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-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells1
* Department of Laboratory Medicine and Pathology and Department of Pediatrics, Center for Immunology, Cancer Center and
Division of Hematology, Oncology, and Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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–/– mice have a significant decrease in Treg numbers. Ectopic expression of foxp3 in a subset of CD4+ T cells rescued Treg development and prevented autoimmunity in IL-2R–/– mice, suggesting that IL-2R-dependent signals regulate foxp3 expression in Tregs. Subsequent analysis of IL-2R-dependent signal transduction pathways established that the transcription factor STAT5 is necessary and sufficient for Treg development. Specifically, T cell-specific deletion of STAT5 prevented Treg development; conversely, reconstitution of IL-2R–/– mice with bone marrow cells expressing an IL-2R mutant that exclusively activates STAT5 restored Treg development. Finally, STAT5 binds to the promoter of the foxp3 gene suggesting that IL-2R-dependent STAT5 activation promotes Treg differentiation by regulating expression of foxp3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, or IL-2R leads to the unexpected development of lethal autoimmune disease (1, 2, 3). Subsequent studies by Malek et al. (4) established that the primary defect in IL-2R-deficient mice was the loss of functional Tregs, as they found that transfer of small numbers of wild-type (WT) Tregs into neonatal IL-2R-deficient mice completely prevented the onset of autoimmune disease. These findings suggested that IL-2/IL-2R signaling plays an important role in Treg biology.
Although IL-2 and IL-2R signaling are critical for the formation or maintenance of a functional Treg population, the exact role that IL-2R signaling plays in Treg biology has not yet been defined. Over the last few years, several studies have reported conflicting evidence about the role of IL-2 or IL-2R subunits in Treg development, homeostasis, or function. For example, Malek et al. (5) used a transgenic mouse line in which IL-2R expression was restricted to the thymus to address the role of IL-2 in Treg development. When these thymus-restricted IL-2R transgenic mice were crossed onto an IL-2R-deficient background, CD4+CD25+ Tregs were restored in both the thymus and spleen, and autoimmunity was prevented. These results suggest that IL-2/IL-2R signaling is required only for the development of Tregs in the thymus and that IL-2 is not required to promote their homeostasis and function in the periphery. In contrast, other work suggested a role for IL-2 in maintaining CD4+CD25+ Treg homeostasis. Specifically, administration of an IL-2-neutralizing Ab to WT mice significantly reduced the population of CD4+CD25+ T cells in the periphery (6). These studies suggested that IL-2 signaling is critical for the survival of Tregs. Conversely, two other groups demonstrated that IL-2–/– mice have either identical (7) or only slightly reduced numbers (8) of Tregs when compared with WT mice, suggesting that IL-2 is not required for Treg development or homeostasis. Finally, several groups have claimed that IL-2 is primarily required for the activation and function of Tregs. For example, studies by Lafaille and colleagues (9) demonstrated that the transfer of CD4+ T cells from IL-2–/– mice could prevent autoimmune disease in a mouse model that normally develops spontaneous experimental autoimmune encephalomyelitis. In contrast, transfer of IL-2R–/– CD4+ T cells did not prevent experimental autoimmune encephalomyelitis in this model, suggesting a requirement for IL-2 to support suppressive capacity. Likewise, using in vitro suppression assays, two separate groups demonstrated that neutralization of IL-2 inhibits Treg suppressor function in vitro (10, 11). Taken together, the published literature reveals ongoing uncertainty regarding the role of IL-2 and the IL-2R in the development, homeostasis, and/or function of Tregs.
Although the past few years have witnessed advances in the functional and phenotypic characterization of Tregs, the reliance on CD25 as a marker to identify these cells is a potential confounding factor because it is also expressed on activated effector T cells. More recently, the transcription factor Foxp3 has been identified as a unique marker for Tregs in both humans and mice (12, 13, 14, 15, 16, 17). Importantly, these studies further documented that Foxp3 expression was sufficient to convert CD4+CD25– T cells into CD4+CD25+ T cells that exhibited potent suppressor function and thereby established Foxp3 as a master regulator of Treg lineage commitment and function. Therefore, to identify how IL-2 signaling affects Treg biology, we used intracellular staining for Foxp3 to more accurately identify Treg populations in mice in which IL-2 signaling has been disrupted. As recently reported (7, 8), we also observed that CD4+Foxp3+ T cells are detectable in IL-2–/– mice. In contrast, we found that IL-2R–/– and 
c–/– mice have drastically reduced populations of CD4+Foxp3+ cells in the thymus and peripheral lymphoid organs. Consistent with these findings, IL-2–/– x IL-15–/– mice show a comparable Treg defect as that observed in IL-2R–/– mice. Thus, we demonstrate that IL-2 and IL-15 are redundant cytokines that can both signal through an IL-2R/common c (c) complex to promote the development of CD4+Foxp3+ Tregs.
Having established that IL-2R-dependent pathways are required for Treg development, we proceeded to identify key downstream effectors of this process. We found that the introduction of Foxp3 via transgenesis into a subset of IL-2R–/– CD4+ T cells restored Treg development and functional Treg suppressor activity, thereby inhibiting activation of the remaining CD4+Foxp3– T cells and preventing autoimmunity in vivo. These results demonstrate that Foxp3 expression alone, and not additional IL-2R-dependent signals, is sufficient for production of Tregs that have inhibitory activity. Furthermore, we provide evidence that IL-2R-dependent STAT5 activation is the key signaling event involved in this process. First, we demonstrate that T cell-specific expression of STAT5 is required for Treg development. Second, we demonstrate that STAT5 activation, achieved via expression of a constitutively active STAT5 transgene, is sufficient to restore CD4+CD25+Foxp3+ Tregs in the thymus of IL-2R–/– mice. Third, reconstitution of IL-2R–/– mice with bone marrow cells expressing IL-2R mutants that can only activate STAT5, and not other IL-2R-dependent signaling pathways, restored a population of CD4+CD25+Foxp3+ Tregs in both the thymus and spleen and was sufficient to prevent T cell activation. Finally, we demonstrate that STAT5 binds specifically to the foxp3 promoter in CD4+CD25+ Tregs. Taken together, our results provide strong evidence that IL-2R-dependent STAT5 activation is required for Treg development and that it does so via direct effects on foxp3 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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IL-2–/–, IL-2R–/–, IL-2R–/–, c–/–, and IL-7R–/– mice were obtained from The Jackson Laboratory and maintained in our breeding colony. In some experiments, IL-2–/– x rag-2+/– and IL-2R–/– x rag-2+/– mice were used; rag-2 heterozygosity had no effect on results since identical findings were obtained with IL-2–/– and IL-2R–/– mice. IL-15–/– and rag-2–/– mice were obtained from Taconic Farms. IL-2–/– x IL-15–/– mice were generated by crossing IL-2–/– x rag2–/– with IL-15–/– mice; STAT5b-CA x IL-2R–/– mice were generated by crossing STAT5b-CA x IL-2R+/– mice with IL-2R–/– x rag-2–/– mice. STAT5a/bFL/FL and CD4-Cre mice were provided by Dr. L. Hennighausen (National Institutes of Health, Bethesda, MD) and Dr. K. Hogquist (University of Minnesota, Minneapolis, MN), respectively. B6.C.H2bm12/KhEg (termed bm12) mice were purchased from The Jackson Laboratory. C57BL/6 mice were purchased from the National Institutes of Health or Taconic Farms. The foxp3 transgenic mice were generated as described (18) and provided by Dr. F. Ramsdell (Zymogenetics, Seattle, WA). All mouse strains used were backcrossed at least 10 generations onto the C57BL/6 genetic background except for STAT5a/bFL/FL mice. Mice used for analysis were 4–6 wk old, unless otherwise noted. The University of Minnesota Institutional Animal Care and Use Committee approved all animal experiments.
Intracellular flow cytometry
Spleen, thymus, and lymph nodes were harvested and single-cell suspensions were generated by disruption with ground glass slides. Before enumeration, RBC were removed from spleen preparations by ammonium chloride lysis. Abs against the following cell surface markers were obtained from eBioscience: CD8 (53-6.7), CD25 (PC61.5), and glucocorticoid-induced TNFR family-related gene (DTA-1). In some experiments, potential contaminating cells were removed by gating out cells expressing B220, CD19, GR-1, CD11b, TER-119, NK1.1, and TCR
. Additional Abs directed at the cell surface molecule CD4 (RM4-5) were obtained from BD Pharmingen. Following surface staining, samples were fixed and permeabilized overnight using the PE anti-mouse/rat Foxp3 staining set obtained from eBioscience. Following fixation, cells were stained with either PE-labeled rat/anti-mouse Foxp3 (FJK-16s) or PE-labeled rat IgG2a,
(P3) isotype control. Labeled cells were then collected and analyzed using a LSRII flow cytometer (BD Biosciences). Analysis of flow cytometry data was conducted using FlowJo software (Tree Star). Staining for intracellular phospho-STAT5 and phospho-Erk was done as previously described (19).
Proliferation assays
For proliferation assays involving IL-2R–/– x foxp3tg mice, CD4+CD25– T cells were isolated from lymph nodes (LN) using a wire mesh to obtain a single-cell suspension. Cells were resuspended in medium consisting of DMEM (BioWhittaker), 10% FBS (HyClone), 50 µM 2-ME (Sigma-Aldrich), 10 mM HEPES buffer, 1 mM sodium pyruvate (Invitrogen Life Technologies), amino acid supplements (1.5 mM L-glutamine, L-arginine, and L-asparagine; Sigma-Aldrich), and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Sigma-Aldrich). Importantly, the percentage of Foxp3+ cells in the sorted CD4+ IL-2R–/– x foxp3tg cells, CD4+foxp3tg cells, and WT CD4+CD25+ cells was roughly equivalent (63–75% Foxp3+). LN cells were depleted of NK cells (hybridoma PK136) and CD8+ T cells (hybridoma 2.43) by incubation with mAb, followed by passage through a goat anti-mouse and goat anti-rat Ig-coated column (Cedarlane Laboratories). CD25+ T cells were depleted using anti-CD25 PE (hybridoma PC61; BD Pharmingen) and MACS anti-PE MicroBeads followed by LS MACS separation columns (Miltenyi Biotec). T cell- and NK cell-depleted stimulator cells were prepared from bm12 spleens, incubated with anti-Thy1.2 mAb (hybridoma 30H-12) and NK mAb (hybridoma PK136) followed by incubation with rabbit complement (Nieffenegger). Cells were irradiated at 30 Gy. CD4 T cells from WT, foxP3tg, IL-2R–/–, and IL-2R–/– x foxP3tg mice were enriched from the spleen using anti-CD4 PE (hybridoma RM4-5) followed by MACS separation. Cells were further purified using a FACSAria sorter to 99% purity. One hundred thousand CD4+CD25– T cells were mixed with equal numbers of bm12 stimulators and indicated numbers of sorted CD4+ T cells from the various strains in 200 µl in 96-well plates. Cultures were incubated for 7 days in DMEM at 37°C and 10% CO2 and pulsed with [3H]thymidine at 1 µCi/well (Amersham Biosciences) for the last 16–18 h of culture. Cells were harvested and counted in the absence of scintillation fluid using a beta plate reader (Packard Instrument) and analyzed in quadruplicates.
Retroviral bone marrow chimeras
The murine WT IL-2R cDNA was used as a template to generate both IL-2R-STAT5 and IL-2R-
AH constructs. In brief, the IL-2R-AH construct encodes for a receptor that is truncated after serine-326. The IL-2R-STAT5 construct was made by overlap extension PCR and encodes a chimeric receptor consisting of the first 326 aa of the murine WT IL-2R construct fused to a sequence encoding the distal aa 494–505. WT-IL-2R, IL-2R-STAT5, and IL-2R-AH cDNAs were cloned into a modified pMIGR retroviral vector and retroviral particles were produced using 293T cells and a replication-deficient helper virus. Four-week-old IL-2R–/– donor mice were treated with 5-fluorouracil to enrich for long-term hemopoietic stem cells, and 4 days posttreatment bone marrow was harvested from donor mice. High titer viral supernatants were used to infect donor bone marrow with either WT-IL-2R, IL-2R-STAT5, or IL-2R-AH constructs as previously described (20). Following retroviral infection, donor bone marrow was introduced into sublethally irradiated rag2–/– recipient mice (650 rad). Eight weeks after reconstitution, thymii and spleens were harvested from recipient mice and analyzed by flow cytometry.
Chromatin immunoprecipitation (ChiP) assays
Spleen and LN were isolated from C57BL/6 mice and single-cell suspensions were obtained by disrupting organs with ground glass slides. Cells were resuspended in 1x PBS containing 2% FBS (Atlas Biologicals). Isolated cells were then enumerated and stained with biotin-labeled CD8 (53-6.7), CD19 (MB19-1), CD11b (M1/70), CD45R (RA3-6B2), DX5 (DX5), Ly6C (RB6-8C5), I-Ab (M5/114.15.2), IgM (eB121-15F9), and Ter-119 obtained from eBioscience and TCR (GL3) obtained from BD Pharmingen. Following Ab staining, the cells were incubated with streptavidin-labeled MicroBeads and fractionated with an LS column (Miltenyi Biotec). The enriched CD4 lymphocytes were then stained with allophycocyanin-labeled CD4 (L3T4) and PE-labeled CD25 (PC61), and sorted into CD4+CD25– and CD4+CD25+ populations to a purity of >95% using a FACSAria (BD Biosciences). Alternatively, the enriched CD4 lymphocytes were stained with PE-labeled CD25 and fractionated into CD4+CD25– and CD4+CD25+ populations using LS columns to a purity of >80%. Following fractionation, CD4+CD25– and CD4+CD25+ populations were fixed with a 1x PBS solution containing 27% formaldehyde, and the mixture was then sonicated to generate DNA fragments of <1 kb. The size of the DNA fragments after sonication was confirmed by running aliquots of sheared DNA on ethidium bromide-stained agarose gels; DNA fragments typically averaged between 250 and 500 bp with no detectable fragments >1 kb. ChiP assays were performed with anti-STAT5 (sc-835; Santa Cruz Biotechnology) or anti-Mek1 (sc-219; Santa Cruz Biotechnology) Abs as previously described (21) using a kit from Upstate Biotechnology. Real-time PCR was performed using the Brilliant QPCR core reagent kit (Stratagene). All PCR were conducted for one cycle at 95°C for 10 min, followed by 45 cycles at 95°C (15 s) and 60°C (90 s). Serial dilutions of genomic DNA were used to obtain a standard curve used to quantify levels of DNA in ChIP assays. Oligonucleotides and TaqMan probes were obtained from Integrated DNA Technologies and are listed as follows: 5' promoter primer, 5'-CTCACTCAGAGACTCGCAGCA-3'; 3' promoter primer, 5'-GCAAGCATGCATATGATCACC-3'; TaqMan promoter probe, FAM-CCAGCCATTCTGAGACTCTCTGATTCTGTG-BHQ1; 5' intron 1 primer, 5'-CAGAAAAATCTGGCCAAGTT-3'; 3'-intron 1 primer, 5'-AGGACCTGAATTGGATATGGT-3'; TaqMan intron 1 probe, FAM-ACCTCCATACAGCTTCTAAGAAACAGTCAAACA-BHQ1; 5' intron 11, 5'-CTGTGAGTTGGAAGCCAG-3'; 3' intron 11 primer, 5'-CTGAGCTATCTGTCCAGTTCC-3'; and TaqMan intron 11 probe, FAM-AACTGTAAATCTGTAATC CCAGCATGTGTACABHQ1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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signaling is critical for Treg development
To investigate the role of IL-2 and IL-2R in Treg development, we compared littermate control mice (LMC) with IL-2–/–, IL-2R–/–, and c–/– mice for the presence of CD4+Foxp3+ cells. As recently reported (7, 8), we observed comparable populations of CD4+Foxp3+ cells in LMC and IL-2–/– mice (Fig. 1A). Importantly, the absolute number of CD4+Foxp3+ cells in IL-2–/– mice was not significantly different from the number of CD4+Foxp3+ cells observed in LMC mice (Fig. 1B). Similar results were found in IL-2R–/– mice (data not shown). In contrast, we found that IL-2R–/– mice have a significant decrease in both the percentage and absolute cell numbers of CD4+Foxp3+ cells in the thymus and in the periphery (Fig. 1, C and D). Specifically, IL-2R–/– mice show a 4-fold decrease in numbers of thymic Tregs relative to LMC; the decrease is even more dramatic in the spleen and LN (27- and 6-fold decrease, respectively, relative to LMC mice). Analysis of c-deficient mice revealed an even more striking defect with a virtual complete absence of Tregs in both the thymus and spleen (Fig. 1, E and F). A potential concern with this latter experiment is that c–/– mice have dramatically reduced numbers of all lymphocyte subsets due to a requirement for c in IL-7R-dependent early B and T cell development (22, 23). Hence, the abrogation of Treg development observed in c–/– mice may be an indirect effect of the paucity of effector T cells in these mice. This is particularly relevant because Sakaguchi and colleagues (6) have provided evidence that IL-2 produced by activated effector T cells may be involved in the maintenance of Tregs. To address this issue, we analyzed Treg development in IL-7R–/– mice. Like c–/– animals, IL-7R–/– mice have an identical block in thymocyte development (at the double-negative (DN)2 to DN3 transition) and exhibit a similar paucity of mature T cells (24). However, as shown in Fig. 1E, IL-7R–/– mice contain a substantial population of CD4+Foxp3+ cells in both the thymus and the periphery that are significantly greater in total cell numbers than the few cells observed in c–/– mice (albeit still reduced in numbers relative to WT mice, Fig. 1F). Importantly, the ratio of CD4+Foxp3+:CD4+Foxp3– T cells in IL-7R–/– mice was greater than or equal to that seen in WT mice, demonstrating that Treg development was not impaired. These results suggest that one or more cytokines that bind both IL-2R and c are required for the development of Tregs.
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/c receptor complex is IL-15. IL-15–/– mice do not develop autoimmunity, and we have observed that they have virtually identical numbers of Tregs as WT mice. In contrast, IL-2–/– x IL-15–/– mice show a comparable decrease in Treg numbers as that observed in IL-2R–/– mice (6-fold 
in the thymus and 12-fold in the spleen; Fig. 2). Thus, IL-2 and IL-15 are redundant cytokines, either of which can signal through an IL-2R/c receptor complex to promote Treg development.
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–/– mice
One potential mechanism by which IL-2R-dependent signals could govern Treg development would be by influencing foxp3 expression. To test this hypothesis, we made use of foxp3 transgenic mice. The foxp3 transgene (made by Ramsdell and colleagues, 18) was derived from a 30.8-kb cosmid encompassing the entire coding region of the foxp3 gene as well as a significant portion of the upstream regulatory elements (18 kb). However, expression of the transgene does not mimic that of the endogenous foxp3 gene, as it is expressed in most CD4+ (75%) and CD8+ (44%) T cells. We intercrossed IL-2R–/– mice with foxp3 transgenic mice (foxp3tg) and observed that the foxp3 transgene effectively restores a large population of CD4+Foxp3+ T cells in IL-2R–/– x foxp3tg mice (Fig. 3A). Notably, expression of the foxp3 transgene in IL-2R–/– mice prevented activation of the remaining CD4+Foxp3– T cells typically seen in IL-2R–/– mice. For example, CD62L expression was restored, and CD69 levels were reduced, to levels seen on WT T cells (Fig. 3B). Furthermore, the lymphoproliferative disorder seen in IL-2R–/– mice was completely inhibited in IL-2R–/– x foxp3tg mice as demonstrated by the reversal of splenomegaly (Fig. 3C) and the decreased total lymphocyte counts observed in IL-2R–/– x foxp3tg mice relative to IL-2R–/– controls (data not shown). Because peripheral T cells in IL-2R–/– x foxp3tg mice express high levels of Foxp3, but are completely incapable of responding to IL-2R-dependent signals, we used them to examine whether IL-2R signaling is required to induce Treg suppressor function in vitro. For these experiments, we isolated WT CD4+CD25+ T cells, CD4+ T cells from foxp3tg mice, and CD4+ T cells from IL-2R–/– x foxp3tg mice and assayed their ability to inhibit proliferation of WT CD4+CD25– responder cells that had been stimulated with allogeneic APCs from bm12 mice. As shown in Fig. 3D, the CD4+ T cells from IL-2R–/– x foxp3tg mice suppressed responder cell proliferation to an equivalent degree as that achieved when using WT CD4+CD25+ T cells. CD4 T cells from foxp3tg mice also inhibited proliferation of WT effector cells, whereas CD4+ T cells from IL-2R–/– mice did not (data not shown). These results demonstrate that Foxp3 expression is sufficient, in the absence of other IL-2R-dependent signals, to effectively inhibit effector T cell proliferation in vitro.
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The IL-2R activates a number of downstream signaling pathways including those involving Erk, PI3K, and STAT5. Having previously demonstrated that STAT5 is preferentially activated in Tregs (25), we hypothesized that STAT5 signaling may play a critical role in Treg development. In fact, we and others have previously demonstrated that mice in which the first exon of STAT5a and STAT5b is disrupted (referred to as STAT5a/bEx1/Ex1 mice) have a decreased number of cells with regulatory activity (26, 27). However, because these studies predated the availability of methods to stain for intracellular foxp3, we reexamined this issue. We found that although STAT5a/bEx1/Ex1 mice do have a reduced percentage of Tregs, substantial numbers of these cells could still be detected. However, we also observed that lymphocytes from these mice express significant amounts of truncated STAT5a/b protein (data not shown). Importantly, this truncated STAT5 protein has recently been shown to exhibit functional activity (28). To circumvent this issue, we made use of mice generated by Hennighausen and colleagues (28), in which the entire STAT5a and STAT5b genes are flanked by loxP sites (referred to as STAT5a/bFL/FL mice), allowing for Cre-mediated deletion of both STAT5a and STAT5b genes. We crossed these animals to CD4-Cre transgenic mice in which Cre protein is expressed in CD4+CD8+ and CD4+ thymocytes, as well as in peripheral CD4+ T cells (29). Consistent with recent reports (30), we observed a significant decrease in CD8+ T cells in the thymus and the periphery of CD4-cre x STAT5a/bFL/FL mice. Notably, CD4+Foxp3+ T cells were also clearly reduced in both the thymus and spleen of CD4-cre x STAT5a/bFL/FL mice when compared with STAT5a/bFL/FL or CD4-cre– x STAT5a/bFL/+ controls (Fig. 4, A–C). Consistent with the reduction in Treg numbers, we observed a significant increase in the percentage of activated CD4+ T cells in these mice based on decreased CD62L and increased CD69 expression (Fig. 4D). However, it was also clear that not all CD4+Foxp3+ Tregs were eliminated in CD4-Cre x STAT5a/bFL/FL mice. One explanation for the remaining Tregs is that STAT5 may not have been efficiently eliminated in that cell lineage. To examine this issue, we stimulated T cells from CD4-Cre x STAT5a/bFL/FL mice with IL-2 and IL-7, which results in STAT5 phosphorylation that can be detected by flow cytometry. Because the intracellular staining protocols for Foxp3 and phospho-STAT5 require different fixation conditions that are not compatible, we used CD25 as a surrogate marker to identify Tregs. Phospho-STAT5 was clearly detected in CD4+CD25–, CD4+CD25+, and CD8+ T cells from LMC (Fig. 4, E and F). However, in CD4-Cre x STAT5a/bFL/FL mice, the majority of CD4+CD25– and CD8+ T cells had deleted STAT5a/b protein (95 and 70%, respectively) as demonstrated by the failure to detect phospho-STAT5 protein following IL-2/IL-7 stimulation. Importantly, our results with loss of STAT5 protein are in perfect agreement with previous findings by Yao et al. (30), who reported that >90% of double-positve thymocytes in CD4-Cre x STAT5a/bFL/FL mice have deleted the STAT5a and STAT5b genes. In contrast, CD4+CD25+ T cells from CD4-Cre x STAT5a/bFL/FL mice exhibited minimal to no deletion of STAT5a/b as demonstrated by their robust ability to induce phospho-STAT5 upon IL-2/IL-7 stimulation in both the thymus and the spleen (Fig. 4, E and F). Similar results were obtained when using glucocorticoid-induced TNFR family-related gene as a cell surface marker to identify Tregs (data not shown). These results indicate that the only remaining Tregs are those that had not deleted STAT5 and thus strongly support a role for STAT5 in the development of Tregs.
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Having demonstrated that STAT5 is required for Treg development, we decided to examine whether STAT5 activation was sufficient to rescue Treg development in IL-2R–/– mice. For these studies, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA) (21, 26, 31). Expression of STAT5b-CA is regulated by a compound promoter element consisting of the lck proximal promoter with the Eµ enhancer inserted into it. We have previously demonstrated that this transgene is expressed early during thymocyte development (at the early T cell progenitor stage) and that it continues to be expressed in both mature CD4+ and CD8+ T cells (21, 26). As shown in Fig. 5, expression of the STAT5b-CA transgene restored a substantial population of CD4+CD25+Foxp3+ Tregs in the thymus of IL-2R–/– mice. Likewise, CD4+Foxp3+ Tregs could still be detected in the spleen of STAT5b-CA x IL-2R–/– mice. However, these Tregs no longer expressed CD25 (Fig. 5B) and could not prevent T cell lymphoproliferation in vivo or animal death (data not shown). Thus, in many respects the CD4+CD25–Foxp3+ Tregs observed in the spleen and lymph nodes of STAT5b-CA x IL-2R–/– mice are very similar to the Tregs observed in IL-2–/– mice, which also fail to express CD25 and do not prevent autoimmunity (8).
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–/– mice is that the lck proximal promoter, which drives expression of the STAT5b-CA transgene, is known to become inactive in mature T cells and typically results in decreased expression of transgenes in mature T cells (32). Indeed, the expression of the STAT5b-CA transgene is quite low in peripheral T cells (26). Moreover, expression of the STAT5b-CA transgene in CD8+ and CD4+Foxp3– T cells renders them more resistant to Treg-mediated suppression (data not shown). Finally, Rudensky and colleagues (8) have shown that Tregs develop in IL-2–/– mice (Fig. 1A) but that they fail to prevent autoimmunity in these mice because they exhibit reduced fitness compared with IL-2–/– effector T cells. We propose that a parallel situation exists in STAT5b-CA x IL-2R–/– mice in which STAT5b-CA x IL-2R–/– Tregs exhibit reduced fitness relative to STAT5b-CA x IL-2R–/– effector T cells. Supporting this proposal, we have recently observed that expression of the STAT5b-CA transgene in c–/– mice restores peripheral T cell numbers including CD4+CD25+Foxp3+ Tregs; importantly, these mice do not exhibit T cell activation, as determined by CD69 and CD62L expression, nor do they develop autoimmune disease (data not shown). Thus, in a situation where all T cells are equally affected (c–/–) STAT5b-CA expression is sufficient to induce a population of functional Tregs that are capable of preventing autoimmune disease.
To circumvent these possible problems, we expressed a chimeric IL-2R construct that selectively activates STAT5 in bone marrow cells from IL-2R–/– mice (Fig. 6A). Reconstitution of rag2–/– mice with these transduced bone marrow cells results in the generation of T cells that should induce STAT5 phosphorylation at physiological levels when exposed to endogenously produced IL-2. Control constructs encoding either the full-length WT IL-2R chain or a signaling defective receptor in which the cytoplasmic tail contained just the S region were also generated and tested using this approach (referred to as WT IL-2R and IL-2R-AH, respectively). We first examined the signaling properties of control and chimeric receptors by introducing them into the cytokine-dependent BAF cell line. IL-2 stimulation of the cells expressing the WT IL-2R chain led to activation of the ERK and STAT5 signaling pathways. In contrast, the chimeric IL-2R-STAT5 receptor only activated STAT5, whereas the signaling defective IL-2R-AH receptor did not activate any of these pathways (data not shown). Thus, the chimeric IL-2R-STAT5 receptor selectively leads to STAT5 activation.
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, IL-2R-STAT5, or IL-2R-AH constructs were then introduced into IL-2R–/– bone marrow using a modified version of the pMIGR retroviral vector (20). Infected IL-2R–/– bone marrow was then transferred into rag2–/– recipients; 9 wk following reconstitution, recipient mice were analyzed for the presence of CD4+Foxp3+ and CD4+CD25+ Treg populations. Cell surface staining for the IL-2R chain demonstrated that equal percentages of splenic CD4+ T cells in these bone marrow chimeric mice expressed the retrovirally driven IL-2R chain (Fig. 6B). Mice reconstituted with the WT IL-2R construct developed a significant population of CD4+Foxp3+ and CD4+ CD25+ cells, indicating that reintroduction of the WT receptor could restore Tregs in IL-2R–/– mice (Fig. 6C and data not shown). Similarly, the IL-2R-STAT5 construct also restored a comparable population of CD4+Foxp3+ Tregs. Importantly, most Tregs in the spleen of these mice expressed high levels of CD25, indicating that they resembled Tregs that are normally observed in WT mice. In contrast, the signaling defective IL-2R-AH construct did not restore a population of CD4+Foxp3+ Tregs.
We next examined T cell activation in these mice. Virtually all T cells (95%) in mice reconstituted with the signaling defective IL-2R-AH construct expressed low levels of CD62L consistent with an activated phenotype (Fig. 6D, right panel). In contrast, T cell activation was greatly diminished in mice reconstituted with either the WT or IL-2R-STAT5 receptor as demonstrated by the increase in the percentage of T cells expressing high levels of CD62L (Fig. 6D, left and center panels). Identical conclusions were reached when using expression of CD69 as a marker of T cell activation (data not shown). Thus, Tregs generated following reconstitution with an IL-2R construct that can only activate STAT5 were capable of preventing T cell activation in vivo (although we cannot rigorously rule out the possibility that these Tregs may exhibit somewhat reduced suppressor function). These results demonstrate that STAT5 activation is sufficient to promote IL-2R-dependent development of CD4+CD25–Foxp3+ and CD4+CD25+Foxp3+ Tregs in both the thymus and peripheral lymphoid organs.
STAT5 binds to the foxp3 gene promoter
The findings that foxp3 expression or STAT5 activation are both sufficient to restore Treg development in IL-2R–/– mice suggested that STAT5 may act by directly regulating foxp3 expression. To examine this issue, we first scanned the murine foxp3 gene for potential STAT5 consensus binding sites. As shown in Fig. 7A, the murine foxp3 gene contains 11 potential STAT5 binding sites. Because functional binding sites should be evolutionarily conserved, we compared the foxp3 gene sequences from murine, human, monkey, and dog species and observed that six of these sites were conserved (Fig. 7B); these include three closely spaced sequences in the foxp3 promoter and three sites located between murine exon-2a and exon-1 (Fig. 7A). To determine whether STAT5 actually binds to these conserved sites we conducted ChIP assays. As seen in Fig. 7C, immunoprecipitation with an anti-STAT5 Ab revealed STAT5 binding to the conserved sites in the promoter of the foxp3 gene in murine CD4+CD25+ T cells. In contrast, no binding could be seen to the three conserved sites in intron 2 or the distal site located in intron 11. No PCR product was observed when ChIP was done using an anti-Mek-1 control Ab. Finally, significant STAT5 binding to the foxp3 gene was not observed in CD4+CD25– T cells, a cell population which is mostly comprised of CD4+Foxp3– T cells (Fig. 7D). Thus, these experiments provide evidence that STAT5 can play a role in regulating foxp3 gene expression.
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and c are required for Treg development. The surprising finding that the IL-2R and c chains are required for Treg development, whereas IL-2 is not, raised the question of what cytokines signal through the IL-2R/c complex to promote Treg differentiation. The only other cytokine known to use the IL-2R chain for signaling is IL-15. Although IL-15–/– mice have no obvious defects in Treg numbers or function, IL-2–/– x IL-15–/– mice have a clear deficit in Tregs, which mimics that seen in IL-2R–/– mice. Thus, our findings point to a previously unappreciated role for IL-15 in Treg development and provide a compelling explanation for the continued presence of Tregs in IL-2–/– mice.
A remaining question is whether other cytokines can contribute to this process as well. It is difficult to directly compare results in c–/– and IL-2R–/– mice due to the additional early blocks in thymocyte development that occur in c null mice. However, the more stringent block in Treg development observed in c–/– vs IL-2R–/– mice is at least consistent with the possibility that other c-using cytokines (IL4, IL-7, IL9, or IL-21) might be able to contribute to STAT5-dependent Treg development. Supporting this conjecture, Fontenot et al. (8) did not report a significant difference in Treg numbers in very young IL-2R–/– mice (2 wk old). Their results differ from our findings in older (4–5 wk of age) IL-2R–/– and IL-2–/– x IL-15–/– mice. The most likely explanation for this difference is that, in very young mice, Tregs will face less competition from naive T cells for binding to IL-7. A similar phenomenon has already been described for naive T cells in young mice, which appear to undergo a homeostatic expansion-like process due to less competition for endogenous IL-7 (35, 36). Thus, in the relatively lymphopenic conditions that exist in very young mice, developing Tregs might still be able to bind IL-7. In older mice, the increased number of naive T cells expressing high levels of IL-7R, coupled with the reduced expression of IL-7R on Tregs relative to other T cell subsets, would make it unlikely that Tregs could effectively compete for and respond to IL-7 signals.
The finding that both IL-2–/– x IL-15–/– mice and IL-2R–/– mice lack Tregs provides strong evidence that signaling pathways downstream of the IL-2R receptor complex entrain Treg differentiation. In this study, we document that STAT5 activation is both necessary and sufficient for IL-2R-dependent Treg development. Previous studies using STAT5a/bEx1/Ex1 mice had provided evidence that STAT5 is required for Treg survival (26, 27). However, the discovery that these mice express a hypomorphic rather than a STAT5 null allele makes it impossible to draw conclusions about the role of STAT5 in Treg development based on studies using these mice (28). Using a conditional STAT5 knockout model, we demonstrate clear differences in the role that STAT5 plays in CD8+ and CD4+Foxp3– T cells vs CD4+Foxp3+ Tregs. Specifically, we observe decreased numbers of CD4+Foxp3– and CD8+ T cells in CD4-Cre x STAT5a/bFL/FL mice (J. Yang, M. A. Burchill, and M. A. Farrar, unpublished results). Importantly, these cell populations also showed efficient deletion of STAT5 (70–95% deletion), indicating that STAT5 affects survival but not development of these naive T cell populations. We also observed a decrease in Treg numbers in CD4-Cre x STAT5a/bFL/FL mice (Fig. 4, A and B). However, virtually none of the remaining Tregs had deleted STAT5, thereby strongly supporting a role for STAT5 in governing development of this cell population. Thus, our results establish that an IL-2R-activated STAT5-dependent signaling pathway plays a critical role in Treg development.
A key question in the field of Treg biology is what are the signals that govern foxp3 transcription. Our findings that either forced foxp3 expression or constitutive STAT5b activation in Tregs could rescue Treg development in the absence of IL-2R signaling suggested that these factors might be involved in a coherent signaling pathway. Specifically, our studies provide evidence that STAT5 binds to conserved sites within the foxp3 promoter and thereby provide a direct link between IL-2R signals and foxp3 expression. A potential concern is that the identified STAT5 binding sites do not exactly match the STAT5b binding consensus motif (TTCXXXGAA). However, it is important to note that the three sites detected in the promoter region are separated by only 7 bp. Previous work by Leonard and colleagues (37) has demonstrated that STAT5 binding sites separated by 6–7 bp allows efficient binding of STAT5 to those sites as STAT5 tetramers rather than dimers. Moreover, they noted that these tetrameric sites frequently diverged from the optimal consensus STAT5-binding motif. Thus, the spacing of the STAT5 binding sites in the foxp3 promoter suggest that formation of STAT5 tetramers may be important for STAT5 binding to the foxp3 promoter.
A recent report by Zorn et al. (38) has also suggested that STAT5 regulates foxp3 expression, although they suggested that the relevant binding sites are those found within the first intron of the foxp3 gene. This conclusion was based on experiments in which they isolated the STAT5 consensus binding sites in the first intron, placed them in a luciferase reporter construct, and introduced this reporter construct along with a plasmid encoding an activated form of STAT5 into fibroblasts. In those studies, they found that the isolated STAT5 binding sites could lead to enhanced luciferase expression in a STAT5-dependent manner. In contrast, our ChIP studies using murine Tregs demonstrate that STAT5 does not bind to the intronic sites in vivo. Thus, we believe that the physiologically relevant STAT5 binding sites are those found within the promoter region of the foxp3 gene and not the first intron. Finally, we note that STAT5 signaling by itself is not sufficient to induce foxp3 expression because STAT5 activation did not convert all T cells into Tregs. Thus, an important question for the future will be the identification of other factors that interact with STAT5 to help initiate or maintain foxp3 gene expression.
In conclusion, our data suggest a model for how IL-2 signals entrain Treg development. Namely, we propose that IL-2/IL-15 signal through an IL-2R/c complex to transmit STAT5-dependent signals that directly impinge on foxp3 transcription. A remaining question is where the IL-2/IL-15 comes from that initiates this process. A simple explanation would be that IL-15 produced constitutively by stromal cells may be sufficient to drive Treg development in the thymus. However, IL-2 must also contribute as IL-15–/– mice show only slight defects, if any, in thymic Treg generation. It is tempting to speculate that this relates to the higher self-reactivity of TCRs expressed by Tregs (39). In this model, the stronger T cell signals delivered by TCRs expressed on developing Tregs may be sufficient to induce IL-2R expression allowing these cells to respond to either autocrine or paracrine IL-2, and thereby inducing STAT5 activation and subsequent foxp3 transcription. Foxp3 expression would then act in conjunction with STAT5 to maintain high level IL-2R expression (thereby allowing Tregs to respond to IL-2 produced by activated effector T cells) and inhibit subsequent TCR-dependent IL-2 secretion in mature Tregs. Although some aspects of this model await experimental verification, it provides a testable hypothesis that links the current elements known to be required for Treg development, namely, the TCR and CD28, the IL-2R complex, and the transcription factor Foxp3.
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1 This work was supported in part by a Pew Scholar Award (to M.A.F.), a Cancer Investigator Award (to M.A.F.), and by National Institutes of Health Grants AI061165 (to M.A.F.) and HL63452, AI34495, and HL56067 (to B.R.B.). M.A.B. is supported by a National Institutes of Health Training Grant (2T32-AI07313) and a gift from the estate of Eli and Dorothy Rosen and Bernard Collins. 
2 M.A.B. and J.Y. contributed equally to the results presented here.
3 Address correspondence and reprint requests to Dr. Michael A. Farrar, Center for Immunology, University of Minnesota, 312 Church Street SE, 6-116 Nils Hasselmo Hall, Minneapolis, MN 55455. E-mail address: farra005{at}tc.umn.edu
4 Abbreviations used in this paper: Treg, regulatory T cell; WT, wild type; LN, lymph node; ChIP, chromatin immunoprecipitation; LMC, littermate control mice; c, common -chain; DN, double negative.
Received for publication July 27, 2006. Accepted for publication October 23, 2006.
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-chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3: 521-530. [Medline]. Science 268: 1472-1476. -deficient mice. Implications for the nonredundant function of IL-2. Immunity 17: 167-178. [Medline] chain. Proc. Natl. Acad. Sci. USA 92: 377-381. -chain of cytokine receptors regulates intrathymic T cell development at multiple stages. J. Immunol. 158: 2592-2599. [Abstract]This article has been cited by other articles:
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 |
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|
|
|
|
|
|
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 G. Lal and J. S. Bromberg Epigenetic mechanisms of regulation of Foxp3 expression Blood, October 29, 2009; 114(18): 3727 - 3735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sakaguchi, K. Wing, Y. Onishi, P. Prieto-Martin, and T. Yamaguchi Regulatory T cells: how do they suppress immune responses? Int. Immunol., October 1, 2009; 21(10): 1105 - 1111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Le Campion, M.-C. Gagnerault, C. Auffray, C. Becourt, M. Poitrasson-Riviere, E. Lallemand, B. Bienvenu, B. Martin, F. Lepault, and B. Lucas Lymphopenia-induced spontaneous T-cell proliferation as a cofactor for autoimmune disease development Blood, August 27, 2009; 114(9): 1784 - 1793. [Abstract] [Full Text] [PDF]
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|
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|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
N. Ouaked, P.-Y. Mantel, C. Bassin, S. Burgler, K. Siegmund, C. A. Akdis, and C. B. Schmidt-Weber Regulation of the foxp3 Gene by the Th1 Cytokines: The Role of IL-27-Induced STAT1 J. Immunol., January 15, 2009; 182(2): 1041 - 1049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 W. Wang, H. D. Edington, U. N.M. Rao, D. M. Jukic, A. Radfar, H. Wang, and J. M. Kirkwood Effects of High-Dose IFN{alpha}2b on Regional Lymph Node Metastases of Human Melanoma: Modulation of STAT5, FOXP3, and IL-17 Clin. Cancer Res., December 15, 2008; 14(24): 8314 - 8320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mazzucchelli, J. A. Hixon, R. Spolski, X. Chen, W. Q. Li, V. L. Hall, J. Willette-Brown, A. A. Hurwitz, W. J. Leonard, and S. K. Durum Development of regulatory T cells requires IL-7R{alpha} stimulation by IL-7 or TSLP Blood, October 15, 2008; 112(8): 3283 - 3292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tsuji-Takayama, M. Suzuki, M. Yamamoto, A. Harashima, A. Okochi, T. Otani, T. Inoue, A. Sugimoto, T. Toraya, M. Takeuchi, et al. The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus J. Immunol., September 15, 2008; 181(6): 3897 - 3905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Vang, J. Yang, S. A. Mahmud, M. A. Burchill, A. L. Vegoe, and M. A. Farrar IL-2, -7, and -15, but Not Thymic Stromal Lymphopoeitin, Redundantly Govern CD4+Foxp3+ Regulatory T Cell Development J. Immunol., September 1, 2008; 181(5): 3285 - 3290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Inaba, M. Steeves, P. Nguyen, and T. L. Geiger In Vivo Suppression of Naive CD4 T Cell Responses by IL-2- and Antigen-Stimulated T Lymphocytes in the Absence of APC Competition J. Immunol., September 1, 2008; 181(5): 3323 - 3335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhu and W. E. Paul CD4 T cells: fates, functions, and faults Blood, September 1, 2008; 112(5): 1557 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kasprzycka, Q. Zhang, A. Witkiewicz, M. Marzec, M. Potoczek, X. Liu, H. Y. Wang, M. Milone, S. Basu, J. Mauger, et al. {gamma}c-Signaling Cytokines Induce a Regulatory T Cell Phenotype in Malignant CD4+ T Lymphocytes J. Immunol., August 15, 2008; 181(4): 2506 - 2512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Doganci, R. Karwot, J. H. Maxeiner, P. Scholtes, E. Schmitt, M. F. Neurath, H. A. Lehr, I-C. Ho, and S. Finotto IL-2 Receptor {beta}-Chain Signaling Controls Immunosuppressive CD4+ T Cells in the Draining Lymph Nodes and Lung during Allergic Airway Inflammation In Vivo J. Immunol., August 1, 2008; 181(3): 1917 - 1926. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Bayer, J. Y. Lee, A. de la Barrera, C. D. Surh, and T. R. Malek A Function for IL-7R for CD4+CD25+Foxp3+ T Regulatory Cells J. Immunol., July 1, 2008; 181(1): 225 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kang, S. J. Huddleston, J. M. Fraser, and A. Khoruts De novo induction of antigen-specific CD4+CD25+Foxp3+ regulatory T cells in vivo following systemic antigen administration accompanied by blockade of mTOR J. Leukoc. Biol., May 1, 2008; 83(5): 1230 - 1239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hennighausen and G. W. Robinson Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B Genes & Dev., March 15, 2008; 22(6): 711 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, H. Takeuchi, Y. Sonobe, S. Jin, T. Mizuno, S. Miyakawa, M. Fujiwara, Y. Nakamura, T. Kato, H. Muramatsu, et al. Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population PNAS, March 11, 2008; 105(10): 3915 - 3920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
L. Strauss, C. Bergmann, M. J. Szczepanski, S. Lang, J. M. Kirkwood, and T. L. Whiteside Expression of ICOS on Human Melanoma-Infiltrating CD4+CD25highFoxp3+ T Regulatory Cells: Implications and Impact on Tumor-Mediated Immune Suppression J. Immunol., March 1, 2008; 180(5): 2967 - 2980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Elias, A. Laurence, T. S. Davidson, G. Stephens, Y. Kanno, E. M. Shevach, and J. J. O'Shea Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway Blood, February 1, 2008; 111(3): 1013 - 1020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. M. Lockyer, E. Tran, and B. H. Nelson STAT5 Is Essential for Akt/p70S6 Kinase Activity during IL-2-Induced Lymphocyte Proliferation J. Immunol., October 15, 2007; 179(8): 5301 - 5308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Sanchez-Guajardo, C. Tanchot, J. T. O'Malley, M. H. Kaplan, S. Garcia, and A. A. Freitas Agonist-Driven Development of CD4+CD25+Foxp3+ Regulatory T Cells Requires a Second Signal Mediated by Stat6 J. Immunol., June 15, 2007; 178(12): 7550 - 7556. [Abstract] [Full Text] [PDF]
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) and IL-2–/– (
) mice. C, Thymii, spleens, and LN were harvested from 4-wk-old C57BL/6 or IL-2R
) mice. Error bars, SEM. Mice used in these experiments were between 4 and 5 wk of age. Data are representative of nine WT C57BL/6 and IL-15–/– mice and four IL-2–/– x IL-15–/– mice.
), or CD4+CD25+ T cells from C57BL/6 mice (
