Cutting Edge: The Natural Ligand for Glucocorticoid-Induced TNF Receptor-Related Protein Abrogates Regulatory T Cell Suppression

Expression and purification of the sGITR-L

Full-length mouse GITR-L was cloned from macrophages with the primers (5′-CATCAGAGAACGAGTTCTAGCCTCAT-3′) and (5′-CTAAGAGATGAATGGTAGATCAGGCAT-3′) that were based on predicted mouse GITR-L sequences found in the genomic database. The sequence of mouse GITR-L, also known as TNFSF18, is available in the GenBank (accession number NM_183391). A vector pSec-Tag-sGITR-L, encoding the extracellular domain (aa 38–73) of mouse GITR-L along with an N-terminal Flag peptide and the Igκ leader sequence was transiently transfected into 293-F cells. The sGITR-L protein was isolated from the supernatant 72 h after transfection using an anti-Flag-agarose column (Sigma-Aldrich, St. Louis, MO) at 4°C. sGITR-L was eluted with 100 μg/ml the Flag peptide (DYKDDDDK; Sigma-Aldrich) and the sample was dialyzed against PBS. The purity of sGITR-L was assessed by SDS-PAGE followed by Western blotting with anti-Flag (Sigma-Aldrich) or by Coomassie blue staining.

Cell surface staining and flow cytometry

293T cells were transiently transfected with pSec-Tag-GITR (provided by Dr. C. Riccardi, Perugia, Italy) or with an empty control vector for 48 h as described previously (9). Transfectant cells were stained with either rat-anti-GITR (R&D Systems, Minneapolis, MN) or sGITR-L followed by either FITC-mouse-anti-rat-IgG or FITC-mouse-anti-Flag (BD PharMingen, San Diego, CA), respectively.

Resting CD4⁺ T cells, purified from the spleen of BALB/c mice (The Jackson Laboratory, Bar Harbor, ME), were stained with either anti-GITR and FITC-mouse-anti-rat-IgG or with sGITR-L and FITC-mouse-anti-Flag. Counterstaining employed PE-anti-CD4 and biotin-anti-CD25 plus streptavidin-Red670 (BD PharMingen). CD4⁺ T cells activated for 3 days using plate-bound anti-CD3, anti-CD28, and IL-2 (BD PharMingen) were stained with PE-anti-CD4 and either FITC-anti-CD25 or sGITR-L plus FITC-mouse-anti-Flag. Cell surface staining was analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA).

NF-κB reporter gene assay

293T cells were transfected with 0.1 μg of the NF-κB reporter gene plasmid pNF-κB-Luc that expresses firefly luciferase under the control of five copies of the NF-κB binding site from mouse E-selectin gene (Stratagene, La Jolla, CA). The same cells were also transfected with pRL-TK vector (0.01 μg) expressing Renilla luciferase (Promega, Madison WI) and with either pSec-Tag-GITR or the mutants GITRΔ215, GITRΔ206, or GITR-RKK. GITRΔ215 has a deletion of aa 216–228 and GITRΔ206 has a deletion of aa 207–228. In GITR-RKK, the point mutations P209R, E210K, and E211K were introduced, altering a potential TNFR-associated factor 1/2/3 binding site PEEER.

The indicated amounts of sGITR-L were added half an hour after transfection and NF-κB activation was assessed after 48 h by the dual luciferase reporter assay (Promega). Firefly luciferase activity was normalized against Renilla luciferase activity and the relative luciferase activity, representative of NF-κB activation, was calculated by subtraction of the activity obtained with an empty vector.

T cell suppression and proliferation assays

Splenocytes from wild-type BALB/c or HA28xTS1 mice (10) were stained with FITC-anti-CD25 and PE-anti-CD4 (BD PharMingen) and sorted by FACS. Purity of CD4⁺25⁺ or CD4⁺25⁻ T cells was >98 and 95% respectively. In polyclonal Treg cell suppression assays (11), CD4⁺25⁻ cells (1 × 10⁵) were cocultured with irradiated T cell-depleted splenocytes (APC), 5 μg/ml anti-CD3, and indicated numbers of CD4⁺25⁺ cells for 3 days. TS1 cells were stimulated with APCs pulsed with the S1 peptide (concentration ranging from 0.003 to 3 μM (10)). To block suppression, a combination of mouse sGITR-L and anti-Flag (Sigma-Aldrich) was added.

Proliferation of purified CD4⁺25⁺ or CD4⁺25⁻ T cells was also induced by different combinations of plate-bound anti-CD3, sGITR-L, and IL-2 for 3 days. Neutralizing Abs (10 μg/ml) directed at IL-2, IL-4, IL-6, IL-10, or IFN-γ (BD PharMingen) along with sGITR-L (2.5 μg/ml) were used in some of the suppression assays.

Ag-specific Treg cell suppression assays were performed as described elsewhere (10). Pooled lymph node cells from TS1 mice were cultured with irradiated splenocytes and 0.3 μM S1 peptide (SFERFEIFPKE) in 96-well plates. CD4⁺25⁺ T cells purified from TS1xHA28 mice were added to the TS1 cell proliferation assays with either sGITR-L (2.5 μg/ml) or IL-2 (10 ng/ml).

Proliferation was assessed by incorporation of [³H]thymidine (1μCi/well; Amersham Biosciences, Piscataway, NJ), which was added for the last 12 h of each culture. Supernatants of cell cultures were also used to determine IL-2 using the ELISA kit from BD PharMingen. The lower limit of detection was 31 pg/ml.

Statistical analysis

Results are expressed as the mean ± SEM Statistical comparisons were performed by a two-tailed paired t test or by ANOVA when unequal n values were present. Values of p < 0.05 were considered to be statistically significant.

GITR-L interacts with GITR on the surface of CD4⁺25⁺ T cells and enhances NF-κB signaling

We cloned a cDNA encoding mouse GITR-L using PCR probes based on mouse genomic sequences that had 60% identity with human GITR-L (12, 13) and RNA from macrophages. Mouse GITR-L is a 173-aa protein (19.7 kDa) with a type II transmembrane topology with an N-terminal cytoplasmic domain (20 aa), a transmembrane region, and a C-terminal extracellular domain (133 aa) containing two potential N-glycosylation sites. We generated a sGITR-L by transfecting a construct encoding a chimeric protein comprising the GITR-L extracellular domain and an N-terminal Flag peptide into 293F cells. Purity of sGITR-L was assessed by either Coomassie blue staining or Western blotting with anti-Flag (Fig. 1⇓A, lanes 1 and 2). Twenty percent of unboiled sGITR-L appeared as trimers in SDS-PAGE (Fig. 1⇓A, lane 3), which indicated that an even larger percentage of the sGITR-L preparation had assembled into homotrimers.

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FIGURE 1.

sGITR-L specifically binds to GITR and augments GITR-induced NF-κB activation. A, sGITR-L was expressed and purified as described. Purity of sGITR-L was assessed by Coomassie blue staining (both boiled and unboiled samples) and Western blotting with anti-Flag (lanes 1, 3, and 2, respectively). B, The specificity of sGITR-L was assessed by staining of GITR-transfected or vector-transfected 293T cells (panels 1, 2, 5, and 6 vs 3, 4, 7, and 8) with either sGITR-L or anti-GITR followed by FITC-labeled secondary Ab (panels 1 and 3 vs 5 and 7, respectively) or the appropriate negative control (panels 2 and 4 vs 6 and 8). y-axis, Number of cells; x-axis, fluorescence intensity. C, Resting splenic CD4⁺ T cells were isolated and sorted into both CD25⁻ and CD25⁺ populations. Whereas only CD4⁺25⁺ resting T cells were positive for GITR expression (panels 1 and 2), activation of CD4⁺ T cells with anti-CD3 induced robust expression of GITR and CD25 (panels 3 and 4, respectively). y-axis, Number of cells; x-axis, fluorescence intensity. D, The ability of sGITR to induce NF-κB via GITR or GITR mutants (described in Materials and Methods) was determined using the dual luciferase reporter assay and the relative luciferase activity was taken as a measure of NF-κB activation. The data are representative of three independent experiments.

sGITR-L binds to 27% of 293T cells transiently transfected with a cDNA encoding mouse GITR, but not nontransfected 293T cells, as judged by staining with sGITR-L and FITC-mouse-anti-Flag (Fig. 1⇑B). The same percentage of the GITR transfectants were stained by anti-GITR (Fig. 1⇑B). Furthermore, sGITR-L and FITC-anti-Flag stained specifically freshly isolated CD4⁺25⁺ T cells, but not CD4⁺25⁻ T cells (Fig. 1⇑C, panels 1 and 2). As expected from studies with anti-GITR (7, 8), CD4⁺ T cells become positive for staining with sGITR-L upon activation with αCD3 (Fig. 1⇑C, panels 3 and 4). Thus, sGITR-L specifically binds to GITR on the surface of Treg cells and of activated naive CD4⁺ cells.

To determine whether mouse GITR-L induced GITR-specific signal transduction, an NF-κB activation-dependent signal transduction assay was used. To this end, sGITR-L was added to 293T cells, which had been cotransfected with mouse GITR and the NF-κB-inducible luciferase reporter pNF-κB-Luc (7). Addition of >0.5 μg/ml sGITR-L augmented GITR-dependent NF-κB activation (Fig. 1⇑D). By contrast, no increase in NF-κB-dependent luciferase activity was detected, when the mutant GITR-Δ206 with a truncated cytoplasmic tail was used (Fig. 1⇑D). Because mutant GITRΔ215 responded to sGITR-L, an important site had to be located between amino acid residues 206 and 215. As the peptide segment between residues 206 and 215 of the mouse GITR cytoplasmic tail contains a potential TNFR-associated factor1/2/3-binding motif, this site was altered. The resulting mutant GITR-RKK in which the sequence ²⁰⁹PEEER²¹³ was changed into ²⁰⁹RKKER²¹³ did not respond to sGITR-L (Fig. 1⇑D). The increased responses of deletion mutant GITRΔ215 indicated the presence of a negative regulatory element in the C-terminal 13 aa of GITR. Taken together, our findings clearly demonstrate that the engagement of GITR by sGITR-L induces physiologically relevant signals.

sGITR-L blocks CD4⁺25⁺ Treg cell suppression

The hallmark of naturally arising CD4⁺25⁺ regulatory T cells is that they suppress proliferation of both Ag-specific CD4⁺25⁻ T cell clones and of autologous polyclonal CD4⁺25⁻ T cells. To examine whether sGITR-L stimulation affects Treg cell suppression, sGITR-L was added to a polyclonal in vitro suppression assay. sGITR-L, in combination with anti-Flag, completely reversed in vitro suppression mediated by CD4⁺25⁺ Treg cells (p < 0.05), Fig. 2⇓A) at a concentration of 2.5 μg/ml (Fig. 2⇓B). Although sGITR-L by itself blocks suppression, addition of anti-Flag enhances its functional capability, most likely by cross-linking of GITR/GITR-L complexes (data not shown). Whereas McHugh et al. (8) reported that preactivated Treg cells are refractory to anti-GITR-mediated abrogation, sGITR-L blocked preactivated CD4⁺25⁺ Treg cell suppression (data not shown). Suppression by Ag-specific Treg cells was also blocked by sGITR-L, as demonstrated by using HA28xTS1 Treg cells (p < 0.005, Fig. 2⇓C). The effect of sGITR-L was comparable to that of adding 10 ng/ml rIL-2 (Fig. 2⇓C). Taken together, these experiments demonstrate that sGITR-L abrogates the in vitro suppressor function of both polyclonal and Ag-specific CD4⁺25⁺ Treg cells.

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FIGURE 2.

sGITR-L abrogates suppression by CD4⁺25⁺ regulatory T cells. A, sGITR-L was able to inhibit the suppression by Treg cells of CD4⁺25⁻ T cell proliferation, as determined by [³H] thymidine incorporation (∗, p < 0.05). However, increasing amounts of sGITR-L do not induce proliferation of CD4⁺25⁻ cells cultured with APCs. B, In addition, sGITR-L is able to abrogate Treg-mediated suppression of TS1 cells in an Ag-specific (S1 peptide concentrations between 0.003 and 3 μM) fashion (10 ) comparable to that of IL-2. C, The data are representative of six independent experiments.

sGITR-L augments proliferation of both CD4⁺25⁺ and CD4⁺25⁻ T cells

To dissect the potential mechanisms through which cells sGITR-L lifted Treg suppression, the effect of sGITR-L on proliferation of CD4⁺25⁺ and CD4⁺25⁻ T cells was examined. Upon a 3-day incubation with increasing amounts of sGITR-L in combination with anti-CD3 and/or rIL-2, DNA synthesis by purified CD4⁺25⁺ T cells dramatically increased (p < 0.01, Fig. 3⇓A). This effect was most pronounced in combination of sGITR-L with both anti-CD3 and IL-2 (p < 0.01), but was also detectable in combination with either anti-CD3 or IL-2 (Fig. 3⇓A). However, neither anti-CD3, IL-2 nor sGITR-L alone induce CD4⁺25⁺ T cell proliferation (Fig. 3⇓A). Addition of sGITR-L did also enhance anti-CD3-induced proliferation of purified CD4⁺25⁺ T cells (p < 0.05, Fig. 3⇓B), which acquire expression of GITR during the 3-day culture (see Fig. 1⇑C). We conclude that sGITR-L is a costimulator which augments proliferation of both CD4⁺25⁺ Treg cells and CD4⁺25⁻ T cells. These findings are indicative that sGITR-L may block suppression by breaking the anergic state of Treg cells and by an indirect mechanism involving CD4⁺ responder cells.

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FIGURE 3.

sGITR-L induces proliferation of both CD4⁺25⁺ and CD4⁺25⁻ T cells. Upon a 3-day incubation with increasing amounts of sGITR-L in combination with anti-CD3 and/or rIL-2 DNA synthesis by purified CD4⁺25⁺ (A) or CD4⁺25⁻ (A) T cells were assessed. [³H]Thymidine was added to cell culture for the last 12 h. The sGITR-L (A) and anti-CD3 (B) concentrations are expressed on a logarithmic scale. These data are representative of four separate experiments.

sGITR-L-induced IL-2 secretion by activated naive CD4⁺ cells contributes to the abrogation of Treg suppression

Because addition of rIL-2 shuts off Treg cell suppression and because IL-6 in the supernatant of LPS-activated dendritic cells may counteract the function of Treg cells (5, 11), the role of selected cytokines on the effect of sGITR-L was examined by addition of blocking Abs. Interestingly, in this assay only anti-IL-2 (p < 0.04), but not anti-IL-4, anti-IL-6, anti-IL-10, or anti-IFN-γ counteracted the blocking effect of sGITR-L (Fig. 4⇓A). This prompted us to determine which of the T cells that participate in the suppression assay produced IL-2 in response to the addition of sGITR-L. As shown in Fig. 4⇓B, IL-2 production was dramatically increased when 2.5 μg/ml sGITR-L was added to a coculture of CD4⁺25⁻ T cells and APCs in the presence of soluble anti-CD3 (p = 0.02). No IL-2 was secreted by CD4⁺25⁺ T cells upon addition of sGITR-L. However, addition of CD4⁺25⁺ Treg cells to the CD4⁺25 ⁻T cell culture suppressed IL-2 production regardless of the presence of sGITR-L (p = 0.02). Taken together, the data demonstrate that stimulation of GITR by sGITR-L abrogates Treg suppression through up-regulation of IL-2 production by CD4⁺25 ⁻T cells. This increase in IL-2 production along with sGITR-L’s ability to augment CD4⁺25⁺ T cell proliferation provides the robust cellular signals that abrogate in vitro suppression.

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FIGURE 4.

sGITR-L induces IL-2 secretion by CD4⁺25⁻ T cells, which in turn affects Treg suppression. A, Neutralizing Abs (10 μg/ml) directed at IL-2, IL-4, IL-6, IL-10, or IFN-γ (BD PharMingen) along with sGITR-L (2.5 μg/ml) were added to the standard suppression assay. Only the addition of anti-IL-2 was able to counteract sGITR-l-mediated blocking of Treg suppression (∗, p = 0.03; ∗∗, p = 0.04). B, sGITR-L (2.5 μg/ml) induces IL-2 production by CD4⁺25⁻ T cells in the presence of APCs and anti-CD3. This IL-2 production was reduced in the presence of CD4⁺25⁺ Treg cells, which do not secrete IL-2 in the presence of sGITR-L. ∗, p = 0.02; ∗∗, p = 0.02. ND, Nondetectable. The data are representative of three independent experiments.

In conclusion, our studies shed light on the cellular and temporal dynamics underlying GITR-L-GITR interactions and their role in reversal of Treg suppression. The natural ligand for GITR controls the activity of Treg cells and activated Th cells. To determine whether GITRT-L governs immune self-tolerance through regulation of Th cell proliferation, in vivo studies will be required. These factors must be borne in mind, if the GITR-L/GITR pathway of signal transduction is to become a target for therapeutic intervention in vaccine development and autoimmune disorders.