IFN- Suppresses STAT6 Phosphorylation by Inhibiting Its Recruitment to the IL-4 Receptor
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免疫学杂志 2005年第3期
Abstract
Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN- played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN- stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN- receptor knockout (IFNGR–/–) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR–/– Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (mev/mev) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR–/– Th1 cells. Our results suggest that IFN- may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.
Introduction
Thelper type 1 cells are central immune components in combating invasion of intracellular pathogens. They help CD8+ T cells to become killer T cells and B cells to produce Abs through their cytokine production, such as IFN-, IL-2, TNF-, and TNF- (lymph toxin). In contrast, uncontrolled Th1 response can cause autoimmune disease (1, 2). Dysregulated Th1 cells found in autoimmune diseases display highly polarized phenotype. Their cytokine-producing profiles remain unchanged even after exposure to Th2-inducing conditions. It is still unsolved how Th1 cells become insensitive to Th2-inducing conditions.
Previously, we reported that committed Th1 cells were insensitive to Th2-inducing conditions; they exhibit strikingly diminished STAT6 phosphorylation in response to IL-4 stimulation (3). IL-4 is the major determinant in directing naive CD4+ T cells to differentiate into Th2 cells (4, 5, 6, 7). It mediates its function by binding and stimulating the IL-4R -chain (8, 9). Ligand-bound IL-4R -chain recruits common -chain and its associated kinase Jak3 (10, 11). Jak3 and IL-4R -chain-associated Jak1 then phosphorylate each other and become activated (12, 13, 14). Activated Jak1 and Jak3 phosphorylate the IL-4R on several tyrosine residues located in the C-terminal portion (15). The phosphorylated IL-4R serve as docking sites to recruit STAT6 as a substrate for activated Jak1 and Jak3 (16). Activation of STAT6 is essential for the induction of GATA3 and c-maf expression that control Th2 polarization (17, 18, 19, 20, 21). Thus, the Jak-STAT6 pathway becomes a logical target to be rendered insensitive to IL-4 stimulation.
We previously showed that Th1 cells that lacked IFN- or IFN- responsiveness retained the ability to respond to IL-4 (22), suggesting that IFN- plays an important role in Th1 cell commitment. Two classes of molecules, Src homology 2 (SH2)3 domain-containing protein tyrosine phosphatase and suppressor of cytokine signaling (SOCS), have been shown to inhibit IL-4-induced STAT6 phosphorylation in various cell types (23, 24, 25, 26). SH2-containing phosphatase (SHP-1) dephosphorylates STAT6 in B cells, macrophages, and fibroblast cells (27, 28, 29). SOCS1 inhibits STAT6 phosphorylation in monocytes, liver cells, and B cells (30, 31, 32). SOCS5 was shown to suppress STAT6 phosphorylation in Th1 cells (33). Whether IFN- can suppress STAT6 phosphorylation in committed CD4+ Th1 cells through the known classes of inhibitors is not clear.
In this study, we analyzed the role of known STAT6 phosphorylation inhibitors in both wild-type (WT) and IFN- receptor knockout (IFNGR–/–) Th1 cells and we did not find evidence to support that these known inhibitors mediated the inhibition of STAT6 phosphorylation. Rather, by using a newly developed pulldown assay, we observed that STAT6 from WT Th1 cells bound less effectively to the phosphorylated IL-4R than that from IFNGR–/– Th1 cells. These results suggest that IFN- mediates inhibition of STAT6 phosphorylation through a novel mechanism.
Materials and Methods
Animals and cell cultures
Mice bearing transgenic TCR- and - chains specific for pigeon cytochrome c peptide 88–104 in association with the I-Ek class II molecule on C57BL/6 background (5CC7) were purchased from The Jackson Laboratory. The 5CC7 mice were crossed to IFN- receptor-deficient mice on C57BL/6 background (from The Jackson Laboratory) to generate 5CC7 IFN- receptor-deficient mice (IFNGR–/–) on C57BL/6 background. Naive CD4+ T cells were prepared, as described elsewhere (22). These cells (1 x 106) were stimulated with 1 μm of cytochrome c peptide (prepared by Alpha Diagnostic International) and 5 x 106 of I-Ek-expressing fibroblast cells (PI-39), irradiated with 2500 rad of gamma-ray, in 10 ml of complete RPMI 1640 medium supplemented with IL-2 (10 U/ml), IL-12 (10 ng/ml), anti-CD28 Ab (3 μg/ml, prepared from hybridoma 37.N.51.1, and provided by J. Allison (University of California, Berkeley, CA)), and anti-IL-4 Ab (11B11; 10 μg/ml) for 3–11 days. The PI-39 cells were provided by R. Germain (National Institutes of Health, Bethesda, MD).
For preparing Th1 cells that were deficient in SHP-1 activity, C57BL/6J-Hcphme-v/Hcphme-v (viable motheaten (mev/mev)) and littermate control (+/–) mice, maintained in The Jackson Laboratory and shipped to Loyola Medical Center animal facility 2 wk before experiments, were used. For priming of Th1 cells, primary stimulation of CD4+ T cells was conducted by culturing 106 CD4+ T cells in the presence of 107 irradiated T-depleted spleen cells from +/– mice with anti-CD3 (2C11; 3 μg/ml), anti-CD28 (37.N.51.1; 3 μg/ml), anti-IL-4 Ab (11B11; 10 μg/ml), IL-2 (10 U/ml), and IL-12 (10 ng/ml; BD Pharmingen) for 7 days. Handling of animals complied with the animal protocol approved by Institute Animal Care and User Committee of Loyola University Medical School.
Immunoprecipitation and Western blotting
To prepare whole cell lysates, cells (1 x 107 for immunoprecipitating STAT6; 5 x 107 for immunoprecipitating IL-4R -chain) were lysed with 0.5 ml of lysis buffer (50 mM HEPES, 0.5% Nonidet P-40, or 1% Brij 97, 5 mM EDTA, 50 mM NaCl, 10 mM Na pyrophosphate, 50 mM NaF) freshly supplemented with 1 mM Na3VO4, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin or complete protease inhibitor tablet (Boehringer Mannheim). To prepare cytosolic lysates, cells (5 x 107) were lysed in 0.5 ml of hypotonic buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 0.2% Nonidet P-40). After incubation on ice for 10 min, lysates were spun down at 1500 rpm for 1 min. Supernatants were collected as cytosolic lysates. For immunoprecipitation, lysates were incubated with 5–10 μg of Abs for 2 h. Immunoprecipitation and Western blot were conducted, as described previously (3).
Northern blot analysis
Total RNA (20 μg) isolated by the guanidinium method was separated in a 1% agarose-formaldehyde gel and transferred onto a nitrocellulose membrane (Nytran; Schleicher & Schuell Microscience). A probe containing a fragment of SOCS1 or SOCS3 cDNA (provided by D. Hilton, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) was labeled with [32P]dCTP by random primer method.
IL-4R -chain staining
Cells (1 x 106) were incubated with 10% goat serum for 5 min to block nonspecific binding. For selected samples, 5 ng/ml IL-4 (BD Pharmingen) was added and incubated on ice for 30 min to inhibit the binding of anti-IL-4R -chain Ab (M1; provided by Amgen) to IL-4R -chain. M1 mAb or a rat isotype control Ig (R&D Systems) were then added to the cells and incubated for 20 min in FACS buffer (3% FCS, 0.1% sodium azide, 1x PBS). Cells were washed with FACS buffer before they were incubated with biotinylated goat Fab against rat IgG (Southern Biotechnology Associates) for 20 min. After wash, samples were incubated with FITC-labeled streptavidin. The stained cells were analyzed by FACS.
Preparation and phosphorylation of IL-4R-GST fusion protein
cDNA encoding the C-terminal portion of the mouse IL-4R -chain (aa 258–810) was ligated in-frame into a GST gene fusion vector (pGEX-6p-2; Amersham Biosciences) to create a IL-4R/GST fusion protein (4RC). To prepare phosphorylated 4RC (4RC-P), 4RC, pATH-v-Abl plasmids (a gift from K. Shuai, University of California, Los Angeles, CA) were cotransformed into Escherichia coli (strain HB101) as described (34). The transformed bacteria were selected on Luria broth plates containing 100 μg/ml ampicillin, 25 μg/ml tetracycline, and 100 μg/ml tryptophan. The selected bacteria were grown overnight in 100 ml of Luria broth culture medium supplemented with ampicillin (100 μg/ml), tetracycline (25 μg/ml), and tryptophan (100 μg/ml). The overnight-grown bacterial culture was expanded into 1 L by adding 900 ml of the same medium and further grew at 37°C for 3 h. Then, 0.2 mM isopropylthio--D-galactoside was added to the cultures, which were incubated at 30°C for another 1.5 h. The cells were collected and resuspended in 1 L of modified M9 medium (1x M9 salts, 0.5% (w/v) casamino acids (Sigma-Aldrich A-2427), 0.1 mM CaCl2, 0.2% glucose, 10 μg/ml thiamine B1) in the presence of isopropylthio--D-galactoside, but no tryptophan. The cell suspension was incubated at 30°C for 1.5 h to induce the expression of the TrpE-v-Abl fusion protein. The 4RC- and v-Abl-expressing cells were harvested, resuspended in 20 ml of ice-cold lysis buffer (PBS, 50 mM EDTA, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin), and sonicated in Sonic Dismembrator Model 60 (Fisher Scientific) at 50% output for 2 min with 30-s intervals every minute. Cell lysates were incubated with 0.5 ml of glutathione Sepharose beads (Amersham Biosciences) at room temperature for 30 min. After extensive wash with PBS, the beads were resuspended in 0.5 ml of lysis buffer and stored at –70°C for later use.
GST pulldown assay
The 4RC-P or unphosphorylated 4RC was expressed in E. coli (strain HB101) and purified with glutathione Sepharose beads. Phosphorylated or unphosphorylated fusion proteins (prepared from 100 ml of bacterial culture) were incubated with 0.5 ml of cell lysates at 4°C for time indicated. After extensive wash, proteins were eluted in 35 μl of 2x SDS loading buffer. A total of 10 μl of the eluted protein was loaded for SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore) for Western blot. The amounts of STAT6 and Jak1 bound to the fusion protein were detected with anti-STAT6 and anti-Jak1 Ab, respectively.
Results
IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells
Previously, we demonstrated that well-differentiated Th1 cells showed impaired STAT6 phosphorylation (3). More recently, we reported that IFN- played an essential role in stabilizing Th1 phenotype (22). However, whether IFN- stabilizes Th1 phenotype by suppressing STAT6 phosphorylation remains unclear. To determine whether IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells, we generated IFNGR and STAT6 double-knockout mice (DKO). We found that DKO Th1 cells failed to differentiate into IL-4-producing cells, as measured by both ELISA and intracellular staining (Fig. 1). These results demonstrated that elimination of STAT6 stabilized IFNGR–/– Th1 cell phenotype, suggesting that down-regulation of STAT6 phosphorylation by IFN- in Th1 cells can be an effective way for Th1 cells to achieve a stable phenotype.
FIGURE 1. IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells. Naive CD4+ T cells from WT, IFNGR–/–, IFNGR, and STAT6 double-knockout (DKO) mice were primed under Th1- or Th2-inducing conditions for 11 days. Differentiated Th1 cells were primed under Th1- or Th2-inducing conditions for an additional 7 days (Th1–2). A, The resultant cells were harvested, washed, and stimulated with PMA/ionomycin overnight in the presence of IL-2. IL-4 production in the supernatants from the overnight stimulated cells was detected by ELISA. B, The resultant cells were harvested, washed, and stimulated with PMA/ionomycin for 6 h in the presence of IL-2 and monensin. IL-4 content in the stimulated cells was detected by intracellular staining and analyzed by FACS.
IFN- inhibits STAT6 phosphorylation in differentiated Th1 cells
To determine whether IFN- suppresses STAT6 phosphorylation in committed Th1 cells, we examined STAT6 phosphorylation in differentiating and differentiated WT and IFNGR–/– Th1 cells. We found that IL-4 induced robust STAT6 phosphorylation in freshly isolated naive CD4+ T cells. After 3–7 days of differentiation under Th1-inducing conditions, both WT and IFNGR–/– Th1 cells showed significantly reduced levels of STAT6 phosphorylation. After 11 days of differentiation under Th1-inducing conditions, STAT6 phosphorylation in WT Th1 cells diminished even more dramatically, which concurred with the timing of Th1 cell commitment. In contrast, STAT6 phosphorylation in IFNGR–/– Th1 cells was restored to the levels comparable to that of naive CD4+ cells (Fig. 2). These biphasic changes in STAT6 phosphorylation during the course of Th1 cell differentiation suggest that the diminution of STAT6 phosphorylation in differentiated Th1 cells may be controlled by two processes. The first phase may be mediated by TCR stimulation, and the second phase may be mediated by IFN-. The data also suggest that IFN- may mediate Th1 cell commitment by inhibiting STAT6 signaling.
FIGURE 2. STAT6 phosphorylation in differentiated WT Th1 cells is inhibited. Naive CD4+ T cells were prepared from WT and IFNGR–/– mice and primed under Th1-inducing conditions for the time indicated. At the end of each culture period, cells were washed and not stimulated or stimulated with IL-4 (5 ng/ml) at room temperature for 10 min. Whole cell lysates were prepared and analyzed for STAT6 amount and phosphorylation by using anti-phosphotyrosine Ab (anti-PY) and anti-STAT6 Abs (anti-STAT6), respectively.
Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells occurs in the cytoplasm
We focused on IFN--mediated inhibition of STAT6 phosphorylation because the timing of inhibition concurred with that of Th1 cell commitment (i.e., Th1 cells resist to Th2-inducing conditions). Inhibition of STAT6 phosphorylation could occur in the cytoplasm or nucleus. To determine this, we cultured WT and IFNGR–/– Th1 cells for 11 days (day 11 WT and IFNGR–/– Th1 cells) and analyzed STAT6 phosphorylation in the cytosolic fraction. We noticed that STAT6 phosphorylation in WT Th1 cells increased only moderately after 1 min of IL-4 stimulation. The STAT6 phosphorylation did not enhance significantly even with longer IL-4 stimulation (Fig. 3). By contrast, STAT6 phosphorylation in differentiated IFNGR–/– Th1 cells markedly elevated after 1 min of IL-4 stimulation, reached peak levels after 5 min, and maintained at high levels even after 30 min of stimulation (Fig. 3). These data suggest that IFN- may inhibit STAT6 phosphorylation in the initiation stage of IL-4R signaling in differentiated WT Th1 cells.
FIGURE 3. Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells occurs in the initiation step of the IL-4R signaling. Naive CD4+ T cells prepared from WT and IFNGR–/– mice were cultured under Th1-inducing conditions for 11 days (day 11). The day 11 WT and IFNGR–/– Th1 cells were not stimulated or stimulated with IL-4 (5 ng/ml) for 1–30 min, as indicated. Cytosolic lysates were prepared, and STAT6 phosphorylation and amount were analyzed.
Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1
To determine whether known inhibitors mediate the inhibition of STAT6 phosphorylation, we examined mRNA and protein expression of SOCS1 and SOCS3. We did not observe significant difference in SOCS1 and SOCS3 expressions between differentiated WT and IFNGR–/– Th1 cells (Fig. 4A). Another known inhibitor, SOCS5, also did not show significant difference in its protein expression between WT Th1 cells and IFNGR–/– Th1 cells (Fig. 4A). SHP-1 is known to suppress STAT6 phosphorylation. However, mev/mev Th1 cells that lack SHP-1 activity did not show enhancement of IL-4-induced STAT6 phosphorylation (Fig. 4B). It was reported that SOCS and SHP-1 inhibited STAT6 phosphorylation through inhibiting their upstream Jak kinase. When we examined phosphorylation of STAT6 and Jak, to our surprise, we found that the phosphorylation of Jak1 and Jak3 was not diminished in differentiated WT Th1 cells (Fig. 4C). Furthermore, IL-4R expression or IL-4R phosphorylation in WT Th1 cells was not different from that in IFNGR–/– Th1 cells (Fig. 4, D and E). This finding is consistent with the idea that those known inhibitors do not mediate the inhibition of STAT6 phosphorylation. Our data suggest that inhibition of STAT6 phosphorylation in WT Th1 cells may involve a novel mechanism.
FIGURE 4. Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1. A, Total RNA and protein (whole cell lysate) were prepared from day 11 WT and IFNGR–/– Th1 cells. A total of 10 μg of total RNA was used for electrophoresis, and SOCS1 and SOCS3 mRNA expression was detected by Northern blot analysis (left panel). A total of 10 μg of protein was used for electrophoresis, and SOCS1, SOCS3, and SOCS5 protein expression was analyzed by Western blot with anti-SOCS1, anti-SOCS3, or anti-SOCS5 Abs (right panel). Numbers indicate the ratio of the protein amount bound to the fusion protein to the amount of the input protein, as determined by densitometry. B, Day 11 mev/mev and +/– Th1 cells were stimulated with IL-4 for 10 min. The phosphorylation of STAT6 was analyzed with anti-phosphotyrosine Ab, and the amount of STAT6 was determined with anti-STAT6 Ab. C, Day 11 WT and IFNGR–/– Th1 cells were stimulated without or with IL-4 for 10 min. Cell lysates were prepared and incubated with anti-STAT6, anti-Jak1, and anti-Jak3 Abs. Phosphorylation of STAT6, Jak1, and Jak3 was analyzed using anti-phosphotyrosine Ab (PY). D, Naive CD4+ T cells and differentiated WT and IFNGR–/– Th1 and Th2 cells were incubated with (dotted line; IL-4 competes for the binding site for M1) or without IL-4 (5 ng/ml) (filled line) for 30 min before addition of M1 anti-IL-4R -chain mAb or a rat isotype control. The stained cells were analyzed by FACS. E, Day 11 WT and IFNGR–/– Th1 cells were not stimulated or stimulated with IL-4 for the time periods indicated. The cells were lysed and analyzed for IL-4R -chain protein phosphorylation with anti-PY Ab and content with anti-IL-4R -chain Ab.
STAT6 in differentiated WT Th1 cells shows a reduced ability to bind phosphorylated IL-4R fusion protein
Because only STAT6 phosphorylation is inhibited, we hypothesized that IFN- may inhibit STAT6 phosphorylation by suppressing its recruitment to the IL-4R. To test this hypothesis, we generated a fusion protein consisting of IL-4R C-terminal and GST protein (4RC). The 4RC fusion protein was either not phosphorylated or phosphorylated (4RC-P) by v-ABL kinase (Fig. 5A). The 4RC-P, but not 4RC fusion protein pulled down STAT6 (Fig. 5B). When using 4RC-P to pull down STAT6, we detected that the amount of STAT6 bound to 4RC-P fusion protein in WT Th1 cell samples reduced 3- to 4-fold compared with that in IFNGR–/– Th1 cell lysates. In contrast, the amounts of Jak1 bound to 4RC-P fusion protein were similar in WT Th1 cells and IFNGR–/– Th1 cells (Fig. 5C).
FIGURE 5. STAT6 in differentiated WT Th1 cells shows a reduced ability to bind to the IL-4R. A, HB101 cells were transformed by the GST vector or 4RC alone or in combination with pATH-v-Abl (+Abl). The expressed proteins were purified by glutathione Sepharose beads and detected by Western blot with anti-GST, anti-IL-4R, or anti-PY Abs. B, Unphosphorylated (4RC) or phosphorylated (4RC-P) fusion proteins (purified from 100 ml of bacterial culture) were used to incubate with cell lysates prepared from spleen cells (3 x 107) at 4°C overnight. Proteins that interacted with the fusion proteins were pulled down with glutathione Sepharose beads. The amount of STAT6 and Jak1 binding to the fusion protein was detected by Western blot with anti-STAT6 Ab or anti-Jak1, respectively. The amount of fusion proteins used in the experiment was quantified by anti-IL-4R Ab. C, Cell lysates prepared from day 11 WT and IFNGR–/– Th1 cells (50 x 106) were incubated with 4RC-P fusion protein at 4°C for 2 and 5 h. The amount of STAT6 and Jak1 being pulled down by the fusion proteins was detected by Western blot with anti-STAT6 and anti-Jak1 Abs, respectively. The amounts of fusion proteins used in the experiment were quantified by Western blot with anti-IL-4R Ab. Numbers indicate the ratio of the protein amount bound to the fusion proteins to the amount of the input protein as determined by densitometry. D, A total of 20 μg of protein from day 11 differentiated WT and IFNGR–/– Th1 and Th2 cells was used for electrophoresis. The amount of SOCS5 and STAT6 was detected by Western blot with anti-SOCS5 or anti-STAT6 Abs, respectively. Numbers indicate the density of each band measured by densitometry.
It has been reported that, compared with WT Th2 cells, WT Th1 cells showed impaired Jak1 phosphorylation and impaired recruitment of Jak1 to the IL-4R (3, 29). To find out how the amounts of Jak1-bound 4RC-P fusion protein in Th1 samples compare with that in Th2 cell samples, we analyzed the binding of Jak1 in Th2 samples. Consistent with previous reports, we showed that the amounts of Jak1 bound to 4RC-P fusion protein in both WT Th1 cells and IFNGR–/– Th1 cell lysates were several-fold less than that in Th2 cell lysates (Fig. 5C). This result raised the possibility that down-regulation of Jak1 binding to the IL-4R can be controlled by an IFN- signaling-independent pathway. To determine whether reduced Jak1 binding to the IL-4R in WT Th1 cells and IFNGR–/– Th1 cells was due to elevated levels of SOCS5, we detected SOCS5 protein expression by Western blot. We showed that Th1 cells contained 4-fold less of SOCS5 protein than Th2 cells (Fig. 5D). Together, these results suggest that down-regulation of Jak1 alone is not sufficient to impair STAT6 phosphorylation to the degree such that Th1 cells become insensitive to IL-4, and that further down-regulation of STAT6 binding to the IL-4R mediated by IFN- may be needed to achieve a state of unresponsiveness to the IL-4 stimulation.
Discussion
In this study, we sought to investigate the mechanism by which IFN- inhibits STAT6 phosphorylation to stabilize Th1 cell phenotype. By analyzing STAT6 phosphorylation and binding of STAT6 to the 4RC-P during Th1 cell development, we demonstrated that IFN- inhibited STAT6 phosphorylation by preventing the recruitment of STAT6 to the IL-4R signaling complex. These findings represent a novel mechanism that negatively regulates STAT6 signaling.
We observed that there existed two phases in which STAT6 phosphorylation was inhibited. These two phases of inhibition may involve two separate mechanisms: a TCR-mediated mechanism and an IFN--mediated mechanism. In the first week of Th1 cell differentiation, we demonstrated that STAT6 phosphorylation was diminished in both WT and IFNGR–/– Th1 cells. This phase of down-regulation might be mediated by TCR stimulation. Previous studies demonstrated that TCR engagement caused down-regulation of STAT6 phosphorylation through activating both protein kinase C-MAPK and calcineurin pathways (35). In the second phase of inhibition (TCR signaling had weakened significantly 11 days after the initial stimulation), STAT6 phosphorylation in the absence of IFN- signaling was restored to the levels comparable to that of naive CD4 T cells, whereas the reduction in STAT6 phosphorylation in WT Th1 cells was maintained. These observations suggest that IFN- produced by Th1 cells may play an important role in suppressing STAT6 phosphorylation in the second phase of inhibition.
It appears that IFN- down-regulates STAT6 phosphorylation in differentiated Th1 cells through a novel mechanism. There are several lines of evidence to support this idea. First, the timing, during which inhibition of STAT6 phosphorylation occurs, did not support the involvement of SOCS in IFN--mediated function. SOCS1 and SOCS3 inhibit STAT6 phosphorylation in monocytes, B cells, and fibroblast cells within a few hours of treatment with IFN- (31, 32), whereas the IFN--mediated inhibition of STAT6 phosphorylation in differentiated WT Th1 cells appears to take several days. Second, this novel mechanism appears to target only the STAT6 molecule, but not the Jak1 or Jak3 molecule. Others and we have reported Jak1 and STAT6 phosphorylation was impaired in Th1 cells (3, 33). More recently, Seki et al. (33) described a molecular mechanism that involved SOCS5 to explain the impairments. They showed that SOCS5 inhibited STAT6 phosphorylation in Th1 cells by competing with Jak1 binding to the box1 region of the IL-4R. However, the study does not explain our observation that IFNGR–/– Th1 cells possessed higher capacity than WT Th1 cells to phosphorylate their STAT6 despite that these two types of cells showed comparable Jak1 and Jak3 phosphorylation. Our findings raise a possibility that down-regulation of Jak1 and down-regulation of STAT6 in Th1 cells can involve more than one mechanism. One is an IFN--independent, but SOCS5-dependent mechanism as reported by Seki et al.; the other is an IFN--dependent, but SOCSs-independent mechanism. In fact, our data examining mRNA and protein expression of SOCS1, SOCS3, and SOCS5 support the latter mechanism.
In addition, inhibition of STAT6 phosphorylation in differentiated Th1 cells does not seem to involve another class of known inhibitors. Our study showed that SHP-1 mutation had no significant effect on STAT6 phosphorylation in differentiated Th1 cells, and SHP-2 is not known to interact with the IL-4R (36). Moreover, if the SH2 domain-containing protein tyrosine phosphatase was involved, one would expect that IL-4R phosphorylation would also be down-regulated. Our data did not show reduced IL-4R phosphorylation in WT Th1 cells.
Rather, our study provides evidence to support that IFN- suppresses STAT6 phosphorylation in differentiated WT Th1 cells by inhibiting its recruitment to the IL-4R, although the mechanism is still unknown. One possibility could be that IFN- signaling might cause posttranslational modification of STAT6, probably by serine/threonine phosphorylation or arginine methylation. Such modification may cause STAT6 conformational changes and consequently impair its binding to the IL-4R. Another possibility could be that IFN- might induce an undefined STAT6-binding protein. The binding of IFN--induced protein to STAT6 may prevent its recruitment to the IL-4R. Recent studies support both ideas. Chen et al. (37) demonstrated that unmethylated STAT6 showed reduced phosphorylation induced by IL-4. Daines et al. (38) showed that STAT6-binding protein existed in cytosolic plasma and blocked its DNA-binding activity. How IFN- reduces STAT6 recruitment to the IL-4R in differentiated Th1 cells would be an important topic that requires further investigation.
Acknowledgments
We thank Dr. Ke Shuai for providing us with pATH-v-Abl plasmid, Dr. Douglas Hilton for SOCS1 and SOCS3 probes, Dr. Leonard D. Shultz for mev/mev mice, and Dr. Ronald Germain for PI-39 cell lines. We thank Yan Su for assistance in the initial phase of this work.
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 study is supported by a grant from the Schweppe Foundation and a RO1 grant (A1 AI 48568) from the National Institutes of Health (to H.H.).
2 Address correspondence and reprint requests to Dr. Hua Huang, Department of Cell Biology, Stritch School of Medicine, Loyola University Chicago, Building 102, Room 5657, 2160 South First Avenue, Maywood, IL 60153. E-mail address: hhuang@lumc.edu
3 Abbreviations used in this paper: SH2, Src homology 2; 4RC, IL-4R/GST fusion protein; 4RC-P, phosphorylated 4RC; SHP-1, SH2-containing phosphatase; SOCS, suppressor of cytokine signaling; WT, wild type.
Received for publication March 25, 2004. Accepted for publication November 17, 2004.
References
De Carli, M., M. M. D’Elios, G. Zancuoghi, S. Romagnani, G. Del Prete. 1994. Human Th1 and Th2 cells: functional properties, regulation of development and role in autoimmunity. Autoimmunity 18:301.
Romagnani, S.. 1996. Development of Th 1- or Th 2-dominated immune responses: what about the polarizing signals?. Int. J. Clin. Lab. Res. 26:83.
Huang, H., W. E. Paul. 1998. Impaired interleukin 4 signaling in T helper type 1 cells. J. Exp. Med. 187:1305.
Swain, S. L., A. D. Weinberg, M. English, G. Huston. 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3796.
Seder, R. A., W. E. Paul, M. M. Davis, B. Fazekas de St. Groth. 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176:1091.
Le Gros, G., S. Z. Ben-Sasson, R. Seder, F. D. Finkelman, W. E. Paul. 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J. Exp. Med. 172:921.
Hsieh, C. S., A. B. Heimberger, J. S. Gold, A. O’Garra, K. M. Murphy. 1992. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.
Ohara, J., W. E. Paul. 1987. Receptors for B-cell stimulatory factor-1 expressed on cells of haematopoietic lineage. Nature 325:537.
Park, L. S., D. Friend, H. M. Sassenfeld, D. L. Urdal. 1987. Characterization of the human B cell stimulatory factor 1 receptor. J. Exp. Med. 166:476.
Russell, S. M., A. D. Keegan, N. Harada, Y. Nakamura, M. Noguchi, P. Leland, M. C. Friedmann, A. Miyajima, R. K. Puri, W. E. Paul, et al 1993. Interleukin-2 receptor chain: a functional component of the interleukin-4 receptor. Science 262:1880.
Kondo, M., T. Takeshita, N. Ishii, M. Nakamura, S. Watanabe, K. Arai, K. Sugamura. 1993. Sharing of the interleukin-2 (IL-2) receptor chain between receptors for IL-2 and IL-4. Science 262:1874.
Witthuhn, B. A., O. Silvennoinen, O. Miura, K. S. Lai, C. Cwik, E. T. Liu, J. N. Ihle. 1994. Involvement of the Jak-3 Janus kinase in signalling by interleukins 2 and 4 in lymphoid and myeloid cells. Nature 370:153.
Ihle, J. N.. 1995. The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv. Immunol. 60:1.
Johnston, J. A., M. Kawamura, R. A. Kirken, Y. Q. Chen, T. B. Blake, K. Shibuya, J. R. Ortaldo, D. W. McVicar, J. J. O’Shea. 1994. Phosphorylation and activation of the Jak-3 Janus kinase in response to interleukin-2. Nature 370:151.
Ryan, J. J., L. J. McReynolds, A. Keegan, L. H. Wang, E. Garfein, P. Rothman, K. Nelms, W. E. Paul. 1996. Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor. Immunity 4:123.
Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, S. L. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701
Zheng, W., R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.
Ho, I. C., D. Lo, L. H. Glimcher. 1998. c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms. J. Exp. Med. 188:1859.
Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for the development of Th2 cells. Immunity 4:313.
Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.
Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.
Zhang, Y., R. Apilado, J. Coleman, S. Ben-Sasson, S. Tsang, J. Hu-Li, W. E. Paul, H. Huang. 2001. Interferon stabilizes the T helper cell type 1 phenotype. J. Exp. Med. 194:165.
Wormald, S., D. J. Hilton. 2004. Inhibitors of cytokine signal transduction. J. Biol. Chem. 279:821.
Krebs, D. L., D. J. Hilton. 2001. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19:378.
Naka, T., M. Narazaki, M. Hirata, T. Matsumoto, S. Minamoto, A. Aono, N. Nishimoto, T. Kajita, T. Taga, K. Yoshizaki, et al 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924.
Masuhara, M., H. Sakamoto, A. Matsumoto, R. Suzuki, H. Yasukawa, K. Mitsui, T. Wakioka, S. Tanimura, A. Sasaki, H. Misawa, et al 1997. Cloning and characterization of novel CIS family genes. Biochem. Biophys. Res. Commun. 239:439
Hanson, E. M., H. Dickensheets, C. K. Qu, R. P. Donnelly, A. D. Keegan. 2003. Regulation of the dephosphorylation of Stat6: participation of Tyr-713 in the interleukin-4 receptor , the tyrosine phosphatase SHP-1, and the proteasome. J. Biol. Chem. 278:3903.
Haque, S. J., P. Harbor, M. Tabrizi, T. Yi, B. R. Williams. 1998. Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction. J. Biol. Chem. 273:33893.
Kamata, T., M. Yamashita, M. Kimura, K. Murata, M. Inami, C. Shimizu, K. Sugaya, C. R. Wang, M. Taniguchi, T. Nakayama. 2003. src homology 2 domain-containing tyrosine phosphatase SHP-1 controls the development of allergic airway inflammation. J. Clin. Invest. 111:109.
Dickensheets, H. L., C. Venkataraman, U. Schindler, R. P. Donnelly. 1999. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc. Natl. Acad. Sci. USA 96:10800.
Losman, J. A., X. P. Chen, D. Hilton, P. Rothman. 1999. Cutting edge: SOCS-1 is a potent inhibitor of IL-4 signal transduction. J. Immunol. 162:3770.(Zan Huang, Junping Xin, J)
Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN- played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN- stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN- receptor knockout (IFNGR–/–) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR–/– Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (mev/mev) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR–/– Th1 cells. Our results suggest that IFN- may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.
Introduction
Thelper type 1 cells are central immune components in combating invasion of intracellular pathogens. They help CD8+ T cells to become killer T cells and B cells to produce Abs through their cytokine production, such as IFN-, IL-2, TNF-, and TNF- (lymph toxin). In contrast, uncontrolled Th1 response can cause autoimmune disease (1, 2). Dysregulated Th1 cells found in autoimmune diseases display highly polarized phenotype. Their cytokine-producing profiles remain unchanged even after exposure to Th2-inducing conditions. It is still unsolved how Th1 cells become insensitive to Th2-inducing conditions.
Previously, we reported that committed Th1 cells were insensitive to Th2-inducing conditions; they exhibit strikingly diminished STAT6 phosphorylation in response to IL-4 stimulation (3). IL-4 is the major determinant in directing naive CD4+ T cells to differentiate into Th2 cells (4, 5, 6, 7). It mediates its function by binding and stimulating the IL-4R -chain (8, 9). Ligand-bound IL-4R -chain recruits common -chain and its associated kinase Jak3 (10, 11). Jak3 and IL-4R -chain-associated Jak1 then phosphorylate each other and become activated (12, 13, 14). Activated Jak1 and Jak3 phosphorylate the IL-4R on several tyrosine residues located in the C-terminal portion (15). The phosphorylated IL-4R serve as docking sites to recruit STAT6 as a substrate for activated Jak1 and Jak3 (16). Activation of STAT6 is essential for the induction of GATA3 and c-maf expression that control Th2 polarization (17, 18, 19, 20, 21). Thus, the Jak-STAT6 pathway becomes a logical target to be rendered insensitive to IL-4 stimulation.
We previously showed that Th1 cells that lacked IFN- or IFN- responsiveness retained the ability to respond to IL-4 (22), suggesting that IFN- plays an important role in Th1 cell commitment. Two classes of molecules, Src homology 2 (SH2)3 domain-containing protein tyrosine phosphatase and suppressor of cytokine signaling (SOCS), have been shown to inhibit IL-4-induced STAT6 phosphorylation in various cell types (23, 24, 25, 26). SH2-containing phosphatase (SHP-1) dephosphorylates STAT6 in B cells, macrophages, and fibroblast cells (27, 28, 29). SOCS1 inhibits STAT6 phosphorylation in monocytes, liver cells, and B cells (30, 31, 32). SOCS5 was shown to suppress STAT6 phosphorylation in Th1 cells (33). Whether IFN- can suppress STAT6 phosphorylation in committed CD4+ Th1 cells through the known classes of inhibitors is not clear.
In this study, we analyzed the role of known STAT6 phosphorylation inhibitors in both wild-type (WT) and IFN- receptor knockout (IFNGR–/–) Th1 cells and we did not find evidence to support that these known inhibitors mediated the inhibition of STAT6 phosphorylation. Rather, by using a newly developed pulldown assay, we observed that STAT6 from WT Th1 cells bound less effectively to the phosphorylated IL-4R than that from IFNGR–/– Th1 cells. These results suggest that IFN- mediates inhibition of STAT6 phosphorylation through a novel mechanism.
Materials and Methods
Animals and cell cultures
Mice bearing transgenic TCR- and - chains specific for pigeon cytochrome c peptide 88–104 in association with the I-Ek class II molecule on C57BL/6 background (5CC7) were purchased from The Jackson Laboratory. The 5CC7 mice were crossed to IFN- receptor-deficient mice on C57BL/6 background (from The Jackson Laboratory) to generate 5CC7 IFN- receptor-deficient mice (IFNGR–/–) on C57BL/6 background. Naive CD4+ T cells were prepared, as described elsewhere (22). These cells (1 x 106) were stimulated with 1 μm of cytochrome c peptide (prepared by Alpha Diagnostic International) and 5 x 106 of I-Ek-expressing fibroblast cells (PI-39), irradiated with 2500 rad of gamma-ray, in 10 ml of complete RPMI 1640 medium supplemented with IL-2 (10 U/ml), IL-12 (10 ng/ml), anti-CD28 Ab (3 μg/ml, prepared from hybridoma 37.N.51.1, and provided by J. Allison (University of California, Berkeley, CA)), and anti-IL-4 Ab (11B11; 10 μg/ml) for 3–11 days. The PI-39 cells were provided by R. Germain (National Institutes of Health, Bethesda, MD).
For preparing Th1 cells that were deficient in SHP-1 activity, C57BL/6J-Hcphme-v/Hcphme-v (viable motheaten (mev/mev)) and littermate control (+/–) mice, maintained in The Jackson Laboratory and shipped to Loyola Medical Center animal facility 2 wk before experiments, were used. For priming of Th1 cells, primary stimulation of CD4+ T cells was conducted by culturing 106 CD4+ T cells in the presence of 107 irradiated T-depleted spleen cells from +/– mice with anti-CD3 (2C11; 3 μg/ml), anti-CD28 (37.N.51.1; 3 μg/ml), anti-IL-4 Ab (11B11; 10 μg/ml), IL-2 (10 U/ml), and IL-12 (10 ng/ml; BD Pharmingen) for 7 days. Handling of animals complied with the animal protocol approved by Institute Animal Care and User Committee of Loyola University Medical School.
Immunoprecipitation and Western blotting
To prepare whole cell lysates, cells (1 x 107 for immunoprecipitating STAT6; 5 x 107 for immunoprecipitating IL-4R -chain) were lysed with 0.5 ml of lysis buffer (50 mM HEPES, 0.5% Nonidet P-40, or 1% Brij 97, 5 mM EDTA, 50 mM NaCl, 10 mM Na pyrophosphate, 50 mM NaF) freshly supplemented with 1 mM Na3VO4, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin or complete protease inhibitor tablet (Boehringer Mannheim). To prepare cytosolic lysates, cells (5 x 107) were lysed in 0.5 ml of hypotonic buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 0.2% Nonidet P-40). After incubation on ice for 10 min, lysates were spun down at 1500 rpm for 1 min. Supernatants were collected as cytosolic lysates. For immunoprecipitation, lysates were incubated with 5–10 μg of Abs for 2 h. Immunoprecipitation and Western blot were conducted, as described previously (3).
Northern blot analysis
Total RNA (20 μg) isolated by the guanidinium method was separated in a 1% agarose-formaldehyde gel and transferred onto a nitrocellulose membrane (Nytran; Schleicher & Schuell Microscience). A probe containing a fragment of SOCS1 or SOCS3 cDNA (provided by D. Hilton, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) was labeled with [32P]dCTP by random primer method.
IL-4R -chain staining
Cells (1 x 106) were incubated with 10% goat serum for 5 min to block nonspecific binding. For selected samples, 5 ng/ml IL-4 (BD Pharmingen) was added and incubated on ice for 30 min to inhibit the binding of anti-IL-4R -chain Ab (M1; provided by Amgen) to IL-4R -chain. M1 mAb or a rat isotype control Ig (R&D Systems) were then added to the cells and incubated for 20 min in FACS buffer (3% FCS, 0.1% sodium azide, 1x PBS). Cells were washed with FACS buffer before they were incubated with biotinylated goat Fab against rat IgG (Southern Biotechnology Associates) for 20 min. After wash, samples were incubated with FITC-labeled streptavidin. The stained cells were analyzed by FACS.
Preparation and phosphorylation of IL-4R-GST fusion protein
cDNA encoding the C-terminal portion of the mouse IL-4R -chain (aa 258–810) was ligated in-frame into a GST gene fusion vector (pGEX-6p-2; Amersham Biosciences) to create a IL-4R/GST fusion protein (4RC). To prepare phosphorylated 4RC (4RC-P), 4RC, pATH-v-Abl plasmids (a gift from K. Shuai, University of California, Los Angeles, CA) were cotransformed into Escherichia coli (strain HB101) as described (34). The transformed bacteria were selected on Luria broth plates containing 100 μg/ml ampicillin, 25 μg/ml tetracycline, and 100 μg/ml tryptophan. The selected bacteria were grown overnight in 100 ml of Luria broth culture medium supplemented with ampicillin (100 μg/ml), tetracycline (25 μg/ml), and tryptophan (100 μg/ml). The overnight-grown bacterial culture was expanded into 1 L by adding 900 ml of the same medium and further grew at 37°C for 3 h. Then, 0.2 mM isopropylthio--D-galactoside was added to the cultures, which were incubated at 30°C for another 1.5 h. The cells were collected and resuspended in 1 L of modified M9 medium (1x M9 salts, 0.5% (w/v) casamino acids (Sigma-Aldrich A-2427), 0.1 mM CaCl2, 0.2% glucose, 10 μg/ml thiamine B1) in the presence of isopropylthio--D-galactoside, but no tryptophan. The cell suspension was incubated at 30°C for 1.5 h to induce the expression of the TrpE-v-Abl fusion protein. The 4RC- and v-Abl-expressing cells were harvested, resuspended in 20 ml of ice-cold lysis buffer (PBS, 50 mM EDTA, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin), and sonicated in Sonic Dismembrator Model 60 (Fisher Scientific) at 50% output for 2 min with 30-s intervals every minute. Cell lysates were incubated with 0.5 ml of glutathione Sepharose beads (Amersham Biosciences) at room temperature for 30 min. After extensive wash with PBS, the beads were resuspended in 0.5 ml of lysis buffer and stored at –70°C for later use.
GST pulldown assay
The 4RC-P or unphosphorylated 4RC was expressed in E. coli (strain HB101) and purified with glutathione Sepharose beads. Phosphorylated or unphosphorylated fusion proteins (prepared from 100 ml of bacterial culture) were incubated with 0.5 ml of cell lysates at 4°C for time indicated. After extensive wash, proteins were eluted in 35 μl of 2x SDS loading buffer. A total of 10 μl of the eluted protein was loaded for SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore) for Western blot. The amounts of STAT6 and Jak1 bound to the fusion protein were detected with anti-STAT6 and anti-Jak1 Ab, respectively.
Results
IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells
Previously, we demonstrated that well-differentiated Th1 cells showed impaired STAT6 phosphorylation (3). More recently, we reported that IFN- played an essential role in stabilizing Th1 phenotype (22). However, whether IFN- stabilizes Th1 phenotype by suppressing STAT6 phosphorylation remains unclear. To determine whether IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells, we generated IFNGR and STAT6 double-knockout mice (DKO). We found that DKO Th1 cells failed to differentiate into IL-4-producing cells, as measured by both ELISA and intracellular staining (Fig. 1). These results demonstrated that elimination of STAT6 stabilized IFNGR–/– Th1 cell phenotype, suggesting that down-regulation of STAT6 phosphorylation by IFN- in Th1 cells can be an effective way for Th1 cells to achieve a stable phenotype.
FIGURE 1. IFNGR–/– Th1 cells depend on STAT6 to develop into IL-4-producing cells. Naive CD4+ T cells from WT, IFNGR–/–, IFNGR, and STAT6 double-knockout (DKO) mice were primed under Th1- or Th2-inducing conditions for 11 days. Differentiated Th1 cells were primed under Th1- or Th2-inducing conditions for an additional 7 days (Th1–2). A, The resultant cells were harvested, washed, and stimulated with PMA/ionomycin overnight in the presence of IL-2. IL-4 production in the supernatants from the overnight stimulated cells was detected by ELISA. B, The resultant cells were harvested, washed, and stimulated with PMA/ionomycin for 6 h in the presence of IL-2 and monensin. IL-4 content in the stimulated cells was detected by intracellular staining and analyzed by FACS.
IFN- inhibits STAT6 phosphorylation in differentiated Th1 cells
To determine whether IFN- suppresses STAT6 phosphorylation in committed Th1 cells, we examined STAT6 phosphorylation in differentiating and differentiated WT and IFNGR–/– Th1 cells. We found that IL-4 induced robust STAT6 phosphorylation in freshly isolated naive CD4+ T cells. After 3–7 days of differentiation under Th1-inducing conditions, both WT and IFNGR–/– Th1 cells showed significantly reduced levels of STAT6 phosphorylation. After 11 days of differentiation under Th1-inducing conditions, STAT6 phosphorylation in WT Th1 cells diminished even more dramatically, which concurred with the timing of Th1 cell commitment. In contrast, STAT6 phosphorylation in IFNGR–/– Th1 cells was restored to the levels comparable to that of naive CD4+ cells (Fig. 2). These biphasic changes in STAT6 phosphorylation during the course of Th1 cell differentiation suggest that the diminution of STAT6 phosphorylation in differentiated Th1 cells may be controlled by two processes. The first phase may be mediated by TCR stimulation, and the second phase may be mediated by IFN-. The data also suggest that IFN- may mediate Th1 cell commitment by inhibiting STAT6 signaling.
FIGURE 2. STAT6 phosphorylation in differentiated WT Th1 cells is inhibited. Naive CD4+ T cells were prepared from WT and IFNGR–/– mice and primed under Th1-inducing conditions for the time indicated. At the end of each culture period, cells were washed and not stimulated or stimulated with IL-4 (5 ng/ml) at room temperature for 10 min. Whole cell lysates were prepared and analyzed for STAT6 amount and phosphorylation by using anti-phosphotyrosine Ab (anti-PY) and anti-STAT6 Abs (anti-STAT6), respectively.
Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells occurs in the cytoplasm
We focused on IFN--mediated inhibition of STAT6 phosphorylation because the timing of inhibition concurred with that of Th1 cell commitment (i.e., Th1 cells resist to Th2-inducing conditions). Inhibition of STAT6 phosphorylation could occur in the cytoplasm or nucleus. To determine this, we cultured WT and IFNGR–/– Th1 cells for 11 days (day 11 WT and IFNGR–/– Th1 cells) and analyzed STAT6 phosphorylation in the cytosolic fraction. We noticed that STAT6 phosphorylation in WT Th1 cells increased only moderately after 1 min of IL-4 stimulation. The STAT6 phosphorylation did not enhance significantly even with longer IL-4 stimulation (Fig. 3). By contrast, STAT6 phosphorylation in differentiated IFNGR–/– Th1 cells markedly elevated after 1 min of IL-4 stimulation, reached peak levels after 5 min, and maintained at high levels even after 30 min of stimulation (Fig. 3). These data suggest that IFN- may inhibit STAT6 phosphorylation in the initiation stage of IL-4R signaling in differentiated WT Th1 cells.
FIGURE 3. Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells occurs in the initiation step of the IL-4R signaling. Naive CD4+ T cells prepared from WT and IFNGR–/– mice were cultured under Th1-inducing conditions for 11 days (day 11). The day 11 WT and IFNGR–/– Th1 cells were not stimulated or stimulated with IL-4 (5 ng/ml) for 1–30 min, as indicated. Cytosolic lysates were prepared, and STAT6 phosphorylation and amount were analyzed.
Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1
To determine whether known inhibitors mediate the inhibition of STAT6 phosphorylation, we examined mRNA and protein expression of SOCS1 and SOCS3. We did not observe significant difference in SOCS1 and SOCS3 expressions between differentiated WT and IFNGR–/– Th1 cells (Fig. 4A). Another known inhibitor, SOCS5, also did not show significant difference in its protein expression between WT Th1 cells and IFNGR–/– Th1 cells (Fig. 4A). SHP-1 is known to suppress STAT6 phosphorylation. However, mev/mev Th1 cells that lack SHP-1 activity did not show enhancement of IL-4-induced STAT6 phosphorylation (Fig. 4B). It was reported that SOCS and SHP-1 inhibited STAT6 phosphorylation through inhibiting their upstream Jak kinase. When we examined phosphorylation of STAT6 and Jak, to our surprise, we found that the phosphorylation of Jak1 and Jak3 was not diminished in differentiated WT Th1 cells (Fig. 4C). Furthermore, IL-4R expression or IL-4R phosphorylation in WT Th1 cells was not different from that in IFNGR–/– Th1 cells (Fig. 4, D and E). This finding is consistent with the idea that those known inhibitors do not mediate the inhibition of STAT6 phosphorylation. Our data suggest that inhibition of STAT6 phosphorylation in WT Th1 cells may involve a novel mechanism.
FIGURE 4. Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1. A, Total RNA and protein (whole cell lysate) were prepared from day 11 WT and IFNGR–/– Th1 cells. A total of 10 μg of total RNA was used for electrophoresis, and SOCS1 and SOCS3 mRNA expression was detected by Northern blot analysis (left panel). A total of 10 μg of protein was used for electrophoresis, and SOCS1, SOCS3, and SOCS5 protein expression was analyzed by Western blot with anti-SOCS1, anti-SOCS3, or anti-SOCS5 Abs (right panel). Numbers indicate the ratio of the protein amount bound to the fusion protein to the amount of the input protein, as determined by densitometry. B, Day 11 mev/mev and +/– Th1 cells were stimulated with IL-4 for 10 min. The phosphorylation of STAT6 was analyzed with anti-phosphotyrosine Ab, and the amount of STAT6 was determined with anti-STAT6 Ab. C, Day 11 WT and IFNGR–/– Th1 cells were stimulated without or with IL-4 for 10 min. Cell lysates were prepared and incubated with anti-STAT6, anti-Jak1, and anti-Jak3 Abs. Phosphorylation of STAT6, Jak1, and Jak3 was analyzed using anti-phosphotyrosine Ab (PY). D, Naive CD4+ T cells and differentiated WT and IFNGR–/– Th1 and Th2 cells were incubated with (dotted line; IL-4 competes for the binding site for M1) or without IL-4 (5 ng/ml) (filled line) for 30 min before addition of M1 anti-IL-4R -chain mAb or a rat isotype control. The stained cells were analyzed by FACS. E, Day 11 WT and IFNGR–/– Th1 cells were not stimulated or stimulated with IL-4 for the time periods indicated. The cells were lysed and analyzed for IL-4R -chain protein phosphorylation with anti-PY Ab and content with anti-IL-4R -chain Ab.
STAT6 in differentiated WT Th1 cells shows a reduced ability to bind phosphorylated IL-4R fusion protein
Because only STAT6 phosphorylation is inhibited, we hypothesized that IFN- may inhibit STAT6 phosphorylation by suppressing its recruitment to the IL-4R. To test this hypothesis, we generated a fusion protein consisting of IL-4R C-terminal and GST protein (4RC). The 4RC fusion protein was either not phosphorylated or phosphorylated (4RC-P) by v-ABL kinase (Fig. 5A). The 4RC-P, but not 4RC fusion protein pulled down STAT6 (Fig. 5B). When using 4RC-P to pull down STAT6, we detected that the amount of STAT6 bound to 4RC-P fusion protein in WT Th1 cell samples reduced 3- to 4-fold compared with that in IFNGR–/– Th1 cell lysates. In contrast, the amounts of Jak1 bound to 4RC-P fusion protein were similar in WT Th1 cells and IFNGR–/– Th1 cells (Fig. 5C).
FIGURE 5. STAT6 in differentiated WT Th1 cells shows a reduced ability to bind to the IL-4R. A, HB101 cells were transformed by the GST vector or 4RC alone or in combination with pATH-v-Abl (+Abl). The expressed proteins were purified by glutathione Sepharose beads and detected by Western blot with anti-GST, anti-IL-4R, or anti-PY Abs. B, Unphosphorylated (4RC) or phosphorylated (4RC-P) fusion proteins (purified from 100 ml of bacterial culture) were used to incubate with cell lysates prepared from spleen cells (3 x 107) at 4°C overnight. Proteins that interacted with the fusion proteins were pulled down with glutathione Sepharose beads. The amount of STAT6 and Jak1 binding to the fusion protein was detected by Western blot with anti-STAT6 Ab or anti-Jak1, respectively. The amount of fusion proteins used in the experiment was quantified by anti-IL-4R Ab. C, Cell lysates prepared from day 11 WT and IFNGR–/– Th1 cells (50 x 106) were incubated with 4RC-P fusion protein at 4°C for 2 and 5 h. The amount of STAT6 and Jak1 being pulled down by the fusion proteins was detected by Western blot with anti-STAT6 and anti-Jak1 Abs, respectively. The amounts of fusion proteins used in the experiment were quantified by Western blot with anti-IL-4R Ab. Numbers indicate the ratio of the protein amount bound to the fusion proteins to the amount of the input protein as determined by densitometry. D, A total of 20 μg of protein from day 11 differentiated WT and IFNGR–/– Th1 and Th2 cells was used for electrophoresis. The amount of SOCS5 and STAT6 was detected by Western blot with anti-SOCS5 or anti-STAT6 Abs, respectively. Numbers indicate the density of each band measured by densitometry.
It has been reported that, compared with WT Th2 cells, WT Th1 cells showed impaired Jak1 phosphorylation and impaired recruitment of Jak1 to the IL-4R (3, 29). To find out how the amounts of Jak1-bound 4RC-P fusion protein in Th1 samples compare with that in Th2 cell samples, we analyzed the binding of Jak1 in Th2 samples. Consistent with previous reports, we showed that the amounts of Jak1 bound to 4RC-P fusion protein in both WT Th1 cells and IFNGR–/– Th1 cell lysates were several-fold less than that in Th2 cell lysates (Fig. 5C). This result raised the possibility that down-regulation of Jak1 binding to the IL-4R can be controlled by an IFN- signaling-independent pathway. To determine whether reduced Jak1 binding to the IL-4R in WT Th1 cells and IFNGR–/– Th1 cells was due to elevated levels of SOCS5, we detected SOCS5 protein expression by Western blot. We showed that Th1 cells contained 4-fold less of SOCS5 protein than Th2 cells (Fig. 5D). Together, these results suggest that down-regulation of Jak1 alone is not sufficient to impair STAT6 phosphorylation to the degree such that Th1 cells become insensitive to IL-4, and that further down-regulation of STAT6 binding to the IL-4R mediated by IFN- may be needed to achieve a state of unresponsiveness to the IL-4 stimulation.
Discussion
In this study, we sought to investigate the mechanism by which IFN- inhibits STAT6 phosphorylation to stabilize Th1 cell phenotype. By analyzing STAT6 phosphorylation and binding of STAT6 to the 4RC-P during Th1 cell development, we demonstrated that IFN- inhibited STAT6 phosphorylation by preventing the recruitment of STAT6 to the IL-4R signaling complex. These findings represent a novel mechanism that negatively regulates STAT6 signaling.
We observed that there existed two phases in which STAT6 phosphorylation was inhibited. These two phases of inhibition may involve two separate mechanisms: a TCR-mediated mechanism and an IFN--mediated mechanism. In the first week of Th1 cell differentiation, we demonstrated that STAT6 phosphorylation was diminished in both WT and IFNGR–/– Th1 cells. This phase of down-regulation might be mediated by TCR stimulation. Previous studies demonstrated that TCR engagement caused down-regulation of STAT6 phosphorylation through activating both protein kinase C-MAPK and calcineurin pathways (35). In the second phase of inhibition (TCR signaling had weakened significantly 11 days after the initial stimulation), STAT6 phosphorylation in the absence of IFN- signaling was restored to the levels comparable to that of naive CD4 T cells, whereas the reduction in STAT6 phosphorylation in WT Th1 cells was maintained. These observations suggest that IFN- produced by Th1 cells may play an important role in suppressing STAT6 phosphorylation in the second phase of inhibition.
It appears that IFN- down-regulates STAT6 phosphorylation in differentiated Th1 cells through a novel mechanism. There are several lines of evidence to support this idea. First, the timing, during which inhibition of STAT6 phosphorylation occurs, did not support the involvement of SOCS in IFN--mediated function. SOCS1 and SOCS3 inhibit STAT6 phosphorylation in monocytes, B cells, and fibroblast cells within a few hours of treatment with IFN- (31, 32), whereas the IFN--mediated inhibition of STAT6 phosphorylation in differentiated WT Th1 cells appears to take several days. Second, this novel mechanism appears to target only the STAT6 molecule, but not the Jak1 or Jak3 molecule. Others and we have reported Jak1 and STAT6 phosphorylation was impaired in Th1 cells (3, 33). More recently, Seki et al. (33) described a molecular mechanism that involved SOCS5 to explain the impairments. They showed that SOCS5 inhibited STAT6 phosphorylation in Th1 cells by competing with Jak1 binding to the box1 region of the IL-4R. However, the study does not explain our observation that IFNGR–/– Th1 cells possessed higher capacity than WT Th1 cells to phosphorylate their STAT6 despite that these two types of cells showed comparable Jak1 and Jak3 phosphorylation. Our findings raise a possibility that down-regulation of Jak1 and down-regulation of STAT6 in Th1 cells can involve more than one mechanism. One is an IFN--independent, but SOCS5-dependent mechanism as reported by Seki et al.; the other is an IFN--dependent, but SOCSs-independent mechanism. In fact, our data examining mRNA and protein expression of SOCS1, SOCS3, and SOCS5 support the latter mechanism.
In addition, inhibition of STAT6 phosphorylation in differentiated Th1 cells does not seem to involve another class of known inhibitors. Our study showed that SHP-1 mutation had no significant effect on STAT6 phosphorylation in differentiated Th1 cells, and SHP-2 is not known to interact with the IL-4R (36). Moreover, if the SH2 domain-containing protein tyrosine phosphatase was involved, one would expect that IL-4R phosphorylation would also be down-regulated. Our data did not show reduced IL-4R phosphorylation in WT Th1 cells.
Rather, our study provides evidence to support that IFN- suppresses STAT6 phosphorylation in differentiated WT Th1 cells by inhibiting its recruitment to the IL-4R, although the mechanism is still unknown. One possibility could be that IFN- signaling might cause posttranslational modification of STAT6, probably by serine/threonine phosphorylation or arginine methylation. Such modification may cause STAT6 conformational changes and consequently impair its binding to the IL-4R. Another possibility could be that IFN- might induce an undefined STAT6-binding protein. The binding of IFN--induced protein to STAT6 may prevent its recruitment to the IL-4R. Recent studies support both ideas. Chen et al. (37) demonstrated that unmethylated STAT6 showed reduced phosphorylation induced by IL-4. Daines et al. (38) showed that STAT6-binding protein existed in cytosolic plasma and blocked its DNA-binding activity. How IFN- reduces STAT6 recruitment to the IL-4R in differentiated Th1 cells would be an important topic that requires further investigation.
Acknowledgments
We thank Dr. Ke Shuai for providing us with pATH-v-Abl plasmid, Dr. Douglas Hilton for SOCS1 and SOCS3 probes, Dr. Leonard D. Shultz for mev/mev mice, and Dr. Ronald Germain for PI-39 cell lines. We thank Yan Su for assistance in the initial phase of this work.
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 study is supported by a grant from the Schweppe Foundation and a RO1 grant (A1 AI 48568) from the National Institutes of Health (to H.H.).
2 Address correspondence and reprint requests to Dr. Hua Huang, Department of Cell Biology, Stritch School of Medicine, Loyola University Chicago, Building 102, Room 5657, 2160 South First Avenue, Maywood, IL 60153. E-mail address: hhuang@lumc.edu
3 Abbreviations used in this paper: SH2, Src homology 2; 4RC, IL-4R/GST fusion protein; 4RC-P, phosphorylated 4RC; SHP-1, SH2-containing phosphatase; SOCS, suppressor of cytokine signaling; WT, wild type.
Received for publication March 25, 2004. Accepted for publication November 17, 2004.
References
De Carli, M., M. M. D’Elios, G. Zancuoghi, S. Romagnani, G. Del Prete. 1994. Human Th1 and Th2 cells: functional properties, regulation of development and role in autoimmunity. Autoimmunity 18:301.
Romagnani, S.. 1996. Development of Th 1- or Th 2-dominated immune responses: what about the polarizing signals?. Int. J. Clin. Lab. Res. 26:83.
Huang, H., W. E. Paul. 1998. Impaired interleukin 4 signaling in T helper type 1 cells. J. Exp. Med. 187:1305.
Swain, S. L., A. D. Weinberg, M. English, G. Huston. 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3796.
Seder, R. A., W. E. Paul, M. M. Davis, B. Fazekas de St. Groth. 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176:1091.
Le Gros, G., S. Z. Ben-Sasson, R. Seder, F. D. Finkelman, W. E. Paul. 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J. Exp. Med. 172:921.
Hsieh, C. S., A. B. Heimberger, J. S. Gold, A. O’Garra, K. M. Murphy. 1992. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.
Ohara, J., W. E. Paul. 1987. Receptors for B-cell stimulatory factor-1 expressed on cells of haematopoietic lineage. Nature 325:537.
Park, L. S., D. Friend, H. M. Sassenfeld, D. L. Urdal. 1987. Characterization of the human B cell stimulatory factor 1 receptor. J. Exp. Med. 166:476.
Russell, S. M., A. D. Keegan, N. Harada, Y. Nakamura, M. Noguchi, P. Leland, M. C. Friedmann, A. Miyajima, R. K. Puri, W. E. Paul, et al 1993. Interleukin-2 receptor chain: a functional component of the interleukin-4 receptor. Science 262:1880.
Kondo, M., T. Takeshita, N. Ishii, M. Nakamura, S. Watanabe, K. Arai, K. Sugamura. 1993. Sharing of the interleukin-2 (IL-2) receptor chain between receptors for IL-2 and IL-4. Science 262:1874.
Witthuhn, B. A., O. Silvennoinen, O. Miura, K. S. Lai, C. Cwik, E. T. Liu, J. N. Ihle. 1994. Involvement of the Jak-3 Janus kinase in signalling by interleukins 2 and 4 in lymphoid and myeloid cells. Nature 370:153.
Ihle, J. N.. 1995. The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv. Immunol. 60:1.
Johnston, J. A., M. Kawamura, R. A. Kirken, Y. Q. Chen, T. B. Blake, K. Shibuya, J. R. Ortaldo, D. W. McVicar, J. J. O’Shea. 1994. Phosphorylation and activation of the Jak-3 Janus kinase in response to interleukin-2. Nature 370:151.
Ryan, J. J., L. J. McReynolds, A. Keegan, L. H. Wang, E. Garfein, P. Rothman, K. Nelms, W. E. Paul. 1996. Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor. Immunity 4:123.
Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, S. L. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701
Zheng, W., R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.
Ho, I. C., D. Lo, L. H. Glimcher. 1998. c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms. J. Exp. Med. 188:1859.
Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for the development of Th2 cells. Immunity 4:313.
Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.
Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.
Zhang, Y., R. Apilado, J. Coleman, S. Ben-Sasson, S. Tsang, J. Hu-Li, W. E. Paul, H. Huang. 2001. Interferon stabilizes the T helper cell type 1 phenotype. J. Exp. Med. 194:165.
Wormald, S., D. J. Hilton. 2004. Inhibitors of cytokine signal transduction. J. Biol. Chem. 279:821.
Krebs, D. L., D. J. Hilton. 2001. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19:378.
Naka, T., M. Narazaki, M. Hirata, T. Matsumoto, S. Minamoto, A. Aono, N. Nishimoto, T. Kajita, T. Taga, K. Yoshizaki, et al 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924.
Masuhara, M., H. Sakamoto, A. Matsumoto, R. Suzuki, H. Yasukawa, K. Mitsui, T. Wakioka, S. Tanimura, A. Sasaki, H. Misawa, et al 1997. Cloning and characterization of novel CIS family genes. Biochem. Biophys. Res. Commun. 239:439
Hanson, E. M., H. Dickensheets, C. K. Qu, R. P. Donnelly, A. D. Keegan. 2003. Regulation of the dephosphorylation of Stat6: participation of Tyr-713 in the interleukin-4 receptor , the tyrosine phosphatase SHP-1, and the proteasome. J. Biol. Chem. 278:3903.
Haque, S. J., P. Harbor, M. Tabrizi, T. Yi, B. R. Williams. 1998. Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction. J. Biol. Chem. 273:33893.
Kamata, T., M. Yamashita, M. Kimura, K. Murata, M. Inami, C. Shimizu, K. Sugaya, C. R. Wang, M. Taniguchi, T. Nakayama. 2003. src homology 2 domain-containing tyrosine phosphatase SHP-1 controls the development of allergic airway inflammation. J. Clin. Invest. 111:109.
Dickensheets, H. L., C. Venkataraman, U. Schindler, R. P. Donnelly. 1999. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc. Natl. Acad. Sci. USA 96:10800.
Losman, J. A., X. P. Chen, D. Hilton, P. Rothman. 1999. Cutting edge: SOCS-1 is a potent inhibitor of IL-4 signal transduction. J. Immunol. 162:3770.(Zan Huang, Junping Xin, J)