An Interaction between Kynurenine and the Aryl Hydrocarbon Receptor Can Generate Regulatory T Cells

This information is current as Joshua D. Mezrich, John H. Fechner, Xiaoji Zhang, Brian P. of October 2, 2021. Johnson, William J. Burlingham and Christopher A. Bradfield J Immunol 2010; 185:3190-3198; Prepublished online 18 August 2010; doi: 10.4049/jimmunol.0903670 http://www.jimmunol.org/content/185/6/3190 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/08/18/jimmunol.090367

Material 0.DC1 http://www.jimmunol.org/ References This article cites 57 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/185/6/3190.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Interaction between Kynurenine and the Aryl Hydrocarbon Receptor Can Generate Regulatory T Cells

Joshua D. Mezrich,* John H. Fechner,* Xiaoji Zhang,* Brian P. Johnson,† William J. Burlingham,* and Christopher A. Bradfield†

The aryl hydrocarbon receptor (AHR) has been known to cause immunosuppression after binding dioxin. It has recently been discovered that the receptor may be central to T cell differentiation into FoxP3+ regulatory T cells (Tregs) versus Th17 cells. In this paper, we demonstrate that kynurenine, the first breakdown product in the IDO-dependent tryptophan degradation pathway, activates the AHR. We furthermore show that this activation leads to AHR-dependent Treg generation. We additionally investigate the dependence of TGF-b on the AHR for optimal Treg generation, which may be secondary to the upregulation of this receptor that is seen in T cells postexposure to TGF-b. These results shed light on the relationship of IDO to the generation of Tregs, in addition to highlighting the central importance of the AHR in T cell differentiation. All tissues and cells were derived Downloaded from from mice. The Journal of Immunology, 2010, 185: 3190–3198.

t has long been recognized that the immune system is in a fine cell differentiation and stability of Tregs, as the ability of Tregs balance between immunity and self-tolerance. The concept of to redifferentiate into Th17 cells in the appropriate inflammatory suppressor T cells playing a role in this balance was first pro- milieu has now been described (8, 9).

I http://www.jimmunol.org/ posed in the 1970s (1). Efforts to identify these cells were gener- Recent publications implicate the aryl hydrocarbon receptor ally unsuccessful, and their very existence was brought into (AHR) as a central player in T cell differentiation. The AHR is best question in the early 1980s by molecular biologists who failed known as the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (di- to locate an elusive suppressor gene in the mouse MHC class II oxin, or TCDD). Activation of the AHR by this environmental locus (2). The suppressor T cell concept was dropped and re- pollutant can lead to a range of toxic endpoints, including hepa- mained out of vogue until it re-emerged as the CD4+CD25+ reg- tocellular damage, epithelial changes, cancer, birth defects, thymic ulatory T cell (Treg), first described in detail by Sakaguchi in 1995 involution, and immunosuppression (10, 11). Although the AHR is (3). Since that time, numerous studies have characterized these well known for its role in toxicology, this receptor has also been cells and the role they play in autoimmunity, control of , shown to play a role in vascular and hematopoietic development by guest on October 2, 2021 and transplant rejection. Identification of FoxP3, a transcription (12, 13). Moreover, several potential endogenous ligands have factor for Treg development, has led to further characterization been shown to bind to the AHR with variable affinity and potency of the importance of regulation (4, 5). More recently, a new Th cell (14). The fact that this receptor has been conserved in evolution lineage, termed Th17, was described (6, 7). These IL-17–secreting (15) and that invertebrate orthologs of the AHR are well preserved cells are thought to play a major role as effectors in autoimmunity and yet do not bind to TCDD (16) is consistent with the idea that and transplant rejection. Interestingly, these new data have led a physiologically relevant endogenous ligand for this receptor investigators to question previously held beliefs about terminal exists (17). In evaluating the mechanisms for immunosuppression seen postexposure to TCDD, it was discovered that activation of the *Division of Transplantation, Department of Surgery and †McArdle Laboratory for AHR with TCDD leads to the generation of Tregs in vitro or in vivo Cancer Research, University of Wisconsin School of Medicine and Public Health, (18), and, alternatively, activation with a different endogenous Madison, WI 53792 ligand, 6-formylindolo[3,2-b]carbazole (FICZ), leads to Th17 cell Received for publication November 13, 2009. Accepted for publication July 8, 2010. formation (19). Although this unusual pharmacology in which one This work was supported by Grant 1UL1RR025011 from the Clinical and Translational AHR agonist diverts T cells toward regulators and another agonist Science Award program of the National Center for Research Resources, National Insti- tutes of Health (to J.D.M.), National Institute on Environmental Health Sciences Grant generates effectors is surprising, multiple studies have confirmed R37ES005703 (to C.A.B.), National Cancer Institute Grant P30CA014520 (to C.A.B.), the importance of the AHR in the generation of Th17 cells both an American Society of Transplant Surgeons-Astellas Faculty Development Award in vitro and in vivo (20, 21). Regarding Treg generation, the direct (to J.D.M.), National Institute of Allergy and Infectious Grant RO1AI066219 (to W.J.B.), and National Institutes of Health Training Grant T32ES007015-32 (to B.P.J.). relationship of regulatory cells to the AHR has been less clear Address correspondence and reprint requests to Dr. Joshua Mezrich, Department of (22). This has led investigators to question whether the AHR truly Surgery, University of Wisconsin School of Medicine and Public Health, H4/784 has a direct effect on the generation of these cells (23, 24). Clinical Science Center, 600 Highland Avenue, Madison, WI 53792-7375. E-mail Our laboratory has focused on the role of indolylic products as address: [email protected] potential endogenous ligands of the AHR (25, 26). Therefore, we The online version of this article contains supplemental material. began to think about the potential for an interaction between the Abbreviations used in this paper: AHR, aryl hydrocarbon receptor; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DRE, dioxin-responsive element; AHR and the IDO pathway. The IDO enzyme catalyzes the rate- FICZ, 6-formylindolo[3,2-b]carbazole; nt, not tested; pDC, plasmacytoid dendritic limiting step of tryptophan degradation along the kynurenine path- cell; qPCR, quantitative PCR; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Treg, way (27). IDO is present and activated in subsets of dendritic cells regulatory T cell; WT, wild-type. (DCs; particularly plasmacytoid DCs, or pDCs) and thought to be Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 central to Treg generation from T cell precursors by DC–T cell in- www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903670 The Journal of Immunology 3191 teractions (28, 29). The exact mechanistic pathway by which IDO ces, San Jose, CA). The relative light unit is the indicator of luciferase leads to Tregs has been debated, and both tryptophan starvation expression level. All experiments were repeated three times, and each sam- and direct effects of tryptophan metabolites (including kynure- ple was tested in triplicate each time. nine) have been proposed (30–32). In addition to a connection Real-time quantitative PCR via indole , the IDO–AHR interaction was particularly Total RNA was extracted using the reagents: RNeasy Mini Kit and RNase- interesting in light of the observation that IDO may be upregulated Free DNase Set (Qiagen, Valencia, CA). A total of 500 ng total RNA in each by the AHR (33, 34) and that kynurenine and related metabolites group was used for RT reaction (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, may be AHR agonists (35–37). CA; or High-capacity cDNA Reverse Transcription Kits, Applied Biosystems, In this report, we demonstrate an important role for kynurenine, Foster City, CA). The relative quantitation PCR for IDO1 (Mm00492586-m1), g the first tryptophan metabolite of the IDO pathway, in Treg gen- GAPDH (4352339-E0806018), and IFN- (Mm99999071-m1) were per- formed in the Applied Biosystems 7900HT Fast Real-Time PCR System eration. We provide evidence that kynurenine activates the AHR at (Applied Biosystems), and TaqMan Universal PCR Master Mix (Applied a dose clinically relevant in humans and leads to Tregs in vitro. The Biosystems) was used as a reaction reagent. The relative quantitation PCR role for the AHR in this process is supported by two observations. for Foxp3, Cyp1a1, Cyp1b1, and GAPDH were processed by the Bio-Rad First, kynurenine does not influence Treg generation in AHR-null iCycler (Bio-Rad) and iQ SYBR Green Supermix (Applied Biosystems) used as the reaction reagent. T cells. Second, kynurenine can be shown to activate the AHR us- ing classical response genes, such as Cyp1a1 and Cyp1b1. In our Isolation of naive CD4 T cells and T cell differentiation model, the AHR in T cells is required for the generation of Tregs by Naive CD4 T cells were isolated from spleens of C57BL/6J and AHR-null mice kynurenine. We further define the importance of the AHR for op- using the CD4 CD62L Isolation Kit (Miltenyi Biotec, Auburn, CA) and an timal generation of Tregs by TGF-b and characterize potential autoMACS. This kit includes a depletion mixture, including the addition of Downloaded from mechanisms for this. a CD25 and an anti-TCRg/d+ Ab. CD62L is expressed on naive T cells and downregulated upon activation. A small subset of central memory T cells also Materials and Methods express CD62L and could be included in this separation. These represent Mice a very small proportion of the final separation, and we will refer to sepa- rated cells as naive T cells. Cells were tested for purity postsorting and con- 2 C57BL/6J wild-type (WT) and BALB/c mice were obtained from The Jack- sistently showed .90% purity for CD4+CD62+CD25 cells. An example of son Laboratory (Bar Harbor, ME). AHR-null (AHR-deficient B6) mice on a analysis of the separations is included in the Supplemental Material (Supple-

C57BL/6J background (13) were bred and maintained under specific path- mental Fig. 2A). Viability at the beginning of culture was typically .98% as http://www.jimmunol.org/ ogen-free conditions. All animal experiments were carried out according seen by trypan blue staining. For quantitative PCR (qPCR) analysis, 2–5 3 105 to institutional guidelines approved by the University of Wisconsin School cells were cultured in each well of a 96- well round-bottom plate coated of Medicine and Public Health Animal Care and Use Committee (Madi- with 0.5 mg/ml anti-CD3 and anti-CD28 overnight and then washed with son, WI). PBS twice before seeding the cells. The naive T cells were maintained in F10 media supplemented with 10% heat-inactivated FBS, 100 mg/ml strepto- Isolation and differentiation of bone marrow-derived DCs mycin, 100 U/ml penicillin, 50 mm 2-ME, 25 mM HEPES, and 2 mM L- glutamine and were treated with 10 nM TCDD, 100 nM FICZ, 2–10 ng/ml The method of murine bone marrow-derived DCs (BMDCs) was performed TGF-b (as specified), 50 mM kynurenine, or 25 mM each kynurenine (hydro- as previously described (38). Briefly, bone marrow was obtained from mice xykynurenine, hydroxyanthranilic acid, anthranilic acid, nicotinamide, and femurs. After RBC lysis, the cells were plated in six-well plates with a 6 quinolinic acid). After 5 d, the cultured cells were harvested for RNA assay. density of 1 3 10 /ml in complete RPMI 1640 media supplemented with by guest on October 2, 2021 Prior to this, cells were checked for viability using live-dead staining with 30 ng/ml GM-CSF. On day 3, nonadherent cells and 75% of culture media flow cytometry. The majority of cells were viable, but dead cells were gated were exchanged for fresh media. On day 6, the cells were either harvested out in flow cytometry analysis. For flow cytometric analysis, purified naive as immature DCs or cultured an additional day to maturity by again ex- T cells were stimulated with the CD3/CD28 T cell Activation/Expander Kit changing 75% of the media, with the addition of 50 ng/ml LPS. A total of (Miltenyi Biotec) for 5 d. As indicated, cultures were supplemented with 80% of the cell population stained positive for CD11c by flow cytometry. recombinant cytokines and reagents: human TGF- b1 (R&D Systems, Min- For analysis of mRNA expression, cells were treated with or without neapolis, MN), mouse IL-6 (20 ng/ml; R&D Systems), kynurenine, FICZ (100 TCDD (10 nM). As mentioned above, 50 ng/ml LPS was used for matu- nM), and AHR antagonist CH-223191 (Calbiochem, San Diego, CA). ration of BMDCs. There is a previous publication that LPS alone can lead to IDO (39). It should be clarified that this only occurred when higher Intracellular FoxP3 and IL-17 cytokine staining doses of LPS (5 mg/ml) were used. This response is dose dependent, as seen by other investigators (33, 40, 41). As an additional control, an LPS To stain for Foxp3, T cells were first surface stained with anti-CD4 and anti- titration was performed with BMDCs as further confirmation, the results of CD25 and then fixed and permeabilized with the Fixation/Permeabilization which are in the Supplemental Materials (Supplemental Fig. 1). No in- buffer (eBioscience, San Diego, CA) for 30 min at 4˚C. Following this, crease in IDO mRNA was seen until at least 100 ng/ml LPS was used in cells were stained with Pacific Blue-conjugated anti-Foxp3. For intra- these assays. cellular IL-17 staining, T cells were first stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 800 ng/ml ionomycin (Sigma-Aldrich) for 4 h in the Luciferase assay presence of GolgiStop (BD Pharmingen, San Diego, CA) for the final 2 h. A mouse hepatoma cell line H1L6.1c3, stably carrying a dioxin-responsive Cells were then fixed and permeabilized with the Fixation/Permeabiliza- element (DRE)-driven firefly luciferase reporter gene [a gift from Dr. Denison, tion buffer (eBioscience) and then stained with PE-conjugated anti–IL-17. University of California, Davis, CA (42)] was maintained with 0.3 mg/ml All Abs were from eBioscience. Flow cytometric analysis was performed G418 in completed DMEM media. Briefly, 0.6 3 106 cells were seeded in using an LSR-II (BD Biosciences). each well of a six-well plate overnight and were then treated with different pDC/T cell coculture concentrations of TCDD, L-tryptophan, L-kynurenine, hydroxykynurenine, hydroxyanthranilic acid, anthranilic acid, nicotinamide, and quinolinic acid Naive CD4+CD252 T cells were isolated from WT and AHR-null mice and for the time specified. All of the kynurenines1 (including kynurenine) were cocultured with BALB/c pDCs isolated using the Miltenyi Mouse pDC purchased from Sigma-Aldrich (St. Louis, MO), and purity was listed at Isolation Kit (Miltenyi Biotec) at a ratio of 20:1 or 10:1 (example of pDC $98% confirmed by HPLC. They were placed in solution in 0.5 M HCL, as separation in Supplemental Fig. 2B). CpG, FICZ, and kynurenine were recommended by the manufacturer for maximum solubility. Cells were added at the start of culture. On day 5, cells were harvested and subjected lysed by lysis buffer (Promega, Madison, WI), and the luciferase assay to flow cytometric analysis. was performed by using a BD monolight 3010 luminometer (BD Bioscien- Results 1Throughout this paper, kynurenine is used specifically to delineate this first trypto- AHR activation in DCs leads to IDO induction phan breakdown product in the kynurenine pathway, catalyzed by the enzyme IDO. Those breakdown products downstream to kynurenine are termed kynurenines and do We first examined the role that the AHR in DCs might play in not include kynurenine itself. directing T cell differentiation. Based on previously published data 3192 AHR, IDO, AND IMMUNITY

(29, 33, 34), we considered the possibility that IDO may be consider that kynurenine could generate Tregs through the AHR. a mediator of the cross talk between these cells. We initially set Using the well-documented technique for Treg generation with out to confirm that IDO could be induced by activation of the TGF-b and Ab stimulation (45), we initially exposed naive CD4+ AHR. Fig. 1 shows that cultured BMDCs, when exposed to the T cells from WT and AHR-null animals to 2 ng/ml TGF-b and AHR agonist TCDD, led to Cyp1a1 induction, confirming acti- analyzed collected mRNA for FoxP3 expression. As seen in Fig. vation of the AHR in these cells. Exposure to TCDD also in- 3A, WT cells generated FoxP3, .40 times the response in AHR creased IDO mRNA levels. DCs produced Cyp1a1 and IDO in nulls. To further support this finding, we performed a similar both immature and mature states, indicating they did not have to experiment exposing WT or AHR-null cells to Ab stimulation be activated to have this response. AHR-null DCs did not exhibit and a higher dose of TGF-b and measured Treg generation by IDO production postexposure to TCDD. In Supplemental Fig. 3, flow cytometry. This is represented in Fig. 3B, in which optimal mRNA for IDO was analyzed at earlier time points than 48 h used Treg populations were generated in WT cells (29.1% in this rep- above to control for any indirect affect that may have led to IDO resentative assay), with a muted response from null cells (11.6% in expression. As early as 7 h postculture, IDO mRNA was generated. this same assay). By titrating doses of TGF-b, we were able to yield increasing numbers of CD25+FoxP3+ cells seen by flow cytometry, Kynurenine activates the AHR, whereas other tryptophan represented graphically in Fig. 3C. When naive CD4+ T cells were breakdown products downstream to kynurenine do not separated from AHR-null animals, increasing doses of TGF-b had Given that IDO can be generated by AHR activation in DCs and that little effect on the generation of Tregs (Fig. 3B,3C). To further test tryptophan breakdown products have been known to generate AHR the importance of AHR-ligand binding in Treg generation, we re- ligands, we examined all of the tryptophan breakdown products of peated Ab stimulation of naive WT T cells with titrating doses of the IDO pathway for their ability to activate the AHR. We employed TGF-b, and this time included an AHR antagonist (CH-223191) Downloaded from a mouse cell line of hepatoma cells, termed Hepa1. These cells have known to competitively bind to the receptor. As seen in Fig. 3D, the been transfected with a luciferase reporter gene fused to the DRE addition of antagonist blocked the increase of Tregs seen by flow (43). We tested each of the commercially available substrates in the cytometry postexposure to TGF-b in vitro. kynurenine pathway, and compared their ability to activate the DRE The above experiments all demonstrate the importance of the with TCDD. Interestingly, kynurenine itself showed the strongest AHR in Ab-stimulated Treg generation via TGF-b. Given our activity (Fig. 2A). Peak activity was at 5 h, with a dose (50 mm) that suspicion that cell–cell contact is important in AHR-dependent http://www.jimmunol.org/ is comparable to levels encountered clinically in humans in areas of Treg generation, we employed an in vitro system separating pDCs inflammation postactivation of IDO (44) (Fig. 2A). All other and exposing them to allogeneic naive CD4+ T cells (pDCs were breakdown products showed less DRE activity, with decreasing derived from BALB/c mice, and naive T cells from C57BL/6J peaks of activity the further down the kynurenine pathway that mice). This system was previously shown to be dependent on products were examined. As seen in Fig. 2B, we further tested IDO for successful generation of Tregs (29). We were able to this cell line using real-time PCR and found that postexposure to repeat the findings that pDCs exposed to CPG led to significant kynurenine in vitro, a substantial increase in Cyp1a1 and Cyp1b1 generation of FoxP3+ Tregs in WT allogeneic naive T cells (Fig. mRNA was seen, confirming activation of the AHR by this ligand. 3E). When naive T cells were isolated from AHR-null mice, a low by guest on October 2, 2021 FICZ was also tested to compare its efficacy as an activator of the percentage of Tregs were identified prior to manipulation. Addi- DRE in comparison with kynurenine. It is a potent ligand, with tion of CpG did increase Treg generation, but the expression was a peak at a later time point (10 h), seen in Fig. 2C. dramatically less robust than in the WT cells (Fig. 3E).

Presence of the AHR is necessary in T cells for optimal + + Kynurenine induces generation of FoxP3 Tregs in an AHR- generation of FoxP3 Tregs dependent manner The observation that IDO can be upregulated in DCs in an AHR- As it is well known that IDO leads to the generation of Tregs, and dependent manner and that kynurenine activates the AHR led us to it appears that kynurenine activates the AHR, the next step was to assess whether kynurenine can directly lead to FoxP3 expression. We first cultured mouse naive CD4+ T cells with Ab stimulation for 5 d in the presence of 10 ng/ml of TGF-b, kynurenine, TCDD, or FICZ. RNA was then harvested and tested for the presence of FoxP3. As seen in Fig. 4A, top panel, only TGF-b and kynurenine led to significant induction of FoxP3 RNA. We analyzed FoxP3 induction in triplicate in 11 separate biological experiments and achieved a fold change of 3.2, which was significantly increased from control with a p value of 0.017. When AHR-null cells were used (Fig. 4A, bottom panel), only TGF-b (10 ng/ml) yielded FIGURE 1. AHR activation in mouse BMDCs leads to IDO. BMDCs significant induction of FoxP3 RNA. To further show that were generated from the bone marrow of C57BL/6J WT and AHR-null kynurenine is leading to FoxP3 mRNA via an interaction with mice as described in Materials and Methods. Cells were harvested on day the AHR and not in some indirect way dependent on TGF-b,we 6 as immature BMDCs or on day 7 as mature BMDCs following addition cultured mouse naive CD4+ T cells as above with Ab stimulation on day 6 of LPS at a dose of 50 ng/ml, a concentration that itself does not either with or without kynurenine. RNA was then harvested and cause IDO expression, confirmed in Supplemental Fig. 1. BMDCs were tested for Cyp1a1, Cyp1b1, and TGF-b. As seen in Fig. 4B, cultured in the presence or absence of TCDD (10 nM) added on day 0 of kynurenine exposure led to significant amounts of Cyp1a1 com- culture. mRNA was isolated from immature or mature BMDCs and assayed for the expression of Cyp1a1 (left panel), a marker of AHR acti- pared with control, but an increase of TGF-b mRNA over control vation, and IDO1 (right panel). Data were normalized to WT control. Post was not seen, making it unlikely that kynurenine is acting in- ANOVA testing comparisons are to cultures without TCDD. Cyp1a1 directly by generating this cytokine. We then performed a similar mRNA was undetectable in all AHR-null PCR reactions. Each graph is experiment, exposing naive T cells to Ab stimulation with and representative of three independent experiments. pppp , 0.001. without kynurenine and after 5 d of culture used flow cytometry The Journal of Immunology 3193

FIGURE 2. Kynurenine, but not other tryptophan breakdown products, directly activates the AHR. Mouse Hepa1 cells that were transfected with lu- ciferase reporter gene fused to the DREs were seeded at 0.6 3 106 cells/well. Cells were then exposed to tryptophan breakdown products along the kynurenine pathway (50 mM, except as indicated) for 4 h except as indicated. Luciferase activity was measured on a luminometer. A, Data were converted Downloaded from as a percent of response to TCDD (10 nM) to determine the dose-response curve (left panel) and time course (right panel) of luciferase activity. B, mRNA was isolated from Hepa1 cells following 2 and 5 h exposure to TCDD (10 nM) and kynurenine (50 mM). qPCR analysis was performed to determine expression levels of Cyp1a1 (upper panel) and Cyp1b1 (lower panel) to confirm activation of the AHR. Post ANOVA testing comparisons are to control. C, Time course of luciferase activity following exposure of Hepa1 cells to FICZ (200 nM) or kynurenine (50 mM). All figures represent one of three independent experiments. ppp , 0.01; pppp , 0.001. http://www.jimmunol.org/ to assess for FoxP3. As seen in Fig. 4C, cells that were not exposed The data in this section highlight that the AHR on T cells is to kynurenine showed minimal FoxP3 expression, but those ex- activated by kynurenine and leads to Treg induction. posed to kynurenine had a significant shift, with 24.4% more b FoxP3 expression in analyzed cells in one representative assay. TGF- upregulates AHR expression, potentiating activation of We performed this experiment six times and observed significant the DRE by kynurenine FoxP3 protein expression in four out of six assays. This was also To better understand the role of the AHR in TGF-b–dependent repeated with titrating doses of kynurenine with or without the Treg generation, we next extracted total RNA from naive T cells, presence of an AHR antagonist, and these data further confirm either fresh or after 20 h or 3 d of culture, and conducted real-time by guest on October 2, 2021 that kynurenine at a dose of 50 mM leads to FoxP3+ T cells, and PCR for AHR expression. Culture conditions included Ab stimu- the AHR antagonist decreased the percentage of FoxP3+ cells both lation with FICZ, kynurenine, or TGF-b. There is AHR expression at baseline and postexposure to kynurenine. at baseline (Fig. 5A), which increases .4-fold at 20 h with expo- We next used the in vitro system separating pDCs and exposed sure to TGF-b and remains .3-fold elevated at 3 d. We addition- them to allogeneic naive CD4+ T cells. As seen in Fig. 4D,inWT ally looked at Cyp1b1 expression at 20 h and 3 d, and, as seen in T cells, the addition of CpG exhibited the greatest expression of Fig. 5A, FICZ and kynurenine led to 20 and 50 times mRNA Tregs, but kynurenine also yielded elevated Treg generation. We production over baseline at 20 h, respectively, with Cyp1b1 levels also tested FICZ, and this ligand not only did not lead to an in- remaining 20 times elevated at 3 d after kynurenine exposure. creased Treg population, but also decreased the percentage of CD4+ Culturing in the presence of TGF-b did lead to a small increase CD25+FoxP3+ cells compared with untreated control. When AHR- in Cyp1b1 (∼4 times over baseline at 20 h), but much less than null mice were used as the source for naive T cells, very few Tregs seen with FICZ or kynurenine. To assess whether AHR upreg- were generated when exposed to pDCs. The addition of kynurenine ulation secondary to TGF-b would potentiate binding of ligands did not cause any enhancement of Treg formation, presumably due to the AHR, we compared the expression of Cyp1a1 and Cyp1b1 to the lack of the AHR on the T cells. As further confirmation, we after kynurenine exposure with and without TGF-b, which is repeated the DC/T cell coculture with a 1:20 ratio of allogeneic represented in Fig. 5B. The response is strongly enhanced after BALB/c DCs to naive C57BL/6J T cells multiple times and present TGF-b exposure, shifting the curve up significantly, implying that the summarized data graphically. As shown in Fig. 4E, the 1:20 TGF-b does potentiate the binding of kynurenine to the AHR ratio yielded generation of Tregs by flow, and kynurenine led to when this ligand is present in the culture. significant Treg generation as compared with untreated cells. When repeated with AHR-null T cells, CpG was able to yield Tregs, but Kynurenine does not lead to Th17 cell generation, whereas kynurenine did not, supporting the dependence of the function of FICZ does kynurenine on the expression of AHR by the T cell. Given that the AHR has also been implicated in the generation of To assess the importance of the AHR on DCs in this model, we Th17 cells when bound to certain ligands (FICZ), we wondered performed this experiment using AHR-null cells as the source for whether IDO pathway products could also favor Th17 morphology pDCs (on a C57BL/6J background), and naive T cells were taken when present in a milieu favoring Th17 generation. We used the from WT mice (BALB/c). As seen in Supplemental Fig. 4, under Th17-generating conditions described previously, based on expo- these circumstances, similar generation of Tregs was seen using sure of naive CD4+ T cells to IL-6 and TGF-b (21). We first null pDCs as WT pDCs, indicating, at least in this model, that repeated the finding that FICZ leads to enhancement of IL-17+ the presence of the AHR is necessary on the T cell, and not the cells (23) (Fig. 6). As mentioned previously, FICZ is thought to act pDC, for optimal Treg generation. primarily through the AHR, confirming that the AHR can promote 3194 AHR, IDO, AND IMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 3. Presence of the AHR is necessary in T cells for optimal generation of FoxP3+ Tregs in Treg-polarizing conditions with and without cell–cell contact. A, Naive CD4 T cells (CD4+CD62L+ T cells) were generated by magnetic bead separation. A total of 5 3 105 cells/well were cultured for 5 d with anti-CD3/CD28 beads in the presence of no or 2 ng/ml TGF-b. qPCR was used to test the generation of FoxP3. pppp , 0.001. B, Naive CD4 T cells (CD4+ by guest on October 2, 2021 CD62L+ T cells) were generated by magnetic bead separation. A total of 5 3 105 cells/well were cultured for 5 d with anti-CD3/CD28 beads in the presence of titrating doses of TGF-b. Flow cytometry was used to analyze for CD25 and intracellular FoxP3. C, Graphical representation of a similar experiment as B, using titrating doses of TGF-b and titrating numbers of T cells per well. D, Similar to C, except some naive cells were exposed to a soluble AHR antagonist. E, Naive CD4+CD252 T cells were isolated from B6 WT and AHR-null mice and cocultured with pDCs isolated from BALB/c mice using the Miltenyi Mouse pDC Isolation Kit (Miltenyi Biotec) at a ratio of 20:1. CpG was added at the start of culture in some experiments. On day 5, cells were harvested and subjected to flow cytometric analysis. Percentages are the fraction of gated live CD4+ cells that were FoxP3/CD25 double positive. Figures are representative of three independent experiments.

T cell differentiation to both Treg and Th17 differentiation de- AHR, the physiologic significance of this needs to be further de- pending on the milieu. We then tested kynurenine and found no fined. IFN-g does stimulate IDO in AHR-null DCs (data not effect on the generation of Th17 cells (Fig. 6). This indicates that shown), and Supplemental Fig. 4 would suggest that Tregs can be activation of the AHR with different ligands can lead to entirely induced by AHR-null pDCs in coculture assays, but the AHR needs different outcomes depending on the surrounding milieu. to be present on T cells for optimal Treg generation. One issue that needs to be addressed is the reliance of TGF-b on Discussion the AHR. Fig. 3 indicates that optimal generation of Tregs by Collectively, the data represent a novel way that IDO, via the TGF-b is dependent on the presence of the AHR, similar to a kynurenine pathway, leads to Treg generation. It is generally be- report published previously (21). This decrease in Treg pro- lieved that T cell differentiation depends on interactions between duction is demonstrated both in T cells obtained from null mice DCs and T cells (46–48), with IDO playing a role in this. The above and also with the use of the AHR antagonist (Fig. 3D). It is un- findings establish a direct relationship between the AHR and the likely that this cytokine binds directly to the AHR, given its struc- fate of T cells in vitro. IDO is generated by pDCs. This leads to ture (we have tested this in a DRE luciferase assay with no tryptophan metabolism and kynurenine formation. Kynurenine response to TGF-b alone). More likely, it is secondary to the binds to the AHR in T cells, leading to differentiation to CD25+ effect demonstrated in Fig. 5, which shows that TGF-b with Ab FoxP3+ T cells. Absence of the AHR in T cells prevents this effect. stimulation leads to an upregulation of the AHR in culture. This Kynurenine was previously identified in a review of AHR ligands effect is seen in the first 24 h in culture and seems to persist at (35), which corresponds to the knowledge that tryptophan metab- least 3 d, according to our data and the literature (21, 49). It is olites can lead to ligands of the AHR; the finding that this ligand– important to note that CD4+CD252 T cells do express the AHR receptor interaction leads to Treg generation is novel. Whereas Fig. prior to its upregulation, which is demonstrated by both Western 1 does indicate that IDO induction by DCs can be stimulated via the blot (21, 49) and DNA microarray (21) and in our own data (Fig. The Journal of Immunology 3195 Downloaded from http://www.jimmunol.org/

FIGURE 4. Kynurenine induces generation of FoxP3+ Tregs in an AHR-dependent manner. A, Naive CD4+CD252 T cells were isolated from B6 WT (top panel) and AHR-null (bottom panel) mice. Cells at varying cell densities (50–200 3 103 cell/well) were cultured in F10 plus 5% FCS in wells coated by guest on October 2, 2021 with anti-CD3/anti-CD28 Abs for 5 d. Human TGF-b, kynurenine, TCDD, and FICZ were added at the start of the culture. mRNA was isolated from harvested cells, and qPCR was performed to determine FoxP3 expression levels. Data are relative to WT or AHR-null cells cultured without TGF-b.The WT experiments were conducted 11 times and are presented as the mean values. Null experiments were conducted three times, with mean values presented. Post ANOVA testing comparisons are against the WT or AHR-null control. pp , 0.05; pppp , 0.001. B, Naive T cells were separated and cultured with Ab stimulation as in A, with and without kynurenine (50 mM) in the culture. mRNA was isolated after 5 d, and qPCR was performed for Cyp1a1, Cyp1b1, and TGF-b. Post ANOVA testing comparisons are against the vehicle control. pppp , 0.001. C, Naive CD4+ CD252 T cells were cultured in the presence of immunomagnetic microbeads coated with anti-CD3/anti-CD28 Abs with (transparent peak) and without (gray peak) kynurenine (50 mM) (top panel). Results were measured by flow cytometry with intracellular FoxP3 staining after 5 d of culture. These results are representative of the protein induction obtained in four of six separate biological assays. C, Varying concentrations of kynurenine 6 AHR antagonist were added at the start of culture. Cells were then harvested and subjected to flow cytometry. Percentages are the fraction of gated live CD4+ cells that are FoxP3 positive. Post ANOVA testing comparisons are against the vehicle control (bottom panel). ppp , 0.01. D, Naive CD4+CD252 T cells were isolated from B6 WT and AHR-null mice and cocultured with pDCs isolated from BALB/c mice using the Miltenyi Mouse pDC Isolation Kit (Miltenyi Biotec) at a ratio of 20:1. CpG, FICZ, and kynurenine were added at the start of culture at the concentrations indicated. On day 5, cells were harvested and subjected to flow cytometric analysis. Percentages are the fraction of gated live CD4+ cells that were FoxP3/CD25 double positive. E, The experiments in D were repeated at the same ratio of pDCs to naive CD4+ T cells (1:20), and results are expressed graphically for WT and null cells. These figures represent three independent experiments. Post ANOVA testing comparisons are against the untreated control. pp , 0.05; ppp , 0.01.

5). This is further demonstrated by the fact that exposure of naive well as the fact that we do see some Cyp1a1 and Cyp1b1 induction T cells to kynurenine or FICZ leads to mRNA transcription of in T cells postexposure to TGF-b (Fig. 5A,5B). Perhaps there is Cyp1a1 and Cyp1b1, which would only occur in the presence of ongoing binding between the AHR and endogenous ligands (which the AHR. It is possible that AHR upregulation alone leads to Treg may include kynurenine in an in vivo system), and blocking the generation after TGF-b exposure, which would correlate with receptor blocks this Treg-generating effect. Although endogenous a previously published experiment in which cotransfection of a ligands may play a role in this differentiation, the effects of kynur- construct coding for mouse AHR into a bacterial artificial chro- enine in our assays far outweigh ligands that may already be present mosome with FoxP3 tagged with a Renilla luciferase reporter led in the media. We tested the role of the AHR with TGF-b in Fig. 5B, to upregulation of Renilla activity (18). Another possibility is that in which kynurenine was titrated in culture with CD4+CD252 the upregulation of the AHR allows endogenous ligands present in T cells either in the presence of or without TGF-b. When this the system (either in media or secreted from cells during inflam- cytokine was present, the response of Cyp1a1 and Cyp1b1 was dra- mation) to bind to the increased receptor with enhanced effect. This matically elevated when exposed to increasing doses of kynurenine, is supported by the data in Fig. 4C, which indicate that the AHR much higher than was seen with TGF-b alone. This would further antagonist reduces the amount of FoxP3+ cells seen at baseline, as support that kynurenine is a ligand of the AHR and that TGF-b 3196 AHR, IDO, AND IMMUNITY

FIGURE 6. FICZ but not kynurenine enhances Th17 cell differentia- tion in vitro. Naive CD4+CD252 T cells were isolated from B6 WT spleens and cultured in the presence of immunomagnetic microbeads coated with anti-CD3/anti-CD28 Abs, TGF-b (4 ng/ml), and IL-6 (20 ng/ ml) for 5 d. FICZ (200 nM) or kynurenine (50 mM) was added at the start of culture. Cells were then stimulated for 6 h in the presence of PMA, ionomycin, and brefeldin A, at which time they were harvested and surfaced stained for CD4. This was followed by intracellular staining for IL-17. Numbers are the percent of live CD4 T cells expressing IL-

17. Solid thick line is experimental histogram, and shaded histogram is Downloaded from control (anti-CD3/CD28, TGF-b, and IL-6 alone). Number above gate is the percent IL-17 positive of experimental histogram. Percent IL-17 posi- tive of control histogram is 15.4. Figure is representative of two inde- pendent experiments.

and stayed in solution throughout the assays. We did find that kynurenine in bicarbonate buffer did not strongly activate the DRE http://www.jimmunol.org/ in the luciferase assay when made fresh, but after a few days in culture displayed strong activity, which may correlate with solu- bility issues. When we examined kynurenine in HCL at 3 wk and FIGURE 5. TGF-b upregulates AHR expression, potentiating activation 7 mo by HPLC, it continued to show minimal breakdown, in- of the DRE by kynurenine. A, Left panel, Total RNA was extracted from dicating there is stability when placed in this solution. It is well naive T cells (separated by magnetic beads), either fresh or after 20 h or 3 d of known in the literature that the isoforms of kynurenine can be culture, and qPCR was performed for AHR expression. Culture conditions modified depending on the surrounding milieu in vivo (50–53), included Ab stimulation, FICZ 200 nM, kynurenine 50 mM, or TGF-b and it is difficult to rule out that this may occur to some degree in 3ng/ml. Right panel, Cyp1b1 mRNA expression was also examined by by guest on October 2, 2021 qPCR at 20 h and 3 d after the same culture conditions. The FICZ sample our assays. Nevertheless, if it is a metabolite of kynurenine was not tested at 3 d. B, To assess whether AHR upregulation secondary to binding to the AHR, it is still formed early in the kynurenine TGF-b would potentiate binding of ligands to the AHR, Cyp1a1 and Cyp1b1 pathway, generated by effects of IDO on tryptophan. This does not expression after kynurenine exposure in culture for 3 d with and without diminish the importance of these findings, still linking IDO and TGF-b was measured. The response is strongly enhanced after TGF-b the kynurenine pathway to the AHR. exposure, shifting the curve up significantly, implying that TGF-b does It is fascinating that some ligands (like FICZ) activate the AHR, potentiate the binding of kynurenine to the AHR when this ligand is as seen by the luciferase assays and mRNA analysis, but do not present in the culture. Post ANOVA testing comparisons are against the lead to FoxP3 expression. Other ligands (kynurenine) activate this vehicle control. pp , 0.05; pppp , 0.001. nt, not tested. same receptor and do lead to FoxP3 on T cells. We have considered how different ligands might activate the same receptor and lead to potentiates the effect of kynurenine binding by increasing the disparate outcomes in protein generation. One hypothesis that we amount of receptor, far beyond what was seen with any ligand are investigating is that kynurenine itself may be enzymatically already present in the media. modified by the cytochrome P450 enzymes that are induced by the A second important question is whether kynurenine undergoes AHR, whereas FICZ may not. The product of this modification may catabolism, and is it actually a metabolite or breakdown product directly lead to FoxP3 induction as opposed to a direct effect of that is binding the AHR. It is possible that kynurenine in solution, kynurenine itself. This would explain this differential effect of like other small molecules, undergoes breakdown through various these AHR ligands. This hypothesis is further supported by the fact mechanisms. We used a 0.5 M solution of HCL to get kynurenine that TGF-b can generate FoxP3 in our assays as early as 3 d, into solution, as per the recommendations of the manufacturer whereas the kynurenine-induced generation is typically not seen regarding maximum solubility. In an effort to test for breakdown of prior to 5 d (data not shown), despite the fact that the AHR is kynurenine, we have conducted HPLC with diode array detection to activated by kynurenine within a few hours. We will test this analyze kynurenine in this solution, as well as in buffered solution theory further by using known inhibitors of Cyp1a1 and Cyp1b1 at a physiologic pH (50–53). In addition, the manufacturer has enzymes [trans-stilbenes (54)] and experimenting with our re- tested kynurenine in HCL by HPLC and found it to be .98% cently generated DRE cluster null mice (55), which have dysfunc- pure, which is similar to our findings (Supplemental Fig. 5). As tional Cyp1a1 and Cyp1a2 enzymes. can be seen in the figure, kynurenine dissolved in HCL at Regarding the concentration of kynurenine used in these 1 d shows minimal decomposition, whereas kynurenine dissolved experiments, the dose chosen was physiologic (44, 56), comparable in buffer shows decreased purity. It was more difficult to dissolve to levels encountered in humans in areas of inflammation. It is also kynurenine in buffered solution, which took up to 8 h at 37˚C, as consistent with observations on the amounts of kynurenine and opposed to kynurenine in HCL, which went rapidly into solution other tryptophan breakdown products generated in vitro by DCs The Journal of Immunology 3197

(57) (5–50 mM range). In areas without an ongoing immune re- 3. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immu- nologic self-tolerance maintained by activated T cells expressing IL-2 receptor sponse, concentrations of the kynurenines are significantly lower, alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes in the nanomolar range, but increase 1000-fold in microenviron- various autoimmune diseases. J. Immunol. 155: 1151–1164. ments of inflammation. The fact that kynurenine concentrations 4. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell de- velopment by the transcription factor Foxp3. Science 299: 1057–1061. in vivo only reach the doses examined in our assays in areas of 5. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, and A. Rudensky. 2002. inflammation could serve as a way to localize the IDO-AHR– Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat. dependent Treg generation. Conceivably, this would allow an im- Immunol. 3: 33–41. 6. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard, mune response to commence, leading to IFN-g and other inflam- C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006. matory cytokines, which would then induce DCs to generate IDO. Transforming growth factor-beta induces development of the T(H)17 lineage. Ultimately, kynurenine levels would build up sufficiently to in- Nature 441: 231–234. 7. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. teract with the AHR and generate Tregs (and AHR expression 2006. TGFbeta in the context of an inflammatory cytokine milieu supports de would be enhanced by TGF-b expression, further potentiating novo differentiation of IL-17-producing T cells. Immunity 24: 179–189. the effects of kynurenine), hence muting the immune response. 8. Afzali, B., G. Lombardi, R. I. Lechler, and G. M. Lord. 2007. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation In areas without inflammation, kynurenine doses would be inad- and autoimmune . Clin. Exp. Immunol. 148: 32–46. equate to generate significant amounts of AHR-dependent Tregs. 9. O’Shea, J. J., and W. E. Paul. 2010. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 327: 1098–1102. These data shed light on the direct mechanism of IDO, which has 10. Schmidt, J. V., L. A. Carver, and C. A. Bradfield. 1993. Molecular character- been controversial. Two theories on the function of IDO in Treg ization of the murine Ahr gene. Organization, promoter analysis, and chromo- generation have been proposed. The first is that IDO leads to somal assignment. J. Biol. Chem. 268: 22203–22209. 11. Mandal, P. K. 2005. Dioxin: a review of its environmental effects and its aryl tryptophan depletion, and this relative starvation leads to cell cycle hydrocarbon receptor biology. J. Comp. Physiol. B 175: 221–230. Downloaded from arrest in some populations, favoring generation of Tregs (32). The 12. Fernandez-Salguero, P., T. Pineau, D. M. Hilbert, T. McPhail, S. S. Lee, second theory is that the tryptophan catabolites themselves have S. Kimura, D. W. Nebert, S. Rudikoff, J. M. Ward, and F. J. Gonzalez. 1995. Immune system impairment and hepatic fibrosis in mice lacking the dioxin- a more direct role in the generation of Tregs, supported by some binding Ah receptor. Science 268: 722–726. observational studies but without any clearly understood mecha- 13. Schmidt, J. V., G. H. Su, J. K. Reddy, M. C. Simon, and C. A. Bradfield. 1996. nism (30, 31). The data in this paper strongly support the second Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc. Natl. Acad. Sci. USA 93: 6731–6736. theory, and experiments to elucidate the exact molecular responses 14. Thomas, R. S., S. G. Penn, K. Holden, C. A. Bradfield, and D. R. Rank. 2002. elicited after binding of kynurenine or FICZ to the AHR are un- Sequence variation and phylogenetic history of the mouse Ahr gene. Pharma- http://www.jimmunol.org/ cogenetics 12: 151–163. derway. It is still plausible that tryptophan depletion continues to 15. Walker, M. K., S. E. Heid, S. M. Smith, and H. I. Swanson. 2000. Molecular char- play a role in the IDO-dependent generation of Tregs, and this acterization and developmental expression of the aryl hydrocarbon receptor from paper does not exclude this. In fact, as mentioned in our Materials the chick embryo. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 126: 305–319. 16. Nguyen, L. P., and C. A. Bradfield. 2008. The search for endogenous activators and Methods section, we did perform the assays with naive T cells of the aryl hydrocarbon receptor. Chem. Res. Toxicol. 21: 102–116. alone exposed to kynurenine in a low tryptophan media (F10), 17. McMillan, B. J., and C. A. Bradfield. 2007. The aryl hydrocarbon receptor sans based on previously published data (32) and our own experience xenobiotics: endogenous function in genetic model systems. Mol. Pharmacol. 72: 487–498. that the yield in this media is superior to when RPMI media (with 18. Quintana, F. J., A. S. Basso, A. H. Iglesias, T. Korn, M. F. Farez, E. Bettelli, higher tryptophan content) is used. We have contemplated that the M. Caccamo, M. Oukka, and H. L. Weiner. 2008. Control of T(reg) and T(H) by guest on October 2, 2021 reason low tryptophan solutions lead to more regulatory cells is 17 cell differentiation by the aryl hydrocarbon receptor. Nature 453: 65–71. 19. Veldhoen, M., K. Hirota, A. M. Westendorf, J. Buer, L. Dumoutier, J. C. Renauld, that tryptophan itself is known to form other potential ligands of and B. Stockinger. 2008. The aryl hydrocarbon receptor links TH17-cell-mediated the AHR, including FICZ. In our in vitro assay, we use a low autoimmunity to environmental . Nature 453: 106–109. 20. Veldhoen, M., K. Hirota, J. Christensen, A. O’Garra, and B. Stockinger. 2009. tryptophan solution with kynurenine as the primary AHR ligand. Natural agonists for aryl hydrocarbon receptor in culture medium are essential Perhaps this shifts the balance toward generation of regulatory for optimal differentiation of Th17 T cells. J. Exp. Med. 206: 43–49. cells, whereas when tryptophan is present to start with, additional 21. Kimura, A., T. Naka, K. Nohara, Y. Fujii-Kuriyama, and T. Kishimoto. 2008. Aryl hydrocarbon receptor regulates Stat1 activation and participates in the ligands can be generated that shift the balance toward an effector development of Th17 cells. Proc. Natl. Acad. Sci. USA 105: 9721–9726. response. It is possible that both mechanisms play a role and in 22. Hauben, E., S. Gregori, E. Draghici, B. Migliavacca, S. Olivieri, fact may explain why DCs are so potent at generating Tregs in M. Woisetschla¨ger, and M. G. Roncarolo. 2008. Activation of the aryl hydro- carbon receptor promotes allograft-specific tolerance through direct and coculture assays (as they generate IDO and deplete the tryptophan dendritic cell-mediated effects on regulatory T cells. Blood 112: 1214–1222. present in the assays). Of course, other mechanisms, including 23. Stockinger, B., M. Veldhoen, and K. Hirota. 2009. Modulation of Th17 de- cell–cell contact and cytokine release, may also be involved. velopment and function by activation of the aryl hydrocarbon receptor—the role of endogenous ligands. Eur. J. Immunol. 39: 652–654. The data in this manuscript further our understanding of the 24. Marshall, N. B., W. R. Vorachek, L. B. Steppan, D. V. Mourich, and emerging role of the AHR as a key player in the differentiation of N. I. Kerkvliet. 2008. Functional characterization and gene expression analysis of CD4+ CD25+ regulatory T cells generated in mice treated with 2,3,7,8- T cells to Tregs, as well as the ultimate balance of regulatory and tetrachlorodibenzo-p-dioxin. J. Immunol. 181: 2382–2391. effector responses in immunity. As this pathway is further charac- 25. Nguyen, L. P., E. L. Hsu, G. Chowdhury, M. Dostalek, F. P. Guengerich, and terized, improved understanding will enhance our knowledge of C. A. Bradfield. 2009. D-amino acid oxidase generates agonists of the aryl hydrocarbon receptor from D-tryptophan. Chem. Res. Toxicol. 22: 1897–1904. T cell differentiation and yield new strategies of modulating the bal- 26. Jo¨nsson, M. E., D. G. Franks, B. R. Woodin, M. J. Jenny, R. A. Garrick, ance of regulation and effector response using ligands of the AHR. L. Behrendt, M. E. Hahn, and J. J. Stegeman. 2009. The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces mul- tiple CYP1 genes via AHR2 in zebrafish. Chem. Biol. Interact. 181: 447–454. Disclosures 27. Stone, T. W., and L. G. Darlington. 2002. Endogenous kynurenines as targets for C.A.B. has served as a scientific consultant to Dow Chemical on issues drug discovery and development. Nat. Rev. Drug Discov. 1: 609–620. related to dioxin toxicity. 28. Mellor, A. L., and D. H. Munn. 2004. IDO expression by dendritic cells: tol- erance and tryptophan catabolism. Nat. Rev. Immunol. 4: 762–774. 29. Chen, W., X. Liang, A. J. Peterson, D. H. Munn, and B. R. Blazar. 2008. The indole- amine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell- References induced adaptive T regulatory cell generation. J. Immunol. 181: 5396–5404. 1. Gershon, R. K. 1974. T cell control of antibody production. Contemp. Top. 30. Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and Immunobiol. 3: 1–40. G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibi- 2. Hedrick, S. M., R. N. Germain, M. J. Bevan, M. Dorf, I. Engel, P. Fink, tion of T and natural killer cell proliferation induced by indoleamine 2,3- N. Gascoigne, E. Heber-Katz, J. Kapp, Y. Kaufmann, et al. 1985. Rearrangement dioxygenase. J. Exp. Med. 196: 459–468. and transcription of a T-cell receptor beta-chain gene in different T-cell subsets. 31. Terness, P., T. M. Bauer, L. Ro¨se, C. Dufter, A. Watzlik, H. Simon, and G. Opelz. Proc. Natl. Acad. Sci. USA 82: 531–535. 2002. Inhibition of allogeneic T cell proliferation by indoleamine 2,3- 3198 AHR, IDO, AND IMMUNITY

dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan 44. Heyes, M. P., C. Y. Chen, E. O. Major, and K. Saito. 1997. Different kynurenine metabolites. J. Exp. Med. 196: 447–457. pathway enzymes limit quinolinic acid formation by various human cell types. 32. Fallarino, F., U. Grohmann, S. You, B. C. McGrath, D. R. Cavener, C. Vacca, Biochem. J. 326: 351–356. C. Orabona, R. Bianchi, M. L. Belladonna, C. Volpi, et al. 2006. The combined 45. Zheng, S. G., J. D. Gray, K. Ohtsuka, S. Yamagiwa, and D. A. Horwitz. 2002. effects of tryptophan starvation and tryptophan catabolites down-regulate T cell Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- receptor zeta-chain and induce a regulatory phenotype in naive T cells. J. Immu- precursors. J. Immunol. 169: 4183–4189. nol. 176: 6752–6761. 46. Razmara, M., B. Hilliard, A. K. Ziarani, Y. H. Chen, and M. L. Tykocinski. 2008. 33. Jux, B., S. Kadow, and C. Esser. 2009. Langerhans cell maturation and contact CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory hypersensitivity are impaired in aryl hydrocarbon receptor-null mice. J. Immu- T cells. Int. Immunol. 20: 471–483. nol. 182: 6709–6717. 47. Zheng, S. G., J. H. Wang, W. Stohl, K. S. Kim, J. D. Gray, and D. A. Horwitz. 34. Vogel, C. F., S. R. Goth, B. Dong, I. N. Pessah, and F. Matsumura. 2008. Aryl 2006. TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 hydrocarbon receptor signaling mediates expression of indoleamine 2,3- and generate adaptive CD4+CD25+ regulatory cells. J. Immunol. 176: 3321– dioxygenase. Biochem. Biophys. Res. Commun. 375: 331–335. 35. Denison, M. S., and S. R. Nagy. 2003. Activation of the aryl hydrocarbon re- 3329. ceptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. 48. Kaiko, G. E., J. C. Horvat, K. W. Beagley, and P. M. Hansbro. 2008. Immuno- Pharmacol. Toxicol. 43: 309–334. logical decision-making: how does the immune system decide to mount a help- 36. DiNatale, B. C., I. A. Murray, J. C. Schroeder, C. A. Flaveny, T. S. Lahoti, er T-cell response? Immunology 123: 326–338. E. M. Laurenzana, C. J. Omiecinski, and G. H. Perdew. 2010. Kynurenic acid is 49. Negishi, T., Y. Kato, O. Ooneda, J. Mimura, T. Takada, H. Mochizuki, a potent endogenous aryl hydrocarbon receptor ligand that synergistically induces M. Yamamoto, Y. Fujii-Kuriyama, and S. Furusako. 2005. Effects of aryl interleukin-6 in the presence of inflammatory signaling. Toxicol. Sci. 115: 89–97. hydrocarbon receptor signaling on the modulation of TH1/TH2 balance. J. 37. Rieber, N., and B. H. Belohradsky. 2010. AHR activation by tryptophan— Immunol. 175: 7348–7356. pathogenic hallmark of Th17-mediated inflammation in eosinophilic fasciitis, 50. Vazquez, S., R. J. Truscott, R. A. O’Hair, A. Weimann, and M. M. Sheil. 2001. A -myalgia-syndrome and toxic oil syndrome? Immunol. Lett. 128: study of kynurenine fragmentation using electrospray tandem mass spectrome- 154–155. try. J. Am. Soc. Mass Spectrom. 12: 786–794. 38. Matheu, M. P., D. Sen, M. D. Cahalan, and I. Parker. 2008. Generation of bone 51. Taylor, L. M., J. Andrew Aquilina, J. F. Jamie, and R. J. Truscott. 2002. UV filter marrow derived murine dendritic cells for use in 2-photon imaging. J. Vis. Exp. 17: pii. instability: consequences for the human lens. Exp. Eye Res. 75: 165–175. Downloaded from 39. Hwu, P., M. X. Du, R. Lapointe, M. Do, M. W. Taylor, and H. A. Young. 2000. 52. Taylor, L. M., J. Andrew Aquilina, J. F. Jamie, and R. J. Truscott. 2002. Glu- Indoleamine 2,3-dioxygenase production by human dendritic cells results in the tathione and NADH, but not ascorbate, protect lens proteins from modification inhibition of T cell proliferation. J. Immunol. 164: 3596–3599. by UV filters. Exp. Eye Res. 74: 503–511. 40. Kimura, A., T. Naka, T. Nakahama, I. Chinen, K. Masuda, K. Nohara, Y. Fujii- 53. Vazquez, S., J. A. Aquilina, J. F. Jamie, M. M. Sheil, and R. J. Truscott. 2002. Kuriyama, and T. Kishimoto. 2009. Aryl hydrocarbon receptor in combination Novel protein modification by kynurenine in human lenses. J. Biol. Chem. 277: with Stat1 regulates LPS-induced inflammatory responses. J. Exp. Med. 206: 4867–4873. 2027–2035. 54. Kim, S., H. Ko, J. E. Park, S. Jung, S. K. Lee, and Y. J. Chun. 2002. Design, 41. Jung, I. D., C. M. Lee, Y. I. Jeong, J. S. Lee, W. S. Park, J. Han, and Y. M. Park. synthesis, and discovery of novel trans-stilbene analogues as potent and selective

2007. Differential regulation of indoleamine 2,3-dioxygenase by lipopolysaccha- http://www.jimmunol.org/ ride and interferon gamma in murine bone marrow derived dendritic cells. FEBS human cytochrome P450 1B1 inhibitors. J. Med. Chem. 45: 160–164. Lett. 581: 1449–1456. 55. Nukaya, M., S. Moran, and C. A. Bradfield. 2009. The role of the dioxin- 42. Rushing, S. R., and M. S. Denison. 2002. The silencing mediator of retinoic acid responsive element cluster between the Cyp1a1 and Cyp1a2 loci in aryl hydro- and thyroid hormone receptors can interact with the aryl hydrocarbon (Ah) re- carbon receptor biology. Proc. Natl. Acad. Sci. USA 106: 4923–4928. ceptor but fails to repress Ah receptor-dependent gene expression. Arch. Bio- 56. Schro¨cksnadel, K., B. Wirleitner, C. Winkler, and D. Fuchs. 2006. Monitoring chem. Biophys. 403: 189–201. tryptophan metabolism in chronic immune activation. Clin. Chim. Acta 364: 43. Andrieux, L., S. Langoue¨t, A. Fautrel, F. Ezan, J. A. Krauser, J. F. Savouret, 82–90. F. P. Guengerich, G. Baffet, and A. Guillouzo. 2004. Aryl hydrocarbon receptor 57. Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, C. Orabona, A. Spreca, activation and cytochrome P450 1A induction by the mitogen-activated protein M. C. Fioretti, and P. Puccetti. 2002. T cell apoptosis by tryptophan catabolism. kinase inhibitor U0126 in hepatocytes. Mol. Pharmacol. 65: 934–943. Cell Death Differ. 9: 1069–1077. by guest on October 2, 2021