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* Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Department of Pathology and Medicine, Columbia University, New York, NY 10032;
Abramson Family Cancer Research Institute, Departments of Cancer Biology and Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
Center for Immunology, Cancer Center, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455
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Introduction |
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-chain (CD25), 
-chain (CD122), and the common 
-chain (CD132). Engagement of the IL-2R on activated T cells initiates a complex signaling program that can induce proliferation, increase survival, as well as prime for activation-induced cell death (2). More recently, studies on IL-2, CD25, and CD122 knockout mice have identified an essential role for IL-2 signals in the development and survival of CD4+CD25+ regulatory T cells (Tregs)4 (3, 4, 5). CD4+CD25+ Tregs are a distinct population of T lymphocytes that have the capacity to dominantly suppress the proliferation of responder T cells in vitro and inhibit autoimmune disease in vivo (6, 7). Despite expression of all three subunits of the high-affinity IL-2R, CD4+CD25+ T cells remain hypoproliferative when stimulated with IL-2 alone. However, combinations of signals, such as TCR and IL-2 or glucocorticoid-induced TNFR and IL-2, result in both the proliferation and the transient loss of suppressive characteristics (8, 9, 10). The molecular mechanism underlying these observations remains unknown.
The current signaling paradigm of IL-2-mediated proliferation and survival in activated T cells requires a coordinated effort between multiple signaling pathways downstream of the IL-2R (11, 12). The IL-2R has no intrinsic catalytic activity and relies on the ligand-mediated heterodimerization of the IL-2R to initiate activation of extrinsic signaling molecules, such as the Janus kinase (JAK). These initial events result in the subsequent activation of the transcription factor STAT5, as well as the recruitment of the phosphatidylinositol 3-kinase (PI3K) and Ras-mitogen-activated protein kinase (MAPK) signaling pathways (1, 13). This complex signaling system ultimately results in the up-regulation of genes that are critical for cell cycle progression and survival. Although IL-2 is required for the development and peripheral survival of CD4+CD25+ T cells, their hypoproliferative response to IL-2 in vitro suggests a differential pattern of IL-2R signaling compared with their activated T lymphocyte counterparts. However, there is currently no information available regarding IL-2R signal transduction in primary CD4+CD25+ T cells.
Given the central role of IL-2 in the biology of CD4+CD25+ T cells we sought to characterize the cellular and molecular responses of these cells to IL-2R signals.
In this report, we demonstrate that CD4+CD25+ T cells have a distinct IL-2R signaling pattern. Engagement of the IL-2R on Tregs results in the activation of the JAK/STAT signaling pathway, but fails to activate downstream targets of the PI3K signaling pathway, such as Akt or p70s6kinase. This failure to activate PI3K/Akt does not abrogate the antiapoptotic effect of IL-2 on Tregs. Examination of IL-2-dependent PI3K signaling in CD4+CD25+ T cells reveals that negative regulation of the PI3K signaling pathway is inversely associated with expression of the lipid phosphatase and tensin homologue deleted on chromosome 10, PTEN. Taken together, these results demonstrate a unique IL-2R signaling pattern, which may define how IL-2 mediates the development and survival of CD4+CD25+ T cells without inducing a significant mitogenic response in vitro.
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Materials and Methods |
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BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions in the animal facilities of the University of Pennsylvania, Philadelphia, PA.
Media, reagents, Abs, and flow cytometry
All cells were grown in RPMI 1640 (Mediatech Cellgro, Herndon, VA) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES (all from Life Technologies, Rockville, MD), 50 µM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). FITC and biotin-anti-CD25 (7D4), FITC and allophycocyanin-anti-CD4 (RM4-5), PE–anti-CD122 (TM-1), PE-anti-CD132 (4G3), Streptavidin-PE, purified anti-B220 (RA3), anti-MHC class II (M/5114), and purified anti-CD3 (2C11) were purchased from BD PharMingen (San Diego, CA). Murine rIL-2 was purchased from R&D Systems (Minneapolis, MN). Cells were analyzed on a Becton Dickinson FACSCaliber (BD Biosciences, Mountain View, CA) using FloJo software (Tree Star, San Carlos, CA). Anti-STAT5, anti-phospho-STAT5, anti-Akt, anti-phospho-Akt, anti-p70s6kinase, anti-phospho-p70s6kinase, anti-PTEN, and anti-actin Abs (all from Cell Signaling Technology, Beverly, MA), and anti-bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA) were used for Western blot analysis.
Cell purification
Lymph node and spleen cells were initially prepared by lysing erythrocytes with ACK lysis buffer (BioWhittaker, Walkersville, MD). Cell preparations were then enriched for T cells by negative selection. Single cell suspensions were incubated with purified anti-B220 (RA3) and anti-MHC class II (M/5114) (BD Pharmingen) at 1 µg/ml for 30 min at 4°C followed by incubation with microbead-conjugated goat anti-rat IgG (Polysciences, Warrington, PA) for 30 min at 4°C. Cell preps were placed on a magnetic stand for 10 min and the negative fraction was harvested. Single cell suspensions of 
3 x 108 were subsequently labeled with biotin-anti-CD25 (7D4), allophycocyanin-anti-CD4 (RM4-5), and Streptavidin-PE and purified by flow cytometry on a FACSVantage Cell Sorter (BD Biosciences). The percentage of CD4+CD25+ T cells was typically 4–6% of the overall T cell pool and the purity of sorted CD4+CD25+ T cells was consistently >95%. These cells functioned as Tregs when assessed for their ability to suppress CD4+CD25– T cell proliferation in vitro coculture assays (data not shown).
Survival assays
FACS-sorted CD4+CD25+ or CD4+CD25– cells (1 x 106 cells/ml) were cultured in complete medium in 48-well plates (Costar, Corning, Corning, NY) with or without 100 U/ml murine rIL-2 (R&D Systems) for 96 h. Absolute cell numbers were determined by counting cells on a hemocytometer using trypan blue (Sigma-Aldrich) exclusion. In addition, cells were stained with the vital dye 7-amino actinomycin D (7-AAD; Calbiochem, La Jolla, CA) and cell viability was assessed by flow cytometry.
CD3 and CD28 stimulation
Latex beads (Interfacial Dynamics, Portland, OR) were coated with either anti-CD3 (2 µg/ml) and/or anti-CD28 (10 µg/ml) as previously described (14), and resuspended at 3 x 106 beads/ml. FACS sorted CD4+CD25+ or CD4+CD25– cells (1 x 106 cells/ml) were rested overnight in complete medium at 37°C of 5% CO2 and then stimulated with anti-CD3 (10 µg/ml) and/or anti-CD28 (10 µg/ml) coated beads for the indicated times at a 3:1 ratio of beads to T cells. Cells were subsequently lysed and analyzed by Western blotting.
IL-2 stimulation and T cell priming plus IL-2 stimulation
FACS sorted CD4+CD25+ or CD4+CD25– cells (1 x 106 cells/ml) were rested overnight in complete medium at 37°C of 5% CO2 and then stimulated with 100 U/ml IL-2 (R&D Systems) for the time indicated, or cultured in 96-well plates (Costar, Corning) with T-depleted irradiated (2500 rads) BALB/c APCs (2 x 106 cells/ml) and 1 µg/ml anti-CD3 for 72 h at 37°C of 5% CO2. Cells were subsequently washed and rested overnight in complete medium, then stimulated with 100 U/ml IL-2 as previously mentioned. Cells were then lysed and analyzed by Western blotting (see below). "Primed" CD4+ T cells referred to in this study indicate CD4+CD25– T cells activated with anti-CD3, with or without anti-CD28 as indicated, to induce expression of the high-affinity IL-2R.
Western blotting analysis
Cells were lysed at 4°C at a concentration of 1 x 104 cells/µl. The lysis buffer was composed of 50 mM Tris-HCL (pH 6.8), 0.2% 2-mercaptoethanol, 20% glycerol, 4% SDS, and 0.001% bromphenol blue (all from Sigma-Aldrich). Cell lysates were clarified by centrifugation at 11,000 x g for 10 min. Supernatants were boiled for 10 min, separated on a 10% SDS-PAGE gel at 1 x 106 cell equivalents/well, and blotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The membranes were blocked overnight in blocking reagent (Boehringer Mannheim, Mannheim, Germany) at 4°C and probed with indicated Abs at 1/1000 dilution overnight at 4°C. Membranes were washed and probed with a HRP-conjugated anti-rabbit or anti-mouse Ab at 1:1000 for 60 min at room temperature. Blots were visualized by ECL (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol and on Hyperfilm ECL (Amersham Pharmacia Biotech). Abs were subsequently stripped off membranes for reprobing using Restore Western Blot Stripping buffer (Pierce, Rockford, IL), and reprobed as previously described.
PI3K assay
CD4+CD25+ T cells were isolated by flow cytometry as previously described. For control purposes, BALB/c total splenocytes were stimulated for 48 h with soluble anti-CD3 before washing and rested in serum-free medium for 4 h. Cells (6 x 106/sample) were stimulated with rIL-2 (R&D Systems) for the indicated time points and subsequently lysed in RIPA buffer at 4°C for 30 min. Debris was separated by centrifugation at 12,000 x g for 15 min at 4°C. To immunoprecipitate PI3K, lysates were incubated overnight at 4°C with 2.5 µg anti-p85 (Upstate Biotechnology, Lake Placid, NY) followed by incubation at 4°C for 4 h with 25 µl of protein A-agarose beads (15, 16). Precipitated protein pellets were washed twice with ice-cold RIPA lysis buffer and twice with ice-cold kinase buffer before resuspension in 20 µl of kinase buffer (40 mM HEPES, pH 7.5, 2 mM EGTA, 6 mM MgCl2, 1 mM DTT, 2.5 mM PMSF, 5 mM NaCl, 0.2 mM EDTA, and 10 µM unlabelled ATP). Lipid substrate (phosphatidylinositol; Matreya, State College, PA) was freshly prepared by the addition of 1% v/w cholic acid and sonication for 5 min on ice. A total of 10 µg of lipid substrate was added to each sample followed by incubation at 25°C for 10 min. This was followed by the addition of 20 µCi (0.74 MBq) of [-32P]ATP per sample and a further incubation of 20 min at 25°C. The reaction was stopped by the addition of 5 N HCl (20 µl) and was then extracted with 160-µl mixture of chloroform-methanol (60:100) and nonaqueous fraction was separated by thin layer chromatography and developed with CHCl3/MeOH/NH4OH/H2O (129:114:15:21). TLC plates were dried at room temperature and visualized by autoradiography.
DNA microarray hybridization and analysis
CD4+CD25+ T cells (3 x 106) were cultured in the presence or absence of rIL-2 (100 U/ml) for 12 h. Alternatively CD4+CD25– T cells were stimulated with plate bound anti-CD3 (10 µg/ml) for 24 h to induce functional IL-2R expression (data not shown) and washed extensively before culture in the presence or absence of rIL-2 (100 U/ml) for a further 12 h. After stimulation RNA was purified using RNeasy columns (Qiagen, Valencia, CA) according to manufacturers instructions. Total RNA was quantitatively amplified using two cycles of standard cDNA synthesis and in vitro transcription as previously described (17). In the second cycle of IVT synthesis, biotin-ribonucleotides were incorporated to produce labeled antisense RNA (Enzo Bioarray; Enzo Diagnostics, Farmingdale, NY). Labeled cRNA was fragmented at 94°C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 30 mM MgOAc, 100 mM KOAc) and subsequently hybridized to MG U74Av2 chips (Affymetrix, Santa Clara, CA) and stained with Streptavidin-PE as per manufacturers instructions. Analysis of DNA microarray data was conducted using Microarray Suite 5.0 software using manufacturer recommended parameters (Affymetrix). Two independent experiments were conducted for each stimulation condition. For alterations in expression to be classified as "real," a specific gene probe was required to be called as increased or decreased with a signal log ratio of at least one in two independent experiments. Fold increase or decrease in expression as shown in Table I is calculated from signal log ratio values.
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FACS-purified CD4+CD25– and CD4+CD25+ cells were cultured either in the presence of rIL-2 (100 U/ml) or plate bound anti-CD3 with or without anti-CD28 for 48 h. After 48 h cells were washed and replated either in the presence or absence of rIL-2 (20 U/ml) for a further 48 h. Cultures were pulsed with [3H]thymidine for the final 16 h and harvested. All proliferation experiments were set up in triplicate and results are expressed as a mean of triplicates. For cell cycle analysis, 1 x 105 freshly isolated or cultured CD4+CD25+ T cells were fixed in 1 ml of 70% cold ethanol, and stored at –20°C overnight. Samples were centrifuged at 250 x g and supernatants were aspirated. Cell pellets were resuspended with 400 µL of a solution containing: 0.5 ml of propidium iodide (20 µg/ml, Sigma-Aldrich), 1000 U of RNase A (10 mg/ml, heat-inactivated in TE buffer, Sigma-Aldrich), and 10 ml of buffer (1 g/ml glucose in 1x PBS). Cells were stained for 30 min at room temp and subsequently analyzed on a FACSCalibur for DNA content.
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Results |
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Recent work has demonstrated that IL-2 signals are required for the maintenance of CD4+CD25+ T cells in vivo (3, 4). To directly determine whether exogenous IL-2 could protect Tregs from apoptosis in vitro, FACS purified CD4+CD25+ or CD4+CD25– T cells were cultured in medium for 96 h with or without exogenous IL-2, subsequently stained with the vital dye 7-AAD, and examined by flow cytometry. Although the addition of IL-2 to CD4+CD25– T cells in culture had little effect on their viability, titration of exogenous IL-2 into cultures demonstrated that <3 U/ml IL-2 markedly increased the survival of CD4+CD25+ T cells (Fig. 1A). Similarly, absolute cell counts determined by trypan blue exclusion on a hemocytometer demonstrated that the addition of IL-2 to CD4+CD25+ T cells in culture over a 96-h period resulted in an 5-fold increase in the number of cells recovered when compared with cells cultured in medium alone (data not shown; p > 0.001). CD4+CD25+ T cells cultured in the presence of exogenous IL-2 exhibited marked cellular enlargement (Fig. 1B) and enhanced cell survival occurred without any appreciable cell division as assessed by CFSE dilution (Fig. 1B, right panel). These observations are consistent with the known inability of IL-2 alone to induce the proliferation of highly purified CD4+CD25+ T cells (7). The ability of IL-2 to promote the survival of CD4+CD25+ Tregs was associated with induction of bcl-xL protein (Fig. 1C), whereas the effects on bcl-2 levels were far more modest, with only a minimal and variable increase seen (Fig. 1D).
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The high-affinity IL-2R is a heterotrimeric complex composed of the -chain (CD25), -chain (CD122), and common -chain (CD132). Binding of IL-2 to the IL-2R initiates a complex signaling cascade that can drive the proliferation of Ag-activated T cells. Although CD4+CD25+ Tregs appear to constitutively express all three subunits of the IL-2R (3, 18 and data not shown), studies on CD4+CD25+ T cells have clearly demonstrated that these cells are hypoproliferative to IL-2 in vitro (7). To better understand the mechanism(s) by which CD4+CD25+ T cells remain hypoproliferative to IL-2 signals, we sought to characterize signaling events downstream of the IL-2R.
Previous studies on IL-2R signaling have shown that activation of both the JAK/STAT and PI3K/Akt pathways are critical for IL-2-induced proliferation (11, 12, 19, 20, 21). Thus, we examined the activation state of STAT5 and Akt downstream of the IL-2R in CD4+CD25+ T cells. To this end, purified CD4+CD25+ T cells or CD4+CD25– T cells, were rested overnight in medium and subsequently stimulated with 100 U/ml IL-2 for 30 min. In addition, to serve as a positive control for IL-2R signaling in this and future experiments, CD4+ T cells (designated "primed CD4+" in the study) were activated with anti-CD3 for a period of 72 h to induce expression of the high-affinity IL-2R, rested overnight, and then stimulated with IL-2 as indicated. Similar to primed CD4+ T cells, we observed expression of phosphorylated STAT5 in response to IL-2 in CD4+CD25+ T cells (Fig. 2A). Primed CD4+ T cells also phosphorylated Akt following incubation with IL-2. In contrast, we could not detect phosphorylated Akt in CD4+CD25+ T cells stimulated with IL-2 for 2, 10, or 30 min (Fig. 2A and data not shown).
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Previous studies on activated T cells have shown that PI3K signaling and activation of Akt are critical for the antiapoptotic effects of IL-2 (22). The ability of IL-2 to increase the viability of CD4+CD25+ T cells (Fig. 1, A and B, left panel) without the apparent activation of Akt (Fig. 2A) suggested that this effect did not require the activation of downstream PI3K targets. To confirm that the IL-2-mediated survival of Tregs was occurring in a PI3K-independent manner, CD4+CD25+ T cells were cultured with 100 U/ml IL-2 in the presence of increasing doses of the PI3K chemical inhibitor LY294002 (23) for 96 h. At concentrations sufficient to inhibit IL-2-mediated activation of Akt as well as proliferation of primed CD4+ T cells (Fig. 2C), we found that LY294002 did not inhibit IL-2-mediated survival of Tregs or primed CD4+ T cells (Fig. 2B).
Distinct transcriptional activity of IL-2-stimulated CD4+CD25+ Tregs
We have observed a distinct pattern of IL-2R signaling in Tregs. To broadly determine whether IL-2R signaling in CD4+CD25+ T cells was capable of initiating transcriptional activity, we compared the gene expression profile of IL-2-stimulated primed CD4+ T cells or CD4+CD25+ T cells. Using cDNA microarray analysis we found that IL-2R signaling altered transcription of significantly fewer genes in CD4+CD25+ cells when compared with primed CD4+ cells (71 vs 229) (Fig. 3A). This included both a reduction in the number of genes up-regulated (67 vs 120), and more strikingly, a large disparity in the number of genes down-regulated (4 vs 109). Further analysis of the gene array data revealed that the expression of 15 gene transcripts was altered both in CD4+CD25+ T cells and in primed CD4+ T cells after stimulation with IL-2 (see Table I). Significantly, of these 15 genes almost one-half (IL-2R, suppressor of cytokine signaling (SOCS)-1 and SOCS-2, cytokine-inducible SH2-containing protein, c-Myc, bcl-2, and TNF-) have been previously characterized to be downstream of JAK/STAT-mediated signaling pathways (24, 25, 26, 27, 28, 29). These data demonstrate that IL-2R signaling in CD4+CD25+ T cells, although differing markedly from that observed in primed CD4+ T cells, is sufficient to initiate transcription and results in the specific up-regulation of several known STAT5-dependent genes.
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Uncoupling of IL-2R signaling in Tregs occurs downstream of PI3K
The previous data indicate that IL-2 stimulation of CD4+CD25+ T cells does not activate downstream targets of the PI3K signaling pathway. Possible explanations for this observation include 1) CD4+CD25+ T cells are incapable of activating PI3K regardless of the stimulus, 2) a failure to activate PI3K specifically downstream of the IL-2R, or 3) downstream mediators of PI3K signaling, such as phosphatidylinositol 3,4,5-trisphosphate (PIP3), are negatively regulated in Tregs. To address these possibilities, we asked whether Akt could be activated in response to other stimuli, such as TCR signals or costimulation. FACS sorted CD4+CD25+ T cells were rested overnight in culture medium and stimulated with anti-CD3, anti-CD3 plus IL-2 or anti-CD3 plus anti-CD28 for 30 min. Cells were subsequently lysed and probed for phosphorylated Akt or phosphorylated STAT5 by Western blot. TCR signals alone or in conjunction with IL-2 did not activate Akt in Tregs (Fig. 4A). However, we could detect phosphorylated Akt in response to anti-CD3 plus anti-CD28 or anti-CD28 alone (Fig. 4A and data not shown), indicating that PI3K can be activated in CD4+CD25+ T cells. STAT5 phosphorylation confirmed our earlier observation that IL-2 is capable of STAT5 activation in CD4+CD25+ T cells. Thus, CD28 cross-linking is capable of activating the PI3K signaling pathway, ruling out an intrinsic defect in PI3K activity in Tregs.
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TCR priming down-regulates PTEN expression and facilitates IL-2-induced proliferation of CD4+CD25+ T cells
CD4+CD25+ T cells are hypoproliferative to either TCR or IL-2R stimulation alone, however both of these signals in combination restores the proliferative capacity of Tregs (7), which can be subsequently maintained by IL-2 alone (our unpublished observation). We have observed that PTEN expression levels inversely correlate with the proliferative response of primed CD4+ T cells to exogenous IL-2 as well as the hypoproliferative phenotype of CD4+CD25+ T cells to IL-2 signals. Therefore, we sought to determine whether TCR stimulation of CD4+CD25+ T cells altered the expression of PTEN. As shown in Fig. 5A, TCR or TCR/CD28 stimulation resulted in the down-regulation of PTEN after 72 h.
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We have demonstrated that TCR activation results in the down-regulation of PTEN and restores the ability of CD4+CD25+ T cells to proliferate to IL-2. Thus, we next asked whether TCR stimulation restored IL-2 mediated PI3K signaling. To this end CD4+CD25+ T cells were activated for 72 h with anti-CD3 and anti-CD28, rested in medium for 6 h and restimulated with IL-2 (Fig. 5C). In contrast to freshly isolated CD4+CD25+ T cells (Fig. 2A), activated CD4+CD25+ T cells phosphorylate Akt in response to IL-2. Taken together, these data demonstrate that TCR stimulation of CD4+CD25+ T cells primes the IL-2R for IL-2-mediated proliferation, coincides with the loss of PTEN, and the restoration of PI3K signaling downstream of the IL-2R.
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Discussion |
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-, -, and common -chain of the IL-2R (3, 18). Despite expression of the high-affinity IL-2R, initial characterization of CD4+CD25+ T cells demonstrated that these cells are hypoproliferative to IL-2 in vitro (7). One explanation for these data would be that Tregs do not express a "functional" IL-2R. However, subsequent studies have shown that CD4+CD25+ T cells require IL-2 and IL-2R signaling for their development and survival (3, 4, 5), suggesting that these cells are responsive to IL-2 signals. The mechanism(s) by which IL-2R signaling on CD4+CD25+ T cells promotes their development and survival, yet maintains their hypoproliferative phenotype is unknown. To that end, we have closely examined the cellular and molecular responses of CD4+CD25+ T cells to IL-2 in vitro. In this report we demonstrate that CD4+CD25+ T cells have a distinct IL-2R signaling pattern, which may in part explain their hypoproliferative phenotype while preserving IL-2-mediated survival. The molecular mechanism(s) that regulate cytokine or growth factor-induced cell cycle progression and survival remain incompletely understood. STAT5a and STAT5b are two closely related transcription factors that are activated by the IL-2R (35), and appear to be required for IL-2-mediated proliferation in activated T cells (12). Examination of STAT5a/STAT5b–/– T lymphocytes demonstrated a specific role for STAT5 in linking IL-2 signals to the cell cycle via the up-regulation of cell cycle proteins such as cyclin D2, cyclin D3, and cdk 6. However, these studies also demonstrated that assembly of the cell cycle machinery and cell cycle progression is not entirely dependent on STAT5 activation. Despite a defect in the expression of some cell cycle machinery, primed STAT5-deficient T cells are able to up-regulate the cell cycle proteins cdk2 and cdk4, as well as down-regulate p27kip in response to IL-2 (12). Thus, efficient cell cycle progression requires coordinated signaling from multiple signaling pathways. Similar to activated T cells, we find IL-2-mediated phosphorylation of STAT5 and up-regulation of the STAT5-dependent G1 cyclin D3 in CD4+CD25+ T cells. Additionally, we find that IL-2 stimulation initiates the transcription of several of STAT5-dependent genes, as well as the cell cycle associated transcription factor c-myc. Thus, we have concluded that the JAK/STAT5 signaling pathway appears to be intact in these cells.
We find that IL-2 stimulation of Tregs increases cellular survival, which coincides with the up-regulation of the antiapoptotic molecule bcl-xL. Chemical inhibitors of PI3K and MAPK signaling did not block the IL-2-mediated survival of CD4+25+ T cells (Fig. 2B and data not shown), thus, we concluded that JAK/STAT5 signaling pathway is critical for this IL-2 effect. Such a role of STAT5 in IL-2-mediated T cell survival has not been previously established. Examination of activated T cells expressing a mutated IL-2R, or from STAT5-deficient mice have shown that STAT5 activation is not necessary for IL-2-induced protection from apoptosis (2, 12). A clear caveat to these studies is that multiple signaling pathways are activated downstream of the IL-2R, and as such, STAT5-mediated survival may act in a redundant manner. In contrast, studies in cell lines expressing chimeric and/or mutated growth factor receptors demonstrated that activation of STAT5 protects cells from apoptosis via the up-regulation of bcl-2 and bcl-xL, independent of cell cycle progression (24). Likewise, further studies on STAT5-deficient mice have shown that activation of STAT5 by growth factors is required for the up-regulation of the antiapoptotic molecule bcl-xL and survival of erythroid progenitors (36).
The PI3K signaling pathway plays a central role in growth factor-mediated cellular functions such as, proliferation, survival, growth, and glucose homeostasis. Activation of PI3K results in the production of 3'-phosphoinositide lipids, such as PIP3 and phosphatidylinositol 4,5-bisphosphate, which serve as lipid second messengers. These second messengers bind to the plextrin homology domains of target proteins and directly recruit a wide variety of downstream effector molecules, such as Akt, PDK1, and Itk. Studies on both the IL-2R and CD28 have demonstrated that PI3K signaling and activation of Akt plays an important role in promoting T cell survival and proliferation (11, 21, 22). Akt (protein kinase B) is a serine/threonine kinase that is a downstream target of PI3K signaling. Activation of Akt is thought to promote survival through a number of mechanisms including up-regulation of bcl-2 (37), phosphorylation of proapoptotic bad, and its subsequent sequestration away from antiapoptotic bcl-2 family members (38) as well as negatively controlling the activity of proapoptotic Forkhead Family transcription factors (39). However, our results indicate that the PI3K/Akt pathway is dispensable in cytokine-mediated survival of Tregs. In support of our observations, we demonstrate that 1) CD4+CD25+ T cells do not activate Akt in response to IL-2, 2) the PI3K chemical inhibitor LY294002 does not block the IL-2-mediated survival of Tregs, and 3) IL-2 leads to significant up-regulation of bcl-xL without significantly altering bcl-2 expression levels in these cells.
Proximal IL-2R signaling in Tregs is sufficiently intact to activate PI3K, however downstream targets such as Akt do not get phosphorylated. Our data suggest that regulation of this signaling pathway occurs downstream of PI3K. The lipid phosphatase PTEN is a ubiquitously expressed protein, which has been characterized as a negative regulator of PI3K-mediated signaling, through its ability to dephosphorylate lipid substrates. PTEN (also referred to as mutated in multiple advanced cancers) is, as its name suggests, a potent tumor suppressor gene that is deleted or mutated in a variety of different forms of cancer (40). Complete inactivation of the gene in mice was found to be embryonic lethal and subsequent studies on hemizygous animals demonstrated a high incidence of spontaneous tumor development, impaired Fas-mediated apoptosis, as well as autoimmunity (41, 42).
In this study we demonstrate that the inability of IL-2 to stimulate signaling pathways downstream of PI3K coincides with relatively high expression of PTEN in Tregs. Moreover down-regulation of PTEN through TCR stimulation appears to be permissive for a restoration of IL-2-mediated PI3K-dependent signaling and proliferative capacity in Tregs. These observations are in agreement with earlier observations that a combination of TCR and IL-2 signals are sufficient to reverse the hypoproliferative phenotype of Tregs (7). It is interesting that such a combination of signals does not appear to significantly activate Akt in the short term, e.g., 30 min (Fig. 4A) perhaps due to maintained PTEN expression at these earlier time points. Indeed we have found that under such stimulation conditions PTEN degradation is detectable after 24 h and we also demonstrate that prolonged activation of Tregs (72 h) facilitates IL-2-dependent signaling downstream of PI3K (Fig. 5C).
IL-2 plays a critical role in the prevention of autoimmunity and the maintenance of lymphocyte homeostasis. One such mechanism for IL-2-mediated tolerance is via the development and survival of CD4+CD25+ T cells (3, 4, 5). In this study, we define a distinct pattern of IL-2R signaling in Tregs, which is regulated by PTEN and differs significantly from the established paradigm of IL-2R signaling. This signaling pattern appears to be crucial for the "anergic" phenotype of these cells to IL-2. Mice that are haploinsufficient for PTEN or have a T cell-specific PTEN deletion develop autoimmune disease (41, 43). In these models, it was demonstrated that effector T cells are hyperresponsive, however the Treg populations were not investigated. Thus, it will be of interest to determine the relative contribution of PTEN to the maintenance of T cell tolerance by specific T cell subsets. Our in vitro data suggest PTEN acts as a negative regulator of IL-2-mediated expansion of Tregs. Whether PTEN plays a role in their suppressive phenotype remains to be investigated. Taken together, these data describe a novel signaling paradigm that explains how IL-2 mediates the development and survival of CD4+CD25+ T cells without inducing a significant mitogenic response.
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Acknowledgments |
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Footnotes |
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2 S.J.B. and P.T.W. contributed equally to this work and should be considered co-first authors.
3 Address correspondence and reprint requests to Dr. Laurence A. Turka, Department of Medicine, University of Pennsylvania, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104-6144. E-mail address: turka{at}mail.med.upenn.edu
4 Abbreviations used in this paper: Treg, regulatory T cell; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; PIP3, phosphatidylinositol 3,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; SHIP, Src homology 2-containing inositol polyphosphate 5-phosphatase; JAK, Janus kinase; 7-AAD, 7-amino actinomycin; SOCS, suppressor of cytokine signaling.
Received for publication October 29, 2003. Accepted for publication February 24, 2004.
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  M. Pierau, S. Engelmann, D. Reinhold, T. Lapp, B. Schraven, and U. H. Bommhardt Protein Kinase B/Akt Signals Impair Th17 Differentiation and Support Natural Regulatory T Cell Function and Induced Regulatory T Cell Formation J. Immunol., November 15, 2009; 183(10): 6124 - 6134. [Abstract] [Full Text] [PDF]
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B. B. L. Pillemer, Z. Qi, B. Melgert, T. B. Oriss, P. Ray, and A. Ray STAT6 Activation Confers upon T Helper Cells Resistance to Suppression by Regulatory T Cells J. Immunol., July 1, 2009; 183(1): 155 - 163. [Abstract] [Full Text] [PDF]
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 S. Andre, D. F. Tough, S. Lacroix-Desmazes, S. V. Kaveri, and J. Bayry Surveillance of Antigen-Presenting Cells by CD4+CD25+ Regulatory T Cells in Autoimmunity: Immunopathogenesis and Therapeutic Implications Am. J. Pathol., May 1, 2009; 174(5): 1575 - 1587. [Abstract] [Full Text] [PDF]
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D. Kamimura and M. J. Bevan Endoplasmic Reticulum Stress Regulator XBP-1 Contributes to Effector CD8+ T Cell Differentiation during Acute Infection J. Immunol., October 15, 2008; 181(8): 5433 - 5441. [Abstract] [Full Text] [PDF]
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 P. Serafini, S. Mgebroff, K. Noonan, and I. Borrello Myeloid-Derived Suppressor Cells Promote Cross-Tolerance in B-Cell Lymphoma by Expanding Regulatory T Cells Cancer Res., July 1, 2008; 68(13): 5439 - 5449. [Abstract] [Full Text] [PDF]
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 S. Bessoles, F. Fouret, S. Dudal, G. S. Besra, F. Sanchez, and V. Lafont IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines J. Leukoc. Biol., July 1, 2008; 84(1): 224 - 233. [Abstract] [Full Text] [PDF]
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C. F. Zheng, G. J. Jones, M. Shi, J. C. D. Wiseman, K. J. Marr, B. M. Berenger, S. M. Huston, M. J. Gill, A. M. Krensky, P. Kubes, et al. Late Expression of Granulysin by Microbicidal CD4+ T Cells Requires PI3K- and STAT5-Dependent Expression of IL-2R{beta} That Is Defective in HIV-Infected Patients J. Immunol., June 1, 2008; 180(11): 7221 - 7229. [Abstract] [Full Text] [PDF]
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 S. Haxhinasto, D. Mathis, and C. Benoist The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells J. Exp. Med., March 17, 2008; 205(3): 565 - 574. [Abstract] [Full Text] [PDF]
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T. L. Sumpter, K. K. Payne, and D. S. Wilkes Regulation of the NFAT pathway discriminates CD4+CD25+ regulatory T cells from CD4+CD25- helper T cells J. Leukoc. Biol., March 1, 2008; 83(3): 708 - 717. [Abstract] [Full Text] [PDF]
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M. Marzec, K. Halasa, M. Kasprzycka, M. Wysocka, X. Liu, J. W. Tobias, D. Baldwin, Q. Zhang, N. Odum, A. H. Rook, et al. Differential Effects of Interleukin-2 and Interleukin-15 versus Interleukin-21 on CD4+ Cutaneous T-Cell Lymphoma Cells Cancer Res., February 15, 2008; 68(4): 1083 - 1091. [Abstract] [Full Text] [PDF]
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 M. Marzec, X. Liu, M. Kasprzycka, A. Witkiewicz, P. N. Raghunath, M. El-Salem, E. Robertson, N. Odum, and M. A. Wasik IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes Blood, February 15, 2008; 111(4): 2181 - 2189. [Abstract] [Full Text] [PDF]
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R. Zeiser, D. B. Leveson-Gower, E. A. Zambricki, N. Kambham, A. Beilhack, J. Loh, J.-Z. Hou, and R. S. Negrin Differential impact of mammalian target of rapamycin inhibition on CD4+CD25+Foxp3+ regulatory T cells compared with conventional CD4+ T cells Blood, January 1, 2008; 111(1): 453 - 462. [Abstract] [Full Text] [PDF]
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C. Quintarelli, J. F. Vera, B. Savoldo, G. M. P. Giordano Attianese, M. Pule, A. E. Foster, H. E. Heslop, C. M. Rooney, M. K. Brenner, and G. Dotti Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes Blood, October 15, 2007; 110(8): 2793 - 2802. [Abstract] [Full Text] [PDF]
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 J. Yates, F. Rovis, P. Mitchell, B. Afzali, J.-S Tsang, M. Garin, R. Lechler, G. Lombardi, and O. Garden The maintenance of human CD4+CD25+ regulatory T cell function: IL-2, IL-4, IL-7 and IL-15 preserve optimal suppressive potency in vitro Int. Immunol., June 1, 2007; 19(6): 785 - 799. [Abstract] [Full Text] [PDF]
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J. Andersson, I. Stefanova, G. L. Stephens, and E. M. Shevach CD4+CD25+ regulatory T cells are activated in vivo by recognition of self Int. Immunol., April 1, 2007; 19(4): 557 - 566. [Abstract] [Full Text] [PDF]
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A. L. Bayer, A. Yu, and T. R. Malek Function of the IL-2R for Thymic and Peripheral CD4+CD25+ Foxp3+ T Regulatory Cells J. Immunol., April 1, 2007; 178(7): 4062 - 4071. [Abstract] [Full Text] [PDF]
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N. K. Crellin, R. V. Garcia, and M. K. Levings Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells Blood, March 1, 2007; 109(5): 2014 - 2022. [Abstract] [Full Text] [PDF]
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L. Arruvito, M. Sanz, A. H. Banham, and L. Fainboim Expansion of CD4+CD25+and FOXP3+ Regulatory T Cells during the Follicular Phase of the Menstrual Cycle: Implications for Human Reproduction J. Immunol., February 15, 2007; 178(4): 2572 - 2578. [Abstract] [Full Text] [PDF]
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M. A. Burchill, J. Yang, C. Vogtenhuber, B. R. Blazar, and M. A. Farrar IL-2 Receptor beta-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells J. Immunol., January 1, 2007; 178(1): 280 - 290. [Abstract] [Full Text] [PDF]
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M. Battaglia, A. Stabilini, B. Migliavacca, J. Horejs-Hoeck, T. Kaupper, and M.-G. Roncarolo Rapamycin Promotes Expansion of Functional CD4+CD25+FOXP3+ Regulatory T Cells of Both Healthy Subjects and Type 1 Diabetic Patients J. Immunol., December 15, 2006; 177(12): 8338 - 8347. [Abstract] [Full Text] [PDF]
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J. L. Buckler, P. T. Walsh, P. M. Porrett, Y. Choi, and L. A. Turka Cutting Edge: T Cell Requirement for CD28 Costimulation Is Due to Negative Regulation of TCR Signals by PTEN J. Immunol., October 1, 2006; 177(7): 4262 - 4266. [Abstract] [Full Text] [PDF]
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E. Zorn, E. A. Nelson, M. Mohseni, F. Porcheray, H. Kim, D. Litsa, R. Bellucci, E. Raderschall, C. Canning, R. J. Soiffer, et al. IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo Blood, September 1, 2006; 108(5): 1571 - 1579. [Abstract] [Full Text] [PDF]
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S. P. Hickman, J. Yang, R. M. Thomas, A. D. Wells, and L. A. Turka Defective Activation of Protein Kinase C and Ras-ERK Pathways Limits IL-2 Production and Proliferation by CD4+CD25+ Regulatory T Cells J. Immunol., August 15, 2006; 177(4): 2186 - 2194. [Abstract] [Full Text] [PDF]
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N. Sugimoto, T. Oida, K. Hirota, K. Nakamura, T. Nomura, T. Uchiyama, and S. Sakaguchi Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis Int. Immunol., August 1, 2006; 18(8): 1197 - 1209. [Abstract] [Full Text] [PDF]
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J. Y.-S. Tsang, N. O. S. Camara, E. Eren, H. Schneider, C. Rudd, G. Lombardi, and R. Lechler Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells J. Leukoc. Biol., July 1, 2006; 80(1): 145 - 151. [Abstract] [Full Text] [PDF]
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S. R. Mayack and L. J. Berg Cutting Edge: An Alternative Pathway of CD4+ T Cell Differentiation Is Induced Following Activation in the Absence of {gamma}-Chain-Dependent Cytokine Signals J. Immunol., February 15, 2006; 176(4): 2059 - 2063. [Abstract] [Full Text] [PDF]
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L. Li, W. R. Godfrey, S. B. Porter, Y. Ge, C. H. June, B. R. Blazar, and V. A. Boussiotis CD4+CD25+ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy Blood, November 1, 2005; 106(9): 3068 - 3073. [Abstract] [Full Text] [PDF]
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B. Acres, M. Gantzer, C. Remy, N. Futin, N. Accart, O. Chaloin, J. Hoebeke, J.-M. Balloul, and S. Paul Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity Cancer Res., October 15, 2005; 65(20): 9536 - 9546. [Abstract] [Full Text] [PDF]
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L.-X. Wang, S. Shu, and G. E. Plautz Host Lymphodepletion Augments T Cell Adoptive Immunotherapy through Enhanced Intratumoral Proliferation of Effector Cells Cancer Res., October 15, 2005; 65(20): 9547 - 9554. [Abstract] [Full Text] [PDF]
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 S. P Hickman and L. A Turka Homeostatic T cell proliferation as a barrier to T cell tolerance Phil Trans R Soc B, September 29, 2005; 360(1461): 1713 - 1721. [Abstract] [Full Text] [PDF]
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F. Baratelli, Y. Lin, L. Zhu, S.-C. Yang, N. Heuze-Vourc'h, G. Zeng, K. Reckamp, M. Dohadwala, S. Sharma, and S. M. Dubinett Prostaglandin E2 Induces FOXP3 Gene Expression and T Regulatory Cell Function in Human CD4+ T Cells J. Immunol., August 1, 2005; 175(3): 1483 - 1490. [Abstract] [Full Text] [PDF]
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M. Battaglia, A. Stabilini, and M.-G. Roncarolo Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells Blood, June 15, 2005; 105(12): 4743 - 4748. [Abstract] [Full Text] [PDF]
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C. T. Duthoit, D. J. Mekala, R. S. Alli, and T. L. Geiger Uncoupling of IL-2 Signaling from Cell Cycle Progression in Naive CD4+ T Cells by Regulatory CD4+CD25+ T Lymphocytes J. Immunol., January 1, 2005; 174(1): 155 - 163. [Abstract] [Full Text] [PDF]
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