IL-17–Producing Vg4 gd T Cells Require Sphingosine 1-Phosphate ...

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Homeostatic and Inflammatory ConditionsTheir Egress from the Lymph Nodes underSphingosine 1-Phosphate Receptor 1 for

T Cells Requireδγ +4γProducing V−IL-17

Sugahara and Kenji ChibaTakemoto, Hiroyuki Utsumi, Atsushi Fukunari, Kunio Yasuhiro Maeda, Noriyasu Seki, Hirotoshi Kataoka, Kana

http://www.jimmunol.org/content/195/4/1408doi: 10.4049/jimmunol.1500599July 2015;

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The Journal of Immunology

IL-17–Producing Vg4+ gd T Cells Require Sphingosine1-Phosphate Receptor 1 for Their Egress from the LymphNodes under Homeostatic and Inflammatory Conditions

Yasuhiro Maeda,* Noriyasu Seki,* Hirotoshi Kataoka,* Kana Takemoto,†

Hiroyuki Utsumi,† Atsushi Fukunari,† Kunio Sugahara,* and Kenji Chiba*

Conventional ab T cells require sphingosine 1-phosphate (S1P) receptor 1 (S1P1) for circulation through the lymph nodes (LN);

however, it is unclear whether gd T cells use similar mechanisms. In this study, we found that treatment with fingolimod (FTY720,

1 mg/kg, orally) markedly reduced not only conventional CD4 T cells but also circulating gd T cells (Vg4+ and Vg42 subsets) in

the blood of mice. In contrast, IL-17+Vg4+, IL-17+Vg42, and IL-172Vg42 subsets were significantly accumulated in the LN after

6 h of FTY720 treatment. By skin application of a synthetic TLR7/8 agonist, Vg4+ gd T cells (IL-17+ and IL-172 subsets) were

accumulated and expanded in the draining LN (DLN), whereas the IL-17+ subset predominantly migrated to the inflamed skin.

FTY720 induced a marked sequestration of IL-17–producing Vg4+ gd T cells in the DLN and inhibited their infiltration into the

inflamed skin. Similarly, FTY720 inhibited infiltration of Vg4+ gd T cells into the CNS by their sequestration into the DLN in

experimental autoimmune encephalomyelitis. Vg4+ gd T cells expressed a significant level of S1P1 and showed a migratory

response toward S1P. FTY720 treatment induced almost complete downregulation of S1P1 expression and S1P responsiveness

in Vg4+ gd T cells. Our findings strongly suggest that IL-17–producing Vg4+ gd T cells require S1P1 for their egress from the LN

under homeostatic and inflammatory conditions. Consequently, inhibition of S1P1-dependent egress of pathogenic IL-17–produc-

ing Vg4+ gd T cells from the DLN may partly contribute the clinical therapeutic effects of FTY720 in relapsing multiple

sclerosis. The Journal of Immunology, 2015, 195: 1408–1416.

Fingolimod (FTY720), the sphingosine 1-phosphate (S1P)receptor modulator, is shown to be highly effective inrelapsing multiple sclerosis (MS) and has been widely used

for the oral therapy of this disease (1, 2). It is well documented thatphosphorylated FTY720 acts as a functional antagonist at S1Preceptor 1 (S1P1) by internalization and degradation of the re-ceptor and inhibits S1P1-dependent lymphocyte egress from thelymph nodes (LN) (3–6). Because it has been suggested that IL-17rather than IFN-g plays a pathogenic role in MS and experimentalautoimmune encephalomyelitis (EAE), a CD4 T cell–dependentanimal model for MS, many studies have focused on understandingthe differentiation and effector functions of IL-17–producing Thcells (Th17 cells) in autoimmunity (7–9). The production of IL-17by peripheral blood T cells stimulated with anti-CD3 and anti-CD28mAbs was markedly reduced (.95% inhibition) in FTY720-administered MS patients, suggesting that FTY720 inhibits egressof Th17 cells from the LN (10, 11). Furthermore, FTY720 showeda marked therapeutic effect on mouse EAE by reducing infiltrationof Th17 cells into the spinal cord (12–15). Based on these data, ithas been thought that the therapeutic effects of FTY720 on MS and

EAE are predominantly due to inhibition of infiltration of patho-genic Th17 cells into the CNS.More recently, however, it has been noted that conventional Th17

cells are not the sole producers of IL-17 because larger amounts ofIL-17 are produced by several types of gd T cells rather than Th17cells without the clonal expansion or additional TCR stimulationrequired for the adaptive response (16–18). Unlike conventionalab T cells, gd T cells preferentially reside in epithelial tissues,including the skin, intestine, lung, and reproductive tract, and theyprovide a first line of host defense against various pathogens (19,20). In MS patients, the frequency of gd T cells was significantlyincreased in the cerebrospinal fluid and blood, and the accumu-lation of Vd2 T cells was found in chronic active lesions, suggestinga pathogenic role of IL-17–producing gd T cells in MS (21, 22).Similarly, a significant infiltration of IL-17–producing gd T cells,particularly the Vg4+ subset, was found in the CNS of EAE mice(17, 23, 24). Although IL-17–producing murine gd T cells mainlyconsist of Vg4+ and Vg6+ subsets (25), it is strongly suggested thatIL-17–producing Vg4+ gd T cells play a pathogenic role in thedevelopment of EAE because activation of this subset by anti-Vg4mAb treatment exacerbated EAE (17, 26–28).It has been reported that thymic gd T cells express S1P1 mRNA

and are accumulated in the thymus of S1P1-deficient mice, indi-cating that gd T cells, similar to ab T cells, require S1P1 for thymicegress (29, 30). Peripheral IL-17–producing Vg4+ gd T cells areshown to be migrated to the draining LN (DLN), considerablyexpand there, and recirculate back to the inflamed skin by appli-cation with a synthetic TLR7/8 agonist to mouse ear skin (31).However, it remains unclear whether gd T cells, including the Vg4+

subset, require S1P1 for their circulation. In this study, we inves-tigated the role of the S1P/S1P1 axis in the circulation of gd T cells,particularly IL-17–producing Vg4+ gd T cells between the blood,LN, and inflamed tissues. We found that FTY720 markedly reduces

*Pharmacology Research Laboratories I, Research Division, Mitsubishi TanabePharma Corporation, Kanagawa 227-0033, Japan; and †Safety Research Laboratory,Research Division, Mitsubishi Tanabe Pharma Corporation, Saitama 335-8505, Japan

Received for publication March 11, 2015. Accepted for publication June 18, 2015.

Address correspondence and reprint requests to Dr. Kenji Chiba, PharmacologyResearch Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation,1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan. E-mail ad-dress: [email protected]

Abbreviations used in this article: DLN, draining LN; EAE, experimental autoim-mune encephalomyelitis; IMQ, imiquimod; LN, lymph node; MOG, myelin oligo-dendrocyte glycoprotein; MS, multiple sclerosis; S1P, sphingosine 1-phosphate;S1P1, S1P receptor type 1; THI, 2-acetyl-4-tetrahydroxybutylimidazole.

Copyright� 2015 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500599

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the numbers of circulating gd T cells and IL-17–producing Vg4+ gdT cells by their sequestration into the LN. Our data clearly dem-onstrated that Vg4+ gd T cells express significant levels of S1P1 andshow a migratory response toward S1P. FTY720 induced almostcomplete downregulation of S1P1 expression in Vg4+ gd T cells andreduced infiltration of Vg4+ gd T cells into the CNS of EAE mice bysequestration of these cells into the DLN. Thus, our findings stronglysuggest that IL-17–producing Vg4+ gd T cells require S1P1 foregress from LN under homeostatic and inflammatory conditions.

Materials and MethodsAnimals

Male BALB/c and C57BL/6 mice were purchased from Charles River Japanand were used at 6–10 wk of age. All animal experiments were performedunder experimental protocols approved by the Ethics Review Committeefor Animal Experimentation of Research Division, Mitsubishi Tanabe Pharma.

Compound treatment

FTY720 and 2-acetyl-4-tetrahydroxybutylimidazole (THI) were synthesizedaccording to the methods described previously (32, 33), dissolved in distilledwater, and given orally every day.

Cell preparation

BALB/c mice were treated with 5 mg 5% imiquimod (IMQ) cream (Beselnacream;Mochida Pharmaceuticals) on the right ear daily for 6 d. Inguinal andauricular DLN were digested with 1.25 mg/ml collagenase type IV (RocheApplied Science) and 200 mg/ml DNase I (Roche Applied Science) for30 min at 37˚C. Whole ear skins were minced and digested with collagenase,DNase I, and 500 mg/ml dispase (Life Technologies) for 1 h. Spleen cellswere prepared by gently disrupting in RPMI 1640 medium (Sigma-Aldrich)using glass slides. Removal of RBC in spleen cells and peripheral blood was

performed using Tris-NH4Cl buffer and an automated TQ-Prep system(Beckman Coulter), respectively. Spinal cords of four mice were minced anddigested with collagenase and DNase I for 1 h. The resulting cell pellets wereresuspended in 30% Percoll (GE Healthcare) and layered on 70% Percoll.After centrifugation at 5003 g for 25 min at 25˚C, cells at the interface werecollected. For migration assays, gd T cells in the auricular LN of IMQ-treated mice were prepared using a TCRg/d+ T cell isolation kit (MiltenyiBiotec). Single-cell suspensions were disaggregated by passage througha 70-mm nylon cell strainer.

Flow cytometry

Single-cell suspensions were stained with the following mAbs (all from BDBiosciences or eBioscience); anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-TCRgd (GL3), anti-Vg4 (UC3-10A6), and anti–IL-17A(TC11-18H10). For S1P1 staining, LN cells were stained with rat anti-S1P1mAb (713412, R&D Systems), biotinylated donkey anti-rat IgG (JacksonImmunoResearch Laboratories), and PE-conjugated streptavidin (eBio-science) (34). For detection of intracellular IL-17, cells were cultured in thepresence of 50 ng/ml PMA (Sigma-Aldrich), 1 mg/ml ionomycin (Sigma-Aldrich), and 2 mM monensin (eBioscience) for 4 h. After blocking by ratanti-mouse CD16/32 mAb (2.4G2; FcR Block, BD Biosciences), the cellswere labeled with surface Ags, treated with Cytofix buffer and Perm/Washbuffer (both from BD Biosciences), and then stained with anti–IL-17 mAb.Samples were acquired on a flow cytometer (LSR II; BD Biosciences). Forcell counting, the number of each cell subset was determined using com-parison with a known number of beads as an internal standard of Flow-Countfluorosphere (Beckman Coulter) by flow cytometry with a Cytomics FC500(Beckman Coulter). The numbers of IL-17+ and IL-172 gd T cells werecalculated by using the absolute number of gd T cells and the frequencies(%) of IL-17+ and IL-172 gd T cells in total gd T cells, respectively.

Immunohistochemistry

Samples of auricular LN from mice were embedded in Tissue-Tek OCTcompound (Sakura FineTek) and fixed with acetone. LN sections werestained with hamster anti-mouse TCRgd polyclonal Ab (GL3; BD Bio-

FIGURE 1. FTY720 modulates circulation of Vg4+ gd T cells through the LN under homeostatic conditions. After a single oral administration of FTY720

(1 mg/kg) or vehicle, the numbers of total lymphocytes, CD4 T cells, Vg4+ gd T cells, and Vg42 gd T cells in the blood (A) and inguinal LN (B) were de-

termined by flow cytometry. (C) Intracellular staining of IL-17 in the LN lymphocytes at 6 h after administration of FTY720. Representative dot plots of IL-17–

expressing Vg4+ gd T cells are shown gated on gd TCR+ cells at 4 h after stimulation with PMA plus ionomycin. (D) The numbers of IL-17+ and IL-172 gd T

cell subsets in the LN. All results are expressed as the means 6 SEM (n = 3). Statistical differences were calculated by a Student t test. *p , 0.05, **p , 0.01.

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sciences), incubated with biotin-goat anti-hamster IgG (Vector Laborato-ries) and peroxidase-conjugated streptavidin (Dako), and colorized with3,39-diaminobenzidine in the presence of peroxidase. Images of the stainedsections were inspected under an Olympus BX51 microscope equippedwith cellSens software (Olympus).

In vivo BrdU labeling

Mice were given 0.8 mg/ml BrdU (Sigma-Aldrich) and 2% glucose in theirdrinking water during IMQ applications. Intracellular BrdU staining wasperformed using a BrdU flow kit (BD Biosciences).

Real time-PCR

Total RNA was isolated from the DLN of mice using TRIzol reagents(Invitrogen). cDNAwas synthesized with a TaqMan reverse transcription kit(Life Technologies) using random hexamers and 0.5mg total RNA. Real-timePCR for the detection of Il2 (Mm00434256_m1), Il4 (Mm00445259_m1),Il7 (Mm01295803_m1), Il9 (Mm00434305_m1), Il15 (Mm00434210_m1),and Il21 (Mm00517640_m1) mRNAwas performed with predesigned TaqMangene expression assays (Life Technologies). For normalization, internalcontrol GAPDH mRNAwas detected using TaqMan rodent GAPDH controlreagents (Life Technologies). Samples were assayed on an ABI Prism 7900

FIGURE 2. FTY720 reduces infiltration of IL-17–producing Vg4+ gd T cells into the inflamed ear skin of mice treated with IMQ. BALB/c mice were treated

with 5 mg 5% IMQ cream on the right ear daily for 6 d. FTY720 (1 mg/kg, orally) or vehicle was administered daily from day 0 to day 6. The numbers of total

lymphocytes, CD4 T cells, Vg4+ gd T cells, and Vg42 gd T cells in the blood (A), ear skin (B), and auricular DLN (C) were determined by flow cytometry on

days 0, 3, and 6. (D) Intracellular staining of IL-17 in the DLN lymphocytes and infiltrated lymphocytes into the ear skin on day 6. Representative dot plots of

IL-17–expressing Vg4+ gd T cells are shown gated on gd TCR+ cells at 4 h after stimulation with PMA plus ionomycin. (E) Numbers of IL-17+ and IL-172 gd

T cell subsets in the ear skin (upper panels) and DLN (lower panels). All results are expressed as the means 6 SEM (n = 4). Statistical differences were

calculated by a Student t test. *p , 0.05, **p , 0.01.

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sequence detector (Life Technologies) and the data were analyzed usingsequence detector software (Life Technologies).

Migration assay

Migration assays were performed according to the method describedpreviously (5). Lymphocytes (106 cells) or gd T cells (105 cells) preparedfrom the DLN of mice applied with IMQ for 6 d were added to the upperwells of 5-mm pore, polycarbonate 24-well tissue culture inserts (Costar).S1P (10 nM; Sigma-Aldrich) was added to the lower chamber. The mi-gration assays were conducted in RPMI 1640 medium with 0.5% fattyacid–free BSA (Sigma-Aldrich) for 4 h at 37˚C in 5% CO2. The numbersof CD4 T cells and Vg4+ gd T cells in the starting and the migratedpopulation were determined by flow cytometry described above, and themigration index was calculated from these values.

EAE induction

C57BL/6 mice were immunized s.c. with 200 mg myelin oligodendrocyteglycoprotein (MOG)35–55 peptide (Peptide Institute) emulsified in CFA(containing 4 mg/ml killed Mycobacterium tuberculosis, H37Ra; Chondrex)on day 0, followed by i.v. injection with 200 ng pertussis toxin (ListBiological Laboratories) on day 0 and day 2 (12, 13). The severity of EAEwas daily monitored and graded on a scale of 0–5 using the followingcriteria: 0, no paralysis; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with in-ability to right; 2, paralysis of one limb; 2.5, paralysis of one limb andweakness of one other limb; 3, complete paralysis of both hindlimbs; 4,moribund state; 5, death (12, 13).

Statistical analysis

The results were expressed as the means6 SEM. Statistical differences werecalculated by a Student t test and considered significant when p , 0.05.

ResultsFTY720 sequesters circulating Vg4+ gd T cells into the LNunder homeostatic conditions

We investigated the influence of FTY720 on the number of Vg4+

gd T cells in the blood of normal BALB/c mice because several gdT cell subsets are known to be circulating in the blood under

homeostatic conditions (35, 36). As shown in Fig. 1A, FTY720(1 mg/kg, a single oral administration) induced a marked reductionof the numbers of CD4 T cells, gd T cells, Vg4+ gd T cells, andVg42 gd T cells with similar time course changes (at 3–24 h afterthe administration). In contrast, the numbers of these T cells (CD4T cells, gd T cells, Vg4+ gd T cells, and Vg42 gd T cells) weresignificantly increased in the peripheral LN at 6 h after FTY720administration (Fig. 1B). The intracellular cytokine stainingrevealed that both IL-17+ and IL-172 gd T cell subsets were ac-cumulated in the LN of mice given FTY720 (Fig. 1C, 1D). Par-ticularly, more than half (55.1%) of Vg4+ gd T cells expressedsignificant levels of intracellular IL-17 and FTY720 increasedthe frequency of IL-17–expressing Vg4+ gd T cells in the LN(Fig. 1C). FTY720 significantly showed an ∼1.5-fold increase inthe number of gd T cells (IL-17+Vg4+, IL-17+Vg42, and IL-172

Vg42 subsets) but not in the number of the IL-172Vg4+ gd T cellsubset. (Fig. 1D). These results suggest that FTY720 sequesterscirculating gd T cells, including IL-17–expressing Vg4+ gdT cells into the LN under homeostatic conditions.

FTY720 reduces infiltration of IL-17–producing Vg4+ gd

T cells into the inflamed skin of mice treated with TLR7/8agonist

It has been reported that IL-17–producing Vg4+ gd T cells areincreased in the inflamed skin and DLN by multiple skin appli-cation of a synthetic TLR7/8 agonist, IMQ (31, 37, 38). Based onthese findings, we examined the time course change of the num-bers of total lymphocytes, CD4 T cells, Vg4+ gd T cells, andVg42 gd T cells in the blood, inflamed skin, and auricular DLNby the consecutive application of IMQ for 6 d. In the blood, thenumbers of total lymphocytes, CD4 T cells, and Vg4+ gd T cellswere increased on day 6 during IMQ applications whereas thosewere significantly reduced by FTY720 treatment (Fig. 2A).

FIGURE 3. FTY720 induces accumulation of

gd T cells around the lymphatic sinus of the

auricular DLN in IMQ-treated mice. FTY720

(1 mg/kg, orally) or vehicle was administered

daily for 6 d. The auricular LN sections from

normal mice (top) and mice treated with IMQ

(middle) or mice treated with IMQ and FTY720

(bottom) were stained with anti-mouse gd-TCR

mAb. Images are representative of four mice from

two independent experiments in each group.

Scale bars, 400 mm (left) and 50 mm (right).

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Although the number of Vg42 gd T cells in the blood was notincreased by IMQ treatment, these T cells were also reduced byFTY720 administration (Fig. 2A). A noticeable infiltration of CD4T cells and Vg4+ gd T cells into the ear skin was induced by IMQapplications, and the infiltration of these T cells was significantlyreduced by FTY720 (Fig. 2B). IMQ applications led to a significantand time-dependent increase in the numbers of total lymphocytes,CD4 T cells, and Vg4+ gd T cells in the DLN, and FTY720 sig-nificantly inhibited the increase of total lymphocytes and CD4T cells (Fig. 2C). In contrast, interestingly, a dramatic increase inthe number of Vg4+ gd T cells was observed in the DLN of IMQ-applied mice treated with FTY720 (Fig. 2C). Alternatively, therewas no clear alteration of Vg42 gd T cell counts in the ear skin orDLN in mice treated with IMQ and/or FTY720 (Fig. 2B, 2C). In-tracellular cytokine staining revealed that FTY720 treatmentshowed 80% reduction in the number of IL-17–expressing Vg4+ gdT cells in the inflamed skin whereas FTY720 induced a 20-foldincrease in the number of the Vg4+ subset in the DLN (Fig. 2D,2E). Based on these results, FTY720 appeared to affect the traf-ficking of IL-17–expressing Vg4+ gd T cells rather than IL-172

Vg4+ gd T cells between the inflamed skin and DLN.

FTY720 sequesters Vg4+ gd T cells into the DLN underinflammatory conditions

Immunohistochemical analyses demonstrated that there were fewgd T cells in the auricular LN of normal mice, whereas a signifi-cant increase in the number of gd T cells was found to be localizedin the T cell area of the auricular DLN by IMQ application(Fig. 3). Consistent with the flow cytometry data shown in Fig. 2C,a marked accumulation of gd T cells was seen in the T cell area

surrounding the lymphatic sinus of the DLN by FTY720 treatment(Fig. 3). These results strongly suggest that FTY720 induces se-questration of IL-17–producing Vg4+ gd T cells into the DLN andthereby inhibiting infiltration of the gd T cells into the inflamedtissues under inflammatory conditions.Next, we investigated whether a marked increase of Vg4+ gd

T cells by FTTY720 selectively occurred in the DLN after IMQapplications. The proportion and total cell number of Vg4+ gdT cells were markedly increased in the auricular DLN but not inthe distal (inguinal) LN by FTY720 administration (Fig. 4A–D).In the spleen, although there was no change in the proportion ofVg4+ gd T cells by treatment with IMQ or FTY720, the number ofVg4+ gd T cells was significantly increased by IMQ application ascompared with normal control and were reduced markedly byFTY720 treatment (Fig. 4E, 4F). From these results, it is highlyprobable that a striking increase of Vg4+ gd T cells by FTY720 isselectively induced in the DLN and is not due to alteration ofsystemic lymphocyte distribution.As shown in Fig. 5A, FTY720 treatment induced a marked

increase in the number of Vg4+ gd T cells but not Vg42 gd T cellsin the DLN on day 6 after IMQ applications. The proportion ofBrdU-incorporated Vg4+ gd T cells in total LN lymphocytes wasnot altered on days 5 and 6 after IMQ application (Fig. 5B). Incontrast, FTY720 treatment induced a significant increase in thenumber of BrdU-incorporated Vg4+ gd T cells in the DLN on day6 (Fig. 5B). As Vg4+ gd T cells were comparably increased in theDLN even by IMQ application for 3 d, we examined the mRNAexpressions of gc cytokines on days 3 and 6 (Fig. 5C). The mRNAexpressions of IL-4 and IL-21 were significantly elevated in theDLN on day 6 after IMQ application and there was no clear

FIGURE 4. FTY720 induces a striking increase of Vg4+ gd T cells in the DLN but not in the distal LN or spleen of IMQ-treated mice. FTY720 (1 mg/kg, orally)

or vehicle was administered daily for 6 d to normal mice or mice treated with IMQ. Frequencies and absolute numbers of the Vg4+ gd T cells in the auricular DLN

(A and B), distal (inguinal) LN (C and D), and spleen (E and F) were determined by flow cytometry. The results are expressed as the mean6 SEM (n = 4). Statistical

differences were calculated by a Student t test. **p , 0.01, normal versus IMQ-treated mice; ##p , 0.01, IMQ-treated versus IMQ/FTY720-treated mice.

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change by FTY720 treatment (Fig. 5D), implying that IL-4 and/orIL-21 may induce proliferation of gd T cells. From these results,it is probable that pathogenic IL-17–producing Vg4+ gd T cells,after their proliferation, can emigrate from the DLN and subse-quently migrate to the inflamed tissues. Because FTY720 mark-edly reduced the infiltration of Vg4+ gd T cells into the inflamedskin (Fig. 2B), it is highly probable that FTY720 sequesters Vg4+

gd T cells into the DLN without affecting their proliferation.

The S1P/S1P1 axis regulates emigration of Vg4+ gd T cellfrom the DLN

It has been widely accepted that T cells, including effector T cells,require S1P1 for efficient egress from the LN (3, 4, 39, 40).However, it remains unknown whether S1P1 regulates circulationand trafficking of gd T cells through the DLN under inflammatoryconditions. Significant levels of S1P1 were expressed in Vg4+ gdT cells as well as CD4 T cells in the DLN of mice with IMQapplication, and the S1P1 expressions in both T cell subsets werealmost completely downregulated by in vivo treatment withFTY720 (Fig. 6A, 6C). As expected, similar to conventional CD4T cells, Vg4+ gd T cells prepared from mice treated with IMQshowed a significant migratory response toward S1P (10 nM), andtheir S1P responsiveness was almost completely inhibited byin vivo treatment with FTY720 (Fig. 6B, 6D). Similarly, by in vivoadministration of an S1P lyase inhibitor, THI (100 mg/kg, orally),

the number of Vg4+ gd T cells was significantly reduced in theblood, increased in the DLN, and decreased in the inflamed skin ofmice treated with IMQ (Fig. 6E). As noted, the S1P1 expression inVg4+ gd T cells was almost completely downregulated by THItreatment (Fig. 6F). Taken together, these results indicate that theS1P/S1P1 axis regulates trafficking of Vg4+ gd T cells from theDLN to inflamed tissues.

FTY720 reduces infiltration of Vg4+ gd T cells into the CNS inmouse EAE

Because Vg4+ gd T cells are known to produce larger amounts ofIL-17 compared with the conventional Th17 cells and are found inthe CNS of mouse EAE (17, 24), it is strongly suggested that IL-17–producing Vg4+ gd T cells play a pathogenic role in EAE.Although FTY720 showed marked therapeutic effects on EAE byinhibiting infiltration of Th17 cells into the CNS (14, 39), itremains unclear whether FTY720 affects trafficking of Vg4+ gd

T cells to the CNS of EAE mice. As shown in Fig. 7A, prophy-lactic administration of FTY720 (1 mg/kg, orally) showed almostcomplete preventative effects on EAE induced by immunizationwith MOG35–55 peptide to C57BL/6 mice. Along with the devel-opment of EAE, significant numbers of infiltrated Vg4+ gd T cellsas well as CD4 T cells were found in the spinal cord of EAE mice,and the infiltration of these T cells into the CNS was of an ex-tremely low level in EAE mice treated with FTY720 (Fig. 7B, 7C,

FIGURE 5. FTY720 induces a marked increase in the number of BrdU-incorporated Vg4+ gd T cells in the DLN of mice treated with IMQ. FTY720 (1 mg/kg,

orally) or vehicle was administered daily for 6 d to mice treated with IMQ. (A) The numbers of gd T cells, Vg4+ gd T cells, and Vg4- gd T cells in the auricular

DLN were determined by flow cytometry on days 5 and 6. (B) IMQ-treated mice were fed with BrdU-containing water (0.8 mg/ml) from day 3 and the frequencies

of BrdU-incorporated Vg4+ gd T cells among total lymphocytes were determined on days 5 and 6. (C) Mice were applied with IMQ once (day 0), three times (days

0–2), or six times (days 0–5). FTY720 or vehicle was administered daily for 6 d to mice treated with IMQ. On day 6, the numbers of Vg4+ gd T cells and CD4

T cells in the DLN were determined by flow cytometry. (D) The mRNA expressions of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 in the DLN were evaluated by real-

time PCR normalized to GAPDH. All results are expressed as the means 6 SEM (n = 4). Statistical differences were calculated by a Student t test. **p , 0.01.

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7E). In the inguinal DLN, significant numbers of Vg4+ gd T cellswere found in EAE mice on days 10–21, although Vg4+ gd T cellswere very rare in mice before immunization (Fig. 7D, 7E). In-terestingly, a more marked increase in the number of Vg4+ gdT cells was seen in the DLN on day 21 in EAE mice treated withFTY720 (Fig. 7D, 7E), suggesting sequestration of these gd

T cells into the DLN. Consequently, our findings imply that thepreventative effects of FTY720 on EAE development may bepartly due to inhibiting egress of pathogenic IL-17–producingVg4+ gd T cells from the DLN via downregulation of S1P1.

DiscussionIt has been well known that FTY720 markedly decreases thenumber of circulating lymphocytes including conventional abT cells in the blood by inhibiting S1P1-dependent lymphocyte

egress from the LN (3, 41). Several years ago, it was initiallydemonstrated that FTY720 is highly effective in the cerebral in-farction in mice, and one possible mechanism was proposed thatthis therapeutic effect is caused by inhibiting infiltration of gd

T cells into the mouse brain (42). However, there was no de-scription or discussion of where the gd T cells come from or whatmolecular mechanisms are underlying in the infiltration of gdT cells into the inflamed lesions. Although it has been reportedthat thymic gd T cells express S1P1 mRNA and S1P1-deficient gdT cells accumulate in the thymus, suggesting S1P1-dependentthymic egress of gd T cells (29, 30), there were few reports onthe effect of FTY720 or other S1P receptor modulators on ho-meostatic circulation or trafficking of gd T cells in the periphery.In this study, we demonstrate that similar to conventional CD4+

ab T cells, circulating Vg4+ gd T cells are markedly reduced in theblood and sequestrated into the LN by administration of FTY720under homeostatic conditions. Furthermore, FTY720 strikingly re-duced infiltration of IL-17–producing Vg4+ gd T cells into theinflamed tissues by their sequestration into the DLN in mice treatedwith TLR7/8 agonist. Vg4+ gd T cells express a significant level ofS1P1 expression on the cell surface and show a migratory response

FIGURE 6. The S1P/S1P1 axis regulates emigration of Vg4+ gd T cells

from the DLN. FTY720 [(A–D) 1 mg/kg, orally], THI [(E and F) 100 mg/kg,

orally], or vehicle was administered daily for 6 d to mice treated with IMQ.

(A, C, and F) The mean fluorescence intensity of S1P1 on CD4 T cells (A)

and Vg4+ gd T cells (C and F) from the auricular DLN was determined by

flow cytometry. Histograms are representative of four mice from two inde-

pendent experiments in each group. Isotype control (shaded), mice treated

with IMQ and FTY720 [(A and C) blue], IMQ and THI [(F) green], or IMQ

and vehicle (red). (B and D) Sorted gd T cells or total lymphocytes from the

DLN of mice treated with IMQ (and FTY720 or vehicle) for 6 d were

performed to migration assays toward 10 nM S1P for 4 h. The percentage of

migration of CD4 T cells (B) and Vg4+ gd T cells (D) was determined by

flow cytometry. (E) The numbers of Vg4+ gd T cells in the blood, auricular

DLN, and ear skin of mice treated with IMQ (and THI or vehicle) were

determined by flow cytometry on day 6. All results are expressed as the

mean 6 SEM. (A, C, E, and F) n = 4. (B and D) n = 3. Statistical differences

were calculated by a Student t test. **p , 0.01.

FIGURE 7. FTY720 reduces infiltration of Vg4+ gd T cells into the spinal

cord of EAE mice. C57BL/6 mice were immunized with MOG35–55 peptide

and CFA. FTY720 (1 mg/kg, orally) or vehicle was administered daily for

21 d. (A) Clinical scores of EAE symptoms are expressed as the means 6SEM (n = 3–4). Statistical differences were calculated by a Mann–Whitney

U test. *p , 0.05, **p , 0.01. (B–E) The numbers of CD4 T cells (B) and

Vg4+ gd T cells in the spinal cord and inguinal DLN (C and D) were de-

termined by flow cytometry on days 10, 14, and 21. The results are expressed

as the means 6 SEM (n = 3–4). Statistical differences were calculated by

a Student t test. *p, 0.05, **p, 0.01. (E) Representative dot plots of Vg4+

gd T cells and CD4 T cells in the spinal cord and DLN on day 21 are shown.

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toward a physiological concentration (10 nM) of S1P. Of note, theexpression of S1P1 and the S1P responsiveness in Vg4+ gd T cellswere almost completely lost in mice treated with FTY720 or THI,an inhibitor of S1P lyase. These results strongly suggest that theS1P/S1P1 axis plays an important role in the egress of IL-17–pro-ducing Vg4+ gd T cells from the LN.Recent analyses using two-photon intravital microscopy revealed

that conventional ab T cells exit LN through cortical sinuses in anS1P1-dependent manner (43, 44). After S1P1-dependent emigrationinto cortical sinuses, T cells become rounded, flow unidirectionallyinto medullary sinuses, and are subsequently flushed into the sub-capsular space and efferent lymph (44). However, it is still unclearwhether Vg4+ gd T cells can exit from the LN with a similarmechanism to conventional ab T cells. Our immunohistochemicalanalyses clearly showed that significant numbers of gd T cellswere found to be located in the T cell area of the DLN by IMQapplications. More marked accumulation of gd T cells were seenin the T cell area surrounding the lymphatic sinus of the DLN byFTY720 treatment. These results strongly support that FTY720induces sequestration of IL-17–producing Vg4+ gd T cells into theDLN and thereby inhibiting infiltration of these gd T cells into thesites of inflammation. Consequently, it is highly probable thatVg4+ gd T cells, similar to conventional ab T cells, exit from LNby an S1P1-dependent mechanism.BrdU-incorporated Vg4+ gd T cells were increased in the DLN

on days 5 and 6 in IMQ-treated mice and significant infiltration ofVg4+ gd T cells was found in inflamed skin on day 6. It has beenreported that IL-2, IL-4, and IL-21 can induce proliferation ofhuman gd T cells (45). Although the mRNA expressions of IL-4and IL-21 were elevated in the DLN on day 6 and these gccytokines may induce proliferation of Vg4+ gd T cells in mice, itwould be necessary to perform in vitro or in vivo studies usingthese cytokines or their neutralizing mAbs. Our results imply thatafter TLR7/8 stimulation, activated Vg4+ gd T cells are migratedto the DLN, are expanded there, and then recirculate back to theinflamed skin. Interestingly, we stably observed that FTY720treatment induced a marked increase of BrdU-incorporated Vg4+

gd T cells in the DLN and strikingly reduced infiltration of Vg4+

gd T cells into the inflamed skin on day 6. When S1P1 isdownregulated by treatment with FTY720, it is highly likely thatVg4+ gd T cells are unable to exit from the DLN and thereforethey are expanded and accumulated there. Consequently, it is pre-sumed that the dramatic increase of Vg4+ gd T cells by FTY720 isdue to sequestration of these cells in the DLN.It has been reported that IL-17–producing Vg4+ gd T cells are

detectable in the CNS prior to the onset of EAE and thus con-tribute to the pathogenesis of EAE (17). Our results clearlydemonstrate that FTY720 inhibits infiltration of Vg4+ gd T cellsinto the spinal cord and shows a marked preventing effect onMOG35–55-induced EAE. We and others have reported previouslythat FTY720 shows marked prophylactic and therapeutic effectsand reduces infiltration of Th17 and Th1 cells into the CNS byinhibiting S1P1-dependent egress of these Th cells in mouse EAE(12–15). Alternatively, recent studies demonstrated that Th17 cellsmay not be a sole producer of IL-17 in mouse EAE, because Vg4+

gd T cells can produce larger amounts of IL-17 compared withTh17 cells (17). It has been impressive that the infiltration of Vg4+

gd T cells in the CNS correlated with disease severity in EAE inSJL/J mice; a significant number of Vg4+ gd T cells were found inthe spinal cord during onset, peak, and relapse of EAE and thesegd T cells were temporarily lost during remission (24). Studies inMS patients have described that accumulation of Vd2 T cells isfound in the chronic active lesions (21). Vg9Vd2 T cells producedlarge amounts of IL-17 in the presence of IL-1b, IL-23, IL-6, and

TGF-b (46). Moreover, it has been reported that the frequency ofgd T cells is significantly increased in the PBL from MS patients,and FTY720 administration resulted in a marked reduction of IL-17 production by PBL stimulated with anti-CD3 and anti-CD28mAbs in vitro (11, 22, 47). Based on these findings, it is highlyprobable that inhibition of S1P1-dependent egress of pathogenicIL-17–producing gd T cells partly contributes to the therapeuticeffects of FTY720 on mouse EAE and relapsing MS.

AcknowledgmentsWe thank Dr. Kunitomo Adachi for synthesizing THI, and Shoji Wada and

Yasuko Ogawa for excellent technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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