Linda B. Steppan

Cutting Edge: Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo- p -dioxin Generates a Population of CD4 + CD25 + Cells with Characteristics of Regulatory T Cells

Activation of the aryl hydrocarbon receptor (AhR) by its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to immune suppression in mice. Although the underlying mechanisms responsible for AhR-mediated immune suppression are not known, previous studies have shown that activation of the AhR must occur within the first 3 days of an immune response and that CD4+ T cells are primary targets. Using the B6-into-B6D2F1 model of an acute graft-vs-host response, we show that activation of AhR in donor T cells leads to the generation of a subpopulation of CD4+ T cells that expresses high levels of CD25, along with CD62Llow, CTLA-4, and glucocorticoid-induced TNFR. These donor-derived CD4+CD25+ cells also display functional characteristics of regulatory T cells in vitro. These findings suggest a novel role for AhR in the induction of regulatory T cells and provide a new perspective on the mechanisms that underlie the profound immune suppression induced by exposure to TCDD.

Influence of the Ah locus on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: Evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppression☆

There are conflicting reports in the literature regarding the role of the Ah locus in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity. The present studies have utilized two congenic strains of C57Bl/6 mice that differ only at this locus to assess its influence on TCDD-induced suppression of antibody responses. Mice were given a single oral dose of TCDD 2 days prior to challenge with sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). The subsequent dose-dependent effects of TCDD on the amount of antibody produced by splenic plasma cells were measured using the hemolytic antibody isotope release assay. In addition, the relative importance of the Ah genotype of lymphoid versus nonlymphoid tissue was examined in adoptive transfer experiments. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced in Ahbb mice by a dose of 0.5 micrograms/kg TCDD and maximally induced by a dose of 2 micrograms/kg. Ahdd mice required 10-fold higher doses of TCDD to induce comparable levels of AHH. The degree of thymic involution and liver hypertrophy induced by TCDD was also influenced by the Ah genotype of the animals. Both Ahbb and Ahdd mice exhibited dose-dependent suppression of the anti-TNP response following TCDD exposure. The ID50 was 7.0 micrograms/kg in Ahbb mice and 30.8 micrograms/kg in Ahdd mice. Suppression of the antibody response to SRBC was also dependent on the Ah locus. The ID50 in Ahbb mice was 0.6 micrograms/kg TCDD. However, an apparent biphasic dose response for suppression of the anti-SRBC response in Ahdd mice suggested the involvement of an Ah-independent component of suppression as well. In adoptive transfer studies, lymphocytes were identified as an Ah-dependent component of the response. The Ah-independent component of the response was not identified, and could be either lymphoid or nonlymphoid in nature. The possibility that T helper cells represent the Ah-independent component is discussed.

Functional Characterization and Gene Expression Analysis of CD4+CD25+ Regulatory T Cells Generated in Mice Treated with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4+ T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4+ cells (TCDD-CD4+ cells) by comparing and contrasting them with both natural regulatory CD4+ T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4+ cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4+ cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4+ cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4+ cells including TGF-β3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4+ cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4+ cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4+ cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.

Aryl Hydrocarbon Receptor-Mediated Perturbations in Gene Expression during Early Stages of CD4(+) T-cell Differentiation.

Activation of the aryl hydrocarbon receptor (AhR) by its prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mediates potent suppression of T-cell dependent immune responses. The suppressive effects of TCDD occur early during CD4+ T-cell differentiation in the absence of effects on proliferation and have recently been associated with the induction of AhR-dependent regulatory T-cells (Treg). Since AhR functions as a ligand-activated transcription factor, changes in gene expression induced by TCDD during the early stages of CD4+ T-cell differentiation are likely to reflect fundamental mechanisms of AhR action. A custom panel of genes associated with T-cell differentiation was used to query changes in gene expression induced by exposure to 1 nM TCDD. CD4+ T-cells from AhR+/+ and AhR−/− mice were cultured with cytokines known to polarize the differentiation of T-cells to various effector lineages. Treatment with TCDD induced the expression of Cyp1a1, Cyp1b1, and Ahrr in CD4+ T-cells from AhR+/+ mice under all culture conditions, validating the presence and activation of AhR in these cells. The highest levels of AhR activation occurred under Th17 conditions at 24 h and Tr1 conditions at 48 h. Unexpectedly, expression levels of most genes associated with early T-cell differentiation were unaltered by AhR activation, including lineage-specific genes that drive CD4+ T-cell polarization. The major exception was AhR-dependent up-regulation of Il22 that was seen under all culture conditions. Independent of TCDD, AhR down-regulated the expression of Il17a and Rorc based on increased expression of these genes in AhR-deficient cells across culture conditions. These findings are consistent with a role for AhR in down-regulation of inflammatory immune responses and implicate IL-22 as a potential contributor to the immunosuppressive effects of TCDD.