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Essential Role of GATA3 for the Maintenance of Type 2 Helper T (Th2) Cytokine Production and Chromatin Remodeling at the Th2 Cytokine Gene Loci

2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês

10.1074/jbc.m403688200

ISSN

1083-351X

Autores

Masakatsu Yamashita, Maki Ukai‐Tadenuma, Takeshi Miyamoto, Kimio Sugaya, Hiroyuki Hosokawa, Akihiro Hasegawa, Motoko Y. Kimura, Masaru Taniguchi, James DeGregori, Toshinori Nakayama,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division. GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division. After antigenic stimulation, naive CD4 T cells differentiate into two distinct helper T cell (Th) 1The abbreviations used are: Th, helper T; TCR, T cell antigen receptor; STAT, signal transducer and activator of transcription; CAR, coxsackie/adenovirus receptor; EGFP, enhanced green fluorescence protein; ChIP, chromatin immunoprecipitation; STAT6-KO, signal transducer and activator of transcription 6-deficient; IL, interleukin; mAb, monoclonal antibody; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; Ad, adenovirus; IFU, infection units; PE, phycoerythrin. subsets, Th1 and Th2 cells (1Mosmann T.R. Coffman R.L. Annu. Rev. Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6879) Google Scholar). Th1 cells produce IFN-γ to control cell-mediated immunity against intracellular pathogens. Th2 cells produce IL-4, IL-5, and IL-13, and are involved in humoral immunity and allergic reactions (2Abbas A.K. Murphy K.M. Sher A. 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Recent studies have identified several transcription factors that control Th2 cell differentiation (8Murphy K.M. Ouyang W. Farrar J.D. Yang J. Ranganath S. Asnagli H. Afkarian M. Murphy T.L. Annu. Rev. Immunol. 2000; 18: 451-494Crossref PubMed Scopus (547) Google Scholar, 14Agarwal S. Rao A. Curr. Opin. Immunol. 1998; 10: 345-352Crossref PubMed Scopus (69) Google Scholar, 15Rengarajan J. Szabo S.J. Glimcher L.H. Immunol. Today. 2000; 21: 479-483Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Among them, GATA3 appears to be a master transcription factor for Th2 cell differentiation. GATA3 is selectively expressed in Th2 cells and its ectopic expression induces Th2 cell differentiation even in the absence of STAT6 (16Zhang D.H. Cohn L. Ray P. Bottomly K. Ray A. J. Biol. Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 17Zheng W. Flavell R.A. 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Med. 2001; 193: 643-650Crossref PubMed Scopus (89) Google Scholar) were reported. Changes in the chromatin structure of the Th2 cytokine (IL-4/IL-5/IL-13) gene loci occur during Th2 cell differentiation (14Agarwal S. Rao A. Curr. Opin. Immunol. 1998; 10: 345-352Crossref PubMed Scopus (69) Google Scholar, 21Bird J.J. Brown D.R. Mullen A.C. Moskowitz N.H. Mahowald M.A. Sider J.R. Gajewski T.F. Wang C.R. Reiner S.L. Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Th2 cell differentiation induced by ectopic expression of GATA3 results in DNA demethylation (21Bird J.J. Brown D.R. Mullen A.C. Moskowitz N.H. Mahowald M.A. Sider J.R. Gajewski T.F. Wang C.R. Reiner S.L. Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar) and the induction of DNase I-hypersensitive sites in the IL-4 gene locus (19Lee H.J. Takemoto N. Kurata H. Kamogawa Y. Miyatake S. O'Garra A. Arai N. J. Exp. Med. 2000; 192: 105-115Crossref PubMed Scopus (340) Google Scholar, 22Takemoto N. Kamogawa Y. Jun Lee H. Kurata H. Arai K.I. O'Garra A. Arai N. Miyatake S. J. Immunol. 2000; 165: 6687-6691Crossref PubMed Scopus (131) Google Scholar). Recently, we and others demonstrated that histone hyperacetylation of the Th2 cytokine gene loci occurs in developing Th2 cells in a Th2-specific and STAT6-dependent manner (23Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 24Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar, 25Fields P.E. Kim S.T. Flavell R.A. J. Immunol. 2002; 169: 647-650Crossref PubMed Scopus (287) Google Scholar). We demonstrated an essential role for GATA3 in Th2-specific histone hyperacetylation (23Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). We generated a precise map of the Th2-specific histone hyperacetylation within the type 2 cytokine gene loci, and identified a 71-bp conserved GATA3 response element (CGRE) 1.6 kbp upstream of the IL-13 locus exon 1 (23Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The conserved GATA3 response element (CGRE) may play a crucial role for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci in developing Th2 cells. However, it is still unclear whether continuous expression of GATA3 is required for the maintenance of the established chromatin remodeling at the Th2 cytokine gene loci. In the present study, we investigated the role for GATA3 in the maintenance of Th2 cytokine production and the remodeled chromatin using a newly established in vitro site-specific recombination system. The loss of GATA3 expression resulted in decreased Th2 cytokine production, reduction of histone hyperacetylation at the IL-5 gene locus, and increased DNA methylation at the IL-4 gene locus. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene loci. Mice—C57BL/6 mice were purchased from SLC (Shizuoka, Japan). STAT6-deficient mice were kindly provided by Dr. Shizuo Akira (Osaka University, Japan) (26Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1285) Google Scholar). Transgenic mice expressing coxsackie/adenovirus receptor under the control of an lck proximal promoter (coxsackie/adenovirus receptor (CAR) Tg mice) has been previously described (27Wan Y.Y. Leon R.P. Marks R. Cham C.M. Schaack J. Gajewski T.F. DeGregori J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13784-13789Crossref PubMed Scopus (70) Google Scholar). All mice used in this study were maintained under specific pathogen-free conditions and were used at 4–6 weeks of age. Animal care was in accordance with the guidelines of Chiba University. Immunofluorescent Staining and Flow Cytometry Analysis—In general, one million cells were stained with antibodies as indicated according to a standard method (28Nakayama T. June C.H. Munitz T.I. Sheard M. McCarthy S.A. Sharrow S.O. Samelson L.E. Singer A. Science. 1990; 249: 1558-1561Crossref PubMed Scopus (122) Google Scholar). Anti-CD4-fluorescein isothiocyanate (RM4–1-FITC) and anti-CD8-PE (53.6–72-PE) were purchased from BD Pharmingen. For detecting hCAR, biotinylated anti-CAR antibody (RmcB) (27Wan Y.Y. Leon R.P. Marks R. Cham C.M. Schaack J. Gajewski T.F. DeGregori J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13784-13789Crossref PubMed Scopus (70) Google Scholar) and Cy5-conjugated avidin were used. For intracellular staining, allophycocyanin-conjugated anti-IFN-γ antibody (XMG1.2; BD Pharmingen), anti-IL-5 antibody (TRFK5; BD Pharmingen), and PE-conjugated anti-IL-4 antibody (11B11; BD Pharmingen) were used (29Yamashita M. Kimura M. Kubo M. Shimizu C. Tada T. Perlmutter R.M. Nakayama T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1024-1029Crossref PubMed Scopus (178) Google Scholar, 30Omori M. Yamashita M. Inami M. Ukai-Tadenuma M. Kimura M. Nigo Y. Hosokawa H. Hasegawa A. Taniguchi M. Nakayama T. Immunity. 2003; 19: 281-294Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Flow cytometry analysis was performed on FACScalibur (BD Biosciences) and results were analyzed with CELLQUEST software (BD Biosciences). In Vitro T Cell Differentiation Culture—Purification and in vitro Th cell differentiation cultures were done as described (23Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 29Yamashita M. Kimura M. Kubo M. Shimizu C. Tada T. Perlmutter R.M. Nakayama T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1024-1029Crossref PubMed Scopus (178) Google Scholar). Splenic CD4 cells were purified using magnetic beads and an Auto-MACS Sorter™ (Miltenyi Biotec), yielding purity of >98%. For Th1 differentiation, the cells (1.5 × 106) were stimulated for 2 days with immobilized anti-TCR mAb (H57–597; BD Pharmingen) and anti-CD28 mAb (37.51; BD Pharmingen) in the presence of IL-2 (25 units/ml), IL-12 (100 units/ml), and anti-IL-4 mAb (11B11, 25% culture supernatant). For Th2 cell differentiation, cells were stimulated with immobilized anti-TCR mAb and anti-CD28 mAb for 2 days in the presence of IL-2 (25 units/ml), IL-4 (100 units/ml), and anti-IFNγ mAb (R4–6A2, 25% culture supernatant). The cells were then transferred to new wells and cultured for another 3 days in the presence of only the cytokines present in the initial culture. In some experiments, two or three cycles of the anti-TCR plus anti-CD28 stimulation were used. Virus Vectors, Infection, and Strategy for Deletion of GATA3 Transgene—The retroviral vector pMX-IRES-EGFP and a Plat-E packaging cell line were kindly provided by Dr. Toshio Kitamura (University of Tokyo, Tokyo, Japan). Retrovirus vectors containing a loxP-flanked EGFP cassette (pMX-loxP-EGFP-loxP) and a loxP-flanked human GATA3-IRES-EGFP cassette (pMX-loxP-GATA3-IRES-EGFP-loxP) were generated using the original pMX-IRES-GFP vector (31Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (438) Google Scholar) (Fig. 1A). The method for the preparation of virus supernatant was described previously (32Kimura M. Koseki Y. Yamashita M. Watanabe N. Shimizu C. Katsumoto T. Kitamura T. Taniguchi M. Koseki H. Nakayama T. Immunity. 2001; 15: 275-287Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). An adenovirus vector containing a Cre recombinase expression cassette (Ad-Cre) was kindly provided by Izumi Saito (University of Tokyo, Tokyo, Japan) (Fig. 1B) (33Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Gene (Amst.). 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar). To investigate the effect of loss of GATA3 expression in differentiated Th2 cells, we established a site-specific recombination system in CD4 T cells cultured in vitro. The strategy of introduction and deletion of the GATA3 transgene is illustrated in Fig. 1C. First, naive CD4 T cells were stimulated under Th1-skewed conditions, and infected with retrovirus vectors containing a loxP-flanked GATA3/IRES/EGFP cassette. Three days later, GFP-positive retrovirus-infected cells were sorted with a FACSVantage (BD Biosciences) flow cytometer and restimulated under the same Th1-skewed conditions of initial stimulation for a further 5 days. After another cycle of 5-day re-stimulation culture under Th1-skewed conditions, the cells were infected with Ad-Cre to delete the GATA3/IRES/EGFP transgene by expressing NLS-tagged Cre recombinase. The preparation of adenovirus supernatant was done as described (33Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Gene (Amst.). 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar). Cell entry by adenovirus involves high-affinity binding of the viral fiber capsid protein to a cellular receptor, CAR. We used CAR Tg mouse T cells to avoid the limited expression of CAR on T cells (27Wan Y.Y. Leon R.P. Marks R. Cham C.M. Schaack J. Gajewski T.F. DeGregori J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13784-13789Crossref PubMed Scopus (70) Google Scholar). In Figs. 4 and 5, a more strict protocol was used. The outline of the protocol is shown in Fig. 4A. Four days after infection of retrovirus vectors containing a loxP-flanked GATA3/IRES/EGFP, cells were stimulated with immobilized anti-TCR and anti-CD28 for 4 h, stained with anti-IL-4 PE detection mAbs using IL-4 Secretion Assay kit (number 130-090-515: Militenyi Biotec), and GFP+IL-4+ cells were sorted with purity >98%. The sorted cells were cultured for 6 days in the presence of cytokines (IL-2 and IL-12), and then another stimulation with anti-TCR and anti-CD28 was performed. Two days later, cells were infected with Ad-Cre. Four days after Ad-Cre infection, GFP– cells were sorted to exclude the small numbers of GFP+ (GATA3) expressing cells remaining in the culture. After T cell expansion by anti-TCR stimulation, analysis was done on day 25. To exclude the effect of endogenously induced GATA3 molecules, we used naive STAT6-deficient CD4 T cells and Th1-skewed culture conditions containing anti-IL-4 mAb throughout the 25-day cultivation.Fig. 5Effect of GATA3 depletion on Th2 cytokine production in in vitro differentiated Th2 cells.A, cytokine production profiles of Th2 cells after depletion of the GATA3 transgene prepared as in Fig. 4A were assessed by cytoplasmic staining (IFN-γ/IL-4 and IL-5/IL-4). The percentages of the cells present in each quadrant are shown. Four independent experiments were done with similar results. B, cytokine production of Th2 cells after depletion of the GATA3 transgene was assessed by ELISA. Four independent experiments were done with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PCR Analysis—The levels of EGFP transgene were assessed by semi-quantitative PCR with a specific primer pairs: forward, GTGAACCGTCAGATCCG-3′ and reverse, 5′-TTACTTGTACAGCTCGTC. Immunoblot Analysis—The amounts of GATA3 and GFP were assessed by immunoblotting with anti-GATA3 mouse mAb, HG3-31 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-GFP antiserum (MBL, Nagoya, Japan) as described (30Omori M. Yamashita M. Inami M. Ukai-Tadenuma M. Kimura M. Nigo Y. Hosokawa H. Hasegawa A. Taniguchi M. Nakayama T. Immunity. 2003; 19: 281-294Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). ELISA for the Measurement of Cytokine Concentration—Cells were stimulated with immobilized anti-TCR (3 μg/ml) in 48-well flat bottom plates (2.5 × 105 cells/well) for 24 h at 37 °C. The production of IL-4, IL-5, IFN-γ, and IL-2 was assessed by ELISA as described previously (30Omori M. Yamashita M. Inami M. Ukai-Tadenuma M. Kimura M. Nigo Y. Hosokawa H. Hasegawa A. Taniguchi M. Nakayama T. Immunity. 2003; 19: 281-294Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The production of IL-13 was evaluated with a mouse IL-13 ELISA kit (R & D Systems) according to the manufacturer's protocol. Reverse Transcriptase-PCR—Total RNA was isolated from cultured cells using the TRIzol reagent. Reverse transcription was carried out with Superscript II RT (Invitrogen). Three-fold serial dilutions of template cDNA were done. The primers used were as described previously (32Kimura M. Koseki Y. Yamashita M. Watanabe N. Shimizu C. Katsumoto T. Kitamura T. Taniguchi M. Koseki H. Nakayama T. Immunity. 2001; 15: 275-287Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed using histone H3 ChIP assay kits (number 17-245; Upstate Biotechnology) and specific primers as described previously (23Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Methylation-specific PCR—Genomic DNA was isolated from 1 × 106 cells by using a Wizard Genomic DNA Purification kit (Promega). Bisulfate treatment of DNA was performed by using a CpGenome DNA Modification kit (Intergen, Purchase, NY). The sequences of primers used for PCR amplification were described previously by Guo et al. (34Guo L. Hu-Li J. Zhu J. Watson C.J. Difilippantonio M.J. Pannetier C. Paul W.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10623-10628Crossref PubMed Scopus (70) Google Scholar). Efficient Adenovirus-mediated Transgene Introduction into CAR Tg CD4 T Cells in Vitro—The aim of this study was to determine the role of GATA3, if any, in the maintenance of the established Th2 phenotype and Th2-type chromatin remodeling. To investigate the effect of loss of GATA3 expression in differentiated Th2 cells, we established a Cre/loxP-mediated site-specific recombination system in T cells. The system constitutes (i) retrovirus-mediated introduction of a loxP-flanked GATA3 transgene for Th2 cell differentiation from naive CD4 T cells, and (ii) subsequent adenovirus-mediated Cre expression to delete the loxP-flanked GATA3 transgene (Fig. 1). Thus, we first evaluated the feasibility of adenovirus-mediated gene transfer in T cells. Naive CD4 T cells express limited amounts of CAR and are known to be resistant to adenovirus infection. To increase the efficiency of adenovirus infection in T cells, we used CAR Tg mice expressing CAR on T cells under the control of the proximal promoter of lck and a CD2 enhancer, in which the majority of CD4 and CD8 T cells in the spleen showed high level cell surface expression of CAR (27Wan Y.Y. Leon R.P. Marks R. Cham C.M. Schaack J. Gajewski T.F. DeGregori J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13784-13789Crossref PubMed Scopus (70) Google Scholar). Freshly prepared CD4 T cells from CAR Tg mice were infected with Ad-EGFP. Two days after infection, the majority of CAR Tg CD4 T cells expressed substantial levels of GFP compared with that of non-Tg B6 CD4 T cells (Fig. 2A). A time course of the GFP expression after Ad-EGFP infection was assessed in CAR CD4 T cells cultured under Th1- or Th2-skewed conditions (Fig. 2B). The expression of GFP peaked on day 3 in either Th1 or Th2-skewed culture conditions. The high-level expression was maintained for at least 4 days after infection. Thus, adenovirus-mediated gene transfer was efficient when using CD4 T cells from CAR Tg mice. Deletion of a loxP-flanked EGFP Transgene by Ad-Cre Infection in Cultured CD4 T Cells—The efficiency of Cre-mediated DNA recombination was next assessed in CAR Tg CD4 T cells using EGFP as an indicator. CAR Tg CD4 T cells were stimulated with anti-TCR mAb plus anti-CD28 mAb and infected with a retrovirus containing a loxP-flanked EGFP cassette (pMX-loxP-EGFP-loxP). GFP-expressing infected cells were sorted, restimulated for 3 days, and then infected with either 1 or 3 × 108 IFU of Ad-Cre as described under "Experimental Procedures." Fig. 3A shows a representative genomic DNA PCR result assessing the amount of EGFP transgene DNA left in CD4 T cells after Ad-Cre infection. The ratio of EGFP/input DNA is shown in Fig. 3B. The EGFP transgene content was significantly decreased 2 days after Ad-Cre infection in a virus-dosage dependent manner. On day 3, the majority of the EGFP transgene was deleted by infection with 3 × 108 IFU of Ad-Cre (Fig. 3A, bottom). These results suggest that the lox-P-flanked EGFP transgene was efficiently deleted in cultured CD4 T cells by Ad-Cre infection when CAR Tg CD4 T cells are used. Concurrently, the expression levels of EGFP protein after Ad-Cre infection were monitored by flow cytometry. To allow comparison with the EGFP-negative T cell control peak, no GFP sorting was done in this particular experiment. Shown are representative flow cytometry histograms (Fig. 3C), and relative intensity data from the GFP-positive peaks after infection with 3 × 108 IFU of Ad-Cre (Fig. 3D). As can be seen, expression levels of GFP in CAR Tg CD4 T cells decreased day by day after Ad-Cre infection, and were approximately one-fifth of the original expression level on day 4 when 3 × 108 IFU of Ad-Cre were used (Fig. 3D). In contrast, only a marginal decrease was observed in non-Tg CD4 T cell cultures. Although almost complete deletion of the EGFP transgene was detected on day 3 post-infection with 3 × 108 IFU of Ad-Cre (see Fig. 3, A and B), a significant amount of GFP protein (∼20–30%) was detected by flow cytometry. This could be explained by the substantially long half-life of the GFP protein. Efficient Depletion of Retrovirus-induced GATA3 Expression by Ad-Cre Infection in in Vitro Differentiated Th2 Cells—To investigate the role of GATA3 in the maintenance of the Th2 phenotype, CD4 T cells from STAT6-deficient CAR Tg mice cultured under Th1-skewed conditions were infected with retroviral vectors containing a lox-P-flanked GATA3/IRES/GFP cassette (pMx-loxP-GATA3-IRES-GFP-loxP). In the STAT6-deficient T cells cultured under Th1-skewed conditions, endogenous GATA3 induction was minimum. The outline of the protocol is shown in Fig. 4A. Four days after infection of the retrovirus vector, the cells were stimulated with immobilized anti-TCR and anti-CD28 mAb, stained with anti-IL-4-PE detection mAbs, and GFP+IL-4+ cells were sorted. Representative GFP/IL-4 profiles are shown in Fig. 4B. The sorted cells were cultured for 6 days in the presence of cytokines (IL-2 and IL-12), and another cycle of stimulation with anti-TCR and anti-CD28 was performed on day 12. Two days later, the cells were infected with Ad-Cre (3 × 108 IFU). Four days after Ad-Cre infection, GFP– cells were sorted to enrich for GATA3 transgene-depleted cells. Th1-skewed conditions were used throughout the 25-day culture. The cultured cells were harvested, and the expression levels of EGFP and GATA3 were assessed to confirm that GATA3 protein is depleted (Fig. 4, C and D). As can be seen, the levels of GFP fluorescence were reduced (Fig. 4C), and the expression levels of GATA3 protein were decreased dramatically (about 10-fold) in the cells infected with Ad-Cre (Fig. 4D). Without Ad-Cre infection, the expression of GATA3 protein was not changed during the last 7-day cultivation (data not shown). The mRNA levels of several transcriptional regulators (GATA3, c-Maf, JunB, and T-bet) in the Th2 cells after ablation of GATA3 were assessed (Fig. 4E). As expected, the mRNA levels of GATA3 were ∼1/10 of those of LacZ-infected control cells. In contrast, essentially no significant change in c-Maf or JunB expression was detected in the Ad-Cre-infected T cells. The expression of T-bet was reduced by the expression of GATA3, and restored by the depletion of GATA3 transgene. Expression of GATA3 Is Required for Th2 Cytokine Production in in Vitro Differentiated Th2 Cells—Cytokine production profiles of the cells prepared in Fig. 4 were assessed by cytoplasmic staining. As can be seen in Fig. 5A, middle panels, more than 40% (39.3 + 3.5%) of the cells infected with pMx-loxP-GATA3-IRES-GFP-loxP were IL-4 producing cells, and more than 30% (19.2 + 18.2%) were IL-5 producing cells. Marginal numbers of IFN-γ producing IL-4 non-producing cells were detected (7.0%). The percentages of IL-4 producing cells were decreased to about 25% (23.1 + 3.8%) after Ad-Cre infection, and those of IL-5 were about 16% (8.1 + 7.9%) (compare the percentages depicted in the middle and right panels in IFN-γ/IL-4 and IL-5/IL-4 profiles). A significant number of IFN-γ producing cells was noted, suggesting that some of the cells become IFN-γ producing cells after depletion of the GATA3 transgene. These results suggest that GATA3 expression is important for the maintenance of Th2 cytokine production. Next, the levels of Th2 cytokines produced in the culture supernatant were determined by ELISA. Little in the way of Th2 cytokines (IL-4, IL-13, and IL-5) were detected in supernatants from non-infected cells cultured under Th1-skewed conditions. In contrast, GATA3-transduced cells produced large amounts of Th2 cytokines and decreased amounts of Th1 cytokines (IFN-γ and IL-2) as previously reported (16Zhang D.H. Cohn L. Ray P. Bottomly K. Ray A. J. Biol. Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 17Zheng W. Flavell R.A. Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1909) Google Scholar, 18Ouyang W. Ranganath S.H. Weindel K. Bhattacharya D. Murphy T.L. Sha W.C. Murphy K.M. Immunity. 1998; 9: 745-755Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 19Lee H.J. Takemoto N. Kurata H. Kamogawa Y. Miyatake S. O'Garra A. Arai N. J. Exp. Med. 2000; 192: 105-115Crossref PubMed Scopus (340) Google Scholar). As expected, the production of Th2 cytokines (IL-4, IL-13, and IL-5) was significantly decreased by Ad-Cre infection (Fig. 5B, bottom). IFN-γ production was moderately restored. These results suggest that the continuous expression of GATA3 is important for the production of IL-4, IL-5, and IL-13 in the in vitro differentiated Th2 cells. GATA3 Is Required for the Maintenance of Hyperacetylation of Histone H3 in the IL-5 Gene Locus but Not in the IL-13/IL-4 Gene Loci—Finally, we assessed the chromatin remodeling status of the Th2 cytokine gene loci after deletion of the GATA3 transgene. Acetylation status of histone H3 (K9/14) in the nucleosomes associated with the Th2 cytokine gene loci was d

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