Receptor Inhibiting Its Recruitment to the IL-4 Suppresses STAT6

IFN-γ Suppresses STAT6 Phosphorylation by Inhibiting Its Recruitment to the IL-4 Receptor

This information is current as Zan Huang, Junping Xin, John Coleman and Hua Huang of September 27, 2021. J Immunol 2005; 174:1332-1337; ; doi: 10.4049/jimmunol.174.3.1332 http://www.jimmunol.org/content/174/3/1332 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IFN-␥ Suppresses STAT6 Phosphorylation by Inhibiting Its Recruitment to the IL-4 Receptor1

Zan Huang,* Junping Xin,† John Coleman,† and Hua Huang2*†

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 Downloaded from 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. The Journal of Immunology, 2005, 174: 1332–1337. http://www.jimmunol.org/

helper type 1 cells are central immune components in of GATA3 and c-maf expression that control Th2 polarization (17– combating invasion of intracellular pathogens. They help 21). Thus, the Jak-STAT6 pathway becomes a logical target to be T CD8ϩ T cells to become killer T cells and B cells to rendered insensitive to IL-4 stimulation. produce Abs through their cytokine production, such as IFN-␥, We previously showed that Th1 cells that lacked IFN-␥ or IL-2, TNF-␣, and TNF-␤ (lymph toxin). In contrast, uncontrolled IFN-␥ responsiveness retained the ability to respond to IL-4 (22), Th1 response can cause autoimmune disease (1, 2). Dysregulated suggesting that IFN-␥ plays an important role in Th1 cell com- Th1 cells found in autoimmune diseases display highly polarized mitment. Two classes of molecules, Src homology 2 (SH2)3 do-

phenotype. Their cytokine-producing profiles remain unchanged main-containing protein tyrosine phosphatase and suppressor of by guest on September 27, 2021 even after exposure to Th2-inducing conditions. It is still unsolved cytokine signaling (SOCS), have been shown to inhibit IL-4-in- how Th1 cells become insensitive to Th2-inducing conditions. duced STAT6 phosphorylation in various cell types (23–26). SH2- Previously, we reported that committed Th1 cells were insensi- containing phosphatase (SHP-1) dephosphorylates STAT6 in B tive to Th2-inducing conditions; they exhibit strikingly diminished cells, macrophages, and fibroblast cells (27–29). SOCS1 inhibits STAT6 phosphorylation in response to IL-4 stimulation (3). IL-4 STAT6 phosphorylation in monocytes, liver cells, and B cells (30– is the major determinant in directing naive CD4ϩ T cells to dif- 32). SOCS5 was shown to suppress STAT6 phosphorylation in ferentiate into Th2 cells (4–7). It mediates its function by binding Th1 cells (33). Whether IFN-␥ can suppress STAT6 phosphory- and stimulating the IL-4R ␣-chain (8, 9). Ligand-bound IL-4R lation in committed CD4ϩ Th1 cells through the known classes of ␣-chain recruits common ␥-chain and its associated kinase Jak3 inhibitors is not clear. (10, 11). Jak3 and IL-4R ␣-chain-associated Jak1 then phosphor- In this study, we analyzed the role of known STAT6 phosphorylate each other and become activated (12–14). Activated Jak1 and ylation inhibitors in both wild-type (WT) and IFN-␥ receptor Jak3 phosphorylate the IL-4R on several tyrosine residues located knockout (IFNGRϪ/Ϫ) Th1 cells and we did not find evidence to in the C-terminal portion (15). The phosphorylated IL-4R serve as support that these known inhibitors mediated the inhibition of docking sites to recruit STAT6 as a substrate for activated Jak1 STAT6 phosphorylation. Rather, by using a newly developed pull- and Jak3 (16). Activation of STAT6 is essential for the induction down assay, we observed that STAT6 from WT Th1 cells bound less effectively to the phosphorylated IL-4R than that from IFNGRϪ/Ϫ Th1 cells. These results suggest that IFN-␥ mediates *Graduate Program in Molecular Biology and †Department of Cell Biology, Stritch inhibition of STAT6 phosphorylation through a novel mechanism. School of Medicine, Loyola University Chicago, Maywood, IL 60153 Received for publication March 25, 2004. Accepted for publication November 17, 2004. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Animals and cell cultures charges. This article must therefore be hereby marked advertisement in accordance ␣ ␤ with 18 U.S.C. Section 1734 solely to indicate this fact. Mice bearing transgenic TCR- and - chains specific for pigeon cytochrome c peptide 88–104 in association with the I-Ek class II molecule on 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, 3 Abbreviations used in this paper: SH2, Src homology 2; 4RC, IL-4R/GST fusion Room 5657, 2160 South First Avenue, Maywood, IL 60153. E-mail address: protein; 4RC-P, phosphorylated 4RC; SHP-1, SH2-containing phosphatase; SOCS, [email protected] suppressor of cytokine signaling; WT, wild type.

C57BL/6 background (5CC7) were purchased from The Jackson Labora- added to the cultures, which were incubated at 30°C for another 1.5 h. The tory. The 5CC7 mice were crossed to IFN-␥ receptor-deficient mice on cells were collected and resuspended in1Lofmodified M9 medium (1ϫ C57BL/6 background (from The Jackson Laboratory) to generate 5CC7 M9 salts, 0.5% (w/v) casamino acids (Sigma-Aldrich A-2427), 0.1 mM ␥ Ϫ/Ϫ ␮ IFN- receptor-deficient mice (IFNGR ) on C57BL/6 background. Na- CaCl2, 0.2% glucose, 10 g/ml thiamine B1) in the presence of isopropy- ϩ ive CD4 T cells were prepared, as described elsewhere (22). These cells lthio-␤-D-galactoside, but no tryptophan. The cell suspension was incu- (1 ϫ 106) were stimulated with 1 ␮m of cytochrome c peptide (prepared by bated at 30°C for 1.5 h to induce the expression of the TrpE-v-Abl fusion Alpha Diagnostic International) and 5 ϫ 106 of I-Ek-expressing fibroblast protein. The 4RC- and v-Abl-expressing cells were harvested, resuspended cells (PI-39), irradiated with 2500 rad of gamma-ray, in 10 ml of complete in 20 ml of ice-cold lysis buffer (PBS, 50 mM EDTA, 1% Triton X-100, 1 RPMI 1640 medium supplemented with IL-2 (10 U/ml), IL-12 (10 ng/ml), mM DTT, 0.2 mM PMSF, 10 ␮g/ml pepstatin A, 10 ␮g/ml leupeptin, 10 anti-CD28 Ab (3 ␮g/ml, prepared from hybridoma 37.N.51.1, and provided ␮g/ml aprotinin), and sonicated in Sonic Dismembrator Model 60 (Fisher by J. Allison (University of California, Berkeley, CA)), and anti-IL-4 Ab Scientific) at 50% output for 2 min with 30-s intervals every minute. Cell (11B11; 10 ␮g/ml) for 3–11 days. The PI-39 cells were provided by R. lysates were incubated with 0.5 ml of glutathione Sepharose beads (Am- Germain (National Institutes of Health, Bethesda, MD). ersham Biosciences) at room temperature for 30 min. After extensive wash For preparing Th1 cells that were deficient in SHP-1 activity, C57BL/ with PBS, the beads were resuspended in 0.5 ml of lysis buffer and stored 6J-Hcphme-v/Hcphme-v (viable motheaten (mev/mev)) and littermate control at Ϫ70°C for later use. (ϩ/Ϫ) 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 CD4ϩ T cells was conducted GST pulldown assay 6 ϩ 7 by culturing 10 CD4 T cells in the presence of 10 irradiated T-depleted The 4RC-P or unphosphorylated 4RC was expressed in E. coli (strain ϩ Ϫ ␮ spleen cells from / mice with anti-CD3 (2C11; 3 g/ml), anti-CD28 HB101) and purified with glutathione Sepharose beads. Phosphorylated ␮ ␮ (37.N.51.1; 3 g/ml), anti-IL-4 Ab (11B11; 10 g/ml), IL-2 (10 U/ml), and or unphosphorylated fusion proteins (prepared from 100 ml of bacterial IL-12 (10 ng/ml; BD Pharmingen) for 7 days. Handling of animals com- culture) were incubated with 0.5 ml of cell lysates at 4°C for time plied with the animal protocol approved by Institute Animal Care and User indicated. After extensive wash, proteins were eluted in 35 ␮lof2ϫ Downloaded from Committee of Loyola University Medical School. SDS loading buffer. A total of 10 ␮l of the eluted protein was loaded for SDS-PAGE and transferred to polyvinylidene difluoride membrane Immunoprecipitation and Western blotting (Millipore) for Western blot. The amounts of STAT6 and Jak1 bound to To prepare whole cell lysates, cells (1 ϫ 107 for immunoprecipitating the fusion protein were detected with anti-STAT6 and anti-Jak1 Ab, STAT6; 5 ϫ 107 for immunoprecipitating IL-4R ␣-chain) were lysed with respectively. 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) http://www.jimmunol.org/ freshly supplemented with 1 mM Na VO , 1 mM PMSF, 10 ␮g/ml apro- Results 3 4 Ϫ Ϫ tinin, 10 ␮g/ml leupeptin, and 10 ␮g/ml pepstatin or complete protease IFNGR / Th1 cells depend on STAT6 to develop into inhibitor tablet (Boehringer Mannheim). To prepare cytosolic lysates, IL-4-producing cells cells (5 ϫ 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% Non- Previously, we demonstrated that well-differentiated Th1 cells idet P-40). After incubation on ice for 10 min, lysates were spun down showed impaired STAT6 phosphorylation (3). More recently, we at 1500 rpm for 1 min. Supernatants were collected as cytosolic lysates. reported that IFN-␥ played an essential role in stabilizing Th1 ␮ For immunoprecipitation, lysates were incubated with 5–10 gofAbs phenotype (22). However, whether IFN-␥ stabilizes Th1 pheno- for 2 h. Immunoprecipitation and Western blot were conducted, as de- type by suppressing STAT6 phosphorylation remains unclear. To

scribed previously (3). by guest on September 27, 2021 determine whether IFNGRϪ/Ϫ Th1 cells depend on STAT6 to de- Northern blot analysis velop into IL-4-producing cells, we generated IFNGR and STAT6 Total RNA (20 ␮g) isolated by the guanidinium method was separated in double-knockout mice (DKO). We found that DKO Th1 cells a 1% agarose-formaldehyde gel and transferred onto a nitrocellulose mem- failed to differentiate into IL-4-producing cells, as measured by brane (Nytran; Schleicher & Schuell Microscience). A probe containing a both ELISA and intracellular staining (Fig. 1). These results dem- fragment of SOCS1 or SOCS3 cDNA (provided by D. Hilton, Walter and onstrated that elimination of STAT6 stabilized IFNGRϪ/Ϫ Th1 cell Eliza Hall Institute of Medical Research, Melbourne, Australia) was la- phenotype, suggesting that down-regulation of STAT6 phosphor- beled with [32P]dCTP by random primer method. ylation by IFN-␥ in Th1 cells can be an effective way for Th1 cells IL-4R ␣-chain staining to achieve a stable phenotype. Cells (1 ϫ 106) were incubated with 10% goat serum for 5 min to block nonspecific binding. For selected samples, 5 ng/ml IL-4 (BD Pharmingen) IFN-␥ inhibits STAT6 phosphorylation in differentiated Th1 cells 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 To determine whether IFN-␥ suppresses STAT6 phosphorylation or a rat isotype control Ig (R&D Systems) were then added to the cells and in committed Th1 cells, we examined STAT6 phosphorylation in incubated for 20 min in FACS buffer (3% FCS, 0.1% sodium azide, 1ϫ differentiating and differentiated WT and IFNGRϪ/Ϫ Th1 cells. We PBS). Cells were washed with FACS buffer before they were incubated found that IL-4 induced robust STAT6 phosphorylation in freshly with biotinylated goat Fab against rat IgG (Southern Biotechnology Asso- ϩ ciates) for 20 min. After wash, samples were incubated with FITC-labeled isolated naive CD4 T cells. After 3–7 days of differentiation un- Ϫ/Ϫ streptavidin. The stained cells were analyzed by FACS. der Th1-inducing conditions, both WT and IFNGR Th1 cells showed significantly reduced levels of STAT6 phosphorylation. Preparation and phosphorylation of IL-4R-GST fusion protein After 11 days of differentiation under Th1-inducing conditions, cDNA encoding the C-terminal portion of the mouse IL-4R ␣-chain (aa STAT6 phosphorylation in WT Th1 cells diminished even more 258–810) was ligated in-frame into a GST gene fusion vector (pGEX- dramatically, which concurred with the timing of Th1 cell com- 6p-2; Amersham Biosciences) to create a IL-4R/GST fusion protein (4RC). mitment. In contrast, STAT6 phosphorylation in IFNGRϪ/Ϫ Th1 To prepare phosphorylated 4RC (4RC-P), 4RC, pATH-v-Abl plasmids (a cells was restored to the levels comparable to that of naive CD4ϩ gift from K. Shuai, University of California, Los Angeles, CA) were co- transformed into Escherichia coli (strain HB101) as described (34). The cells (Fig. 2). These biphasic changes in STAT6 phosphorylation transformed bacteria were selected on Luria broth plates containing 100 during the course of Th1 cell differentiation suggest that the dim- ␮g/ml ampicillin, 25 ␮g/ml tetracycline, and 100 ␮g/ml tryptophan. The inution of STAT6 phosphorylation in differentiated Th1 cells may selected bacteria were grown overnight in 100 ml of Luria broth culture be controlled by two processes. The first phase may be mediated medium supplemented with ampicillin (100 ␮g/ml), tetracycline (25 ␮g/ ml), and tryptophan (100 ␮g/ml). The overnight-grown bacterial culture by TCR stimulation, and the second phase may be mediated by was expanded into1Lbyadding 900 ml of the same medium and further IFN-␥. The data also suggest that IFN-␥ may mediate Th1 cell grew at 37°C for 3 h. Then, 0.2 mM isopropylthio-␤-D-galactoside was commitment by inhibiting STAT6 signaling. 1334 IFN-␥ SUPPRESSES RECRUITMENT OF STAT6 TO IL-4R

FIGURE 3. Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells occurs in the initiation step of the IL-4R signaling. Naive CD4ϩ T cells prepared from WT and IFNGRϪ/Ϫ mice were cultured under Th1- inducing conditions for 11 days (day 11). The day 11 WT and IFNGRϪ/Ϫ 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.

STAT6 phosphorylation in differentiated IFNGRϪ/Ϫ 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 Downloaded from signaling in differentiated WT Th1 cells.

Inhibition of STAT6 phosphorylation in differentiated WT Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1 FIGURE 1. IFNGRϪ/Ϫ Th1 cells depend on STAT6 to develop into ϩ Ϫ/Ϫ IL-4-producing cells. Naive CD4 T cells from WT, IFNGR , IFNGR, To determine whether known inhibitors mediate the inhibition of http://www.jimmunol.org/ and STAT6 double-knockout (DKO) mice were primed under Th1- or STAT6 phosphorylation, we examined mRNA and protein expres- Th2-inducing conditions for 11 days. Differentiated Th1 cells were primed sion of SOCS1 and SOCS3. We did not observe significant dif- under Th1- or Th2-inducing conditions for an additional 7 days (Th1–2). A, ference in SOCS1 and SOCS3 expressions between differentiated The resultant cells were harvested, washed, and stimulated with PMA/ WT and IFNGRϪ/Ϫ Th1 cells (Fig. 4A). Another known inhibitor, ionomycin overnight in the presence of IL-2. IL-4 production in the su- SOCS5, also did not show significant difference in its protein ex- pernatants from the overnight stimulated cells was detected by ELISA. B, Ϫ Ϫ pression between WT Th1 cells and IFNGR / Th1 cells (Fig. The resultant cells were harvested, washed, and stimulated with PMA/ ionomycin for6hinthepresence of IL-2 and monensin. IL-4 content in the 4A). SHP-1 is known to suppress STAT6 phosphorylation. How- v v stimulated cells was detected by intracellular staining and analyzed by ever, me /me Th1 cells that lack SHP-1 activity did not show FACS. enhancement of IL-4-induced STAT6 phosphorylation (Fig. 4B). It by guest on September 27, 2021 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 Inhibition of STAT6 phosphorylation in differentiated WT Th1 found that the phosphorylation of Jak1 and Jak3 was not dimin- cells occurs in the cytoplasm ished in differentiated WT Th1 cells (Fig. 4C). Furthermore, IL-4R We focused on IFN-␥-mediated inhibition of STAT6 phosphory- expression or IL-4R phosphorylation in WT Th1 cells was not lation because the timing of inhibition concurred with that of Th1 different from that in IFNGRϪ/Ϫ Th1 cells (Fig. 4, D and E). This cell commitment (i.e., Th1 cells resist to Th2-inducing conditions). finding is consistent with the idea that those known inhibitors do Inhibition of STAT6 phosphorylation could occur in the cyto- not mediate the inhibition of STAT6 phosphorylation. Our data plasm or nucleus. To determine this, we cultured WT and suggest that inhibition of STAT6 phosphorylation in WT Th1 cells IFNGRϪ/Ϫ Th1 cells for 11 days (day 11 WT and IFNGRϪ/Ϫ may involve a novel mechanism. Th1 cells) and analyzed STAT6 phosphorylation in the cytosolic fraction. We noticed that STAT6 phosphorylation in WT STAT6 in differentiated WT Th1 cells shows a reduced ability to Th1 cells increased only moderately after 1 min of IL-4 stim- bind phosphorylated IL-4R fusion protein ulation. The STAT6 phosphorylation did not enhance signifi- Because only STAT6 phosphorylation is inhibited, we hypothe- cantly even with longer IL-4 stimulation (Fig. 3). By contrast, sized 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 FIGURE 2. STAT6 phosphorylation in differentiated WT Th1 cells is samples reduced 3- to 4-fold compared with that in IFNGRϪ/Ϫ inhibited. Naive CD4ϩ T cells were prepared from WT and IFNGRϪ/Ϫ Th1 cell lysates. In contrast, the amounts of Jak1 bound to 4RC-P mice and primed under Th1-inducing conditions for the time indicated. At Ϫ/Ϫ the end of each culture period, cells were washed and not stimulated or fusion protein were similar in WT Th1 cells and IFNGR Th1 stimulated with IL-4 (5 ng/ml) at room temperature for 10 min. Whole cell cells (Fig. 5C). lysates were prepared and analyzed for STAT6 amount and phosphoryla- It has been reported that, compared with WT Th2 cells, WT Th1 tion by using anti-phosphotyrosine Ab (anti-PY) and anti-STAT6 Abs (anti- cells showed impaired Jak1 phosphorylation and impaired recruit- STAT6), respectively. ment of Jak1 to the IL-4R (3, 29). To find out how the amounts of The Journal of Immunology 1335

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 Downloaded from vector or 4RC alone or in combination with pATH-v-Abl (ϩAbl). 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, Un- phosphorylated (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 ϫ 107) at 4°C overnight. Proteins that inter- FIGURE 4. Inhibition of STAT6 phosphorylation in differentiated WT

acted with the fusion proteins were pulled down with glutathione Sepha- http://www.jimmunol.org/ Th1 cells does not appear to involve SOCS1, SOCS3, SOCS5, or SHP-1. rose beads. The amount of STAT6 and Jak1 binding to the fusion protein A, Total RNA and protein (whole cell lysate) were prepared from day 11 was detected by Western blot with anti-STAT6 Ab or anti-Jak1, respec- WT and IFNGRϪ/Ϫ Th1 cells. A total of 10 ␮g of total RNA was used for tively. The amount of fusion proteins used in the experiment was quantified electrophoresis, and SOCS1 and SOCS3 mRNA expression was detected Ϫ Ϫ by anti-IL-4R Ab. C, Cell lysates prepared from day 11 WT and IFNGR / by Northern blot analysis (left panel). A total of 10 ␮g of protein was used Th1 cells (50 ϫ 106) were incubated with 4RC-P fusion protein at 4°C for for electrophoresis, and SOCS1, SOCS3, and SOCS5 protein expression 2 and 5 h. The amount of STAT6 and Jak1 being pulled down by the fusion was analyzed by Western blot with anti-SOCS1, anti-SOCS3, or anti- proteins was detected by Western blot with anti-STAT6 and anti-Jak1 Abs, SOCS5 Abs (right panel). Numbers indicate the ratio of the protein amount respectively. The amounts of fusion proteins used in the experiment were bound to the fusion protein to the amount of the input protein, as deter- v v quantified by Western blot with anti-IL-4R Ab. Numbers indicate the ratio mined by densitometry. B, Day 11 me /me and ϩ/Ϫ Th1 cells were stim- by guest on September 27, 2021 of the protein amount bound to the fusion proteins to the amount of the ulated with IL-4 for 10 min. The phosphorylation of STAT6 was analyzed input protein as determined by densitometry. D, A total of 20 ␮g of protein with anti-phosphotyrosine Ab, and the amount of STAT6 was determined Ϫ Ϫ from day 11 differentiated WT and IFNGR / Th1 and Th2 cells was used with anti-STAT6 Ab. C, Day 11 WT and IFNGRϪ/Ϫ Th1 cells were stim- for electrophoresis. The amount of SOCS5 and STAT6 was detected by ulated without or with IL-4 for 10 min. Cell lysates were prepared and Western blot with anti-SOCS5 or anti-STAT6 Abs, respectively. Numbers incubated with anti-STAT6, anti-Jak1, and anti-Jak3 Abs. Phosphorylation indicate the density of each band measured by densitometry. of STAT6, Jak1, and Jak3 was analyzed using anti-phosphotyrosine Ab (PY). D, Naive CD4ϩ T cells and differentiated WT and IFNGRϪ/Ϫ 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 and that further down-regulation of STAT6 binding to the IL-4R before addition of M1 anti-IL-4R ␣-chain mAb or a rat isotype control. The mediated by IFN-␥ may be needed to achieve a state of unrespon- Ϫ/Ϫ stained cells were analyzed by FACS. E, Day 11 WT and IFNGR Th1 siveness to the IL-4 stimulation. 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. 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 Jak1-bound 4RC-P fusion protein in Th1 samples compare with STAT6 to the 4RC-P during Th1 cell development, we demon- that in Th2 cell samples, we analyzed the binding of Jak1 in Th2 strated that IFN-␥ inhibited STAT6 phosphorylation by preventing samples. Consistent with previous reports, we showed that the the recruitment of STAT6 to the IL-4R signaling complex. These amounts of Jak1 bound to 4RC-P fusion protein in both WT Th1 findings represent a novel mechanism that negatively regulates cells and IFNGRϪ/Ϫ Th1 cell lysates were several-fold less than STAT6 signaling. that in Th2 cell lysates (Fig. 5C). This result raised the possibility We observed that there existed two phases in which STAT6 that down-regulation of Jak1 binding to the IL-4R can be con- phosphorylation was inhibited. These two phases of inhibition may trolled by an IFN-␥ signaling-independent pathway. To determine involve two separate mechanisms: a TCR-mediated mechanism whether reduced Jak1 binding to the IL-4R in WT Th1 cells and and an IFN-␥-mediated mechanism. In the first week of Th1 cell IFNGRϪ/Ϫ Th1 cells was due to elevated levels of SOCS5, we differentiation, we demonstrated that STAT6 phosphorylation was detected SOCS5 protein expression by Western blot. We showed diminished in both WT and IFNGRϪ/Ϫ Th1 cells. This phase of that Th1 cells contained 4-fold less of SOCS5 protein than Th2 down-regulation might be mediated by TCR stimulation. Previous cells (Fig. 5D). Together, these results suggest that down-regula- studies demonstrated that TCR engagement caused down-regulation of Jak1 alone is not sufficient to impair STAT6 phosphorylation of STAT6 phosphorylation through activating both protein tion to the degree such that Th1 cells become insensitive to IL-4, kinase C-MAPK and calcineurin pathways (35). In the second 1336 IFN-␥ SUPPRESSES RECRUITMENT OF STAT6 TO IL-4R phase of inhibition (TCR signaling had weakened significantly 11 Acknowledgments days after the initial stimulation), STAT6 phosphorylation in the We thank Dr. Ke Shuai for providing us with pATH-v-Abl plasmid, absence of IFN-␥ signaling was restored to the levels comparable Dr. Douglas Hilton for SOCS1 and SOCS3 probes, Dr. Leonard D. Shultz to that of naive CD4 T cells, whereas the reduction in STAT6 for mev/mev mice, and Dr. Ronald Germain for PI-39 cell lines. We thank phosphorylation in WT Th1 cells was maintained. These observa- Yan Su for assistance in the initial phase of this work. tions suggest that IFN-␥ produced by Th1 cells may play an important role in suppressing STAT6 phosphorylation in the second References 1. De Carli, M., M. M. D’Elios, G. Zancuoghi, S. Romagnani, and G. Del Prete. phase of inhibition. 1994. Human Th1 and Th2 cells: functional properties, regulation of development It appears that IFN-␥ down-regulates STAT6 phosphorylation in and role in autoimmunity. Autoimmunity 18:301. differentiated Th1 cells through a novel mechanism. There are sev- 2. 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. eral lines of evidence to support this idea. First, the timing, during 3. Huang, H., and W. E. Paul. 1998. Impaired interleukin 4 signaling in T helper which inhibition of STAT6 phosphorylation occurs, did not sup- type 1 cells. J. Exp. Med. 187:1305. ␥ 4. Swain, S. L., A. D. Weinberg, M. English, and G. Huston. 1990. IL-4 directs the port the involvement of SOCS in IFN- -mediated function. development of Th2-like helper effectors. J. Immunol. 145:3796. SOCS1 and SOCS3 inhibit STAT6 phosphorylation in monocytes, 5. Seder, R. A., W. E. Paul, M. M. Davis, and B. Fazekas de St. Groth. 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine- B cells, and fibroblast cells within a few hours of treatment with ϩ producing potential of CD4 T cells from T cell receptor transgenic mice. J. Exp. IFN-␥ (31, 32), whereas the IFN-␥-mediated inhibition of STAT6 Med. 176:1091. phosphorylation in differentiated WT Th1 cells appears to take 6. Le Gros, G., S. Z. Ben-Sasson, R. Seder, F. D. Finkelman, and W. E. Paul. 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and several days. Second, this novel mechanism appears to target only IL-4 are required for in vitro generation of IL-4-producing cells. J. Exp. Med. Downloaded from the STAT6 molecule, but not the Jak1 or Jak3 molecule. Others 172:921. 7. Hsieh, C. S., A. B. Heimberger, J. S. Gold, A. O’Garra, and K. M. Murphy. 1992. and we have reported Jak1 and STAT6 phosphorylation was im- Differential regulation of T helper phenotype development by interleukins 4 and paired in Th1 cells (3, 33). More recently, Seki et al. (33) de- 10 in an ␣␤ T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA scribed a molecular mechanism that involved SOCS5 to explain 89:6065. 8. Ohara, J., and W. E. Paul. 1987. Receptors for B-cell stimulatory factor-1 ex- the impairments. They showed that SOCS5 inhibited STAT6 pressed on cells of haematopoietic lineage. Nature 325:537. 9. Park, L. S., D. Friend, H. M. Sassenfeld, and D. L. Urdal. 1987. Characterization

phosphorylation in Th1 cells by competing with Jak1 binding to http://www.jimmunol.org/ of the human B cell stimulatory factor 1 receptor. J. Exp. Med. 166:476. the box1 region of the IL-4R. However, the study does not 10. Russell, S. M., A. D. Keegan, N. Harada, Y. Nakamura, M. Noguchi, P. Leland, Ϫ Ϫ explain our observation that IFNGR / Th1 cells possessed M. C. Friedmann, A. Miyajima, R. K. Puri, W. E. Paul, et al. 1993. Interleukin-2 ␥ higher capacity than WT Th1 cells to phosphorylate their receptor chain: a functional component of the interleukin-4 receptor. Science 262:1880. STAT6 despite that these two types of cells showed comparable 11. Kondo, M., T. Takeshita, N. Ishii, M. Nakamura, S. Watanabe, K. Arai, and Jak1 and Jak3 phosphorylation. Our findings raise a possibility K. Sugamura. 1993. Sharing of the interleukin-2 (IL-2) receptor ␥ chain between receptors for IL-2 and IL-4. Science 262:1874. that down-regulation of Jak1 and down-regulation of STAT6 in 12. Witthuhn, B. A., O. Silvennoinen, O. Miura, K. S. Lai, C. Cwik, E. T. Liu, and Th1 cells can involve more than one mechanism. One is an J. N. Ihle. 1994. Involvement of the Jak-3 Janus kinase in signalling by inter- ␥ leukins 2 and 4 in lymphoid and myeloid cells. Nature 370:153. IFN- -independent, but SOCS5-dependent mechanism as re- 13. Ihle, J. N. 1995. The Janus protein tyrosine kinase family and its role in cytokine by guest on September 27, 2021 ported by Seki et al.; the other is an IFN-␥-dependent, but signaling. Adv. Immunol. 60:1. SOCSs-independent mechanism. In fact, our data examining 14. Johnston, J. A., M. Kawamura, R. A. Kirken, Y. Q. Chen, T. B. Blake, K. Shibuya, J. R. Ortaldo, D. W. McVicar, and J. J. O’Shea. 1994. Phosphory- mRNA and protein expression of SOCS1, SOCS3, and SOCS5 lation and activation of the Jak-3 Janus kinase in response to interleukin-2. Na- support the latter mechanism. ture 370:151. 15. Ryan, J. J., L. J. McReynolds, A. Keegan, L. H. Wang, E. Garfein, P. Rothman, In addition, inhibition of STAT6 phosphorylation in differenti- K. Nelms, and W. E. Paul. 1996. Growth and gene expression are predominantly ated Th1 cells does not seem to involve another class of known controlled by distinct regions of the human IL-4 receptor. Immunity 4:123. inhibitors. Our study showed that SHP-1 mutation had no signif- 16. Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, and S. L. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 icant effect on STAT6 phosphorylation in differentiated Th1 cells, Stat. Science 265:1701. and SHP-2 is not known to interact with the IL-4R (36). Moreover, 17. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is neces- sary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587. if the SH2 domain-containing protein tyrosine phosphatase was 18. Ho, I. C., D. Lo, and L. H. Glimcher. 1998. c-maf promotes T helper cell type 2 involved, one would expect that IL-4R phosphorylation would also (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms. J. Exp. Med. 188:1859. be down-regulated. Our data did not show reduced IL-4R phos- 19. Kaplan, M. H., U. Schindler, S. T. Smiley, and M. J. Grusby. 1996. Stat6 is phorylation in WT Th1 cells. required for mediating responses to IL-4 and for the development of Th2 cells. Rather, our study provides evidence to support that IFN-␥ sup- Immunity 4:313. 20. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, presses STAT6 phosphorylation in differentiated WT Th1 cells by K. Nakanishi, N. Yoshida, T. Kishimoto, and S. Akira. 1996. Essential role of inhibiting its recruitment to the IL-4R, although the mechanism is Stat6 in IL-4 signalling. Nature 380:627. ␥ 21. Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, still unknown. One possibility could be that IFN- signaling might R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al. 1996. Lack cause posttranslational modification of STAT6, probably by of IL-4-induced Th2 response and IgE class switching in mice with disrupted serine/threonine phosphorylation or arginine methylation. Such Stat6 gene. Nature 380:630. 22. Zhang, Y., R. Apilado, J. Coleman, S. Ben-Sasson, S. Tsang, J. Hu-Li, modification may cause STAT6 conformational changes and con- W. E. Paul, and H. Huang. 2001. Interferon ␥ stabilizes the T helper cell type 1 sequently impair its binding to the IL-4R. Another possibility phenotype. J. Exp. Med. 194:165. ␥ 23. Wormald, S., and D. J. Hilton. 2004. Inhibitors of cytokine signal transduction. could be that IFN- might induce an undefined STAT6-binding J. Biol. Chem. 279:821. protein. The binding of IFN-␥-induced protein to STAT6 may pre- 24. Krebs, D. L., and D. J. Hilton. 2001. SOCS proteins: negative regulators of vent its recruitment to the IL-4R. Recent studies support both cytokine signaling. Stem Cells 19:378. 25. Naka, T., M. Narazaki, M. Hirata, T. Matsumoto, S. Minamoto, A. Aono, ideas. Chen et al. (37) demonstrated that unmethylated STAT6 N. Nishimoto, T. Kajita, T. Taga, K. Yoshizaki, et al. 1997. Structure and func- showed reduced phosphorylation induced by IL-4. Daines et al. tion of a new STAT-induced STAT inhibitor. Nature 387:924. 26. Masuhara, M., H. Sakamoto, A. Matsumoto, R. Suzuki, H. Yasukawa, K. Mitsui, (38) showed that STAT6-binding protein existed in cytosolic T. Wakioka, S. Tanimura, A. Sasaki, H. Misawa, et al. 1997. Cloning and char- plasma and blocked its DNA-binding activity. How IFN-␥ reduces acterization of novel CIS family genes. Biochem. Biophys. Res. Commun. 239:439. STAT6 recruitment to the IL-4R in differentiated Th1 cells would 27. Hanson, E. M., H. Dickensheets, C. K. Qu, R. P. Donnelly, and A. D. Keegan. be an important topic that requires further investigation. 2003. Regulation of the dephosphorylation of Stat6: participation of Tyr-713 in The Journal of Immunology 1337

the interleukin-4 receptor ␣, the tyrosine phosphatase SHP-1, and the proteasome. 33. Seki, Y., K. Hayashi, A. Matsumoto, N. Seki, J. Tsukada, J. Ransom, T. Naka, J. Biol. Chem. 278:3903. T. Kishimoto, A. Yoshimura, and M. Kubo. 2002. Expression of the suppressor 28. Haque, S. J., P. Harbor, M. Tabrizi, T. Yi, and B. R. Williams. 1998. Protein- of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent activation and Th2 differentiation. Proc. Natl. Acad. Sci. USA 99:13003. signal transduction. J. Biol. Chem. 273:33893. 34. ten Hoeve, J., M. de Jesus Ibarra-Sanchez, Y. Fu, W. Zhu, M. Tremblay, 29. Kamata, T., M. Yamashita, M. Kimura, K. Murata, M. Inami, C. Shimizu, M. David, and K. Shuai. 2002. Identiﬁcation of a nuclear Stat1 protein tyrosine K. Sugaya, C. R. Wang, M. Taniguchi, and T. Nakayama. 2003. src homology 2 phosphatase. Mol. Cell. Biol. 22:5662. domain-containing tyrosine phosphatase SHP-1 controls the development of al- 35. Zhu, J., H. Huang, L. Guo, T. Stonehouse, C. J. Watson, J. Hu-Li, and W. E. Paul. lergic airway inﬂammation. J. Clin. Invest. 111:109. 2000. Transient inhibition of interleukin 4 signaling by T cell receptor ligation. J. Exp. Med. 192:1125. 30. Dickensheets, H. L., C. Venkataraman, U. Schindler, and R. P. Donnelly. 1999. 36. Huang, H., and W. E. Paul. 2000. Protein tyrosine phosphatase activity is re- Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by quired for IL-4 induction of IL-4 receptor ␣-chain. J. Immunol. 164:1211. inducing SOCS-1 gene expression. Proc. Natl. Acad. Sci. USA 96:10800. 37. Chen, W., M. O. Daines, and G. K. Hershey. 2004. Methylation of STAT6 mod- 31. Losman, J. A., X. P. Chen, D. Hilton, and P. Rothman. 1999. Cutting edge: ulates STAT6 phosphorylation, nuclear translocation, and DNA-binding activity. SOCS-1 is a potent inhibitor of IL-4 signal transduction. J. Immunol. 162:3770. J. Immunol. 172:6744. 32. Venkataraman, C., S. Leung, A. Salvekar, H. Mano, and U. Schindler. 1999. 38. Daines, M. O., R. P. Andrews, W. Chen, S. A. El-Zayaty, and G. K. Hershey. Repression of IL-4-induced gene expression by IFN-␥ requires Stat1 activation. 2003. DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by an J. Immunol. 162:4053. interaction with a detergent-sensitive factor. J. Biol. Chem. 278:30971. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021