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CD25+ Regulatory CD4+ T CellsTNF Receptor in Both Conventional and Costimulation via Glucocorticoid-Induced

Sakaguchi, Isao Ishikawa and Miyuki AzumaShimonHashiguchi, Tomohisa Nishioka, Takeshi Takahashi,

Fumiko Kanamaru, Pornpan Youngnak, Masaaki

http://www.jimmunol.org/content/172/12/73062004; 172:7306-7314; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/12/7306.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Costimulation via Glucocorticoid-Induced TNF Receptor inBoth Conventional and CD25� Regulatory CD4� T Cells1

Fumiko Kanamaru,* † Pornpan Youngnak,* Masaaki Hashiguchi,* Tomohisa Nishioka,‡

Takeshi Takahashi,‡ Shimon Sakaguchi,‡ Isao Ishikawa,† and Miyuki Azuma 2*

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially athigh levels on CD25�CD4� regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITRis also expressed on conventional CD4� and CD8� T cells, and its expression is enhanced rapidly after activation. In this reportwe show that the GITR provides a potent costimulatory signal to both CD25� and CD25� CD4� T cells. GITR-mediatedstimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of bothCD25�CD4� and CD25�CD4� T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation wasfurther confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokineproduction, such as IL-2, IFN-�, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessedby translocation of NF-�B components. GITR costimulation showed a potent ability to produce high amounts of IL-10, whichresulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potentand unique costimulator for an early CD4� T cell activation. The Journal of Immunology, 2004, 172: 7306–7314.

T he TNF and TNF receptor superfamilies (TNFRSF)3 reg-ulate diverse biological functions, including cell prolifer-ation, differentiation, and survival (1–5). The glucocorti-

coid-induced TNF receptor (GITR) (TNFR18), which is a newmember of the TNFRSF, is overexpressed on T cells after dexa-methasone treatment or TCR stimulation (6, 7). GITR is a type Itransmembrane protein with three cysteine-rich pseudorepeats inthe extracellular domain and striking homology in its intracellulardomain to a subgroup of the TNFRSF, which includes CD27,OX40, and 4-1BB (6, 7). These molecules lack the death domain,which is required for the induction of apoptosis, and they mediateintracellular signaling by recruiting TNF receptor-associated factor(TRAF) proteins to their cytoplasmic domains (6, 7). Interestingly,all these molecules are highly induced on T cells after activation,and provide strong costimulatory signals for T cells when ligatedwith their respective ligands or agonistic Abs (8–10). Initial stud-ies have revealed that GITR gene-transfected cells induce resis-tance to anti-CD3-induced apoptosis, which suggests involvementin the regulation of TCR-mediated apoptosis (6, 7). Recently,GITR-deficient mice were generated, the T cells of which exhib-ited higher proliferative responses, IL-2 production, IL-2 receptor

expression, and activation-induced cell death in response to anti-CD3 stimulation, which suggests a regulatory role for GITR in Tcell activation and apoptosis (11).

Interestingly, GITR is expressed predominantly onCD25�CD4� regulatory T (Treg) cells, and the mAb or polyclonalAb directed against GITR abrogates Treg cell-mediated suppres-sion both in vitro and in vivo (12, 13). It is generally believed thatthe reversal of suppression by anti-GITR Ab is mediated by theinfluence of the Ab on Treg cells, and that GITR signaling is ableto break the immunological self-tolerance mediated by Treg cells.However, several questions remain to be answered regarding themechanism behind these activities. One of the key issues relates toGITR function in conventional T cells. GITR is also expressed onconventional T cells (12, 13), and the Con A-induced proliferativeresponses of CD25�CD4� T cells from CD28-deficient mice areenhanced in the presence of anti-GITR mAb (12). In this study weinvestigated the costimulatory capacity of GITR for CD4� T cellsusing anti-GITR mAb.

Materials and MethodsMice

Specific pathogen-free, 6-wk-old, female BALB/c mice were purchasedfrom Japan Charles River Breeding Laboratories (Kanagawa, Japan). Theywere maintained in the animal facility of Tokyo Medical and Dental Uni-versity (Tokyo, Japan) and used at 7–10 wk of age. All procedures wereapproved by the animal care and use committee of Tokyo Medical andDental University.

Monoclonal Abs and flow cytometry

Hybridomas against CD3 (145-2C11, hamster IgG), I-Ab,d,q (M5/114, ratIgG2b), CD24 (J11d, rat IgM), CD45R/B220 (RA3-3A1, rat IgM), CD4(RL172.4, rat IgM), and CD8 (3.155, rat IgM) were obtained from Amer-ican Type Culture Collection (Manassas, VA). A hybridoma against GITR(DTA-1, rat IgG2a) was generated as described previously (12). ThesemAbs were purified from ascites (14, 15) for T cell functional assay orwere used as culture supernatants for purification of T cells. Biotinylationof anti-GITR mAb was performed by a standard method in our laboratory.Control rat IgG and hamster IgG were obtained from BD PharMingen (SanDiego, CA). Anti-CD28 (PV-1, hamster IgG) (16) and anti-NK (DX5, rat

Departments of *Molecular Immunology and†Periodontal Diseases, GraduateSchool, Tokyo Medical and Dental University, Tokyo, Japan; and‡Department ofExperimental Pathology, Institute for Frontier Medical Sciences, Kyoto University,Kyoto, Japan

Received for publication November 12, 2003. Accepted for publication April 7, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a Grant-in-Aid for Scientific Research from the Min-istry of Education, Culture, Sports, and Science of Japan.2 Address correspondence and reprint requests to Dr. Miyuki Azuma, Department ofMolecular Immunology, Graduate School, Tokyo Medical and Dental University,1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address:miyuki.mim@tmd.ac.jp3 Abbreviations used in this paper: TNFRSF, TNF receptor superfamily; DC, den-dritic cell; GFP, green fluorescence protein; GITR, glucocorticoid-induced TNF;GITRL, GITR ligand; MFI, mean fluorescence intensity; NIK, NF-�B-inducing ki-nase; TRAF, TNF receptor-associated factor; Treg, regulatory T cell; m, mouse.

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IgM) mAbs were provided by Drs. R. Abe (Research Institute for Biolog-ical Science, University of Tokyo, Tokyo, Japan) and L. Lanier (Universityof California, San Francisco, CA), respectively. PE-conjugated anti-CD3(145-2C11, hamster IgG), anti-CD69 (H1.2F3, hamster IgG), and anti-CD25 (PC61, rat IgG) mAbs; biotinylated anti-CD25 (7D4, rat IgM) mAb;FITC-conjugated anti-CD4 (GK1.5, rat IgG2b) and anti-CD8 (53-6.7, ratIgG) mAbs; and allophycocyanin-conjugated anti-CD4 (L3T4, rat IgG)mAb as well as appropriate fluorochrome-conjugated control hamster andrat Ig were obtained from BD PharMingen or eBioscience (San Diego,CA). For biotinylated mAbs, PE- and allophycocyanin-streptavidin (BDPharMingen) were used as the second-step reagents. Immunofluorescenceand flow cytometry were performed using FACSCalibur and CellQuestsoftware (BD Biosciences, San Jose, CA).

Isolation of T cells

Splenocytes from BALB/c mice were incubated with a hybridoma super-natant mixture that contained anti-I-A, anti-CD24, anti-CD45R, anti-NK(DX5), and anti-CD8 mAbs, and then were treated with rabbit complement(Cedarlane, Hornby, Canada) to deplete the APCs, CD8� T, and NK cells,as described previously (17). The purity of �90% CD3�CD4� I-A� cellswas confirmed by flow cytometry, and these cells were used as CD4� Tcells. For selected experiments, CD4� T cells were stained with PE-con-jugated anti-CD25 mAb, incubated with anti-PE microbeads (Miltenyi Bio-tec, Bergisch Gladbach, Germany), and then sorted into CD25�CD4� andCD25�CD4� T cell populations using the MACS system, according to themanufacturer’s protocol (Miltenyi Biotec). The purity levels of theCD25�CD4� and CD25�CD4� T cell fractions were �95 and �93%,respectively.

T cell proliferation and cytokine production

Purified CD4�, CD25�CD4�, or CD25�CD4� T cells (2 � 105/wells)were stimulated with combinations of immobilized anti-CD3 mAb (0.25–2.0 �g/ml) and either immobilized or soluble anti-GITR, anti-CD28 mAb,or control IgG (0.01–20 �g/ml) in flat-bottom, 96-well plates for 72 h. Forneutralization of secreted cytokines, 10 �g/ml each of anti-IL-2 (JES6-1A12, rat IgG) or anti-IL-10 (JES5-2A5, rat IgG) mAb was added at thestart of the assay. All mAbs were obtained from BD PharMingen. Thecultures were pulsed for the final 18 h with [3H]thymidine (0.5 �Ci/well;DuPont/NEN, Boston, MA) and were harvested on a 96-well plate har-vester (Skatron, Liver, Norway). The incorporated radioactivity was mea-sured using a microplate beta counter (Micro � Plus; Wallac, Turku, Fin-land). Supernatants from similar cultures were collected after 24 and 48 hfor assessment of cytokine production by ELISA. The ELISAs for murineIL-2, IFN-�, IL-4, and IL-10 were performed using ELISA kits (Ready-SET-Go; eBiosciences) according to the protocols recommended by themanufacturer.

CFSE labeling and flow cytometry

Purified CD4� T cells were labeled with CFSE (Molecular Probes, Eugene,OR) as described previously (18). The CFSE-labeled CD4� T cells (1 �106/well) were stimulated with immobilized anti-CD3 mAb (5 �g/ml) inthe presence of control rat IgG or anti-GITR mAb (1 �g/ml) in 48-wellplates for the indicated periods. The cells were then collected, and 100,000events/sample were acquired in flow cytometry. Distinct peaks in the pop-ulations of proliferating cells were determined by the sequential halving ofthe CFSE intensity.

Anti-CD3-induced redirected cytotoxicity assay

The murine mastocytoma cell line P815, which expresses Fc�RII, was usedas the target cell. Anti-CD3-induced redirected cytotoxicity was measuredby the JAM test, as described previously (19, 20). In brief, purified CD4�

T cells were cocultured for 6 h with [3H]thymidine-labeled P815 targets(5000/well) in the presence of anti-CD3 mAb (2C11; 2 �g/ml) and anti-GITR mAb (10 �g/ml). The cells were harvested, the radioactivity wasmeasured as described above, and the percentage of specific cytotoxicitywas calculated as described previously (19).

GITR ligand (GITRL)-P815 transfectants and costimulationassay

The GITR-Ig fusion protein and GITRL cDNA in pGEM-T vector wereprovided by Dr. T. Nishioka (details will be described elsewhere). Themouse GITRL (mGITRL) in pGEM was subcloned into the internal ribo-some entry site 2-enhanced green fluorescence protein (GFP) expressionvector (BD Biosciences). P815 cells were transfected with 10 �g ofmGITRL/internal ribosome entry site 2-enhanced GFP by electroporation

and then drug-selected by 1.0 mg/ml G418 as described previously (21).The cells were cloned, and the GFP-positive cells were selected by flowcytometry. The cell surface expression of GITRL was confirmed by thestaining with GITR-Ig, followed by PE-conjugated anti-human IgG (CaltagLaboratories, Burlingame, CA).

Purified CD25�CD4� and CD25�CD4� T cells (2 � 105/wells) werecocultured with either mytomycin C-treated parental P815 or mGITRL-transfected P815 cells in the presence of soluble anti-CD3 mAb (0.25 �g/ml) for 48 and 72 h. The proliferative responses were assessed as describedabove.

Preparation of cytosolic and nuclear extracts andimmunoblotting

Purified CD4� T cells were stimulated with anti-CD3 and/or anti-GITRmAb for 24 h. After washing, cells were solubilized in lysis buffer con-taining 0.6% IGEPAL, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2,10 mMKCl, 0.5 mM DTT, 2 �g/ml aprotinin, and 0.01 mM PMSF. Supernatantswere used as cytosolic proteins (22). The pellets were extracted with vig-orous agitation at 4°C in the buffer containing 20 mM HEPES (pH 7.9),0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mMDTT, and 2 �g/ml aprotinin. Protein amounts for cytosolic and nuclearextracts were assessed by bicinchoninic acid protein assay kit (Pierce,Rockford, IL). Twenty-five micrograms each of cytosolic or nuclear ex-tracts was subjected to 7.5% SDS-PAGE, with subsequent electrophoretictransfer to polyvinylidene difluoride membranes. After blocking with PBScontaining 1% BSA and 5% skim milk (Difco, Detroit, MI), the mem-branes were incubated with rabbit anti-c-Rel (sc-70), rabbit anti-p50 (sc-114), or mouse anti-p65 (sc-8008) Ab, followed by HRP-conjugated goatanti-rabbit IgG (Cell Signaling Technology, Beverly, MA) or goat anti-mouse IgG Ab (Upstate Biotechnology, Lake Placid, NY), and then de-veloped with ECL (Amersham, Arlington Heights, IL). All primary Abswere obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

ResultsSubstantial expression of GITR on conventional CD4� andCD8� T cells

We first examined the expression of GITR on CD3� T cells insplenocytes, both before and after stimulation with anti-CD3 andanti-CD28 mAbs. GITR was substantially expressed on freshlyisolated CD4� and CD8� T cells, and its expression was stronglyenhanced after activation (Fig. 1A). Most CD4� and CD8� T cellsexpressed GITR at high levels after activation. A kinetic studyrevealed that GITR expression on T cells was induced rapidly onlyafter 6 h of stimulation and peaked within 24 h of activation (Fig.1B). Additional long term activation did not further increase thelevel of GITR expression (data not shown). In freshly isolatedsplenocytes, the mean fluorescence intensity (MFI) for GITR onCD4� T cells was higher than that on CD8� T cells; this differencewas consistent throughout the activation period (Fig. 1, A and B).Consistent with previous reports (12, 13), the expression of GITRon CD25�CD4� Treg cells was �5-fold higher than that onCD25�CD4� T cells before activation (Fig. 1C), but all CD4� Tcells induced high levels of GITR after the 24-h activation (Fig.1B). Our results confirmed the constitutive expression of GITR onboth CD4� and CD8� T cells, the predominant expression ofGITR on CD4� T cells, and the rapid enhancement of GITR ex-pression on both CD4� and CD8� T cells by activation signals.

Costimulation of CD4� T cells by anti-GITR mAb

McHugh et al. (13) demonstrated that a polyclonal anti-GITR Abcostimulated the proliferative responses of CD25�CD4� T cells,but not those of CD25�CD4� T cells, in conjunction with IL-2. Incontrast, we reported previously that the addition of anti-GITRmAb (DTA-1) produced a 2- to 3-fold enhancement of the prolif-erative responses of CD25�CD4� T cells from wild-type orCD28-deficient mice in the presence of lower concentrations ofanti-CD3 mAb (�0.5 �g/ml) or Con A (12). To investigate inmore depth the costimulatory function of GITR, we examined the

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proliferative responses of CD4� T cells that were induced in con-junction with anti-GITR mAb DTA-1. Purified CD4� T cells fromBALB/c mice were stimulated in the presence of high, moderate,and low doses (2.0, 0.5, and 0.25 �g/ml) of immobilized (coated)anti-CD3 mAb and graded amounts of soluble anti-GITR mAb.When CD4� T cells were stimulated with the low (0.25 �g/ml)dose of anti-CD3 mAb, the addition of anti-GITR mAb did notinduce significant proliferative responses in the CD4� T cells (Fig.2A). However, when the CD4� T cells were stimulated with theappropriate suboptimal dose (0.5 �g/ml) of anti-CD3 mAb,DTA-1 augmented efficiently the proliferative responses of CD4�

T cells in a dose-dependent manner. These effects were also ob-served when DTA-1 was used in the immobilized form (data notshown). At the high dose (2 �g/ml) of anti-CD3 mAb, no enhanc-ing effect of anti-GITR mAb was seen at low doses of anti-GITRmAb; indeed, the proliferative responses appeared to be inhibitedby high doses (�1 �g/ml) of anti-GITR mAb. GITR is expressedpredominantly on CD25�CD4� Treg cells (12, 13). Therefore, tospecify the effects of anti-GITR mAb on CD25�CD4� andCD25�CD4� T cells, we first compared the effects of anti-GITRmAb on whole CD4� and CD25�-depleted CD4� T cell fractions.

The proliferative responses of both CD4� and CD25�CD4� Tcells were augmented in a similar dose-dependent manner by anti-GITR mAb (Fig. 2B). We then examined directly the effect ofanti-GITR mAb on the proliferative responses of CD25�CD4� Tcells. Consistent with previous observations (23–25), theCD25�CD4� T cells were clearly hyporesponsive after anti-CD3(0.5 and 2.0 �g/ml) stimulation (Fig. 2C) compared with conven-tional CD4� T cells (Fig. 2A). Surprisingly, the proliferative re-sponses of the CD25�CD4� T cells that were stimulated withanti-CD3 mAb were also enhanced dramatically by the addition ofanti-GITR mAb (Fig. 2C). These results demonstrate that ligationof GITR by anti-GITR mAb costimulates the proliferation of bothCD25�CD4� and CD25�CD4� T cells.

To further confirm the costimulatory function of anti-GITRmAb, we examined the induction of activation Ags CD69 andCD25 by anti-GITR mAb stimulation. Anti-CD3 mAb stimulationalone rapidly induced, at 6 h, the early activation Ag CD69; thiseffect was enhanced further by anti-GITR mAb up to 18 h (Fig.3A). The expression of CD25 (IL-2R�) was also induced by anti-CD3 stimulation, and the MFI of cells that were stimulated withanti-GITR mAb was consistently higher. These results indicate

FIGURE 1. Expression of GITR on CD4� and CD8� T cells. A, Freshly isolated splenocytes (upper panels) and activated splenocytes (lower panels)were stimulated with immobilized anti-CD3 mAb (5 �g/ml) and anti-CD28 mAb (1 �g/ml) for 3 days and stained with FITC-conjugated anti-CD4 oranti-CD8 mAbs and biotinylated anti-GITR mAb, followed by PE-streptavidin and allophycocyanin-conjugated anti-CD3 mAb or the appropriate fluo-rochrome-conjugated control Ig. The cells were analyzed by flow cytometry. An electronic gate was set on the CD3� lymphocytes; the respective expressionlevels of GITR vs CD4 or CD8 are presented as dot plots. B, Kinetics of GITR expression. Splenocytes were stimulated as described above, and kineticchanges in GITR expression of the CD4� and CD8� T cells were examined. An electronic gate was set on the CD3�CD4� or CD3�CD8� lymphocytes;the histograms show GITR expression (bold lines) along with the control staining (dotted lines) at the indicated time points. The MFI values are indicatedin the upper right of each panel. Data shown are representative of three independent experiments that gave similar results. C, Comparison of GITRexpression between freshly isolated conventional and regulatory CD4� T cells. Purified CD4� T cells were stained with FITC-conjugated anti-CD4 mAb,PE-conjugated anti-CD25 mAb, and biotinylated anti-GITR mAb, followed by allophycocyanin-streptavidin, or with the appropriate fluorochrome-con-jugated control Igs. An electronic gate was set on either CD25�CD4� (a) or CD25�CD4� (b) cells, and the histograms for GITR expression (bold lines)are shown along with the control staining (dotted lines).

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that anti-GITR mAb stimulation efficiently induces early (�24 h)activation of CD4� T cells, as assessed by CD69 and CD25 in-duction. We also examined the effects of anti-GITR stimulation oncell division using CFSE-labeled CD4� T cells. Although cell di-vision was not observed 24 h after activation (data not shown), thecells that were stimulated with anti-GITR mAb showed a progres-sive increase in the number of divided cells between 48 and 72 hcompared with the control culture (Fig. 3B). Note that the additionof anti-GITR mAb did not affect the frequency of early apoptosis.

Costimulation of CD4� T cells by anti-GITR mAb enhancesnuclear translocation of the c-Rel/NF-�B component

To determine whether the addition of anti-GITR mAb affects sig-naling events in CD4� T cells, we examined nuclear translocationof the NF-�B family molecules, as most TNFRSF members, in-cluding GITR, induce activation of NF-�B (7, 26–28). PurifiedCD4� T cells were stimulated with anti-CD3 mAb alone or to-gether with anti-GITR mAb for 24 h. Proteins from cytosolic andnuclear fractions were immunoblotted for p50, p65, and c-Rel.Stimulation with anti-CD3 mAb alone induced NF-�B members,p50, p65, and c-Rel, in both cytosolic and nuclear fractions (Fig.4). The amounts of p50, p65, and c-Rel in the nuclear extracts wereclearly enhanced by the stimulation with anti-GITR mAb, althoughthose in the cytosolic fractions were not affected. In particular, thetranslocation of c-Rel, which is a critical NF-�B member for IL-2gene activation (29), was increased 3-fold. These results indicatedthat anti-GITR mAb together with a suboptimal anti-CD3 stimu-lation enhances signaling to T cells and promotes the activationand translocation of NF-�B. Note that this was caused by the only24-h costimulation. Coligation of GITR with TCR/CD3 promotesthe NF-�B signaling cascade, which may result in IL-2 promotoractivation.

Comparison of the costimulatory effects of CD28 and GITR

CD28 is a well-characterized potent costimulatory molecule thatinduces various T cell effector functions, such as proliferative re-sponses, cytokine production, and cytotoxicity (21, 30). We per-formed a parallel assay with anti-CD28 mAb. Similar to the effectsseen with anti-GITR mAb, anti-CD28 mAb costimulated the pro-liferative responses of CD4� T cells in conjunction with 1 �g/mlanti-CD3 mAb (Fig. 5A). However, in the case of stimulation with0.25 �g/ml anti-CD3 mAb, only anti-CD28 mAb efficiently co-stimulated the proliferative responses.

We also examined the combined effects of anti-CD28 plus anti-GITR mAbs on the proliferative responses of CD4� T cells. In thepresence of a constant dose (0.5 �g/ml) of anti-CD28 mAb, theaddition of anti-GITR mAb further augmented, in a dose-depen-dent manner, the proliferation of the CD4� T cells (Fig. 5B). At aconstant dose (1 �g/ml) of anti-GITR mAb, similar additional co-stimulatory effects were observed for anti-CD28 mAb. These re-sults show GITR-mediated synergy with CD28 costimulation inCD4� T cells, which suggests the existence of different signalingpathways for CD28- and GITR-mediated costimulation.

Analysis of the supernatants from CD4� T cell cultures dem-onstrated that both anti-GITR and anti-CD28 mAbs enhanced theproduction of IL-2, IFN-�, IL-4, and IL-10 (Fig. 6A). The enhanc-ing effect of anti-GITR mAb was especially obvious for IL-10production. Consistent with the results for cell proliferation, thecostimulatory effects of anti-GITR mAb on IL-2, IFN-�, and IL-4production were inferior to those induced by anti-CD28 mAb. Toexplore how the secreted IL-10 contributed to the proliferativeresponses, we examined the effects of neutralization of IL-2 orIL-10 on GITR-induced proliferation. Neutralization of IL-2 effi-ciently inhibited both proliferative responses stimulated with anti-CD3 mAb alone and together with anti-GITR mAb (Fig. 6B). Sur-prisingly, the addition of anti-IL-10 mAb significantly enhancedGITR-mediated proliferation. This enhancing effect by anti-IL-10mAb was only seen in the proliferation stimulated with anti-GITRmAb, not in the proliferation stimulated with anti-CD3 alone orwith anti-CD3 and anti-CD28 mAbs (data not shown). The en-hanced effects by the neutralization of IL-10 were seen in bothCD25�CD4� and CD25�CD4� T cells. These results suggest thatIL-2 and IL-10 cytokines that were induced by GITR costimula-tion have opposing actions on proliferation. The lesser potency ofGITR-mediated costimulation in proliferation may result in thereverse action by IL-10 that was also induced by GITR-mediatedcostimulation. Our results demonstrate that GITR acts on CD4� Tcells as a costimulatory molecule to induce proliferation, but ex-hibits a unique profile in cytokine production.

Anti-GITR mAb stimulation induces rapid cytotoxicity

To assess the role of GITR costimulation in the generation of CTL,we examined anti-CD3-induced redirected cytotoxicity againstFc�R-bearing P815 cells in a 6-h JAM test (20). CD28 (30, 31) orCD137 (20) costimulation enhanced anti-CD3-induced redirected

FIGURE 2. GITR promotes CD4� T cell proliferative responses. A, CD4� T cells (2 � 105/well) were stimulated for 72 h with immobilized anti-CD3mAb (E, 2 �g/ml; F, 0.5 �g/ml; f, 0.25 �g/ml) and the indicated amounts of soluble anti-GITR (DTA-1) mAb. B, CD4� (F) and CD25�CD4� (�) Tcells (2 � 105/well) were stimulated for 72 h with 0.5 �g/ml immobilized anti-CD3 mAb in the presence of graded doses of anti-GITR mAb. C,CD25�CD4� T cells (2 � 105/well) were stimulated for 72 h with immobilized anti-CD3 mAb (‚, 2 �g/ml; Œ, 0.5 �g/ml) and the titrated anti-GITR mAb.The proliferative responses for the final 18 h were measured by [3H]thymidine incorporation. The proliferative responses of all CD4� T cells withoutanti-CD3 mAb in the presence or the absence of anti-GITR mAb were always �1000 cpm (data not shown). Data shown are representative of threeindependent experiments with similar results.

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cytotoxicity of CD4� T cells against P815. Cytotoxicity was ef-ficiently induced in CD4� T cells by the addition of a suboptimaldose (2 �g/ml) of anti-CD3 mAb after 6 h of culture, and theaddition of anti-GITR mAb significantly enhanced anti-CD3-redirected cytotoxicity (Fig. 7). At a high dose (10 �g/ml) ofanti-CD3, the addition of anti-GITR mAb was no longer effectivein the generation of cytotoxicity (data not shown). These resultsindicate that GITR costimulation is capable of rapidly inducingcytotoxicity in CD4� T cells under certain conditions of TCRstimulation.

GITRL costimulates anti-CD3-induced proliferation of CD4� Tcells

To confirm the costimulatory function of GITR, mGITRL cDNAwas transfected into P815 cells, and stable transfectants expressingGITRL on their cell surface were generated. The expression ofGFP and GITRL is shown in Fig. 8A. To determine whether

GITRL-P815 cells were functionally competent to activate CD4�

T cells, purified CD25�CD4� T cells and CD25�CD4� T cellswere cocultured with either parental P815 or GITRL-P815 cells inthe presence of anti-CD3 mAb (0.25 �g/ml). When CD25�CD4�

T cells were stimulated by culture with GITRL-P815 cells, weobserved a pronounced effect on anti-CD3-induced proliferationcompared with culture with parental P815 cells (Fig. 8B). Consis-tent with the results using anti-GITR mAb as shown in Fig. 2C, theproliferative responses of CD25�CD4� T cells were enhanced ef-ficiently when GITRL-P815 cells were cocultured. The enhancedeffects were more prominent at 48 h than at 72 h of culture (notshown), and this enhanced proliferation was inhibited by the ad-dition of GITR-Ig to a similar level as the proliferation stimulatedwith parental P815 cells (data not shown). These results suggestthat the binding of a natural ligand, GITRL, to GITR on bothconventional CD4� T cells and CD25� Treg cells costimulatesanti-CD3-induced proliferation.

FIGURE 3. GITR promotes early activation and division of CD4� T cells. A, CD4� T cells were stimulated with immobilized anti-CD3 mAb (5 �g/ml)in the presence of soluble anti-GITR mAb or control rat IgG (1 �g/ml) for the indicated periods of time. The cells were stained with FITC-conjugatedanti-CD4 mAb and either PE-conjugated anti-CD69 mAb or biotinylated anti-CD25 mAb, followed by PE-streptavidin or the appropriate fluorochrome-conjugated control Ig, then were analyzed by flow cytometry. An electronic gate was placed on the CD4� lymphocytes; the expression levels of CD69 andCD25 are presented as histograms (bold lines). Histograms of the control Ig-stained cells are overlaid (broken lines). The MFI values are indicated in theupper right of each panel. Data shown are representative of three experiments with similar results. B, CFSE-labeled CD4� T cells were stimulated underthe conditions described above. The cells were harvested at 24 h (not shown), 48 h, and 72 h poststimulation for flow cytometry. The undivided parentalgeneration of CD4� T cells shows the peak of highest CFSE fluorescence in each histogram. The results shown are representative of three independentexperiments.

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DiscussionWe demonstrate that GITR exerts a potent costimulatory functionin CD4� T cells. Immobilized or soluble anti-GITR (DTA-1) mAbefficiently promoted proliferative responses; production of cyto-kines, such as IL-2, IFN-�, IL-4, and IL-10; generation of cyto-toxicity; expression of activation Ags; and cell cycle progression,all of which were induced with the appropriate dosage of anti-CD3mAb. In addition, the costimulation with anti-GITR mAb for 24 hclearly accelerated the intranuclear translocation of NF-�B com-ponents in CD4� T cells. The costimulatory function of GITR wasfurther confirmed by the binding of GITRL to GITR on bothCD25�CD4� and CD25�CD4� T cells.

Previous reports have demonstrated the regulation of TCR-in-duced apoptosis by anti-GITR mAb (6, 7). T cell responses to TCRstimulation, such as proliferation, IL-2/IL-2R expression, and ac-tivation-induced cell death, are promoted in GITR-deficient mice(11). In addition, GITR is predominantly expressed onCD25�CD4� Treg cells, and the Ab against GITR abrogates theirregulatory function (12, 13). All these observations highlight theregulatory role of GITR on CD25�CD4� Treg cells.

In this study we have clearly demonstrated another crucial func-tion of GITR, i.e., costimulation. Compared with CD28-mediatedcostimulation, the optimum range for TCR signaling within whichGITR costimulation is effective seems to be limited, as the co-stimulatory effect of anti-GITR mAb was not seen at either high orlow levels of CD3 stimulation. Although GITR expression pre-dominated on CD25�CD4� T cells, freshly isolated conventionalCD4� and CD8� T cells also constitutively expressed GITR at asignificant level, and its expression was rapidly up-regulated afteractivation. Therefore, it is possible that the cell surface GITR thatis induced earlier on naive and activated T cells can transducecostimulatory signals for early T cell activation.

These findings are not surprising, as most TNFRSF memberspossess costimulatory functions for T cells (1–5). The GITR in thecytoplasmic domain shares a striking homology with CD27 and4-1BB (6). Both molecules have been reported as either costimu-lating T cell activation and promoting cell survival or inducingapoptosis (8, 32). 4-1BB associates with the protein tyrosine ki-nase p56lck (33) and transmits signals through the TRAF2-NF-�B-inducing kinase (NIK) pathway, which results in the activation of

NF-�B (26). CD27 signals also activate NF-�B and stress-acti-vated protein kinase/c-Jun N-terminal kinase through the TRAF2/TRAF5-NIK pathway (27) and involve the protein tyrosine kinasecascade. Similarly, GITR signaling has been shown to involveTRAF2-NIK pathway-mediated activation of NF-�B (7). A recentreport demonstrated that the cells coexpressing GITR and GITRLor stimulation of GITR� cells with soluble GITRL led to activa-tion of NF-�B, and this was reduced by anti-GITR Ab (34). Ourresults also showed enhancement of intranuclear translocation ofc-Rel, which is a critical NF-�B member for IL-2 gene activation(29). Similar to our observations, costimulation by CD28, which isa potent costimulator for naive T cells, induced greater amounts oftranslocation of c-Rel/p50 complex to the nuclear (35). Further-more, similar to CD27 and 4-1BB, binding of the proapoptoticprotein Siva to the cytoplasmic domain of GITR has been shown(36). Thus, GITR may have similar functions to CD27 and 4-1BB,and these functions probably depend on the specific signal trans-duction of these molecules. In addition, more recently several

FIGURE 4. Translocation of NF-�B components by GITR costimula-tion. CD4� T cells were stimulated with immobilized anti-CD3 mAb (2�g/ml) alone or together with anti-GITR mAb (10 �g/ml) for 24 h. Thecytosolic and nuclear fractions from unstimulated and activated CD4� Tcells were separated by SDS-PAGE and immunoblotted with anti-p50, anti-p65, and anti-c-Rel Abs. The values under the bands show the relativedensities measured by densitograph. The results are representative of threeexperiments.

FIGURE 5. Comparative analyses of costimulation with anti-CD28 andanti-GITR mAbs. A, CD4� T cells were stimulated with immobilized anti-CD3 mAb (E, 0.25 �g/ml; ‚, 1.0 �g/ml) and the indicated amounts ofeither anti-GITR (F and Œ) or anti-CD28 (E and ‚) mAb. B, CD4� T cellswere stimulated with immobilized anti-CD3 mAb (0.5 �g/ml), the indi-cated amount of either anti-GITR mAb or control rat IgG (rIg), and eitheranti-CD28 mAb or control hamster IgG (hIg). The proliferative responseswere measured as described in Fig. 2. Representative data from three in-dependent experiments are shown.

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spliced variant forms of GITR with functionally different proper-ties have been identified (37). Variable levels of these splicingproducts on T cells may cause differential activation of intracel-lular pathways, resulting in differences in T cell functions.

Among the several cytokines that were enhanced by GITR-me-diated costimulation, IL-10 may play a unique role. GITR costimu-lation induced preferentially high amounts of IL-10, and the IL-10produced counter-regulated the action of IL-2, which was also in-duced by GITR costimulation. It seems likely that the regulatoryfunction of GITR in the TCR-induced stimulation that was re-ported previously (6, 7, 11) is dependent upon the action of IL-10.IL-10 has multifunctions to stimulate and to regulate immune re-sponses (38). IL-10 directly regulates T cells by inhibiting theirability to produce IL-2 and to proliferate (39, 40). In contrast,IL-10 also has immunostimulatory effects by inhibiting T cell ap-optosis (41). The immunostimulatory or immunosuppressive prop-erties of IL-10 may be controlled in part by the activation state ofT cells mediated by TCR and costimulatory signals.

GITRL has been identified in humans (7, 28, 42) and just re-cently in mice (34, 43). In humans, the expression of GITRLmRNA has been observed in the small intestine, ovary, testis, andkidney, but not in T cells; furthermore, cell surface expression ofGITRL on vascular endothelial cells has been reported (7, 28, 42).In mice, the studies using polyclonal anti-GITRL Ab or solubleGITRL showed constitutive expression of GITRL on immatureand mature dendritic cells (DC), and macrophages (34, 43). Inaddition to the variable expression and forms of GITR on T cells,the selective expression of GITRL on lymphoid and nonlymphoidtissue cells might influence the function of GITR in T cell activa-tion and regulation. In particular, CD25�CD4� Treg cells ex-pressed constitutively high GITR; therefore, GITR-mediated co-stimulation to the Treg cells may predominate in othercostimulation. Supporting this speculation, our results usingGITRL transfectants showed the preferentially sensitive and ear-lier responses of Treg cells to GITR costimulation. All previous

reports demonstrated the abrogation of Treg function by the addi-tion of anti-GITR mAb (12) or recombinant GITRL (43). How canwe explain the reason why GITR costimulation abrogated the sup-pressive function of Treg cells? We previously reported that highdoses of IL-2 or CD28 costimulation induced the proliferation ofTreg cells, but simultaneously abrogated their suppressive function(23, 44). Moreover, the transfer of such hyperproliferative Tregcells induced various autoimmune diseases in syngeneic athymicnude mice (44). These results suggested that the exhibition of sup-pressive function required the anergic/hypoproliferative state ofTreg cells. The GITR-mediated costimulation by anti-GITR mAbor GITRL may put Treg cells in an active/hyperproliferative state,

FIGURE 7. GITR costimulates anti-CD3-induced redirected cytotoxic-ity against P815. CD4� T cells were cocultured with [3H]thymidine-la-beled P815 cells in the presence of anti-CD3 mAb (2 �g/ml) and either 10�g/ml anti-GITR mAb (F) or control rat IgG (E) for 6 h at the indicatedE:T cell ratio, and cytotoxicity was measured. No cytotoxicity was ob-served in the absence of anti-CD3 mAb (data not shown). Values are themean � SD from four independent experiments. �, Statistically differentfrom control Ig (p � 0.05).

FIGURE 6. GITR costimulates cytokineproduction by CD4� T cells. A, CD4� Tcells were stimulated with immobilized anti-CD3 mAb (1 �g/ml) and the indicatedamount of anti-GITR, anti-CD28 mAb, orcontrol rat IgG. Cytokine production for IL-2after 24 h and that for IFN-�, IL-10, and IL-4after 48 h of culture were measured byELISA. The proliferative responses of thesame cultures were assessed after 72 h ofculture. Representative data from three inde-pendent experiments are shown. B, CD4�

(2 � 105 cells/well; a), CD25�CD4� (2 �105 cells/well; b), or CD25�CD4� (2 � 105

cells/well; c) T cells were stimulated withimmobilized anti-CD3 mAb (a and b, 1.0�g/ml; c, 2.0 �g/ml) and either 5.0 �g/mlcontrol rat IgG (�) or anti-GITR mAb (f) inthe presence of 10 �g/ml neutralizing anti-IL-2, anti-IL-10 mAb, or control rat IgG.The proliferative responses for the final 18 hof the 72-h culture were measured as de-scribed in Fig. 2. a, Values are the mean �SE from three independent experiments. �,Statistically significant (p � 0.05). b and c,Representative data from two independentexperiments are shown.

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and this may result in abrogation of the suppressive function ofTreg cells. Recent reports suggested that proliferation and activa-tion of Treg cells could be controlled by mature DC in an IL-2-dependent manner (45, 46). It is likely that GITR-GITRL-medi-ated costimulation may be involved in the interaction of Treg cellswith DC. Further studies are now underway to clarify the contri-bution of GITR-GITRL costimulation to the interactions of Treg orconventional CD4� T cells with DC.

In this report we highlight GITR function as a costimulatorymolecule for T cell activation in both conventional andCD25�CD4� T cells. Among an array of T cell costimulatoryreceptors, GITR and CD28 alone are expressed constitutively onnaive and resting T cells. The constitutive expression of GITR onconventional CD4� T cells may play an important role in the ini-tiation of T cell activation; in addition, GITR expressed on Tregcells may play a crucial role in the maintenance of peripheral tol-erance. The interactions between GITR and its ligand during im-mune responses may regulate diverse biological functions in Tcells, such as proliferation, activation, differentiation, and cellsurvival.

AcknowledgmentsWe thank Drs. R. Abe (Research Institute for Biological Science Univer-sity of Tokyo, Tokyo, Japan) and L. Lanier (University of California, SanFrancisco, CA) for mAbs.

References1. Gruss, H. J., and S. K. Dower. 1995. Tumor necrosis factor ligand superfamily:

involvement in the pathology of malignant lymphomas. Blood 85:3378.2. Locksley, R. M., N. Killeen, and M. J. Lenardo. 2001. The TNF and TNF re-

ceptor superfamilies: integrating mammalian biology. Cell 104:487.3. Chan, K. F., M. R. Siegel, and J. M. Lenardo. 2000. Signaling by the TNF

receptor superfamily and T cell homeostasis. Immunity 13:419.4. Screaton, G., and X. N. Xu. 2000. T cell life and death signalling via TNF-

receptor family members. Curr. Opin. Immunol. 12:316.5. Armitage, R. J. 1994. Tumor necrosis factor receptor superfamily members and

their ligands. Curr. Opin. Immunol. 6:407.6. Nocentini, G., L. Giunchi, S. Ronchetti, L. T. Krausz, A. Bartoli, R. Moraca,

G. Migliorati, and C. Riccardi. 1997. A new member of the tumor necrosis factor/

nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.Proc. Natl. Acad. Sci. USA 94:6216.

7. Gurney, A. L., S. A. Marsters, R. M. Huang, R. M. Pitti, D. T. Mark,D. T. Baldwin, A. M. Gray, A. D. Dowd, A. D. Brush, A. D. Heldens, et al. 1999.Identification of a new member of the tumor necrosis factor family and its re-ceptor, a human ortholog of mouse GITR. Curr. Biol. 9:215.

8. Kobata, T., K. Agematsu, J. Kameoka, S. F. Schlossman, and C. Morimoto. 1994.CD27 is a signal-transducing molecule involved in CD45RA� naive T cell co-stimulation. J. Immunol. 153:5422.

9. DeBenedette, M. A., N. R. Chu, K. E. Pollok, J. Hurtado, W. F. Wade,B. S. Kwon, and T. H. Watts. 1995. Role of 4-1BB ligand in costimulation of Tlymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp.Med. 181:985.

10. Akiba, H., H. Oshima, K. Takeda, M. Atsuta, H. Nakano, A. Nakajima,C. Nohara, H. Yagita, and K. Okumura. 1999. CD28-independent costimulationof T cells by OX40 ligand and CD70 on activated B cells. J. Immunol. 162:7058.

11. Ronchetti, S., G. Nocentini, C. Riccardi, and P. P. Pandolfi. 2002. Role of GITRin activation response of T lymphocytes. Blood 100:350.

12. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, and S. Sakaguchi. 2002. Stim-ulation of CD25�CD4� regulatory T cells through GITR breaks immunologicalself-tolerance. Nat. Immunol. 3:135.

13. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach,M. Collins, and M. C. Byrne. 2002. CD4�CD25� immunoregulatory T cells:gene expression analysis reveals a functional role for the glucocorticoid-inducedTNF receptor. Immunity 16:311.

14. Nakajima, A., M. Azuma, S. Kodera, S. Nuriya, A. Terashi, M. Abe, S. Hirose,T. Shirai, H. Yagita, and K. Okumura. 1995. Preferential dependence of autoan-tibody production in murine lupus on CD86 costimulatory molecule. Eur. J. Im-munol. 25:3060.

15. Nuriya, S., H. Yagita, K. Okumura, and M. Azuma. 1996. The differential role ofCD86 and CD80 co-stimulatory molecules in the induction and the effectorphases of contact hypersensitivity. Int. Immunol. 8:917.

16. Sperling, A. I., P. S. Linsley, T. A. Barrett, and J. A. Bluestone. 1993. CD28-mediated costimulation is necessary for the activation of T cell receptor-��� Tlymphocytes. J. Immunol. 151:6043.

17. Sakurai, J., J. Ohata, K. Saito, H. Miyajima, T. Hirano, T. Kohsaka, S. Enomoto,K. Okumura, and M. Azuma. 2000. Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft- versus-host disease by an enhanced expansion ofregulatory CD8� T cells. J. Immunol. 164:664.

18. Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division byflow cytometry. J. Immunol. Methods 171:131.

19. Matzinger, P. 1991. The JAM test: a simple assay for DNA fragmentation andcell death. J. Immunol. Methods 145:185.

20. Ebata, T., S. Mogi, Y. Hata, J. I. Fujimoto, H. Yagita, K. Okumura, andM. Azuma. 2001. Rapid induction of CD95 ligand and CD4� T cell-mediatedapoptosis by CD137 (4-1BB) costimulation. Eur. J. Immunol. 31:1410.

21. Azuma, M., M. Cayabyab, D. Buck, J. H. Phillips, and L. L. Lanier. 1992. CD28interaction with B7 costimulates primary allogeneic proliferative responses andcytotoxicity mediated by small, resting T lymphocytes. J. Exp. Med. 175:353.

FIGURE 8. GITR ligand-transfected P815 cells costimulates anti-CD3-induced proliferative responses of CD4� T cells. A, Parental P815 (plain lines, b) andGITRL-P815 (bold lines, c) cells were pretreated with Fc-block (BD PharMingen) and stained with either GITR-Ig or control human IgG (broken lines), followedby PE-conjugated anti-human IgG. Samples were analyzed by flow cytometry. The expression of GFP (a) and that of GITRL (b and c) are shown as histograms.B, Purified CD25�CD4� or CD25�CD4� T cells (2 � 105/wells) were cocultured with mytomycin C-treated P815 (E) or GITRL-P815 (F) stimulator cells atthe indicated responder/stimulator (R/S) ratio in the presence of soluble anti-CD3 mAb (0.25 �g/ml). The proliferative responses for the final 18 h of the 48- or72-h culture were measured as described in Fig. 2. Representative data for CD25�CD4� T cells after 72 h and for CD25�CD4� T cells after 48 h from threeindependent experiments are shown. The proliferative responses of CD25�CD4� and CD25�CD4� T cells with or without anti-CD3 mAb in the absence of P815cells were always �2000 and 200 cpm, respectively (data not shown).

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22. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcriptioninitiation by RNA polymerase II in a soluble extract from isolated mammaliannuclei. Nucleic Acids Res. 11:1475.

23. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata,J. Shimizu, and S. Sakaguchi. 1998. Immunologic self-tolerance maintained byCD25�CD4� naturally anergic and suppressive T cells: induction of autoimmunedisease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.

24. Thornton, A. M., and E. M. Shevach. 1998. CD4�CD25� immunoregulatory Tcells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2production. J. Exp. Med. 188:287.

25. Kuniyasu, Y., T. Takahashi, M. Itoh, J. Shimizu, G. Toda, and S. Sakaguchi.2000. Naturally anergic and suppressive CD25�CD4� T cells as a functionallyand phenotypically distinct immunoregulatory T cell subpopulation. Int. Immu-nol. 12:1145.

26. Arch, R. H., and C. B. Thompson. 1998. 4-1BB and OX40 are members of atumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bindTNF receptor-associated factors and activate nuclear factor �B. Mol. Cell. Biol.18:558.

27. Akiba, H., H. Nakano, S. Nishinaka, M. Shindo, T. Kobata, M. Atsuta,C. Morimoto, C. F. Ware, N. L. Malinin, D. Wallach, et al. 1998. CD27, amember of the tumor necrosis factor receptor superfamily, activates NF-�B andstress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, andNF-�B-inducing kinase. J. Biol. Chem. 273:13353.

28. Kwon, B., K. Y. Yu, J. Ni, G. L. Yu, I. K. Jang, Y. J. Kim, L. Xing, D. Liu,S. X. Wang, and B. S. Kwon. 1999. Identification of a novel activation-inducibleprotein of the tumor necrosis factor receptor superfamily and its ligand. J. Biol.Chem. 274:6056.

29. Kontgen, F., R. J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, andS. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects inlymphocyte proliferation, humoral immunity, and interleukin-2 expression.Genes Dev. 9:1965.

30. Lanier, L. L., S. O’Fallon, C. Somoza, J. H. Phillips, P. S. Linsley, K. Okumura,and M. Azuma. 1995. CD80(B7) and CD86(B70) provide similar costimulatorysignals for T cell proliferation, cytokine production and generation of cytotoxicT lymphocytes. J. Immunol. 154:97.

31. Azuma, M., and L. L. Lanier. 1995. The role of CD28 co-stimulation in thegeneration of cytotoxic T lymphocytes. Curr. Top. Microbiol. Immunol. 198:59.

32. Vinay, D. S., and B. S. Kwon. 1998. Role of 4-1BB in immune responses. Semin.Immunol. 10:481.

33. Kim, Y. J., K. E. Pollok, Z. Zhou, A. Shaw, J. B. Bohlen, M. Fraser, andB. S. Kwon. 1993. Novel T cell antigen 4-1BB associates with the protein ty-rosine kinase p56lck1. J. Immunol. 151:1255.

34. Yu, K. Y., H. S. Kim, S. Y. Song, S. S. Min, J. J. Jeong, and B. S. Youn. 2003.Identification of a ligand for glucocorticoid-induced tumor necrosis factor recep-tor constitutively expressed in dendritic cells. Biochem. Biophys. Res. Commun.310:433.

35. Zhou, X. Y., Y. Yashiro-Ohtani, M. Nakahira, W. R. Park, R. Abe, T. Hamaoka,M. Naramura, H. Gu, and H. Fujiwara. 2002. Molecular mechanisms underlyingdifferential contribution of CD28 versus non-CD28 costimulatory molecules toIL-2 promoter activation. J. Immunol. 168:3847.

36. Spinicelli, S., G. Nocentini, S. Ronchetti, L. T. Krausz, R. Bianchini, andC. Riccardi. 2002. GITR interacts with the pro-apoptotic protein Siva and inducesapoptosis. Cell Death Differ. 9:1382.

37. Nocentini, G., S. Ronchetti, A. Bartoli, S. Spinicelli, D. Delfino, L. Brunetti,G. Migliorati, and C. Riccardi. 2000. Identification of three novel mRNA splicevariants of GITR. Cell Death Differ. 7:408.

38. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. In-terleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683.

39. de Waal Malefyt, R., H. Yssel, and J. E. de Vries. 1993. Direct effects of IL-10on subsets of human CD4� T cell clones and resting T cells: specific inhibitionof IL-2 production and proliferation. J. Immunol. 150:4754.

40. Taga, K., H. Mostowski, and G. Tosato. 1993. Human interleukin-10 can directlyinhibit T-cell growth. Blood 81:2964.

41. Cohen, S. B., J. B. Crawley, M. C. Kahan, M. Feldmann, and B. M. Foxwell.1997. Interleukin-10 rescues T cells from apoptotic cell death: association with anupregulation of Bcl-2. Immunology 92:1.

42. Kwon, B., B. S. Youn, and B. S. Kwon. 1999. Functions of newly identifiedmembers of the tumor necrosis factor receptor/ligand superfamilies in lympho-cytes. Curr. Opin. Immunol. 11:340.

43. Tone, M., Y. Tone, E. Adams, S. F. Yates, M. R. Frewin, S. P. Cobbold, andH. Waldmann. 2003. Mouse glucocorticoid-induced tumor necrosis factor recep-tor ligand is costimulatory for T cells. Proc. Natl. Acad. Sci. USA 100:15059.

44. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, T. Otsuka, andS. Skaguchi. 1999. Thymus and autoimmunity: production of CD25�CD4� nat-urally anergic and suppressive T cell as akey function of the thymus in main-taining immunologic self-tolerance. J. Immunol. 162:5317.

45. Oldenhove, G., M. de Heusch, G. Urbain-Vansanten, J. Urbain, C. Maliszewski,O. Leo, and M. Moser. 2003. CD4� CD25� regulatory T cells control T helpercell type 1 responses to foreign antigens induced by mature dendritic cells invivo. J. Exp. Med. 198:259.

46. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, andR. M. Steinman. 2003. Direct expansion of functional CD25� CD4� regulatoryT cells by antigen-processing dendritic cells. J. Exp. Med. 198:235.

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