the in vivo proliferation of antigen-specific CD8+ T cells.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 4. Proliferation is reduced in influenza virus-specific CD8+ T cells from mice exposed to TCDD. B6 and B10 mice were gavaged with vehicle or TCDD one day prior to infection with influenza virus. In vivo proliferation was assessed by adding BrdU to drinking water (0.8 mg/ml) three days prior to sacrificing mice. Mice were sacrificed on days 3 through 12, and MLN cells were isolated and stained with FITC-labeled anti-BrdU and APC-labeled anti-CD8 antibodies as described in the Materials and Methods. Additionally, PE-labeled anti-Vß11 antibody was used to detect influenza virus-specific CD8+ T cells from B10 mice infected with A/Mempis/102/72, whereas PE-labeled DbNP366 tetramers were used to stain influenza virus-specific CD8+ T cells from B6 mice infected with A/HKx31. Graphs represent the number of BrdU+Vß11+CD8+ T cells (A) and BrdU+ DbNP366+ CD8+ T cells (B). Error bars depict the SEM. On every day tested, each treatment group consisted of 6–8 B10 mice or 3–7 B6 mice. Results are representative of two separate experiments for each strain of mouse. Asterisk indicates a significant difference when compared to vehicle group (p ≤ 0.05).

 
Optimal in Vitro Restimulation Fails to Elicit Maximal Responsiveness in Influenza Virus-Specific CD8+ T Cells from TCDD-Treated Mice
The observation that TCDD suppressed the in vivo proliferation of influenza virus-specific CD8+ T cells suggested that these cells may have become anergic, which would prevent them from responding adequately to viral challenge. To address this possibility, we tested the ability of virus-specific CD8+ T cells from infected, TCDD-exposed mice to proliferate and produce IFN{gamma} when restimulated ex vivo with antigen and IL-2. These culture conditions have been shown to be sufficient for stimulating the proliferation and development of effector functions in primed CD8+ T cells (Curtsinger et al., 1998Go; Gschoesser et al., 2002Go; Murali-Krishna et al., 1998Go). As expected, restimulation with NP366 peptide and exogenous IL-2 induced maximal IFN{gamma} production in MLN cells from vehicle-treated mice, compared to the addition of either NP366 peptide or IL-2 alone (Fig. 5Go). In contrast to restimulated cells from vehicle-treated mice, stimulation with NP366 peptide and IL-2 failed to elicit IFN{gamma} production by MLN cells from TCDD-exposed mice. In fact, the levels of IFN{gamma} in cultures from TCDD-treated mice were similar to those from naïve mice. Diminished levels of IFN{gamma} were not simply due to fewer CD8+ T cells in the cultures from TCDD-exposed mice, as the number of antigen-specific CD8+ T cells added to the wells was equivalent in vehicle and TCDD cultures (data not shown). Thus, these data demonstrate that optimal stimulation conditions fail to induce IFN{gamma} production by influenza virus-specific CD8+ T cells from TCDD-treated mice.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 5. Exogenous NP peptide and IL-2 do not stimulate IFN{gamma} production by influenza virus-specific CD8+ T cells from TCDD-treated mice. B6 and B10 mice were treated with vehicle or TCDD one day prior to intranasal infection with influenza virus strains A/HKx31 or A/Memphis/102/72. On days 3 to 9 post infection, MLN cells were isolated and restimulated in vitro with 1 µM NP366 peptide and/or 12.5 U/ml IL-2. Supernatants were collected after 24 h of culture and IFN{gamma} levels were measured by ELISA. Data represent mean IFN{gamma} concentration ± SEM (pg/ml). For each treatment group, cells from 5 to 6 mice were assessed per day. Asterisk indicates a significant difference when compared to vehicle group (p ≤ 0.05), and # indicates the p value was ≤ 0.06.

 
To further assess the functional capacity of influenza virus-specific CD8+ T cells from TCDD-exposed mice, we measured the proliferation of these cells in response to NP366 peptide and IL-2. Addition of IL-2 clearly induced the proliferation of cultured MLN cells from vehicle-treated, influenza virus-infected mice (Fig. 6Go). Although providing IL-2 did induce some proliferation in cells from TCDD-exposed mice, the level was about half of that observed in cells from vehicle-treated mice.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 6. Proliferation is only partially restored in restimulated MLN cultures from TCDD-exposed mice. Mice were exposed to vehicle or TCDD and infected with influenza virus as described in Figure 5Go. MLN cells were restimulated in vitro for 72 h in the presence of 1 µM NP366 peptide and/or 12.5 U/ml IL-2. 3H-thymidine was added at 2 µCi/well during the last 24 h. Data represent mean c.p.m. ± SEM from triplicate wells. For each treatment group, cells from 5 to 6 mice were assessed per day. Asterisk indicates a significant difference when compared to vehicle group (p ≤ 0.05).

 
Given that proliferation was detected in cultures that contained IL-2, both in the presence and absence of NP366 peptide, it was possible that IL-2 could drive the proliferation not only of antigen-specific CD8+ T cells, but also of CD4+ T cells and of CD8+ T cells that were not NP-specific. Thus, we accounted for the relative contribution of proliferating CD4+ T cells and non-antigen-specific CD8+ T cells to the overall levels of proliferation measured in the cultures. To do this, we compared the number of CD4+, CD8+, and Vß11+CD8+ T cells at the beginning of the culture period to the number of cells recovered after 72 h of culture. We found that proliferation in the cultures was not due to the expansion of CD4+ T cells, as the addition of NP366 peptide and IL-2 did not support the expansion of these cells in cultures from either vehicle- or TCDD-treated mice (Fig. 7AGo). In contrast, the number of CD8+ T cells from vehicle-treated cultures increased as much as two-fold under the same stimulation conditions (Fig. 7BGo). However, no net increase in the number of CD8+ T cells was observed in cultures from TCDD-exposed mice.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 7. Exogenous NP peptide and IL-2 do not support maximum proliferation of influenza virus-specific CD8+ T cells from TCDD-exposed mice. B10 mice were treated with vehicle or TCDD one day prior to infection with A/Memphis/102/72. MLN cells were collected, counted and restimulated for 72 h in the presence of 1 µM NP366 peptide and 12.5 U/ml IL-2. At the end of the culture period, cells were counted and stained with antibodies specific for CD8, Vß11, and CD4. Graphs depict the mean number of CD4+ (A), CD8+ (B), and Vß11+CD8+ (C) T cells in the wells at the beginning and end of the 72-hour culture period. Error bars represent the SEM. For each treatment group, 3 to 5 mice were used per day. Asterisk indicates a significant difference when compared to the vehicle treatment group after 72 h (p ≤ 0.05).

 
We also accounted for the proliferation of NP-specific CD8+ T cells in cultures stimulated with peptide and IL-2. The number of Vß11+CD8+ T cells recovered from cultures from vehicle-treated mice was two- to three-times higher than from TCDD-treated mice (Fig. 7CGo). Given that the percentage of Vß11+CD8+ T cells in the MLN is equivalent among vehicle- and TCDD-treated mice (data not shown), suppressed expansion of these cells in cultures from TCDD-exposed mice cannot simply be due to the addition of fewer NP-specific CD8+ T cells to the wells. Thus, these data clearly show that restimulation only partially restored the expansion of influenza virus-specific CD8+ T cells from TCDD-exposed mice. In summary, using both IFN{gamma} production and proliferation as endpoints, our data indicate that exposure to TCDD induced a state of hyporesponsiveness in influenza virus-specific CD8+ T cells that could not be overcome even when optimal stimulation conditions were provided.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous studies have demonstrated that T cell-dependent immune responses are impaired following exposure to TCDD, yet the mechanisms by which TCDD induces T cell dysfunction are poorly understood. In the present study, we investigated how exposure to TCDD alters the fate of antigen-specific T cells activated in vivo. Specifically, we addressed mechanisms by which influenza virus-specific CD8+ T cells fail to generate an adequate CTL response in TCDD-exposed mice. We used clonotypic antibodies and MHC class I-restricted tetramers to characterize the effects of TCDD on the fate of influenza virus-specific CD8+ T cells upon antigen challenge. Using these tools, we found that exposure to TCDD suppressed the development of CTL in the lymph nodes of mice infected with influenza virus. This was evidenced by decreased expansion of Vß11+CD8+ and DbNP366+CD8+ T cells, diminished production of IFN{gamma} by CD8+ T cells, and a reduction in the number of influenza virus-specific CD8+ T cells bearing a CTLe phenotype. These findings are consistent with previously reported effects of TCDD on T cells during infection with influenza virus, including our observations that exposure to TCDD reduced the number of CTL in the lung of virus-infected mice and suppressed the ability of restimulated MLN cells to kill virus-infected target cells and produce IFN{gamma} (Warren et al., 2000Go).

These suppressive effects of TCDD on CD8+ T cells from influenza virus-infected mice are also strikingly consistent with the impaired CTL response observed in TCDD-exposed mice challenged with allogeneic P815 tumor cells. For example, exposure of mice to TCDD prior to injection with P815 cells impaired the expansion of CD8+ T cells in the spleen, reduced the number of cells expressing a CTLe phenotype, and suppressed splenic CTL activity (De Krey and Kerkvliet, 1995Go; Kerkvliet et al., 1996Go). Moreover, these suppressive effects were accompanied by reduced levels of IFN{gamma} and IL-2, which were produced exclusively by CD8+ T cells in this model (Kerkvliet et al., 1996Go). The similarities between CTL suppression during infection with influenza virus and in the P815 tumor allograft model indicate that TCDD impairs the response of CD8+ T cells to two distinct types of antigen and suggest that suppression of the CTL response in these models likely occurs via a common mechanism.

To explain why there were fewer functional antigen-specific T cells in TCDD-treated mice, we addressed the possibility that exposure to TCDD increased apoptosis in influenza virus-specific CD8+ T cells. In other words, we tried to find evidence that in vivo exposure to TCDD promoted clonal deletion. However, when we examined influenza virus-specific CD8+ T cells directly ex vivo, we found no evidence of enhanced apoptosis. This finding is supported by our observation in separate studies that exposure to TCDD did not alter intracellular bcl-2 levels in influenza virus-specific CD8+ T cells (data not shown). The lack of direct evidence for enhanced apoptosis in our model system is consistent with that of Prell et al.(2000)Go, who found that exposure to TCDD did not enhance apoptosis in CD8+ T cells during challenge with allogeneic P815 tumor cells. It also concurs with a recent report in which TCDD treatment did not enhance apoptosis in CD4+ T cells activated with anti-CD3 (Dearstyne and Kerkvliet, 2002Go). However, our findings contrast with reports by Pryputniewicz et al.(1998)Go and Camacho et al.(2001)Go, who found that exposure to TCDD increased apoptosis in activated lymphoid cells from anti-CD3-treated mice. Discrepancies between our findings and these studies may be due to differences in the amount of TCDD administered (10 µg/kg versus 50 µg/kg, respectively). Alternatively, it could be due to differences in methodology, as the latter studies measured apoptosis in T cells after a 24-hour in vitro culture period.

While we did not observe an effect of TCDD on apoptosis, the proliferation of influenza virus-specific CD8+ T cells was clearly impaired in TCDD-exposed mice. Interestingly, suppressed proliferation of virus-specific CD8+ T cells was not evident in TCDD-treated mice until day 5 post infection. This delay in suppression was observed not only in the proliferative response of virus-specific CD8+ T cells, but also in the development of CTLe in the lymph node. Similar kinetics have been described for the suppressive effects of TCDD on adoptively transferred DO11.10 CD4+ T cells in OVA-challenged mice (Shepherd et al., 2000Go). Specifically, this study showed that the initial expansion of OVA-specific CD4+ T cells was not altered in TCDD-treated recipient mice. However, in contrast to vehicle-treated mice, the initial expansion of antigen-specific cells was followed by a decrease in the number of these cells in spleens from TCDD-exposed mice. Taken together, these findings raise the possibility that exposure to TCDD does not suppress the initial activation and proliferation of antigen-specific T cells, but rather alters these cells, or the local lymph node environment, such that these functions are not sustained.

One possible explanation for the diminished function of virus-specific CD8+ T cells in TCDD-exposed mice is that IL-2 levels in the lymph nodes of TCDD-treated mice are insufficient for sustaining a CD8+ T cell-mediated response. Indeed, we have found that exposure to TCDD reduces IL-2 production in restimulated MLN cells, and the timing of this suppression occurs between 5 and 9 days post infection, which coincides with the impaired proliferation of influenza virus-specific CD8+ T cells (Warren et al., 2000Go). Furthermore, decreased IL-2 levels may contribute to the diminished CTL response in TCDD-exposed mice challenged with allogeneic tumor cells, as in vivo administration of IL-2 on days 7 to 9 was found to restore the cytolytic activity of isolated spleen cells (Prell et al., 2000Go). Recent studies indicate that the role of IL-2 in maintaining the CTL response may be more complex than previously believed. For example, it has been postulated that activated CD8+ T cells pass through a regulatory "checkpoint" referred to as activation-induced nonresponsiveness, which converts an initially helper-independent CTL response to a response that requires IL-2 produced by CD4+ T cells (Deeths et al., 1999Go; Schwartz, 1990Go; Shrikant and Mescher, 1999Go; Tham and Mescher, 2001Go). In light of this, it is feasible that reduced levels of IL-2 in TCDD-exposed mice may interfere with the sustained response of the virus-specific CTL, leaving the cells in a state of unresponsiveness.

The idea that exposure to TCDD induces a state of nonresponsiveness in virus-specific CD8+ T cells is supported by our observation that providing antigen and exogenous IL-2 failed to induce IFN{gamma} production and only partially restored the proliferative response of virus-specific CD8+ T cells from TCDD-exposed mice. These characteristics are consistent with recent reports describing anergy in CD8+ T cells in vivo (Blish et al., 1999Go; Dubois et al., 1998Go ; Gray et al., 1999Go; Preckel et al., 2001Go; Xiong et al., 2001Go). For example, Dubois et al.(1998)Go demonstrated that multiple peptide injections in vivo induced CD8+ T cell tolerance characterized by decreased in vivo proliferation that was only partially restored upon in vitro restimulation. Although hyporesponsiveness in the virus-specific CD8+ T cells from TCDD-treated mice could theoretically be explained by a TCDD-induced downregulation of the TCR or IL-2 receptor, we and others have found no evidence that exposure to TCDD alters the expression of these receptors (Neumann et al., 1993Go, and our unpublished observations). Interestingly, neither proliferation nor IFN{gamma} production was suppressed when MLN cells from TCDD-exposed mice were stimulated with anti-CD3 (data not shown), which indicates that these cells are capable of responding to polyclonal stimulation. Moreover, it suggests that exposure to TCDD may impair the ability of influenza virus-specific CD8+ T cells to receive or respond to signals delivered through the TCR or through the IL-2R.

Consistent with the notion that treatment with TCDD induces a state of nonresponsiveness in antigen-specific CD8+ T cells, exposure to TCDD has been shown to alter the activity of APC. Thus, it is possible that the suppressive effects of TCDD on virus-specific CD8+ T cells are due to the impaired ability of APC to stimulate T cells in the lymph node. In support of this, Prell and Kerkvliet (1997)Go reported that injection of B7-transfected allogeneic P815 tumor cells restored the CTL response in TCDD-exposed mice, indicating that impaired costimulation may be one mechanism by which exposure to TCDD suppresses the CTL response. However, on a per cell basis, exposure to TCDD has been shown to increase, not decrease, the expression of costimulatory molecules such as B7-2 on splenic dendritic cells (DC), and was found to enhance the ability of DC to stimulate allogeneic T cells (Vorderstrasse and Kerkvliet, 2001Go). An explanation for this paradoxical effect of TCDD on DC is that exposure to TCDD promotes activation-induced cell death in DC. Two recent studies have provided experimental evidence in support of this hypothesis. First, a report by Vorderstrasse and Kerkvliet (2001)Go demonstrated that exposure to TCDD decreased the total number of DC recovered from the spleen. Second, Ruby et al.(2002)Go recently found that exposure of a dendritic cell line to TCDD suppressed the function of NF-kB/Rel, a transcription factor important for DC maturation and survival (Rescigno et al., 1998Go). In light of these findings, it is possible that treatment with TCDD impairs the survival and/or alters the function of DC presenting viral antigen in the lymph node. Such circumstances could reduce the number of influenza virus-specific CD8+ T cells that are stimulated. Alternatively, exposure to TCDD could cause DC in the lymph node to deliver either partial or improper signals, thereby inducing anergy in the responding CD8+ T cells.

In summary, the present findings provide new information regarding possible mechanisms by which exposure to TCDD impairs T cell-mediated immune responses. By identifying and monitoring influenza virus-specific CD8+ T cells with clonotypic antibodies and tetramers, we examined how exposure to TCDD alters the fate of antigen-specific CD8+ T cells responding to in vivo challenge. Our data indicate that the impaired CTL response in TCDD-exposed mice is due to the induction of hyporesponsiveness in influenza virus-specific CD8+ T cells. Collectively, these results strengthen the hypothesis that exposure to TCDD impedes the proper and sufficient activation of antigen-specific T cells, thereby hindering the clonal expansion and differentiation of CD8+ T cells into effector CTL. Given that the molecular events involved in T cell activation during infection with influenza virus are similar to those events elicited during infection with other pathogens, our findings expand the current understanding of how TCDD affects T cell activation in general.


    ACKNOWLEDGMENTS
 
The authors are especially grateful to Drs. Peter Doherty and Stephen Turner for helpful comments and generously providing the DbNP366 tetramers. We also thank Dr. Demetrius Moskophidis for providing the initial stock of A/Memphis/102/72 and Dr. Graeme Price for helpful discussions regarding the plaque assay. Additionally, we wish to acknowledge Dr. Beth Vorderstrasse for critical review of this manuscript and Minh-Chau Nguyen for excellent technical assistance. This research was supported by grants from the National Institutes for Environmental Health Sciences (RO1-ES10619) and Washington State University (New Faculty Seed Grant). K.A.M. is a recipient of pre-doctoral fellowships from the American Foundation for Pharmaceutical Education and the Pharmaceutical Research and Manufacturers of America Foundation.


    NOTES
 
1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6534. Fax: (509) 335-5902. E-mail: bpl{at}mail.wsu.edu Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allan, W., Tabi, Z., Cleary, A., and Doherty, P. C. (1990). Cellular events in the lymph node and lung of mice with influenza. Consequences of depleting CD4+ T cells. J. Immunol. 144, 3980–3986.[Abstract/Free Full Text]

Barrett, T., and Inglis, S. (1985). Growth, purification and titration of influenza viruses. In Virology: A Practical Approach (B. W. J. Mahy, Ed.), pp. 119–150. IRL Press, Oxford.

Belz, G. T., Xie, W., Altman, J. D., and Doherty, P. C. (2000). A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge. J. Virol. 74, 3486–3493.[Abstract/Free Full Text]

Birnbaum, L. S. (1986). Distribution and excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin in congenic strains of mice which differ at the Ah locus. Drug Metab. Dispos. 14, 34–40.[Abstract]

Blish, C. A., Dillon, S. R., Farr, A. G., and Fink, P. J. (1999). Anergic CD8+ T cells can persist and function in vivo. J. Immunol. 163, 155–164.[Abstract/Free Full Text]

Burleson, G. R., Lebrec, H., Yang, Y. G., Ibanes, J. D., Pennington, K. N., and Birnbaum, L. S. (1996). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundam. Appl. Toxicol. 29, 40–47.[CrossRef][ISI][Medline]

Camacho, I. A., Hassuneh, M. R., Nagarkatti, M., and Nagarkatti, P. S. (2001). Enhanced activation-induced cell death as a mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity in peripheral T cells. Toxicology 165, 51–63.[CrossRef][ISI][Medline]

Clark, D. A., Sweeney, G., Safe, S., Hancock, E., Kilburn, D. G., and Gauldie, J. (1983). Cellular and genetic basis for suppression of cytotoxic T cell generation by haloaromatic hydrocarbons. Immunopharmacology 6, 143–153.[CrossRef][ISI][Medline]

Curtsinger, J. M., Lins, D. C., and Mescher, M. F. (1998). CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C-) to TCR/CD8 signaling in response to antigen. J. Immunol. 160, 3236–3243.[Abstract/Free Full Text]

De Krey, G. K., and Kerkvliet, N. I. (1995). Suppression of cytotoxic T lymphocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin occurs in vivo, but not in vitro, and is independent of corticosterone elevation. Toxicology 97, 105–112.[CrossRef][ISI][Medline]

Dearstyne, E. A., and Kerkvliet, N. I. (2002). Mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced decrease in anti-CD3-activated CD4(+) T cells: The roles of apoptosis, Fas, and TNF. Toxicology 170, 139–151.[CrossRef][ISI][Medline]

Deckhut, A. M., Allan, W., McMickle, A., Eichelberger, M., Blackman, M. A., Doherty, P. C., and Woodland, D. L. (1993). Prominent usage of V beta 8.3 T cells in the H-2Db-restricted response to an influenza A virus nucleoprotein epitope. J. Immunol. 151, 2658–2666.[Abstract/Free Full Text]

Deeths, M. J., Kedl, R. M., and Mescher, M. F. (1999). CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation. J. Immunol. 163, 102–110.[Abstract/Free Full Text]

Dubois, P. M., Pihlgren, M., Tomkowiak, M., Van Mechelen, M., and Marvel, J. (1998). Tolerant CD8 T cells induced by multiple injections of peptide antigen show impaired TCR signaling and altered proliferative responses in vitro and in vivo. J. Immunol. 161, 5260–5267.[Abstract/Free Full Text]

Flynn, K. J., Belz, G. T., Altman, J. D., Ahmed, R., Woodland, D. L., and Doherty, P. C. (1998). Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8, 683–691.[ISI][Medline]

Flynn, K. J., Riberdy, J. M., Christensen, J. P., Altman, J. D., and Doherty, P. C. (1999). In vivo proliferation of naive and memory influenza-specific CD8(+) T cells. Proc. Natl. Acad. Sci. U.S.A. 96, 8597–8602.[Abstract/Free Full Text]

Gray, C. M., Lawrence, J., Schapiro, J. M., Altman, J. D., Winters, M. A., Crompton, M., Loi, M., Kundu, S. K., Davis, M. M., and Merigan, T. C. (1999). Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). J. Immunol. 162, 1780–1788.[Abstract/Free Full Text]

Gschoesser, C., Almanzar, G., Hainz, U., Ortin, J., Schonitzer, D., Schild, H., Saurwein-Teissl, M., and Grubeck-Loebenstein, B. (2002). CD4(+) and CD8(+) mediated cellular immune response to recombinant influenza nucleoprotein. Vaccine 20, 3731–3738.[CrossRef][ISI][Medline]

Hou, S., and Doherty, P. C. (1993). Partitioning of responder CD8+ T cells in lymph node and lung of mice with Sendai virus pneumonia by LECAM-1 and CD45RB phenotype. J. Immunol. 150, 5494–5500.[Abstract/Free Full Text]

Jones, L. A., Chin, L. T., Longo, D. L., and Kruisbeek, A. M. (1990). Peripheral clonal elimination of functional T cells. Science 250, 1726–1729.[ISI][Medline]

Kerkvliet, N. I. (2002). Recent advances in understanding the mechanisms of TCDD immunotoxicity. Int. Immunopharmacol. 2, 277–291.[CrossRef][ISI][Medline]

Kerkvliet, N. I., Baecher-Steppan, L., Shepherd, D. M., Oughton, J. A., Vorderstrasse, B. A., and DeKrey, G. K. (1996). Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Immunol. 157, 2310–2319.[Abstract]

Kerkvliet, N. I., and Burleson, G. R. (1994). Immunotoxicity of TCDD and Related Halogenated Aromatic Hydrocarbons. Raven Press, New York.

Kyburz, D., Aichele, P., Speiser, D. E., Hengartner, H., Zinkernagel, R. M., and Pircher, H. (1993). T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides. Eur. J. Immunol. 23, 1956–1962.[ISI][Medline]

Lang, D. S., Becker, S., Clark, G. C., Devlin, R. B., and Koren, H. S. (1994). Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro. Arch. Toxicol. 68, 296–302.[CrossRef][ISI][Medline]

Lawrence, B. P., Leid, M., and Kerkvliet, N. I. (1996). Distribution and behavior of the Ah receptor in murine T lymphocytes. Toxicol. Appl. Pharmacol. 138, 275–284.[CrossRef][ISI][Medline]

Lundberg, K., Dencker, L., and Gronvik, K. O. (1992). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the activation of antigen-specific T-cells in mice. Int. J. Immunopharmacol. 14, 699–705.[CrossRef][ISI][Medline]

Mobley, J. L., and Dailey, M. O. (1992). Regulation of adhesion molecule expression by CD8 T cells in vivo. I. Differential regulation of gp90MEL-14 (LECAM-1), Pgp-1, LFA-1, and VLA-4 alpha during the differentiation of cytotoxic T lymphocytes induced by allografts. J. Immunol. 148, 2348–2356.[Abstract/Free Full Text]

Murali-Krishna, K., Altman, J. D., Suresh, M., Sourdive, D. J., Zajac, A. J., Miller, J. D., Slansky, J., and Ahmed, R. (1998). Counting antigen-specific CD8 T cells: A reevaluation of bystander activation during viral infection. Immunity 8, 177–187.[ISI][Medline]

Neumann, C. M., Oughton, J. A., and Kerkvliet, N. I. (1993). Anti-CD3-induced T-cell activation–II. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Int. J. Immunopharmacol. 15, 543–550.[CrossRef][ISI][Medline]

Palmer, M. S., Bentley, A., Gould, K., and Townsend, A. R. (1989). The T cell receptor from an influenza-A specific murine CTL clone. Nucleic Acids Res. 17, 2353.[ISI][Medline]

Preckel, T., Hellwig, S., Pflugfelder, U., Lappin, M. B., and Weltzien, H. U. (2001). Clonal anergy induced in a CD8+ hapten-specific cytotoxic T-cell clone by an altered hapten-peptide ligand. Immunology 102, 8–14.[CrossRef][ISI][Medline]

Prell, R. A., Dearstyne, E., Steppan, L. G., Vella, A. T., and Kerkvliet, N. I. (2000). CTL hyporesponsiveness induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin: Role of cytokines and apoptosis. Toxicol. Appl. Pharmacol. 166, 214–221.[CrossRef][ISI][Medline]

Prell, R. A., and Kerkvliet, N. I. (1997). Involvement of altered B7 expression in dioxin immunotoxicity: B7 transfection restores the CTL but not the autoantibody response to the P815 mastocytoma. J. Immunol. 158, 2695–2703.[Abstract]

Prell, R. A., Oughton, J. A., and Kerkvliet, N. I. (1995). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on anti-CD3-induced changes in T-cell subsets and cytokine production. Int. J. Immunopharmacol. 17, 951–961.[CrossRef][ISI][Medline]

Price, G. E., Gaszewska-Mastarlarz, A., and Moskophidis, D. (2000). The role of alpha/beta and gamma interferons in development of immunity to influenza A virus in mice. J. Virol. 74, 3996–4003.[Abstract/Free Full Text]

Pryputniewicz, S. J., Nagarkatti, M., and Nagarkatti, P. S. (1998). Differential induction of apoptosis in activated and resting T cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its repercussion on T cell responsiveness. Toxicology 129, 211–226.[CrossRef][ISI][Medline]

Rescigno, M., Martino, M., Sutherland, C. L., Gold, M. R., and Ricciardi-Castagnoli, P. (1998). Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188, 2175–2180.[Abstract/Free Full Text]

Rhile, M. J., Nagarkatti, M., and Nagarkatti, P. S. (1996). Role of Fas apoptosis and MHC genes in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity of T cells. Toxicology 110, 153–167.[CrossRef][ISI][Medline]

Ruby, C. E., Leid, M., and Kerkvliet, N. I. (2002). 2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses tumor necrosis factor-alpha and anti-CD40-induced activation of NF-kappaB/Rel in dendritic cells: p50 homodimer activation is not affected. Mol. Pharmacol. 62, 722–728.[Abstract/Free Full Text]

Schwartz, R. H. (1990). A cell culture model for T lymphocyte clonal anergy. Science 248, 1349–1356.[ISI][Medline]

Shepherd, D. M., Dearstyne, E. A., and Kerkvliet, N. I. (2000). The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11. 10 transgenic CD4(+) T cells in adoptively transferred mice. Toxicol. Sci. 56, 340–350.[Abstract/Free Full Text]

Shrikant, P., and Mescher, M. F. (1999). Control of syngeneic tumor growth by activation of CD8+ T cells: Efficacy is limited by migration away from the site and induction of nonresponsiveness. J. Immunol. 162, 2858–2866.[Abstract/Free Full Text]

Taylor, P. M., Meager, A., and Askonas, B. A. (1989). Influenza virus-specific T cells lead to early interferon gamma in lungs of infected hosts: Development of a sensitive radioimmunoassay. J. Gen. Virol. 70, 975–978.[Abstract]

Tham, E. L., and Mescher, M. F. (2001). Signaling alterations in activation-induced nonresponsive CD8 T cells. J. Immunol. 167, 2040–2048.[Abstract/Free Full Text]

Tomonari, K., and Lovering, E. (1988). T-cell receptor-specific monoclonal antibodies against a V beta 11- positive mouse T-cell clone. Immunogenetics 28, 445–451.[ISI][Medline]

Tough, D. F., and Sprent, J. (1994). Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179, 1127–1135.[Abstract]

Townsend, A. R., Taylor, P. M., Melief, C. J., and Askonas, B. A. (1983). Diversity in the interaction of influenza-A-specific cytotoxic T cells with H-2Kb mutant targets. Immunogenetics 17, 543–549.[CrossRef][ISI][Medline]

van Engeland, M., Nieland, L. J., Ramaekers, F. C., Schutte, B., and Reutelingsperger, C. P. (1998). Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1–9.[CrossRef][ISI][Medline]

Van Parijs, L., and Abbas, A. K. (1998). Homeostasis and self-tolerance in the immune system: Turning lymphocytes off. Science 280, 243–248.[Abstract/Free Full Text]

Vorderstrasse, B. A., and Kerkvliet, N. I. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin affects the number and function of murine splenic dendritic cells and their expression of accessory molecules. Toxicol. Appl. Pharmacol. 171, 117–125.[CrossRef][ISI][Medline]

Vorderstrasse, B. A., Steppan, L. B., Silverstone, A. E., and Kerkvliet, N. I. (2001). Aryl hydrocarbon receptor-deficient mice generate normal immune responses to model antigens and are resistant to TCDD-induced immune suppression. Toxicol. Appl. Pharmacol. 171, 157–164.[CrossRef][ISI][Medline]

Warren, T. K., Mitchell, K. A., and Lawrence, B. P. (2000). Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the humoral and cell-mediated immune responses to influenza A virus without affecting cytolytic activity in the lung. Toxicol. Sci. 56, 114–123.[Abstract/Free Full Text]

Webb, S., Morris, C., and Sprent, J. (1990). Extrathymic tolerance of mature T cells: Clonal limination as a consequence of immunity. Cell 63, 1249–1256.[ISI][Medline]

Wynford-Thomas, D., and Williams, E. D. (1986). Use of bromodeoxyuridine for cell kinetic studies in intact animals. Cell Tissue Kinet. 19, 179–182.[ISI][Medline]

Xiong, Y., Luscher, M. A., Altman, J. D., Hulsey, M., Robinson, H. L., Ostrowski, M., Barber, B. H., and MacDonald, K. S. (2001). Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8(+) T cells with a defect in effector function that is reversible on extended interleukin-2 incubation. J. Virol. 75, 3028–3033.[Abstract/Free Full Text]