CCR6 Regulates the Migration of Inflammatory and Regulatory T Cells Tomohide Yamazaki, Xuexian O. Yang, Yeonseok Chung, Atsushi Fukunaga, Roza Nurieva, Bhanu Pappu, Natalia This information is current as Martin-Orozco, Hong Soon Kang, Li Ma, Athanasia D. of September 25, 2021. Panopoulos, Suzanne Craig, Stephanie S. Watowich, Anton M. Jetten, Qiang Tian and Chen Dong J Immunol 2008; 181:8391-8401; ;

doi: 10.4049/jimmunol.181.12.8391 Downloaded from http://www.jimmunol.org/content/181/12/8391

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCR6 Regulates the Migration of Inflammatory and Regulatory T Cells1

Tomohide Yamazaki,* Xuexian O. Yang,* Yeonseok Chung,* Atsushi Fukunaga,* Roza Nurieva,* Bhanu Pappu,* Natalia Martin-Orozco,* Hong Soon Kang,‡ Li Ma,§ Athanasia D. Panopoulos,* Suzanne Craig,† Stephanie S. Watowich,* Anton M. Jetten,‡ Qiang Tian,§ and Chen Dong2*

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-␤ and requires two nuclear receptors, ROR␣ and ROR␥. Th17 cells also express the ␤ ␥

CCR6 ligand CCL20, which is induced synergistically by TGF- and IL-6, which requires STAT3, ROR and IL-21. Th17 cells, Downloaded from by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration. The Journal of Immunology, 2008, 181: 8391–8401.

he CD4ϩ Th cells are central organizers in immune re- diseases, such as animal models for multiple sclerosis and rheu- http://www.jimmunol.org/ sponses. Naive T cells, upon activation by APCs, differ- matoid arthritis (5, 6, 11, 12). Neutralizing IL-17 strongly inhibited T entiate into -expressing effector Th cells, which experimental autoimmune encephalomyelitis (EAE)3 (5, 6). have been historically classified into Th1 and Th2 lineages based Th17 differentiation is potently driven by TGF-␤ and IL-6 (13– on their cytokine secretion and immune regulatory function (1, 2). 15), which is reinforced by IL-23 (16, 17). Recently, IL-21 was Th1 cells regulate cellular and Ag presentation through reported as an autocrine factor induced by IL-6 to regulate Th17 secreting IFN-␥, while Th2 cells mediate the humoral and allergic differentiation (17–19). For cytokine-mediated Th17 differentia- responses by producing IL-4, IL-5, and IL-13. Th1 and Th2 cells tion, STAT3 was found to be a necessary (16, differ not only in their immune function but also in their migratory 20). STAT3 may function by regulating the expression of lineage- regulation; they express distinct receptors which me- specific master transcription factor(s), such as ROR␥t and ROR␣, by guest on September 25, 2021 diate their selective recruitments into tissues depending on the two orphan nuclear receptors that were recently shown to regulate types of pathogen infections or immune responses (3). Th17 differentiation (21, 22). In addition to Th1 and Th2, a third subset of Th cells, namely, In contrast to -promoting Th17 cells, -de- Th17, was recently identified (4–6). Th17 cells were first marked rived natural regulatory T cells (nTreg) represent a unique sub- by IL-17 production and subsequently were found to also secret population of CD4ϩ T cells that inhibits T cell proliferation and IL-17F and IL-22 (5, 7–9). IL-17 has been previously found to be autoimmune responses (23). The hallmark of nTreg cells is the involved in host defenses against bacterial infection (10). Re- expression of Foxp3 transcription factor, which serves as a master cently, Th17 cells have been strongly associated in autoimmune regulator of Treg cell development and function (24). Under the influence of TGF-␤, Foxp3 can be also induced in naive T cells’ periphery and the resulting inducible Treg (iTreg) cells exhibit the *Department of Immunology, †Veterinary Medicine & Surgery, M.D. Anderson Cancer same suppressive phenotype as nTreg (24). Interestingly, both ‡ Center, Houston, TX 77030; Cell Biology Section, Laboratory of Respiratory Biology, ␤ National Institute on Environmental Sciences, National Institutes of Health, Research iTreg and Th17 cell development depends on TGF- . Thus, there Triangle Park, NC 27709; and §Institute for Systems Biology, Seattle, WA 98103 is not only functional antagonism between Th17 cells and Treg Received for publication July 23, 2008. Accepted for publication October 20, 2008. cells in autoimmunity, but also a reciprocal regulation in the gen- ␤ The costs of publication of this article were defrayed in part by the payment of page eration of these cells. Although TGF- induces Foxp3 expression, charges. This article must therefore be hereby marked advertisement in accordance IL-6 and IL-21 inhibit this regulation and along with TGF-␤ drive with 18 U.S.C. Section 1734 solely to indicate this fact. Th17 differentiation. 1 This work was supported by research grants from the National Institutes of Despite recent progress on understanding the regulation and Health (to C.D.), an Intramural Research Program of the National Institute on Environmental Health Sciences, National Institutes of Health (to A.M.J.), the function of Treg and Th17 cells, it is unclear how their recruit- Leukemia and Lymphoma Society (to C.D.) and M.D. Anderson Cancer Center (to ments into inflammatory sites are regulated. In this study, we re- C.D. and S.S.W.), and the Gillson Longenbaugh Foundation (to S.S.W.). T.Y. port that both Treg and Th17 cells express CCR6. Th17 cells also received a fellowship from the Odyssey Program and The Kimberly-Clark Foun- dation Award for Scientific Achievement at MD Anderson Cancer Center. R.N. is express the CCR6 ligand CCL20, through which Th17 promotes a recipient of a Scientist Development Grant from the American Heart Associa- the migration of Th17 and Treg cells. CCR6 deficiency in T cells tion. C.D. is a Trust Fellow of the MD Anderson Cancer Center, a Cancer Re- search Institute Investigator, a Leukemia and Lymphoma Society Scholar, and an American Lung Association Career Investigator. 2 Address correspondence and reprint requests to Dr. Chen Dong, Department of 3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyeli- Immunology and Center for Cancer Immunology Research, University of Texas tis; nTreg, natural regulatory T; Treg, regulatory T; iTreg, inducible Treg; KO, knock- M.D. Anderson Cancer Center, Houston, TX 77030. E-mail address: cdong@ out; BM, bone marrow; MOG, myelin oligodendrocyte glycoprotein; LN, lymph mdanderson.org node; WT, wild type. www.jimmunol.org 8392 CCR6-MEDIATED MIGRATION OF Th17 AND Treg CELLS decreases the susceptibility to autoimmune diseases. Lack of tonicity; 2, wobbly gait; 3, hind limb paralysis; 4, hind limb and forelimb CCR6 in Th17 cells inhibits their own as well as Treg recruitment paralysis; and 5, death. into inflammatory tissues. Similarly, CCR6 on Treg cells is also Th17 transfer EAE important for their recruitment into inflammatory tissues. Our data thus indicate an essential role of CCR6 in Treg and Th17 migration C57BL/6 or CCR6 KO mice were immunized s.c. at the dorsal flanks with 150 ␮gofMOG peptide emulsified in CFA. The following day, per- in autoimmune disease and suggest that Th17 induces both ampli- 35–55 tussis toxin was injected i.p. at a dose of 500 ng/mouse. Seven days after fication and inhibition mechanisms on inflammatory responses immunization, a single-cell suspension of spleen cells was prepared from via CCR6. the mice and cultured with MOG35–55 peptide and IL-23 for 7 days. MOG- reactive CD4ϩ T cells were purified by AutoMACS (Miltenyi Biotec) with anti-CD4 microbeads (Miltenyi Biotec). Purified CD4ϩ T cells (Ͼ 95% Materials and Methods ϩ ϩ CD3 CD4 ;4ϫ 106) were adoptively transferred into C57BL/6 mice Mice followed by MOG immunization on the second day. Pertussis toxin was C57BL/6 mice, CCR6 knockout (KO), Rag1 KO, B6.SJL-PtprcaPepcb/ injected i.p. on the following day. Signs of EAE were assigned scores on BoyJ (CD45.1) mice were purchased from The Jackson Laboratory. ROR␥ a scale of 1–5 as mentioned above. KO, ROR␣/␥ double mutant mice, STAT3 conditional KO, and IL-21 KO mice have been previously described (16, 19, 22). All animal experiments Treg transfer in EAE have been conducted under the animal protocols approved by M.D. Ander- CD4ϩCD25high Treg cells from wild-type (WT) or CCR6Ϫ/Ϫ mice were son Cancer Center Institutional Animal Care and Usage Committee. isolated by FACS sorting. Isolated Treg cells were adoptively transferred into RAG1Ϫ/Ϫ (0.5 ϫ 106/mouse along with 4.5 ϫ 106 naive T cells) or T cell differentiation ϩ CD45.1 (1 ϫ 106/mouse) mice. The mice were immunized with MOG Downloaded from CD4ϩCD25ϪCD62LhighCD44low cells were isolated by FACS sorting as peptide with CFA on the second day. Pertussis toxin was injected on the described before (16, 19). Naive CD4ϩ T cells were stimulated with anti- following day. CD3 (3 ␮g/ml) and anti-CD28 (2 ␮g/ml). For Th1 differentiation, IL-12 (10 ng/ml) and anti-IL-4 (5 ␮g/ml; 11B11) were used additionally. For Th2 Statistical analysis differentiation, IL-4 (10 ng/ml) and anti-IFN-␥ (5 ␮g/ml; XMG 1.2) were Significant differences between two groups were analyzed by the Mann- used additionally. For Th17 cell differentiation, IL-6 (50 ng/ml; Pepro- Whitney U test. Values of p Ͻ 0.05 and p Ͻ 0.01 were considered Tech), TGF-␤ (10 ng/ml; PeproTech), IL-23 (50 ng/ml; R&D Systems), significant. anti-IL-4 (5 ␮g/ml; 11B11), and anti-IFN-␥ (5 ␮g/ml; XMG 1.2) were used http://www.jimmunol.org/ additionally. For Treg differentiation, TGF-␤ (10 ng/ml), anti-IL-4 (5 ␮g/ ml; 11B11), and anti-IFN-␥ (5 ␮g/ml; XMG 1.2) were used additionally. Results To examine intracellular cytokine staining, at 4 days of the culture, the CCR6 is highly expressed on Th17 cells cells were washed and restimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and GolgiPlug (BD Biosciences) for 5 h. The cells were perme- In a gene expression profiling analysis of in vitro-differentiated abilized with a Cytofix/Cytoperm kit (BD Biosciences). Th1, Th2, and Th17 cells, we found that the expression of CCR6 was significantly elevated in Th17 cells compared with Th1 or Th2 Quantitative real-time PCR cells (data not shown). We thus confirmed CCR6 expression in Total RNA was prepared from T cells with the use of TRIzol reagent Th1, Th2, and Th17 cells. Assessed by real-time RT-PCR, Th17 (Invitrogen). cDNAs were synthesized with Superscript reverse transcrip- cells, but not Th1 and Th2 cells, highly expressed CCR6 mRNA by guest on September 25, 2021 tase and oligo(dT) primers (Invitrogen) and gene expression was examined with a Bio-Rad iCycler Optimal System using a iQ SYBER Green real- (Fig. 1A). IL-17 mRNA was also specifically expressed in Th17 time PCR kit (Bio-Rad). The primers used were (5Ј–3Ј): CCR6: forward, cells, which indicated proper differentiation of Th17 cells in vitro. CCTCACATTCTTAGGACTGGAGC and reverse, GGCAATCAGAGCT We further analyzed which cytokine regulates CCR6 expression CTCGGA and CCL20: forward, ATGGCCTGCGGTGGCAAGCGTCTG in Th17 cells. Time-dependent levels of CCR6 mRNA were ana- and reverse, TAGGCTGAGGAGGTTCACAGCCCT. The data were nor- lyzed in T cells after treatment with various Th17-regulating cy- malized to ␤-actin gene expression as a reference. tokines for 1, 2, or 5 days. TGF-␤, but not any other tested cyto- In vitro migration assay kine, rapidly and strongly induced CCR6 mRNA expression in T A migration assay was performed in 24-well plates (Costar) carrying Tr- cells at early time points of T cell activation such as days 1 and 2 answell-permeable supports with a 3-␮m polycarbonate membrane for T (Fig. 1B). IL-6 or IL-21 did not further enhance this induction by cells. Recombinant CCL20 or the supernatants collected from Th17 cells TGF-␤. These results indicate that TGF-␤ is a main factor for restimulated with anti-CD3 for 3 days were placed on the lower chamber. ϩ induction of CCR6 expression in Th cells. On day 5, the expres- Th1, Th2, Th17, Treg, or naive CD4 T cells from CCR6 KO mice or sion of CCR6 mRNA was reduced in T cells. Although T cells C57BL/6 mice were placed on the upper wells of Transwell membranes ␤ containing 100 ␮l of RPMI 1640 medium. A total of 5 ϫ 105 T cells was treated with TGF- alone still exhibited elevated levels of CCR6 ␤ incubated for7hat37°C and 5% CO2 atmosphere. T cells on the upper mRNA expression, cells treated with both TGF- and IL-6 showed surface of membranes were completely removed by washing with distilled the highest CCR6 and IL-17 mRNA expression (Fig. 1B). These water. Migrated cells were fixed with methanol and stained with Giemsa results indicate that although CCR6 is highly expressed by Th17, (Sigma-Aldrich). Migrated cells were determined by counting the number of T cells stained with Giemsa in 10 randomly selected nonoverlapping its induction kinetics and cytokine regulation appear to differ from fields of the wells under light microscope. those of IL-17. ROR␥t and ROR␣ are key transcription factors mediating Th17 EAE induction cell differentiation (20–22). We thus assessed whether they regu- Bone marrow (BM) cells from female C57BL/6 or CCR6 KO mice were late CCR6 expression in Th17 cells. ROR␥-deficient, ROR␣/ prepared by flushing the femurs and tibiae with PRMI 1640 medium (Life ROR␥ doubly mutant, or their littermate control T cells were dif- Technologies). After washing them with ice-cold PBS twice, the cells (5 ϫ 6 ferentiated into Th17 cells, and CCR6 mRNA expression was 10 ) were resuspended with sterile PBS and injected i.v. into female Rag1 ␥ KO mice (6–7 wk old) that had been irradiated (750 rad) with a cesium examined as above. ROR -deficient T cells exhibited enhanced irradiator. Around 5 wk later, reconstituted mice that had normal numbers CCR6 mRNA expression compared with their WT controls (Fig. of CD4 and CD8 in the blood were used for EAE induction. For induction 1C). Also, T cells carrying an ROR␣ gene mutation did not have of EAE, mice were immunized s.c. at the dorsal flanks with 150 ␮gof impaired CCR6 expression either (data not shown). However, T MOG35–55 peptide (MEVGWYRSPFSROVHLYRNGK) emulsified in ␣ ␥ CFA on days 0 and 7. Pertussis toxin was given i.p. on days 1 and 8 with cells defective in both ROR and ROR greatly reduced CCR6 a dose of 500 ng/mouse. Clinical EAE symptoms were scored according to mRNA expression, indicating that these two nuclear receptors are a 1–5 scale as followed: 0, no symptoms; 1, limp tail or waddling with tail redundant in CCR6 regulation. The Journal of Immunology 8393 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. CCR6 is highly expressed by Th17 cells. A, In vitro-differentiated Th1, Th2, and Th17 cells were restimulated with anti-CD3 for4hfor real-time RT-PCR analysis. The data are expressed as the mean Ϯ SD of triplicate samples. Data shown were repeated twice with consistent results. B, FACS-sorted naive T cells were cultured with the indicated for 1, 2, and 5 days. After restimulation with anti-CD3, CCR6 and IL-17 expression was analyzed by real-time PCR. The data are expressed as the mean Ϯ SD of triplicate samples. Data shown were repeated twice with consistent results. C, Naive CD4ϩ T cells from ROR␥ KO, ROR␣/␥ double mutant, IL-21 KO, or WT mice were differentiated under Th17 conditions for 4 days. CCR6 expression was analyzed by real-time PCR. Real-time PCR results in A–C were normalized to Actb gene expression and relative gene expression levels are indicated. The data are expressed as the mean Ϯ SD of triplicate samples. A–C, The expression level in Th0 of WT or KO cells was set at 1. Data shown were repeated twice with consistent results. D, CCR6 expression was analyzed with allophycocyanin-labeled anti-CCR6 or allophycocyanin-labeled rat IgG in Th1, Th2, or Th17 cells. E, CCR6 expression on Th17 cells in the spleen, LN, and CNS of mice with EAE diseases. C57BL/6 mice were immunized with MOG peptide to induce EAE. The cells from spleen, LN, and CNS of mice that developed EAE were restimulated with MOG peptide for 1 day. CCR6 expression was analyzed with allophycocyanin-labeled anti-IFN-␥ and PE-labeled IL-17A in IL-17ϩ or IFN-␥ϩ CD4ϩ T cells. The data are expressed as the mean Ϯ SD of three mice. The results shown are a representative of at least two independent experiments.

We also assessed the effect of IL-21, an autocrine Th17 differ- tion of mouse Th17 cells express CCR6. Altogether, CCR6 is entiation inducer, on CCR6 regulation. IL-21-deficient T cells ex- highly expressed on Th17 cells in vitro and in vivo. hibited more enhanced CCR6 expression than WT cells when ex- posed to Th17 differentiation conditions (Fig. 1C). These results CCR6 expression on Treg cells indicate that IL-21 would not be necessary in CCR6 expression. Since TGF-␤ induces Foxp3 expression in T cells, we thus assessed In addition to mRNA expression, CCR6 protein expression was CCR6 expression in Treg cells. CD4ϩCD25ϪCD62LhighCD44low na- assessed in in vitro-differentiated Th1, Th2, and Th17 cells. CCR6 ive T cells were isolated by FACS sorting and differentiated into was only expressed in a small percentage of Th1 and Th2 cells, but iTreg cells with anti-CD3 and anti-CD28 with TGF-␤, anti-IFN-␥, was highly expressed in Th17 cells (Fig. 1D). In addition, CCR6 and anti-IL-4 for 5 days. Those activated with just anti-CD3/CD28 expression was assessed on IL-17- or IFN-␥-producing cells in the (Th0) served as controls. A high level of CCR6 mRNA expression spleen, draining lymph node (LN), or CNS of mice in which ex- was found in iTreg cells (Fig. 2A), when compared with naive perimental encephalomyelitis (EAE) diseases were induced. CCR6 CD4ϩ T cells and Th0 cells. CCR6 expression was also found on expression was detected on IL-17ϩ, but not IFN-␥ϩ cells in the iTreg cells at the protein level (Fig. 2B). In addition, CCR6 mRNA spleen, LN, and CNS (Fig. 1E). Although CCR6 appears to be was also highly expressed in sorted CD4ϩC25ϩ nTreg cells (Fig. expressed by all Th17 cells in humans, it appears that only a por- 2A). CD4ϩCD25ϩ cells in the spleen and peripheral LN, most of 8394 CCR6-MEDIATED MIGRATION OF Th17 AND Treg CELLS

ated in vitro (data not shown), confirming that CCL20 is indeed produced by Th17 cells. After anti-CD3 restimulation, Th17 cells produced CCL20 protein in a time-dependent manner (Fig. 3B). Thus, mouse Th17 cells, similar to their human counterparts (28, 29), express CCL20. To understand CCL20 regulation in T cells, naive T cells were cultured with various cytokines for up to 5 days and then analyzed for CCL20 mRNA expression. Although TGF-␤ or IL-6 alone weakly induced the expression of CCL20 mRNA on days 1 and 2, the combination of TGF-␤ and IL-6 or IL-21 exhibited strong syn- ergy in inducing CCL20 expression (Fig. 3C). CCL20 expression was maintained in T cells in the presence of TGF-␤/IL-6 or TGF- ␤/IL-21 combination on day 5 after activation (Fig. 3C). To determine the mechanisms underlying CCL20 expression in Th17 cells, the effect of Th17 differentiation regulating transcrip- tional factors such as ROR␣, ROR␥t, and STAT3(16) was as- sessed. Naive T cells from STAT3-deficient, ROR␥-deficient, both ROR␣/␥-deficient mice, or their WT controls were differentiated

under Th17 conditions and CCL20 expression was analyzed by Downloaded from real-time RT-PCR. CCL20 mRNA expression was abolished in STAT3-deficient T cells (Fig. 3D). ROR␥-deficient as well as ROR␣/␥ mutant T cells also showed greatly reduced CCL20 mRNA expression (Fig. 3E). Conversely, ROR␥ overexpression in T cells enhanced CCL20 mRNA expression (Fig. 3E).

Because CCL20 mRNA expression was regulated by IL-21 http://www.jimmunol.org/ along with TGF-␤, we also evaluated the effect of IL-21 deficiency on CCL20 expression in T cells. CCL20 mRNA expression was substantially decreased in IL-21-deficient T cells when they were activated under Th17-polarizing conditions (Fig. 3F). Our above results indicated that in addition to CCR6, Th17 cells also highly expressed its ligand CCL20. CCR6 and CCL20 ex- pression appears to be differentially regulated by cytokines and transcription factors. by guest on September 25, 2021 CCL20 does no regulate Th17 differentiation or their cytokine production A role has been reported for in costimulation and sub- ␥ FIGURE 2. CCR6 expression on Treg cells. A, CCR6 or Foxp3 expres- sequent IL-2, IFN- , or IL-4 production as well as proliferation of ϩ T cells (30). We assessed whether CCL20 acts on Th17 differen- sion was analyzed in cDNA of naive CD4 T cells, nTreg, Th0, and iTreg ϩ cells by real-time PCR. Results were normalized to Actb gene expression tiation and cytokine production of Th17 cells. Naive CD4 T cells and the relative gene expression levels are indicated. The expression level were differentiated into Th1 or Th17 cells with or without recom- in Th0 was set at 1. The data are expressed as the mean Ϯ SD of triplicate binant CCL20. The frequency of IFN-␥- or IL-17-producing cells samples. Data shown were repeated twice with consistent results. B, iTreg was not significantly different between T cells with or without ϩ ϩ cells were analyzed for the expression of CCR6 in gated CD4 CD25 recombinant CCL20 (Fig. 4A). To assess the CCL20 effect in cy- cells. Data shown were repeated twice with consistent results. C, The ex- ϩ ϩ tokine production from Th17 cells, T cells cultured with Th17 pression of CCR6 in nTreg cells was analyzed in gated CD4 CD25 cells differentiation condition for 4 days were restimulated with anti- in the spleen and popliteal LN. The results shown are a representative of CD3 or anti-CD3 and recombinant CCL20. No significant differ- two independent experiments. ence was found in IL-17 or IL-17F production between T cells with or without recombinant CCL20 (Fig. 4B). Also, WT or CCL6-deficient naive CD4ϩ T cells were cultured in Th17 differ- ϩ which were Foxp3 , expressed CCR6 protein as well (Fig. 2C). entiation conditions. The frequency of Th17-producing cells was Thus, in addition to Th17 cells, Treg cells generated in vitro and in not different between WT T cells and CCR6-defficient T cells, vivo also express CCR6, consistent with a previous report by which indicated that CCR6-deficient T cells did not have impair- Kleinewietfeld et al. (25). ment in Th17 differentiation (Fig. 4C). These results indicated that CCL20 has no effect on Th17 differentiation and cytokine produc- CCL20 is expressed in Th17 but not Treg cells tion of Th17 cells. CCL20, also called MIP-3␣, is the only ligand for CCR6 (26). In addition to CCR6, we also attempted to analyze the expression of Th17 cells regulate the migration of Th17 and Treg cells CCL20 in Th17 and Treg cells. CCL20 mRNA was specifically through CCR6 expressed in Th17 cells, but not Treg and other effector Th subsets We thus focused on the function of CCR6 in T cell directional (Fig. 3A). Moreover, we used a Th17 reporter mouse we recently chemotaxis (31). To determine the function of CCR6 in Th17 and reported, in which a red fluorescent protein sequence was inserted Treg cells, their migratory responses to CCL20 were first exam- into the IL-17F gene (27). By real-time PCR, CCL20 mRNA was ined by a Transwell assay. The chemotaxis was measured using the strongly expressed in red fluorescent protein-positive cells gener- method described by Boyden (32). Migration of Th17, but not Th1 The Journal of Immunology 8395 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CCL20 is specifically expressed by Th17 cells. A, Th1, Th2, Th17, nTreg, and iTreg cells were analyzed for CCL20 expression by real-time PCR. Real-time PCR results were normalized to Actb gene expression and relative gene expression levels are indicated. The expression level in iTreg was set at 1. The data are expressed as the mean Ϯ SD of triplicate samples. Data shown were repeated twice with consistent results. B, In vitro-differentiated Th17 cells were restimulated with anti-CD3 for up to 72 h. The cell culture supernatants were analyzed for CCL20 production by ELISA at the indicated p Ͻ 0.01. C, Naive ,ء .time points. The data are expressed as the mean Ϯ SD of triplicate wells. Data shown were repeated twice with consistent results T cells were cultured with the indicated cytokines for 5 days. After restimulation with anti-CD3, CCL20 expression was analyzed by real-time PCR. The expression level in was set at 1. The data are expressed as the mean Ϯ SD of triplicate samples. Data shown were repeated twice with consistent results. D–F, Naive CD4ϩ T cells from STAT3 KO (D), ROR␥ KO (E), ROR␣/␥ KO (E), or IL-21 KO mice (F) and their appropriate control mice were cultured for Th17 differentiation for 4 days. CCL20 expression was analyzed by real-time PCR after restimulation of T cells with anti-CD3. E, CCL20 expression was assessed in CD4ϩ T cells overexpressing ROR␥t with real-time PCR. Real-time PCR results in C–F were normalized to Actb gene expression and relative gene expression levels are indicated. The expression levels in Th0 of WT cells were set at 1. The data are expressed as the mean Ϯ SD of triplicate samples. The results shown (D–F) are a representative of two independent experiments. or Th2 cells, was detected in response to recombinant CCL20 (Fig. abolished in CCR6-deficient Th17 and Treg cells (Fig. 5D). This 5A). Likewise, nTreg cells, but not naive CD4ϩ T cells, also result indicates that Th17 cells produce CCL20 to attract other showed significant migration to CCL20 (Fig. 5B). CCR6-deficient Th17 as well as Treg cells through CCR6. Th17 or Treg cells completely lost the ability to migrate to CCL20 (Fig. 5C). This result indicated that the migration of Th17 and Treg CCR6 regulates migration and function of Th17 cells in EAE cells in response to CCL20 is dependent on CCR6. Although the populations of Th17 and Treg cells in lymphoid tis- Since Th17 cells express CCL20, we reasoned that activated sues have been well characterized, the distribution of Th17 and Th17 cells may regulate the migration of Th17 or Treg cells. To Treg cells in nonlymphoid tissues and the underlying mechanisms address this hypothesis, Th17, Treg, or Th1 cells from C57BL/6 or were poorly understood. To determine whether CCR6 plays any CCR6-deficient mice were tested with the culture supernatants role for the tissue localization of Th17 and Treg cells, CD4ϩ cells from anti-CD3-stimulated Th17 cells in Transwell plates. CCL20 that express IL-17 or Foxp3 were analyzed in a variety of tissues (5 ng/ml) was detected in the culture supernatant of stimulated in WT or CCR6-deficient mice on C57BL/6 background. The pop- Th17 (data not shown). WT Th17 and Treg, but not Th1, cells ulation of Treg cells was significantly reduced in the mesenteric migrated in response to the culture supernatants of activated Th17 LN (by 25%), Peyer’s patch (by 47%), and lung (by 56%). On the cells (Fig. 5D). However, this migratory response was completely other hand, the population of Th17 cells was also significantly 8396 CCR6-MEDIATED MIGRATION OF Th17 AND Treg CELLS Downloaded from

FIGURE 5. The migration of Th17 and Treg cells is mediated by CCR6 and CCL20 interaction. A, Transwell chemotaxis assay on Th1, Th2, and

Th17 cells in the presence of CCL20. The migrated cells were analyzed at http://www.jimmunol.org/ 7 h of culture. Data shown were repeated twice with consistent results. B, The migration of naive CD4ϩ T cells and nTreg cells was evaluated in response to CCL20. Data shown were repeated twice with consistent re- sults. C, WT or CCR6-deficient Th1, Th17, and Treg cells were examined in chemotaxis assays in response to CCL20. Data shown were repeated FIGURE 4. CCL20 does not regulate the differentiation or cytokine pro- ϩ twice with consistent results. D, The culture supernatants of anti-CD3- duction of Th17 cells. A, FACS-sorted naive CD4 T cells were stimulated stimulated Th17 cells were used for chemotaxis assay. The migration of ␮ ␮ with anti-CD3 (3 g/ml) and anti-CD28 (2 g/ml) under Th1 conditions Th1, Th17, or Treg cells from C57BL/6 or CCR6 KO mice was measured. ␮ (20 ng/ml IL-12 and 5 g/ml anti-IL-4) or Th17 conditions (10 ng/ml The result shown is representative of two independent experiments. All

␤ ␮ ␥ ␮ by guest on September 25, 2021 .p Ͻ 0.01 ,ء .TGF- , 50 ng/ml IL-6, 30 ng/ml IL-23, 5 g/ml anti-IFN- , and 5 g/ml data are expressed as the mean Ϯ SD of triplicate wells anti-IL-4) in the presence or absence of CCL20 (10 ng/ml) for 4 days. Intracellular cytokine staining was performed with allophycocyanin-la- beled anti-IFN-␥ and PE-labeled IL-17 after permeabilization. The results are representative of three independent experiments. B, In vitro- differen- KO mice exhibited significantly reduced EAE disease compared tiated Th17 cells were restimulated with anti-CD3 (3 ␮g/ml) with or with- with C57BL/6 mice in the early phase of EAE. However, CCR6 out CCL20 (10 ng/ml) for 3 days. The production of IL-17 and IL-17F in KO mice caught up with C57BL/6 mice in EAE disease (Fig. 6A). the culture supernatants was measured by ELISA. The data has no statis- Since CCR6 is also expressed by non-T cells, to determine CCR6 tical difference between T cells in the presence of CCL20 and absence of function directly in T cells during inflammatory diseases, Rag1Ϫ/Ϫ CCL20 (IL-17, p ϭ 0.322; IL-17F, p ϭ 0.391). Similar results were ob- mice were reconstituted with BM cells from WT or CCR6-defi- ϩ tained from three independent experiments. C, Naive CD4 T cells from cient mice. Five weeks later, CD4ϩ or CD8ϩ T cell populations in Ϫ/Ϫ WT or CCR6 mice were differentiated to Th17 cells with anti-CD3 (3 the blood of BM-transferred mice were analyzed by flow cytom- ␮ ␮ ␤ g/ml) and anti-CD28 (2 g/ml) in the presence of TGF- (10 ng/ml), IL-6 etry. The frequencies of CD4ϩ or CD8ϩ cell populations were (50 ng/ml), IL-23 (30 ng/ml), anti-IFN-␥ (5 ␮g/ml), and anti-IL-4 (5 ␮g/ similar between mice transferred with WT and KO BM and also ml). Four days after culture, the cells were analyzed for Th17 differentia- Ϯ tion in flow cytometry by staining with allophycocyanin-labeled IFN-␥ and similar to those of C57BL/6 mice (C57BL/6 CD4: 38.9 6.9%, ϭ ϭ PE-labeled IL-17. The data show no statistical difference between T cells p 0.457 compared with WT CD4, p 0.088 compared with KO from WT mice and CCR6Ϫ/Ϫ mice (IFN-␥ϩ cells, p ϭ 0.050; IL-17ϩ cells, CD4; WT CD4: 39.3 Ϯ 9.4% p ϭ 0.125 compared with KO CD4; p ϭ 0.144). Data shown are based on three mice in each group. The results KO CD4: 45.1 Ϯ 4.6%; C57BL/6 CD8: 18.9% Ϯ 1.5% p ϭ 0.309 are representative of two independent experiments. compared with WT CD8, p ϭ 0.370 compared with KO CD8; WT CD8: 17.3 Ϯ 4.1%, p ϭ 0.212 compared with KO CD8; KO CD8: 16.4 Ϯ 3.8%). Mice with good constitution of were reduced in the mesenteric LN (by 50%), Peyer’s patch (by 36%), immunized with MOG peptide and CFA to induce EAE. CCR6- and lung (by 59%) (data not shown). These results suggested that deficient chimeric mice showed significantly reduced EAE disease CCR6 is important for the tissue distribution of Th17 and Treg compared with WT chimeric mice (Fig. 6B). Because the disease cells in naive mice. score in CCR6-deficient mice caught up to the disease score in WT Th17 cells have been recently shown as a major pathogenic T mice in the later time point, the other chemokines expressed by cell subset in various autoimmune diseases, including EAE (12). Th17 and the inflammatory site might help the migration of Th17 Although there has been considerable progress on the regulation of cells. To understand the underlying causes of reduced disease in Th17 differentiation programs, regulation on Th17 migration to the CCR6-deficient chimeric mice, infiltrating cells in the CNS and the inflammatory tissues has been unclear. To assess the CCR6 effect cytokine production by CD4ϩ cells in both CNS and spleen were in inflammatory tissue, CCR6 KO and control C57BL/6 mice were analyzed. Although CD4ϩ or CD11bϩ cell percentages in the CNS immunized with MOG peptide with CFA to induce EAE. CCR6 were not different (data not shown), the absolute cell numbers of The Journal of Immunology 8397 Downloaded from http://www.jimmunol.org/

FIGURE 6. CCR6 deficiency in lymphocytes leads to inhibited EAE diseases. A, CCR6Ϫ/Ϫ or C57BL/6 mice (five mice in each group) were immunized with MOG peptide to induce EAE. Disease scores in each group were evaluated. The data are shown as the mean Ϯ SD. The data represent one of two by guest on September 25, 2021 experiments with similar results. B–K, Rag1Ϫ/Ϫ chimeric mice reconstituted with BM cells from C57BL/6 or CCR6Ϫ/Ϫ mice were immunized with MOG peptide to induce EAE. B, Disease scores in five mice in each group were evaluated. The data are expressed as the mean Ϯ SD of five mice. The data represent one of two experiments with similar results. C and D, The CNS-infiltrating cells were analyzed by staining with PerCP-labeled anti-CD4 and FITC-labeled anti-CD11b. The analysis was done on five mice in each group. E, The population of CD4ϩCD25ϩFoxp3ϩ cells was analyzed in the cells from the CNS. The analysis was done on five mice in each group. F–H, IL-17ϩ or IFN-␥ϩ cells in mononuclear cells in the CNS were analyzed in a CD4ϩ gate. The analysis was done on five mice in each group. Representative data from flow cytometry are shown in F. The data of cell percentage are expressed as the mean Ϯ SD of five mice. The absolute cell numbers of IL-17ϩ cell and IFN-␥ϩ cells were shown in G and H. I–K, IL-17ϩ or IFN-␥ϩ cells in spleen cells were analyzed in a CD4ϩ gate. The data from flow cytometry was shown in I. The analysis was done on five mice in each group. The data of cell percentages are expressed as the mean Ϯ SD of five mice. The absolute cell numbers of IL-17ϩ cell and IFN-␥ϩ cells were shown in J and K. The analysis was done on five mice in each group. B–J, Circles represent individual mice. The horizontal lines represent the mean. The results shown are representative .p Ͻ 0.05 ,ءء p Ͻ 0.01 and ,ء .of two independent experiments

CD4ϩ or CD11bϩ infiltrating cells were significantly reduced in peptide and MOG-specific T cell responses were measured. No the CCR6Ϫ/Ϫ chimeric mice (Fig. 6, C and D), supporting reduced significant difference was found in MOG-specific T cell prolifer- CNS inflammation in these mice. Absolute cell numbers of Treg ation or IL-2, IL-17, and IFN-␥ production between WT and KO cells, but not the percentages of Foxp3ϩ Treg cells in total cells cells (data not shown). Thus, immune responses against MOG pep- in CNS (data not shown), were also significantly decreased in the tide were similar between WT and KO chimeric mice. These re- KO chimeric mice (Fig. 6E). The percentages of IL-17ϩ, but not sults indicated that although there was no defective expression of IFN-␥ϩ, cells were significantly reduced in the CNS of the KO IL-17 in the spleen in the KO chimeric mice, Th17 cells appeared chimeric mice (Fig. 6F), indicating that CCR6 may be more im- to be reduced in both numbers and percentages in the CNS. portant for Th17 than Th1 cells. However, the absolute cell num- The above results suggest that CCR6-mediated Th17 migration bers of IL-17ϩ or IFN-␥ϩ cells in the CNS were both reduced in to inflamed tissues may be important for driving CNS inflamma- the KO chimeric mice (Fig. 6, G and H). Next, we examined T tion. To directly confirm the role of CCR6 on Th17 cell migration cells in the spleen by restimulation with MOG peptide and intra- and function in EAE, C57BL/6 and CCR6 KO were immunized cellular staining. The percentages of MOG-specific IFN-␥ϩ T cells with MOG peptide and Ag-specific Th17 cells were expanded in in the spleen were significantly increased in the KO chimeric mice vitro with MOG and IL-23 and adoptively transferred into C57/ while IL-17ϩ cells were similar between WT and KO chimeric BL/6 recipient mice. After expansion by MOG and IL-23, mice (Fig. 6I). Moreover, there appeared to be more total IL-17ϩ C57BL/6 or CCR6-deficient T cells contained ϳ40% of IL-17ϩ and IFN-␥ϩ cell numbers in the spleens of KO chimeric mice (Fig. cells, 5% of IFN-␥ϩ cells, and 10% of Foxp3ϩ cells, respectively. 6, J and K). In addition, spleen cells were cultured with MOG The recipient mice were subsequently immunized with MOG and 8398 CCR6-MEDIATED MIGRATION OF Th17 AND Treg CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. CCR6Ϫ/Ϫ Th17 cells fail in induction of EAE. A, IL-23-cultured MOG-reactive CD4ϩ T cells from C57BL/6 or CCR6 KO mice were adoptively transferred into five C57BL/6 mice in each group. The mice were immunized with MOG to induce EAE. A, Disease scores in five mice in each group were evaluated. The data are shown as the mean Ϯ SD of five mice. B and C, CD4ϩ (B) or CD11bϩ cells (C) in the mononuclear cells from CNS were analyzed in five mice in each group by staining with PerCP-labeled anti-CD4 and FITC-labeled anti-CD11b. D, CD4ϩCD25ϩFoxp3ϩ cells were by guest on September 25, 2021 analyzed in the cells from the CNS of five mice in each group. E–G, IL-17ϩ or IFN-␥ϩ cells in the CNS of five mice in each group were analyzed in mononuclear cells in a CD4ϩ gate. The data from flow cytometry are shown in E. The data of cell percentages are expressed as the mean Ϯ SD of five mice. The absolute cell numbers of IL-17ϩ cell and IFN-␥ϩ cells in the CNS of five mice in each group were shown in F and G. H–J, IL-17ϩ or IFN-␥ϩ cells in spleen cells of five mice in each group were analyzed in a CD4ϩ gate. The data from flow cytometry are shown in H. The data of cell percentages are expressed as the mean Ϯ SD of five mice. The absolute cell numbers of IL-17ϩ cells and IFN-␥ϩ cells in spleen cells of five mice in each group are shown in I and J. B–J, Circles represent individual mice. The horizontal lines represent the mean. All results are representative of two independent .p Ͻ 0.05 ,ءء p Ͻ 0.01 and ,ء .experiments

CFA to boost the MOG-specific transferred cells in vivo. The de- that CCR6-deficient autoreactive Th17 cells failed in migration to velopment of EAE was greatly reduced in mice receiving KO the CNS, which resulted in blockade of EAE development. Th17 cells than those injected with WT cells (Fig. 7A). The num- bers of infiltrating CD4ϩ cells in the CNS were greatly reduced in CCR6 regulates the migration of Treg cells to inflamed site mice receiving CCR6-deficient T cells, but there was no significant To understand the function of CCR6 in Treg cell migration in the difference in CD11bϩ cells (Fig. 7, B and C). The percentages of autoimmune disease, Rag1Ϫ/Ϫ mice were transferred with the mix- CD4ϩ cells in the total CNS cells were also significantly decreased ture of WT naive CD4ϩ T cells from CD45.1-congenic mice and in the KO Th17 cell-transferred mice (data not shown). In contrast, WT Treg or CCR6Ϫ/Ϫ Treg cells. The mice were immunized with the absolute numbers of Treg cells (Fig. 7D) as well as their per- MOG to induce EAE. No significant difference was observed in centages in total cells of the CNS (data not shown) also showed the clinical scores between mice receiving WT and CCR6Ϫ/Ϫ Treg significant reduction. In addition, IL-17ϩ or IFN-␥ϩ cells in the cells (data not shown). However, the frequency of Treg cells in the CNS were reduced in percentages (Fig. 7E) and in total numbers CNS was significantly lower in mice transferred with CCR6Ϫ/Ϫ in KO T cell-transferred mice (Fig. 7, F and G). In spleen, al- Treg (data not shown). Also, the absolute cell numbers of though the percentages of IL-17ϩ cells were less in KO T cell- CCR6Ϫ/Ϫ Treg cells in the CNS was significantly less than WT transferred mice (Fig. 7H), the total numbers of IL-17ϩ cells were Treg cells (Fig. 8A). In contrast, the frequencies (data not shown) not significantly different between two experimental groups (Fig. and absolute cell numbers of Treg cells in the spleen were not 7I). In addition, there was no difference in the percentages of significantly different (Fig. 8B), although Treg cells appeared to be IFN-␥ϩ cells in the spleen (Fig. 7H). Although the absolute cell increased in the spleen of CCR6Ϫ/Ϫ Treg cell-transferred mice numbers of IFN-␥ϩ cells were not significantly different in KO T (Fig. 8B). cell-transferred mice, IFN-␥ϩ cells appear to be more in the spleen CCR6 function in the migration of Treg cells to the inflamed sites of KO T cell-transferred mice (Fig. 7J). These results indicated was also investigated in a different way. We transferred Treg from The Journal of Immunology 8399

(9.14 Ϯ 2.36%; Fig. 8E). Therefore, small populations of Treg cells express IL-17, regardless of CCR6 expression. These results indicated that CCR6 plays a critical role in Treg cells by mediating the migration of Treg cells to the inflamed sites.

Discussion Our study presented here has demonstrated that CCR6 expressed on Th17 and Treg cells plays a critical role in Th17 and Treg migration to inflammatory sites. In the absence of CCR6, Th17 and Treg cells were impaired in infiltration into the CNS during EAE disease. Differentiation of naive CD4ϩ T cells to Th17 cells is mediated by TGF-␤ and IL-6 signaling via STAT3, ROR␥t, and ROR␣ tran- scription factors, which is further enhanced and maintained by IL-23 and IL-21. Among Th1, Th2, and Th17 subsets, Th17 cells expressed both CCR6 and CCL20, which were also recently re- ported in human Th17 cells (28, 29). However, these two mole- cules appear to have different regulatory mechanisms. The expres-

sion of CCL20 on Th17 cells is regulated similarly as IL-17. It is Downloaded from induced by TGF-␤ along with IL-6 or IL-21, in which both ROR␥ and STAT3 were required. However, CCR6 expression in Th17 cells does not require ROR␥, due to a redundant function of ROR␣. In addition, CCR6 was induced by TGF-␤ alone. Since FIGURE 8. CCR6 regulates the distribution of Treg cells in EAE. A and TGF-␤ also induces Foxp3-expressing iTreg cells, we went on and ϩ Ϫ/Ϫ

B, WT naive CD4 T cells were mixed with either WT or CCR6 Treg found expression of CCR6 in both iTreg and nTreg cells, consis- http://www.jimmunol.org/ Ϫ/Ϫ cells and transferred into five RAG1 mice in each group. The mice were tent with a previous report (25). CCR6 expression remains or even immunized with MOG peptide to induce EAE. A, The absolute cell number Ϫ/Ϫ ϩ ϩ is increased in IL-21 cells, possibly due to the expression by of CNS-infiltrating Treg cells was analyzed in CD4 Foxp3 gate. The iTreg cells. Expression of CCR6 in Th17 and Treg cells not only data of absolute cell number are expressed as the mean Ϯ SD of five supports their intimate relationship, but also has a biological sig- mice. B, The absolute cell number of Treg cells in the spleen was analyzed in the CD4ϩFoxp3ϩ gate. The data of absolute cell number are expressed nificance in regulation of coordinated localization of pro- and anti- as the mean Ϯ SD of five mice. C and D, The distribution of Treg cells in inflammatory T cells. the CD45.1ϩ congenic mice developing EAE. CD45.2ϩ WT Treg cells Th17 cells have been implicated in various inflammatory dis- (1 ϫ 106/mouse) or CCR6Ϫ/Ϫ CD45.2ϩ Treg cells (1 ϫ 106/mouse) were eases that affect different target organs, including EAE, rheumatoid transferred into CD45.1ϩ congenic mice. The mice were immunized with arthritis, allergic pulmonary inflammation, and (35, 36). by guest on September 25, 2021 MOG peptide to induce EAE. C, The absolute cell number of CNS-infil- Once primed in lymphoid organs, how effector Th17 cells are re- trating CD45.2ϩ Treg cells derived from WT or CCR6Ϫ/Ϫ mice were an- ϩ cruited in large numbers to these inflammatory tissues has been alyzed by staining with anti-CD45.2 and anti-Foxp3 in CD4 gate. The unclear. Our results indicate an essential function of CCR6 in the Ϯ data of absolute cell number are expressed as the mean SD of two mice. organization of inflammatory reactions. Chemotaxis assays dem- D, The absolute cell number of CD45.2ϩ Treg cells from WT or CCR6Ϫ/Ϫ onstrated that Th17 cells and Treg cells migrated in response to mice in the spleen was analyzed by staining with anti-CD45.2 and anti- Foxp3 in the CD4ϩ gate. The data of absolute cell number are expressed CCL20 in a CCR6-dependent manner, indicating that CCR6 on as the mean Ϯ SD of two mice. E, Foxp3-GFP mice (three mice in each both cells are functional. Moreover, Th17 cells, by producing group) were immunized with MOG/CFA to induce EAE. After the mice CCL20, could also attract other Th17 cells via CCR6. In addition reached EAE disease score 3, the cells in the CNS were harvested and to a Th17 source, CCL20 could be also induced in nonhemopoietic analyzed for CCR6 and IL-17 expression. All data shown are representa- cells by IL-17 (6), dependent on Act1 adaptor protein (37). There- ,p Ͻ 0.05. fore, we propose that the first wave of inflammatory Th17 cells ,ءء p Ͻ 0.01 and ,ء .tive of two independent experiments once entered into the target tissues and become reactivated by local APCs, will express CCL20 and IL-17. The latter will induce either WT or CCR6Ϫ/Ϫ mice, that are CD45.2, into C57BL/6 mice CCL20 expression by other residential cell types. Production of congenic for CD45.1. These recipient mice were further immunized CCL20 leads to further recruitment of other CCR6-expressing with MOG and CFA to induce EAE. No significant difference in the Th17 cells and sustained chronic inflammation. Memory Th17 clinical scores was found in the mice transferred with WT or cells with different antigenic specificity may be recruited into the CCR6Ϫ/Ϫ Treg cells (data not shown). Similar to the result in Fig. 8, inflammatory site, possibly resulting in an spreading. Our A and B, the CCR6Ϫ/Ϫ donor’s Treg cells showed significantly lower hypothesis is supported by our EAE experiments, where CCR6- percentages (data not shown) and the absolute cell numbers than their deficient MOG-specific Th17 cells did not initiate CNS inflamma- WT controls in the CNS (Fig. 8C). In contrast, no significant differ- tion. Th17 cells were greatly reduced in CNS but still were found ence was observed in the frequencies (data not shown) and absolute in large numbers in spleen. Thus, CCR6 is important for Th17 cell numbers of Treg cells in the spleen (Fig. 8D). migration to inflammatory tissues and may mediate an amplifying It has been reported that human Treg cells produce IL-17 (33). regulation to sustain inflammatory responses. To assess IL-17 production in CNS-infiltrating CCR6ϩ Treg cells In contrast, it has been reported that Treg cells inhibit various of EAE mice, Foxp3-GFP mice (34) were induced EAE and when autoimmune diseases, such as EAE (38, 39), diabetes (40), colitis they reached EAE disease score 3 analyzed for their IL-17 pro- (41), gastritis (42), and collagen-induced arthritis (43). In EAE, duction in CCR6ϩ Treg cells in the CNS. Approximately 10% of Ag-specific Treg cells have been recently reported to accumulate CNS-infiltrating CCR6ϩ Treg cells produced IL-17 (10.84 Ϯ in the CNS along with effector T cells (44), the underlying mech- 4.57%) and 9% of CCR6Ϫ Treg cells in the CNS produced IL-17 anism of which is still unknown. In this study, we showed that in 8400 CCR6-MEDIATED MIGRATION OF Th17 AND Treg CELLS addition to Th17 cells, Treg cells also expressed CCR6, although 8. Liang, S. C., X. Y. Tan, D. P. Luxenberg, R. Karim, K. Dunussi-Joannopoulos, they did not express CCL20. Treg cells migrated toward to M. Collins, and L. A. Fouser. 2006. (IL)-22 and IL-17 are coex- pressed by Th17 cells and cooperatively enhance expression of antimicrobial CCL20-producing Th17 in vitro, which was completely dependent peptides. J. Exp. Med. 203: 2271–2279. on CCR6. In vivo during EAE disease, not only Th17 but also Treg 9. Zheng, Y., D. M. Danilenko, P. Valdez, I. Kasman, J. Eastham-Anderson, J. Wu, and W. Ouyang. 2007. Interleukin-22, a TH17 cytokine, mediates IL-23-induced cell numbers were reduced in the CNS, indicating that Treg mi- dermal inflammation and acanthosis. Nature. 445: 648–651. gration into inflammatory tissues is also mediated by CCR6. Since 10. Kolls, J. K., and A. Linden. 2004. Interleukin-17 family members and inflam- in mice receiving CCR6Ϫ/Ϫ Th17 cells, the host Treg cells could mation. Immunity 21: 467–476. 11. Dong, C., and R. I. Nurieva. 2003. Regulation of immune and autoimmune re- still express CCR6, lack of Treg cells in the CNS supports a role sponses by ICOS. J. Autoimmun. 21: 255–260. of Th17 cells in initiation of inflammatory responses and in the 12. Murphy, C. A., C. L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, recruitment of Treg cells. In this case, the absence of CCL20 rather R. A. Kastelein, J. D. Sedgwick, and D. J. Cua. 2003. Divergent pro- and anti- inflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. than CCR6 expression is important in inhibition of Treg cell mi- J. Exp. Med. 198: 1951–1957. Ϫ/Ϫ gration. In the result of CCR6 Treg cell transfer in EAE, 13. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, CCR6Ϫ/Ϫ Treg cells were greatly reduced in their migration into and V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238. the CNS during EAE disease. The clinical disease score of EAE 14. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard, was not different between the mice transferred with WT Treg and C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006. CCR6Ϫ/Ϫ Treg at this stage. It has been reported that migrated Transforming growth factor-␤ induces development of the TH17 lineage. Nature 441: 231–234. Treg cells to the CNS cannot regulate encephalitogenic effector T 15. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. cells in EAE-developing mice. TNF-␣ and IL-6 produced by en- 2006. TGF␤ in the context of an inflammatory cytokine milieu supports de novo cephalitogenic effector T cells in the CNS suppress the function of differentiation of IL-17-producing T cells. Immunity 24: 179–189. Downloaded from 16. Yang, X. O., A. D. Panopoulos, R. Nurieva, S. H. Chang, D. Wang, Treg cells in the CNS (44). It is possible that transferred Treg cells S. S. Watowich, and C. Dong. 2007. STAT3 regulates cytokine-mediated gen- were also suppressed Treg function by encephalitogenic effector T eration of inflammatory helper T cells. J. Biol. Chem. 282: 9358–9363. cells in the CNS of EAE-developing mice. Our study does not 17. Zhou, L., I. I. Ivanov, R. Spolski, R. Min, K. Shenderov, T. Egawa, D. E. Levy, W. J. Leonard, and D. R. Littman. 2007. IL-6 programs TH-17 cell differentiation formally rule out the importance of other chemokine receptors in by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat. Im- trafficking of Treg cells, considering CCR4 has also been reported munol. 8: 967–974. 18. Korn, T., E. Bettelli, W. Gao, A. Awasthi, A. Jager, T. B. Strom, M. Oukka, and to be expressed on Treg cells (45). Th1 cells were also reduced in http://www.jimmunol.org/ Ϫ/Ϫ V. K. Kuchroo. 2007. IL-21 initiates an alternative pathway to induce proinflam- numbers in mice receiving CCR6 Th17 cells, suggesting that matory TH17 cells. Nature 448: 484–487. Th17 may regulate Th1 cell migration in vivo through an uniden- 19. Nurieva, R., X. O. Yang, G. Martinez, Y. Zhang, A. D. Panopoulos, L. Ma, tified mechanism. Our data not only demonstrate expression of K. Schluns, Q. Tian, S. S. Watowich, A. M. Jetten, and C. Dong. 2007. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature CCR6 on Treg cells and its possible function in Treg cell local- 448: 480–483. ization and migration, but also suggest that CCL20-CCR6 inter- 20. Laurence, A., C. M. Tato, T. S. Davidson, Y. Kanno, Z. Chen, Z. Yao, R. B. Blank, F. Meylan, R. Siegel, L. Hennighausen, et al. 2007. Interleukin-2 action in EAE, by recruiting Treg cells, initiates a feedback anti- signaling via STAT5 constrains generation. Immunity 26: inflammatory mechanism in the CNS. This adds a complexity in 371–381. the regulation of inflammatory responses in autoimmunity. One 21. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, ␥ D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor ROR t directs by guest on September 25, 2021 would be cautious about targeting CCR6 in autoimmune diseases the differentiation program of proinflammatory IL-17ϩ T helper cells. Cell 126: as this may also interfere with the natural healing process of the 1121–1133. hosts mediated by Treg cells. 22. Yang, X. O., B. P. Pappu, R. Nurieva, A. Akimzhanov, H. S. Kang, Y. Chung, L. Ma, B. Shah, A. D. Panopoulos, K. S. Schluns, et al. 2008. T helper 17 lineage In summary, our current work reveals that CCR6 is expressed on differentiation is programmed by orphan nuclear receptors ROR␣ and ROR␥. not only Th17 cells but also Treg cells. Th17 cells express CCL20 Immunity 28: 29–39. to promote migration of Th17 and Treg cells via CCR6. Our results 23. Wing, K., Z. Fehervari, and S. Sakaguchi. 2006. Emerging possibilities in the development and function of regulatory T cells. Int. Immunol. 18: 991–1000. indicate an important, previously unknown role of CCL20-CCR6 24. Zheng, Y., and A. Y. Rudensky. 2007. Foxp3 in control of the interaction in coordinating the migration of both proinflammatory lineage. Nat. Immunol. 8: 457–462. and Treg cells in autoimmune diseases, which will benefit our 25. Kleinewietfeld, M., F. Puentes, G. Borsellino, L. Battistini, O. Rotzschke, and K. Falk. 2005. CCR6 expression defines regulatory effector/memory-like cells understanding of tissue inflammation and autoimmunity. within the CD25ϩCD4ϩ T-cell subset. Blood 105: 2877–2886. 26. Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, and J. M. Farber. 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T Disclosures cells and determines responsiveness to inflammatory protein 3␣. The authors have no financial conflict of interest. J. Immunol. 162: 186–194. 27. Yang, X. O., R. Nurieva, G. J. Martinez, H. S. Kang, Y. Chung, B. P. Pappu, B. Shah, S. H. Chang, K. S. Schluns, S. S. Watowich, et al. 2008. Molecular References antagonism and plasticity of regulatory and inflammatory T cell programs. Im- 1. Dong, C., and R. A. Flavell. 2000. Cell fate decision: T-helper 1 and 2 subsets in munity 29: 44–56. immune responses. Arthritis Res. 2: 179–188. 28. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, and F. Sallusto. 2007. 2. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune 1␤ and 6 but not transforming growth factor-␤ are essential for the system: the T helper grows up. Genes Dev. 14: 1693–1711. differentiation of -producing human T helper cells. Nat. Immunol. 3. Dong, C., and R. A. Flavell. 2001. Th1 and Th2 cells. Curr. Opin. Hematol. 8: 8: 942–949. 47–51. 29. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein, 4. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, et al. 2007. Development, K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4ϩ effector cytokine profile and function of human interleukin 17-producing helper T cells. T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Nat. Immunol. 8: 950–957. Immunol. 6: 1123–1132. 30. Taub, D. D., S. M. Turcovski-Corrales, M. L. Key, D. L. Longo, and 5. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, W. J. Murphy. 1996. Chemokines and T lymphocyte activation, I: ␤ chemokines J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23 costimulate human T lymphocyte activation in vitro. J. Immunol. 156: drives a pathogenic T cell population that induces autoimmune inflammation. 2095–2103. J. Exp. Med. 201: 233–240. 31. Richards, G. R., R. M. Millard, M. Leveridge, J. Kerby, and P. B. Simpson. 2004. 6. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, Quantitative assays of chemotaxis and chemokinesis for human neural cells. As- L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells say Drug Dev. Technol. 2: 465–472. regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: 32. Boyden, S. 1962. The chemotactic effect of mixtures of and on 1133–1141. polymorphonuclear leucocytes. J. Exp. Med. 115: 453–466. 7. Chung, Y., X. Yang, S. H. Chang, L. Ma, Q. Tian, and C. Dong. 2006. Expression 33. Koenen, H. J., R. L. Smeets, P. M. Vink, E. van Rijssen, A. M. Boots, and and regulation of IL-22 in the IL-17-producing CD4ϩ T lymphocytes. Cell Res. I. Joosten. 2008. Human CD25highFoxp3pos regulatory T cells differentiate into 16: 902–907. IL-17-producing cells. Blood 112: 2340–2352. The Journal of Immunology 8401

34. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, and diabetes expresses L-selectin and high levels of CCR7. J. Immunol. 169: A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead 2461–2465. transcription factor . Immunity 22: 329–341. 41. Singh, B., S. Read, C. Asseman, V. Malmstrom, C. Mottet, L. A. Stephens, 35. Bettelli, E., M. Oukka, and V. K. Kuchroo. 2007. TH-17 cells in the circle of R. Stepankova, H. Tlaskalova, and F. Powrie. 2001. Control of intestinal inflam- immunity and autoimmunity. Nat. Immunol. 8: 345–350. mation by regulatory T cells. Immunol. Rev. 182: 190–200. 36. Dong, C. 2006. Diversification of T-helper-cell lineages: finding the family root 42. Asano, M., M. Toda, N. Sakaguchi, and S. Sakaguchi. 1996. Autoimmune disease of IL-17-producing cells. Nat. Rev. Immunol. 6: 329–333. as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. 37. Chang, S. H., H. Park, and C. Dong. 2006. Act1 adaptor protein is an immediate Med. 184: 387–396. and essential signaling component of interleukin-17 receptor. J. Biol. Chem. 281: 43. Huehn, J., K. Siegmund, J. C. Lehmann, C. Siewert, U. Haubold, M. Feuerer, 35603–35607. G. F. Debes, J. Lauber, O. Frey, G. K. Przybylski, et al. 2004. Developmental 38. Hori, S., M. Haury, A. Coutinho, and J. Demengeot. 2002. Specificity require- stage, phenotype, and migration distinguish naive- and effector/memory-like ments for selection and effector functions of CD25ϩ4ϩ regulatory T cells in CD4ϩ regulatory T cells. J. Exp. Med. 199: 303–313. anti-myelin basic protein T cell receptor transgenic mice. Proc. Natl. Acad. Sci. 44. Korn, T., J. Reddy, W. Gao, E. Bettelli, A. Awasthi, T. R. Petersen, USA 99: 8213–8218. B. T. Backstrom, R. A. Sobel, K. W. Wucherpfennig, T. B. Strom, et al. 2007. 39. Kohm, A. P., P. A. Carpentier, H. A. Anger, and S. D. Miller. 2002. Cutting edge: Myelin-specific regulatory T cells accumulate in the CNS but fail to control CD4ϩCD25ϩ regulatory T cells suppress antigen-specific autoreactive immune autoimmune inflammation. Nat. Med. 13: 423–431. responses and central nervous system inflammation during active experimental 45. Iellem, A., M. Mariani, R. Lang, H. Recalde, P. Panina-Bordignon, F. Sinigaglia, autoimmune encephalomyelitis. J. Immunol. 169: 4712–4716. and D. D’Ambrosio. 2001. Unique chemotactic response profile and specific 40. Szanya, V., J. Ermann, C. Taylor, C. Holness, and C. G. Fathman. 2002. The expression of chemokine receptors CCR4 and CCR8 by CD4ϩCD25ϩ regulatory subpopulation of CD4ϩCD25ϩ splenocytes that delays adoptive transfer of T cells. J. Exp. Med. 194: 847–853. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021