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Originally published online as doi:10.1189/jlb.0906583 on January 2, 2007

Published online before print January 2, 2007
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(Journal of Leukocyte Biology. 2007;81:1102-1110.)
© 2007 by Society for Leukocyte Biology

IL-4 induces a wide-spectrum intracellular signaling cascade in CD8+ T cells

Ana Acacia de Sa Pinheiro*, Alexandre Morrot*, Sumana Chakravarty*, Michael Overstreet*, Jay H. Bream*, Pablo M. Irusta{dagger},1 and Fidel Zavala*,1,2

* Department of Molecular Microbiology and Immunology, Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA; and
{dagger} Department of Human Science, Georgetown University Medical Center, Georgetown University, Washington, DC, USA

2 Correspondence: Department of Molecular Microbiology and Immunology, Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205, USA. E-mail: fzavala{at}jhsph.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-4 has distinct effects on the differentiation and functional properties of CD8+ T cells. In vivo studies have shown that it is critical for the development of protective memory responses against tumors and infections by Leishmania and Plasmodium parasites. The intracellular signaling events mediated by IL-4/IL-4 receptor (IL-4R) interactions on CD4+ T cells have been studied extensively; however, the nature of IL-4-induced signaling on CD8+ T cells has not been characterized. Using naïve, activated, as well as differentiated CD8+ T cells, we show that IL-4 has a strong in vivo and in vitro antiapoptotic effect on activated and resting CD8+ T cells. We demonstrate that IL-4 induces the phosphorylation of the IL-4R, which is followed by the activation of at least two distinct intracellular signaling cascades: the Jak1/STAT6 and the insulin receptor substrate/PI-3K/protein kinase B pathways. We also found that IL-4 induces the Jak3-mediated phosphorylation and nuclear migration of STAT1, STAT3, and STAT5 in naïve, activated, as well as differentiated, IFN-{gamma}-producing CD8+ T cells. The induction of this broad signaling activity in CD8+ T cells coincides with a transcriptional activity of suppressors of cytokine signaling genes, which are decreased significantly in comparison with CD4+ T cells. To our knowledge, this report constitutes the first comprehensive analysis of the signaling events that shape CD8+ T cell responses to IL-4.

Key Words: cytokine signaling • CTL • JAK/STAT • PI-3K


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-4, a member of the {gamma}-chain receptor cytokine family, is considered a crucial mediator of CD4+ Th2 T cell differentiation and suppression of IFN-{gamma}-producing CD4+ Th1 cells [1 ]. This cytokine also plays a pivotal role in differentiation of B cells, controlling the specificity of IgG class-switching and the development of memory B cells. In addition, several studies indicate that IL-4 helps to sustain the growth and prolongs the survival of CD4+ T and B cells [1 ]. To exert these functions, IL-4 binds and signals through a heterodimeric receptor complex formed by the IL-4 receptor (IL-4R) {alpha}-chain and an accessory chain, {gamma}-common chain (Type I receptor) or IL-13R{alpha}1 chain (Type II receptor) [2 , 3 ].

IL-4 also modulates important functions of CD8+ T cells, which compared with CD4+ T cells, undergo distinct differentiation pathways and exhibit radically different functional properties. Early in vitro studies described a number of effects of IL-4 on CD8+ T cells, including the induction of IL-4 secretion and enhancement of IL-2-induced proliferation [4 5 6 ]. Other studies suggested a role for IL-4 in the in vitro development of cytotoxic T cells [7 , 8 ], and some studies indicated that IL-4 could decrease cytolytic activity in vitro [9 ]. This cytokine was also shown to be necessary for the in vitro generation of memory CD8+ T cells [10 ]. It is important that in vivo studies using a murine model of mammary and colon carcinoma showed that cytotoxic T lymphocyte-mediated tumor immunity was abrogated or did not develop in the absence of IL-4 [11 ]. More recently, vaccination studies in mice using Leishmania donovani demonstrated that the development of CD8+ T cell-mediated, protective responses against this parasite was fully dependent on IL-4 [12 ]. Our studies with the rodent malaria parasite Plasmodium yoelii, using parasite-specific TCR transgenic CD8+ T cells, revealed a critical role of IL-4 in the generation of memory CD8+ T cell responses against the liver stages of this parasite [13 ]. Using IL-4R–/– mice, we demonstrated that IL-4 acts directly on activated, antiparasite CD8+ T cells through the IL-4R and promotes the survival of memory CD8+ T cells belonging to the effector/peripheral subset homing to nonlymphoid organs [14 ]. In these studies, we also found lower levels of the antiapoptotic molecule Bcl-xL in activated IL-4R–/– CD8+ T cells compared with wild-type cells, suggesting a role for IL-4 in the induction of intracellular mechanisms that rescue CD8+ T cells from apoptosis.

The intracellular signaling induced by IL-4/IL-4R interactions on CD4+ T cells has been characterized in great detail. The binding of IL-4 to the Type I receptor in CD4+ T cells induces the activation of the nonreceptor tyrosine kinase Jak1, which promotes the phosphorylation of STAT6. In addition, IL-4 activates the insulin receptor substrate-2 (IRS-2) pathway, which subsequently induces the activation of PI-3K. The Jak1/STAT6 and IRS-2/PI-3K pathways mediate IL-4-dependent Th2 differentiation and production of Th2 cytokines [15 , 16 ]. Both pathways are required for the proliferative activity of CD4+ T cells induced by IL-4, but they do not seem to participate in the IL-4-dependent rescue from apoptosis [16 ]. IL-4 can also induce a Jak3-dependent activation of STAT1 and STAT5, but this effect appears to be observed only in differentiated CD4+ Th2 cells [17 18 19 20 ]. Although the effects of IL-4 on CD4+ T cells have been examined extensively, the signaling events induced by IL-4 on CD8+ T cells have not been studied, in spite of increasing evidence indicating an important role for this cytokine in CD8+ T cell function and differentiation.

In this report, we describe studies aimed at identifying the signaling pathways activated by IL-4 in CD8+ T cells. Similar to CD4+ T cells, IL-4 induced the activation of the IRS-2/PI-3K/protein kinase B (PKB) and Jak1/STAT6 pathways in naïve, activated and differentiated, IFN-{gamma}-producing CD8+ T cells. It is surprising that IL-4 also induced Jak3-dependent activation of STAT1, STAT3, and STAT5 in CD8+ T cells, irrespective of their differentiation status. This broad activation of STAT family members coincided with reduced transcriptional levels of intrinsic suppressors of cytokine signaling (SOCS) in CD8+ T cells stimulated with IL-4 when compared with similarly treated CD4+ T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and parasites
BALB/c mice were purchased from Taconic (Germantown, NY, USA). The generation of P. yoelii-specific transgenic mice expressing a TCR specific for the SYVPSAEQI epitope using C57BL/6 oocytes was described elsewhere [21 ]. Original founders were crossed to BALB/c mice, and the F20 backcross generation was used for the experiments. The Institutional Animal Care and Use Committee of Johns Hopkins University (Baltimore, MD, USA) approved experiments with mice.

Adoptive transfer of transgenic cells and immunization
Spleen cells were obtained from SYVPSAEQI-specific TCR transgenic mice, and after purification, 106 tetramer+/CD8+ T cells were used for adoptive transfer experiments. Before transfer, the total number of transgenic cells in the spleen was determined by FACS analysis after staining with anti-CD8 antibodies and SYVPSAEQI tetramers. Immunization with P. yoelii (17X NL strain) sporozoites was done by i.v. injection of {gamma}-irradiated parasites.

T cell purification and in vitro activation
Spleen CD8+ T cells were purified through mouse T cell enrichment columns according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Briefly, splenocytes were depleted of CD4+ and B220+ cells by negative selection using FITC-labeled anti-CD4 and anti-B220 antibodies (BD PharMingen, San Jose, CA, USA) plus antifluorescein-conjugated magnetic beads (Miltenyi Biotec). The CD8+ T cells were finally purified from the pass-through by positive selection using PE-labeled, anti-CD8 antibody and anti-PE-conjugated magnetic beads (Miltenyi Biotec). Purification of CD4+ T cells was performed using the same protocol but using FITC-labeled, anti-CD8 antibodies in the negative selection and PE-labeled, anti-CD4 antibodies for positive selection (BD PharMingen). The purity of the respective T cell populations was 98% or higher. For the signaling experiments, the purified cells were cultured in vitro for 48 h at 37°C/5% CO2 in DMEM supplemented with 50 µM 2-ME, antibiotics, and 10% FBS in the presence of plate-bound, anti-CD3 (3 µg/ml) and soluble, anti-CD28 (3 µg/ml) antibodies (BD PharMingen) and 2% EL-4 supernatant as a source of IL-2 (30 U/mL). The cells were then divided in two flasks and expanded for 2 additional days. Some experiments were performed using Y26 cells, a memory T cell clone specific for malaria epitope SYVPSAEQI. These cells produce IFN-{gamma} but no IL-4. A total of 1 x 106 cells was cocultured with 1 x 106 A20 (B cell melanoma) cells loaded with the SYVPSAEQI peptide and 20 x 106 splenocytes in DMEM supplemented with 10% FCS and 2% EL-4 supernatant as described above.

Immunoprecipitation and Western blotting
Cultured cells were washed twice in DMEM alone and deprived of serum and cytokine for 3 h prior to stimulation with IL-4 (10 ng/ml; BD PharMingen) for different times as indicated. Cells were then harvested and lysed on ice for 20 min in 200 µL lysis buffer (20 mM Tris-HCl, pH 7.3, 50 mM NaCl, 2 mM DTT, 4 mM EGTA, 1% Nonidet-40, 10% glycerol) freshly supplemented with phosphatase and protease inhibitors (10 mM NaF, 1 mM Na3VO4, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 16 mM Pefabloc). Cell lysates were then cleared by centrifugation at 12,000 rpm for 10 min at 4°C, and the protein concentration of the supernatants was determined by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). In immunoprecipitation assays, lysates were incubated with 4 µg anti-IL-4R{alpha} antibody (BD PharMingen) overnight at 4°C. Immunocomplexes were precipitated with Protein A/G Plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h and eluted by boiling in the presence of 2x SDS-PAGE loading buffer. Proteins were resolved on SDS 7.5% or 10% premade acrylamide gels (Bio-Rad) and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were probed with primary, specific antibodies followed by HRP-labeled secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and visualized with SuperSignal West Pico substrate (Pierce, Rockford, IL, USA). For some experiments, detection sensibility was enhanced with Signal Femto substrate (Pierce). The probed membranes were stripped with Restore and Stripping solution (Pierce) for 20 min at 37°C and reprobed with a second antibody of choice to detect total levels of protein. All antiphospho-specific antibodies, anti-PKB, and anti-STAT1-, anti-STAT3-, and anti-STAT5-specific antibodies were purchased from Cell Signaling Technology. Anti-STAT6 antibody was obtained from BD PharMingen.

Isolation of nuclear and cytoplasmic fractions
Nuclear and cytoplasmic extracts from cells were obtained by using NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce) following the manufacturer’s instructions. Briefly, 200 µl ice-cold CER I reagent, supplemented with inhibitors, as described above, was added to the pellet of 5 x 106 cells. After 10 min of incubation on ice, 11 µL ice-cold CER II reagent was added to each tube and incubated on ice for an additional 1 min. Samples were centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatant containing the cytoplasmic fraction was transferred rapidly to a fresh, prechilled tube. The insoluble pellet was resuspended in 50 µL ice-cold NER reagent, freshly supplemented with the same inhibitors described above. The samples were vortexed vigorously and incubated on ice for 40 min. The nuclear extract was collected by centrifugation at 15,000 rpm for 10 min at 4°C.

Flow cytometry analysis
Annexin V-FITC, in combination with propidium iodide (PI), was used to quantitatively determine the percentage of cells undergoing apoptosis, as described previously [22 ]. Resting CD8+ T cells were cultured for different days in DMEM supplemented with 10% FCS in the presence or absence of IL-4 (10 ng/mL). Alternatively, cells were preincubated or not with Jak Inhibitor I (Jak Inh I) or Jak3 Inhibitor I (Jak3 Inh I; Calbiochem, San Diego, CA, USA) or LY294002 (Sigma Chemical Co., St. Louis, MO, USA) for 30 min prior to IL-4 addition. After treatments, 105 cells were resuspended in 1x binding buffer (BD PharMingen) and incubated with Annexin V-FITC for 15 min at room temperature in the dark, followed by PI staining. Cells were analyzed within 1 h in a FACSCalibur flow cytometer, and CELLQuest software (Becton Dickinson, San Diego, CA, USA) was used to analyze the data. Early apoptotic cells were stained with Annexin V alone, whereas late apoptotic cells were stained with Annexin V and PI.

Quantitative real-time PCR analysis
RNA was isolated using TRIzol® reagent (Invitrogen, San Diego, CA, USA), and first-strand cDNA synthesis was performed using RNA (2 µg), RT, and random hexamers, following the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR analysis was performed in ABI 7500 (Applied Biosystems) using cDNA as template; TaqMan Assays-on-Demand gene expression assay mix specific for SOCS1, SOCS2, SOCS3, or cytokine-inducible Src homology 2 (SH2) domain-containing protein (CIS); and TaqMan Universal PCR master mix (Applied Biosystems). SOCS copies per cell were calculated by extrapolation from standard curves generated using SOCS plasmid cDNA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4 exerts an antiapoptotic effect on CD8+ T cells in vitro
Our previous studies in mice immunized with malaria parasites demonstrated that in vivo-activated CD8+ T cells lacking the IL-4R undergo an accelerated contraction phase, which leads to the virtual disappearance of effector/peripheral memory CD8+ T cells residing in nonlymphoid organs. These antigen-activated, IL-4R-deficient CD8+ T cells expressed decreased levels of the antiapoptotic molecule Bcl-xL, thus suggesting an important in vivo, antiapoptotic effect of IL-4 [14 ].

We have extended these studies using an established IFN-{gamma}-producing CD8+ T cell clone (Y26) specific for the SYVPSAEQI epitope of the circumsporozoite protein of Plasmodium yoelii and confirmed the prosurvival effect of IL-4 in vitro. As shown in Figure 1A , Y26 cells supplemented with IL-4 remain alive in culture for a period of several days. In contrast, Y26 cells incubated without IL-4 underwent a progressive loss in viability, detectable at 24 h after cytokine withdrawal and becoming more evident by Day 6, when 98% of the cells were dead (Fig. 1A) . Flow cytometry analysis for binding of Annexin V to these cells was fully consistent with these results (Fig. 1B) , and a large number of cells (85%) became Annexin V+, i.e., apoptotic on Day 4 in the absence of IL-4 compared with only 14% when cells were incubated with IL-4. This effect appears to be limited to the survival of cells by preventing apoptosis, as IL-4 has no effect on the proliferation of CD8+ T cells, in sharp contrast to IL-2, which strongly promotes proliferation of these cells (Supplemental Fig. 1A).


Figure 1
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  		Figure 1. IL-4 exerts an antiapoptotic effect on CD8+ T cells. (A) Seven to 10 days after stimulation with the peptide SYVPSAEQI, as described [23 ], cloned CD8+ T cells (Y26) were incubated in DMEM supplemented with 10% FCS in the presence (•) or absence ({circ}) of IL-4 (10 ng/mL). At various time-points, the viability of cells was assessed using the Trypan blue dye exclusion test. These results are representative of two independent experiments. (B) Cells were stained with Annexin V-FITC, followed by PI for flow cytometry analysis. These results are representative of three independent experiments. (C) BALB/c mice received 106 tetramer+/CD8+ T cells and were immunized later with P. yoelii sporozoites. Twenty-five days after immunization, memory CD8+/tetramer+ T cells were purified from the spleen by magnetic sorting, and the purified cells were cultured in DMEM supplemented with 10% FCS in the presence or absence of IL-4 (10 ng/mL). On two subsequent days after treatment, cells were harvested and stained with Annexin V-FITC and analyzed by flow cytometry. These results are representative of three independent experiments.

 
Similar results were obtained in ex vivo experiments, in which naïve, transgenic CD8+ T cells expressing a TCR specific for the parasite epitope SYVPSAEQI were transferred into normal mice, which were immunized subsequently with irradiated malaria parasites. The primed CD8+ T cells were purified from the spleens by magnetic sorting and then incubated in vitro in the presence or absence of IL-4. Two days after incubation, we observed a high proportion of cells undergoing apoptosis (as indicated by increased binding of Annexin V), only when the growth medium lacked IL-4 (Fig. 1C) . These results are consistent with our in vivo studies, which demonstrated that CD8+ T cells lacking the IL-4R proliferate normally but undergo an accelerated rate of apoptosis [14 ]. Besides supporting the notion that IL-4 has an important, antiapoptotic function in CD8+ T cells, our results show that the prosurvival effects of this cytokine can be recapitulated in vitro. Incubation of CD8+ T cells with IL-4 also induced an increased expression of surface molecules such as the tyrosine phosphatases CD45 receptor A, B, and C, which are involved in TCR signaling (Supplementary Fig. 1B), as well as modulation of chemokine receptors, such as CCR5 and CCR7 (Supplementary Fig. 1C). Although the in vivo significance of these findings has yet to be determined, it is clear that IL-4 induces a pleiotropic response in CD8+ T cells.

IL-4 induces phosphorylation of the IL-4R{alpha} chain and activates the IRS-2/PI-3K/PKB pathway in CD8+ T cells
To characterize the initial signaling events involved in the response of CD8+ T cells to IL-4, we first studied the phosphorylation status of IL-4R after incubation with IL-4. Western blot analysis of immunoprecipitated IL-4R probed with antiphosphotyrosine antibodies confirmed that IL-4 induces tyrosine phosphorylation of the IL-4R{alpha} chain in CD8+ T cells (Fig. 2A ). Thus, similar to what has been reported in other cell types, IL-4 induces IL-4R activation in CD8+ T cells, a step necessary to recruit signaling molecules that bind directly to the activated complex.


Figure 2
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  		Figure 2. IL-4 induces phosphorylation of the IL-4R{alpha} chain and activation of the IRS/PI-3K/PKB pathway. Splenic CD8+ T cells purified from BALB/c mice were stimulated with immobilized antibodies against CD3 and CD28 (5 µg/mL each) for 48 h and maintained in culture. On Day 4 after activation, cells were serum-starved for 3 h and incubated with or without IL-4 (10 ng/mL). (A) Cell lysates were immunoprecipitated (IP) with anti-IL-4R{alpha} antibody followed by Western blot analysis (WB) using the same anti-IL-4R{alpha} antibody (bottom panel). The same filter was stripped and reprobed for phosphotyrosine using mouse antiphosphotyrosine (pTYR; top panel) and anti-IRS-2 mAb (middle panel). These results are representative of two independent experiments. (B) Cells were treated with IL-4 for 5 min after a 30-min pretreatment with Wortmannin (1 µM, Lane 4) or PI-3K inhibitor LY294002 (25 µM, Lane 5). Cells incubated without IL-4 (Lane 1) or with IL-4 alone for 5 min (Lane 2) or 10 min (Lane 3) served as controls. Immunoblots with anti-pSer473-PKB and anti-pThr308-PKB were performed on total protein separated by SDS-PAGE. Filters were then reprobed with anti-PKB antibody, and total PKB was quantified by densitometry using National Institutes of Health (NIH; Bethesda, MD, USA) Image software. Changes in levels of pSer473-PKB and pThr308-PKB in the presence or absence of specific inhibitors are plotted as percentage of maximum stimulation by IL-4 in the absence of inhibitors. These results are representative of two independent experiments.

 
Two major IL-4R signaling pathways involving IRS-2 and Jak/STAT6 molecules have been described. These proteins bind to the activated cytokine receptor and induce a series of molecular events that regulate target genes that affect cell proliferation and survival [24 ]. We investigated the recruitment of IRS-2 to the IL-4R complex in IL-4-stimulated CD8+ T cells by immunoprecipitating IL-4R{alpha} followed by immunoblotting using anti-IRS-2 antibodies. As shown in Figure 2A , addition of IL-4 to CD8+ T cell cultures results in increased association between IRS-2 and IL-4R{alpha}, indicating that IL-4 activates the IRS-2/PI-3K/PKB pathway in these cells.

IL-4 induces the activation of PKB in CD8+ T cells via the phosphorylation of PKB residues Ser473 and Thr308
Recruitment of IRS-2 to the IL-4R provides additional docking sites for downstream signaling intermediates such as the regulatory subunit of PI-3K, which leads to the activation of the PI-3K/PKB pathway. To determine whether PKB is activated by IL-4 in CD8+ T cells, we used mAb that recognize phosphorylated residues Ser473 and Thr308, which are a hallmark of PKB activation. As shown in Figure 2B , incubation of naïve, activated CD8+ T cells with IL-4 results in the phosphorylation of Ser473 and Thr308 residues of PKB. The phosphorylation of both residues shows a temporal increase reaching maximal levels at approximately 5 min after cytokine addition, suggesting that the activation of PKB in CD8+ T cells is an early event in the response to IL-4. Similar results were observed with the differentiated CD8+ T cell line Y26 (Supplemental Fig. 2).

PI-3K activation is reported to be an upstream event in PKB phosphorylation because of its ability to induce rapid and transient formation of phosphoinositides phosphorylated in Position 3, which serve as docking sites for pleckstrin homology domain proteins, such as PKB, in the plasma membrane [1 ]. To determine whether IL-4-induced PKB phosphorylation in CD8+ T cells is dependent on PI-3K activity, we used two selective, chemically unrelated inhibitors of PI-3K (LY294002 and Wortmannin) to block PI-3K function [25 ]. CD8+ T cells were pretreated for 30 min with LY294002 or Wortmannin prior to incubation with IL-4 for 5 min, and activation of PKB was assessed by immunoblot using the anti-pSer473 and -pThr308 phospho-specific antibodies. Under these conditions, both inhibitors completely abolished the IL-4-induced phosphorylation of PKB residues Ser473 and Thr308, indicating that these events are indeed PI-3K-dependent (Fig. 2B) . Taken together, these results indicate that exposure of CD8+ T cells to IL-4 leads to the activation of the IRS-2/PI-3K/PKB pathway.

Induction of the Jak/STAT pathway by IL-4 in CD8+ T cells
As Jak1 associates with IL-4R in CD4+ T cells [26 ], we analyzed the time course of Jak1 activation in CD8+ T cells after incubation with IL-4. As shown in Figure 3 , the phosphorylated form of Jak1, i.e., activated Jak1, was detected in cell lysates as early as 5 min after IL-4 incubation, and levels of phosphorylation declined after 15 min.


Figure 3
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  		Figure 3. IL-4 induces phosphorylation of Jak1 in CD8+ T cells, which when purified from BALB/c splenocytes by magnetic sorting, were stimulated with anti-CD3 and anti-CD28 before stimulation with IL-4, as described in Figure 2 . Cell lysates were subjected to SDS-PAGE followed by Western blotting with antiphospho-Jak1 (pJak1)-specific antibody. Filters were stripped and reprobed with anti-Jak1 antibodies to quantify total protein levels. Densitometry of the bands was performed using NIH Image software. Histograms depict temporal accumulation of phosphorylated Jak1 as a percentage of maximal stimulation by IL-4. Identical results were obtained in two independent experiments.

 
STAT proteins are well-characterized substrates of phosphorylated Jaks, and STAT6 is the prototypic IL-4-induced transcription factor downstream of the IL-4R in CD4+ T cells. The role of STATs in regulating target genes in CD8+ T cells, however, remains unclear. To identify STAT activation profiles in response to IL-4 in CD8+ T cells, protein extracts from cytoplasmic and nuclear fractions of cells incubated in the presence or absence of IL-4 were analyzed at different time-points by Western blotting using antibodies specific for different phospho-STAT proteins. The results showed that phosphorylated STAT6 was present in the cytoplasm and nucleus of IL-4-stimulated CD8+ T cells (Fig. 4A ). A nearly identical activation pattern is found in IL-4-stimulated CD4+ T cells, which display Jak-dependent phosphorylation of STAT6 in the cytoplasm, followed by its detachment from the receptor, dimerization, and translocation to the nucleus.


Figure 4
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  		Figure 4. IL-4 induces phosphorylation of various STAT proteins in CD8+ T cells. (A) CD8+ T cells were stimulated as described in Figure 2 , and cytoplasmic and nuclear extracts were prepared from total cell lysates. After SDS-PAGE, Western blotting was performed using antiphospho (p)-STAT6, antiphospho-STAT1, antiphospho-STAT3, and antiphospho-STAT5 antibodies. Filters were reprobed with specific antibodies against nonphosphorylated forms of the corresponding STAT proteins to measure levels of total protein. Identical results were obtained in three independent experiments. (B) Differentiated cloned CD8+ T cells (Y26) specific for the malaria epitope SYVPSAEQI were stimulated in vitro with antigen as described in Materials and Methods. In the resting phase, cells were challenged with IL-4 after 3 h starvation, as described in Figure 2 . Western blotting against STAT proteins was performed as described in A. Identical results were obtained in two independent experiments. (C) Purified CD8+ T cells were pretreated or not with Jak Inh I (100 nM) or Jak3 Inh I (100 µM) for 30 min prior to challenge with IL-4 as described in Figure 2 . Western blotting was performed on total cell lysates separated by SDS-PAGE, using specific antibodies against phosphorylated STAT1, STAT3, STAT5, and STAT6. Filters were reprobed with anti-STAT1 or anti-STAT6 to assess total protein levels. Identical results were obtained in two independent experiments.

 
An unexpected and intriguing finding was that in addition to STAT6, we found IL-4-induced phosphorylation of STAT1, STAT3, and STAT5 in naïve, activated CD8+ T cells (Fig. 4A) . We did not, however, detect the activation of STAT2 or STAT4. As shown, all activated STAT proteins translocated to the nucleus. It is important that before adding IL-4 to CD8+ T cells, the cells were centrifuged and resuspended in fresh culture medium to eliminate the possibility that other cytokines present in the supernatant might affect the action of IL-4. In addition, to confirm the specificity of the IL-4-induced activation of different STAT proteins in CD8+ T cells, a control sample was included in which IL-4 was neutralized before addition to the cell suspension using an IL-4-specific antibody (11B11). After antibody neutralization, the phosphorylation of STAT1, STAT3, STAT5, and STAT6 was abolished, thus confirming the cytokine specificity of STAT activation (Supplemental Fig. 3). Identical results were obtained after IL-4 stimulation of differentiated CD8+ T cells (Y26; Fig. 4B ). The Y26 cells are a fully differentiated Tc1 line derived from a mouse, which was hyperimmunized with parasites expressing the epitope SYVPSAEQI. As described previously [23 ], these cloned T cells exhibit typical features of Tc1 cell types, i.e., produce high levels of IFN-{gamma} and TNF-{alpha} and traces of IL-2 and do not secret IL-4. Their phenotype is typical of memory cells, including CD44high CD62Llow CD122high CCR7high. These cells produce perforin, are strongly cytotoxic in vitro, and exhibit a strong, antiparasite activity in vivo when transferred into mice challenged with parasites.

In hematopoietic cells, Jak proteins that associate with the Type I IL-4R include not only Jak1, but also Jak3. To determine whether Jak3 was involved in IL-4-dependent STAT activation in CD8+ T cells, we analyzed the phosphorylation of various STATs in the presence of two potent inhibitors of Jak proteins, Jak Inh I, a broad specificity inhibitor that blocks Jak1 and Jak3 activation [27 ], and Jak3 Inh I, which is specific for Jak3 [28 ]. As shown in Figure 4C , the phosphorylation of all STATs was blocked by Jak Inh I, as expected, in view of its broad specificity. However, treatment of cells with Jak3 Inh I abolished only the activation of STAT1, STAT3, and STAT5 but did not prevent STAT6 phosphorylation. These results strongly suggest that IL-4 induces the activation of Jak1 and Jak3 in CD8+ T cells, the latter being responsible for the activation of STAT1, -3, and -5 but not STAT6, which seems to be Jak1-dependent. Attempts to demonstrate phosphorylation of Jak3 by Western blot analysis proved to be technically challenging. The results using commercially available anti-Jak3 antibodies were inconclusive, as it was not possible to differentiate IL-4-induced Jak3 phosphorylation consistently from background levels.

As different signaling intermediates downstream of the IL-4R complex are activated in CD8+ T cells in response to IL-4, we investigated their respective roles in the antiapoptotic effect of IL-4 following the approach described in previous other studies using signaling inhibitors [29 ]. We found that 24 and 48 h after incubation of CD8+ T cells with the nonspecific Jak Inh I (100 nM), the specific Jak3 Inh I (100 µM), or the PI-3K inhibitor LY294002 (25 µM), the antiapoptotic effect of IL-4 was partially blocked (Supplemental Fig. 4). These data raise the possibility that the effect of IL-4 in sustaining the survival of activated CD8+ T cells may require the activation of the IRS-2/PI-3K/PKB and the Jak/STAT pathways. Studies in other cell types also suggest that Jak/STAT as well as IRS-2/PI-3K pathways mediate the generation of signals involved in growth and cell survival [30 31 32 33 ].

Low-level transcription of SOCS genes in CD8+ T cells
In view of our findings indicating that IL-4 induces the simultaneous activation of several STAT proteins in CD8+ T cells, we examined the transcriptional status of SOCS genes in these cells. The SOCS family of intracellular proteins, particularly CIS, SOCS1, SOCS2, and SOCS3, modulates IL-4 signaling in CD4+ T cells, and it is believed that they suppress cytokine-induced signaling pathways by inhibiting Jak activity or STAT phosphorylation directly [34 , 35 ]. Using a quantitative real-time RT-PCR assay, we compared SOCS transcription levels in CD8+ and CD4+ T cells in the absence or presence of IL-4 and found that under the same experimental conditions, CD8+ T cells displayed a greatly diminished induction of CIS, SOCS1, -2, and -3 gene transcription in comparison with CD4+ T cells (Fig. 5 ). These results raise the possibility that the differences in signaling that appear to exist between CD8+ and CD4+ T cells may be in part a result of a decreased inhibitory activity of SOCS in CD8+ T cells. Thus, CD8+ T cells may not be subject to the same strong pressure for the development of cell subsets that is seen in CD4+ T cells.


Figure 5
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  		Figure 5. Low-level transcription of SOCS genes in CD8+ T cells. CD8+ and CD4+ T cells purified from BALB/c splenocytes by magnetic sorting were stimulated with anti-CD3 and anti-CD28, as described in Figure 2 . On Day 4, cell samples were serum-starved for 3 h and incubated with or without IL-4 (10 ng/mL) for 60 min. Cells were then lysed with TRIzol reagent, and total RNA was isolated and reverse-transcribed. cDNA templates were used to quantify mRNA levels of various SOCS family members by real-time PCR using TaqMan-specific primers. SOCS mRNA levels were normalized to the housekeeping gene GAPDH. Values are plotted after conversion to number of copies per cell, assuming 10 pg total RNA per typical mammalian cell. Histograms represent the mean of triplicate samples ± SEM. Statistical significance was evaluated using a Student’s t-test (*, P<0.01; **, P<0.001; ***, P<0.0001). Comparable results were obtained in three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although signal transduction triggered by IL-4 has been studied extensively in CD4+ T cells, the signaling events induced by this cytokine in CD8+ T cells have not been characterized. To our knowledge, this report constitutes the first comprehensive analysis of the molecular events that shape a CD8+ T cell response to IL-4. Although some aspects of IL-4-induced signaling in CD8+ T cells recapitulate previous findings in CD4+ T cells, our studies have also revealed certain features that appear to be unique to CD8+ T cells. It is surprising that IL-4R engagement on these cells seems to recruit a wider spectrum of signaling intermediates than is typically observed in naïve CD4+ T cells stimulated with IL-4.

Our results show that IL-4-induced signaling in CD8+ T cells involves tyrosine phosphorylation of the IL-4R and subsequent binding of IRS-2 to the activated receptor complex, which can, in turn, act as a cytosolic docking site for downstream SH2 domain-containing signaling molecules [36 ], such as PI-3K. Indeed, IL-4 induces PI-3K activity, as shown by Western blotting analysis, demonstrating the specific phosphorylation of PKB, a downstream substrate of PI-3K, at residues Ser473 and Thr308. This notion received further support by the results of experiments indicating that PKB phosphorylation was inhibited by Wortmannin and LY294002, two independent PI-3K inhibitors.

IL-4 also activates the Jak/STAT pathway in CD8+ T cells, which is perhaps the best-characterized IL-4-driven signaling cascade in CD4+ T cells. In CD8+ T cells, IL-4 signaling results in the activation of STAT6 [37 , 38 ], following a distinct pattern of tyrosine phosphorylation and nuclear migration. Jak1, not Jak3, seems to be the primary kinase mediating this process, as Jak3 inhibition does not affect STAT6 phosphorylation. A most surprising finding is that STAT1, STAT3, and STAT5 are also activated in naïve, activated and differentiated CD8+ T cells in response to IL-4, as demonstrated by Western blot analysis using antiphospho-specific antibodies for each STAT protein. Unlike STAT6 phosphorylation, the activation of STAT1, STAT3, and STAT5 is blocked by Jak3 inhibition. It is important that the extensive STAT activation induced by IL-4 is abolished completely after neutralization of this cytokine with anti-IL-4 mAb, demonstrating that the observed effects are mediated strictly by IL-4.

The IL-4-induced activation of STATs other than STAT6, although unexpected, is not entirely without precedent, as this effect of IL-4 has been reported in other cell types including differentiated Th2 CD4+ T cells. IL-4 was found to be a potent inducer of STAT6 and STAT1 activation in Th2 but not in Th1 CD4+ T cells [17 ]. In a different study, IL-4-induced STAT5 was shown to be necessary for the expansion of Th2 CD4+ T cells [20 ]. Therefore, IL-4 appears to induce the activation of STAT6 alone in naïve-activated CD4+ T cells, and the activation of STAT1 and STAT5 is clearly detectable only in differentiated CD4+ T cells of the Th2 subtype. The activation by IL-4 of other STAT proteins, such as STAT5a and STAT5b, has also been observed in a pro-B cell line (Ba/F3), where they acquire specific DNA-binding properties by a mechanism dependent on the {gamma}c chain [19 ]. Other studies describe an IL-4-dependent, antiapoptotic role for the STAT3 transcription factor in human B cells [39 ], malignant glioma cells [40 ], and normal and tumor lung fibroblasts [41 ], although no such role for STAT3 has been defined in CD4+ T cells.

It is intriguing that CD8+ T cells respond to IL-4 stimulation with extensive and simultaneous activation of different STAT pathways, although the effect of IL-4 appears to be more restricted in CD4+ T cells. Cytokine-induced signal transduction is regulated by different classes of intracellular inhibitors, among which the SOCS gene family features prominently in response to IL-4, controlling the Jak/STAT pathway [34 , 35 ]. CIS, SOCS1, SOCS2, and SOCS3 are constitutively expressed in naïve CD4+ T cells, and their gene transcription is differentially regulated by IL-4 [17 ]. Th2-polarized cells stimulated with IL-4 respond with a strong activation of STAT1, SOCS1, and SOCS3, suggesting a tight control of signaling activity [17 ]. Our study indicates that CD8+ T cells, compared with CD4+ T cells, have lower basal, transcriptional levels of SOCS, which do not increase appreciably after IL-4 stimulation, indirectly suggesting a diminished, inhibitory activity on intracellular signaling by SOCS molecules in these cells.

The physiological consequence of the IL-4-mediated, wide-spectrum signaling in CD8+ T cells has yet to be fully elucidated. This is a rather complex and challenging task, as indicated by studies performed during decades on CD4+ T cells in which the functional roles of the different signaling pathways induced by IL-4 still remain controversial and are yet to be clarified. The results of previous in vivo and in vitro studies describing multiple effects of IL-4 on CD8+ T cells suggest that this cytokine may play a number of yet unsuspected roles in the development of CD8+ T cell responses. A role for IL-4 in ensuring the survival of memory CD8+ T cells may be particularly crucial, as CD8+ T cells undergo an intense post-priming proliferation phase, which is followed immediately by an abrupt contraction phase, in which most activated cells die, presumably as a result of apoptosis. Wide-spectrum signaling pathways, which translate in the generation of antiapoptotic mediators, would be an efficient, physiological mechanism to ensure an efficient development of memory populations. Alternatively, IL-4 might direct discrete specializations within activated CD8+ T cell populations to enable effective memory differentiation. Further studies are required to fully unravel the overarching impact of IL-4 on the activity of CD8+ T cells in different animal systems.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant AI44375. A. A. S. P. received a fellowship from CNPq Brazil (PDE, 200169/04-1). The authors thank Dr. Diane Griffin for her comments and discussion of the manuscript.


   FOOTNOTES
 
1 These authors co-directed this study. Back

Received September 21, 2006; revised November 3, 2006; accepted December 7, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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