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Departments of
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Molecular Immunology and
Periodontal Diseases, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; and
Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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Abstract |
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, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-
B components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, GITR is expressed predominantly on CD25+CD4+ regulatory T (Treg) cells, and the mAb or polyclonal Ab directed against GITR abrogates Treg cell-mediated suppression both in vitro and in vivo (12, 13). It is generally believed that the reversal of suppression by anti-GITR Ab is mediated by the influence of the Ab on Treg cells, and that GITR signaling is able to break the immunological self-tolerance mediated by Treg cells. However, several questions remain to be answered regarding the mechanism behind these activities. One of the key issues relates to GITR function in conventional T cells. GITR is also expressed on conventional T cells (12, 13), and the Con A-induced proliferative responses of CD25–CD4+ T cells from CD28-deficient mice are enhanced in the presence of anti-GITR mAb (12). In this study we investigated the costimulatory capacity of GITR for CD4+ T cells using anti-GITR mAb.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free, 6-wk-old, female BALB/c mice were purchased from Japan Charles River Breeding Laboratories (Kanagawa, Japan). They were maintained in the animal facility of Tokyo Medical and Dental University (Tokyo, Japan) and used at 7–10 wk of age. All procedures were approved by the animal care and use committee of Tokyo Medical and Dental University.
Monoclonal Abs and flow cytometry
Hybridomas against CD3 (145-2C11, hamster IgG), I-Ab,d,q (M5/114, rat IgG2b), CD24 (J11d, rat IgM), CD45R/B220 (RA3-3A1, rat IgM), CD4 (RL172.4, rat IgM), and CD8 (3.155, rat IgM) were obtained from American Type Culture Collection (Manassas, VA). A hybridoma against GITR (DTA-1, rat IgG2a) was generated as described previously (12). These mAbs were purified from ascites (14, 15) for T cell functional assay or were used as culture supernatants for purification of T cells. Biotinylation of anti-GITR mAb was performed by a standard method in our laboratory. Control rat IgG and hamster IgG were obtained from BD PharMingen (San Diego, CA). Anti-CD28 (PV-1, hamster IgG) (16) and anti-NK (DX5, rat IgM) mAbs were provided by Drs. R. Abe (Research Institute for Biological Science, University of Tokyo, Tokyo, Japan) and L. Lanier (University of 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, rat IgG) mAbs; and allophycocyanin-conjugated anti-CD4 (L3T4, rat IgG) mAb as well as appropriate fluorochrome-conjugated control hamster and rat Ig were obtained from BD PharMingen or eBioscience (San Diego, CA). For biotinylated mAbs, PE- and allophycocyanin-streptavidin (BD PharMingen) were used as the second-step reagents. Immunofluorescence and flow cytometry were performed using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA).
Isolation of T cells
Splenocytes from BALB/c mice were incubated with a hybridoma supernatant 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– cells was confirmed by flow cytometry, and these cells were used as CD4+ T cells. For selected experiments, CD4+ T cells were stained with PE-conjugated anti-CD25 mAb, incubated with anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and then sorted into CD25–CD4+ and CD25+CD4+ T cell populations using the MACS system, according to the manufacturer’s protocol (Miltenyi Biotec). The purity levels of the CD25–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 x 105/wells) were stimulated with combinations of immobilized anti-CD3 mAb (0.25–2.0 µg/ml) and either immobilized or soluble anti-GITR, anti-CD28 mAb, or control IgG (0.01–20 µg/ml) in flat-bottom, 96-well plates for 72 h. For neutralization of secreted cytokines, 10 µg/ml each of anti-IL-2 (JES6-1A12, rat IgG) or anti-IL-10 (JES5-2A5, rat IgG) mAb was added at the start of the assay. All mAbs were obtained from BD PharMingen. The cultures were pulsed for the final 18 h with [3H]thymidine (0.5 µCi/well; DuPont/NEN, Boston, MA) and were harvested on a 96-well plate harvester (Skatron, Liver, Norway). The incorporated radioactivity was measured using a microplate beta counter (Micro 
Plus; Wallac, Turku, Finland). Supernatants from similar cultures were collected after 24 and 48 h for assessment of cytokine production by ELISA. The ELISAs for murine IL-2, IFN-, IL-4, and IL-10 were performed using ELISA kits (Ready-SET-Go; eBiosciences) according to the protocols recommended by the manufacturer.
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 x 106/well) were stimulated with immobilized anti-CD3 mAb (5 µg/ml) in the presence of control rat IgG or anti-GITR mAb (1 µg/ml) in 48-well plates for the indicated periods. The cells were then collected, and 100,000 events/sample were acquired in flow cytometry. Distinct peaks in the populations of proliferating cells were determined by the sequential halving of the CFSE intensity.
Anti-CD3-induced redirected cytotoxicity assay
The murine mastocytoma cell line P815, which expresses FcRII, was used as the target cell. Anti-CD3-induced redirected cytotoxicity was measured by 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 µg/ml) and anti-GITR mAb (10 µg/ml). The cells were harvested, the radioactivity was measured as described above, and the percentage of specific cytotoxicity was calculated as described previously (19).
GITR ligand (GITRL)-P815 transfectants and costimulation assay
The GITR-Ig fusion protein and GITRL cDNA in pGEM-T vector were provided by Dr. T. Nishioka (details will be described elsewhere). The mouse GITRL (mGITRL) in pGEM was subcloned into the internal ribosome entry site 2-enhanced green fluorescence protein (GFP) expression vector (BD Biosciences). P815 cells were transfected with 10 µg of mGITRL/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 flow cytometry. The cell surface expression of GITRL was confirmed by the staining with GITR-Ig, followed by PE-conjugated anti-human IgG (Caltag Laboratories, Burlingame, CA).
Purified CD25–CD4+ and CD25+CD4+ T cells (2 x 105/wells) were cocultured with either mytomycin C-treated parental P815 or mGITRL-transfected P815 cells in the presence of soluble anti-CD3 mAb (0.25 µg/ml) for 48 and 72 h. The proliferative responses were assessed as described above.
Preparation of cytosolic and nuclear extracts and immunoblotting
Purified CD4+ T cells were stimulated with anti-CD3 and/or anti-GITR mAb for 24 h. After washing, cells were solubilized in lysis buffer containing 0.6% IGEPAL, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2,10 mM KCl, 0.5 mM DTT, 2 µg/ml aprotinin, and 0.01 mM PMSF. Supernatants were used as cytosolic proteins (22). The pellets were extracted with vigorous 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 mM DTT, and 2 µg/ml aprotinin. Protein amounts for cytosolic and nuclear extracts were assessed by bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Twenty-five micrograms each of cytosolic or nuclear extracts was subjected to 7.5% SDS-PAGE, with subsequent electrophoretic transfer to polyvinylidene difluoride membranes. After blocking with PBS containing 1% BSA and 5% skim milk (Difco, Detroit, MI), the membranes 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 goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) or goat anti-mouse IgG Ab (Upstate Biotechnology, Lake Placid, NY), and then developed with ECL (Amersham, Arlington Heights, IL). All primary Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the expression of GITR on CD3+ T cells in splenocytes, both before and after stimulation with anti-CD3 and anti-CD28 mAbs. GITR was substantially expressed on freshly isolated CD4+ and CD8+ T cells, and its expression was strongly enhanced after activation (Fig. 1A). Most CD4+ and CD8+ T cells expressed GITR at high levels after activation. A kinetic study revealed that GITR expression on T cells was induced rapidly only after 6 h of stimulation and peaked within 24 h of activation (Fig. 1B). Additional long term activation did not further increase the level of GITR expression (data not shown). In freshly isolated splenocytes, the mean fluorescence intensity (MFI) for GITR on CD4+ T cells was higher than that on CD8+ T cells; this difference was consistent throughout the activation period (Fig. 1, A and B). Consistent with previous reports (12, 13), the expression of GITR on CD25+CD4+ Treg cells was 5-fold higher than that on CD25–CD4+ T cells before activation (Fig. 1C), but all CD4+ T cells induced high levels of GITR after the 24-h activation (Fig. 1B). Our results confirmed the constitutive expression of GITR on both CD4+ and CD8+ T cells, the predominant expression of GITR on CD4+ T cells, and the rapid enhancement of GITR expression on both CD4+ and CD8+ T cells by activation signals.
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McHugh et al. (13) demonstrated that a polyclonal anti-GITR Ab costimulated the proliferative responses of CD25+CD4+ T cells, but not those of CD25–CD4+ T cells, in conjunction with IL-2. In contrast, we reported previously that the addition of anti-GITR mAb (DTA-1) produced a 2- to 3-fold enhancement of the proliferative responses of CD25–CD4+ T cells from wild-type or CD28-deficient mice in the presence of lower concentrations of anti-CD3 mAb (<0.5 µg/ml) or Con A (12). To investigate in more depth the costimulatory function of GITR, we examined the proliferative responses of CD4+ T cells that were induced in conjunction with anti-GITR mAb DTA-1. Purified CD4+ T cells from BALB/c mice were stimulated in the presence of high, moderate, and low doses (2.0, 0.5, and 0.25 µg/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 µg/ml) dose of anti-CD3 mAb, the addition of anti-GITR mAb did not induce significant proliferative responses in the CD4+ T cells (Fig. 2A). However, when the CD4+ T cells were stimulated with the appropriate suboptimal dose (0.5 µg/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 observed when DTA-1 was used in the immobilized form (data not shown). At the high dose (2 µg/ml) of anti-CD3 mAb, no enhancing effect of anti-GITR mAb was seen at low doses of anti-GITR mAb; indeed, the proliferative responses appeared to be inhibited by high doses (>1 µg/ml) of anti-GITR mAb. GITR is expressed predominantly on CD25+CD4+ Treg cells (12, 13). Therefore, to specify the effects of anti-GITR mAb on CD25–CD4+ and CD25+CD4+ T cells, we first compared the effects of anti-GITR mAb on whole CD4+ and CD25+-depleted CD4+ T cell fractions. The proliferative responses of both CD4+ and CD25–CD4+ T cells were augmented in a similar dose-dependent manner by anti-GITR mAb (Fig. 2B). We then examined directly the effect of anti-GITR mAb on the proliferative responses of CD25+CD4+ T cells. Consistent with previous observations (23, 24, 25), the CD25+CD4+ T cells were clearly hyporesponsive after anti-CD3 (0.5 and 2.0 µg/ml) stimulation (Fig. 2C) compared with conventional CD4+ T cells (Fig. 2A). Surprisingly, the proliferative responses of the CD25+CD4+ T cells that were stimulated with anti-CD3 mAb were also enhanced dramatically by the addition of anti-GITR mAb (Fig. 2C). These results demonstrate that ligation of GITR by anti-GITR mAb costimulates the proliferation of both CD25–CD4+ and CD25+CD4+ T cells.
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) was also induced by anti-CD3 stimulation, and the MFI of cells that were stimulated with anti-GITR mAb was consistently higher. These results indicate that anti-GITR mAb stimulation efficiently induces early (<24 h) activation of CD4+ T cells, as assessed by CD69 and CD25 induction. We also examined the effects of anti-GITR stimulation on cell division using CFSE-labeled CD4+ T cells. Although cell division was not observed 24 h after activation (data not shown), the cells that were stimulated with anti-GITR mAb showed a progressive increase in the number of divided cells between 48 and 72 h compared with the control culture (Fig. 3B). Note that the addition of anti-GITR mAb did not affect the frequency of early apoptosis.
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B component
To determine whether the addition of anti-GITR mAb affects signaling events in CD4+ T cells, we examined nuclear translocation of the NF-B family molecules, as most TNFRSF members, including GITR, induce activation of NF-B (7, 26, 27, 28). Purified CD4+ T cells were stimulated with anti-CD3 mAb alone or together with anti-GITR mAb for 24 h. Proteins from cytosolic and nuclear 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 were clearly enhanced by the stimulation with anti-GITR mAb, although those in the cytosolic fractions were not affected. In particular, the translocation of c-Rel, which is a critical NF-B member for IL-2 gene activation (29), was increased 3-fold. These results indicated that anti-GITR mAb together with a suboptimal anti-CD3 stimulation enhances signaling to T cells and promotes the activation and translocation of NF-B. Note that this was caused by the only 24-h costimulation. Coligation of GITR with TCR/CD3 promotes the NF-B signaling cascade, which may result in IL-2 promotor activation.
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CD28 is a well-characterized potent costimulatory molecule that induces various T cell effector functions, such as proliferative responses, cytokine production, and cytotoxicity (21, 30). We performed a parallel assay with anti-CD28 mAb. Similar to the effects seen with anti-GITR mAb, anti-CD28 mAb costimulated the proliferative responses of CD4+ T cells in conjunction with 1 µg/ml anti-CD3 mAb (Fig. 5A). However, in the case of stimulation with 0.25 µg/ml anti-CD3 mAb, only anti-CD28 mAb efficiently costimulated the proliferative responses.
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Analysis of the supernatants from CD4+ T cell cultures demonstrated that both anti-GITR and anti-CD28 mAbs enhanced the production of IL-2, IFN-, IL-4, and IL-10 (Fig. 6A). The enhancing effect of anti-GITR mAb was especially obvious for IL-10 production. Consistent with the results for cell proliferation, the costimulatory effects of anti-GITR mAb on IL-2, IFN-, and IL-4 production were inferior to those induced by anti-CD28 mAb. To explore how the secreted IL-10 contributed to the proliferative responses, we examined the effects of neutralization of IL-2 or IL-10 on GITR-induced proliferation. Neutralization of IL-2 efficiently inhibited both proliferative responses stimulated with anti-CD3 mAb alone and together with anti-GITR mAb (Fig. 6B). Surprisingly, the addition of anti-IL-10 mAb significantly enhanced GITR-mediated proliferation. This enhancing effect by anti-IL-10 mAb was only seen in the proliferation stimulated with anti-GITR mAb, not in the proliferation stimulated with anti-CD3 alone or with anti-CD3 and anti-CD28 mAbs (data not shown). The enhanced effects by the neutralization of IL-10 were seen in both CD25–CD4+ and CD25+CD4+ T cells. These results suggest that IL-2 and IL-10 cytokines that were induced by GITR costimulation have opposing actions on proliferation. The lesser potency of GITR-mediated costimulation in proliferation may result in the reverse action by IL-10 that was also induced by GITR-mediated costimulation. Our results demonstrate that GITR acts on CD4+ T cells as a costimulatory molecule to induce proliferation, but exhibits a unique profile in cytokine production.
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To assess the role of GITR costimulation in the generation of CTL, we examined anti-CD3-induced redirected cytotoxicity against FcR-bearing P815 cells in a 6-h JAM test (20). CD28 (30, 31) or CD137 (20) costimulation enhanced anti-CD3-induced redirected cytotoxicity of CD4+ T cells against P815. Cytotoxicity was efficiently induced in CD4+ T cells by the addition of a suboptimal dose (2 µg/ml) of anti-CD3 mAb after 6 h of culture, and the addition of anti-GITR mAb significantly enhanced anti-CD3-redirected cytotoxicity (Fig. 7). At a high dose (10 µg/ml) of anti-CD3, the addition of anti-GITR mAb was no longer effective in the generation of cytotoxicity (data not shown). These results indicate that GITR costimulation is capable of rapidly inducing cytotoxicity in CD4+ T cells under certain conditions of TCR stimulation.
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To confirm the costimulatory function of GITR, mGITRL cDNA was transfected into P815 cells, and stable transfectants expressing GITRL on their cell surface were generated. The expression of GFP 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 cells were cocultured with either parental P815 or GITRL-P815 cells in the presence of anti-CD3 mAb (0.25 µg/ml). When CD25–CD4+ T cells were stimulated by culture with GITRL-P815 cells, we observed a pronounced effect on anti-CD3-induced proliferation compared with culture with parental P815 cells (Fig. 8B). Consistent with the results using anti-GITR mAb as shown in Fig. 2C, the proliferative responses of CD25+CD4+ T cells were enhanced efficiently when GITRL-P815 cells were cocultured. The enhanced effects were more prominent at 48 h than at 72 h of culture (not shown), and this enhanced proliferation was inhibited by the addition of GITR-Ig to a similar level as the proliferation stimulated with parental P815 cells (data not shown). These results suggest that the binding of a natural ligand, GITRL, to GITR on both conventional CD4+ T cells and CD25+ Treg cells costimulates anti-CD3-induced proliferation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, and IL-10; generation of cytotoxicity; expression of activation Ags; and cell cycle progression, all of which were induced with the appropriate dosage of anti-CD3 mAb. In addition, the costimulation with anti-GITR mAb for 24 h clearly accelerated the intranuclear translocation of NF-B components in CD4+ T cells. The costimulatory function of GITR was further confirmed by the binding of GITRL to GITR on both CD25+CD4+ and CD25–CD4+ T cells. Previous reports have demonstrated the regulation of TCR-induced apoptosis by anti-GITR mAb (6, 7). T cell responses to TCR stimulation, such as proliferation, IL-2/IL-2R expression, and activation-induced cell death, are promoted in GITR-deficient mice (11). In addition, GITR is predominantly expressed on CD25+CD4+ Treg cells, and the Ab against GITR abrogates their regulatory function (12, 13). All these observations highlight the regulatory role of GITR on CD25+CD4+ Treg cells.
In this study we have clearly demonstrated another crucial function of GITR, i.e., costimulation. Compared with CD28-mediated costimulation, the optimum range for TCR signaling within which GITR costimulation is effective seems to be limited, as the costimulatory effect of anti-GITR mAb was not seen at either high or low levels of CD3 stimulation. Although GITR expression predominated on CD25+CD4+ T cells, freshly isolated conventional CD4+ and CD8+ T cells also constitutively expressed GITR at a significant level, and its expression was rapidly up-regulated after activation. Therefore, it is possible that the cell surface GITR that is induced earlier on naive and activated T cells can transduce costimulatory signals for early T cell activation.
These findings are not surprising, as most TNFRSF members possess costimulatory functions for T cells (1, 2, 3, 4, 5). The GITR in the cytoplasmic domain shares a striking homology with CD27 and 4-1BB (6). Both molecules have been reported as either costimulating T cell activation and promoting cell survival or inducing apoptosis (8, 32). 4-1BB associates with the protein tyrosine kinase 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-activated protein kinase/c-Jun N-terminal kinase through the TRAF2/TRAF5-NIK pathway (27) and involve the protein tyrosine kinase cascade. Similarly, GITR signaling has been shown to involve TRAF2-NIK pathway-mediated activation of NF-B (7). A recent report demonstrated that the cells coexpressing GITR and GITRL or stimulation of GITR+ cells with soluble GITRL led to activation of NF-B, and this was reduced by anti-GITR Ab (34). Our results also showed enhancement of intranuclear translocation of c-Rel, which is a critical NF-B member for IL-2 gene activation (29). Similar to our observations, costimulation by CD28, which is a potent costimulator for naive T cells, induced greater amounts of translocation of c-Rel/p50 complex to the nuclear (35). Furthermore, similar to CD27 and 4-1BB, binding of the proapoptotic protein 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 transduction of these molecules. In addition, more recently several spliced variant forms of GITR with functionally different properties have been identified (37). Variable levels of these splicing products on T cells may cause differential activation of intracellular pathways, resulting in differences in T cell functions.
Among the several cytokines that were enhanced by GITR-mediated costimulation, IL-10 may play a unique role. GITR costimulation induced preferentially high amounts of IL-10, and the IL-10 produced counter-regulated the action of IL-2, which was also induced by GITR costimulation. It seems likely that the regulatory function of GITR in the TCR-induced stimulation that was reported previously (6, 7, 11) is dependent upon the action of IL-10. IL-10 has multifunctions to stimulate and to regulate immune responses (38). IL-10 directly regulates T cells by inhibiting their ability to produce IL-2 and to proliferate (39, 40). In contrast, IL-10 also has immunostimulatory effects by inhibiting T cell apoptosis (41). The immunostimulatory or immunosuppressive properties of IL-10 may be controlled in part by the activation state of T cells mediated by TCR and costimulatory signals.
GITRL has been identified in humans (7, 28, 42) and just recently in mice (34, 43). In humans, the expression of GITRL mRNA has been observed in the small intestine, ovary, testis, and kidney, but not in T cells; furthermore, cell surface expression of GITRL on vascular endothelial cells has been reported (7, 28, 42). In mice, the studies using polyclonal anti-GITRL Ab or soluble GITRL showed constitutive expression of GITRL on immature and mature dendritic cells (DC), and macrophages (34, 43). In addition to the variable expression and forms of GITR on T cells, the selective expression of GITRL on lymphoid and nonlymphoid tissue cells might influence the function of GITR in T cell activation and regulation. In particular, CD25+CD4+ Treg cells expressed constitutively high GITR; therefore, GITR-mediated costimulation to the Treg cells may predominate in other costimulation. Supporting this speculation, our results using GITRL transfectants showed the preferentially sensitive and earlier responses of Treg cells to GITR costimulation. All previous reports demonstrated the abrogation of Treg function by the addition of anti-GITR mAb (12) or recombinant GITRL (43). How can we explain the reason why GITR costimulation abrogated the suppressive function of Treg cells? We previously reported that high doses of IL-2 or CD28 costimulation induced the proliferation of Treg cells, but simultaneously abrogated their suppressive function (23, 44). Moreover, the transfer of such hyperproliferative Treg cells induced various autoimmune diseases in syngeneic athymic nude mice (44). These results suggested that the exhibition of suppressive function required the anergic/hypoproliferative state of Treg cells. The GITR-mediated costimulation by anti-GITR mAb or GITRL may put Treg cells in an active/hyperproliferative state, and this may result in abrogation of the suppressive function of Treg cells. Recent reports suggested that proliferation and activation of Treg cells could be controlled by mature DC in an IL-2-dependent manner (45, 46). It is likely that GITR-GITRL-mediated costimulation may be involved in the interaction of Treg cells with DC. Further studies are now underway to clarify the contribution of GITR-GITRL costimulation to the interactions of Treg or conventional CD4+ T cells with DC.
In this report we highlight GITR function as a costimulatory molecule for T cell activation in both conventional and CD25+CD4+ T cells. Among an array of T cell costimulatory receptors, GITR and CD28 alone are expressed constitutively on naive and resting T cells. The constitutive expression of GITR on conventional CD4+ T cells may play an important role in the initiation of T cell activation; in addition, GITR expressed on Treg cells may play a crucial role in the maintenance of peripheral tolerance. The interactions between GITR and its ligand during immune responses may regulate diverse biological functions in T cells, such as proliferation, activation, differentiation, and cell survival.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Miyuki Azuma, Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: miyuki.mim{at}tmd.ac.jp
3 Abbreviations used in this paper: TNFRSF, TNF receptor superfamily; DC, dendritic cell; GFP, green fluorescence protein; GITR, glucocorticoid-induced TNF; GITRL, GITR ligand; MFI, mean fluorescence intensity; NIK, NF-B-inducing kinase; TRAF, TNF receptor-associated factor; Treg, regulatory T cell; m, mouse.
Received for publication November 12, 2003. Accepted for publication April 7, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T lymphocytes. J. Immunol. 151:6043.[Abstract]
B. Mol. Cell. Biol. 18:558.B and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-B-inducing kinase. J. Biol. Chem. 273:13353.This article has been cited by other articles:
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 L. R. Guerin, J. R. Prins, and S. A. Robertson Regulatory T-cells and immune tolerance in pregnancy: a new target for infertility treatment? Hum. Reprod. Update, September 1, 2009; 15(5): 517 - 535. [Abstract] [Full Text] [PDF]
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 R. W. van Olffen, N. Koning, K. P. J. M. van Gisbergen, F. M. Wensveen, R. M. Hoek, L. Boon, J. Hamann, R. A. W. van Lier, and M. A. Nolte GITR Triggering Induces Expansion of Both Effector and Regulatory CD4+ T Cells In Vivo J. Immunol., June 15, 2009; 182(12): 7490 - 7500. [Abstract] [Full Text] [PDF]
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 F. Duan, Y. Lin, C. Liu, M. E. Engelhorn, A. D. Cohen, M. Curran, S. Sakaguchi, T. Merghoub, S. Terzulli, J. D. Wolchok, et al. Immune Rejection of Mouse Tumors Expressing Mutated Self Cancer Res., April 15, 2009; 69(8): 3545 - 3553. [Abstract] [Full Text] [PDF]
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Y. Kamimura, H. Iwai, J. Piao, M. Hashiguchi, and M. Azuma The Glucocorticoid-Induced TNF Receptor-Related Protein (GITR)-GITR Ligand Pathway Acts As a Mediator of Cutaneous Dendritic Cell Migration and Promotes T Cell-Mediated Acquired Immunity J. Immunol., March 1, 2009; 182(5): 2708 - 2716. [Abstract] [Full Text] [PDF]
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M. Bonelli, A. Savitskaya, C.-W. Steiner, E. Rath, J. S. Smolen, and C. Scheinecker Phenotypic and Functional Analysis of CD4+CD25-Foxp3+ T Cells in Patients with Systemic Lupus Erythematosus J. Immunol., February 1, 2009; 182(3): 1689 - 1695. [Abstract] [Full Text] [PDF]
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Y. Zhan, S. Gerondakis, E. Coghill, D. Bourges, Y. Xu, J. L. Brady, and A. M. Lew Glucocorticoid-Induced TNF Receptor Expression by T Cells Is Reciprocally Regulated by NF-{kappa}B and NFAT J. Immunol., October 15, 2008; 181(8): 5405 - 5413. [Abstract] [Full Text] [PDF]
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 M. Hashiguchi, H. Kobori, P. Ritprajak, Y. Kamimura, H. Kozono, and M. Azuma From the Cover: Triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a counter-receptor for B7-H3 and enhances T cell responses PNAS, July 29, 2008; 105(30): 10495 - 10500. [Abstract] [Full Text] [PDF]
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H. Nishikawa, T. Kato, M. Hirayama, Y. Orito, E. Sato, N. Harada, S. Gnjatic, L. J. Old, and H. Shiku Regulatory T Cell-Resistant CD8+ T Cells Induced by Glucocorticoid-Induced Tumor Necrosis Factor Receptor Signaling Cancer Res., July 15, 2008; 68(14): 5948 - 5954. [Abstract] [Full Text] [PDF]
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A. Joetham, S. Matsubara, M. Okamoto, K. Takeda, N. Miyahara, A. Dakhama, and E. W. Gelfand Plasticity of Regulatory T Cells: Subversion of Suppressive Function and Conversion to Enhancement of Lung Allergic Responses J. Immunol., June 1, 2008; 180(11): 7117 - 7124. [Abstract] [Full Text] [PDF]
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R. Tao, L. Wang, K. M. Murphy, C. C. Fraser, and W. W. Hancock Regulatory T Cell Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells J. Immunol., May 15, 2008; 180(10): 6649 - 6655. [Abstract] [Full Text] [PDF]
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 B. Liu, Z. Li, S. P. Mahesh, S. Pantanelli, F. S. Hwang, W. O. Siu, and R. B. Nussenblatt Glucocorticoid-induced Tumor Necrosis Factor Receptor Negatively Regulates Activation of Human Primary Natural Killer (NK) Cells by Blocking Proliferative Signals and Increasing NK Cell Apoptosis J. Biol. Chem., March 28, 2008; 283(13): 8202 - 8210. [Abstract] [Full Text] [PDF]
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 P. Hu, R. S. Arias, R. E. Sadun, Y.-C. Nien, N. Zhang, H. Sabzevari, M.E. C. Lutsiak, L. A. Khawli, and A. L. Epstein Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer Clin. Cancer Res., January 15, 2008; 14(2): 579 - 588. [Abstract] [Full Text] [PDF]
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P. Zhou, L. L'italien, D. Hodges, and X. M. Schebye Pivotal Roles of CD4+ Effector T cells in Mediating Agonistic Anti-GITR mAb-Induced-Immune Activation and Tumor Immunity in CT26 Tumors J. Immunol., December 1, 2007; 179(11): 7365 - 7375. [Abstract] [Full Text] [PDF]
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S. Ronchetti, G. Nocentini, R. Bianchini, L. T. Krausz, G. Migliorati, and C. Riccardi Glucocorticoid-Induced TNFR-Related Protein Lowers the Threshold of CD28 Costimulation in CD8+ T Cells J. Immunol., November 1, 2007; 179(9): 5916 - 5926. [Abstract] [Full Text] [PDF]
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 S. Tuyaerts, S. Van Meirvenne, A. Bonehill, C. Heirman, J. Corthals, H. Waldmann, K. Breckpot, K. Thielemans, and J. L. Aerts Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+CD25+ regulatory T cells J. Leukoc. Biol., July 1, 2007; 82(1): 93 - 105. [Abstract] [Full Text] [PDF]
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S. P. Hilchey, A. De, L. M. Rimsza, R. B. Bankert, and S. H. Bernstein Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25- and CD4+CD25- T Cells J. Immunol., April 1, 2007; 178(7): 4051 - 4061. [Abstract] [Full Text] [PDF]
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H. Igarashi, J. Piao, Y. Cao, M. Hashiguchi, H. Iwai, T. Amagasa, S. Sakaguchi, and M. Azuma GITRL-mediated costimulation in CD25-conventional and CD25+ regulatory CD4 T cells J. Immunol., April 1, 2007; 178(MeetingAbstracts): S141 - S141.
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A. Berhanu, J. Huang, S. C. Watkins, H. Okada, and W. J. Storkus Treatment-Enhanced CD4+Foxp3+ Glucocorticoid-Induced TNF Receptor Family RelatedHigh Regulatory Tumor-Infiltrating T Cells Limit the Effectiveness of Cytokine-Based Immunotherapy J. Immunol., March 15, 2007; 178(6): 3400 - 3408. [Abstract] [Full Text] [PDF]
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C. Hubeau, I. Apostolou, and L. Kobzik Targeting of CD25 and Glucocorticoid-Induced TNF Receptor Family-Related Gene-Expressing T Cells Differentially Modulates Asthma Risk in Offspring of Asthmatic and Normal Mother Mice J. Immunol., February 1, 2007; 178(3): 1477 - 1487. [Abstract] [Full Text] [PDF]
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 L Santucci, M Agostini, S Bruscoli, A Mencarelli, S Ronchetti, E Ayroldi, A Morelli, M Baldoni, and C Riccardi GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice Gut, January 1, 2007; 56(1): 52 - 60. [Abstract] [Full Text] [PDF]
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 G. W. Stone, S. Barzee, V. Snarsky, C. A. Spina, J. D. Lifson, V. K. B. Pillai, R. R. Amara, F. Villinger, and R. S. Kornbluth Macaque Multimeric Soluble CD40 Ligand and GITR Ligand Constructs Are Immunostimulatory Molecules In Vitro Clin. Vaccine Immunol., November 1, 2006; 13(11): 1223 - 1230. [Abstract] [Full Text] [PDF]
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T. Ramirez-Montagut, A. Chow, D. Hirschhorn-Cymerman, T. H. Terwey, A. A. Kochman, S. Lu, R. C. Miles, S. Sakaguchi, A. N. Houghton, and M. R. M. van den Brink Glucocorticoid-Induced TNF Receptor Family Related Gene Activation Overcomes Tolerance/Ignorance to Melanoma Differentiation Antigens and Enhances Antitumor Immunity. J. Immunol., June 1, 2006; 176(11): 6434 - 6442. [Abstract] [Full Text] [PDF]
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A. D. Cohen, A. Diab, M.-A. Perales, J. D. Wolchok, G. Rizzuto, T. Merghoub, D. Huggins, C. Liu, M. J. Turk, N. P. Restifo, et al. Agonist Anti-GITR Antibody Enhances Vaccine-Induced CD8+ T-Cell Responses and Tumor Immunity. Cancer Res., May 1, 2006; 66(9): 4904 - 4912. [Abstract] [Full Text] [PDF]
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J. Kim, W. S. Choi, H. Kang, H. J. Kim, J.-H. Suh, S. Sakaguchi, and B. Kwon Conversion of Alloantigen-Specific CD8+ T Cell Anergy to CD8+ T Cell Priming through In Vivo Ligation of Glucocorticoid-Induced TNF Receptor J. Immunol., May 1, 2006; 176(9): 5223 - 5231. [Abstract] [Full Text] [PDF]
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H. J. Kim, H. Y. Kim, B. K. Kim, S. Kim, and D. H. Chung Engagement of Glucocorticoid-Induced TNF Receptor Costimulates NKT Cell Activation In Vitro and In Vivo J. Immunol., March 15, 2006; 176(6): 3507 - 3515. [Abstract] [Full Text] [PDF]
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 G. W. Stone, S. Barzee, V. Snarsky, K. Kee, C. A. Spina, X.-F. Yu, and R. S. Kornbluth Multimeric Soluble CD40 Ligand and GITR Ligand as Adjuvants for Human Immunodeficiency Virus DNA Vaccines J. Virol., February 15, 2006; 80(4): 1762 - 1772. [Abstract] [Full Text] [PDF]
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 A. J. M. van Oosterhout and N. Bloksma Regulatory T-lymphocytes in asthma Eur. Respir. J., November 1, 2005; 26(5): 918 - 932. [Abstract] [Full Text] [PDF]
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 K. Ko, S. Yamazaki, K. Nakamura, T. Nishioka, K. Hirota, T. Yamaguchi, J. Shimizu, T. Nomura, T. Chiba, and S. Sakaguchi Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells J. Exp. Med., October 3, 2005; 202(7): 885 - 891. [Abstract] [Full Text] [PDF]
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S. Suvas, B. Kim, P. P. Sarangi, M. Tone, H. Waldmann, and B. T. Rouse In Vivo Kinetics of GITR and GITR Ligand Expression and Their Functional Significance in Regulating Viral Immunopathology J. Virol., September 15, 2005; 79(18): 11935 - 11942. [Abstract] [Full Text] [PDF]
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L.-F. Lu, D. C. Gondek, Z. A. Scott, and R. J. Noelle NF{kappa}B-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation J. Immunol., August 1, 2005; 175(3): 1651 - 1657. [Abstract] [Full Text] [PDF]
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E. M. Esparza and R. H. Arch Glucocorticoid-Induced TNF Receptor Functions as a Costimulatory Receptor That Promotes Survival in Early Phases of T Cell Activation J. Immunol., June 15, 2005; 174(12): 7869 - 7874. [Abstract] [Full Text] [PDF]
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E. M. Esparza and R. H. Arch Glucocorticoid-Induced TNF Receptor, a Costimulatory Receptor on Naive and Activated T Cells, Uses TNF Receptor-Associated Factor 2 in a Novel Fashion as an Inhibitor of NF-{kappa}B Activation J. Immunol., June 15, 2005; 174(12): 7875 - 7882. [Abstract] [Full Text] [PDF]
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T. M. Brusko, C. H. Wasserfall, A. Agarwal, M. H. Kapturczak, and M. A. Atkinson An Integral Role for Heme Oxygenase-1 and Carbon Monoxide in Maintaining Peripheral Tolerance by CD4+CD25+ Regulatory T Cells J. Immunol., May 1, 2005; 174(9): 5181 - 5186. [Abstract] [Full Text] [PDF]
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M. D. Taylor, L. LeGoff, A. Harris, E. Malone, J. E. Allen, and R. M. Maizels Removal of Regulatory T Cell Activity Reverses Hyporesponsiveness and Leads to Filarial Parasite Clearance In Vivo J. Immunol., April 15, 2005; 174(8): 4924 - 4933. [Abstract] [Full Text] [PDF]
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J. Shimizu, R. Iida, Y. Sato, E. Moriizumi, A. Nishikawa, and Y. Ishida Cross-Linking of CD45 on Suppressive/Regulatory T Cells Leads to the Abrogation of Their Suppressive Activity In Vitro J. Immunol., April 1, 2005; 174(7): 4090 - 4097. [Abstract] [Full Text] [PDF]
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H. He, R. J. Messer, S. Sakaguchi, G. Yang, S. J. Robertson, and K. J. Hasenkrug Reduction of Retrovirus-Induced Immunosuppression by In Vivo Modulation of T Cells during Acute Infection J. Virol., November 1, 2004; 78(21): 11641 - 11647. [Abstract] [Full Text] [PDF]
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, 2 µg/ml; •, 0.5 µg/ml; 
, 0.25 µg/ml) and the indicated amounts of soluble anti-GITR (DTA-1) mAb. B, CD4+ (•) and CD25–CD4+ (
) T cells (2 x 105/well) were stimulated for 72 h with 0.5 µg/ml immobilized anti-CD3 mAb in the presence of graded doses of anti-GITR mAb. C, CD25+CD4+ T cells (2 x 105/well) were stimulated for 72 h with immobilized anti-CD3 mAb (
, 2 µg/ml; 
, 0.5 µg/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 without anti-CD3 mAb in the presence or the absence of anti-GITR mAb were always <1000 cpm (data not shown). Data shown are representative of three independent experiments with similar results.
