Cutting Edge: The Common γ-Chain Is an Indispensable Subunit of the IL-21 Receptor Complex

Cells and Abs

Cells used in this paper were of the human T cell leukemia virus-I-transformed human T cell line ED40515⁻ (20), which lacks expression of γ_c, and ED40515⁻-derived transfectants, EDγ-15, EDγ-16, EDtSHγ-4, and ED-AV-7R.cl2 as reported previously (8, 21, 22). EDγ-15 and EDγ-16 cells express wild-type human γ_c. EDtSHγ-4 and ED-AV-7R.cl2 cells express a mutant γ_c deleted of C-terminal 50 aa and a mutant γ_c with one amino acid substitution at the extracellular region (from Ala¹⁵⁶ to Val), respectively; both of them were derived from XSCID patients. These cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, penicillin, and streptomycin.

An anti-human IL-2R γ-chain mAb (TUGh4) was described previously (23). Anti-Janus kinase (JAK)1, -JAK2, -JAK3, -Tyk2, -STAT1, -STAT3, and -STAT5 polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific polyclonal Abs, anti-phospho-STAT1 (Y701), -STAT3 (Y705), and -STAT5 (Y694), and P-Tyr-100, which is an anti-phosphotyrosine monoclonal Ab, were purchased from Cell Signaling Technology (Beverly, MA). HRP-labeled anti-mouse IgG and HRP-labeled anti-rabbit IgG Abs used as secondary Abs were purchased from KPL (Gaithersburg, MD).

Immunofluorescence staining and binding of IL-21 to ED40515⁻ and EDγ-16 cells

Immunofluorescence staining of cells with biotin-conjugated TUGh4 was conducted as described previously (8). Recombinant human IL-21 was conjugated to N-hydroxysulfosuccinimide-long chain-biotin (Pierce, Rockford, IL). Cells were incubated with 100 ng/ml biotin-conjugated IL-21 for 60 min at 4°C. After washing, the cells were incubated with streptavidin-PE (BD Biosciences, San Jose, CA) for 20 min at 4°C to be visualized. The cells were then subjected to a flow cytometer (FACSCalibur; BD Immunocytometry Systems, Mountain View, CA). Incubation with 2 μg/ml unlabeled IL-21 before the addition of the biotinylated IL-21 provided the background control staining.

Immunoprecipitation and immunoblot assay

Immunoprecipitation and immunoblot assay were described previously (22, 24). Cells were stimulated with 10 ng/ml IL-21 or 1 nM IL-2 for 10 min for immunoprecipitation of the JAKs and for 30 and 60 min for phosphospecific immunoblot assay of the STATs.

EMSA

EMSA was conducted as described previously (24). Double-stranded ³²P-labeled M67-SIE sequence (5′-GTGCATTTCCCGTAAATCTTGTCTACAATTC-3′) and β-casein-SIE sequence (5′-TGTGGACTTCTTGGAAT TAAGGGACTTTTG-3′) were used as probes for STAT1/3 and STAT5, respectively.

Cross-linking experiment

Cells were incubated with 100 ng/ml of biotinylated IL-21 or IL-2 for 1 h on ice. Cells were washed with ice-cold PBS and then suspended in ice-cold cross-linking buffer (1 mM MgCl₂-PBS pH 8.3). Disuccinimidyl suberate (DSS; Pierce) was added at a concentration of 100 μg/ml followed by another 30-min incubation on ice. Cells were harvested and lysed for immunoprecipitation as described above. HRP-conjugated streptavidin (Amersham Pharmacia Biotech, Little Chalfont, U.K.) was used for the detection of biotinylated IL-21 or IL-2.

IL-21 binds to ED40515⁻ cell line negative for γ_c

To investigate the possible involvement of γ_c in formation of the IL-21R complex, we first tested IL-21R expression on the ED40515⁻ cell line, which is reportedly defective for γ_c expression on cell surfaces (20). We conducted flow cytometry on this cell line using biotinylated human IL-21. ED40515⁻ cells and also EDγ-16 cells, which stably express the exogenous wild-type γ_c, bind IL-21 (Fig. 1⇓, bottom). The specificity of biotinylated IL-21 binding to its receptor was controlled by a blocking experiment with an excess of unlabeled IL-21. These results suggested that IL-21 can bind to its receptor without γ_c. RT-PCR showed the expression of IL-21R mRNA in the ED40515⁻ cell line (data not shown). The expression of γ_c on each cell line is shown (Fig. 1⇓, top).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Binding of IL-21 to ED40515⁻ and EDγ-16 cells. Cells were incubated with 10 μg/ml biotin-conjugated TUGh4 (anti-human γ_c mAb) (top, thick lines) or 100 ng/ml biotin-conjugated IL-21 (bottom, thick lines) followed by streptavidin-PE and subjected to flow cytometric analysis. Thin lines represent control staining of biotinylated TUGh4 in the presence of 300 μg/ml unlabeled TUGh4 (top) or control staining of biotinylated IL-21 in the presence of 2 μg/ml unlabeled IL-21 (bottom).

IL-21 activates JAK1 and JAK3 in the presence of γ_c

Next, we examined whether IL-21 requires γ_c for its intracellular signal transduction. All the cytokines whose receptors belong to the cytokine receptor family induce activation of JAKs with various combinations among the JAK family tyrosine kinases. The IL-21R and the γ_c were reported to associate with JAK1 and JAK3, respectively (19, 21, 25, 26, 27). Hence, we examined tyrosine phosphorylation of the JAKs as a result of their activation. We immunoprecipitated JAK1, JAK2, JAK3, and Tyk2 before and after IL-21 or IL-2 stimulation and then immunoblotted with P-Tyr-100. IL-21 as well as IL-2 apparently induced phosphorylation of JAK1 in EDγ-16 cells but not in ED40515⁻ cells (Fig. 2⇓A). Phosphorylation of JAK3 was detectable in these cell lines even before stimulation, and was significantly enhanced in EDγ-16 cells after stimulation with either IL-21 or IL-2 (Fig. 2⇓B). Similar results were obtained in EDγ-15, which is another cell line expressing the exogenous wild-type γ_c (data not shown). These data clearly demonstrate that IL-21 absolutely requires the γ_c for its signal transduction. The constitutive tyrosine phosphorylation of JAK3 was previously reported with other human T cell leukemia virus-I-infected cell lines (28). Phosphorylation of Tyk2 was undetectable, and we detected weak phosphorylation of JAK2 in EDγ-16 cells upon stimulation with either IL-21 or IL-2. Because the expression level of JAK2 was quite low, the weak bands may reflect physiologically significant activation of JAK2.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

IL-21 induces JAK1 and JAK3 activation only in the presence of γ_c. ED40515⁻ and EDγ-16 cells were stimulated with either IL-2 or IL-21 as indicated for 10 min. The cell lysates were immunoprecipitated with anti-JAK1, anti-JAK2 (A), anti-JAK3, or anti-Tyk2 (B) Abs. Phosphotyrosine was detected by P-Tyr-100 (top). The filters were reprobed with the anti-JAK1, -JAK2, -JAK3, or -Tyk2 Ab, respectively, to determine the amount of JAKs in each lane (bottom). The positions of each JAK are indicated as arrows.

IL-21 induced activation of STATs in EDγ-16 cells but not in ED40515⁻ cells

STATs are also phosphorylated and activated for their transcriptional activities upon cytokine stimulation (29). Hence, we examined IL-21-mediated phosphorylation of STAT1, STAT3, and STAT5 with EDγ-16 and ED40515⁻ cells. The cells were stimulated with IL-21, and their extracts were then immunoblotted with Abs specific for tyrosine-phosphorylated STAT1, STAT3, and STAT5. IL-21 as well as IL-2 clearly induced tyrosine phosphorylation of STAT1 in EDγ-16 cells but not in ED40515⁻ cells (Fig. 3⇓A). Phosphorylation of STAT3 was detectable in these cell lines even before stimulation, and was significantly enhanced in EDγ-16 cells after stimulation with either IL-21 or IL-2 (Fig. 3⇓B). Although IL-2 induced significant tyrosine phosphorylation of STAT5, IL-21 produced a weak phosphorylation of STAT5 (Fig. 3⇓C). A high concentration of IL-21 (100 ng/ml) was not able to enhance the phosphorylation of STAT5 (data not shown). To confirm these results, we conducted EMSA with the EDγ-16 cell extracts. Both IL-2 and IL-21 activated STAT1 activity. The DNA binding activity of STAT3 was observed without stimulation; however, upon IL-2 AND IL-21 stimulation, the binding activity was increased (Fig. 3⇓D). Whereas IL-2 activated STAT5, IL-21 induced a weak activation of STAT5. The specificity of the bands for the STATs was verified with supershift assays using specific Abs. In the presence of anti-STAT1, -STAT3, or -STAT5, the bands almost disappeared, respectively, and formed supershifted bands in the cases of anti-STAT3 and -STAT5. These results were consistent with those of immunoblots using phosphotyrosine-specific Abs. Homodimerization of the IL-21R is reported to activate STAT5 but not STAT3 (19). This observation seems inconsistent with our present results. Although the IL-21R was reported to have a consensus motif for STAT3 binding in its C-terminal tail (18), it is not clear which tyrosine residue is responsible for the activation of STAT5. Further study will need to identify critical regions for the activation of each STAT.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

STAT activation with IL-21 in wild-type or mutant γ_c-expressing cells. ED40515⁻, EDγ-16, EDtSHγ-4, and ED-AV-7R.cl2 were stimulated with either IL-2 or IL-21 as indicated. Tyrosine-phosphorylated STATs were detected with anti-phospho STAT1 (pY701) (A), anti-phospho STAT3 (pY705) (B), and anti-phospho STAT5 (pY694) (C) Abs (top). These filters were reprobed with anti-STAT1, -STAT3, and -STAT5 Abs, respectively, to determine the amount of STAT in each lane (bottom). The positions of each STAT are indicated as arrows. Whole cell extracts of EDγ-16 cells stimulated with either IL-2 or IL-21 were incubated with ³²P-labeled M67-SIE probe (D, lanes 1–9) or ³²P-labeled β-casein-SIE probe (D, lanes 10–15) in the presence or absence of anti-STAT Abs as indicated. Supershifted bands are indicated by ∗ for STAT3 or ∗∗ for STAT5. Nonspecific bands are indicated by arrows.

Next we tested γ_c mutants for their signal-transducing activities as a component of the IL-21R complex. We used tSH and AV mutants of the γ_c, which were derived from XSCID patients whose phenotypes were a typical and a NK-positive atypical XSCID, respectively (8, 22). tSH mutant lacks C-terminal 50 aa with a result of frame shift mutation, and we reported previously that tSH mutant had no function on IL-2-induced signal transduction (8). As expected, tSH mutant also had no function on IL-21-induced STAT activation. In contrast, AV mutant has an amino acid substitution, Ala¹⁵⁶ to Val on the extracellular domain of γ_c (8). We reported that AV mutant expressing ED40515⁻ subline, ED-AV-7R.cl2, was selectively impaired in their responses to IL-4 or IL-7, although responses to IL-2 or IL-15 were relatively maintained (22). IL-21-induced STAT3 activation through AV mutant γ_c was comparable with wild-type γ_c, however, activation of STAT1 was impaired. These results suggest that IL-21 as well as IL-2 require both of the extracellular and intracytoplasmic regions of the γ_c for the signal transduction.

Sharing of the γ_c with the IL-21R complex

IL-21 was revealed to bind to ED40515⁻ cells negative for γ_c expression (Fig. 1⇑), suggesting the dispensability of the γ_c for IL-21 binding. However, because the γ_c was found to be required for the IL-21 signaling, we speculated that the γ_c is an effector subunit of the IL-21R complex. To examine this possibility, we attempted to detect IL-21 in the γ_c receptor complex. ED40515⁻ and EDγ-16 cells were incubated with biotinylated IL-21 or biotinylated IL-2 and cross-linked with a chemical cross-linker (DSS). Their lysates were immunoprecipitated with anti-γ_c mAb (TUGh4). The immunoprecipitates were then separated with SDS-PAGE and transferred to a membrane, followed by detection with HRP-labeled-streptavidin. Fig. 4⇓ shows biotinylated-IL-21-γ_c complex ∼85 kDa in EDγ-16 cells but not in ED40515⁻ cells. This molecular size is comparable with that of γ_c (64–70 kDa) plus IL-21 (15 kDa). Similar results were obtained with biotinylated IL-2. These results suggest that the IL-21R complex contains the γ_c as an essential component like the IL-2R complex. In this paper, we clearly demonstrated that γ_c is an indispensable receptor subunit for IL-21 as well as IL-2, IL-4, IL-7, IL-9, and IL-15.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Sharing of the γ_c with the IL-21R complex. ED40515⁻ and EDγ-16 cells were incubated with either biotinylated IL-2 or IL-21, respectively, for 1 h on ice. These ligands were chemically cross-linked to the receptors with DSS as described in Materials and Methods. The cell lysates were immunoprecipitated with (γ) or without (C) anti-γ_c Ab. Biotinylated-IL-2 or IL-21-associated γ_c was detected with HRP-conjugated streptavidin. The position of biotinylated IL-2- or IL-21-associated γ_c is indicated by the arrow.