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and FunctionDifferentiationInhibit Th17 but Not Th1 Cell

Induced Regulatory T Cells−Nitric Oxide

Foo Y. LiewAkio Mitani, Peter Pushparaj, Mohammed H. Alqahtani andC. Alves-Filho, Sandra Y. Fukada, Daniela Nascimento, Wanda Niedbala, Anne-Gaelle Besnard, Hui R. Jiang, Jose

http://www.jimmunol.org/content/191/1/164doi: 10.4049/jimmunol.12025802013;

2013; 191:164-170; Prepublished online 29 MayJ Immunol 

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0.DC1http://www.jimmunol.org/content/suppl/2013/05/29/jimmunol.120258

Referenceshttp://www.jimmunol.org/content/191/1/164.full#ref-list-1

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The Journal of Immunology

Nitric Oxide–Induced Regulatory T Cells Inhibit Th17but Not Th1 Cell Differentiation and Function

Wanda Niedbala,* Anne-Gaelle Besnard,* Hui R. Jiang,*,† Jose C. Alves-Filho,*,‡

Sandra Y. Fukada,*,‡ Daniela Nascimento,*,‡ Akio Mitani,*,x Peter Pushparaj,*,{

Mohammed H. Alqahtani,{ and Foo Y. Liew*

NO is a free radical with pleiotropic functions. We have shown earlier that NO induces a population of CD4+CD25+Foxp32

regulatory T cells (NO-Tregs) that suppress the functions of CD4+CD252 effector T cells in vitro and in vivo. We report in this

study an unexpected finding that NO-Tregs suppressed Th17 but not Th1 cell differentiation and function. In contrast, natural

Tregs (nTregs), which suppressed Th1 cells, failed to suppress Th17 cells. Consistent with this observation, NO-Tregs inhibited the

expression of retinoic acid–related orphan receptor gt but not T-bet, whereas nTregs suppressed T-bet but not retinoic acid–

related orphan receptor gt expression. The NO-Treg–mediated suppression of Th17 was partially cell contact–dependent and was

associated with IL-10. In vivo, adoptively transferred NO-Tregs potently attenuated experimental autoimmune encephalomyelitis.

The disease suppression was accompanied by a reduction of Th17, but not Th1 cells in the draining lymph nodes, and a decrease in

the production of IL-17, but an increase in IL-10 synthesis. Our results therefore demonstrate the differential suppressive function

between NO-Tregs and nTregs and indicate specialization of the regulatory mechanism of the immune system. The Journal of

Immunology, 2013, 191: 164–170.

Nitric oxide mediates a variety of biological functions,including vascular relaxation, platelet aggregation, neu-rotransmission, as well as microbicidal, tumoricidal, and

immune regulation (1–5). As such, NO is associated with some ofthe most important immunopathologies such as rheumatoid ar-thritis, diabetes, systemic lupus erythematosus, multiple sclerosis,and septic shock (6–10). NO is derived from the guanidino nitrogenatom(s) and molecular oxygen in a reaction catalyzed by the en-zyme NO synthase. There are three forms of NO synthase: theneuronal form, the inducible form, and the endothelial-derivedform. Neuronal NO synthase and endothelial-derived NO syn-thase produce amounts of NO required for physiological functions.Inducible NO synthase normally exists at a low level, is activatedby a number of immunological stimuli such as IFN-g, TNF-a, and

LPS, and catalyzes the high output of NO, which can be cytotoxicand kill intracellular pathogens.We have previously shown that a low dose of NO selectively en-

hances Th1 cell differentiation and expansion under Th1 polar-izing conditions. This was mediated by enhancing the expressionof IL-12Rb2 through a cGMP-dependent pathway (11). We havealso reported that NO can induce a subset of CD4+CD25+Foxp32

regulatory T cells (NO-Tregs) from CD4+CD252 T cells via p53,IL-2, and OX40 in a cGMP-independent manner (12). NO-Tregssuppress the proliferation and expansion of CD4+CD252 effectorcells in vitro and markedly attenuate the effector cell–mediatedcolitis and collagen-induced arthritis in the mouse in an IL-10–dependent manner. More recently, we reported that NO can alsodirectly suppress the differentiation and function of polarizedhuman and mouse Th17 cells via the downregulation of the arylhydrocarbon receptor leading to attenuation of experimental au-toimmune encephalomyelitis (EAE) (13). These results thereforeindicate that NO is a key player in the modulation of inflamma-tory disease. To further explore the role of NO in immune regu-lation, we investigated the potential role of NO-Tregs in affectingthe differentiation and function of different subsets of Th cells,particularly Th17 and Th1 cells.Th17 cells are now defined as a distinct subset of Th cells that

produce IL-17 with a signature transcription factor retinoid acid–related orphan receptor (ROR)gt. Th17 cells are associated withpathogenesis of human autoimmune diseases, including multiplesclerosis, rheumatoid arthritis, psoriasis, and inflammatory boweldisease (14–17). Thus, there likely exist rigorous endogenouscontrol mechanisms to limit Th17 proliferation and function.We report in this study that NO-Tregs effectively suppress the

differentiation and expansion of Th17 but not Th1 cells. Unex-pectedly, we found that natural Tregs (nTregs), although effectivein suppressing Th1, are relatively ineffective in suppression ofTh17. NO-Tregs suppress Th17 via a cell contact–dependent mech-anism and also partially via IL-10. In vivo, NO-Tregs markedlyattenuate EAE in association with the reduction of Th17 but not

*Centre of Immunology, Institute of Infection, Immunity and Inflammation, Univer-sity of Glasgow, Glasgow G12 8TA, United Kingdom; †Strathclyde Institute ofPharmacy and Biomedical Science, University of Strathclyde, Glasgow G4 0RE,United Kingdom; ‡Department of Pharmacology, School of Medicine of RibeiraoPreto, University of Sao Paulo, Sao Paulo 14049-900, Brazil; xDepartment of Peri-odontology, School of Dentistry, Aichi Gakuin University, Nagoya 464-8651, Japan;and {Center of Excellence in Genomic Medicine Research, King Abdulaziz Univer-sity, Jeddah 21589, Saudi Arabia

Received for publication September 13, 2012. Accepted for publication April 26,2013.

This work was supported by the Wellcome Trust, the Medical Research Council(United Kingdom), the European Union, and the Highly Cited Grant of the KingAbdulaziz University, Saudi Arabia.

Address correspondence and reprint requests to Prof. Foo Y. Liew and Dr. WandaNiedbala, Centre of Immunology, Institute of Infection, Immunity and Inflammation,University of Glasgow, 120 University Place, Glasgow G12 8TA, U.K. E-mail ad-dresses: foo.liew@glasgow.ac.uk (F.Y.L.) and wanda.niedbala@glasgow.ac.uk (W.N.)

The online version of this article contains supplemental material.

Abbreviations used in this article: DLN, draining lymph node; EAE, experimentalautoimmune encephalomyelitis; LN, lymph node; MOG, myelin oligodendrocyteglycoprotein; NOC-18, (Z)-1-[2-(2-aminoethyl)-N-(2-ammononioethyl)amino]diazen-1-ium-1,2-diolate; NO-Treg, NO-induced regulatory T cell; nTreg, natural regulatoryT cell; ROR, retinoic acid–related orphan receptor; Treg, regulatory T cell.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

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Th1. Taken together, these results support a key role of NO in theregulation of the immune system and further indicate compartmen-talization of the regulatory mechanism in inflammatory disease.

Materials and MethodsMice and reagents

Wild-type C57BL/6 and BALB/c mice were purchased from Harlan Olac.Il102/2 mice of the C57BL/6 background were obtained from the Na-tional Institute of Medical Research (London, U.K.). Males and femalesmice were used at the age of 6–10 wk. All animal experiments wereperformed according to the University of Glasgow guidelines and theUnited Kingdom Home Office regulations. (Z)-1-[2-(2-aminoethyl)-N-(2-ammononioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18, also knownas DETA-NONOate) was purchased from Enzo Life Sciences (Exeter, U.K.).Anti-CTLA4 was purchased from eBioscience.

Cell culture

CD4+, CD4+CD252, and CD4+CD25+ T cells were purified from the poolof spleens and lymph nodes of naive mice by negative selection (routinepurity.98%) using an autoMACS (Miltenyi Biotec). Culture medium wasRPMI 1640 supplemented with 10% (v/v) FCS (Lonza), 2 mM L-gluta-mine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.05 M 2-ME. Toobtain NO-Tregs, CD4+CD252 T cells were cultured in medium at 2.5 3104 cells/200 ml in 96-well round-bottom plates (Nunc) with soluble anti-CD3 (1 mg/ml; R&D Systems) and equal numbers of APCs (mitomycin C–treated spleen cells) for 5–6 d in the presence of 200 mM NOC-18 added atthe beginning of culture. The cells were then harvested, washed, and addedat graded numbers to other cell cultures as indicated. For Th17 cell dif-ferentiation, CD4+ T cells (5 3 105 cells/ml) were cultured in round-bottom 96-well plates with equal numbers of APCs and soluble anti-CD3 (1 mg/ml), TGF-b (1 ng/ml), IL-6 (10 ng/ml), IL-1b (10 ng/ml),anti–IFN-g (10 mg/ml), and anti–IL-4 (10 mg/ml) for up to 5 d. For Th1cell differentiation, CD4+ T cells were cultured in round-bottom 96-wellplates with equal numbers of APCs in the presence of soluble anti-CD3 (1mg/ml), IL-12 (10 ng/ml), and anti–IL-4 (10 mg/ml) for 4 d. For Th2 celldifferentiation, CD4+ T cells (5 3 105 cells/ml) were cultured in 96-wellround-bottom plates with equal numbers of APCs, anti-CD3 (1 mg/ml), IL-4 (10 ng/ml), and anti–IFN-g (10 mg/ml) for 4 d. For Th9 cell differen-tiation, CD4+ T cells (5 3 105 cells/ml) were cultured for up to 6 d in flat-bottom 96-well plates with plate-bound anti-CD3 (3 mg/ml) and solubleanti-CD28 (1.5 mg/ml), TGF-b (5 ng/ml), and IL-4 (10 ng/ml). For sup-pression assay, NO-Tregs or nTregs were added at the beginning of the

polarization culture or established T cells at various Treg/effector cellratios. The mixed cell populations were cultured for up to 6 d in 96-wellround-bottom plates. In some experiments, NO-Tregs were treated withmitomycin C before being added to the suppression culture. In otherexperiments, NO-Tregs were placed in the upper well and the effector cellsin the bottom well of a Transwell (Corning) culture. All cytokines and Abswere obtained from R&D Systems. Supernatants were collected and cy-tokine concentrations were determined by ELISA, and the cells wereharvested for FACS analysis. Cell division was determined by labelingCD4+ T cells with CFSE (Molecular Probes), and fluorescence density wasdetermined by FACS.

EAE induction and clinical evaluation

C57BL/6 mice were immunized s.c. on the back with 100 mg myelin ol-igodendrocyte glycoprotein (MOG)35–55 peptide (Sigma-Genosys) in 50 mlPBS emulsified with an equal volume of CFA (total 125 mg Mycobacte-rium tuberculosis, strain H37RA; Difco, Detroit MI). Each mouse alsoreceived i.p. 100 ng/200 ml pertussis toxin (Sigma-Aldrich, Poole, U.K.) inPBS on days 0 and 2 after immunization. EAE was scored according toa 0–5 scale as follows: 0, no clinical sign; 1, complete loss of tail tone; 2,hind limb weakness; 3, hind limb paralysis; 4, forelimb involvement; 5,moribund.

Histological and cellular evaluation of EAE

Mice were euthanized in a CO2 chamber and their spinal cords flushed outwith PBS by hydrostatic pressure using a syringe attached to an 18-gaugeneedle; the brains were also harvested. For histology analysis, the tissueswere then placed in 10% buffered formalin fixative overnight before beingtransferred to 75% ethanol. Tissues were then embedded in paraffin wax.Spinal cord and brain sections were stained with standard H&E. For FACSanalysis, brains and spinal cords were cut into small pieces and digestedwith collagenase A (2 mg/ml; Roche Diagnostics) and DNase (1 mg/ml;Sigma-Aldrich) at 37˚C for 40 min. Mononuclear cells were isolated bypassing the tissue through a cell strainer (100 mM), followed by a Percollgradient (70/30%) centrifugation. Mononuclear cells were removed fromthe interphase, washed, and resuspended in culture medium for furtheranalysis. Draining lymph nodes (DLNs) were also harvested, passedthrough a cell strainer, and resuspended in culture medium for cellularanalysis ex vivo by FACS.

FACS analysis

For intracellular cytokine staining, the cells were restimulated for 4 h with50 ng/ml PMA and 500 ng/ml ionomycin (both from Sigma-Aldrich) in the

FIGURE 1. NO-Tregs suppress the polarization of Th17. CD4+ T cells from BALB/c mice were labeled with CFSE and cultured under Th17 polarizing

conditions in the presence of graded ratio of NO-Tregs. (A) The cells were cultured with added anti–IL-2 and harvested on day 4 of culture and analyzed for

IL-17 and CFSE by FACS. The effector cells were gated for CFSE+ cells only. Results are representative of three experiments. (B) The percentage of IL-17+

cells cultured in the absence of anti–IL-2 in the presence or absence of NO-Tregs at a 1:1 ratio were pooled from six experiments (data are means 6 SEM).

(C) Concentrations of IL-17A in the culture supernatant were determined by ELISA. Supernatants were collected on day 4 of culture set up as in (A). Data

are means6 SEM pooled from three experiments. (D) FACS analysis of RORgt expression in CD4+ cells following 4-d culture under Th17 conditions with

or without NO-Tregs at 1:1 ratio. **p , 0.01, ***p , 0.001.

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presence of GolgiStop (BD Bioscience). The cells were first stained forCD4, then permeabilized with Perm/Fix solution (eBioscience), and finallystained with anti–IL-17A, anti–IFN-g, anti–IL-4, anti–IL-9, anti-RORgt,anti–T-bet, or anti-GATA3 (all from eBioscience). Data were acquiredusing a FACSCalibur (BD Biosciences) and analyzed using FlowJo soft-ware (Tree Star). Isotype-matched Abs (directly conjugated) were used ascontrols.

ELISA

ELISAs were carried out with paired Abs for murine IL-9, IL-10, and IL-17A and IFN-g (all from BD Biosciences) according to the manufacturer’sinstructions. Sensitivity of the assays was ,20 pg/ml.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5.0. Compar-isons between two groups were performed using a two-tailed unpairedStudent t test. Multiple groups were compared using a two-way ANOVAfollowed by a Bonferroni posttest. Values for all measurements are ex-pressed as means 6 SEM. A p value ,0.05 was considered statisticallysignificant. All experiments were performed at least twice.

ResultsNO-Tregs suppress the induction of Th17 cells

We first investigated the possibility that NO-Tregs may inhibit theinduction of Th17 cells. CD4+CD252 T cells were purified fromthe lymph node and spleen of BALB/c mice. The cells were po-larized to NO-Tregs with soluble anti-CD3 Ab and mitomycin C–treated spleen cells (as APCs) in the presence of 200 mM NOC-18(a stable NO donor; 100 mM NOC-18 constantly release 200 nMNO with t1/2 of 20 h) (18) as described previously (12). The cellswere harvested on day 5 or 6 and checked for the NO-Treg phe-notypes, which were CD4+CD25+Foxp32, producing IL-10 butnot IFN-g, IL-4, IL-17, or IL-2 (Ref. 12 and data not shown). NO-Tregs were washed and added at the beginning of the culture ofpolarization to Th17 cells. Naive CD4+ T cells were labeled withCFSE and cultured with APCs, anti-CD3/CD28 plus TGF-b, IL-6,IL-1b, IL-23, anti–IFN-g, and anti–IL-4 with graded CD4+/NO-Treg ratios. The replication of Th17 cells and the production ofIL-17 were determined on day 4 by FACS and ELISA, respec-tively. NO-Tregs inhibited the polarization of Th17 cells in a cellratio–dependent manner (Fig. 1A, 1B). The inhibition of Th17polarization was confirmed by the reduction in the concentrationof IL-17A in the culture supernatant (Fig. 1C). Furthermore, NO-Tregs also inhibited the expression of RORgt, the signature tran-scription factor of Th17 (19) (Fig. 1D). These results indicatethat NO-Tregs may be potent Tregs for Th17 development.

NO-Tregs suppress Th17 but not Th1 cell differentiation,whereas nTregs do the opposite

We next compared the suppressive function of NO-Tregs withnTregs. NO-Tregs were prepared as above and nTregs (CD4+

CD25+) were purified from the spleen and LN of naive BALB/cmice. CD4+ T cells were labeled with CFSE and polarized to Th17as above in the presence of NO-Tregs or nTregs for 4 d. WhereasNO-Tregs strongly suppressed the differentiation of Th17 cells,nTregs had only a modest effect (Fig. 2A). Furthermore, whereasNO-Tregs markedly inhibited the number of IL-17+RORgt+ Tcells, nTregs did not (Fig. 2B). We then compared the suppres-sive effect of NO-Tregs and nTregs on Th1 cell differentiation.Naive CD4+ T cells were polarized to Th1 cells in cultures con-taining anti-CD3/CD28 plus IL-12 and anti–IL-4 in the presenceof NO-Tregs or nTregs. As expected, nTregs potently suppressedthe differentiation of IFN-g+ T cells (Fig. 2C) and the expressionof T-bet on these cells, the signature transcription factor of Th1.In contrast, NO-Tregs failed to affect the differentiation of Th1cells and T-bet expression (Fig. 2D). These results therefore

demonstrate a differential effect of the suppressive functions ofNO-Tregs and nTregs.

NO-Tregs suppress established Th17 but not Th1 cells

We next investigated the relative suppressive effect of NO-Tregsand nTregs on established Th17 and Th1 cells. Th17 and Th1cells were cultured with two rounds of polarization as above,harvested on day 4, washed, and recultured with NO-Tregs ornTregs at various Treg/T cell (Th17 and Th1) ratios as described inMaterials and Methods. Cells were harvested on day 4 and wereanalyzed by FACS (for cytokine and transcription factor expres-sion) and by ELISA (for cytokines in the culture supernatants).Cell proliferation was assessed on day 3 by [3H]thymidine uptake.NO-Tregs potently decreased the percentage of IL-17+RORgt+

cells, whereas nTregs registered only a modest and not significantinhibition of Th17 cells (Fig. 3A). This differential suppressiveeffect was evident across a range of Treg/ Th17 ratio (Fig. 3B) andwas supported by the results of the relative concentrations of IL-17A in the culture supernatants (Fig. 3C) and cellular proliferation(Fig. 3D). Under the present culture conditions, NO-Tregs are∼16-fold more effective (on cell–for-cell basis) than nTregs in

FIGURE 2. NO-Tregs suppress Th17 but not Th1 whereas nTregs in-

hibit Th1 but not Th17 differentiation. (A) CD4+ T cells were labeled with

CFSE and polarized to Th17 in the presence of NO-Tregs or nTregs at

a 1:1 ratio. Cells were harvested on day 4 of culture and analyzed for

CFSE dilution by FACS. Data are representative of three experiments. (B)

CD4+ T cells were polarized under Th17 conditions in the presence of NO-

Tregs or nTregs as above and stained for IL-17 and RORgt. Data are

representative of three experiments. Similar results were obtained on days

3 and 5. (C) CD4+ T cells were labeled with CFSE and polarized to Th1 in

the presence of NO-Tregs or nTregs at a 1:1 ratio. Cells were harvested on

day 4 of culture and analyzed for CFSE dilution by FACS. Data are rep-

resentative of three experiments. (D) CD4+ T cells were polarized under

Th1 conditions in the presence NO-Tregs or nTregs. The cells were har-

vested on day 4 and stained for IFN-g and T-bet. Data are representative of

three experiments. Similar results were obtained on days 3 and 5.

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suppressing Th17 cells. In contrast, whereas nTregs potently de-creased the percentage of established Th1 (IFNg+/T-bet+) cells,NO-Tregs did not (Fig. 3E). Again, these differential suppressiveeffects of NO-Tregs and nTregs were observed over a range ofTreg/Th1 ratios (Fig. 3F) and confirmed by the concentrations ofIFN-g in the culture supernatants (Fig. 3G) and by cellular pro-liferation (Fig. 3H). Under these culture conditions, nTregs are∼8-fold more effective than NO-Tregs in suppressing Th1. Wealso examined the relative suppressive effects of NO-Tregs andnTregs on established Th2 cells and Th9 cells. NO-Tregs andnTregs were equally suppressive against Th2 (Supplemental Fig.1) and Th9 cells (Supplemental Fig. 2). Taken together, theseresults corroborate the finding that NO-Tregs and nTregs havedifferential suppressive effects on the proinflammatory Th17 andTh1 cells.

Th17 suppression by NO-Tregs partially depends on cellcontact and IL-10

We next investigated the mechanism by which NO-Tregs inhibitthe differentiation of Th17 cells. We first tested the potential roleof CTLA4, which has been reported to be associated with thesuppressive activity of nTregs (20). NO-Tregs were added to thepolarization culture of Th17 cells at 1:1 ratio in the presence ofa neutralizing anti-CTLA4 Ab. The suppressive effect of NO-Tregs was not affected by anti-CTLA4 (Fig. 4A). We also testedthe requirement of metabolically active NO-Tregs in the sup-pressive function. NO-Tregs were pretreated with mitomycin C,washed, and then added to the polarizing culture of Th17 cells.The mitomycin C treatment did not affect the suppressive activityof NO-Tregs (Fig. 4A). These results were confirmed by the rel-ative concentrations of IL-17A in the culture supernatants (Fig.4B). Because mitomycin C did not affect the function of NO-Tregs, we tested the possibility that NO-Tregs assert their sup-pressive activity via a cell contact–dependent mechanism. Th17cells and NO-Tregs were cultured together or separated bya semipermeable membrane in a Transwell culture under Th17

polarizing conditions. The suppressive effect of NO-Tregs waspartially reversed when cultured in a Transwell system (Fig. 4C).This result was also evident in the level of IL-17A in the culturesupernatant (Fig. 4D). IL-10 has been shown to be important innTreg-mediated suppression of effector cells (21). We thereforeascertained the potential role of IL-10 in NO-Treg–mediated sup-pression of Th17 cells. NO-Tregs were derived from WT or IL-102/2 mice of the BALB/c background and cultured with CD4+

T cells under Th17 polarizing conditions. NO-Tregs from IL-102/2

mice were significantly less suppressive than those from the WTmice (Fig. 4E, 4F). These results therefore indicate that the sup-pression of Th17 cells by NO-Tregs requires both cell contact andthe presence of IL-10.

NO-Tregs attenuate EAE

Finally, we investigated the role of NO-Tregs in vivo by using theEAE model, a disease in mice closely associated with Th17 (15,22–24). EAE was induced in C57BL/6 mice by priming withMOG35–55 peptide. The EAE mice were injected i.p. with 1 3 106

NO-Tregs on day 10 after immunization, when the disease becameapparent. Mice given NO-Tregs developed significantly less se-vere EAE compared with the control mice injected with PBSalone (Fig. 5A). The disease attenuation by NO-Tregs was con-firmed by histological examination, which showed a marked re-duction of leukocyte infiltration in the submeningeal area of theCNS of the mice receiving NO-Tregs compared with the controlmice (Fig. 5B). The DLN cells were harvested on day 20 andanalyzed by FACS ex vivo. The LN of the NO-Tregs recipientsshowed a marked reduction in the frequency of IL-17+ T cellscompared with the PBS control mice. There was, however, nosignificant difference in the frequency of IFN-g+ or IL-17+/IFN-g+

T cells between the two groups of mice (Fig. 5C–E). We alsoanalyzed the CNS cells of these mice by FACS. There was a sig-nificant reduction of the frequency of CD4+IL-17+ (Th17) cellsin the NO-Treg recipients compared with controls (Fig. 5F–H).There was, however, no difference in the frequency of CD4+IFN-

FIGURE 3. NO-Tregs suppress established Th17 but not established Th1 whereas nTregs inhibit established Th1 but not established Th17. (A–D) CD4+

T cells were polarized to Th17. After two rounds of culture, the cells were recultured for up to 6 d with anti-CD3 and IL-23 in the presence of a graded ratio of

NO-Tregs or nTregs. Cells were stained for IL-17 and RORgt on day 4 (A, B). The concentration of IL-17A in the culture supernatant was determined by ELISA

(C). Cell proliferation of individual cell types or in a combination was determined by [3H]thymidine uptake on day 3 (D). (E–H) CD4+ T cells were polarized

under Th1 conditions. After two rounds of polarization, the cells were recultured with anti-CD3 in the presence of NO-Tregs or nTregs for up to 6 d. The cells

were harvested on day 4 and stained for IFN-g and T-bet (E, F). IFN-g in the culture supernatant was determined by ELISA (G). Cell proliferation was de-

termined by [3H]hymidine uptake (H). All data are representative of at least three experiments. Vertical bars are SEM. *p , 0.05, **p , 0.01.

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g+ (Th1) and IL-17+IFN-g+ double-positive cells in the CNS ofthe two groups (data not shown). Serum from NO-Treg recipientsalso produced less IL-17A but more IL-10 compared with the cellsfrom the control mice (Fig. 5I). These results therefore show thatNO-Tregs are able to attenuate EAE and that this effect is asso-ciated with the suppression of Th17 cells but not Th1 cells.

DiscussionThe existence and function of Tregs are arguably one of the mostsatisfying explanations of the maintenance of homeostasis of theimmune system. Their key roles in immune regulation demand thepossibility that Tregs exist in different forms to meet the challengesof distinct subsets of effector cells, mediating over exuberance ofimmune stimulations. Thus, in addition to nTregs, several otherforms of Tregs have been described. Tregs are now known to beinduced from CD4+CD252 T cells by specific Ags in humans andmice (25–27). Tregs with a Th1-like phenotype have also beenreported (28), whereas Tregs induced by IL-10 (Tr1) mediatedtheir suppressive function in the absence of Foxp3 (29). A subsetof TGF-b–induced Tregs (Th3) has also been described (30). NO-Tregs are distinct from these subsets of Tregs in several importantaspects. NO-Tregs are CD4+CD25+, Foxp32, GITR+, and CD27+,with a Th2-like phenotype, and they ameliorate inflammatorydiseases in an IL-10–dependent manner (12). NO-Tregs differfrom nTregs in that NO-Tregs do not express Foxp3. NO-Tregs arealso different from Th3 in that NO-Tregs do not produce TGF-b.NO-Tregs are distinct from Th2 because Th2 are not suppressiveagainst CD4+CD252 effector cells. NO-Tregs are also different

from Tr1 in that the induction of NO-Tregs is IL-10–independent.Importantly, note that IL-10 is the only detectable cytokine pro-duced by NO-Tregs (12). This finding excludes the possibility thatNO-Tregs may suppress Th17 via the production of IL-2, whichhas been shown to be a key inhibitor of Th17 differentiation (31).nTregs have been reported to suppress Th17 (32). However, this

suppression required a relatively high nTreg/Th17 ratio. In ourhands, on a cell-for-cell basis, NO-Tregs are at least 16-fold morepowerful than nTregs in suppressing Th17. In sharp contrast,nTregs are ∼8-fold more effective than NO-Tregs in suppressingTh1. The exact molecular mechanism for this reciprocal oppositeeffect is not clear, but the suppressions parallel the effect on therespective signature transcription factors of Th17 (RORgt) andTh1 (T-bet). Also note that despite intensive research for morethan a decade, the molecular mechanism for nTreg-mediatedsuppression of effector cells remains elusive and controversial.NO-Tregs suppress Th17 in a cell contact–dependent manner andare partially dependent on IL-10. We have explored other possi-bilities by blocking the usual suspects (ICAM-1, LFA-1, OX40)with negative results (data not shown). It may be that IL-10 needsto act in a short range, effective only when NO-Tregs and Th17are in close contact.The 200–400 nM dose of NO used in our study is likely to occur

in vivo in sites of acute infection and inflammation (18) and hasbeen used routinely in experiments in vitro (4, 13). Althoughhuman macrophages produced only a low level of NO, humans doproduce substantial amounts of NO comparable to those of rodentsin vivo, and the role of NO and inducible NO synthase in humans

FIGURE 4. NO-Tregs suppress Th17 in a cell contact–dependent manner. (A) CD4+ T cells were polarized to Th17 in the presence of NO-Tregs,

mitomycin C–treated NO-Tregs, or NO-Tregs plus anti-CTLA4 Ab. The cells were harvested on day 4 and stained for IL-17 and RORgt. IL-17A con-

centration in the culture supernatants was determined by ELISA (B). (C) CD4+ T cells were polarized to Th17 together with NO-Tregs or separated from

NO-Tregs in Transwells. The cells were harvested on day 4 and stained for IL-17 and RORgt. IL-17A concentration in culture supernatants was determined

by ELISA (D). (E) CD4+ T cells were polarized to Th17 in the presence of NO-Tregs derived from Il102/2 or wild-type (WT) mice. Cells were harvested on

day 4 and stained for IL-17 and RORgt. IL-17A concentration in the culture supernatants was determined by ELISA (F). All experiments were repeated at

least three times. Vertical bars are SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

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is not in dispute. It has been recently reported that NO is producedin large amounts in the mouse by nonhematopoietic stroma cellssuch as fibroblastic reticular cells and lymphatic endothelial cells(33). The production of NO by these cell types in humans remainsto be explored. Thus, it is likely that the findings reported in thisstudy are equally applicable to humans.We have demonstrated that NO-Tregs are effective in attenu-

ating ongoing EAE. We have elected to test NO-Tregs in EAEbecause both Th17 and Th1 have been implicated in this diseasemodel (22–24, 34). NO-Treg–mediated EAE attenuation is ac-companied by a marked reduction in the frequency of Th17 cellsin the DLN. In contrast, the frequency of Th1 cells remains un-changed. These results are consistent with the notion that NO-Tregsselectively suppress Th17 but not Th1 in vivo. It has been welldocumented that nTregs can suppress EAE probably through thesuppression of Th1 cells (reviewed in Refs. 23, 24). We have dem-onstrated earlier that Th17 and Th1 cell infiltration in the DLN andspleen reflect the infiltration of these cells in the CNS (35). Also notethat the disease attenuation by NO-Tregs was incomplete, com-patible with a role for other effector cells such as Th1 in EAE. Forthis matter, we did not test the effect of NO-Tregs from IL-102/2

mice, as the EAE model would not be sensitive enough to measurethis partial effect.

Data reported in this study provide important functional char-acterization of NO-Tregs, which selectively suppress Th17 overTh1 cells. Additionally, we demonstrate the contrasting function ofnTregs, which selectively suppress Th1 over Th17. The molecularmechanism involved is likely the selective inhibition of RORgtby NO-Tregs, and the reduction of T-bet expression by nTregs.nTregs exist under physiological conditions for the maintenance ofimmune balance. A high dose of NO is induced during infections,which also activate Th17 cells that can lead to a range of in-flammatory autoimmune disorders. The selective suppression ofTh17 by NO-Tregs would ensure the rapid and economic localsuppression of excessive Th17 to avoid collateral damage, whileleaving the general surveillance nature of Th1 intact. Our findingof the compartmentalization of Treg activities demonstrates animportant biological function that may be of relevance to theevolution of the immune system.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 5. NO-Tregs attenuate EAE and reduce the frequency of Th17 but not Th1 cells in vivo. C57BL/6 mice were immunized with MOG peptide to

induce EAE. Mice were injected i.v. with NO-Tregs (1 3 106 cells) on day 10 after immunization (red arrow). Disease development was monitored daily

and expressed as mean clinical score (A). Mice were sacrificed on day 30, the spinal cords were collected and fixed, and sections were stained with H&E

(B). Original magnification 3100 and 3400 (insets). (C–E) DLNs were collected 20 d after immunization and analyzed ex vivo by FACS. IL-17+ and IFN-

g+ cells were gated for CD4+ cell populations. Data are representative of 10 mice per group in two independent experiments. (F–H) In an additional

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ELISA. Vertical bars are SEM (n = 5). *p , 0.05, ***p , 0.001.

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