Akt Is a Neutral Amplifier for Th Cell Differentiation
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m309063200
ISSN1083-351X
AutoresYutaka Arimura, Fumiko Shiroki, Shingo Kuwahara, Hidehito Kato, Umberto Dianzani, Takehiko Uchiyama, Junji Yagi,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoBoth CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4+ T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4+ T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4+ T cells. E40K also facilitated Th1 differentiation in CD4+ T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation. Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4+ T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4+ T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4+ T cells. E40K also facilitated Th1 differentiation in CD4+ T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation. Naive CD4+ helper T (Th) 1The abbreviations used are: Th, helper T; ICOS, inducible costimulator; EGFP, enhanced green fluorescent protein; HIg, hamster IgG; NF-κB, nuclear factor of κB; NF-AT, nuclear factor of activated T cells; IFN, interferon; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; TCR, T cell receptor; Ab, antibody; mAb, monoclonal antibody; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; ERK, extracellular signal-regulated kinase; APC, antigen-presenting cell. 1The abbreviations used are: Th, helper T; ICOS, inducible costimulator; EGFP, enhanced green fluorescent protein; HIg, hamster IgG; NF-κB, nuclear factor of κB; NF-AT, nuclear factor of activated T cells; IFN, interferon; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; TCR, T cell receptor; Ab, antibody; mAb, monoclonal antibody; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; ERK, extracellular signal-regulated kinase; APC, antigen-presenting cell. cells proliferate and differentiate into at least two functionally distinct subsets, Th1 cells, which produce interferon-γ (IFN-γ), IL-2, and tumor necrosis factor-β (TNF-β), and Th2 cells, which produce IL-4, IL-5, IL-6, Il-10, and IL-13 (1O'Garra A. 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Ozkaynak E. Coyle A.J. Nat. Immunol. 2001; 2: 605-611Crossref PubMed Scopus (244) Google Scholar). Both CD28 and ICOS have a YXXM motif for recruitment of phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, whereas a YXNX motif for the Src homology 2 domain of Grb2 family members and a PXXP motif for the Src homology 3 domain of various molecules are present only in CD28. The YXNX motif seems to be responsible for IL-2 production (19Harada Y. Ohgai D. Watanabe R. Okano K. Koiwai O. Tanabe K. Toma H. Altman A. Abe R. J. Exp. Med. 2003; 197: 257-262Crossref PubMed Scopus (80) Google Scholar). Cross-linking of CD28 or ICOS with TCR/CD3 leads to activation of PI3K (4Coyle A.J. Lehar S. Lloyd C. Tian J. Delaney T. Manning S. Nguyen T. Burwell T. Schneider H. Gonzalo J.A. Gosselin M. Owen L.R. Rudd C.E. Gutierrez-Ramos J.C. Immunity. 2000; 13: 95-105Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 20Stein P.H. Fraser J.D. Weiss A. Mol. Cell. 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Immunol. 1995; 25: 526-532Crossref PubMed Scopus (85) Google Scholar, 28Shi J. Cinek T. Truitt K.E. Imboden J.B. J. Immunol. 1997; 158: 4688-4695PubMed Google Scholar). Kane et al. (29Kane L.P. Andres P.G. Howland K.C. Abbas A.K. Weiss A. Nat. Immunol. 2001; 2: 37-44Crossref PubMed Scopus (268) Google Scholar) have demonstrated that Akt can provide the costimulatory signal for activation of the IL-2 promoter, which is indistinguishable from the CD28 costimulatory signal. They further showed that enforced Akt expression in primary CD4+ T cells from CD28-deficient mice restored Th1, but not Th2, cytokine production upon restimulation. Previously, we showed Akt phosphorylation to be enhanced by engagement of CD28 and even more strongly by that of ICOS. Since several reports including ours have shown that the engagement of ICOS has a preferential effect on Th2 development, Akt may be involved in Th2 development as well under certain circumstances. Furthermore, the question of whether Akt is differentially required for CD28 and ICOS costimulation remains to be answered. In this report, to address the above questions, we have explored the role of Akt in T cell activation costimulated by CD28 and ICOS, and in Th cell differentiation, by examining the effect of enforced expression of Akt in primary CD4+ T cells under various Th-differentiating conditions. Animals—BALB/c and C57BL/6 mice were obtained from Japan SLC (Hamamatsu, Japan). IL-4 knock-out mice with a BALB/c background were purchased from Jackson Laboratories (Bar Harbor, ME) (30Noben-Trauth N. Kohler G. Burki K. Ledermann B. Trans. Res. 1996; 5: 487-491Crossref PubMed Scopus (109) Google Scholar). All mice were bred and used in accordance with the guidelines of the Institute of Laboratory Animals, Tokyo Women's Medical University. Abs and Reagents—The C398.4A mAb specific for ICOS/H4 has been described previously (8Buonfiglio D. Bragardo M. Redoglia V. Vaschetto R. Bottarel F. Bonissoni S. Bensi T. Mezzatesta C. Janeway Jr., C.A. Dianzani U. Eur. J. Immunol. 2000; 30: 3463-3467Crossref PubMed Scopus (36) Google Scholar, 9Yagi J. Dianzani U. Kato H. Okamoto T. Katsurada T. Buonfiglio D. Miyoshi-Akiyama T. Uchiyama T. J. Immunol. 1999; 163: 3083-3091PubMed Google Scholar). mAb to CD28 (37.51) (31Gross J.A. Callas E. Allison J.P. J. Immunol. 1992; 149: 380-388PubMed Google Scholar) was kindly provided by Dr. J. Allison (University of California at Berkeley, Berkeley, CA). mAbs to I-Ab/d (28-16-8S), CD3 (145-2C11), CD8 (83.12.5), and Thy1.2 (HO13) have been described previously (32Yagi J. Nakata M. Uchiyama T. Nishikawa M. Mizushima Y. Nishioka K. Ito K. Yagita H. Okumura K. Janeway Jr, C.A. Yamamoto K. J. Immunol. 1994; 152: 3833-3841PubMed Google Scholar). The following Abs were used: biotinylated anti-CD28 (37.51), PE-labeled anti-CD3 (145-2C11), PE-CD4 (RM4-4), biotin-labeled anti-CD69 (H1.2F3), fluorescein isothiocyanate-anti-CD44 (IM7), biotin-anti-CD44 (IM7), biotin-anti-CD62L (MEL-14), and biotin-anti-CD11a (LFA-1 α chain, M17/4) mAbs (BD Biosciences); goat anti-hamster IgG (anti-HIg) (ICN Pharmaceuticals, Aurora, OH); PE-streptavidin, streptavidin-Cy-Chrome (BD Biosciences); rabbit polyclonal anti-Akt, anti-phospho-Akt (serine 473), and anti-phospho-ERK (Thr-202, Tyr-204) Abs (Cell Signaling Technology, Beverly, MA); rabbit polyclonal anti-GFP (FL), anti-c-Maf (M-153), goat polyclonal anti-Actin (I-19) Abs, and mouse anti-GATA-3 (HG3–31) mAb (Santa Cruz Biotechnology, Santa Cruz, CA); and mouse anti-Bcl-XL mAb (Transduction Laboratories, Lexington, KY). Rabbit polyclonal Ab to c-Rel (C) and murine mAbs to NF-ATc1 (7A6) and NF-ATc2 (4G6-G5) were obtained from Santa Cruz, and c-Myb mAb (clone 1-1) was from Upstate Biotechnology (Lake Placid, NY). Construction of Expression Vectors—Akt cDNA was amplified by reverse transcriptase-PCR from total RNA of BALB/c mouse spleen cells by using specific primer pair 5′-GGCCACGGATACCATGAACG-3′ with a BamHI linker and 5′-GGCCTCAGGCTGTGCCACTG-3′ with an EcoRI linker. After double digestion with BamHI and EcoRI, the fragment was ligated into BamHI/EcoRI sites of the bicistronic retroviral vector pMX-IRES-enhanced green fluorescent protein (EGFP) (a gift from Dr. Y. Kamogawa, University of Tokyo, Tokyo, Japan) (33Lee H.J. Takemoto N. Kurata H. Kamogawa Y. Miyatake S. O'Garra A. Arai N. J. Exp. Med. 2000; 192: 105-115Crossref PubMed Scopus (334) Google Scholar), and the nucleotide sequence of Akt was verified. To create the constitutively active and kinase-inactive forms of Akt (34Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar), mutagenesis was conducted with the GeneEditor™ in vitro site-directed mutagenesis system (Promega, Madison, WI) according to manufacturer's instructions. The primers used for mutagenesis were as follows: 5′-ACAGGCCGCTACTATGCCATGATGATCCTCAAGAAGGAGGTCATC-3′ for the kinase-inactive form (K179M) and 5′-GGCACCTTTATTGGCTACAAGAAACGGCCTCAGGATGTGGATCAG-3′ for the active form (E40K) of Akt. GATA-3 cDNA (35Ko L.J. Yamamoto M. Leonard M.W. George K.M. Ting P. Engel J.D. Mol. Cell. Biol. 1991; 11: 2778-2784Crossref PubMed Scopus (224) Google Scholar), a gift from Dr. D. Engel (Northwestern University), was inserted at the EcoRI site of pMX-IRES-EGFP, and the retroviral expression vector for T-bet (pMSCV-T-bet) (36Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2669) Google Scholar) was a generous gift from Dr. L. Glimcher (Harvard University). Retrovirus-mediated Gene Transfer and in Vitro Th Cell Differentiation—Splenic CD4+ T cells and APCs were prepared as described previously (13Arimura Y. Kato H. Dianzani U. Okamoto T. Kamekura S. Buonfiglio D. Miyoshi-Akiyama T. Uchiyama Yagi J. Int. Immunol. 2002; 14: 555-566Crossref PubMed Scopus (60) Google Scholar). To obtain naive T cells, splenic CD4+ T cells were stained with anti-CD44 and anti-CD62L Abs and enriched by cell sorting with an Epics ALTRA flow cytometer (Coulter Immunology, Hialeah, FL). The purity of the CD44lowCD62Lhigh cells obtained was >99%. To overexpress the Akt constructs in mouse primary CD4+ T cells, retrovirusmediated gene transfer was conducted as described previously (37Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (223) Google Scholar). Briefly, plasmid expression vectors were transfected into packaging cells for retrovirus, Plat-E (a gift from Dr. T. Kitamura, University of Tokyo) (38Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1328) Google Scholar) by the calcium phosphate method. The culture supernatant containing recombinant retrovirus was collected at 1 or 2 days after transfection. To achieve infection, 2 × 106/ml CD4+ T cells (0.5 × 106/ml for naive CD4+ T cells) that had been preactivated with anti-CD3 mAb (2 μg/ml) plus syngenic APCs 1 day before were incubated with virus-containing supernatant from Plat-E supplemented with anti-CD3 (1 μg/ml), polybrene (10 μg/ml, Sigma), and human rIL-2 (50 units/ml, a gift from Shionogi Co., Osaka, Japan), and centrifuged for 1 h at 1900 rpm and incubated for 4 h at 37 °C. Then, an equal volume of fresh Dulbecco's modified Eagle's medium was added. The following day, the cells were reinfected as above. One day later, the infected cells were expanded/rested in the presence of rIL-2 (100 units/ml) for an additional 2 days. To polarize CD4+ T cells in vitro, 2.5 ng/ml of murine rIL-12 (Genzyme Techne, Framingham, MA) and 3 μg/ml of anti-IL-4 mAb (11B11, ATCC, Rockville, MD) were added to the culture for Th1 cells, and 2 ng/ml of murine rIL-4 (BD Biosciences) and 3 μg/ml of anti-IL-12 mAb (C17.8, Genzyme Techne) for Th2 cells, during the period of priming/infection. When required, EGFP+ (retrovirus-infected) cells were enriched by cell sorting. The purity of harvested EGFP+ cells exceeded 93%. Stimulation, Cytokine Assay, Intracellular Cytokine Staining, and Western Blot—1 × 105 CD4+ T cells were cultured for 24 h in a 96-well flat-bottomed culture plate serially coated with anti-CD3 (0.07 μg/ml) and anti-CD28, anti-ICOS, or control HIg (3 μg/ml), as described previously (13Arimura Y. Kato H. Dianzani U. Okamoto T. Kamekura S. Buonfiglio D. Miyoshi-Akiyama T. Uchiyama Yagi J. Int. Immunol. 2002; 14: 555-566Crossref PubMed Scopus (60) Google Scholar). The IL-2 concentrations in culture supernatants were determined as proliferation of IL-2-dependent CTLL-2 cells in a bioassay. IL-4 and IFN-γ in culture supernatants were quantitated by sandwich ELISA according to the manufacturer's instructions (BD Biosciences). Results are expressed as the mean ± S.D. of triplicate cultures, and the data were analyzed for variance by a one-way analysis of variance followed by Tukey-Kramer's test for multiple comparisons. Results were considered significant if p < 0.01. Intracellular cytokine staining was performed as described previously (39Chen L. Koyanagi M. Fukada K. Imanishi K. Yagi J. Kato H. Miyoshi-Akiyama T. Zhang R. Miwa K. Uchiyama T. J. Immunol. 2002; 168: 3817-3824Crossref PubMed Scopus (22) Google Scholar). For surface staining, CD4+ T cells collected after expansion/resting with rIL-2 for 2 days were stained with PE-labeled mAbs or combinations of biotinylated mAbs and PE-streptavidin and then analyzed with the Epics XL flow cytometer (Coulter Immunology). Transcription factors were analyzed by extracting of nuclear protein with a reagent NE-PER™ (Pierce) according to manufacturer's instruction and performing Western blot analysis as described previously (13Arimura Y. Kato H. Dianzani U. Okamoto T. Kamekura S. Buonfiglio D. Miyoshi-Akiyama T. Uchiyama Yagi J. Int. Immunol. 2002; 14: 555-566Crossref PubMed Scopus (60) Google Scholar, 40Arimura Y. Ogimoto M. Mitomo K. Katagiri T. Yamamoto K. Volarevic S. Mizuno K. Yakura H. J. Biol. Chem. 2001; 276: 8550-8556Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Enforced Expression and Kinase Activity of Akt Constructs—To investigate the role of Akt in CD4+ T cells, we prepared expression constructs for three types of Akt using a bicistronic retrovirus vector and achieved retrovirus-mediated transduction of the Akt constructs into mouse primary CD4+ T cells. The constitutively active form (E40K) and the wild-type (WT) of Akt were highly phosphorylated in BALB/c CD4+ T cells before and after restimulation, although the phosphorylation level was slightly higher in the E40K-infected cells than in WT-infected cells (Fig. 1A). On the other hand, the kinase-inactive form (K179M) of Akt did not show enhanced phosphorylation. These observations indicate the level of phosphorylation to be proportional to the kinase activity of the Akt construct itself. The protein expression levels of Akt and EGFP in WT and E40K were also much higher than those of K179M (Figs. 1 and 2). These differences in expression level do not appear to reflect the difference in virus titer produced but do reflect the kinase activy itself because they were observed even when the infectivity with K179M was higher than that with E40K (data not shown). In addition, as a consequence of the up-regulation of kinase activity, Bcl-XL expression, which is involved in anti-apoptosis through Akt (41Jones R.G. Parsons M. Bonnard M. Chan V.S.F. Yeh W.-C. Woodgett J.R. Ohashi P.S. J. Exp. Med. 2000; 191: 1721-1733Crossref PubMed Scopus (285) Google Scholar), was up-regulated in WT- and E40K-infected CD4+ T cells from both C57BL/6 and BALB/c mice (Fig. 1B). By contrast, the activity of mitogen-activated protein kinase, ERK, was not affected by enforced expression of Akt (Fig. 1A). Consequently, the effects of Akt are limited and specific to particular downstream molecules of the Akt pathway examined.Fig. 2Cellular phenotypes of BALB/c CD4+ T cells introduced with Akt constructs. A, E40K and K179M were used for transfection. EGFP+ cells were enriched by cell sorting. The same number of cells from each cell type was incubated in the presence of 100 units/ml IL-2 and examined for cell aggregation by phase contrast microscopy (left) and for EGFP intensity by fluorescence microscopy (right). The dark scattered clumps in the phase contrast image represent cell aggregates. B, the cells infected with E40K (shaded histogram) or K179M (open histogram) were examined for surface molecules and cell size (FS) by flow cytometry. C, the E40K cells (shaded histogram), K179M cells (open histogram with bold line), and empty vector control cells (open histogram with thin line) were examined for CD69 expression in an activated state and rested state following 2 days in the presence of 100 units/ml IL-2. These figures are representative of at least four independent experiments with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cellular Phenotypes Induced by the Constitutively Active Form of Akt—Following Akt infection, we gated EGFP+ cells to monitor infected cells and examined the phenotypes of cells with the active or the inactive form of Akt. In the cells transfected with E40K, but not K179M, increases in the number and size of cell aggregates were evident (Fig. 2A), suggesting upregulation/activation of integrins. E40K up-regulated several activation/adhesion molecules, CD69, LFA-1, and to a lesser extent, CD44, as well as increasing cell size (forward scatter (FS)) of both BALB/c and C57BL/6 CD4+ T cells, although these changes were more marked in BALB/c cells (Fig. 2, B and C, and data not shown). CD69 expression in E40K-transfected activated CD4+ T cells was consistently higher than in K179M- and empty vector-transfected cells, and a high level of CD69 expression was sustained in rested CD4+ T cells (Fig. 2C). In contrast, the expression levels of CD3 and CD4 did not differ between these transfectants. Consequently, these findings indicate that the Akt signal is involved in T cell activation and may account for the costimulation mediated by CD28 and ICOS. Akt Promotes Th1 Cell Differentiation of C57BL/6 CD4+ T Cells—In the next experiments, we explored the effects of Akt on cytokine production by CD4+ T cells prepared from C57BL/6 mice. CD4+ T cells infected with Akt constructs were enriched by cell sorting and assessed for cytokine production upon restimulation. In accordance with a previous report by another group (29Kane L.P. Andres P.G. Howland K.C. Abbas A.K. Weiss A. Nat. Immunol. 2001; 2: 37-44Crossref PubMed Scopus (268) Google Scholar), transfection with WT and E40K markedly enhanced IFN-γ production in response to anti-CD3 plus anti-CD28 and anti-CD3 plus anti-ICOS, as compared with an empty control vector, whereas K179M had no effect (Fig. 3A). Furthermore, E40K significantly augmented IFN-γ production upon restimulation even with anti-CD3 Ab alone, corroborating that the effects of costimulation could be achieved by Akt alone in the absence of anti-CD28 (29Kane L.P. Andres P.G. Howland K.C. Abbas A.K. Weiss A. Nat. Immunol. 2001; 2: 37-44Crossref PubMed Scopus (268) Google Scholar). IL-2 was also increased in these cells, showing the same pattern as IFN-γ. However, the effect on IL-4 production was very small or negligible. There are two possible interpretations of the results. One is that Akt simply transactivated Th1 cytokine production, and the other is that Akt promoted Th1 differentiation resulting in elevated IFN-γ production upon restimulation. To test these possibilities, we conducted intracellular cytokine staining after anti-CD3 plus anti-CD28 stimulation. In WT- and E40K-infected CD4+ T cells, the percentage of IFN-γ-producing cells was elevated, and the ratio of IFN-γ+ to IL-4+ cells was much higher as compared with the control vector (Fig. 3, B and C, top). These changes were more marked in the EGFP+ (retrovirus-infected) cells than in the EGFP- cells (Fig. 3C, bottom). The EGFP- cells also appeared to be affected to a lesser extent by transfected Akt through the environmental cytokines produced in the same wells in a paracrine manner. In contrast, K179M-infected cells did not show any change in numbers of IFN-γ-producing cells. The difference in the percentage of IL-4-producing cells was essentially negligible in all transfectants. In addition, ICOS and CD28 surface expression levels were each consistent among transfectants (data not shown). Consequently, these results indicate that Akt activity facilitates Th1 cell differentiation of C57BL/6 CD4+ T cells. Akt Promotes Th2 Cell Differentiation of BALB/c CD4+ T Cells—We further examined whether the above findings obtained with Th1-prone C57BL/6 mice were applicable to Th2-prone BALB/c mice. Unexpectedly, infection of BALB/c CD4+ T cells with WT and E40K Akt constructs enhanced IL-4 production, as compared with the control, in response to anti-CD3 plus anti-CD28 (Fig. 4A). To a lesser extent, WT and E40K increased IL-4 production in response to anti-CD3 plus anti-ICOS. E40K resulted in costimulation of IL-4 production upon restimulation with anti-CD3 Ab alone. In contrast, IFN-γ production in WT and E40K cells was reduced by about half in response to both types of stimulation. IL-2 production was also reduced in WT to the same extent as IFN-γ. K179M had no effect on cytokine production. Intracellular cytokine staining yielded essentially the same results in which the percentages of IL-4-producing cells were increased and the ratios of IFN-γ+ to IL-4+ cells were much lower in EGFP+ WT- and E40K-transfected cells than in EGFP+ K179M-transfected cells, although variation was observed (Fig. 4, B and C). These changes in EGFP- cells were less marked than those in EGFP+ cells (Fig. 4C, bottom). In addition, ICOS expression of E40K was much higher than that of K179M, although CD28 expression was slightly decreased (Fig. 4D). Moreover, expressions of the Th2-specific transcription factors GATA-3 (42Zheng W. Flavell R.A. Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1867) Google Scholar) and c-Maf (43Kim J.I. Ho I.C. Grusby M.J. Glimcher L.H. Immunity. 1999; 10: 745-751Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) were induced in WT and E40K cells and correlated well with the amount of IL-4 produced by each transfectant (Fig. 4E). Taken together, these findings indicate enforced expression of Akt in BALB/c CD4+ T cells to dir
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