Suppressor of Cytokine Signaling-1 Regulates Signaling in Response to Interleukin-2 and Other {gamma}c-dependent Cytokines in Peripheral T Cells*

Ann L. Cornish {ddagger}, Mark M. Chong, Gayle M. Davey, Rima Darwiche, Nicos A. Nicola {ddagger}, Douglas J. Hilton {ddagger}, Thomas W. Kay, Robyn Starr {ddagger} and Warren S. Alexander {ddagger} §

From the The Walter and Eliza Hall Institute of Medical Research and {ddagger}Cooperative Research Centre for Cellular Growth Factors, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia

Received for publication, March 24, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressor of cytokine signaling-1 (SOCS-1) is an essential regulator of cytokine signaling. SOCS-1-/- mice die before weaning with a complex disease characterized by fatty degeneration and necrosis of the liver. This disease is mediated by interferon (IFN) {gamma} as neonatal mortality fails to occur in SOCS-1-/-IFN{gamma}-/- mice. However, the immune system of healthy SOCS-1-/-IFN{gamma}-/- mice is dysregulated with a reduced ratio of CD4:CD8 T cells and increases in some aspects of T cell activation. SOCS-1-/-IFN{gamma}-/- mice also die before their wild type and IFN{gamma}-/- counterparts with a range of inflammatory conditions including pneumonia, gut infiltration, and skin ulceration, suggesting that SOCS-1 controls not only IFN{gamma} signaling, but also other immunoregulatory factors. This study shows that T cells from SOCS-1-deficient mice display hypersensitivity to cytokines that act through the {gamma}c receptor. SOCS-1 expression is induced by interleukin (IL) 2, IL-4, IL-7, and IL-15, and SOCS-1-deficient T cells show increased proliferation and prolonged survival in response to IL-2 and IL-4. Furthermore, IL-2 induced increased STAT5 phosphorylation and CD44 expression in SOCS-1-deficient T cells compared with controls. Hypersensitivity to {gamma}c-dependent cytokines may contribute to abnormal T cell function, as well as the pathology observed in mice lacking SOCS-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressor of cytokine signaling-1 (SOCS-1)1 is an important negative regulator of cytokine signaling. Studies in vitro have shown that SOCS-1 can be induced by and inhibit signaling initiated by a wide range of cytokines including interleukin-2 (IL-2), IL-4, IL-6, growth hormone, leukemia inhibitory factor, prolactin, interferon {alpha}/{beta} (IFN{alpha}/{beta}), and IFN{gamma} (reviewed in Ref. 1). Studies using mice lacking SOCS-1, however, have suggested a more specific role for SOCS-1 in vivo. SOCS-1-deficient mice die before weaning with a complex neonatal disease characterized by fatty degeneration of the liver, lymphocyte activation, and hematopoietic infiltration in several tissues (2, 3). This disease is dependent on IFN{gamma} as mice lacking both SOCS-1 and IFN{gamma} do not develop liver disease and survive in apparent health into adult life (4). Disease in SOCS-1–/– mice is characterized by increased production of IFN{gamma} resulting in higher levels of IFN{gamma} in the serum, and hypersensitivity to IFN{gamma} (5, 6). Although IFN{gamma} plays a key role in neonatal disease in SOCS-1/ mice, mice lacking both SOCS-1 and IFN{gamma} die prematurely with a variety of inflammatory conditions including skin ulceration, pneumonia, and hematopoietic infiltration of the gut (7). This suggests that in vivo SOCS-1 not only controls IFN{gamma} signaling, but also regulates other aspects of immune cell function.

Recently, we have shown that SOCS-1 influences T cell homeostasis (8). Mice lacking SOCS-1 have a decreased ratio of CD4:CD8 T cells, and expression of the activation marker CD44 is increased on T cells (5, 9, 10). Furthermore, proliferation in vivo of both CD4+ and CD8+ cells is increased in peripheral lymphoid organs of SOCS-1/IFN{gamma}/ mice. These defects occur not only in sick SOCS-1/ mice but are evident also in healthy SOCS-1/IFN{gamma}/ mice (8). The apparent activation state of T cells in SOCS-1-deficient mice is antigen-independent. T cell receptor (TCR)-transgenic OT-I SOCS-1/ mice, in which T cells respond specifically to an exogenous antigen, ovalbumin, still appear activated despite the absence of ovalbumin stimulation (8).

Cytokines have been shown previously to be important regulators of T cell homeostasis (reviewed in Ref. 11). Altered ratios of CD4:CD8 T cells can occur in instances where cytokine signaling is dysregulated. For example, IL-12 can increase the numbers of CD8+ cells in the thymus (12), and IFN{alpha}/{beta} can promote the survival of CD8+ T cells over that of CD4+ T cells (13). Indeed, in the absence of SOCS-1, IL-12 responses are dysregulated, with IL-12 inducing increased T cell proliferation and natural killer cell activity (14). However, even in the absence of IL-12 signaling in SOCS-1/STAT4/ mice, T cell function is still perturbed with a decreased CD4:CD8 ratio and increases in apparent activation (14). This suggests that, even though uncontrolled IL-12 signaling may contribute to T cell defects, other immunoregulatory factors are also important.

Cytokines that appear to play the most pivotal roles in controlling T cell function are those that signal through the common {gamma} chain receptor subunit ({gamma}c), that is IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (15, 16). Of these IL-2, IL-4, IL-7, and IL-15 show clear roles in T cell proliferation and survival (1721). Moreover, altered {gamma}c-dependent cytokine signaling can lead to changes in the ratio of CD4:CD8 T cells within the mouse. Excess IL-7, for example, induces a decreased CD4:CD8 ratio (22, 23), whereas the converse is evident in IL-15R-deficient mice (24).

To explore the mechanism underlying the decreased ratio of CD4:CD8 T cells and the increased proliferation and CD44 expression by T cells in mice lacking SOCS-1, responses to the {gamma}c-dependent cytokines IL-2, IL-4, IL-7, and IL-15 were investigated in T cells from healthy SOCS-1/IFN{gamma}/ mice. Relative to control cells, T cells from mice lacking SOCS-1 displayed increased sensitivity to {gamma}c-dependent cytokines, particularly IL-2. Thus, SOCS-1 appears not only to regulate IFN{gamma} signaling in vivo, but may also control responses to {gamma}c-dependent cytokines in peripheral T cells.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation and Maintenance of Mice
SOCS-1/IFN{gamma}/ mice were generated as described previously on a mixed 129/Sv and C57BL/6 genetic background (2, 4). IFN{gamma}/ mice on an inbred C57BL/6 background (C57BL/6-IFN{gamma}tm1Ts) were obtained from Jackson Laboratories via Monash University (25). For studies on a syngeneic genetic background, SOCS-1/ mice were backcrossed at least 10 generations to C57BL/6 mice and then mated with IFN{gamma}/ mice to produce mice deficient for both SOCS-1 and IFN{gamma}. These mice had an identical phenotype to SOCS-1/IFN{gamma}/ mice generated on a mixed 129/Sv and C57BL/6 genetic background, as described previously (4). SOCS-1lox/+ mice were generated as described (26). Briefly, the SOCS-1 coding region was replaced by a SOCS-1 coding region flanked by LoxP sites. Immediately after the 3' LoxP site, the human CD4 reporter gene (hCD4) was inserted. These mice were mated with CMV-Cre mice (27), allowing deletion of SOCS-1 in most tissues. CMV-Cre mice were backcrossed onto the C57BL/6 genetic background for at least 10 generations.

Mice were genotyped for SOCS-1 and IFN{gamma} alleles by Southern blot analysis of genomic DNA obtained from tail tips as described (4). Mice from matings with Cre transgenic mice were genotyped for their SOCS-1 allele as described (26). Briefly, mice were genotyped by Southern blot analysis from tail tips to discriminate between the SOCS-1+, SOCS-1, and SOCS-1lox alleles. Recombination of the SOCS-1lox allele was also confirmed by Southern blotting. Cre transgenic mice were genotyped by PCR as described (26).

Purification of T Cell Populations
Lymphoid cells were isolated from pooled inguinal, brachial, axillary, submandibular, and mesenteric lymph nodes from SOCS-1/IFN{gamma}/ or IFN{gamma}/ mice. Cells were sorted into CD4+CD44lo, CD4+CD44hi, CD8+CD44lo, or CD8+CD44hi T cell populations by FACS. Cells were stained with mAb for CD44, CD4 and CD8 (BD Pharmingen, San Diego, CA), each fraction was gated and live cells sorted by FACS. An aliquot of each population was re-analyzed by FACS to confirm the accuracy of the sort.

Cytokine Responses
Isolated cell populations were plated into 96-well plates at 5 x 104 cells/well in 100 µl/well of RPMI medium containing 10% (v/v) heat-inactivated fetal calf serum, 50 µM 2-mercaptoethanol, 20 µg/ml anti-mouse IL-2 (anti-mIL-2; R&D Systems, Minneapolis, MN), and various concentrations of recombinant human IL-2, and mouse IL-4, IL-7, and IL-15 (rhIL-2, rmIL-7, rmIL-15, Peprotech, Rocky Hill, NJ; rmIL-4, R&D Systems).

Proliferation Analysis—After 3 days of culture 1 µCi/well of [3H]thymidine (Amersham Biosciences) was added to each well for 17 h. After washing, cell associated radioactivity was measured using a scintillation counter (PerkinElmer Life Sciences).

Survival and CD44 Up-regulation—After 3.5 days of culture (i.e. at the same time as the proliferation assays were harvested), cells were stained with propidium iodide and mAb to CD44, and analyzed by FACS. Live cells were identified by the exclusion of propidium iodide.

SOCS-1 Expression
As part of the SOCS-1lox targeting strategy, SOCS-1 was replaced by the hCD4 reporter gene that is only expressed from the SOCS-1 promoter upon Cre recombination of the flanked LoxP sites. Cells from pooled lymph nodes and spleen were incubated with 10 µg/ml plate-bound anti-CD3 (clone 145–2C11), 5 units/ml rhIL-2 (rhIL-2; McKesson HBOC, Rockville, MD), 5 µg/ml rmIL-4, 2.5 ng/ml rmIL-7, and 10 ng/ml rhIL-15 for either 4 or 20 h. For some of the samples, 0.5 µg/ml neutralizing antibodies specific for IL-2 and/or IFN{gamma} (clone HB170) were added. Cells were stained with antibodies to CD4, CD8, and hCD4 and analyzed by FACS. Expression of hCD4 was used as a surrogate marker of SOCS-1 transcriptional activity.

Expression of Cytokine Receptors
Lymph node suspensions from either SOCS-1/IFN{gamma}/ or IFN{gamma}/ mice were analyzed by FACS for expression of cytokine receptors using the following antibodies: biotinylated anti-IL-4R (R&D Systems), biotinylated anti-IL-7R{alpha} and anti-{gamma}c chain receptor, and phycoerythrin-conjugated anti-IL-2R{alpha} and -R{beta} (BD Pharmingen).

Western Analysis
T cells were isolated from pooled lymph nodes and spleen of SOCS-1/IFN{gamma}/ or control IFN{gamma}/ mice by nylon wool purification and incubated with various concentrations of rhIL-2, mIL-7, or rhIL-15 for 30 min. Western analysis was performed as previously described (28). Briefly, cells were lysed using KALB buffer containing 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 2 µg/ml aprotinin, 5 mM NaF, and 2 mM Na3VO4. Protein extracts were resolved on a 7.5% polyacrylamide gel and transferred to nitrocellulose membrane. STAT5 activation was detected using a mouse mAb specific for phosphorylated STAT5A/B (clone ST5P-4A9; Zymed Laboratories Inc., South San Francisco, CA). Membranes were stripped with 20 mM Tris-HCl, pH 8.8, 1.5 M glycine, and 200 mM 2-mercaptoethanol, and reprobed with mouse mAb to STAT5A and STAT5B (clones ST5a-2H2 and ST5b-10G1 respectively; Zymed Laboratories Inc.) as loading controls.

Intracellular Calcium Flux in Response to TCR Cross-linking
Intracellular calcium flux was monitored as previously described (29). Briefly, indo-1/AM (Molecular Probes, Eugene, OR)-loaded lymph node cells were stained with mAb for CD4, CD8, and CD44 as described above and then stained with hamster anti-mouse CD3 (clone 145–2C11) at 50 µg/ml. Samples were run on the FACS machine for ~30 s to obtain a baseline reading, followed by TCR stimulation and calcium flux induction after cross-linking the anti-CD3 Ab with 25 µg/ml anti-hamster IgG (BD Pharmingen). Changes in intracellular calcium concentrations were monitored for 4 – 6 min by calculating the ratio of the fluorescence emissions of T cells at 425 nm and 530 nm by FACS.

Statistical Analyses
Data from in vitro assays were analyzed using a two-tailed Student's t test for independent events. Bonferroni adjustments for multiple testing were included.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SOCS-1 Expression Is Up-regulated by {gamma}c-dependent Cytokines—SOCS-1 expression has been shown to be induced by a number of cytokines (reviewed in Ref. 1), including members of the {gamma}c-dependent cytokine family IL-2 and IL-4 (3032). Recently, conditional SOCS-1-deficient mice were produced using the Cre-Lox system (26). These mice were crossed to CMV-Cre mice resulting in widespread deletion of SOCS-1. SOCS-1lox/+ CMV-Cre mice were used to assess SOCS-1 expression in response to {gamma}c-dependent cytokines. In these mice, the reporter gene hCD4 is under the regulation of the SOCS-1 promoter and thus provides a surrogate measure of SOCS-1 expression.

SOCS-1/hCD4 expression was up-regulated in T cells by the {gamma}c-dependent cytokines IL-2, IL-4, IL-7, and IL-15 (Fig. 1). Induction of SOCS-1 expression in response to IL-4 and IL-7 was seen in both CD4+ and CD8+ cells, whereas expression was restricted to CD8+ cells in response to IL-2 and IL-15 (Fig. 1). SOCS-1 expression was most strongly induced by IL-4. Ligation of the TCR through anti-CD3 binding also strongly induced SOCS-1 expression in both T cell subsets (Fig. 1), consistent with recent data (33). This expression, however, appears to be largely because of autocrine cytokine production by these cells in response to anti-CD3, as cytokine-induced SOCS-1 expression was almost completely abrogated in the presence of neutralizing antibodies to IL-2 and IFN{gamma} (Fig. 1).



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 1.
SOCS-1 expression is induced by {gamma}c-dependent cytokines. T cells expressing the hCD4 reporter under control of the SOCS-1 promoter from SOCS-1lox/+ CMV-Cre mice were treated with the indicated stimuli and analyzed by FACS.

 

Increased Proliferation of SOCS-1/IFN{gamma}/ T Cells in Response to {gamma}c-dependent Cytokines—We then examined whether biological responses to {gamma}c-dependent cytokines were perturbed in T cells from SOCS-1/IFN{gamma}/ mice compared with IFN{gamma}/ mice. SOCS-1/IFN{gamma}/ mice display immune defects, including a decreased ratio of CD4:CD8 T cells and increased expression of the activation marker CD44, which complicate direct comparisons with control cells. To overcome this difficulty, T cells were sorted into the following subsets, CD4+CD44lo, CD4+CD44hi, CD8+CD44lo, and CD8+CD44hi. T cells were treated with hIL-2, mIL-7, mIL-4, or mIL-15 (with endogenous mIL-2 effects blocked by a neutralizing anti-mIL-2 antibody) and proliferation was measured by [3H]thymidine incorporation (Fig. 2). SOCS-1/IFN{gamma}/ T cells were more sensitive than control cells to IL-2, with robust proliferation evident at 2–20 ng/ml, doses at which little response was observed for control IFN{gamma}/ T cells (Fig. 2A). The increase in proliferation was greatest in the CD8+CD44hi fraction, although significant increases were also seen in the CD8+CD44lo T cell subset. IL-4 also induced significantly greater proliferation in SOCS-1/IFN{gamma}/ CD8+ T cells compared with controls (Fig. 2B). Comparatively low levels of proliferation were induced by IL-7 and no difference in proliferation was observed between T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice (Fig. 2C). IL-15 induced proliferation only at the highest concentration used, 200 ng/ml, and proliferation was increased modestly in SOCS-1/IFN{gamma}/ CD8+CD44lo T cells (Fig. 2D).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2.
Increased proliferation of SOCS-1-/-IFN{gamma}-/- T cells in response to {gamma}c-dependent cytokines. T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice were sorted into the indicated subsets and stimulated with IL-2 (A), IL-4 (B), IL-7 (C), or IL-15 (D). Cellular proliferation was monitored by [3H]thymidine incorporation after 3.5 days, n = 3, *, p < 0.05.

 

Increased Survival of SOCS-1/IFN{gamma}/ T Cells in Response to {gamma}c-dependent Cytokines—Survival of T cells was compared by propidium iodide exclusion after treatment with IL-2, IL-4, IL-7, and IL-15 (Fig. 3). In general, cell survival was promoted by all 4 cytokines examined, with IL-4 and IL-7 having the strongest effect. IL-2 mainly induced survival of CD8+ cells, and survival of CD8+CD44hi cells from SOCS-1/IFN{gamma}/ mice appeared to be modestly enhanced compared with those from control IFN{gamma}/ mice (Fig. 3A). IL-4-induced survival was increased significantly in SOCS-1/IFN{gamma}/ T cells, and this difference was most pronounced in CD4+ cells (Fig. 3B). In contrast, there was no difference in IL-7-induced survival (Fig. 3C) and only minor increases in IL-15-induced survival were observed in SOCS-1/IFN{gamma}/ CD8+ T cells (Fig. 3D).



View larger version (23K):
[in this window]
[in a new window]
 
FIG. 3.
Increased survival of SOCS-1-/-IFN{gamma}-/- T cells in response to {gamma}c-dependent cytokines. T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice were sorted into the indicated subsets and stimulated with IL-2 (A), IL-4 (B), IL-7 (C), or IL-15 (D). Cellular survival was determined as a percentage of live cells (negative for propidium iodide staining) by FACS after 3.5 days, n = 3, *, p < 0.05.

 

Increased CD44 Expression by SOCS-1/IFN{gamma}/ T Cells in Response to {gamma}c-dependent Cytokines—Mice lacking SOCS-1 express high levels of CD44 on peripheral T cells, which is most pronounced in cells of the CD8+ lineage. This occurs not only in sick SOCS-1/ mice, but also in healthy SOCS-1/IFN{gamma}/ mice and in TCR-transgenic OT-I SOCS-1/ mice not exposed to a specific antigen, suggesting that up-regulation occurs independently of disease, IFN{gamma}, and specific TCR stimulation by antigen (8). CD44 up-regulation was examined on sorted CD44lo T cells from healthy SOCS-1/IFN{gamma}/ mice after {gamma}c-dependent cytokine stimulation. In control cells, only minor changes in CD44 expression were induced by treatment with these cytokines. In SOCS-1/IFN{gamma}/ T cells, however, CD44 expression was strongly induced by IL-2 and to a lesser extent IL-15, but not by IL-4 or IL-7 (Fig. 4). This was only evident in CD8+ T cells (Fig. 4, A, D, and E).



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 4.
Increased CD44 expression by SOCS-1-/-IFN{gamma}-/- T cells in response to {gamma}c-dependent cytokines. T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice were sorted into the indicated subsets and treated with IL-2 (A), IL-4 (B), IL-7 (C), or IL-15 (D), n = 3, *, p < 0.05. E, expression of CD44 by CD8+CD44lo cells following cytokine treatment for 3.5 days.

 

Elevated Responses to {gamma}c-dependent Cytokines Are Not because of Increased Receptor Expression—To assess whether the changes in sensitivity to {gamma}c-dependent cytokines were due merely to changes in receptor expression, the expression of receptor subunits by SOCS-1-deficient T cells was examined (Fig. 5). The receptor subunit {gamma}c is shared by the IL-2, IL-4, IL-7, and IL-15 cytokine receptor complexes, whereas IL-2R{beta} is required for signaling by IL-2 and IL-15, IL-7R{alpha} is specific for IL-7 and IL-2R{alpha} for IL-2 signaling. There appeared to be a higher level of IL-2R{beta} expression on SOCS-1-deficient CD8+ T cells (Fig. 5). From previous studies, it is known that IL-2R{beta} expression is increased on CD44hi T cells, especially from the CD8+ lineage (20, 34). When T cells were separated into CD44lo and CD44hi populations, no difference was observed in IL-2R{beta} expression from CD8+ T cell subpopulations from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice. Therefore, the difference observed in the level of expression of IL-2R{beta} in the total CD8+ population correlated with increased numbers of CD8+CD44hi T cells in SOCS-1/IFN{gamma}/ mice. Expression of the receptor subunit {gamma}c and the IL-4R differed slightly on CD8+ T cells, however, no difference was observed when CD44lo and CD44hi CD8+ cells were examined separately. No differences in IL-2R{alpha} or IL-7R{alpha} expression levels by T cells were observed between control and SOCS-1-deficient populations. Thus, differences in cytokine sensitivity cannot be attributed to changes in receptor expression levels.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 5.
The expression of {gamma}c-dependent cytokine signaling receptor components is not increased on SOCS-1-/-IFN{gamma}-/- T cells. Lymph node T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice were examined by FACS for the expression of IL-2R{alpha}, IL-2R{beta}, IL-4R, IL-7R{alpha}, and {gamma}c.

 

Increased STAT5 Phosphorylation in Response to {gamma}c-dependent Cytokines—Given the enhanced biological responses of SOCS-1-deficient T cells to {gamma}c-dependent cytokines, biochemical studies were performed to determine whether loss of SOCS-1 resulted in increased downstream signaling after {gamma}c-dependent cytokine stimulation. As SOCS-1 acts to negatively regulate the JAK-STAT pathway, the activation status of STAT5 in response to {gamma}c-dependent cytokines was observed in T cells lacking SOCS-1. STAT5 phosphorylation was enhanced in cells lacking SOCS-1 in response to IL-2 and IL-7. STAT5 phosphorylation in SOCS-1-deficient cells was both more intense and occurred in response to lower concentration of these cytokines (Fig. 6, A and B). Phosphorylation of STAT5 in response to IL-15 was also more intense in SOCS-1 deficient cells, but the increase was less pronounced than for IL-2 and IL-7 (Fig. 6C). These results were also seen in T cells lacking SOCS-1 derived from SOCS-1lox/ lck-Cre mice, compared with SOCS-1-replete T cells from control SOCS-1lox/ mice (data not shown).



View larger version (55K):
[in this window]
[in a new window]
 
FIG. 6.
Increased STAT5 phosphorylation in response to {gamma}c dependent cytokines in SOCS-1-deficient T cells. T cells from SOCS-1/IFN{gamma}/ and IFN{gamma}/ mice were treated with IL-2, IL-7, or IL-15 at the indicated concentrations for 30 min. STAT5 phosphorylation was determined by Western blotting, and compared with total STAT5 levels.

 

TCR Signaling Is Not Enhanced in SOCS-1/IFN{gamma}/ T Cells—T cell activation occurs through the specific interaction of the TCR and antigen presented by major histocompatibility complex molecules upon antigen presenting cells. This interaction results in a number of downstream signaling events to induce specific gene activation through the transcription factors NF{kappa}B, NFAT, and AP-1. These signaling events include kinase cascades activating the mitogen-activated protein kinase pathway and increases in intracellular calcium flux. Interestingly, studies in vitro have identified several components implicated in the TCR signaling pathway, namely Syk, CD3{zeta}, and Tec, as potential binding partners of SOCS-1 (35, 36). In addition, reconstitution of the TCR signaling pathway using overexpression of Syk and a chimeric CD8/CD3{zeta} protein in 293T cells was inhibited by the addition of SOCS-1 (35). SOCS-1, therefore, may negatively regulate signals directly downstream of the TCR. In this case, SOCS-1-deficient cells may be partially activated in the absence of cytokine, and may be more responsive to mitogenic signals.

To determine whether increased sensitivity of SOCS-1-deficient cells to {gamma}c-dependent cytokines was secondary to defective TCR signaling, we examined calcium flux in response to TCR ligation, an early event in T cell activation, in T cells lacking SOCS-1. T cells were gated upon their expression of the markers CD4, CD8, and CD44 and intracellular calcium flux monitored in response to anti-CD3 ligation of the TCR. There was no difference in the performance of each T cell subset to stimulation of the TCR (Fig. 7). It seems unlikely, therefore, that increased responses to {gamma}c-dependent cytokines is secondary to unregulated signaling through the TCR.



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 7.
Intracellular calcium flux in response to TCR ligation is not increased in SOCS-1-deficient T cells. Indo-1-loaded lymph node cells were incubated with anti-CD3, analyzed by FACS briefly to obtain a baseline reading, and then anti-CD3 antibody was cross-linked with an anti-hamster antibody at the indicated time point to induce TCR signaling. Changes in intracellular calcium concentrations were monitored by calculating the ratio of the fluorescence emissions at 425 and 530 nm by FACS.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
T cells from healthy SOCS-1/IFN{gamma}/ mice display features of T cell activation including enhanced expression of CD44 and increased T cell proliferation in vivo (8). Classical activation of T cells occurs through antigen stimulation of the TCR. Despite having apparent features of activation, however, T cells from healthy SOCS-1/IFN{gamma}/ mice show little change in CD25 and CD69 expression, and exhibit no significant effector function such as cytotoxic activity (8). Increases in activation marker expression could reflect dysregulated TCR signaling in SOCS-1-deficient cells, but we found no evidence that SOCS-1 regulates signals directly downstream of the TCR. In addition, T cells from TCR-transgenic SOCS-1/ mice show increases in CD44 expression in the absence of specific antigen stimulation of the TCR (8).

Cytokines that signal through the {gamma}c receptor are critical for normal T cell development and function of the immune system (15) and hence are prime candidates for mediating these T cell defects. {gamma}c-dependent cytokines share common receptor subunits and exhibit redundancy in many of their activities, however, they also exhibit non-redundant functions. For example, IL-2, IL-4, IL-7, and IL-15 are all important for T cell proliferation, however, IL-2 is a particularly potent T cell mitogen, IL-4 is a key cytokine mediating Th2 functions of CD4+ cells, IL-7 is critical for the survival of thymocyte progenitors and naïve T cells, and IL-15 is important for the homeostasis of memory CD8+ T cells (reviewed by Ref. 15). We have demonstrated that in peripheral T cells, SOCS-1 is induced by stimulation with {gamma}c-dependent cytokines. To assess responses to {gamma}c-dependent cytokines in the absence of SOCS-1, we measured proliferation, survival, CD44 expression, and STAT activation in SOCS-1/IFN{gamma}/ T cells in response to IL-2, IL-4, IL-7, and IL-15.

The major finding from this study is that T cells lacking SOCS-1 are hypersensitive to IL-2. Using in vitro approaches, we showed that IL-2 treatment was able to induce immune changes similar to those seen in SOCS-1/IFN{gamma}/ mice in vivo. T cells from healthy SOCS-1/IFN{gamma}/ mice displayed increases in proliferation, up-regulation of CD44, and slight increases in survival in response to IL-2. Furthermore, IL-2-induced proliferation of CD8+ cells was much stronger than that of CD4+ cells, suggesting that this response to IL-2 may contribute to the perturbed CD4:CD8 T cell ratio in SOCS-1/IFN{gamma}/ mice.

Consistent with a role for SOCS-1 in the negative regulation of IL-2 signaling, several studies have shown a biochemical interaction between SOCS-1 and the IL-2 signaling pathway. SOCS-1 is postulated to act via two separate (although not necessarily mutually exclusive) mechanisms to inhibit JAK activity. The first is by direct binding to a phosphotyrosine residue in the activation loop of the JAK followed by inhibition of kinase activity (37), and the second is by targeting the JAKs for degradation via the ubiquitin-proteasome pathway (38). SOCS-1 has been shown to bind to IL-2R{beta} and can inhibit IL-2 signaling if overexpressed in IL-2-responsive cells (30). SOCS-1 has also been shown to associate with and inhibit the kinase activity of JAK1 and JAK3, through which the {gamma}c-dependent cytokines signal (30). Moreover, IL-2 signaling is regulated by proteasomal degradation possibly by targeting JAK1 or JAK3 to ubiquitin-mediated degradation (30, 39).

Interestingly, hIL-2 treatment in mice results in phenotypic changes similar to SOCS-1/ mice, including fatty degeneration and necrosis of the liver, tissue infiltration by hematopoietic cells, and thrombocytopenia (40). Therefore, uncontrolled IL-2 signaling, in addition to effects on T cell regulation, may contribute to these IFN{gamma}-dependent pathologies in SOCS-1/ mice.

T cell proliferation and survival were also increased in SOCS-1/IFN{gamma}/ T cells in response to IL-4. This finding is consistent with previous studies that have found SOCS-1 to regulate IL-4, both in vitro and in vivo (3, 4143). Although not as potent as IL-2 for inducing proliferation, IL-4 strongly sustained the survival of SOCS-1/IFN{gamma}/ T cells. Small increases in T cell proliferation, survival, and up-regulation of CD44 were observed also in SOCS-1/IFN{gamma}/ CD8+ T cells in response to IL-15. IL-15 was shown to be an important factor controlling the survival of CD8+ T cells with the generation of mice lacking IL-15 or IL-15R{alpha}, which have reduced numbers of memory CD8+ cells (24, 44). In addition, mice lacking the IL-15R{alpha} subunit have an increased CD4:CD8 ratio, whereas IL-15-transgenic mice have a reduced CD4:CD8 ratio (24, 45).

Biological responses to IL-7 were unaltered in peripheral T cells from SOCS-1/IFN{gamma}/ mice. This contrasts with the hypersensitivity of thymocytes to IL-7 in SOCS-1-deficient mice (26) and overexpression studies where IL-7 responses are inhibited by SOCS-1 (46, 47). Interestingly, SOCS-1 was induced after IL-7 treatment in T cells and T cells lacking SOCS-1 showed increased STAT5 phosphorylation, although no changes in biological responses were apparent. The lack of increased biological sensitivity to IL-7 in this study may reflect differences in the role of IL-7 in the thymus compared with the periphery (4850) and/or may imply that deregulated responses to IL-7 exist in biological responses that were not examined. IL-7 was effective at supporting the survival of T cells in vitro, but was a very poor proliferative stimulus. It has been noted that IL-7 is unable to induce the proliferation of isolated T cells in vitro (51), possibly because a specific micro-environment is required for the appropriate display of IL-7 to responding cells (52). STAT5 phosphorylation, however, was induced strongly in these cells. Previous work has shown that IL-7-induced proliferation and survival are not mediated through STAT5, but rather through the phosphatidylinositol 3-kinase/protein kinase B pathway, whereas IL-7-induced T cell differentiation in the thymus requires STAT5 (53, 54). It is possible, therefore, that SOCS-1 may regulate only the STAT5-dependent functions of IL-7, which have yet to be defined in mature, peripheral T cells.

Collectively, these data indicate that increased signaling by {gamma}c-dependent cytokines can induce T cell changes in vitro similar to those seen in SOCS-1/IFN{gamma}/ mice in vivo. The most profound changes were seen in response to IL-2, suggesting that hypersensitivity to this cytokine in the absence of SOCS-1 may contribute to the immune defects seen in these mice.


    FOOTNOTES
 
* This work was supported by the National Health and Medical Research Council, Canberra, Australia, National Institutes of Health Grant CA-22556, the Australian Federal Government Cooperative Research Centres Program, and National Health and Medical Research Council Program Grant 257500. 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. Back

§ To whom correspondence should be addressed: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia. Tel.: 61-3-9345-2555; Fax: 61-3-93452616; E-mail: alexandw{at}wehi.edu.au.

1 The abbreviations used are: SOCS-1, suppressor of cytokine signaling-1; IL, interleukin; IFN, interferon; TCR, T cell receptor; {gamma}c, common {gamma} chain receptor subunit; h, human; m, mouse; r, recombinant; FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody; STAT, signal transducers and activators of transcription; JAK, Janus kinase. Back


    ACKNOWLEDGMENTS
 
We thank Janelle Mighall and Sally Cane for first class technical assistance and Gabriela Panoschi, Andrew Naughton, Kathy Hanzinikolas, Catherine Tilbrook, and Katya Gray for expert animal husbandry.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alexander, W. S. (2002) Nat. Rev. Immunol. 2, 410 – 416[Medline] [Order article via Infotrieve]
  2. Starr, R., Metcalf, D., Elefanty, A. G., Brysha, M., Willson, T. A., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14395–14399[Abstract/Free Full Text]
  3. Naka, T., Matsumoto, T., Narazaki, M., Fujimoto, M., Morita, Y., Ohsawa, Y., Saito, H., Nagasawa, T., Uchiyama, Y., and Kishimoto, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15577–15582[Abstract/Free Full Text]
  4. Alexander, W. S., Starr, R., Fenner, J. E., Scott, C. L., Handman, E., Sprigg, N. S., Corbin, J. E., Cornish, A. L., Darwiche, R., Owczarek, C. M., Kay, T. W., Nicola, N. A., Hertzog, P. J., Metcalf, D., and Hilton, D. J. (1999) Cell 98, 597– 608[Medline] [Order article via Infotrieve]
  5. Marine, J. C., Topham, D. J., McKay, C., Wang, D., Parganas, E., Stravopodis, D., Yoshimura, A., and Ihle, J. N. (1999) Cell 98, 609 – 616[Medline] [Order article via Infotrieve]
  6. Brysha, M., Zhang, J. G., Bertolino, P., Corbin, J. E., Alexander, W. S., Nicola, N. A., Hilton, D. J., and Starr, R. (2001) J. Biol. Chem. 276, 22086 –22089[Abstract/Free Full Text]
  7. Metcalf, D., Mifsud, S., Di Rago, L., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 943–948[Abstract/Free Full Text]
  8. Cornish, A. L., Davey, G. M., Metcalf, D., Purton, J. F., Corbin, J. E., Greenhalgh, C. J., Darwiche, R., Wu, L., Nicola, N. A., Godfrey, D. I., Heath, W. R., Hilton, D. J., Alexander, W. S., and Starr, R. (2003) J. Immunol. 170, 878 – 886[Abstract/Free Full Text]
  9. Metcalf, D., Di Rago, L., Mifsud, S., Hartley, L., and Alexander, W. S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9174 –9179[Abstract/Free Full Text]
  10. Bullen, D. V., Darwiche, R., Metcalf, D., Handman, E., and Alexander, W. S. (2001) Immunology 104, 92–98[CrossRef][Medline] [Order article via Infotrieve]
  11. Jameson, S. C. (2002) Nat. Rev. Immunol. 2, 547–556[Medline] [Order article via Infotrieve]
  12. Godfrey, D. I., Kennedy, J., Gately, M. K., Hakimi, J., Hubbard, B. R., and Zlotnik, A. (1994) J. Immunol. 152, 2729 –2735[Abstract/Free Full Text]
  13. Marrack, P., Kappler, J., and Mitchell, T. (1999) J. Exp. Med. 189, 521–530[Abstract/Free Full Text]
  14. Eyles, J. L., Metcalf, D., Grusby, M. J., Hilton, D. J., and Starr, R. (2002) J. Biol. Chem. 277, 43735– 43740[Abstract/Free Full Text]
  15. He, Y. W., and Malek, T. R. (1998) Crit. Rev. Immunol. 18, 503–524[Medline] [Order article via Infotrieve]
  16. Habib, T., Senadheera, S., Weinberg, K., and Kaushansky, K. (2002) Biochemistry 41, 8725– 8731[CrossRef][Medline] [Order article via Infotrieve]
  17. Leung, D. T., Morefield, S., and Willerford, D. M. (2000) J. Immunol. 164, 3527–3534[Abstract/Free Full Text]
  18. Schluns, K. S., Kieper, W. C., Jameson, S. C., and Lefrancois, L. (2000) Nat. Immunol. 1, 426 – 432[CrossRef][Medline] [Order article via Infotrieve]
  19. Geginat, J., Sallusto, F., and Lanzavecchia, A. (2001) J. Exp. Med. 194, 1711–1719[Abstract/Free Full Text]
  20. Ku, C. C., Murakami, M., Sakamoto, A., Kappler, J., and Marrack, P. (2000) Science 288, 675– 678[Abstract/Free Full Text]
  21. Goldrath, A. W., Sivakumar, P. V., Glaccum, M., Kennedy, M. K., Bevan, M. J., Benoist, C., Mathis, D., and Butz, E. A. (2002) J. Exp. Med. 195, 1515–1522[Abstract/Free Full Text]
  22. Mertsching, E., Burdet, C., and Ceredig, R. (1995) Int. Immunol. 7, 401– 414[Abstract]
  23. Geiselhart, L. A., Humphries, C. A., Gregorio, T. A., Mou, S., Subleski, J., and Komschlies, K. L. (2001) J. Immunol. 166, 3019 –3027[Abstract/Free Full Text]
  24. Lodolce, J. P., Boone, D. L., Chai, S., Swain, R. E., Dassopoulos, T., Trettin, S., and Ma, A. (1998) Immunity 9, 669 – 676[Medline] [Order article via Infotrieve]
  25. Dalton, D. K., Pitts-Meek, S., Keshav, S., Figari, I. S., Bradley, A., and Stewart, T. A. (1993) Science 259, 1739 –1742[Medline] [Order article via Infotrieve]
  26. Chong, M. M. W., Cornish, A. L., Darwiche, R., Stanley, E. G., Purton, J. F., Godfrey, D. I., Hilton, D. J., Starr, R., Alexander, W. S., and Kay, T. W. H. (2003) Immunity, 18, 475– 487[Medline] [Order article via Infotrieve]
  27. Schwenk, F., Baron, U., and Rajewsky, K. (1995) Nucleic Acids Res. 23, 5080 –5081[Medline] [Order article via Infotrieve]
  28. Nicholson, S. E., Willson, T. A., Farley, A., Starr, R., Zhang, J. G., Baca, M., Alexander, W. S., Metcalf, D., Hilton, D. J., and Nicola, N. A. (1999) EMBO J. 18, 375–385[Abstract/Free Full Text]
  29. Newton, K., Kurts, C., Harris, A. W., and Strasser, A. (2001) Curr. Biol. 11, 273–276[CrossRef][Medline] [Order article via Infotrieve]
  30. Sporri, B., Kovanen, P. E., Sasaki, A., Yoshimura, A., and Leonard, W. J. (2001) Blood 97, 221–226[Abstract/Free Full Text]
  31. Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., Akira, S., and Kishimoto, T. (1997) Nature 387, 924 –929[CrossRef][Medline] [Order article via Infotrieve]
  32. Starr, R., Willson, T. A., Viney, E. M., Murray, L. J., Rayner, J. R., Jenkins, B. J., Gonda, T. J., Alexander, W. S., Metcalf, D., Nicola, N. A., and Hilton, D. J. (1997) Nature 387, 917–921[CrossRef][Medline] [Order article via Infotrieve]
  33. Diehn, M., Alizadeh, A. A., Rando, O. J., Liu, C. L., Stankunas, K., Botstein, D., Crabtree, G. R., and Brown, P. O. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11796 –11801[Abstract/Free Full Text]
  34. Zhang, X., Sun, S., Hwang, I., Tough, D. F., and Sprent, J. (1998) Immunity 8, 591–599[Medline] [Order article via Infotrieve]
  35. Matsuda, T., Yamamoto, T., Kishi, H., Yoshimura, A., and Muraguchi, A. (2000) FEBS Lett. 472, 235–240[CrossRef][Medline] [Order article via Infotrieve]
  36. Ohya, K., Kajigaya, S., Yamashita, Y., Miyazato, A., Hatake, K., Miura, Y., Ikeda, U., Shimada, K., Ozawa, K., and Mano, H. (1997) J. Biol. Chem. 272, 27178 –27182[Abstract/Free Full Text]
  37. Yasukawa, H., Misawa, H., Sakamoto, H., Masuhara, M., Sasaki, A., Wakioka, T., Ohtsuka, S., Imaizumi, T., Matsuda, T., Ihle, J. N., and Yoshimura, A. (1999) EMBO J. 18, 1309 –1320[Abstract/Free Full Text]
  38. Zhang, J. G., Farley, A., Nicholson, S. E., Willson, T. A., Zugaro, L. M., Simpson, R. J., Moritz, R. L., Cary, D., Richardson, R., Hausmann, G., Kile, B. J., Kent, S. B., Alexander, W. S., Metcalf, D., Hilton, D. J., Nicola, N. A., and Baca, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2071–2076[Abstract/Free Full Text]
  39. Yu, C. L., and Burakoff, S. J. (1997) J. Biol. Chem. 272, 14017–14020[Abstract/Free Full Text]
  40. Gately, M. K., Anderson, T. D., and Hayes, T. J. (1988) J. Immunol. 141, 189 –200[Abstract/Free Full Text]
  41. Losman, J. A., Chen, X. P., Hilton, D., and Rothman, P. (1999) J. Immunol. 162, 3770 –3774[Abstract/Free Full Text]
  42. Haque, S. J., Harbor, P. C., and Williams, B. R. (2000) J. Biol. Chem. 275, 26500 –26506[Abstract/Free Full Text]
  43. Naka, T., Tsutsui, H., Fujimoto, M., Kawazoe, Y., Kohzaki, H., Morita, Y., Nakagawa, R., Narazaki, M., Adachi, K., Yoshimoto, T., Nakanishi, K., and Kishimoto, T. (2001) Immunity 14, 535–545[CrossRef][Medline] [Order article via Infotrieve]
  44. Kennedy, M. K., Glaccum, M., Brown, S. N., Butz, E. A., Viney, J. L., Embers, M., Matsuki, N., Charrier, K., Sedger, L., Willis, C. R., Brasel, K., Morrissey, P. J., Stocking, K., Schuh, J. C., Joyce, S., and Peschon, J. J. (2000) J. Exp. Med. 191, 771–780[Abstract/Free Full Text]
  45. Marks-Konczalik, J., Dubois, S., Losi, J. M., Sabzevari, H., Yamada, N., Feigenbaum, L., Waldmann, T. A., and Tagaya, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11445–11450[Abstract/Free Full Text]
  46. Trop, S., De Sepulveda, P., Zuniga-Pflucker, J. C., and Rottapel, R. (2001) Blood 97, 2269 –2277[Abstract/Free Full Text]
  47. Fujimoto, M., Naka, T., Nakagawa, R., Kawazoe, Y., Morita, Y., Tateishi, A., Okumura, K., Narazaki, M., and Kishimoto, T. (2000) J. Immunol. 165, 1799 –1806[Abstract/Free Full Text]
  48. Akashi, K., Kondo, M., and Weissman, I. L. (1998) Immunol. Rev. 165, 13–28[Medline] [Order article via Infotrieve]
  49. Fry, T. J., and Mackall, C. L. (2001) Trends Immunol. 22, 564 –571[CrossRef][Medline] [Order article via Infotrieve]
  50. Vissinga, C. S., Fatur-Saunders, D. J., and Takei, F. (1992) Exp. Hematol. 20, 998 –1003[Medline] [Order article via Infotrieve]
  51. Surh, C. D., and Sprent, J. (2002) Microbes Infect. 4, 51–56[CrossRef][Medline] [Order article via Infotrieve]
  52. Dummer, W., Ernst, B., LeRoy, E., Lee, D., and Surh, C. (2001) J. Immunol. 166, 2460 –2468[Abstract/Free Full Text]
  53. Corcoran, A. E., Smart, F. M., Cowling, R. J., Crompton, T., Owen, M. J., and Venkitaraman, A. R. (1996) EMBO J. 15, 1924 –1932[Abstract]
  54. Pallard, C., Stegmann, A. P., van Kleffens, T., Smart, F., Venkitaraman, A., and Spits, H. (1999) Immunity 10, 525–535[Medline] [Order article via Infotrieve]