How T cells take developmental decisions by using the aryl hydrocarbon receptor to sense the environment

For the last 25 y or so, T helper (Th)-cell responses have been classified according to either Th1 or Th2 depending on whether these T cells produce IFN-γ or IL-4. While Th1 cells produce IFN-γ and induce macrophage-driven inflammation, Th2 cells secrete IL-4 and drive eosinophilic inflammation. Following the discovery of Th17 cells whose signature cytokine is IL-17A, a vigorous debate on the classification of Th-cell lineages has been unleashed. In addition to IL-17, Th17 cells secrete IL-17F, IL-21, and IL-22. The main argument for Th17 cells to be distinct from Th1 and Th2 cells was that they could be generated in the absence of Th1-associated (T-bet, STAT1, and STAT4) and Th2-associated (STAT6) transcription factors, respectively (reviewed in 1). However, the fact that Th17 cells appear unstable in vivo and the unresolved issues on the role of IL-23 for either their differentiation or stabilization, as well as the discovery of even more “T-cell lineages” like Th9 and Th22, have again spurred the debate on the plasticity of Th cells. The classification into Th1, Th2, and Th17 is still and foremost based on the distinct profile of cytokines that are produced by these T-cell subsets. In an effort to characterize other genes that would be differentially expressed in Th-cell subsets, the aryl hydrocarbon receptor (Ahr) gene was recognized to be silent in Th1 and Th2 cells but highly transcribed in Th17 cells (2, 3). In a study published in PNAS, Quintana et al. (4) investigate the functional relevance of AHR expression by specific Th-cell subsets in a disease model of CNS autoimmunity.

The AHR system is a ligand-driven transcription factor system. AHR is a basic helix–loop–helix protein that is sequestered within a protein complex in the cytosol unless it is bound by one of its membrane-permeating ligands. On ligand binding, the ligand–AHR complex translocates to the nucleus, where it dimerizes with the AHR nuclear translocator and binds to specific response elements close to the promoters of its target genes (reviewed in 5). The AHR system has been well characterized in terms of its major target genes, Cyp1a1 and Cyp1b1, in hepatocytes. On activation of AHR, these oxygenases are up-regulated and increase the intracellular availability of oxidizing agents. However, at the same time, cytochrome oxidases are believed to be the main metabolizing enzymes of natural or endogenous AHR ligands, and thus provide an intrinsic negative feedback loop resulting in only transient induction of Cyp1a1 and Cyp1b1. Notably, AHR is also the receptor for a series of xenobiotic agents like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or polychlorinated biphenyls that cannot be metabolized and lead to permanent activation of AHR target genes. These ligands only exist in an industrialized world. Therefore, it is hard to imagine that they could have provided the selection pressure for the high degree of conservation of the AHR system during evolution (6). Yet, the AHR system has a role in embryonic development and was even hijacked by the immune system in the vertebrate cosmos.

AHR is expressed in cells of the innate immune system, such as dendritic cells, macrophages, natural killer cells, and lymphoid tissue inducer-like cells (7, 8). In contrast to other danger-sensing systems, such as the TLR pathways, AHR signals are thought to convey intrinsic metabolic or oxidative stress in a cell type-specific manner (9). Engagement of AHR with different ligands modulates the expression of surface molecules of dendritic cells and the secretion of cytokines with either proinflammatory or tolerogenic net effects (4, 9).

As an alternative sensing system of the cellular milieu, AHR is particularly interesting because it is differentially expressed in various T-cell subsets and could have a direct effect on the shaping of specific Th-cell responses (Fig. 1). In committed Th17 cells, activation of AHR with 6-formylindolo-(3,2-b)-carbazole induces the production of IL-22 (2). Accordingly, Th17 cells differentiated from AHR-deficient T cells (disrupted exon 2 of Ahr) lack production of IL-22 and are also unable to up-regulate IL-22 in response to IL-23. Thus, it is possible that AHR modulates the responsiveness of Th17 cells to IL-23, although a direct impact of AHR deficiency on the expression of the IL-23 receptor (IL-23R) has not been formally tested. IL-22 is a member of the IL-10 family of cytokines. Its receptor is a heterodimer, which is composed of IL-10R2 and IL-22R. The IL-22R is not expressed on lymphocytes but on epithelial cells and specific parenchymal cells, and IL-22 has an important role in maintaining the integrity of epithelial barriers (10). Thus, at epithelial barriers and in certain parenchymal organs, Th17 cells may have a protective and beneficial role because of their production of IL-22. Interestingly, the T cell-restricted AHR pathway may also be involved in inducing IL-10. IL-27, a cytokine produced by innate immune cells under inflammatory conditions, is specifically able to switch on IL-10 production in T cells. It has been proposed that IL-10–producing T regulatory-1 (Tr-1) cells develop on stimulation with TGF-β plus IL-27 (11, 12). Mechanistically, IL-27 induces AHR, and AHR cooperates with the transcription factor c-Maf to transactivate the Il10 gene (13, 14) (Fig. 1). This mechanism may be an important way to limit exaggerated immunopathology during ongoing tissue inflammation.

Fig. 1.

Although Th1 (TH1) and Th2 (TH2) cells fail to up-regulate AHR, this ligand-driven transcription factor is expressed in Th17 (TH17) cells, Foxp3⁺ Tregs, and Tr-1 cells. Ligation of AHR with particular ligands, such as TCDD, 6-formylindolo-(3,2-b)-carbazole (FICZ), ITE, or endogenous ligands, likely promotes and stabilizes the transcriptional program of the AHR-expressing T-cell subsets (in the text and refs. 2, 3, 13, 14). It is interesting to hypothesize (and with the available tools, it can now be tested) whether AHR ligation might also convert precommitted precursors of one T-cell lineage into another T-cell subset, and thus exhibit truly immunomodulatory properties. TC, T cell.

Thus, the best evidence that the AHR pathway is directly involved in tuning the phenotype of activated T cells exists for Th17 cells and Tr-1 cells (Fig. 1). Th17 cells and Tr-1 cells can be differentiated from naive T cells with TGF-β plus IL-6 or IL-27, respectively. It is possible that the presence of TGF-β is required for the up-regulation of AHR (15). Consistent with this idea, AHR is expressed in Th17 cells that have been differentiated with TGF-β plus IL-6 but not in Th17 cells that have been generated in an alternative manner in the absence of TGF-β (16). Neither is AHR-expressed in Th1 or Th2 cells, for the differentiation of which TGF-β has a profound inhibitory effect. Th17 cells have a close ontogenetic relationship with Foxp3⁺ regulatory T cells (Tregs) (17). Tregs and Th17 cells share common factors necessary for their differentiation. Whereas TGF-β induces Foxp3⁺ in naive T cells, IL-6 abrogates the TGF-β–driven induction of Foxp3 and TGF-β and IL-6 cooperate to initiate the expression of ROR-γt, which is an essential member of the transcription factor complex that transactivates IL-17. TGF-β plus IL-6 also lead to the expression of AHR, and it is very likely that AHR will then sense environmental cues to modulate further development of the functional phenotype of a given T cell. Specific AHR ligands (e.g., high affinity vs. low affinity) might skew the fate of a T cell in specific niches toward either the induced Foxp3⁺ Treg phenotype or the Th17 phenotype (3).

In their recent study, Quintana et al. (4) provide evidence that activation of AHR is able to influence the generation of induced Foxp3⁺ Tregs in vivo. Mice that express a low-affinity variant of AHR (AHR^d) harbor fewer Foxp3⁺ Tregs in their mesenteric lymph nodes, most likely because of reduced conversion of conventional T cells into Tregs. Low-affinity ligation of AHR^d by endogenous ligands might fail to limit STAT1 activation, which, in turn, will impair both Treg and Th17 generation, a phenomenon that has

Quintana et al. investigate the functional relevance of AHR expression by specific Th-cell subsets.

also been described in AHR-deficient mice. In pharmacological interventions with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), a natural agonist of AHR, Foxp3⁺ Tregs could be induced in vivo. Thus, pharmacological manipulation of the AHR system with nontoxic ligands is a potent means to modulate fate decisions of developing T cells in vivo.

What are the real endogenous ligands for AHR? A number of natural substances have been identified as ligands for AHR, including indigoids, equilenin, arachidonic acid metabolites, heme metabolites, tryptophan metabolites, and ITE (5). Some natural ligands with extremely high affinity to AHR appear to be generated in the gastrointestinal tract from dietary compounds. For example, the secondary metabolite of glucobrassicin, indole-3 carbinol, and its higher order derivatives, indolo[3,2-b]carbazole (ICZ), 3,3′-diindolylmethane, and 2-(indol-3-ylmethyl)-3,3′-diindolylmethane, are generated naturally from cruciferous plants like broccoli in the stomach, and ICZ binds to AHR with an affinity of 1.9 × 10⁻¹⁰ M, which is only two orders of magnitude lower than the affinity of TCDD. It is likely that these metabolites contribute to shape the gut immune milieu in vivo. These are testable hypotheses that have to be addressed in future studies.

Taken together, the discovery of the importance of the AHR system for the development of Th-cell subsets provides a molecular platform to explain how dietary components, the composition of the commensal gut flora, or the exposure to industrial chemicals (by the generation of specific AHR ligands) might have an impact on fate decisions of T cells, and thus constitute a framework for the overall immunological susceptibility to autoimmunity or chronic inflammation of a given individual. Further investigation of this ligand-driven transcription factor system might not only allow us to understand how endogenous ligands drive fate decisions of T cells but enable us to exploit this potentially “druggable” system for intelligent immunomodulatory interventions in autoimmunity, chronic inflammation, and tumor immunology.