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
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ABSTRACT |
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Key Words: TCDD (dioxin); immunosuppression; CD8+ T lymphocytes; antigen-specific; tetramer, influenza virus; anergy; mouse; lymph node; in vivo.
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INTRODUCTION |
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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, 2002). Suppression of the cytotoxic T lymphocyte (CTL) response has also been reported in mice exposed to TCDD (Clark et al., 1983
; Kerkvliet et al., 1996
; Warren et al., 2000
). For example, using a P815 tumor allograft model, Kerkvliet et al.(1996)
showed that exposure to TCDD dose-responsively suppressed splenic cytolytic activity, decreased the production of interleukin-2 (IL-2) and interferon-gamma (IFN
) by CD8+ T cells, and reduced the number of splenocytes bearing an effector CTL (CTLe) phenotype, defined as CD8+CD44hiCD62Ll°(Hou and Doherty, 1993
; Mobley and Dailey, 1992
). 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., 2000
). 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
production and cytolytic activity in restimulated lymph node cells (Warren et al., 2000
).
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 1995; Lang et al., 1994
; Lawrence et al., 1996
; Lundberg et al., 1992
). 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., 1996
; Lundberg et al., 1992
; Prell et al., 1995
; Pryputniewicz et al., 1998
; Rhile et al., 1996
). 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., 1996). 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., 2000
). 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., 1993
; Palmer et al., 1989
; Tomonari and Lovering 1988
; Townsend et al., 1983
). Furthermore, CD8+ T cells specific for the immunodominant epitope of influenza virus nucleoprotein (NP366374) 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.
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MATERIALS AND METHODS |
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Mice.
C57Bl/6 (B6) and C57Bl/10 (B10) female mice (68 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., 2000; Flynn et al., 1998
). 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, 1988
).
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, 1994), this dose of TCDD is not overtly toxic and is well below the LD50 in C57Bl mice (>120 µg/kg; Birnbaum, 1986
).
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., 1998). 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., 2000). 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. NP366374 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 510 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 by ELISA. Wells containing MLN cells from naive mice and wells with only DC2.4 cells were included as negative controls.
Measurement of IFN production.
Supernatants from ex vivo restimulated MLN cells were collected after 24 h. IFN levels were measured using a sandwich enzyme-linked immunosorbant assay (ELISA) with reagents purchased from BD Pharmingen. The ELISA was performed according to the manufacturers protocol. Briefly, 96-well plates (Nunc Maxisorp Immunoplates) were coated with 2µg/ml rat anti-mouse IFN
antibody (R4-6A2) diluted in 0.1 M NaHCO3 (pH 8.3). Samples or serially diluted recombinant murine IFN
standards were added to the plate in duplicate. IFN
was detected using 1 µg/ml biotinylated rat anti-mouse IFN
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., 1999; Wynford-Thomas and Williams, 1986
). 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, 1994
), 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 Fishers least significant difference multigroup comparison. Data were considered significantly different at p ≤ 0.05.
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RESULTS |
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DISCUSSION |
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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, 1995; Kerkvliet et al., 1996
). Moreover, these suppressive effects were accompanied by reduced levels of IFN
and IL-2, which were produced exclusively by CD8+ T cells in this model (Kerkvliet et al., 1996
). 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), 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, 2002
). However, our findings contrast with reports by Pryputniewicz et al.(1998)
and Camacho et al.(2001)
, 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., 2000). 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., 2000). 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., 2000
). 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., 1999
; Schwartz, 1990
; Shrikant and Mescher, 1999
; Tham and Mescher, 2001
). 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 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., 1999
; Dubois et al., 1998
; Gray et al., 1999
; Preckel et al., 2001
; Xiong et al., 2001
). For example, Dubois et al.(1998)
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., 1993
, and our unpublished observations). Interestingly, neither proliferation nor IFN
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) 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, 2001
). 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)
demonstrated that exposure to TCDD decreased the total number of DC recovered from the spleen. Second, Ruby et al.(2002)
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., 1998
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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