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The Journal of Immunology, 2007, 179, 5916 -5926
Copyright © 2007 by The American Association of Immunologists, Inc.
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Glucocorticoid-Induced TNFR-Related Protein Lowers the Threshold of CD28 Costimulation in CD8+ T Cells1

Simona Ronchetti2, Giuseppe Nocentini2, Rodolfo Bianchini, L. Tibor Krausz, Graziella Migliorati and Carlo Riccardi3

Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, and IBiT Foundation, Perugia, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD28 is well characterized as a costimulatory molecule in T cell activation. Recent evidences indicate that TNFR superfamily members, including glucocorticoid-induced TNFR-related protein (GITR), act as costimulatory molecules. In this study, the relationship between GITR and CD28 has been investigated in murine CD8+ T cells. When suboptimal doses of anti-CD3 Ab were used, the absence of GITR lowered CD28-induced activation in these cells whereas the lack of CD28 did not affect the response of CD8+ T cells to GITR costimulus. In fact, costimulation of CD28 in anti-CD3-activated GITR–/– CD8+ T cells resulted in an impaired increase of proliferation, impaired protection from apoptosis, and an impaired rise of activation molecules such as IL-2R, IL-2, and IFN-{gamma}. Most notably, CD28-costimulated GITR–/– CD8+ T cells revealed lower NF-{kappa}B activation. As a consequence, up-regulation of Bcl-xL, one of the major target proteins of CD28-dependent NF-{kappa}B activation, was defective in costimulated GITR–/– CD8+ T cells. What contributed to the response to CD28 ligation in CD8+ T cells was the early up-regulation of GITR ligand on the same cells, the effect of which was blocked by the addition of a recombinant GITR-Fc protein. Our results indicate that GITR influences CD8+ T cell response to CD28 costimulation, lowering the threshold of CD8+ T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
An effective T cell activation can be achieved by the integration of two signals, one coming from the TCR and the other from costimulatory molecules. Signal 1 is delivered through TCR upon the recognition of Ag in the form of a complex within MHC molecules on APCs and is essential for proliferation and differentiation. Signal 2 is relayed through costimulatory molecules whose ligands are expressed by APCs and is necessary to up-regulate macromolecule synthesis, promote cell cycle progression, augment cell survival, and induce effector functions (1).

Two major families of costimulatory molecules have been identified: the Ig superfamily, which comprises CD28, the prototype of costimulatory molecules, and the TNFR superfamily (TNFRSF),4 which includes members such as glucocorticoid-induced TNFR-related protein (GITR), 4-1BB, OX40, and CD27 that share a similar cytoplasm domain and thus constitute a subfamily inside the TNFRSF called the GITR subfamily (2, 3, 4, 5).

CD28 was the first costimulatory molecule to be identified and it has been shown to synergize with the TCR to lower the threshold of T cell activation, which is not attainable by ligation of the TCR alone (6). The finding that in CD28–/– mice CTLs can still be induced after infection with lymphocytic choriomeningitis virus (7) suggested that other costimulatory molecules, such as TNFRSF members, are able to costimulate T cells. For example, anti-4-1BB Ab can promote expansion of CD28–/– T cells in response to influenza virus (8), and 4-1BBL, together with anti-CD3, induces higher IL-2 production and cell proliferation. However, double knockout mice for 4-1BBL and CD28 (9) mount primary and secondary CD8+ responses in vivo, suggesting that other coactivating molecules participate in T cell activation. The interplay between TNFRSF members and CD28 functions differently in different T cell subsets as suggested, for example, by the preferential activity of 4-1BB and OX40 in CD8+ and CD4+ T cells, respectively (3, 10, 11).

There is a body of evidence that CD28 and TNFRSF members share some, but not all, signaling intermediates with TCR. As a consequence, the outcome of cosignaling can be quantitative, leading to the accumulation of common signaling intermediates, or qualitative, involving distinct nonoverlapping signals that are all necessary for T cell activation (12). In addition, the same signaling intermediates can be regulated by multiple mechanisms in a spatial- and temporal-dependent manner (6).

GITR is a TNFRSF member known to regulate T cell activation (13, 14). GITR expression increases during in vitro T cell activation (13, 15, 16) and exerts a costimulatory function leading to an increased TCR-dependent cell proliferation (15, 16, 17, 18, 19). A number of in vivo experimental models confirm its costimulatory effect in CD4+ and CD8+ T cells (20, 21, 22, 23).

Some evidence suggests that the effects of GITR triggering are different in CD4+ and CD8+ T subsets. In vitro experiments demonstrate that GITR costimulation of CD4+ T cells is evidenced when low doses of anti-CD3 Ab are used, whereas GITR costimulation of CD8+ T cells is detected with high anti-CD3 Ab doses (16). In addition, in CD4+ T cells full activation of both TCR and GITR decreases cell proliferation due to activation-induced cell death (19, 24). GITR expression is much less dependent on CD28 signaling in CD8+ than in CD4+ T cells, where GITR appears to be a signal secondary to CD28 activation (16).

In conclusion, although previous reports indicate that CD28 regulates GITR expression and cooperates in the regulation of CD4+ T cell activation, the relationship between GITR and CD28 in CD8+ T cells remains still to be defined (15, 24). In this article we present results concerning the costimulatory activity of GITR in CD8+ T cells, comparing the phenotypic and molecular effects of GITR and CD28 triggering in wild-type (GITR+/+) and GITR-knockout (GITR–/–) CD8+ T cells. We find a defect in the proliferative response to CD28 coligation in GITR–/– CD8+ T lymphocytes that correlates with a defect in cytokine production, NF-{kappa}B activation, and Bcl-xL expression, thus suggesting that CD28 needs GITR-derived signaling to fully costimulate CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

GITR–/– and GITR+/+ mice on a Sv129 background (25), kept under specific pathogen-free conditions, were sacrificed between 6 and 10 wk of age. CD28tm1Mak/J mice were purchased from The Jackson Laboratory. Animal care was in compliance with regulations in Italy (Decreto Ministeriale 116192), Europe (Official Journal of European Contract Law 358/1 12/18/1986), and the United States (Animal Welfare Assurance No. A594-01, Department of Health and Human Services), and the study was approved by the Italian Ministero della Salute.

Cells

CD4+ and CD8+ T lymphocytes were obtained from axillary, brachial, maxillary, and inguinal lymph node lymphocytes. Briefly, CD4+ and CD8+ separation was preceded by the depletion of B cells, which were removed after incubation with anti-B220-PE Ab (Miltenyi Biotec) followed by anti-PE microbead separation onto LD columns (Miltenyi Biotec). Flow-through cells were incubated with CD4 or CD8 microbeads (Miltenyi Biotec) and eluted from LS columns according to the manufacturer’s instructions. The magnetically retained cells were shown to be >98% CD4+ or CD8+ cells by flow cytometry. CD8+ cells were >99% CD3+.

To obtain CD4+CD25– and CD8+CD25–, B cells were removed as specified above. CD25+ cells were removed after incubation with anti-CD25-PE Ab (Miltenyi Biotec) followed by anti-PE microbead separation onto LD columns (Miltenyi Biotec). Flow-through cells were incubated with CD4 or CD8 microbeads as specified above. The magnetically retained cells were shown to be >98% CD4+CD25– or CD8+CD25– T cells by flow cytometry.

Macrophages were taken from the peritoneal cavity of mice treated for 4 days with thioglycollate by flushing with cold MEM. Cells were then resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, streptomycin (100 µg/ml), 10 mM HEPES, 0.1% nonessential amino acids, 1 mM sodium pyruvate, and 50 µM 2-ME and plated onto 96-well plate at a concentration of 1 x 104 cells/well. After 2 h at 37°C, cells were first washed with warm PBS to remove nonadherent cells and then cultured together with CD4+ or CD8+ T cells (5 x 104 cells/well) for 72 h.

Flow cytometry

Harvested CD8+ T cells were spun down and washed with PBS before the addition of Abs. Rat GITR-PE mAb (clone 108619) and its isotype control, rat IgG2A (R & D Systems), were added undiluted to the cells (10 µl/sample) and kept at 4°C for 30 min; anti-CD28-PE mAb (clone 37.51) was added diluted 1/6 while its isotype, rat IgG2A-PE (BD Pharmingen), was added diluted 1/3; anti-CD25(IL-2R)-FITC mAb (clone 7D4) was added to the samples diluted 1/25 while its isotypic control, FITC-rat IgM was added diluted 1/12 (BD Pharmingen). Anti-GITR ligand (GITRL)-PE mAb (clone eBioYGL386) was added to the samples undiluted; its isotypic control, PE-Rat IgG1, was added undiluted (eBioscience). All samples were stained with the Abs for 30 min at 4°C. Flow cytometric analysis was conducted on a Beckman Coulter EPICS XL-MCL running EXPO32 ADC analysis software.

Proliferation assay

CD8+ T cells (0.5 x 106 cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, streptomycin (100 µg/ml), 10 mM HEPES, 0.1% nonessential amino acids, 1 mM sodium pyruvate, and 50 µM 2-ME. Goat anti-GITR Ab (2 µg/ml) (R & D Systems), anti-CD3{epsilon} mAb (clone 145-2C11; BD Pharmingen), and anti-CD28 mAb (clone 37.51; BD Pharmingen) were either cross-linked onto a 96-well plate over night as stated in the figure legends or added in soluble form as reported in some experiments. When cross-linked, the anti-CD3 and anti-CD28 Ab concentration was 0.5 µg/ml unless specified differently. Where indicated, recombinant murine IL-2 (PeproTech) was added at a final concentration of 100 U/ml. (Murine)GITR-(human)Fc (Alexis) and (human)Fc-isotype control (Alexis) were added at a final concentration of 2 µg/ml.

[3H]Thymidine incorporation assay

[3H]Thymidine (Amersham International) at 2.5 µCi per well was added to the cultures at the specified times. After an over night culture, cells were harvested with a multiple suction-filtration apparatus (Mash II) on a fiberglass filter (BioWhittaker) and counted in a beta counter apparatus (Packard) (26).

Apoptosis

To study apoptosis, propidium iodide in hypotonic solution was added to each sample as described previously (27, 28). Apoptosis was analyzed by flow cytometry with a BD Biosciences FACScan running LYSIS II software.

CFSE labeling

Purified CD8+ T cells were resuspended at 1 x 106 cells/ml in prewarmed (37°C) PBS with 0.1% BSA. Freshly prepared CFSE (Molecular Probes) was added to a final concentration of 10 µM and the cells were incubated for 10 min at 37°C. Excess CFSE was quenched by adding 10 volumes of ice-cold RPMI 1640 medium containing 10% FBS and incubating the cells for 5 min on ice. CFSE-labeled cells were then washed three times with RPMI 1640 medium containing 10% FBS and cultured with the indicated stimuli.

ELISA

Supernatants from 72 h-cultures were analyzed for the quantification of IL-2 or IFN-{gamma} as previously described (17).

Real-time PCR

Purified CD8+ T lymphocytes from activated GITR+/+ mice were homogenized in 1 ml of TRIzol reagent (Invitrogen Life Technologies). Total RNA was isolated according to the manufacturer’s instructions. Reverse transcription of total RNA (1 µg) was performed with random primers and SuperScript II (Invitrogen Life Technologies). PCR experiments were performed using specific primers for GITRL: 5'-CGAGTCCTGCATGGTTAA-3' (forward) and 5'-TCAGCTTCCCATCAGATGTC-3' (reverse). PCR was done in the CHROMO 4 (MJ Research Bio-Rad) using the DyNAmo HS SYBR Green quantitative PCR kit (Finnzymes). Gene expression was quantitated relatively to the expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1) evaluated in separate tubes.

Immunofluorescence

Plates (24-well) were coated with anti-CD3 mAb (0.5 µg/ml) or anti-CD3 plus anti-CD28 (0.5 µg/ml) Abs overnight at 4°C. CD8+ T cells were plated at a final concentration of 0.5 x 106 cells/ml and cultured at 37°C for the indicated times. Cells were then cytospun for 5 min at 400 rpm onto glass slides and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature After an additional washing with PBS, cells were permeabilized with cold methanol at –20°C for 7 min. After washing with PBS again, cells were blocked with blocking buffer (1% glycine and 3% BSA in PBS) for 1 h at room temperature and subsequently incubated with an anti-p65 Ab (Santa Cruz Biotechnology) for 1 h at room temperature. For detection, Texas Red (Molecular Probes) was added in blocking buffer for 1 h at room temperature. To stain nuclei, DAPI (1 ng/ml) was added for an additional 10 min. After washing with PBS, cells were covered with the mounting medium and the slides were viewed on a Leitz Dialux 20 fluorescence microscope.

Immunoblotting

Plates (24-well) were coated with anti-CD3 (0.5 µg/ml) or anti-CD3 plus anti-CD28 (0.5 µg/ml) overnight at 4°C. CD8+ cells were plated at a final concentration of 0.5 x 106 cells/ml and cultured at 37°C for the indicated times. Cells were then harvested, washed, and lysed in lysis buffer for 30 min on ice. After centrifuging, the cleared lysates were resolved by electrophoresis with 10–15% SDS-polyacrylamide gels. The proteins were then transferred onto nitrocellulose membranes that were hybridized with rabbit anti-I{kappa}B{alpha} Ab (Cell Signaling, New England Biolabs) and rabbit anti-Bcl-x Ab (R&D Systems). Immunoreactive protein bands were visualized using HRP-conjugated goat anti-rabbit IgG (Pierce Endogen) followed by enhanced chemoluminescence. Densitometric analysis was done with a Kodak 1D, version 3.5.0 software.

EMSA

Nuclear extracts from 5 x 107 cells were obtained as previously described (25). The gel shift assay was performed by using the Promega gel shift assay system according to the manufacturer’s instructions. After electrophoresis for 2.5 h at room temperature and 10 V/cm, the gel was dried and the separated protein-DNA complexes were subjected to quantitative analysis using an Instant Imager autoradiography system (Packard BioScience). The following dsDNA oligonucleotide (Promega) for NF-{kappa}B was used in EMSA analysis, either as a labeled or a competitor cold probe: 5'-AGTTGAGGGGACTTTCCCAGGC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD28-dependent coactivation is impaired in GITR–/– CD8+ T lymphocytes

We investigated GITR contribution to CD8+ and CD4+ T cell activation by using two experimental approaches: T cell activation by anti-CD3 Abs on a cellular layer and T cell activation by plastic-adhered anti-CD3 Abs.

First, we stimulated GITR+/+ and GITR–/– CD4+ and CD8+ T cells with anti-CD3 Ab on a layer of macrophages. Incorporation of [3H]thymidine in GITR–/– CD4+ T cells was similar to that of GITR+/+ cells (Fig. 1A, left panel), suggesting that GITR expression is not crucial for CD4+ T cell activation. Conversely, [3H]thymidine uptake of GITR–/– CD8+ T cells was significantly lower than that of GITR+/+ cells (Fig. 1A, right panel), indicating that GITR costimulus is essential for CD8+ T cells. We then performed experiments to evaluate whether the defective activation of GITR–/– CD8+ T cells was due to GITR-CD28 interplay. CD4+ and CD8+ from both GITR+/+ and GITR–/– mice were cultured on a feeder of irradiated splenocytes and stimulated with anti-CD3 Ab with or without anti-CD28 Ab. Fig. 1B shows that anti-CD28 costimulus had a similar effect in both GITR+/+ and GITR–/– CD4+ T cells (left panel), but failed in fully activating GITR–/– CD8+ T cells (right panel), indicating that GITR is important for CD28 costimulation in CD8+ T cells.


Figure 1
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  		FIGURE 1. Defective [3H]thymidine uptake of GITR–/– CD8+ T cells. A, [3H]Thymidine uptake of CD4+ and CD8+ purified T cells from GITR+/+ (open columns) or GITR–/– (filled columns) mice cultured on a layer of macrophages (derived from the respective mice) and activated by soluble anti-CD3 Ab (0.5 µg/ml) for 72 h. B, [3H]Thymidine uptake of CD4+ and CD8+ purified T cells cultured on a layer of irradiated splenocytes (derived from the respective mice) and activated by either soluble anti-CD3 ({alpha}-CD3) Ab (0.5 µg/ml) or soluble anti-CD3 plus soluble anti-CD28 ({alpha}-CD28) Abs (0.5 µg/ml) for 72 h. Results are the mean ± SD of four independent experiments. °, p > 0.05; **, p < 0.01; GITR–/– vs GITR+/+.

 
We also performed experiments using plastic-adhered Abs, either anti-CD3 alone or in combination with anti-CD28. Fig. 2A demonstrates that GITR–/– CD8+ T lymphocytes were defective in the proliferative response to CD28 costimulation (right panel) while GITR–/– CD4+ T cells were not (left panel). Moreover, the removal of CD25+ cells in both CD4+ and CD8+ T lymphocytes did not alter the difference observed between GITR+/+ and GITR–/– CD8+ T cells (Fig. 2B). To analyze the CD8+ defect deeper, we used various concentrations of plate-bound anti-CD3 Ab (0.5–5 µg/ml) combined with various amounts of plate-bound anti-CD28 Ab (0.1–5 µg/ml). As shown in Fig. 2C, the defect in the proliferative response of GITR–/– CD8+ T cells was observed at anti-CD3 Ab concentrations of 0.5 and 1 µg/ml combined with several of the anti-CD28 Ab concentrations tested (left and right panel, respectively). At the highest concentration of anti-CD3 Ab, 5 µg/ml, no difference in proliferation was observed between GITR–/– and GITR+/+ costimulated CD8+ T lymphocytes (data not shown), suggesting that GITR plays a role when TCR is not fully triggered.


Figure 2
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  		FIGURE 2. Defective [3H]thymidine uptake of GITR–/– CD8+ T cells costimulated by plate-bound Abs. A, [3H]Thymidine uptake of CD4+ and CD8+ purified T cells from GITR+/+ (open columns) or GITR–/– (filled columns) mice activated by either plate-bound anti-CD3 ({alpha}-CD3) Ab (0.5 µg/ml) or plate-bound anti-CD3 plus plate-bound anti-CD28 ({alpha}-CD28) Abs (0.5 µg/ml) for 72 h. B, [3H]Thymidine uptake of CD4+CD25– and CD8+CD25– purified T cells from GITR+/+ (open columns) or GITR–/– (filled columns) mice activated by either plate-bound anti-CD3 Ab (0.5 µg/ml) or plate-bound anti-CD3 plus plate-bound anti-CD28 Abs (0.5 µg/ml) for 72 h. C, [3H]Thymidine uptake of GITR+/+ (open triangles) and GITR–/– (filled triangles) CD8+ T cells activated by plate-bound anti-CD3 (0.5 or 1 µg/ml, as specified) plus various concentrations of plate-bound anti-CD28 (0.1–5 µg/ml) for 72 h. In each experimental condition, anti-CD28 Ab alone was not able to induce proliferation (not shown). Results are the mean ± SD of three of five independent experiments. °, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; GITR–/– vs GITR+/+.

 
We also measured the number of live cells after long term culture. CD8+ T lymphocytes were activated by anti-CD3 Ab (0.5 µg/ml) alone or in combination with various amounts of anti-CD28 Ab. As shown in Fig. 3A, the number of viable cells was reduced in CD28-costimulated GITR–/– as compared with GITR+/+ cells at anti-CD28 Ab concentrations from 0.5 to 5 µg/ml. Conversely, an equal number of cells was observed in GITR+/+ and GITR–/– T cells when 1 µg/ml anti-CD3 was used together with various concentrations of anti-CD28 Ab (data not shown).


Figure 3
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  		FIGURE 3. Defective proliferation and activation of GITR–/– CD8+ T cells costimulated by CD28. A, Live cell counts after 7 days of culture with plate-bound anti-CD3 ({alpha}-CD3) (0.5 µg/ml) alone or plus various concentrations of plate-bound anti-CD28 ({alpha}-CD28) (0.1–5 µg/ml). Results show the mean ± SD of four independent experiments. B, CFSE-labeled CD8+ GITR+/+ (upper panels) and GITR–/– (lower panels) CD8+ T cells stimulated by plate-bound 0.5 µg/ml anti-CD3 Ab (thin line) alone or in combination with plate-bound 0.5 µg/ml anti-CD28 Ab (bold line) for 72 h. Percentages of proliferating T cells in this representative experiment are shown. Significance (GITR–/– vs GITR+/+) was calculated considering the three experiments performed. C, Flow cytometric analysis of propidium iodide (PI)-stained GITR+/+ (upper panels) and GITR–/– (lower panels) CD8+ T cells for the measurement of apoptosis. Cells have been activated for 72 h by plate-bound 0.5 µg/ml anti-CD3 Ab in the presence or absence of plate-bound 0.5 µg/ml anti-CD28 Ab. Percentages of apoptotic cells in this representative experiment are shown. Significance (GITR–/– vs GITR+/+) was calculated considering the three experiments performed. D, Flow cytometric analysis of IL-2R expression in GITR+/+ (top histograms) and GITR–/– (bottom histograms) CD8+ T cells, activated by either plate-bound 0.5 µg/ml anti-CD3 Ab or plate-bound anti-CD3 plus plate-bound 0.5 µg/ml anti-CD28 Abs after 72 h of culture. Shaded histograms represent isotypic controls, black lines represent stimulated cells. Percentages of positive cells in this representative experiment are shown. Significance (GITR–/– vs GITR+/+) was calculated considering the three experiments performed. E, ELISA evaluation of IL-2 and IFN-{gamma} released in the supernatants of 72-h cultured CD8+ T lymphocytes activated by plate-bound 0.5 µg/ml anti-CD3 Ab (open columns) or plate-bound anti-CD3 plus 0.5 µg/ml anti-CD28 Abs (gray columns). Results are the mean ± SD of three independent experiments. °, p > 0.05; *, p < 0.05; **, p < 0.01; GITR–/– vs GITR+/+.

 
In activated T cells, changes in cell number may be due to the modulation of either cell proliferation and/or apoptosis. To verify whether differences in cell cycle occurred in CD28-costimulated GITR–/– CD8+ T lymphocytes, we used the CFSE labeling technique in 72-h activated cells. Anti-CD3 stimulation yielded similar numbers of divisions and numbers of cells that had undergone divisions in GITR+/+ and GITR–/– lymphocytes while anti-CD28 costimulation induced a lower number of cells that had divided in GITR–/– as compared with GITR+/+ lymphocytes, although the number of divisions remained unaffected (Fig. 3B).

We next asked whether CD28 coligation could prevent GITR–/– CD8+ T cells from undergoing apoptosis. We therefore measured the percentage of apoptotic cells by propidium iodide assay following 72 h of activation. A similar percentage of apoptotic cells was found in GITR–/– cells treated with anti-CD3 and anti-CD3 plus anti-CD28 Abs (Fig. 3C) while, as expected, a significantly lower apoptosis was detected in GITR+/+ T cells costimulated with anti-CD28 Ab.

The effects of CD28 on cell proliferation and death are mediated by the induction of several activation molecules, including IL-2R as well as IL-2 and IFN-{gamma} (7, 29, 30, 31). We therefore performed experiments to evaluate the CD28-dependent induction of IL-2R, IL-2, and IFN-{gamma} in GITR–/– CD8+ T cells. Results clearly indicate a defect in IL-2R expression, as evaluated by flow cytometry (Fig. 3D), and in IL-2 and IFN-{gamma} production, as evaluated by ELISA (Fig. 3E). In summary, the above results indicate a defect in coactivating function of CD28 in GITR–/– as compared with GITR+/+ CD8+ T cells.

GITR-dependent coactivation is normal in CD8+ CD28–/– T lymphocytes

Because GITR–/– CD8+ T cells have a defective response to CD28, we investigated whether CD28–/– CD8+ T cells had the same defective response to GITR costimulation. As demonstrated previously (32), CD28–/– CD8+ T cells proliferated less than CD28+/+ cells following TCR activation. However, when cells were costimulated with anti-GITR Ab the proliferation of CD28–/– CD8+ T lymphocytes increased, similarly to what occurred with CD28+/+ T cells (Fig. 4A). In fact, following GITR costimulation the increase of [3H]thymidine uptake was 58% for CD28+/+ T cells and 83% for CD28–/– T cells. Moreover, IL-2R was up-regulated in response to GITR costimulation, as measured by flow cytometry (Fig. 4B), on both CD28–/– and CD28+/+ T cells. These results indicate that in CD8+ T cells GITR acts as a costimulatory molecule also in the absence of CD28.


Figure 4
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  		FIGURE 4. Effective [3H]thymidine uptake and activation of CD28–/– CD8+ T cells costimulated by GITR. A, [3H]Thymidine uptake after 72 h-activation by plate-bound anti-CD3 ({alpha}-CD3) Ab (0.5 µg/ml) or plate-bound anti-CD3 plus anti-GITR ({alpha}-GITR) (2 µg/ml) Abs of CD28–/– and CD28+/+ CD8+ T cells. B, Flow cytometric evaluation of IL-2R on CD28+/+ and CD28–/– CD8+ T cells activated for 48 and 72 h with plate-bound anti-CD3 Ab or plate-bound anti-CD3 plus anti-GITR Abs. Results are the mean ± SD of three independent experiments. °, p > 0.05; #, p < 0.05; ##, p < 0.01; anti-CD3/anti-GITR cotreated vs anti-CD3-treated.

 
GITR and CD28: reciprocal up-regulation upon reciprocal costimulation

The above-described data suggest that CD28 can only weakly costimulate CD8+ T cells in the absence of GITR, whereas GITR can fully costimulate CD8+ T cells in the absence of CD28. To find out the reason for this phenomenon, we started investigating how GITR and CD28 signaling reciprocally modulate their expression in GITR+/+ CD8+ T lymphocytes. To this purpose, we analyzed GITR expression at various time points following CD3 or CD3 plus CD28 stimulation in GITR+/+ CD8+ T cells (Fig. 5A). Following anti-CD3 Ab treatment, GITR expression kept on increasing over time until the last time point analyzed (72 h). A difference in GITR expression between anti-CD3- and anti-CD3/anti-CD28-treated cells was observed after 48 h when the median fluorescence intensity (MFI) of GITR-stained cells was >3-fold higher in cells treated with anti-CD3 plus anti-CD28 Abs. However, after 72 h GITR expression was similar in both conditions (MFI 97 vs 70), suggesting that CD28 accelerates GITR expression in the first hours and that high levels of GITR in CD8+ T cells can be reached independently of CD28 costimulation, even following treatment with a low anti-CD3 Ab concentration (0.5 µg/ml). The use of a higher dose of anti-CD28 Ab (2 µg/ml) yielded similar results (data not shown).


Figure 5
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  		FIGURE 5. GITR and CD28 up-regulation following GITR+/+ CD8+ T cell activation. A, Flow cytometric analysis of GITR expression in anti-CD3 ({alpha}-CD3)-activated (shaded areas) and anti-CD3 plus anti-CD28 ({alpha}-CD28)-activated (thick line) CD8+ T lymphocytes at various time points. Dotted histograms represent isotypic controls. MFI values are shown (thin numbers for anti-CD3-activated cells and bold numbers for anti-CD3 plus anti-CD28-activated cells) and the percentages of positive cells are reported in parentheses. The result from one representative experiment of three is given. B, Flow cytometric analysis of CD28 expression in anti-CD3-activated (thin line) and anti-CD3 plus anti-GITR ({alpha}-GITR)-activated (thick line) CD8+ T lymphocytes at various concentrations of anti-CD3 Ab and at various time points. Gray histograms represent isotypic controls. MFI values and percentages of positive cells are shown as in A. The result from one representative experiment of three is given.

 
We also investigated the kinetics of CD28 up-regulation in GITR+/+ CD8+ T cells stimulated with anti-CD3 Ab or anti-CD3 plus anti-GITR Abs. At a suboptimal concentration of anti-CD3 Ab (0.5 µg/ml), the percentage of CD28+ cells increased upon GITR costimulus, reached a peak at 48 h of activation, and then decreased (Fig. 5B). At each time point analyzed the MFI and the percentage of CD28+ cells were higher in GITR costimulated T cells as compared with anti-CD3-stimulated T cells, and the levels of CD28 expression never reached those obtained with anti-CD3 plus anti-GITR Abs. The up-regulation of CD28 following GITR costimulation was observed only using 0.5 µg/ml anti-CD3 Ab. In fact, CD28 up-regulation by anti-CD3 Ab at 2 µg/ml was higher than that obtained with 0.5 µg/ml anti-CD3 Ab, and GITR costimulation (even at higher concentrations; data not shown) could not further increase CD28 expression (Fig. 5B). These results suggest that GITR costimulus contributes to a full and sustained CD28 up-regulation in CD8+ T lymphocytes during cell activation when TCR is suboptimally stimulated.

GITRL is expressed in activated CD8+ T cells

To explain the contribution of GITR in CD28 costimulation of purified CD8+ T cells in an experimental system without APC (expressing GITRL), we looked for the expression of GITRL in resting and activated GITR+/+ CD8+ T cells (Fig. 6A). GITRL expression was found as early as 6 h after CD3/CD28 costimulation but was rapidly down-regulated thereafter so that it was almost undetectable after 12 h. Flow cytometric studies confirmed GITRL expression (Fig. 6B), suggesting that GITR can be stimulated by its ligand in our in vitro system in an autocrine manner soon after T cell activation.


Figure 6
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  		FIGURE 6. GITRL expression in activated CD8+ T cells and GITR-Fc blocking activity. A, GITRL expression evaluated by real-time PCR in anti-CD3 ({alpha}-CD3)-activated and anti-CD3/anti-CD28 ({alpha}-CD28)-activated GITR+/+ CD8+ T lymphocytes. HPRT1 was used as housekeeping gene. The result from one representative experiment of four is given. S.L., Significance level. B, Flow cytometric analysis of GITRL expression after 6 h of anti-CD3 and anti-CD3 plus anti-CD28 activation. Gray histograms represent isotypic controls. MFI values are shown and the percentages of positive cells are reported in parentheses. The result from one representative experiment of two is given. C, [3H]thymidine uptake of CD8+ purified T cells from GITR+/+ (open columns) or GITR–/– (filled columns) mice activated by either plate-bound anti-CD3 Ab (0.5 µg/ml) or plate-bound anti-CD3 plus plate-bound anti-CD28 Abs (0.5 µg/ml) for 72 h in the presence or absence of GITR-Fc and Fc-isotypic control (2 µg/ml). Results are the mean ± SD of three independent experiments. °, p > 0.05; *, p < 0.05; **, p < 0.01; GITR–/– vs GITR+/+. #, p < 0.05; Fc-GITR-treated costimulated GITR+/+ CD8+ T cells (column 5, counting from the left) vs costimulated GITR+/+ CD8+ T cells (column 3) and Fc-isotype treated costimulated GITR+/+ CD8+ T cells (column 7).

 
To prove that GITR actually stimulated GITRL in our experimental system, we added a recombinant GITR protein (GITR-Fc) that binds GITRL and prevents GITR engagement by its ligand. Results shown in Fig. 6B clearly demonstrate that GITRL was effectively engaging GITR during CD28 costimulation, because the proliferation of CD28-costimulated GITR+/+ cells in the presence of GITR-Fc was similar to that seen in CD28-costimulated GITR–/– cells. The effect observed is not due to GITRL triggering by GITR-Fc as observed in other experimental systems (33), because the fusion protein had no effect in GITR–/– cells (Fig. 6C).

In summary, the above results demonstrate that stimulation of GITR by its ligand cooperates with CD28 for a full and functional CD8+ T lymphocyte activation.

CD28 signaling and IL-2-driven pathways are intact in CD8+ GITR–/– T cells

The lack of GITR–/– CD8+ T cell response to CD28 costimulation might reside in a lower level of CD28 expression in GITR–/– mice. However, we did not find significant differences in CD28 expression between resting and anti-CD3-activated GITR+/+ and GITR–/– CD8+ T cells (data not shown). We therefore investigated whether a defect of CD28 signaling occurred in GITR–/– CD8+ T cells. No significant differences were observed neither in Akt phosphorylation levels (data not shown), a kinase downstream CD28, nor in ERK phosphorylation (data not shown). The JNK pathway was also activated at comparable levels in GITR+/+ and GITR–/– CD8+ T cells (data not shown), thus suggesting that the defect in proliferation response is due to differences downstream these signaling pathways.

The dependence of GITR effects on the IL-2/IL-2R system is controversial. Stephens et al. (16) found that GITR–/– T cells had an impaired IL-2 responsiveness due to their inability to express IL-2R in the presence of Treg cells. However, we and others have shown that GITR costimulation induces the proliferation of CD4+CD25+ Treg cells in absence of IL-2 (15, 17, 34). In contrast, CD28 costimulation increases IL-2R expression that, in turn, promotes IL-2 production. Therefore, we questioned whether the weakened costimulatory effect of CD28 in GITR–/– CD8+ T cells could be attributed to a defect in the IL-2R response. To this end, we tested whether exogenous IL-2 could restore GITR–/– T cell proliferation (Fig. 7). Results indicate that GITR–/– T cells are able to proliferate in response to exogenous IL-2 and that in GITR–/– CD8+ T cells the defective response to CD28-costimulation is not due to an alteration in IL-2R signaling.


Figure 7
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  		FIGURE 7. IL-2 rescues CD28-costimulation of GITR–/– CD8+ T cells. [3H]Thymidine uptake of GITR+/+ and GITR–/– CD8+ T cells after 72 h-activation by plate-bound anti-CD3 ({alpha}-CD3) and anti-CD28 ({alpha}-CD28) Abs with or without IL-2 (100 U/ml). The result from one representative experiment of two is given and values are the mean of five separate counts. °, p > 0.05; **, p < 0.01; GITR–/– vs GITR+/+.

 
GITR is crucial for CD28-mediated NF-{kappa}B activation

One of the CD28-induced pathways involves NF-{kappa}B (35, 36). Because GITR originates signals leading to NF-{kappa}B nuclear translocation in CD4+ T cells (15, 37), GITR could supposedly contribute to CD28-induced NF-{kappa}B activation for the CD8+ T cell response, resulting in a possible defective NF-{kappa}B nuclear translocation in the absence of GITR. To explore this possibility, we analyzed the nuclear translocation of NF-{kappa}B in GITR–/– and GITR+/+ CD8+ T cells by immunofluorescence. CD8+ T cells were activated for 15 h with anti-CD3 Ab alone or in combination with anti-CD28 Ab and stained for the p65 NF-{kappa}B subunit. Results indicated that NF-{kappa}B nuclear translocation was similar in anti-CD3-activated cells of GITR–/– and GITR+/+ mice but was impaired in GITR–/– cells costimulated by anti-CD28 Ab, where a lower number of cells with translocated NF-{kappa}B were found, as compared with costimulated GITR+/+ cells (Fig. 8A).


Figure 8
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  		FIGURE 8. Lower level of NF-{kappa}B activation in coactivated GITR–/– CD8+ T lymphocytes. A, Immunofluorescence assay for NF-{kappa}B nuclear translocation in GITR+/+ and GITR–/– CD8+ T lymphocytes, either naive (a) or activated for 15 h with plate-bound anti-CD3 Ab (b) or plate-bound anti-CD3 plus anti-CD28 Abs (c). 4',6'-Diamidino-2-phenylindole (DAPI) staining was used to identify nuclei; NF-{kappa}B (red) and DAPI (blue) images have been merged to evidence cells with superimposed red and blue stainings. Arrows point to cells in which NF-{kappa}B translocated into the nucleus. Pictures from one representative experiment of three are given. Images were captured using a x100/1.25 numerical aperture oil objective. d, Nuclei positive for NF-{kappa}B staining have been counted and the percentages expressed as mean ± SD of the three independent experiments as shown in the graph. °, p > 0.05; **, p < 0.01; GITR–/– vs GITR+/+. B, EMSA assay of nuclear extracts of GITR+/+ and GITR–/– CD8+ T lymphocytes, either naive or activated for 15 h with plate-bound anti-CD3 ({alpha}-CD3) Ab or plate-bound anti-CD3 plus anti-CD28 ({alpha}-CD28) Abs. A graph with the net counts of each lane is also reported (right panel). The result from one representative experiment of two is given. C, Western blot analysis of I{kappa}B{alpha} expression in GITR+/+ and GITR–/– CD8+ T lymphocytes activated by anti-CD3 Ab or anti-CD3 plus anti-CD28 Abs for 3 and 24 h. beta-Tubulin Western blot is also shown for equal gel loading. A densitometric evaluation of each blot was performed and the mean ± SD of three independent experiments is reported. °, p > 0.05; *, p < 0.05; GITR–/– vs GITR+/+.

 
These results were confirmed by direct measurement of NF-{kappa}B in the nucleus by using the EMSA. Levels of NF-{kappa}B in the nucleus of GITR–/– CD8+ T cells were much lower than those in GITR+/+ cells in response to anti-CD3 plus anti-CD28 Ab stimulation (Fig. 8B), confirming immunofluorescence assay results. To rule out the possibility of a constitutive difference in the NF-{kappa}B levels, we measured the amount of the NF-{kappa}B subunits p52 and p65 at the basal level in both genotypes. Western blot analysis revealed an equal expression of both subunits (data not shown).

In its latent form, NF-{kappa}B is found in a complex with I{kappa}B{alpha} in the cytoplasm. To further confirm the defect in NF-{kappa}B nuclear translocation in CD28-costimulated GITR–/– CD8+ T lymphocytes under the aforementioned conditions, we indirectly evaluated NF-{kappa}B activation by measuring I{kappa}B{alpha} protein levels. Western blotting results showed that costimulated GITR–/– CD8+ T cells contain slightly more I{kappa}B{alpha} than GITR+/+ cells after 24 h of activation (Fig. 8C). These results provide evidence that in CD8+ T lymphocytes GITR contributes to the activation of NF-{kappa}B in response to CD28 coligation.

Bcl-xL expression is defective in CD28-costimulated GITR–/– T cells

CD28 induces clonal expansion and promotes the expression of the anti-apoptotic protein Bcl-xL in an NF-{kappa}B-dependent manner (38, 39). Given the defect in NF-{kappa}B activation and the increased apoptosis levels in GITR–/– cells, we hypothesized that Bcl-xL expression could be compromised. Analysis by Western blotting revealed that CD28 coligation increased Bcl-xL expression over anti-CD3 Ab stimulation in GITR+/+ but not in GITR–/– CD8+ T lymphocytes (Fig. 9). These results suggest that the defect in NF-{kappa}B activation in costimulated GITR–/– CD8+ T cells causes a reduction of Bcl-xL expression that, in turn, can account for their decreased survival (Fig. 2A).


Figure 9
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  		FIGURE 9. Lower expression levels of Bcl-xL in coactivated GITR–/– CD8+ T lymphocytes. Western blot analysis of Bcl-xL expression in GITR+/+ and GITR–/– CD8+ T lymphocytes, activated by anti-CD3 ({alpha}-CD3) Ab or anti-CD3 plus anti-CD28 ({alpha}-CD28) Abs for 24 h. beta-Tubulin Western blot is also shown for equal gel loading. A densitometric evaluation of each blot was performed and the mean ± SD of three independent experiments is reported. °, p > 0.05; **, p < 0.01; GITR–/– vs GITR+/+.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We here demonstrate that GITR–/– CD8+ T cells have a defect in activation response, including NF-{kappa}B activation, following CD28 costimulation. T cell costimulation involves a variety of molecules that possess distinct activities in specific T cell subpopulations and our results show that GITR–/– CD8+ T lymphocytes respond defectively to CD28 costimulation. Contrary to CD8+ T cells, GITR–/– and GITR+/+ CD4+ T cells are equally costimulated by anti-CD28 Ab. Similar results have been reported for other TNFR and TNF superfamily members such as TNFR2 (40) and LIGHT (41).

In the past years, many studies focused on the effects of GITR stimulation in CD4+ T cells, including CD4+CD25+ suppressor T cells. However, recent in vivo studies have revealed a specific effect of GITR in CD8+ T cells. In a bone marrow transplantation model, GITR stimulation enhanced alloreactive CD8+CD25– T cell proliferation while it decreased the proliferation of alloreactive CD4+CD25– cells (42). In another murine model of chronic graft-vs-host disease, an anti-GITR Ab, while inducing apoptosis of CD4+ donor cells, prevented donor CD8+ T cell anergy by inducing activation (43). Moreover, GITR triggering potentiates CD8+-mediated immune response to tumors, a response partially independent of CD4+ T cells (44). Our findings correlate with these in vivo results, demonstrating that GITR triggering plays a specific role in CD8+ T cell activation.

Kohm et al. (24) demonstrated that GITR up-regulation following activation was dependent on CD28 engagement in CD4+ T cells. Stephens et al. (16) compared the effect of CD28 costimulation in CD4+ and CD8+ T cells and showed a difference in the modulation of GITR expression at the time point studied. We focused our attention on CD8+ T cells and here show that GITR up-regulation is only weakly dependent on CD28 costimulation while CD28 up-regulation is dependent on GITR ligation. Interestingly, in a microarray study with CD8+ T cells GITR was one of the genes directly up-regulated upon TCR ligation (45), further suggesting that, in CD8+ T cells, CD28 costimulation is dispensable for GITR up-regulation.

In our plate-bound model, a culture system in which only purified CD8+ T cells are present, GITR was stimulated by its ligand. In fact, GITRL is expressed in activated CD8+ T lymphocytes soon after stimulation and then rapidly down-regulated. The functional relevance of GITR stimulation by activation-induced GITRL is demonstrated by the observation that the addition of a recombinant GITR protein (GITR-Fc) to GITR+/+ cells gave results similar to those observed in GITR–/– cells. Therefore, in vivo CD8+ T lymphocytes can be costimulated by GITRL expressed on APCs as previously reported (19) and also by GITRL expressed on activated CD8+ T cells. This finding is in line with those regarding other members of the TNFRSF, whose ligands are expressed in activated T cells such as, for example, OX40L, CD30L, and CD27L (46, 47, 48).

IL-2 is induced by costimulation, either by CD28 or GITR (17, 30). In GITR–/– CD8+ T lymphocytes, IL-2, like IFN-{gamma} and IL-2R, does not reach levels comparable to that found in GITR+/+ T cells. Because IL-2 is a critical growth factor for T cells, we anticipated that IL-2 could be one of the limiting factors to achieve a full activation of GITR–/– CD8+ T lymphocytes. In fact, the GITR–/– defect was reversed by the addition of exogenous rIL-2, thus suggesting that GITR engagement lowers the threshold of CD8+ T cell activation through IL-2 production and that the IL-2R pathway is intact in GITR–/– CD8+ T cells.

CD28 costimulation regulates IL-2 production by well known transduction signals. Akt, as well MAPK or JNK, showed equal activation levels in CD28-costimulated GITR–/– and GITR+/+ cells, suggesting that GITR signaling does not interfere with those CD28-activated pathways. On the contrary, differences in NF-{kappa}B nuclear translocation were found. This effect may be explained by considering that both CD28 and GITR act synergistically with TCR to effectively activate NF-{kappa}B (15, 35, 49). NF-{kappa}B can be activated by a variety of signals, and CD28-derived as well as GITR-derived signals could converge. It is likely that in CD8+ T cells the cooperation between GITR and CD28 activation is functionally relevant as suggested by in vivo studies (42, 43, 44). However, GITR signaling loses its relevance when high doses of anti-CD3 Abs are used in vitro, suggesting that GITR lowers the costimulation threshold of CD8+ T cells.

As a consequence of reduced NF-{kappa}B activation, the expression of its target proteins including Bcl-xL, a critical factor for survival of activated T cells (50, 51), was impaired. In GITR–/– CD8+ T lymphocytes costimulated by anti-CD28 Ab, Bcl-xL expression did not increase like in GITR+/+ cells, accounting for the decreased protection from anti-CD3-induced apoptosis. Overall, our findings suggest that NF-{kappa}B, regulating the expression of survival and activation genes (Bcl-xL, IL-2), allows CD28 to signal in the presence of GITR costimulation.

The lack of GITR stimulation raises the threshold of T cell activation in GITR–/– CD8+ T cells treated with anti-CD3 and anti-CD28. Such a finding would suggest a reciprocal defect in CD28–/– CD8+ T cells under GITR costimulation. However, in CD28–/– CD8+ T cells GITR is able to elicit costimulatory effects similar to those observed in CD28+/+ CD8+ T cells, suggesting that costimulation by GITR is thoroughly independent of CD28. These results are in agreement with studies performed with other TNFRSF members (52, 53, 54).

Costimulatory GITR activity has been demonstrated in many laboratories in vitro and in vivo in T cells and in cells of innate immunity such as NK cells (20, 22, 23, 55, 56). However, in an in vitro experimental model using splenocytes and lymph node T cells, we have shown a significantly higher proliferative response of GITR–/– as compared with GITR+/+ cells (14). Although the mechanism responsible for this effect has not been clarified, the possibility that the GITR/GITRL system regulates the activity of cells other than effector T lymphocytes should be considered. As an example, we and others have shown that GITR costimulus is also active in isolated CD4+CD25+ Treg cells. Therefore, GITR costimulation can induce a proliferative response that results in an increase in Treg cell number (17, 57). In this regard, GITR costimulation can weaken Treg cell suppressive activity as measured by in vitro assays, but this effect could be transient and due to an increase in IL-2 production that masks the actual suppressive activity (58). Moreover, it has been shown that GITR and GITRL can be expressed in APCs such as macrophages, B lymphocytes, and dendritic cells (4). We have recently shown in a Candida albicans infection model that GITR, expressed on CD4+CD25+ cells, inhibits dendritic cell function and IL-12 production, thus indicating that GITR/GITRL interaction can contribute to the inhibition of APC activity and, consequently, the inhibition of Th1 immune response (21). Although the tolerogenic activity can be ascribed to an indirect effect on T cells that modulate dendritic cell activity through cytokine release (59), it is clear that GITR/GITRL interaction can influence the activity of cells other than T lymphocytes including APCs and cause the inhibition of effector cell response (33). Therefore, the balance of the effects of GITR/GITRL interaction on different components of the immune system could determine the final outcome of immune response and differ from one experimental system to another.

The results described in this article indicate that GITR is important in CD28-driven activation of CD8+ T cells, lowering the threshold of T cell activation, and warrants deeper investigations in various in vitro and in vivo experimental models.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a research grant from the Italian Association for Cancer Research (AIRC) in Milan, Italy. Back

2 S.R. and G.N. equally contributed to this work. Back

3 Address correspondence and reprint requests to Dr. Carlo Riccardi, Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Perugia, Via del Giochetto, Perugia. E-mail address: riccardi{at}unipg.it Back

4 Abbreviations used in this paper: TNFRSF, TNFR superfamily; GITR, glucocorticoid-induced TNFR-related protein; GITRL, GITR ligand; MFI, median fluorescence intensity. Back

Received for publication October 12, 2006. Accepted for publication August 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Daniel, P. T., A. Kroidl, S. Cayeux, R. Bargou, T. Blankenstein, B. Dorken. 1997. Costimulatory signals through B7.1/CD28 prevent T cell apoptosis during target cell lysis. J. Immunol. 159: 3808-3815. [Abstract]
  2. Spinicelli, S., G. Nocentini, S. Ronchetti, L. T. Krausz, R. Bianchini, C. Riccardi. 2002. GITR interacts with the pro-apoptotic protein Siva and induces apoptosis. Cell Death Differ. 9: 1382-1384. [Medline]
  3. Watts, T. H.. 2005. TNF/TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23: 23-68. [Medline]
  4. Krausz, L. T., R. Bianchini, S. Ronchetti, K. Fettucciari, G. Nocentini, C. Riccardi. 2007. GITR-GITRL system, a novel player in shock and inflammation. Sci. World J. 7: 533-566.
  5. Nocentini, G., S. Ronchetti, S. Cuzzocrea, C. Riccardi. 2007. GITR/GITRL: more than an effector T cell co-stimulatory system. Eur. J. Immunol. 37: 1165-1169. [Medline]
  6. Acuto, O., F. Michel. 2003. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 3: 939-951. [Medline]
  7. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261: 609-612. [Abstract/Free Full Text]
  8. Halstead, E. S., Y. M. Mueller, J. D. Altman, P. D. Katsikis. 2002. In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses. Nat. Immunol. 3: 536-541. [Medline]
  9. DeBenedette, M. A., T. Wen, M. F. Bachmann, P. S. Ohashi, B. H. Barber, K. L. Stocking, J. J. Peschon, T. H. Watts. 1999. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol. 163: 4833-4841. [Abstract/Free Full Text]
  10. Shuford, W. W., K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho, C. P. Larsen, et al 1997. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186: 47-55. [Abstract/Free Full Text]
  11. Sugamura, K., N. Ishii, A. D. Weinberg. 2004. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat. Rev. Immunol. 4: 420-431. [Medline]
  12. Croft, M.. 2003. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?. Nat. Rev. Immunol. 3: 609-620. [Medline]
  13. Nocentini, G., L. Giunchi, S. Ronchetti, L. T. Krausz, A. Bartoli, R. Moraca, G. Migliorati, 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: 6216-6221. [Abstract/Free Full Text]
  14. Ronchetti, S., G. Nocentini, C. Riccardi, P. P. Pandolfi. 2002. Role of GITR in activation response of T lymphocytes. Blood 100: 350-352. [Abstract/Free Full Text]
  15. Kanamaru, F., P. Youngnak, M. Hashiguchi, T. Nishioka, T. Takahashi, S. Sakaguchi, I. Ishikawa, M. Azuma. 2004. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J. Immunol. 172: 7306-7314. [Abstract/Free Full Text]
  16. Stephens, G. L., R. S. McHugh, M. J. Whitters, D. A. Young, D. Luxenberg, B. M. Carreno, M. Collins, E. M. Shevach. 2004. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J. Immunol. 173: 5008-5020. [Abstract/Free Full Text]
  17. Ronchetti, S., O. Zollo, S. Bruscoli, M. Agostini, R. Bianchini, G. Nocentini, E. Ayroldi, C. Riccardi. 2004. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur. J. Immunol. 34: 613-622. [Medline]
  18. Esparza, E. M., R. H. Arch. 2005. Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phases of T cell activation. J. Immunol. 174: 7869-7874. [Abstract/Free Full Text]
  19. Tone, M., Y. Tone, E. Adams, S. F. Yates, M. R. Frewin, S. P. Cobbold, H. Waldmann. 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Natl. Acad. Sci. USA 100: 15059-15064. [Abstract/Free Full Text]
  20. Cuzzocrea, S., E. Ayroldi, R. Di Paola, M. Agostini, E. Mazzon, S. Bruscoli, T. Genovese, S. Ronchetti, A. P. Caputi, C. Riccardi. 2005. Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis. FASEB J. 19: 1253-1265. [Abstract/Free Full Text]
  21. Agostini, M., E. Cenci, E. Pericolini, G. Nocentini, G. Bistoni, A. Vecchiarelli, C. Riccardi. 2005. The glucocorticoid-induced tumor necrosis factor receptor-related gene modulates the response to Candida albicans infection. Infect. Immun. 73: 7502-7508. [Abstract/Free Full Text]
  22. Santucci, L., M. Agostini, S. Bruscoli, A. Mencarelli, S. Ronchetti, E. Ayroldi, A. Morelli, M. Baldoni, C. Riccardi. 2007. GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice. Gut 56: 52-60. [Abstract/Free Full Text]
  23. Cuzzocrea, S., S. Ronchetti, T. Genovese, E. Mazzon, M. Agostini, R. Di Paola, E. Esposito, C. Muia, G. Nocentini, C. Riccardi. 2007. Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation. FASEB J. 21: 117-129. [Abstract/Free Full Text]
  24. Kohm, A. P., J. R. Podojil, J. S. Williams, J. S. McMahon, S. D. Miller. 2005. CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms. Cell. Immunol. 235: 56-64. [Medline]
  25. Ayroldi, E., G. Migliorati, S. Bruscoli, C. Marchetti, O. Zollo, L. Cannarile, F. D’Adamio, C. Riccardi. 2001. Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor {kappa}B. Blood 98: 743-753. [Abstract/Free Full Text]
  26. Ayroldi, E., O. Zollo, A. Bastianelli, C. Marchetti, M. Agostini, R. Di Virgilio, C. Riccardi. 2007. GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling. J. Clin. Invest. 117: 1605-1615. [Medline]
  27. Migliorati, G., I. Nicoletti, M. C. Pagliacci, L. D’Adamio, C. Riccardi. 1993. Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis. Blood 81: 1352-1358. [Abstract/Free Full Text]
  28. Riccardi, C., I. Nicoletti. 2006. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1: 1458-1461. [Medline]
  29. Reichert, P., R. L. Reinhardt, E. Ingulli, M. K. Jenkins. 2001. Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells. J. Immunol. 166: 4278-4281. [Abstract/Free Full Text]
  30. Kane, L. P., P. G. Andres, K. C. Howland, A. K. Abbas, A. Weiss. 2001. Akt provides the CD28 costimulatory signal for up-regulation of IL-2 and IFN-{gamma} but not TH2 cytokines. Nat. Immunol. 2: 37-44. [Medline]
  31. Rochford, R., J. E. Riggs, A. Clavo, D. N. Ernst, M. V. Hobbs. 2004. Differential effects of CD28 costimulation upon cytokine production by CD4+ and CD8+ T cells. Immunobiology 209: 513-522. [Medline]
  32. DeBenedette, M. A., A. Shahinian, T. W. Mak, T. H. Watts. 1997. Costimulation of CD28– T lymphocytes by 4-1BB ligand. J. Immunol. 158: 551-559. [Abstract]
  33. Grohmann, U., C. Volpi, F. Fallarino, S. Bozza, R. Bianchi, C. Vacca, C. Orabona, M. L. Belladonna, E. Ayroldi, G. Nocentini, et al 2007. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Nat. Med. 13: 579-586. [Medline]
  34. Ji, H. B., G. Liao, W. A. Faubion, A. C. Abadia-Molina, C. Cozzo, F. S. Laroux, A. Caton, C. Terhorst. 2004. Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J. Immunol. 172: 5823-5827. [Abstract/Free Full Text]
  35. Kane, L. P., J. Lin, A. Weiss. 2002. It’s all rel-ative: NF-{kappa}B and CD28 costimulation of T-cell activation. Trends Immunol. 23: 413-420. [Medline]
  36. Coudronniere, N., M. Villalba, N. Englund, A. Altman. 2000. NF-{kappa}B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-{tau}. Proc. Natl. Acad. Sci. USA 97: 3394-3399. [Abstract/Free Full Text]
  37. Esparza, E. M., R. H. Arch. 2004. TRAF4 functions as an intermediate of GITR-induced NF-{kappa}B activation. Cell Mol. Life Sci. 61: 3087-3092. [Medline]
  38. Boise, L. H., A. J. Minn, P. J. Noel, C. H. June, M. A. Accavitti, T. Lindsten, C. B. Thompson. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3: 87-98. [Medline]
  39. Khoshnan, A., C. Tindell, I. Laux, D. Bae, B. Bennett, A. E. Nel. 2000. The NF-{kappa}B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J. Immunol. 165: 1743-1754. [Abstract/Free Full Text]
  40. Kim, E. Y., H. S. Teh. 2001. TNF type 2 receptor (p75) lowers the threshold of T cell activation. J. Immunol. 167: 6812-6820. [Abstract/Free Full Text]
  41. Liu, J., C. S. Schmidt, F. Zhao, A. J. Okragly, A. Glasebrook, N. Fox, E. Galbreath, Q. Zhang, H. Y. Song, S. Na, D. D. Yang. 2003. LIGHT-deficiency impairs CD8+ T cell expansion, but not effector function. Int. Immunol. 15: 861-870. [Abstract/Free Full Text]
  42. Muriglan, S. J., T. Ramirez-Montagut, O. Alpdogan, T. W. Van Huystee, J. M. Eng, V. M. Hubbard, A. A. Kochman, K. H. Tjoe, C. Riccardi, P. P. Pandolfi, et al 2004. GITR activation induces an opposite effect on alloreactive CD4+ and CD8+ T cells in graft-versus-host disease. J. Exp. Med. 200: 149-157. [Abstract/Free Full Text]
  43. Kim, J., W. S. Choi, H. Kang, H. J. Kim, J. H. Suh, S. Sakaguchi, B. Kwon. 2006. Conversion of alloantigen-specific CD8+ T cell anergy to CD8+ T cell priming through in vivo ligation of glucocorticoid-induced TNF receptor. J. Immunol. 176: 5223-5231. [Abstract/Free Full Text]
  44. Cohen, A. D., A. Diab, M. A. Perales, J. D. Wolchok, G. Rizzuto, T. Merghoub, D. Huggins, C. Liu, M. J. Turk, N. P. Restifo, et al 2006. Agonist anti-GITR antibody enhances vaccine-induced CD8+ T-cell responses and tumor immunity. Cancer Res. 66: 4904-4912. [Abstract/Free Full Text]
  45. Verdeil, G., D. Puthier, C. Nguyen, A. M. Schmitt-Verhulst, N. Auphan-Anezin. 2006. STAT5-mediated signals sustain a TCR-initiated gene expression program toward differentiation of CD8 T cell effectors. J. Immunol. 176: 4834-4842. [Abstract/Free Full Text]
  46. Miura, S., K. Ohtani, N. Numata, M. Niki, K. Ohbo, Y. Ina, T. Gojobori, Y. Tanaka, H. Tozawa, M. Nakamura, et al 1991. Molecular cloning and characterization of a novel glycoprotein, gp34, that is specifically induced by the human T-cell leukemia virus type I transactivator p40tax. Mol. Cell. Biol. 11: 1313-1325. [Abstract/Free Full Text]
  47. Shimozato, O., K. Takeda, H. Yagita, K. Okumura. 1999. Expression of CD30 ligand (CD153) on murine activated T cells. Biochem. Biophys. Res. Commun. 256: 519-526. [Medline]
  48. Tesselaar, K., L. A. Gravestein, G. M. van Schijndel, J. Borst, R. A. van Lier. 1997. Characterization of murine CD70, the ligand of the TNF receptor family member CD27. J. Immunol. 159: 4959-4965. [Abstract]
  49. Esparza, E. M., T. Lindsten, J. M. Stockhausen, R. H. Arch. 2006. Tumor necrosis factor receptor (TNFR)-associated factor 5 is a critical intermediate of costimulatory signaling pathways triggered by glucocorticoid-induced TNFR in T cells. J. Biol. Chem. 281: 8559-8564. [Abstract/Free Full Text]
  50. Sperling, A. I., J. A. Auger, B. D. Ehst, I. C. Rulifson, C. B. Thompson, J. A. Bluestone. 1996. CD28/B7 interactions deliver a unique signal to naive T cells that regulates cell survival but not early proliferation. J. Immunol. 157: 3909-3917. [Abstract]
  51. Tang, Q., J. A. Smith, G. L. Szot, P. Zhou, M. L. Alegre, K. J. Henriksen, C. B. Thompson, J. A. Bluestone. 2003. CD28/B7 regulation of anti-CD3-mediated immunosuppression in vivo. J. Immunol. 170: 1510-1516. [Abstract/Free Full Text]
  52. Saoulli, K., S. Y. Lee, J. L. Cannons, W. C. Yeh, A. Santana, M. D. Goldstein, N. Bangia, M. A. DeBenedette, T. W. Mak, Y. Choi, T. H. Watts. 1998. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J. Exp. Med. 187: 1849-1862. [Abstract/Free Full Text]
  53. Hendriks, J., Y. Xiao, J. Borst. 2003. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J. Exp. Med. 198: 1369-1380. [Abstract/Free Full Text]
  54. Akiba, H., H. Oshima, K. Takeda, M. Atsuta, H. Nakano, A. Nakajima, C. Nohara, H. Yagita, K. Okumura. 1999. CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells. J. Immunol. 162: 7058-7066. [Abstract/Free Full Text]
  55. Nocentini, G., C. Riccardi. 2005. GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily. Eur. J. Immunol. 35: 1016-1022. [Medline]
  56. Hanabuchi, S., N. Watanabe, Y. H. Wang, Y. H. Wang, T. Ito, J. Shaw, W. Cao, F. X. Qin, Y. J. Liu. 2006. Human plasmacytoid predendritic cells activate NK cells through glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL). Blood 107: 3617-3623. [Abstract/Free Full Text]
  57. Ramirez-Montagut, T., A. Chow, D. Hirschhorn-Cymerman, T. H. Terwey, A. A. Kochman, S. Lu, R. C. Miles, S. Sakaguchi, A. N. Houghton, M. R. van den Brink. 2006. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J. Immunol. 176: 6434-6442. [Abstract/Free Full Text]
  58. Shevach, E. M., G. L. Stephens. 2006. The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity?. Nat. Rev. Immunol. 6: 613-618. [Medline]
  59. Mahesh, S. P., Z. Li, B. Liu, R. N. Fariss, R. B. Nussenblatt. 2006. Expression of GITR ligand abrogates immunosuppressive function of ocular tissue and differentially modulates inflammatory cytokines and chemokines. Eur. J. Immunol. 36: 2128-2138. [Medline]



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