TY - JOUR
AU - Kerkvliet, Nancy I.
AB - Abstract TCDD is a potent immunotoxicant that suppresses adaptive immunity by mechanisms that are not well defined. To gain insight at the level of the T cell, we used the DO11.10 transgenic T-cell receptor (TCR) mouse model in an adoptive transfer approach to characterize the influence of TCDD on the responsiveness of antigen-specific CD4+ T cells in vivo. Flow cytometry was used to track the response of the OVA-specific transgenic CD4+ T cells in syngeneic recipients using an antibody specific for the transgenic TCR (KJ1-26 [KJ]). Consistent with a previous report, exposure of the recipient mice to TCDD (15 μg/kg po) did not alter the initial expansion of the CD4+KJ+ T cells in the spleen following immunization with OVA but resulted in a significant decline in the number of cells present on and after day 4. The degree of decline was dependent on the dose of TCDD. On day 3 after OVA injection, a higher percentage of the CD4+KJ+ T cells in the spleens of TCDD-treated mice had down-regulated the expression of CD62L, a phenotype associated with T-cell activation. Also on day 3, an increased number of CD4+KJ+ T cells were found in the blood of TCDD-treated mice. However, as in the spleen, the number of CD4+KJ+ T cells in the blood rapidly declined on day 4. CD4+KJ+ T cells in both the spleen and blood of TCDD-treated mice failed to up-regulate CD11a, an adhesion molecule important for sustained interaction between T cells and DC whereas the up-regulation of the adhesion molecule CD49d was not altered. Based on analysis of cell division history, CD4+KJ+ T cells in vehicle-treated mice continued to divide through day 4 whereas CD4+KJ+ T cells in TCDD-treated mice showed no further division after day 3. Increased annexin V staining on CD4+KJ+ T cells in TCDD-treated mice was also observed but not until days 5 and 6. Fas-deficient CD4+KJ+ T cells were depleted from the spleen of TCDD-treated mice in a manner similar to wild-type CD4+KJ+ T cells, suggesting that Fas signaling does not play a critical role in this model. On the other hand, gene array analysis of purified CD4+KJ+ T cells on day 3 showed that the expression of several genes associated with cell survival/death were altered by TCDD. Taken together, the results are consistent with our hypothesis that TCDD provides an early but inappropriate activation signal to the antigen-specific T cells that allows, and possibly enhances, the initial activation and proliferation of the T cells, yet at the same time, interferes with the vital expression of certain adhesion/costimulatory molecules that serve to enhance the survival of the T cells. These changes result in truncated proliferation, increased T-cell death, and suppression of the adaptive immune response. TCDD, T cell, activation, apoptosis, CD11a, CD62L T helper cells (CD4+ T cells) are a central component in the adaptive immune response to foreign antigen. When CD4+ T cells recognize and bind antigenic peptide presented with major histocompatibility complex class II molecules on dendritic cells (DC), they become activated, clonally expand, and differentiate into effector T cells. The primary effector function of CD4+ T cells is to support the survival and conditioning of dendritic cells, the differentiation and survival of cytotoxic T lymphocytes (CTL), and the development of antibody-producing B cells. Without sufficient expansion and prolonged survival of CD4+ T cells, adaptive immune responses are severely impaired. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant that possesses a profound capacity to suppress adaptive immune responses (Kerkvliet, 2002; Kerkvliet and Burleson, 1994). The immunotoxic effects of TCDD are mediated through binding to the aryl hydrocarbon receptor (AhR), which then translocates to the nucleus where it dimerizes with the AhR nuclear translocator (ARNT) (Mimura and Fujii-Kuriyama, 2003; Schmidt and Bradfield, 1996; Vorderstrasse et al., 2001). The AhR-ARNT heterodimer functions as a transcription factor, binding to specific sequences of DNA called dioxin response elements (DRE). Alterations in DRE-regulated gene transcription are thought to underlie most, if not all, of the toxic responses induced by TCDD. Recent studies using a graft-vs.-host model indicate that AhR expression in both CD4+ and CD8+ T cells is required for full suppression of T-cell-mediated immunity by TCDD (Kerkvliet et al., 2002). However, the specific changes induced in T cells by activation of the AhR that result in suppression of their responsiveness have not been established. Several animal models have been developed for the exclusive study of antigen-specific T cells. One such model is the DO11.10 adoptive transfer model that was developed by Kearney et al. (1994) and is used to study antigen-specific CD4+ T cells in vivo. DO11.10 mice express a transgenic T cell receptor (TCR) that is specific for chicken ovalbumin (OVA) peptide 323-339 in the context of I-Ad (Murphy et al., 1990). Since nearly all CD4+ T cells in the DO11.10 mice express the transgenic TCR, a more physiologic frequency of antigen-specific T cells is achieved by adoptively transferring a small number of OVA-specific T cells into syngeneic Balb/c mice. The OVA-specific CD4+ T cells are identified after adoptive transfer using the KJ1-26 antibody, which recognizes the transgenic TCR. In addition, the majority of the CD4+ T cells in the Balb/c mice that do not respond to OVA can be identified as the CD4+KJ− population, allowing for the differentiation between antigen-specific and nonspecific effects within the same animal. Previous studies using the DO11.10 adoptive transfer model have shown that activated antigen-specific CD4+ T cells expand normally in the spleen during the first three days of the response but then prematurely decline in mice treated with 15 μg TCDD/kg body weight (Shepherd et al., 2000). This premature loss of CD4+ T cells was associated with a decrease in the production of anti-OVA IgM and IgG antibodies. However, phenotypic analysis of the activated CD4+ T cells revealed only small changes in the expression of several early activation markers, providing little insight into how TCDD was causing the premature contraction of the T cell response. In the studies reported here, we have examined in greater detail the effects of TCDD on the activation, proliferation, and survival of antigen-specific T cells. Multi-color flow cytometry was used to compare the OVA-induced activation of adoptively transferred CD4+KJ+ T cells in the spleen and blood of vehicle- and TCDD-treated mice. Cell division of the adoptively transferred T cells was measured by labeling the DO11.10 spleen cells with 5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) prior to adoptive transfer. Altered T-cell survival was examined by annexin V/SytoxGreen staining, and the specific role of Fas-mediated signaling in the loss of T cells was addressed by adoptively transferring OVA-specific T cells from Fas-deficient D011.10-lpr/lpr (DO11-lpr) mice. To identify other signaling pathways that may be altered in T cells by TCDD, changes in the expression profile of several genes associated with cell death or survival were analyzed in purified CD4+KJ+ T cells from vehicle- and TCDD-treated mice. MATERIALS AND METHODS Animals. Balb/c mice (male and female) were purchased from either the Jackson Laboratory (Bar Harbor, ME) or ATL (Kent, WA) at 6–8 weeks of age. DO11.10 transgenic mice (originally provided by Dr. Marc Jenkins, University of Minnesota Medical School, Minneapolis, MN) were bred and maintained in the Laboratory Animal Resources Facility, Oregon State University (Corvallis, OR). DO11-lpr mice were generated by crossing DO11.10 mice with Balb/c-lpr/lpr mice (provided by Dr. Jan Erikson, Wistar Institute, Philadelphia, PA), and were bred and maintained in the Laboratory Animal Science Center, Boston University Medical Center (Boston, MA). All mice were housed in front of laminar flow units and were fed standard rodent chow and tap water ad libitum. All mice were maintained according to the National Research Council guidelines. Reagents. All cell culture reagents were purchased from GibcoBRL (Grand Island, NY) except for fetal bovine serum (FBS), which was purchased from Hyclone (Ogden, UT). Phycoerythrin (PE)-anti-CD4 (clone RM4-5), CyChrome (CY)-anti-CD4 (clone H129.19), PE-anti-CD95 (Fas; clone Jo2), PE-KJ1-26 (DO11.10 hybridoma), fluorescein isothiocyanate (FITC)-anti-CD11a (clone M17/4), PE-anti-CD62L (clone MEL-14), PE-anti-CD49d (clone R1-2), CY-anti-CD45 (clone 30-F11), and PE-streptavidin were purchased from BD Biosciences Pharmingen (San Jose, CA). FITC-KJ1-26 and biotinylated KJ1-26 were purchased from Caltag (Burlingame, CA). Red613-streptavidin was purchased from GibcoBRL. ECD-streptavidin was purchased from Immunotech (Marseille, France). TCDD exposure. TCDD (Cambridge Isotope Laboratories, Inc., Woburn, MA) was dissolved in anisole and diluted in peanut oil to 1.5 μg/ml. Balb/c recipient mice were given a single po dose of 15 μg TCDD/kg body weight or a similarly prepared vehicle solution. For the dose-response experiments, both donor DO11.10 and Balb/c recipient mice were given a single po dose of 0, 0.5, 5, or 15 μg TCDD/kg body weight. Adoptive transfer of DO11.10 cells. One day after treatment with TCDD or vehicle, Balb/c recipient mice (sex- and age-matched) were injected intravenously with DO11.10 spleen cells containing 3–5 × 106 CD4+KJ+ T cells as previously described (Shepherd et al., 2000). In each experiment, a single pool of DO11.10 spleen cells was used to inject both vehicle- and TCDD-treated mice, thereby eliminating any experimental variation due to different preparations of antigen-specific CD4+ T cells. For the experiments in which the role of Fas signaling was examined, spleens from both DO11-lpr and DO11.10-wildtype littermate (DO11-wt) mice were collected at Boston University Medical Center and shipped overnight on ice to Oregon State University. The spleens were received the following day and processed into single-cell suspensions. The viability of the spleen cells was similar to freshly collected spleen cells (>90%), based on the exclusion of trypan blue and by flow cytometric analysis of forward and side scatter. Because there is an excessive proliferation of CD4−CD8− T cells in lpr mice beginning at six weeks of age, CD4+ spleen cells were enriched from both DO11-lpr and DO11-wt mice by magnetic cell-sorting prior to adoptive transfer. Spleen cells were labeled with anti-CD4 MicroBeads (Miltenyi Biotech, Auburn, CA) and then sorted using an autoMACS automated magnetic cell-sorter (Miltenyi Biotech) according to the manufacturer's recommendations. The purity of CD4+ T cells was 85–90%, as determined by flow cytometry. Immunization with OVA. Balb/c recipient mice were immunized with 2 mg OVA emulsified in complete Freund's adjuvant by ip injection two days after the adoptive transfer of DO11.10 cells. At various times after immunization the mice were sacrificed and the spleens were collected. In some experiments peripheral blood was also collected. CFSE-labeling of DO11.10 cells. CFSE (Molecular Probes, Eugene, OR) was used to monitor the proliferation of CD4+KJ+ T cells in response to OVA in vivo (Lyons and Parish, 1994). Spleen cells from DO11.10 mice were labeled with 10 μM CFSE at room temperature for 8 mins. After washing, the cells were adoptively transferred into Balb/c recipients as described above. Flow cytometry. Spleens were processed into single-cell suspensions using the frosted ends of glass slides. For most experiments, splenic red blood cells were removed by hypotonic lysis. For studies in which apoptosis was being assessed, the red blood cells were lysed using ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2). Blood samples were collected by heart puncture (500 μl/mouse) using a syringe coated with heparin and stored on ice. All cell preparations were enumerated using a Coulter Counter (Coulter Electronics, Hialeah, FL). Aliquots of spleen or blood cells were washed and resuspended in phosphate buffered saline containing 1.0% bovine serum albumin and 0.1% sodium azide. Binding of the antibodies to Fc receptors was blocked with rat IgG, hamster IgG, or normal mouse serum (Jackson Immunoresearch Labs, Inc., West Grove, PA). The cells were stained with anti-CD4 and KJ antibodies along with antibodies to one or two of the following markers: CD11a, CD49d, CD62L, and Fas. Separate aliquots of the cells were stained with isotype-matched immunoglobulin to determine nonspecific antibody binding. For analysis of peripheral blood lymphocytes (PBL), the lymphocyte population was identified by side scatter and staining with anti-CD45. The majority of the red blood cells were removed after staining using FACS lysing solution (BD Biosciences, San Jose, CA). For some experiments, after surface staining, the cells were stained for apoptosis and viability using the Vybrant apoptosis assay kit from Molecular Probes (Eugene, OR) according to the manufacturer's instructions. The kit contains Alexa Fluor 488-labeled-annexin V, which binds to phosphatidylserine residues in the outer leaflet of the cell membrane, and SytoxGreen, a fluorescent viability stain. Apoptotic cells are then differentiated from dead cells by the intensity of the fluorescence, with dead cells exhibiting higher fluorescence than apoptotic cells. Data were collected on freshly stained cells using a Coulter XL flow cytometer (Coulter Electronics, Hialeah, FL). The total number of events analyzed for each sample was determined by first gating on the live cells and then monitoring the CD4+KJ+ population until 3000–5000 CD4+KJ+ cells had been collected. The data were analyzed using Winlist software (Verity Software House, Topsham, ME). Analysis of cell division history by fluorescence of CFSE. Forward scatter and CFSE-fluorescence associated with the CD4+KJ+ population were displayed in a two-parameter density plot. Electronic regions were drawn to identify each cell division based on the 50% decrease in the mean channel fluorescence of CFSE, which results when the cells divide. The mean number of cell divisions for each animal was calculated by taking the weighted average of the cell divisions: Σ ([percentage of cells within divisionN/100] × N), where N is the number of cell divisions from 0 to 7. Isolation of CD4+KJ+ T cells for gene array analysis. Spleens from vehicle- or TCDD-treated Balb/c recipient mice (n = 6–7/group) that had been previously injected with D011.10 spleen cells were pooled and processed three days after immunization with OVA as described above. T cells were labeled with biotinylated KJ antibody followed by anti-biotin MicroBeads (Miltenyi Biotech) and enriched using an autoMACS automated magnetic cell-sorter according to the manufacturer's recommendations. The enriched KJ+ cells were then labeled with PE-streptavidin and CY-anti-CD4 antibody and sorted on a MoFlo high-performance cell-sorter (DakoCytomation, Fort Collins, CO) to a purity of ≥ 95% CD4+KJ+ T cells. RNA preparation and gene array. Total RNA was isolated from purified CD4+KJ+ T cells using RNeasy Mini columns (Qiagen, Valencia, CA) according to the manufacturer's protocol. The RNA was quantified and checked for purity by measuring the absorbance at 260 nm and 280 nm. In addition, a 1.5% agarose gel was run to ensure little or no DNA contamination. The RNA samples were analyzed using the mouse apoptosis Q series GEArray (SuperArray, Inc., Bethesda, MD) following the manufacturer's protocol. The 96 genes represented on the array are shown in Table 1. Chemiluminescence was measured using a Kodak ImageStation 440CF (Eastman Kodak Company, Rochester, NY). The digital images were then quantitated using ImageQuant software (Molecular Dynamics, Piscataway, NJ). TABLE 1 Genes Represented on the Mouse Apoptosis Q Series Array Gene/domain family   Genes   Bcl-2  Bad, Bak, Bar-like, Bax, Bcl-2, Bcl-w, Bcl-x, Bfl-1,Bid, Bik, Bim, Blk, Bok, Hrk, Mcl-1, Nip3  CARD  Apaf-1, Arc, ASC, Bcl-10, Bcl-2-L-10, Nop30-like  Caspase  caspase 1, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, caspase 11, caspase 12, caspase 14  CIDE  CIDE-A, CIDE-B, DFF40, DFFA  DD/DED  Casper, CRADD, DAPkinase, FADD, MyD88, RIP, Flash  IAP  Bruce, IAP1, IAP2, NAIP1, NAIP2, NAIP5, survivin, XIAP  ATM/p53  ATM, chk1, chk2, gadd45, Hus1, Mdm2, p21, p53, RPA  TNF  4-1BBL, April, CD27L, CD30L, CD40L, DR3L, FasL, HVEM-L, LTβ, OX40-L, TNFα, TNFβ, TRAIL, TRANCE  TNFR  4-1BB, CD27, CD30, CD40, DR3, DR5, DR6, Fas, LTαR, OPG, OX40, RANK, TNFR1, TNFR2  TRAF  TANK, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRIP  Gene/domain family   Genes   Bcl-2  Bad, Bak, Bar-like, Bax, Bcl-2, Bcl-w, Bcl-x, Bfl-1,Bid, Bik, Bim, Blk, Bok, Hrk, Mcl-1, Nip3  CARD  Apaf-1, Arc, ASC, Bcl-10, Bcl-2-L-10, Nop30-like  Caspase  caspase 1, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, caspase 11, caspase 12, caspase 14  CIDE  CIDE-A, CIDE-B, DFF40, DFFA  DD/DED  Casper, CRADD, DAPkinase, FADD, MyD88, RIP, Flash  IAP  Bruce, IAP1, IAP2, NAIP1, NAIP2, NAIP5, survivin, XIAP  ATM/p53  ATM, chk1, chk2, gadd45, Hus1, Mdm2, p21, p53, RPA  TNF  4-1BBL, April, CD27L, CD30L, CD40L, DR3L, FasL, HVEM-L, LTβ, OX40-L, TNFα, TNFβ, TRAIL, TRANCE  TNFR  4-1BB, CD27, CD30, CD40, DR3, DR5, DR6, Fas, LTαR, OPG, OX40, RANK, TNFR1, TNFR2  TRAF  TANK, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRIP  Note. For a full description of each gene, see http://www.ncbi.nlm.nih.gov/omim/ (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University [Baltimore, MD] and National Center for Biotechnology Information, National Library of Medicine [Bethesda, MD], 2000). View Large Statistical analysis. All experiments were independently conducted at least twice. Results from a representative experiment are presented as the mean ± SEM of 3–6 animals/group unless stated otherwise. Analysis of variance modeling was performed using Statview Statistical software (Abacus Concepts, Inc., Berkeley, CA). Comparisons between means were made using Fisher's least significant difference multiple comparison t-test or Student's t-test. Values of p ≤ 0.05 were considered statistically significant. RESULTS Premature Loss of CD4+KJ+ T Cells from TCDD-Treated Mice Is Dose-Dependent Previous studies documented the expansion and decline of CD4+KJ+ spleen cells in vehicle- and TCDD-treated mice on days 1, 3, 5, 7, and 10 after immunization with OVA. In mice treated with an immunosuppressive dose of TCDD (15 μg/kg), normal expansion was observed on day 3 followed by a significant reduction in the number of CD4+KJ+ spleen cells beginning on day 5 and persisting through day 10 (Shepherd et al., 2000). In order to establish the early time-course of the effects of TCDD on CD4+ T cells, adoptively transferred CD4+KJ+ T cells in the spleen were monitored daily on days 2 through 6 following immunization with OVA. As shown in Figure 1A, the percentage and number of CD4+KJ+ T cells in the spleen of vehicle-treated mice increased following immunization with OVA, peaking on day 4 and day 5, respectively. Exposure to TCDD (15 μg/kg) significantly reduced both the percentage and number of CD4+KJ+ T cells in the spleen beginning on day 4 and continuing through day 6. These data confirm and extend the results of Shepherd et al. (2000). FIG. 1. View largeDownload slide TCDD decreases the percentage and number of CD4+KJ+ spleen cells beginning on day 4 after immunization with OVA. (A) Mice were dosed with 15 μg TCDD/kg body weight (filled circles) or vehicle (open circles) three days prior to immunization with OVA. Spleen cells were collected daily beginning on day 2 after immunization with OVA. The spleen cells were then stained with anti-CD4 and KJ antibodies as described in Materials and Methods and analyzed by flow cytometry. Each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. (B) Mice were dosed with 0, 0.5, 5, or 15 μg TCDD/kg body weight and the number of CD4+KJ+ spleen cells was determined on day 5 after immunization with OVA. Each bar represents the mean ± SEM (n = 6/group). Statistically significant differences (p ≤ 0.05) between groups are indicated by different letters above each bar. FIG. 1. View largeDownload slide TCDD decreases the percentage and number of CD4+KJ+ spleen cells beginning on day 4 after immunization with OVA. (A) Mice were dosed with 15 μg TCDD/kg body weight (filled circles) or vehicle (open circles) three days prior to immunization with OVA. Spleen cells were collected daily beginning on day 2 after immunization with OVA. The spleen cells were then stained with anti-CD4 and KJ antibodies as described in Materials and Methods and analyzed by flow cytometry. Each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. (B) Mice were dosed with 0, 0.5, 5, or 15 μg TCDD/kg body weight and the number of CD4+KJ+ spleen cells was determined on day 5 after immunization with OVA. Each bar represents the mean ± SEM (n = 6/group). Statistically significant differences (p ≤ 0.05) between groups are indicated by different letters above each bar. To determine if the depletion of CD4+KJ+ spleen cells was dose-dependent, mice were treated with 0, 0.5, 5, or 15 μg TCDD/kg body weight and the number of CD4+KJ+ T cells in the spleen was determined on day 5. As shown in Figure 1B, the number of CD4+KJ+ spleen cells decreased in a dose-dependent manner and was statistically significant at doses of TCDD ≥5 μg/kg. A 15 μg/kg dose of TCDD was used in all subsequent experiments. TCDD Influences the Number of CD4+KJ+ T Cells in the Blood Activated T cells can be found circulating in the blood following exposure to antigen. To determine if the decline in splenic CD4+KJ+ T cells induced by TCDD reflected a systemic effect, blood samples were taken from vehicle- and TCDD-treated mice on days 2 through 5 postimmunization and examined for the presence of CD4+KJ+ T cells. As shown in Figure 2, the percentage and number of CD4+KJ+ T cells in the blood increased in vehicle-treated mice between days 2 and 3, followed by a modest decline on days 4 and 5. In TCDD-treated mice, a similar pattern was observed, however both the increase and decrease of CD4+KJ+ T cells were more pronounced. In fact, the total number of CD4+KJ+ T cells in the blood on day 3 was 36% higher in TCDD-treated mice while on day 4 there were 39% fewer CD4+KJ+ T cells in the blood (Fig. 2). The significant increase in CD4+KJ+ T cells in the blood of TCDD-treated mice on day 3 was an unexpected observation. However, similar results have been observed in two additional experiments. FIG. 2. View largeDownload slide TCDD increases, and then decreases, the percentage and number of CD4+KJ+ T cells in the blood after immunization with OVA. Mice were dosed with TCDD (filled circles) or vehicle (open circles) as described in Figure 1A. Blood lymphocytes were stained on days 2–5 as described in the Materials and Methods. Each data point is the mean ± SEM of five mice. *p ≤ 0.05 compared to vehicle on the same day. FIG. 2. View largeDownload slide TCDD increases, and then decreases, the percentage and number of CD4+KJ+ T cells in the blood after immunization with OVA. Mice were dosed with TCDD (filled circles) or vehicle (open circles) as described in Figure 1A. Blood lymphocytes were stained on days 2–5 as described in the Materials and Methods. Each data point is the mean ± SEM of five mice. *p ≤ 0.05 compared to vehicle on the same day. TCDD Selectively Alters the Expression of Activation Molecules on CD4+KJ+ T Cells The activation of T cells is characterized by changes in the expression of numerous co-stimulatory and adhesion molecules on the cell surface. We examined the kinetics of expression of three important activation molecules, CD62L, CD11a, and CD49d, on CD4+KJ+ T cells in the spleen after OVA injection. We also examined expression of CD11a and CD49d on CD4+KJ+ T cells in the blood of the same animals (CD62L expression was not detected on CD4+KJ+ or CD4+KJ− cells in the blood, which was likely related to technical problems). CD62L is a surface protein that is expressed at a high level on naïve T cells and is gradually shed from the surface of activated T cells as they divide. As shown in Figure 3A, CD62L expression was reduced on a majority of the antigen-specific CD4+KJ+ T cells in the spleen of vehicle-treated mice on day 3 after OVA injection when compared to CD4+KJ− T cells. Over time, the percentage of CD4+KJ+ T cells that had fully down-regulated CD62L expression increased from approximately 50% on day 3 to 70% on day 6 in vehicle-treated mice while no change was seen in the CD4+KJ− T cells. Interestingly, the percentage of CD4+KJ+ T cells that had fully down-regulated CD62L was significantly increased by TCDD exposure and was already at 72% on day 3. The higher percentage of CD62Lneg cells in TCDD-treated mice was maintained through day 6 despite the fact that the total number of CD4+KJ+ T cells had significantly decreased during this time. TCDD did not alter the percentage of CD4+KJ− T cells that were CD62Lneg over the same time period (Fig. 3B), indicating that the influence of TCDD on CD62L expression was related to antigenic activation. These data suggest that more CD4+KJ+ T cells are being activated earlier in TCDD-treated mice. FIG. 3. View largeDownload slide TCDD enhances the down-regulation of CD62L on CD4+KJ+ spleen cells but not on CD4+KJ− T cells. Mice were dosed with TCDD (solid line and filled circles) or vehicle (dotted line and open circles) as described in Figure 1A. The expression of CD62L on CD4+KJ+ T cells (A) or CD4+KJ− T cells (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (left-hand panels). The bars on the histograms indicate the region used to identify the CD62Lneg population. The expression of CD62L was also measured on days 2–6 (right-hand panels; same animals as in Figure 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. FIG. 3. View largeDownload slide TCDD enhances the down-regulation of CD62L on CD4+KJ+ spleen cells but not on CD4+KJ− T cells. Mice were dosed with TCDD (solid line and filled circles) or vehicle (dotted line and open circles) as described in Figure 1A. The expression of CD62L on CD4+KJ+ T cells (A) or CD4+KJ− T cells (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (left-hand panels). The bars on the histograms indicate the region used to identify the CD62Lneg population. The expression of CD62L was also measured on days 2–6 (right-hand panels; same animals as in Figure 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. Increased expression of CD11a on the cell surface is another characteristic of T-cell activation. As shown in Figure 4A, the mean channel fluorescence (MCF) of CD11a was already increased on CD4+KJ+ T cells in the spleen of vehicle-treated mice on day 2 compared to CD4+KJ− T cells, and it remained elevated through day 5. Unexpectedly, exposure to TCDD appeared to prevent the up-regulation of CD11a on CD4+KJ+ T cells in the spleen and expression remained at the level found on CD4+KJ− T cells throughout the time-course (Fig. 4A). The expression of CD11a on CD4+KJ− spleen cells increased slightly over time, probably as a result of bystander activation via cytokines. This antigen-nonspecific increase in CD11a expression was also significantly decreased in TCDD-treated mice on days 5 and 6 of the response (Fig. 4A). FIG. 4. View largeDownload slide The OVA-induced up-regulation of CD11a on CD4+KJ+ T cells in the spleen and blood is blocked by TCDD. Mice were dosed with TCDD or vehicle as described in Figure 1A. The expression of CD11a on CD4+KJ+ T cells or CD4+KJ− T cells in the spleen (A) or blood (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (upper panels), where vehicle = dotted line, TCDD = solid line. Isotype staining was on the baseline. In the lower panels, the time course of the expression of CD11a is shown (vehicle = open circle, TCDD = filled circle; same animals as in Fig. 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. FIG. 4. View largeDownload slide The OVA-induced up-regulation of CD11a on CD4+KJ+ T cells in the spleen and blood is blocked by TCDD. Mice were dosed with TCDD or vehicle as described in Figure 1A. The expression of CD11a on CD4+KJ+ T cells or CD4+KJ− T cells in the spleen (A) or blood (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (upper panels), where vehicle = dotted line, TCDD = solid line. Isotype staining was on the baseline. In the lower panels, the time course of the expression of CD11a is shown (vehicle = open circle, TCDD = filled circle; same animals as in Fig. 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. CD11a expression on CD4+KJ+ T cells in the blood is shown in Figure 4B. In vehicle-treated mice, CD11a expression was increased on days 3 and 4 after immunization with OVA and then returned to the baseline level expressed on CD4+KJ− T cells. As in the spleen, no up-regulation of CD11a on the CD4+KJ+ T cells in the blood of TCDD-treated mice was observed throughout the time-course and expression levels remained similar to the level expressed on CD4+KJ− T cells (Fig. 4B). The expression of CD49d on CD4+KJ+ T cells in both the spleen (Fig. 5A) and blood (Fig. 5B) was elevated on all days examined when compared to the level expressed by CD4+KJ− T cells. In contrast to the other activation molecules, TCDD had no effect on the expression of CD49d on CD4+KJ+ or CD4+KJ− T cells (Fig. 5). FIG. 5. View largeDownload slide TCDD does not affect the expression of CD49d on CD4+KJ+ T cells in the spleen or blood. Mice were dosed with TCDD or vehicle as described in Figure 1A. The expression of CD49d on CD4+KJ+ T cells or CD4+KJ− T cells in the spleen (A) or blood (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (upper panels), where vehicle = dotted line, and TCDD = solid line. Isotype staining was on the baseline. In the lower panels, the time course of the expression of CD49d is shown (vehicle = open circle, TCDD = filled circle; same animals as in Figure 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. FIG. 5. View largeDownload slide TCDD does not affect the expression of CD49d on CD4+KJ+ T cells in the spleen or blood. Mice were dosed with TCDD or vehicle as described in Figure 1A. The expression of CD49d on CD4+KJ+ T cells or CD4+KJ− T cells in the spleen (A) or blood (B) was determined by flow cytometry as described in the Materials and Methods. Representative histograms from day 3 are shown (upper panels), where vehicle = dotted line, and TCDD = solid line. Isotype staining was on the baseline. In the lower panels, the time course of the expression of CD49d is shown (vehicle = open circle, TCDD = filled circle; same animals as in Figure 1A), where each data point is the mean ± SEM of four mice, except the vehicle group on day 2 and the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. Taken together, the enhanced down-regulation of CD62L expression and the lack of up-regulation of CD11a expression on CD4+KJ+ T cells in TCDD-treated mice suggest that TCDD is selectively altering early activation events in CD4+ T cells. TCDD Prematurely Terminates the Proliferation of CD4+KJ+ T Cells Sufficiently prolonged clonal expansion of the antigen-specific CD4+ T-cell population is necessary for the generation of an adaptive immune response. The systemic decline in the antigen-specific CD4+KJ+ T-cell population between day 3 and day 4 in TCDD-treated mice suggested that exposure to TCDD may be inducing premature termination of clonal expansion. To test this hypothesis, D011.10 T cells were labeled with CFSE prior to adoptive transfer into Balb/c mice, and spleen cells were collected on days 3 and 4 after immunization with OVA for analysis of cell division history. As shown in Figure 6, naïve CD4+KJ+ T cells from nonimmunized mice did not divide, whereas nearly all of the CD4+KJ+ T cells from both TCDD- and vehicle-treated mice immunized with OVA had divided one or more times by day 3. By day 4 the CD4+KJ+ spleen cells from vehicle-treated mice had divided further, as indicated by the additional decrease in the fluorescence intensity of CFSE compared to day 3. In contrast, in TCDD-treated mice the CFSE fluorescence distribution on day 4 was similar to day 3, suggesting that no additional cell division had occurred. When the mean number of divisions for the CD4+KJ+ spleen cells was calculated (Fig. 6B), the CD4+KJ+ cells from both vehicle- and TCDD-treated mice had undergone an average of four divisions by day 3. By day 4, the average number of cell divisions had increased to five for vehicle-treated mice but was unchanged in TCDD-treated mice. These results suggest that premature termination of cell division may contribute to the reduced number of activated CD4+KJ+ T cells in the spleen of TCDD-treated mice. FIG. 6. View largeDownload slide TCDD inhibits the continued cycling of CD4+KJ+ spleen cells after day 3. Mice were dosed with TCDD or vehicle as described in Figure 1A. Spleen cells from DO11.10 mice were labeled with CFSE prior to adoptive transfer into Balb/c mice. On days 3 and 4 postimmunization, spleen cells were stained with anti-CD4 and KJ antibodies as described in the Materials and Methods. (A) Representative density plots of naïve, vehicle-, or TCDD-treated CFSE-labeled CD4+KJ+ spleen cells from day 3 and day 4 are shown. Numbers above the histograms indicate the number of division cycles. (B) The mean number of cell divisions was calculated on day 3 and day 4 for vehicle- (open bars) and TCDD-treated (filled bars) mice as described in the Materials and Methods. *p ≤ 0.05 compared to all other groups (n = 4 mice/group/day). FIG. 6. View largeDownload slide TCDD inhibits the continued cycling of CD4+KJ+ spleen cells after day 3. Mice were dosed with TCDD or vehicle as described in Figure 1A. Spleen cells from DO11.10 mice were labeled with CFSE prior to adoptive transfer into Balb/c mice. On days 3 and 4 postimmunization, spleen cells were stained with anti-CD4 and KJ antibodies as described in the Materials and Methods. (A) Representative density plots of naïve, vehicle-, or TCDD-treated CFSE-labeled CD4+KJ+ spleen cells from day 3 and day 4 are shown. Numbers above the histograms indicate the number of division cycles. (B) The mean number of cell divisions was calculated on day 3 and day 4 for vehicle- (open bars) and TCDD-treated (filled bars) mice as described in the Materials and Methods. *p ≤ 0.05 compared to all other groups (n = 4 mice/group/day). TCDD Decreases the Survival of CD4+KJ+ T Cells The decline in the number of CD4+KJ+ T cells in TCDD-treated mice could also be due to enhanced cell death. In order to address this possibility, the percentage of cells expressing an apoptotic or dead phenotype was determined using annexin V and SytoxGreen, respectively. Figure 7A shows the typical staining pattern of annexin V and SytoxGreen on spleen cells. Annexin V+ cells typically fall within the third log-decade, while the SytoxGreen+ cells can be found in the fourth log-decade. As shown in Figure 7C, the percentage of vehicle-treated CD4+KJ+ T cells expressing an apoptotic phenotype appeared to increase slightly over time but remained within the same low range as seen in the CD4+KJ− T cells. Treatment with TCDD significantly increased the percentage of apoptotic CD4+KJ+ T cells on days 5 and 6 after immunization with OVA, without affecting the CD4+KJ− T cells. Interestingly, in this study, annexin V+ cells were detected after gating on the CD4+KJ+ T cells but few, if any SytoxGreen+ cells were seen (Fig. 7B). This contrasts with the results of a previous study using 7-amino actinomycin D (7-AAD) as a viability stain, wherein TCDD significantly increased the percentage of CD4+KJ+ T cells that stained with both annexin V and 7-AAD rather than annexin V alone (data not shown). The reason for these different results is not known. In either case, increased apoptosis or death was not apparent until days 5 and 6, suggesting that the major decline in T cells between day 3 and day 4 may be precipitated by a cause other than enhanced cell death. FIG. 7. View largeDownload slide TCDD exposure increases the percentage of CD4+KJ+ spleen cells with an apoptotic phenotype. Mice were treated with TCDD or vehicle as described in Figure 1A. The percentage of CD4+KJ+ T cells or CD4+KJ− T cells displaying an apoptotic phenotype and their viability was determined by flow cytometry as described in the Materials and Methods. (A) The typical staining pattern for annexin V and SytoxGreen on ungated spleen cells is shown. (B) Representative histograms from day 5 are shown, where vehicle = dotted line and TCDD = solid line. The bars on the histograms indicate the region used to identify the AnnexinV+SytoxGreen+ population. (C) The apoptotic phenotype of CD4+KJ+ T cells over time is shown (vehicle = open circles, TCDD = filled circles; same animals as in Fig. 1A), where each data point is the mean ± SEM of four mice, except the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. FIG. 7. View largeDownload slide TCDD exposure increases the percentage of CD4+KJ+ spleen cells with an apoptotic phenotype. Mice were treated with TCDD or vehicle as described in Figure 1A. The percentage of CD4+KJ+ T cells or CD4+KJ− T cells displaying an apoptotic phenotype and their viability was determined by flow cytometry as described in the Materials and Methods. (A) The typical staining pattern for annexin V and SytoxGreen on ungated spleen cells is shown. (B) Representative histograms from day 5 are shown, where vehicle = dotted line and TCDD = solid line. The bars on the histograms indicate the region used to identify the AnnexinV+SytoxGreen+ population. (C) The apoptotic phenotype of CD4+KJ+ T cells over time is shown (vehicle = open circles, TCDD = filled circles; same animals as in Fig. 1A), where each data point is the mean ± SEM of four mice, except the TCDD group on day 4, where n = 3. *p ≤ 0.05 compared to vehicle on the same day. Fas Is Not Required for the Loss of CD4+KJ+ T Cells Previously published studies have implicated Fas/FasL (CD95/CD178) in the effects of TCDD on T cells (Camacho et al., 2001, 2002; Dearstyne and Kerkvliet, 2002). To determine if Fas signaling in the T cell was necessary for the decreased number of CD4+KJ+ T cells, splenocytes from DO11-lpr mice, which lack functional Fas, were used for adoptive transfer. As shown in Figure 8, the expansion of CD4+KJ+ T cells from DO11-lpr mice and DO11-wt mice was similar in vehicle-treated mice through day 5 following immunization with OVA. Treatment with TCDD induced the same level of depletion of DO11-lpr and DO11-wt CD4+KJ+ spleen cells on day 5 (56 and 57% decrease, respectively, Fig. 8), suggesting that Fas signaling in the T cells was not required for the decreased number of CD4+KJ+ T cells. FIG. 8. View largeDownload slide Fas expression on CD4+KJ+ T cells is not required for the deletion of CD4+KJ+ cells in the spleen induced by TCDD. Mice were dosed with TCDD (filled circles) or vehicle (open circles) as described in Figure 1A. Spleen cells from DO11-lpr or DO11-wt mice were adoptively transferred as described in the Materials and Methods. The number of CD4+KJ+ spleen cells was determined by flow cytometry as described in the Materials and Methods. Each data point is the mean ± SEM of five mice. *p ≤ 0.05 compared to vehicle on the same day. FIG. 8. View largeDownload slide Fas expression on CD4+KJ+ T cells is not required for the deletion of CD4+KJ+ cells in the spleen induced by TCDD. Mice were dosed with TCDD (filled circles) or vehicle (open circles) as described in Figure 1A. Spleen cells from DO11-lpr or DO11-wt mice were adoptively transferred as described in the Materials and Methods. The number of CD4+KJ+ spleen cells was determined by flow cytometry as described in the Materials and Methods. Each data point is the mean ± SEM of five mice. *p ≤ 0.05 compared to vehicle on the same day. To further address a possible effect of TCDD on Fas signaling, the expression of Fas on CD4+KJ+ T cells in the spleen was assessed. As shown in Figure 9, the median channel fluorescence (MedianCF) of Fas was increased on CD4+KJ+ T cells as early as one day after immunization with OVA, and continued to increase through day 4 in vehicle-treated mice. Interestingly, the expression of Fas was increased to a lesser degree in mice treated with TCDD, starting on day 3 (Fig. 9), and this reduction was evident through day 10 (data not shown). Minimal changes in the surface expression of Fas were seen on CD4+KJ− T cells following immunization with OVA and TCDD had no effect on the expression of Fas on these cells (Fig. 9). Taken together, these data support the conclusion that Fas signaling in the T cell is not involved in the loss of the CD4+KJ+ T cells in mice treated with TCDD. FIG. 9. View largeDownload slide TCDD decreases the expression of Fas on CD4+KJ+ spleen cells. The expression of Fas was determined by flow cytometry on the CD4+KJ+ T cells and CD4+KJ− T cells from mice treated with vehicle (open circles) or TCDD (filled circles) as described in the Materials and Methods and Figure 1A. Representative histograms from day 4 are shown. Each data point is the mean ± SEM of three mice. *p ≤ 0.05 compared to vehicle on the same day. FIG. 9. View largeDownload slide TCDD decreases the expression of Fas on CD4+KJ+ spleen cells. The expression of Fas was determined by flow cytometry on the CD4+KJ+ T cells and CD4+KJ− T cells from mice treated with vehicle (open circles) or TCDD (filled circles) as described in the Materials and Methods and Figure 1A. Representative histograms from day 4 are shown. Each data point is the mean ± SEM of three mice. *p ≤ 0.05 compared to vehicle on the same day. TCDD Alters the Expression Profile of Genes Involved in Cell Death and Survival Because Fas did not appear to play a significant role in the depletion of the CD4+KJ+ T cells in the spleen, a gene array was used to determine if TCDD influenced other pathways of cell survival (Table 1). Day 3 postimmunization was selected for analysis based on the rationale that changes on day 3 would best predict the precipitating cause of the decline in the number of CD4+KJ+ T cells that occurs during the next 24 h. In addition, analysis on day 3 avoided the complication of T-cell depletion having already occurred in TCDD-treated mice. Two independent experiments were conducted using CD4+KJ+ T cells purified from the spleens of 6–7 vehicle- or TCDD-treated mice. For each experiment, the expression of each gene was calculated as a percentage of the housekeeping gene, GAPDH. The results of the two experiments were then averaged (n = 2). As shown in Figure 10, the expression of 21 genes was increased at least two-fold in the CD4+KJ+ T cells from the spleens of TCDD-treated mice. 4–1BBL was not detected in the vehicle-treated samples so the expression relative to GAPDH was zero. Of the 21 genes shown in Figure 10, all but four (caspase 1, caspase 8, cide b, and rpa) had nonoverlapping standard deviations (SD). The remaining 75 genes were unaffected by TCDD or were increased in one experiment and decreased in the other. FIG. 10. View largeDownload slide Apoptosis- and survival-related genes are differentially expressed in purified CD4+KJ+ spleen cells from TCDD- (filled bars) and vehicle-treated (open bars) mice on day 3 postimmunization. Mice were dosed with TCDD or vehicle as described in Figure 1A. The array was prepared using RNA from purified CD4+KJ+ T cells pooled from 6–7 mice as described in the Materials and Methods. Data shown represent the mean mRNA expression (± SD) as a percentage of the housekeeping gene, GAPDH, from two independent experiments. FIG. 10. View largeDownload slide Apoptosis- and survival-related genes are differentially expressed in purified CD4+KJ+ spleen cells from TCDD- (filled bars) and vehicle-treated (open bars) mice on day 3 postimmunization. Mice were dosed with TCDD or vehicle as described in Figure 1A. The array was prepared using RNA from purified CD4+KJ+ T cells pooled from 6–7 mice as described in the Materials and Methods. Data shown represent the mean mRNA expression (± SD) as a percentage of the housekeeping gene, GAPDH, from two independent experiments. DISCUSSION TCDD is a highly potent immunosuppressive chemical that impairs adaptive immune responses by mechanisms that are not understood. Although engagement of the AhR in both CD4+ and CD8+ T cells was recently shown to be required for suppression of the CD4+-dependent CD8+-mediated CTL response in a graft-vs.-host model (Kerkvliet et al., 2002), the cellular changes that underlie altered T-cell responsiveness have not been defined. Previous studies using the DO11.10 adoptive transfer model have shown that the antigen-specific CD4+KJ+ T cells expand normally in TCDD-treated mice following immunization with OVA but are prematurely lost from the spleen (Shepherd et al., 2000). In the present studies, we have tracked the antigen-specific response of adoptively transferred CD4+KJ+ T cells in the spleen and blood of vehicle- and TCDD-treated mice in order to identify changes in the T cells that precede the loss of activated CD4+KJ+ T cells. One of the earliest changes induced by TCDD was a block in the up-regulation of CD11a, which could have several ramifications in terms of suppressed immune responses. CD11a is a β2 integrin that is expressed on activated T cells and is critical for maintaining contact between T cells and antigen presenting cells, thus allowing the T cells to become fully activated (Bachmann et al., 1997; Bleijs et al., 2000; Cai et al., 1997). A failure to sufficiently up-regulate CD11a in TCDD-treated mice could result in a reduced ability to maintain contact with dendritic cells (DC) leading to a loss of adequate co-stimulation or sufficiently prolonged survival signals. The loss of these DC-derived signals could result in the passive death of the activated T cells. It is important to note that basal expression of CD11a was not reduced by TCDD. Thus, the initial interaction between the DC and T cells did not appear to be impeded, consistent with the seemingly normal OVA-induced proliferation of the CD4+KJ+ T cells in the first three days of the response. As in the spleen, CD11a expression was elevated on CD4+KJ+ T cells in the blood of vehicle-treated mice on day 3 after immunization and this up-regulation was similarly blocked in TCDD-treated mice. In studies where CD11a was blocked with monoclonal antibodies, the localization of T cells at the site of antigen was impaired (Hamann et al., 2000; Issekutz, 1993), suggesting that CD11a is involved in the emigration of activated T cells from the blood into sites of inflammation. The lack of increased CD11a expression on CD4+KJ+ T cells in the blood of TCDD-treated mice could impede the emigration of the cells out of the blood and into the tissues, resulting in increased numbers of CD4+KJ+ T cells in the blood. This would be consistent with the finding of significantly more CD4+KJ+ T cells in the blood of TCDD-treated mice on day 3 after immunization. However, it is also possible that the unimpeded up-regulation of CD49d, the ligand for endothelial VCAM-1, on T cells from TCDD-treated mice was sufficient for extravasation. Further studies are necessary to resolve the fate of activated T cells in the blood of TCDD-treated mice. The possibility that TCDD exposure enhanced the early activation of CD4+KJ+ T cells was suggested by the increased percentage of T cells that down-regulated expression of CD62L. CD62L is a selectin that is involved in the homing of naïve T cells to the lymph nodes (Arbones et al., 1994; Bradley et al., 1994). During activation, CD62L expression decreases as a result of both altered transcriptional rates and proteolytic cleavage from the T-cell surface, thus preventing activated T cells from homing to the lymph nodes (Borland et al., 1999; Preece et al., 1996). Following immunization with OVA, CD62L expression on CD4+KJ+ T cells was decreased on the majority of cells from both vehicle- and TCDD-treated mice. However, the degree of down-regulation was significantly greater on cells from TCDD-treated mice on all days examined except day 2 with a higher percentage showing a complete loss of CD62L expression. Data from day 2 were difficult to interpret owing to the transient down-regulation of CD62L on the CD4+KJ− cells as well as the CD4+KJ+ cells. This likely reflected a transient nonspecific response to the Freund's adjuvant used in the immunization. The down-regulation of CD62L has been shown to occur progressively during the initial rounds of T cell division following exposure to antigen (Gudmundsdottir et al., 1999). Since TCDD promoted CD62L down-regulation, it suggests that TCDD was promoting CD4+KJ+ T-cell division. However, cell division history was similar in CFSE-labeled CD4+KJ+ T cells from vehicle and TCDD-treated mice on day 3, suggesting that CD62L expression may be uncoupled from cell division in TCDD-treated mice. In other studies in the laboratory, we have looked at the association between CD62L expression and cell division using an acute graft-vs.-host model of T-cell function. We found that CD62L expression on CD4+ T cells from TCDD-treated mice was, in fact, correlated with cell division but the degree of decrease in expression was greater with each cell division (unpublished observations). This enhanced down-regulation resulted in no expression of CD62L on the CD4+ T cells from TCDD-treated mice after division 4 whereas T cells from vehicle-treated mice still expressed low levels of CD62L (unpublished observations). The mechanism underlying this effect of TCDD is not currently known. Another possible mechanism for the loss of T cells in TCDD-treated mice is premature termination of T-cell proliferation. Consistent with this hypothesis, the proliferation of activated CD4+KJ+ T cells from TCDD-treated mice appeared to terminate on day 3, whereas cells from vehicle-treated mice continued to divide through day 4. Similar to our results, studies by Mitchell and Lawrence (2003) showed that treatment with TCDD did not inhibit the proliferation of influenza virus-specific CD8+ T cells early in the response but significantly reduced the proliferation after day 5. Since the early cycling of activated T cells is not reduced by TCDD, the delay in cell cycle arrest could be dependent on the induction of proteins regulated by the AhR. TCDD has been shown to alter the expression of several cell cycle regulators, including p27kip1 and retinoblastoma protein, in other cell types (Ge and Elferink, 1998; Kolluri et al., 1999; Puga et al., 2000, 2002; Rininger et al., 1997). However, it should be noted that interpretation of the cell cycling data is complicated by the fact that the CD4+KJ+ T-cell population analyzed on day 4 was already decreased in TCDD-treated mice. Thus, it is possible that TCDD selectively depleted the cells that had divided four or more times. Further studies will be necessary to determine if TCDD induces cell-cycle arrest in antigen-specific T cells and the mechanism underlying this effect. The finding that CD4+KJ+ T cells from TCDD-treated mice exhibited increased annexin V staining on day 5 and 6 post-immunization suggests that T-cell survival was also impaired by TCDD. Reduced T-cell survival could result from ligation of death receptors or reduction in survival signals. Many of the survival signals for CD4+ T cells are derived from activated DC that express ligands for co-stimulatory and cytokine receptors on the T cells. Based on previous studies showing that TCDD exposure reduces the number of DC in the spleen (Vorderstrasse and Kerkvliet, 2001), it is possible that the loss of DC plays a role in decreased T-cell survival. It has also been reported that TCDD induces apoptosis of activated T cells by a process involving Fas signaling (Camacho et al., 2001, 2002). In these reports, lymph node T cells were obtained from mice that had been injected in the footpad with anti-CD3 antibody or Staphylococcal enterotoxin A (SEA) along with Freund's adjuvant. Apoptosis was evaluated by terminal dUTP nick-end labeling (TUNEL), and was apparent only after culturing the lymph node cells in vitro for 24 h. To determine if Fas signaling contributed to the loss of antigen-specific CD4+KJ+ T cells following OVA injection, CD4+KJ+ T cells from DO11.10 mice that had been backcrossed to Fas-deficient Balb/c-lpr/lpr mice were used for adoptive transfer. The results of this study showed that TCDD caused the depletion of both Fas-deficient and Fas-expressing CD4+KJ+ T cells in a similar manner, indicating that depletion was independent of Fas signaling. This conclusion was supported by the finding that TCDD decreased, rather than increased, cell surface expression of Fas on wildtype CD4+KJ+ T cells beginning on day 3 post-immunization. The apparent discrepancy in the role of Fas in T-cell depletion mediated by TCDD in the D011.10 model compared to previous studies may be due to differences in how the T cells were activated, or to possible confounding effects of the lymphoproliferation that occurs with the lpr mutation. These mice undergo uncontrolled proliferation of CD4−CD8− T cells that could confound the effects of TCDD by disturbing the normal frequency and number of single-positive CD4+ and CD8+ T cells in the secondary lymphoid organs. In our study, by adoptively transferring only the CD4+ T cells from the DO11-lpr mice into normal Balb/c mice, any confounding effect of the CD4−CD8− T cells was avoided. Since Fas expression on the T cells did not appear to be involved in the loss of CD4+KJ+ T cells from the spleens of TCDD-treated mice, we used a gene array approach to determine if TCDD changed the expression pattern of other apoptosis- or survival-related genes in CD4+KJ+ T cells. The majority of the 21 genes that increased in expression in the CD4+KJ+ T cells from TCDD-exposed mice were pro-apoptotic, although no members of the Bcl-2 family were affected by TCDD. Several members of the TNF/TNF receptor superfamilies, such as TNFα, TNFR1, TNFR2, TRAIL, and FasL were increased, which agreed with previous studies that analyzed mRNA preparations from whole spleens (Zeytun et al., 2002). The mRNA level of Fas on day 3 was not affected by exposure to TCDD, which is consistent with the minimal change in the expression of Fas on the cell surface, as measured by flow cytometry. Although TNF receptor expression was altered, TNF-signaling did not appear to be involved in the TCDD-dependent depletion of T cells from anti-CD3 treated mice (Dearstyne and Kerkvliet, 2002). Several genes whose products are involved in the signal transduction of apoptosis were also increased, including caspases 1 and 8, as well as genes whose products are involved in cell cycle arrest, such as DAP kinase, Hus 1, CHK 2, RPA 3, and p53. CIDE-B and DFF40, whose gene products are responsible for DNA fragmentation, were also increased in the CD4+KJ+ T cells from TCDD-treated mice. Paradoxically, there were also increases in several genes generally associated with increased survival of activated T cells. These pro-survival genes were all members of the TNF/TNF receptor superfamilies: TRANCE, CD27L, 4-1BB, 4-1BBL, CD30, and CD30L. The up-regulation of these pro-survival genes could reflect an attempt by the CD4+KJ+ T cells to enhance their interaction with dendritic cells. The results of the gene array experiments need to be confirmed at the level of protein expression before specific conclusions can be made about the importance of any changes in gene expression, particularly because many of these gene products are not regulated at the level of transcription. However, it is interesting to note that analysis of the same RNA samples in a separate array showed CYP1A1 expression in CD4+KJ+ T cells from TCDD-treated but not vehicle-treated mice (data not shown), demonstrating direct effects of TCDD on the CD4+KJ+ T cells. In summary, the studies presented here addressed several questions about the fate of antigen-specific T cells responding to antigen in the presence of TCDD. Figure 11 summarizes a potential pathway for the effects of TCDD on activated antigen-specific T cells and illustrates the hypothesis that TCDD-dependent changes in activation early during the response lead to premature loss of adhesion between the antigen-specific T cells and DC, which in turn leads to a loss of survival signals for both cells, and contracted proliferation and increased death of activated T cells. The specific lack of CD11a up-regulation on activated T cells in the blood could also result in decreased emigration of T helper cells into sites of antigen deposition. Current studies are aimed at identifying the specific cascade of events that is disrupted by TCDD during T-cell activation that lead to altered activation and survival of antigen-specific T cells. FIG. 11. View largeDownload slide A possible model for the effects of TCDD on the activation and survival of antigen-specific T cells. Exposure to TCDD enhances the activation of T cells and DC following exposure to antigen. Subsequently, the expression of several activation markers is changed, possibly resulting in a premature loss of adhesion between the T cell and DC, a premature cessation of proliferation, and increased cell death. Ultimately, the effects of TCDD onT-cell proliferation and survival both contribute to the reduced number of activated antigen-specific T cells in the spleen. *A significant decrease in the number of splenic DC has been shown previously (Vorderstrasse and Kerkvliet, 2001), and we have also observed a TCDD-dependent increase in the death of DC in vitro (manuscript in preparation). FIG. 11. View largeDownload slide A possible model for the effects of TCDD on the activation and survival of antigen-specific T cells. Exposure to TCDD enhances the activation of T cells and DC following exposure to antigen. Subsequently, the expression of several activation markers is changed, possibly resulting in a premature loss of adhesion between the T cell and DC, a premature cessation of proliferation, and increased cell death. Ultimately, the effects of TCDD onT-cell proliferation and survival both contribute to the reduced number of activated antigen-specific T cells in the spleen. *A significant decrease in the number of splenic DC has been shown previously (Vorderstrasse and Kerkvliet, 2001), and we have also observed a TCDD-dependent increase in the death of DC in vitro (manuscript in preparation). 1 Present address: Center for Environmental Health Sciences, Department of Biomedical & Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812 We thank Julie Oughton for technical support and data analysis, as well as critical review of this manuscript. 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TI - Early Consequences of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure on the Activation and Survival of Antigen-Specific T Cells
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfh245
DA - 2004-08-30
UR - https://www.deepdyve.com/lp/oxford-university-press/early-consequences-of-2-3-7-8-tetrachlorodibenzo-p-dioxin-exposure-on-LJyyszuFDu
SP - 129
EP - 142
VL - 82
IS - 1
DP - DeepDyve
ER -