Gene Transcription and Switch Recombination STAT6 Is Required for ...

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STAT6 Is Required for IL-4-Induced Germline Ig Gene Transcription and Switch Recombination1 Leslie A. Linehan,* Wendy D. Warren,* Patricia A. Thompson,2* Michael J. Grusby,† and Michael T. Berton3* Transcription of the germline Cg1 and Ce Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline g1 and e transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline Cg1 and Ce genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline g1 or e transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to Sg1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline g1 transcript expression and switch recombination to Sg1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells. The Journal of Immunology, 1998, 161: 302–310.

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sotype switching occurs in B lymphocytes by a deletional recombination mechanism termed switch recombination (reviewed in Ref. 1). This process joins the assembled VDJ gene segment from the expressed Cm/Cd Ig CH gene with one of the downstream CH genes to create a newly expressed heavy chain gene. Switch recombination occurs within highly repetitive sequences known as switch regions (S)4 found upstream of each of the CH genes except Cd. Many studies have demonstrated a near perfect correlation between prior transcriptional activation of downstream CH genes and their subsequent switch recombination. The transcription of germline CH genes produces stable, yet apparently sterile, RNA transcripts (germline transcripts) that initiate upstream of the S and include an upstream exon (Ig, Ie, etc.) that is spliced to the associated CH exons. Although numerous studies, *Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78284; and †Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, MA 02115 Received for publication October 29, 1997. Accepted for publication February 26, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Wendy Will Case Foundation and the American Cancer Society (IM-680B) to M.T.B., and National Institutes of Health Grant AI40171 and a gift from the Mathers Foundation to M.J.G. M.J.G. is a Scholar of the Leukemia Society of America. 2

Current address: National Center for Toxicological Research, Division of Molecular Epidemiology, 3900 NCTR Rd, Jefferson, AR 72079. 3 Address correspondence and reprint requests to Dr. Michael T. Berton, Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7758. E-mail address: [email protected] 4 Abbreviations used in this paper: S, switch region; DC-PCR, digestion-circularization PCR; CD40L, CD40 ligand; CD40L/Sf9, CD40L-baculovirus-infected Sf9 cells; nAChRe, nicotinic acetylcholine receptor b subunit gene; a-d-dex, anti-IgD antibodies conjugated to dextran; C/EBP, CCAAT box enhancer binding proteins; EMSA, electrophoretic mobility shift assays.

Copyright © 1998 by The American Association of Immunologists

including I region promoter knockouts and replacements, have suggested a causal link between germline Ig gene transcription and the targeting of switch recombination (2– 6), unequivocal demonstration of this link has proved to be difficult. It is well established that certain cytokines regulate isotype switching in vivo and in vitro and that these same cytokines also stimulate germ-line Ig CH gene transcription (1, 7). In the mouse, IL-4 stimulates germline g1 and e Ig gene transcription (2, 8 –10) and, in combination with the appropriate costimulus such as LPS or CD40L, induces switch recombination to Sg1 and Se (11–15). Several studies have also demonstrated that CD40 signaling can synergize with cytokine signals to stimulate enhanced transcription of germline Ig genes (16 –19). CD40-mediated signaling is also presumed to be necessary to promote B cell proliferation and activation of the switch recombinase (1). Indeed, IL-4 and CD40L are sufficient to induce switching to IgG1 and IgE in vitro (20, 21). Thus, both cytokines and CD40-mediated signals are important for optimal activation of germline Ig gene transcription and efficient targeting of switch recombination. The regulation of germline Ig CH gene transcription by cytokines has been the focus of intense investigation, and several transcription factors have been implicated (1). IL-4 has been shown to activate the transcription factor STAT6 in a variety of cell types, and it is believed to be a primary mediator of IL-4-responsive gene expression (22–25). Following IL-4R stimulation, cytoplasmic STAT6 monomers are phosphorylated on tyrosine 641 by receptor-associated Janus kinases 1 and 3 (26 –28). This leads to homodimerization and translocation of activated STAT6 to the nucleus (25, 28). Recently, several laboratories have demonstrated that STAT6 contains a carboxyl-terminal transactivation domain (28 –31). The germline g1 and e Ig gene proximal promoters both contain at least one STAT6-binding site, and IL-4-induced DNAbinding protein complexes (termed NF-IL-4-g1 and NF-IL-4, respectively) with characteristics similar to STAT6 have been shown 0022-1767/98/$02.00

The Journal of Immunology to bind in vitro to these sites (24, 32, 33). Functional analyses of germline g1 and e promoter-reporter gene constructs containing mutated STAT6 sites have also suggested that binding of these complexes to the STAT6 sites are necessary for IL-4-responsive promoter activity in transiently transfected B cell lines (19, 33). Recently, B cells from STAT6 knockout (STAT62/2) mice were shown to have significantly impaired serum IgE and IgG1 responses when immunized with conventional T-dependent Ags (34 –36), but an essential role for STAT6 in the IL-4-induced activation of germ-line transcription and switch recombination at the endogenous germline Ig Cg1 and Ce genes has yet to be directly demonstrated. In the present study, the role of STAT6 in regulating germline g1 and e transcription and switch recombination to Sg1 was examined in B lymphocytes from STAT62/2 mice. The results are the first to demonstrate that B cells from STAT6-deficient mice do not make detectable germline g1 or e transcripts in response to IL-4 and do not undergo efficient switch recombination to Sg1 in response to CD40 ligand (CD40L) and IL-4. STAT6-deficient B cells are also impaired in their ability to switch to Sg1 in response to stimulation in vitro with anti-IgD Abs conjugated to dextran (a-d-dex) in the presence of IL-4 and IL-5, an in vitro model for T-independent isotype switching. These studies directly demonstrate that STAT6 is necessary for IL-4-mediated induction of endogenous germline g1 and e Ig genes and for IL-4-induced switch recombination to Sg1 in primary B lymphocytes, and provide novel evidence for the causal link between germline Ig gene transcription and the targeting of switch recombination.

Materials and Methods Mice STAT62/2 mice were derived as described previously (34). The STAT62/2 mice used in this study were backcrossed five or six times onto the BALB/c background. Control littermates (1/1) and STAT62/2 mice were housed in laminar flow isolation hoods in a specific pathogen-free environment and were used at 10 to 24 wk of age.

Cytokines and Abs Murine rIL-4 was affinity purified from culture supernatants of Sf9 cells infected with a recombinant baculovirus containing the murine IL-4 gene (provided by Dr. William Paul, National Institute of Allergy and Infectious Diseases, Bethesda, MD). One unit of IL-4 activity is defined as the amount required for half-maximal stimulation of [3H]thymidine incorporation by the HT-2 thymoma cell line in a 100-ml culture as described previously (37). Recombinant IL-5 was purchased from Genzyme (Cambridge, MA) and added to B cell cultures at a final concentration of 50 to 100 U/ml. Anti-IgD conjugated to dextran (a-d-dex) was a gift from Dr. Clifford Snapper (Uniformed Services University of the Health Sciences, Bethesda, MD) and was used in cultures at a final concentration of 3 ng/ml as previously described (38). Rabbit antisera to STAT6 were a gift from Dr. Steven McKnight (Tularik, South San Francisco, CA). Polyclonal rabbit STAT-specific Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

CD40L-baculovirus expression system Wild-type Autographica californica nuclear polyhedrosis virus and recombinant baculovirus stocks were propagated in the Spodoptera frugiperda cell line, Sf9. The recombinant mouse CD40L baculovirus used in this study has been described in detail previously (19). Sf9 cells were maintained at 27°C in TNM-FH medium (Invitrogen, San Diego, CA) containing 10% FBS and antibiotics as described by Summers and Smith (39). For optimal expression, Sf9 cells were infected with purified CD40L-baculovirus at a multiplicity of infection of 10 PFU/cell. CD40L-baculovirusinfected Sf9 cells (CD40L/Sf9) were harvested at 5 or 6 days postinfection and stored at 280°C in TNM-FH complete medium containing 10% DMSO.

Preparation and culture of B cells B cells were prepared from the spleens of STAT62/2 mice or control littermates. Erythrocytes were lysed in Tris-buffered ammonium chloride,

303 and T cells removed by treatment with monoclonal anti-Thy-1.2 (HO.13.4) (40) at 4°C and subsequently with baby rabbit complement (Pel-Freeze Biologicals, Rogers, AR) at 37°C. Where indicated, resting B cells (.1.085 g/cm3) were isolated from the 68/100% interface of discontinuous Percoll gradients according to the method of Layton et al. (41) and were 85 to 90% surface Ig1 as determined by fluorescent staining and FACS analysis. In some experiments, surface IgG12 B cells were obtained by electronic cell sorting of Percoll-fractionated B cells utilizing a FACStar Plus (Becton Dickinson, Mountain View, CA). Briefly, Percoll-fractionated, dense B cells (2 3 107/ml) were first incubated for 5 min at 4°C with anti-CD16 (2.5 mg/ml) (mAb 2.4G2; PharMingen, San Diego, CA) (42) to prevent cytophilic binding of the anti-IgG1 Ab. B cells were then incubated for 30 min with FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL). The stained cells were washed extensively, resuspended at 5 3 106 cells/ml, and sorted under sterile conditions. Dead cells and macrophages were gated out based on their characteristic forward and side scatter profiles. Immediate postsort analysis demonstrated that the sorted population contained .99% surface IgG12 B cells. B cells were cultured at 0.2 to 2.0 3 106 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 25 mM HEPES, 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and 50 mM 2-ME. All cultures were incubated at 37°C in a 6% CO2 atmosphere. B cell proliferation was measured during the last 16 h of a 64-h culture by addition of 1 mCi [3H]thymidine per well of triplicate cultures in a 96-well plate. B cells were harvested on glass fiber filters (Skatron, Sterling, VA) and [3H]thymidine incorporation was determined by liquid scintillation counting.

Semiquantitative RT-PCR Total cellular RNA was prepared from B cell cultures with TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s recommended protocol. RNA preparations were quantified by UV spectrophotometry and electrophoresed on 1% agarose minigels to assess RNA integrity and verify quantity. RT-PCR was performed in single reaction tubes using the GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA) as described previously (19). Briefly, reverse transcription of RNA (0.5 mg) was performed in a final volume of 20 ml containing 13 PCR buffer II (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 5 mM MgCl2, 1 mM dCTP, 1 mM dGTP, 1 mM TTP, 0.8 mM dATP, 20 U RNase inhibitor, 2.5 mM random hexamer primers, and 50 U reverse transcriptase. PCR amplifications were performed by addition of 80 ml of a master mix containing 1.25 mM (Ig1 and Ie PCR) or 2.5 mM (HGPRT PCR) MgCl2, 13 PCR buffer II, 2.0 mCi [a-32P]dATP (3000 Ci/mmol, DuPont, Wilmington, DE) 2.5 U AmpliTaq DNA polymerase, and 0.2 to 0.5 mM of each PCR primer. Germline e primers were selected from the upstream Ie exon and downstream Ce1 exon to amplify a 325-bp DNA product as reported previously (19): upstream primer, 59-GCACAGGGGGCAGAAGAT-39 (nucleotides 799 – 816) (43); downstream primer, 59-CGTTGAATGATGGAGGAT-39 (nucleotides 377–394) (44). Germline g1 primers have been described (45) and were selected from the Ig1 exon and the Cg1 hinge region: upstream primer, 59-CAGATCTTTGAGTCATCCTATCACG-39 (nucleotides 1673–1697) (46), downstream primer, 59-TACATATGCAAGGCTTA-39 (nucleotides 766 –782) (47). All RNAs were also amplified with primers specific for the housekeeping gene HGPRT (48) to control for sample to sample variation in RNA isolation and integrity, RNA input, and reverse transcription: upstream primer, 59-GTTGGATACAGGCCAGACTTT GTTG-39 (nucleotides 514 –538); downstream primer, 59-GATTCAACTT GCGCTCATCTTAGGC-39 (nucleotides 652– 678). Both PCR amplification cycle number and RNA input were varied over a broad range for each pair of primers to establish PCR conditions that resulted in a linear correlation between RNA input and PCR product and that would therefore allow reliable comparison of the relative levels of germline Ig transcripts and HGPRT mRNA in different samples (see Fig. 3A). The following reaction conditions were chosen as optimal: Ie and HGPRT PCRs were incubated at 95°C for 3 min for 1 cycle, then at 95°C for 45 s, 54°C for 45 s, and 72°C for 2 min for 25 and 20 cycles, respectively; Ig1 PCRs were incubated at 95°C for 1.5 min, 47°C for 2 min, and 72°C for 2 min for 28 cycles. All PCR amplifications were performed at least twice with multiple sets of experimental RNAs. PCR products were analyzed on 8% polyacrylamide gels. Gels were dried and exposed to x-ray film or to a phosphor screen.

Preparation of nuclear extracts B cells (1 to 1.5 3 106/ml) were cultured for 16 h in medium alone or with IL-4 (1000 U/ml). Cells were harvested and disrupted on ice by hypotonic lysis in 20 mM HEPES, pH 7.9, 10 mM KCl, and 1.5 mM MgCl2. Nuclei were extracted as described previously (19, 49) by incubation for 30 min

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at 4°C in 20 mM HEPES, pH 7.9, 300 mM KCl, 1.5 mM MgCl2, and 20% glycerol with gentle mixing. All buffers included the protease inhibitors PMSF (0.2 mM) or AEBSF [4-(2-aminoethyl)-benzenesulfonylfluoride, HCl] (0.2 mM) (Calbiochem, San Diego, CA), leupeptin (50 mg/ml), aprotinin (2 mg/ml), and DTT (0.5 mM). Extracts were quick frozen in liquid nitrogen and stored in aliquots at 280°C. Protein concentrations were determined by the Bradford method (50) (Bio-Rad Laboratories, Richmond, CA).

Electrophoretic mobility shift assays (EMSA) Binding reactions were performed in 20 ml of binding buffer (15 mM HEPES, pH 7.9, 30 to 50 mM KCl, and 12% glycerol) with 10 mg of nuclear extract, 0.5 mg of poly (dI-dC) (Pharmacia, Piscataway, NJ), and 60,000 cpm of [a-32P]-labeled DNA probe. Complementary oligonucleotides containing the NF-IL-4-g1 binding site (59-catgCATTCACAT GAAG-39) were annealed as previously described (32), end-labeled with [a-32P]dNTPs (3000 Ci/mmol; NEN, Boston, MA) by the Klenow fragment of DNA polymerase I, and purified on 8% native polyacrylamide gels for use as a probe in the EMSA. Binding reactions were preincubated without probe for 15 min at room temperature followed by an additional 15-min incubation with probe. For Ab supershift experiments, Abs were added for an additional 30-min incubation prior to probe addition. DNAbinding reactions were electrophoresed at 4°C and 250 V for 1 to 2 h on 4% nondenaturing polyacrylamide gels. Gels were prerun for 1 h at 100 V in recirculating 0.53 Tris-borate-EDTA buffer. Dried gels were exposed to x-ray film at 280°C or to a phosphor screen (Molecular Dynamics, Sunnyvale, CA).

Purification of genomic DNA Genomic DNA was purified from cultured B cells by standard methods. Briefly, B cells were lysed overnight at 37°C in 0.5% SDS, 10 mM TrisHCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, 50 mg/ml RNase A, and 0.2 mg/ml proteinase K. The lysate was extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and once with an equal volume of chloroform-isoamyl alcohol. DNA was ethanol precipitated, pelleted, rinsed twice with 70% ethanol, air dried, and dissolved in 10 mM Tris-HCl, pH 7.6, and 1 mM EDTA. DNA concentration was determined by UV absorption at 260 nm and 280 nm.

Digestion-circularization PCR (DC-PCR) DC-PCR was performed as described in detail by Chu et al. (51). Genomic DNA samples (2 mg/100 ml) were digested overnight with EcoRI (Promega, Madison, WI), diluted to 1.8 mg/ml, and ligated overnight at 16°C with T4 DNA (Promega) to circularize the EcoRI fragments. The ligated samples were then dialyzed against distilled H2O for 5 to 10 min through 0.05 mM filters (type VM; Millipore, Bedford, MA). Sm/Sg1 DC-PCR was performed on each DNA sample with a 59 Sm primer (59-GGC CGGTCGACGGAGACCAATAATCAGAGGGAAG-39) and a 39 Sg1 primer (59-GCGCCATCGATGGAGAGCAGGGTCTCCTGGGTAGG39). Ligated DNA (5 ng) was amplified in 20 ml of Sm/Sg1 PCR buffer (2.0 mM MgCl2, 10 mM Tris-HCl, pH 9.0, 50 mM KCl), 0.4 mM 59 Sm, and 39 Sg1 primers, 200 mM of each dNTP, 0.2 ml of a-[P32]dATP (3000 Ci/mmol) (NEN DuPont) and 0.5 U Taq DNA polymerase (Perkin-Elmer, Norwalk, CT). Amplification of Sm/Sg1 circles yields a 219-bp PCR product. To monitor amplification efficiency in each tube, an equivalent amount (25 copies) of an exogenous Sm/Sg1 plasmid substrate (p4AP) (51) was also included in each reaction. Amplification of this substrate by the Sm and Sg1 primers yields a 265-bp PCR product. To monitor sample-to-sample variation in DNA isolation, EcoRI digestion and ligation, DNA samples were also amplified with primers for the nicotinic acetylcholine receptor b subunit gene (nAChRe) (59-GGCCGGTCGACAGGCGCGCACTGAC ACCACTAAG-39 and 59-GCGCCATCGATGGACTGCTGTGGGTTTCA CCCAG-39) as described (51). Ligated DNA (1.8 ng) was amplified in 20 ml of nAChRe PCR buffer (1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, and 50 mM KCl), 0.4 mM 59 and 39 nAChRe primers, 200 mM of each dNTP, 0.1 ml of a-[P32]dATP (3000 Ci/mmol), and 0.5 U Taq DNA polymerase. Amplification of genomic DNA with the nAChRe primers yields a 753-bp PCR product. Amplification efficiency of the nAChRe PCR was also monitored by including in each tube an equivalent amount (25 copies) of an exogenous nAChRe plasmid substrate (p2AO) (51) that yielded a 490-bp PCR product (data not shown). All DNA amplifications were performed as follows: 94°C for 6 min, 5 cycles at 94°C for 1 min, 65°C for 1 min, and 72°C for 2 min; 30 cycles at 94°C for 1 min, 68°C for 1 min, and 72°C for 2 min; 72°C for 7 min. The PCR products were analyzed on 8% polyacrylamide gels and the dried gels were exposed to a phosphor screen for subsequent analysis on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The amount of DNA substrate included in each DC-PCR and the

FIGURE 1. STAT6-deficient B cells proliferate normally in response to CD40 stimulation. Percoll-fractionated, resting splenic B cells prepared from STAT62/2 mice and STAT61/1 control mice were cultured for 64 h in medium alone, LPS (20 mg/ml), or with the indicated ratios of B cells: CD40L/Sf9 cells (shown in parentheses). [3H]Thymidine incorporation was measured during the last 16 h of the culture period as described in Materials and Methods and expressed as the mean cpm (6SD) of triplicate cultures. number of PCR amplification cycles were chosen such that a linear correlation was obtained between DNA input and PCR product output. This allowed the relative levels of nAchRe and Sm/Sg1 products amplified from different DNA samples to be compared in a semiquantitative fashion (see Fig. 4B).

Results

STAT62/2 B cells proliferate normally in response to CD40 stimulation Preliminary analyses of the immune responsiveness of STAT62/2 mice suggested possible defects in T-dependent serum IgG1 and IgE expression, but no additional analysis was performed to characterize these defects in more detail (34 –36). We therefore investigated the effect of STAT6 deficiency on isotype switching in B cells directly, and chose to do so in an in vitro “T-dependent” system we have described previously (19). In this system, stimulation of normal splenic B cells in vitro with baculovirus-expressed CD40L plus IL-4 and IL-5 leads to proliferation, germline g1 and e Ig transcript expression, and switching to IgG1 and IgE. Others have obtained similar results by stimulating mouse and human B cells with soluble CD40L or anti-CD40 Abs plus cytokines (16, 21, 52–55). To begin to assess the role of STAT6 in this system, we tested the ability of STAT62/2 B cells to proliferate in response to CD40 stimulation since B cell proliferation is believed to be required for switch recombination (1). Representative results shown in Figure 1 demonstrate that STAT62/2 B cells proliferated vigorously in response to stimulation with CD40L and to the same degree as control B cells. The small positive effect of IL-4 costimulation, reported previously (19, 21), was not observed in the STAT62/2 B cell cultures (data not shown). Thus, the previously observed defects in isotype switching to IgG1 and IgE in STAT6deficient mice immunized with T-dependent Ags is not due to an intrinsic inability of the B cells to proliferate in response to CD40mediated signals. STAT6 is a component of NF-IL-4-g1 and is required for NF-IL-4-g1 activation We previously characterized an IL-4-induced nuclear DNA-binding factor (termed NF-IL-4-g1) that binds in vitro to an IL-4-responsive element in the germline g1 promoter (32). An apparently similar IL-4-induced complex has been shown to bind to the germline e promoter (24, 33). These nuclear factors are believed to be required for IL-4-induced germline g1 and e Ig gene transcription

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FIGURE 2. STAT6 is a component of NF-IL-4-g1 and is required for NF-IL-4-g1 activation. A, Nuclear extracts were prepared from STAT61/1 B cells cultured in the presence or absence of IL-4 (1000 U/ml), as described in Materials and Methods. NF-IL-4-g1 activity was assayed in each extract by EMSA using a double-stranded oligonucleotide probe containing the germline g1 GAS (IFN-g activation site) described previously (23, 32). Rabbit Abs (1 mg) specific for individual STATs or normal rabbit Ig (NRbIg) were added as indicated to individual binding reactions for 30 min prior to probe addition. The NF-IL-4-g1 complex and the supershifted complex are indicated by arrows. One of two independent experiments with similar results is shown. B, Nuclear extracts were prepared from STAT62/2 and STAT 61/1 B cells cultured in the presence or absence of IL-4 (1000 U/ml). NF-IL-4-g1 activity was assayed in each extract by EMSA using the germline g1 GAS probe. The NF-IL-4-g1 complex is indicated. One of three independent experiments with similar results is shown.

based on functional analyses of their respective binding sites in transient reporter gene expression systems (19, 33). The binding sites for these factors contain exact matches with the human STAT6 consensus sequence (TTCN4GAA) (56, 57), although the g1- and e-binding sites differ in the sequence of the N4 spacer region. These factors also resemble the STATs in that they are rapidly phosphorylated on tyrosine and translocated to the nucleus following IL-4 stimulation (24, 32). To demonstrate that NF-IL4-g1 does indeed contain STAT6, we performed EMSA with nuclear extracts prepared from normal BALB/c B cells cultured with or without IL-4 and a double-stranded oligonucleotide probe containing the NF-IL-4-g1-binding site (Fig. 2A, lanes 1 and 2). A panel of STAT-specific Abs was used to attempt to block or supershift the NF-IL-4-g1 complex. NF-IL-4-g1 was completely supershifted to a slower mobility in the gel only by the STAT6specific Ab (lane 8). Similar results were obtained with two other STAT6-specific antisera (data not shown). To directly determine if STAT6 is required for NF-IL-4-g1 formation and activation, we also stimulated B cells prepared from STAT62/2 mice with IL-4 and assayed nuclear and cytoplasmic extracts for NF-IL-4-g1 activity by EMSA. Figure 2B demonstrates that NF-IL-4-g1 was not present in the nuclei of STAT62/2 B cells (compare lanes 2 and 4) nor was it present in the cytoplasm of these cells (data not shown).

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FIGURE 3. Germline g1 and e Ig transcript expression in STAT6-deficient B cells. A, Linear regression analysis of the relationship between RNA input and specific PCR product output for the germline g1, germline e, and HGPRT RT-PCRs. Twofold serial dilutions of total cellular RNA prepared from splenic B cells (106/ml) stimulated for 16 h with CD40L/Sf9 (105/ml) and IL-4 (500 U/ml) were amplified in single-tube RT-PCRs with primers specific for Ig1 (Œ), Ie (■), or HGPRT (F) cDNAs as described in Materials and Methods. PCR products were analyzed on 8% polyacrylamide gels and dried gels were exposed to a phosphor screen. Quantification of specific Ig1, Ie, and HGPRT PCR products was performed by volume integration with ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and linear regression analysis was performed by SigmaPlot for Windows (Jandel Scientific Software, San Rafael, CA); r2, correlation coefficient. B, Splenic B cells (106/ml) from STAT62/2 and STAT61/1 mice were cultured for 16 h with medium, IL-4 (500 U/ml), CD40L/Sf9 (105/ml), or with IL-4 and CD40L/Sf9. Total cellular RNA was prepared from the cultured cells and 0.5 mg was assayed for relative amounts of Ig1 and Ie Ig transcripts by the semiquantitative RT-PCR shown in A and described in Materials and Methods. HGPRT mRNA expression was determined for each sample in separate RT-PCR reactions as a control for RNA input. A representative experiment of three performed is shown.

The integrity of these extracts was verified by assessing binding to a nuclear factor-kB probe (data not shown). Together, these results demonstrate that NF-IL-4-g1 contains STAT6 and requires STAT6 for activation. STAT6 is required for IL-4-induced but not CD40L-induced germline g1 and e Ig transcript expression The germline g1 Ig gene has been shown to be regulated at the transcriptional level (58), and several studies utilizing transiently

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GERMLINE TRANSCRIPTS AND S RECOMBINATION IN STAT62/2 B CELLS transfected B cell lines have suggested an important role for STAT6 in the IL-4-mediated activation of the germline g1 and e Ig promoters (32, 33, 59). To test this directly in nontransformed B cells, we studied the induction of endogenous germline g1 and e transcripts in splenic B cells from STAT62/2 mice. Total RNA was purified from control or STAT62/2 B cells cultured 16 h in medium alone, IL-4, CD40L/Sf9, or IL-4 and CD40L/Sf9, and analyzed by a semiquantitative RT-PCR with primers specific for the Ig1 and Ie transcripts as described previously (19). Levels of HGPRT mRNA were determined for each sample to serve as a control for RNA integrity and input. A linear correlation between RNA input and PCR product for each RT-PCR demonstrated that the relative amounts of germline g1, germ-line e, and HGPRT mRNA present in different RNA samples could be reliably compared in a semiquantitative fashion (Fig. 3A). As shown in Figure 3B and reported previously (19), IL-4 and CD40L each induced significant but low level germline g1 transcript expression in wildtype B cells, and together acted synergistically to induce significantly higher levels of this RNA (lanes 2 to 4). Similarly, CD40L alone induced low level germline e transcript expression and acted in synergy with IL-4 to induce a much greater level of these transcripts. Although not observed in the experiment shown, we have found that IL-4 alone induces low but detectable germline e transcript expression in approximately three out of five experiments as we have reported previously (19). In contrast to these results in wild-type B cells, IL-4 did not induce detectable levels of germline g1 or e transcripts in STAT62/2 B cells (Fig. 3, lane 6). In addition, no synergy was observed between IL-4 and CD40L in the STAT62/2 B cells (compare lanes 7 and 8). The induction of germline transcripts by CD40-mediated signaling was completely unaffected by the STAT6 deficiency, consistent with our previous studies that indicated that CD40-mediated induction of germline g1 transcripts was independent of IL-4 signaling (19). Thus, STAT6 is necessary for IL-4-mediated induction of endogenous germ-line g1 and e transcription and for synergy with CD40-mediated signals in primary B cells, but it is not necessary for CD40mediated induction of germline transcripts. STAT6 is required for efficient T-dependent switch recombination to Sg1 Analysis of serum IgG1 levels in STAT62/2 mice immunized with conventional T-dependent Ags such as DNP-OVA and Nippostrongylus brasiliensis indicated a possible defect in T-dependent switching to IgG1 in these animals (35). To test this possibility directly at the DNA level, we used the DC-PCR technique developed by Chu et al. (51) to detect actual switch recombination in control and STAT62/2 B cells stimulated in vitro with CD40L and IL-4. The DC-PCR strategy is outlined in Figure 4A and the detailed methodology is described in Materials and Methods. Briefly, high m.w. genomic DNA prepared from B cells induced in culture

FIGURE 4. STAT6 is required for switch recombination to Sg1 induced by CD40L and IL-4. A, Schematic diagram of DC-PCR method. Switch regions are indicated by hatched rectangles; Sm/Sg1 PCR primers a and b, and nAchRe PCR primers c and d are indicated by small horizontal arrows; RI, EcoRI sites. B, Linear regression analysis of the relationship between genomic DNA input and amplified nAChRe DC-PCR product. Twofold serial dilutions of genomic DNA from wild-type B cells stimulated with CD40L/Sf9 and IL-4 were amplified with nAchRe primers as described in Materials and Methods. DC-PCR products were electrophoresed on 8%

polyacrylamide gels, and dried gels were exposed to a phosphor screen. Quantification and linear regression analysis was performed as described in the legend for Figure 3; r2, correlation coefficient. Similar results (r2 5 0.9909) were obtained for the Sm/Sg1 DC-PCR (data not shown). C, DC-PCR analysis of Sm/Sg1 switch recombination in wild-type and STAT62/2 B cells stimulated in vitro with CD40L/Sf9 and IL-4. STAT61/1 and STAT62/2 B cells (2.5–5 3 105 cells/ml) were cultured with CD40L/Sf9 (105 cells/ml), CD40L/ Sf9 and IL-4 (1000 U/ml), or LPS (20 mg/ml) for 4 days. Freshly isolated, unstimulated B cells were also analyzed as controls as indicated. Genomic DNA was isolated, digested with EcoRI, ligated and used as a template for DC-PCR as described in detail in Materials and Methods. The nAchRe, p4AP control, and Sm/Sg1 PCR products and their respective sizes are indicated. One of three experiments with similar results is shown.

The Journal of Immunology

FIGURE 5. STAT6 is required for IL-4-induced switch recombination to Sg1 in the a-d-dex T-independent culture system. DC-PCR analysis of Sm/Sg1 switch recombination was performed with genomic DNA prepared from wild-type and STAT6-deficient B cells stimulated in vitro with a-ddex (3 ng/ml), a-d-dex and IL-5 (100 U/ml), or a-d-dex, IL-5 and IL-4 (1000 U/ml) for 4 days. Unstimulated B cells were also analyzed as controls as indicated. The nAchRe, p4AP control, and Sm/Sg1 PCR products and their respective sizes are indicated. One of three independent experiments with similar results is shown.

to switch to Sg1 was digested with EcoRI to create DNA fragments that contain the Sm/Sg1 recombination junctions from rearranged chromosomes. The digested DNA fragments were ligated under conditions that promote circularization, and the region spanning the ligated EcoRI sites (the circle “joint”) was amplified using primers complementary to sequences near the EcoRI site 59 of Sm and the EcoRI site 39 of Sg1. The PCR product is of uniform size regardless of exactly where rearrangement takes place within Sm and Sg1, and the proper size PCR product (219 bp) can only be generated from rearranged DNA containing Sm/Sg1 junctions,

307 since in unrearranged DNA the primer targets are on separate DNA circles and no amplification is possible. As a control for effects of all the DNA manipulations and for DNA input, each sample was also analyzed with primers specific for a template not dependent on rearrangement for its creation, the nonrearranging nAChRe gene (51). Additionally, an exogenous PCR substrate was also included in the reactions to control for variations in amplification efficiency from tube to tube. All PCR amplifications were performed in the presence of [a-32P]dATP, the products were visualized by gel electrophoresis, and relative amounts quantified following exposure to a phosphor screen as described in Materials and Methods. The semiquantitative nature of the DC-PCR was verified by demonstrating a linear relationship between genomic DNA input and the nAChRe and Sm/Sg1 PCR products over a range of genomic DNA input (Fig. 4B and data not shown). For these studies, Percoll-fractionated, resting splenic B cells were cultured in vitro for 4 days in the presence of various stimuli as indicated in Figure 4C. Genomic DNA was purified from each culture and analyzed for switching to Sg1 by DC-PCR. The combination of CD40L and IL-4 or LPS and IL-4 induced switch recombination between Sm and Sg1 as evidenced by the readily detectable 219 bp PCR product indicative of recombination (Fig. 4C, lanes 3 and 4). These results clearly demonstrate that switch recombination to Sg1 is induced by CD40L and IL-4 in wild-type B cells in this culture system. Although not observed in the experiment shown, the switch PCR product was occasionally detected at very low levels in unstimulated B cells and in B cells stimulated with CD40L alone, suggesting that a small fraction of the starting B cell population may have already switched to Sg1 in vivo. In contrast to the results with wild-type B cells, switch recombination to Sg1 in response to IL-4 and CD40L or LPS was significantly reduced in STAT62/2 B cells and was not detectable in the experiment shown (Fig. 4C, lanes 7 and 8). Titrations of increased amounts of genomic DNA into the DC-PCR assay indicated that switch recombination occurred at least 20-fold less frequently in STAT62/2 B cells than in wild-type B cells, and that there was no apparent difference in the levels of Sm/Sg1 PCR product in STAT62/2 B cells stimulated with CD40L alone or with CD40L and IL-4 (data not shown). The switch product detected in STAT62/2 B cells by DC-PCR at higher DNA input was possibly derived from a small fraction of B cells that had already undergone STAT6-independent switching in vivo rather than de novo switching induced by CD40L and IL-4. This would be consistent with the previous observation that STAT62/2 mice can make IgG1 in response to T-dependent Ags, albeit at 10-fold reduced levels (35). STAT6 is also required for switching to Sg1 in an in vitro model for T-independent switching

FIGURE 6. STAT6 is required for high level expression of germline g1 transcripts in response to a-d-dex, IL-5, and IL-4. Splenic B cells (106/ml) from STAT62/2 and STAT61/1 mice were cultured for 16 h with medium, a-d-dex (3 ng/ml), a-d-dex and IL-5 (100 U/ml), or a-d-dex, IL-5, and IL-4 (1000 U/ml). Total cellular RNA was prepared from the cultured cells and assayed for relative amounts of germline g1 Ig transcripts by semiquantitative RT-PCR as described in Materials and Methods. HGPRT mRNA expression was also determined for each sample in separate RTPCR reactions as a control for RNA input. PCR products were analyzed on 8% polyacrylamide gels and dried gels were exposed to a phosphor screen. One of two independent experiments with similar results is shown.

Costimulation of murine B cells in vitro with cytokines and a-ddex has been studied extensively as a model for B cell responses to T-independent type II Ags (38). In vitro polyclonal stimulation with a-d-dex in combination with IL-4 and IL-5 has been shown to induce switch recombination to IgG1 (60); and in two different in vitro systems, IL-5 has been shown to be necessary for activating switch recombination to Sg1 in B cells stimulated with anti-Ig, while IL-4 is required for transcriptional activation of the germline g1 Ig gene (13, 60). Interestingly, immunization of mice with unconjugated, soluble anti-IgD Abs results in a vigorous induction of IgG1 isotype switching and serum IgG1 expression (61), and this induction is apparently independent of STAT6 expression as demonstrated recently in STAT62/2 mice (34, 36). To further explore the role of STAT6 in the B cell response to polyclonal stimulation with anti-IgD, we tested whether

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STAT62/2 B cells could undergo switch recombination to Sg1 in the in vitro a-d-dex system described previously (60). Percolled, sIgG12 B cells from control or STAT62/2 mice were cultured for 4 days with a-d-dex (3 ng/ml) in the presence or absence of IL-5 and/or IL-4 as described previously (60). Genomic DNAs were prepared and tested by DC-PCR for switching to Sg1. As shown in Figure 5 and reported previously (60), the combination of a-d-dex, IL-5, and IL-4 consistently induced a significantly greater level of Sm/Sg1 recombination than did a-d-dex and IL-5 only in wildtype B cells (compare lanes 3 and 4). In contrast, induction of switch recombination in STAT62/2 B cells by a-d-dex, IL-5, and IL-4 was significantly reduced relative to that induced by a-d-dex and IL-5 in the experiment shown (Fig. 5, compared lanes 7 and 8), and was completely inapparent in two additional independent experiments with B cells from different STAT62/2 mice (data not shown). The slight difference in the levels of the Sm/Sg1 PCR product from STAT61/1 and STAT62/2 B cells stimulated with a-d-dex and IL-5 (lanes 3 and 7) was not reproducible in two additional independent experiments. Titrations of genomic DNA from control and STAT62/2 B cells in the DC-PCR indicated a 10 to 20-fold lower level of Sm/Sg1 recombination in STAT62/2 B cells stimulated with a-d-dex, IL-5, and IL-4 relative to the level observed in control B cells (data not shown). Thus, the significant increase in Sm/Sg1 recombination induced by IL-4 in primary B cells costimulated with a-d-dex and IL-5 is also dependent on STAT6. The low level of switched PCR product reproducibly detected in both STAT61/1 and STAT62/2 B cells stimulated with a-d-dex and IL-5 (lanes 3 and 7) may have derived from de novo switch recombination in culture or from a small fraction of B cells that had already undergone switching to Sg1 in vivo. A similar low level was also sometimes observed in B cells stimulated with a-ddex alone and, as noted in the experiments with CD40L/Sf9, in unstimulated B cells as well. To correlate the observed effects of STAT6-deficiency on switch recombination in this model system to the effects on germline g1 transcript expression, RT-PCR was performed with RNA from B cell cultures similarly stimulated with a-d-dex and cytokines. Figure 6 demonstrates, as has been reported previously (60), that a-ddex alone can induce germline g1 transcript expression and that this expression is not enhanced by treatment with IL-5 (compare lanes 1 to 3, and 5 to 7). The addition of IL-4, however, resulted in a dramatic increase in germ-line g1 transcript expression in control B cells, whereas the levels in STAT62/2 B cells were equivalent to the levels observed upon stimulation with a-d-dex alone or in combination with IL-5 (compare lanes 4, 7, and 8). These data demonstrate that IL-4-mediated, high level expression of germline g1 transcripts and IL-4-induced Sm/Sg1 recombination in the a-d-dex model system are both dependent on STAT6, and suggest that the unimpaired switching to IgG1 previously reported in STAT62/2 mice immunized with soluble anti-IgD probably occurs by a distinct mechanism (34, 36).

Discussion STAT6 is a transcription factor believed to be critically involved in regulating IL-4-responsive genes, and recent analyses of the expression of several IL-4-regulated gene products (CD23, MHC class II, Thy-1) in STAT6 knockout mice have confirmed its important role (23, 25, 34 –36). In vitro DNA-binding assays and studies employing promoter-reporter gene constructs transiently transfected into B cell lines have strongly suggested that STAT6 is also necessary for IL-4-induced transcription of the unrearranged g1 and e Ig genes prior to switch recombination at these loci in murine B cells. A role for STAT6, however, in regulating endog-

enous germline g1 and e Ig gene transcription and switch recombination in primary B cells has yet to be directly demonstrated. Recent analyses of serum Ig isotype responses in STAT62/2 mice revealed defects in IgG1 and IgE expression, but the effects of STAT6 deficiency on germ-line transcript expression and switch recombination were not investigated (34 –36). We therefore explored the role of STAT6 in regulating these events by studying B cells from STAT62/2 mice. Several findings relevant to the role of STAT6 in isotype switching emerged from these studies: 1) STAT6 is a required component of the DNA-binding protein complex, NF-IL-4-g1, shown previously to be activated in IL-4-stimulated B cells and to bind in vitro to an IL-4 responsive GAS (IFN-g activation site) element in the germline g1 Ig gene promoter (32); 2) STAT6 is required for IL-4-mediated induction of germline g1 and e Ig transcripts, but is not required for CD40mediated induction of these genes; 3) switch recombination to Sg1 in response to stimulation by CD40L and IL-4 is highly dependent on STAT6; and 4) STAT6 is also required for IL-4-mediated germline g1 transcript expression and efficient switching to Sg1 in the a-d-dex model system for T-independent B cell responses (38, 62). These studies have identified the mechanisms responsible for the previously observed defects in serum IgG1 and IgE responses in STAT62/2 mice (34 –36) and provide direct evidence of a role for STAT6 in mediating IL-4-induced germline transcription and switch recombination in normal B cells. We and others have previously described IL-4-induced factors in B cells that bind specifically to IL-4 response elements in the germline g1 and e promoters in vitro (24, 32, 33, 59, 63). These factors are latent in the cytoplasm and, upon IL-4 stimulation, are rapidly phosphorylated on tyrosine and translocated to the nucleus, all properties that strongly suggested identity with STAT6 (22, 23, 25). The results presented herein firmly establish that STAT6 is a necessary component of NF-IL-4-g1 since EMSA supershift experiments with anti-STAT6 Abs clearly demonstrated the presence of STAT6 in the NF-IL-4-g1 complex and since no complex was induced by IL-4 in STAT6-deficient B cells. Recently, rSTAT6 has been shown to transactivate the mouse and human germline e Ig IL-4-response elements in reporter gene constructs, and an autonomous transactivation domain has been identified in its C terminus (28 –31). One study has also recently shown that a truncation mutant of human STAT6, lacking the transactivation domain, can act in a dominant negative fashion to partially inhibit the induction of endogenous germline e transcripts in a human Burkitt’s lymphoma B cell line, supporting a role for STAT6 as a necessary factor in the expression of the endogenous germline e Ig gene (30). It has also been shown, however, that single or multimerized STAT6-binding sites from the germline e promoter are unable to confer IL-4 responsiveness to a minimal promoter unless adjacent sequences containing a C/EBP site are included (28, 30, 33). We have also observed that the multimerized STAT6 site from the murine germline g1 promoter, including an overlapping C/EBP site (32, 59), fails to confer detectable IL-4 responsiveness to a minimal promoter transfected into the BCL1-3B3 and M12.4.1 murine B cell lines (K. Roberts and M. T. Berton, unpublished observations). Thus, although STAT6 is necessary for germline g1 and e transcription as shown in this study, it may require cooperation with other factors bound at adjacent regulatory sites in order to activate the basal transcription machinery. Our studies clearly demonstrate that B cells deficient in STAT6 do not undergo switch recombination to Sg1 efficiently in response to CD40L and IL-4, a stimulus designed to model T-dependent responses. The significant reduction in switch recombination to Sg1 observed in these studies correlated with a concomitant loss of germline g1 transcript expression in response to IL-4 in the

The Journal of Immunology STAT6-deficient B cells. The fact that switch recombination does not appear to be induced to a significant degree above background in B cells stimulated with CD40L alone, despite the induction of low levels of germline transcripts, suggests the possibility that CD40 signaling alone is not sufficient for activation of the recombinase or, as we have suggested previously, that a minimum threshold of germline transcript expression is required for switch recombination to be effectively targeted and/or activated (19). Although we have not directly tested switch recombination to Se, we have shown that no germline transcripts are made in response to IL-4 and that there is no synergistic activation with the combination of IL-4 and CD40L. Since IgE is completely undetectable in STAT6-deficient mice (34 –36), we assume that switch recombination to Se is also completely abrogated, but this remains to be demonstrated. It has recently been shown that IgE can be induced in IL-4-deficient mice (64 – 66), suggesting a possible IL-4-independent pathway for STAT6 activation. Alternatively, a STAT6independent mechanism for IgE expression could exist that has yet to be identified in the STAT6-deficient mice. In vivo immunization with anti-IgD was shown to induce normal IgG1 responses in STAT6-deficient mice (34, 36). Our studies have demonstrated, however, that IL-4-dependent switching to Sg1 in response to in vitro stimulation with anti-IgD conjugated to dextran and the combination of IL-5 and IL-4 is significantly impaired in STAT6-deficient B cells, indicating that efficient switching in this in vitro model for T-independent responses is also dependent on STAT6. We also demonstrated that high level expression of germline g1 transcripts correlates with efficient switching to IgG1 in this system, as both IL-4-dependent germline g1 transcript expression and switch recombination to Sg1 are blocked in STAT6-deficient B cells. The mechanism by which in vivo immunization with soluble anti-IgD induces normal levels of switching to IgG1 in the absence of STAT6 remains to be defined and is particularly intriguing since anti-IgD has been shown previously to induce abnormally low levels of IgG1 in IL-4-deficient mice (66, 67). Although we did not observe a significant STAT6independent effect of IL-4 in either in vitro system employed in our studies, the previous in vivo studies suggest that such a mechanism may be activated in response to certain immunogens. Numerous I region promoter knockouts and replacements have provided support for the hypothesis that germline Ig gene transcription is necessary for switch recombination to be targeted to a specific switch region (4 – 6, 58). The interpretation of these experiments has been complicated, however, by the relatively large disruptions made in the I regions and associated switch regions and the resultant possibility that additional important sequences, possibly recombination enhancers, were also disrupted. By taking advantage of the lack of a necessary transcription factor in STAT6-deficient B cells, rather than disruption of the germline g1 Ig gene, our studies provide novel evidence in support of a causal link between germline Ig gene transcription, and/or the expression of germline transcripts, and switch recombination. Although the lack of STAT6 could possibly disrupt switch recombination at other levels, such as through negative effects on B cell proliferation or on the induction or activation of the switch recombinase, we consider this very unlikely. In support of this view, we have shown that STAT6-deficient B cells proliferate vigorously in response to stimulation with CD40L and IL-4. Moreover, switching to IgG1 is normal in STAT6-deficient mice immunized with anti-IgD, and switching to other Ig isotypes, besides IgE, is also unimpaired (34 –36). In summary, our studies directly demonstrate that STAT6 is required for IL-4-mediated transcriptional activation of the endogenous germline g1 and e Ig genes in primary B lymphocytes and

309 confirm previous studies performed in transformed B cell lines. These studies further demonstrate that STAT6 is required for IL4-dependent switch recombination and that this requirement is most likely the result of its critical role in activating germline Ig gene transcription. Additional studies of isotype switching in STAT6-deficient mice will be necessary to elucidate the factors involved in STAT6-independent switching to IgG1 and to understand the biologic significance of such a mechanism.

Acknowledgments We thank Dr. Steven McKnight for STAT6 antisera, Dr. Clifford Snapper for a-d-dex, Mr. Charles Thomas for expert FACS sorting and analysis, and Dr. Charles Chu for reagents and helpful advice concerning the DCPCR assay.

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