== We (3) and others (4,5,7,10) have shown that AHR activation in vivo can expand the FoxP3+Tregcompartment; thus, we investigated the effect of ITE on FoxP3+Treg. FoxP3+Tregthat suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3+Tregdifferentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders. Regulatory T cells (Treg) that express the transcription factor FoxP3 control immune autoreactivity in healthy individuals (1). FoxP3+Tregare generated in the thymus (natural Treg, nTreg) and also in the periphery (induced Treg, iTreg). The importance of FoxP3+Tregfor immunoregulation is highlighted by the immune disorders that result from their depletion or loss of function in both mice and humans (1). Conversely, the induction of FoxP3+Tregis viewed as a promising approach for the treatment of immune-mediated disorders (2). We (3) and others (48) have found that the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) controls the differentiation of Treg, Tr1 cells (9), and IL-17producing T cells (Th17) in vitro and in vivo. AHR activation by its high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo results in the expansion of the CD4+CD25+Foxp3+Tregcompartment (3). These CD4+CD25+Foxp3+Tregare functional and suppress the development of experimental autoimmune encephalomyelitis (EAE) (3), experimental autoimmune Sirt6 uveoretinitis (7), and spontaneous autoimmune diabetes (10). TCDD is a valuable tool to study the immunological effects of AHR activation, but TCDD is not a natural AHR ligand and its toxicity rules out its therapeutic use. Thus, it is not yet known whether there is a physiological role for AHR in FoxP3+Treg, and whether nontoxic AHR ligands Fasudil HCl (HA-1077) exist which can expand FoxP3+Tregin vivo to treat autoimmunity. To address these questions, we used mice carrying a GFP Fasudil HCl (HA-1077) reporter infoxp3and a mutant AHR protein with reduced affinity for its ligands. In addition, we investigated the effect and mechanisms of action of the nontoxic mucosal AHR ligand 2-(1H-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on FoxP3+Tregboth in vitro and in vivo in the model Fasudil HCl (HA-1077) of EAE. == Results == == AHR Activation by Endogenous Ligands Promotes the Differentiation of FoxP3+iTreg. == FoxP3+Tregare classified as FoxP3+nTreg(generated in the thymus) and FoxP3+iTreg(generated in the periphery) (1). The gut-associated lymphoid tissue is a major physiological site for the differentiation of FoxP3+iTreg(11). To investigate the physiological role of AHR on FoxP3+iTregdifferentiation, we generated AHR-d Foxp3gfpmice and analyzed the frequency of FoxP3+Tregin the thymus and mesenteric lymph nodes (MLN). AHR-d Foxp3gpfmice carry a GFP reporter in thefoxp3gene (12), and harbor the d allele ofahr(AHR-d), which codes for an AHR protein with reduced affinity for its ligands (13). We found a significant reduction in the frequency of FoxP3+Tregcells in the MLN of AHR-d Foxp3gfpmice (Fig. S1A); no difference was detected in the frequency of thymic FoxP3+Treg(Fig. S1B). WT and AHR-d Foxp3+Tregshowed comparable suppressive activity in vitro (Fig. S2). To determine whether the reduced frequency of FoxP3+Tregin the MLN of AHR-d Foxp3gfpmice resulted from an impaired generation of FoxP3+iTregin vivo, we used a transfer model (14). CD4+FoxP3:GFPT cells from naive AHR-d Foxp3gfpor Foxp3gfpmice were Fasudil HCl (HA-1077) transferred to RAG-1-deficient mice, and the reconstituted mice were immunized with MOG3555(14). We found a significant reduction in the frequency of CD4+FoxP3:GFP+Tregin mice that received AHR-d FoxP3:GFPT cells (Fig. S1C). These results suggest a physiological role for endogenous AHR ligands in the generation of FoxP3+iTregin vivo. Given our results on the role of AHR on FoxP3+iTregin vivo, we analyzed the role of AHR on the differentiation of FoxP3+iTregtriggered in vitro with TGF-1 and IL-2 (3,15). Naive CD4+T cells from AHR-d Foxp3gfpmice showed a significant impairment in their differentiation into FoxP3+Treg(Fig. S1D), with or without antigen presenting cells (APCs). Thus, AHR activation by endogenous ligands in T cells affects FoxP3+iTregdifferentiation. We investigated the mechanism.