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GABPβ2 Is Dispensible for Normal Lymphocyte Development but Moderately Affects B Cell Responses

2008; Elsevier BV; Volume: 283; Issue: 36 Linguagem: Inglês

10.1074/jbc.m804487200

1083-351X

Xuefang Jing, Dongmei Zhao, Thomas J. Waldschmidt, Hai‐Hui Xue,

Immune Response and Inflammation

GA-binding protein (GABP) is the only Ets family transcription factor that functions as a heterodimer. The GABPα subunit binds to DNA, and the GABPβ subunit possesses the ability to transactivate target genes. Inactivation of GABPα caused embryonic lethality and defective lymphocyte development and immune responses. There are 3 isoforms of the GABPβ subunit, but whether they have distinct functions has not been addressed. In this study, we selectively ablated the expression of GABPβ2 using a gene trap strategy. GABPβ2-deficient mice were viable and had normal T and B cell development, suggesting that loss of GABPβ2 is compensated for by other GABPβ isoforms during these processes. GABPβ2-deficient T cells can be activated and proliferate similarly to wild-type controls. In contrast, B cells lacking GABPβ2 showed 2–3-fold increases in proliferation in response to B cell receptor stimulation. In addition, GABPβ2-deficient mice exhibited moderately increased antibody production and germinal center responses when challenged with T-dependent antigens. These results indicate that albeit GABPβ isoforms are redundant in lymphocyte development, GABPβ2 has a distinct role in restraining B cell expansion and humoral responses. GA-binding protein (GABP) is the only Ets family transcription factor that functions as a heterodimer. The GABPα subunit binds to DNA, and the GABPβ subunit possesses the ability to transactivate target genes. Inactivation of GABPα caused embryonic lethality and defective lymphocyte development and immune responses. There are 3 isoforms of the GABPβ subunit, but whether they have distinct functions has not been addressed. In this study, we selectively ablated the expression of GABPβ2 using a gene trap strategy. GABPβ2-deficient mice were viable and had normal T and B cell development, suggesting that loss of GABPβ2 is compensated for by other GABPβ isoforms during these processes. GABPβ2-deficient T cells can be activated and proliferate similarly to wild-type controls. In contrast, B cells lacking GABPβ2 showed 2–3-fold increases in proliferation in response to B cell receptor stimulation. In addition, GABPβ2-deficient mice exhibited moderately increased antibody production and germinal center responses when challenged with T-dependent antigens. These results indicate that albeit GABPβ isoforms are redundant in lymphocyte development, GABPβ2 has a distinct role in restraining B cell expansion and humoral responses. GA-binding protein (GABP) 2The abbreviations used are:GABPGA-binding proteinILinterleukinSRBCsheep red blood cellsGCgerminal centerBCRB cell receptorTLRtoll-like receptorLPSlipopolysaccharideCFSEcarboxy-fluorescein diacetate succinimidyl esterELISAenzyme-linked immunosorbent assayWTwild typeDNdouble negativeRACErapid amplification of cDNA endsHAhemagglutinin. is a member of the Ets family transcription factors and is comprised of two subunits, GABPα and GABPβ (1Rosmarin A.G. Resendes K.K. Yang Z. McMillan J.N. Fleming S.L. Blood Cells Mol. Dis. 2004; 32: 143-154Crossref PubMed Scopus (162) Google Scholar). GABPα contains a DNA binding Ets domain, which is conserved among all the Ets factors and is of ∼85 amino acids in length. The Ets domain assumes a winged helix-loop-helix configuration and binds preferentially to a purine-rich consensus DNA sequence containing GGA(A/T). On the other hand, GABPβ cannot bind DNA but contains 4 ankyrin repeats in its N terminus, which mediate the protein-protein interaction with GABPα. GABPβ also contains a nuclear localization signal, which targets the GABPα/β dimer into the nucleus. Transactivation activity of the GABPα/β complex is considered to reside in the C terminus of the GABPβ subunit, but the exact location has not been unequivocally mapped. GA-binding protein interleukin sheep red blood cells germinal center B cell receptor toll-like receptor lipopolysaccharide carboxy-fluorescein diacetate succinimidyl ester enzyme-linked immunosorbent assay wild type double negative rapid amplification of cDNA ends hemagglutinin. The GABPα/β complex has versatile roles in regulating basic cellular functions and tissue-specific functions (1Rosmarin A.G. Resendes K.K. Yang Z. McMillan J.N. Fleming S.L. Blood Cells Mol. Dis. 2004; 32: 143-154Crossref PubMed Scopus (162) Google Scholar, 2Sharrocks A.D. Nat. Rev. Mol. 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Cancer Res. 1997; 57: 3145-3148PubMed Google Scholar), and in synaptic function at the neuromuscular junction (9O'Leary D.A. Noakes P.G. Lavidis N.A. Kola I. Hertzog P.J. Ristevski S. Mol. Cell Biol. 2007; 27: 3470-3480Crossref PubMed Scopus (28) Google Scholar, 10Ravel-Chapuis A. Vandromme M. Thomas J.L. Schaeffer L. EMBO J. 2007; 26: 1117-1128Crossref PubMed Scopus (38) Google Scholar). In lymphocytes, GABP activates an interleukin (IL)-2 enhancer (11Avots A. Hoffmeyer A. Flory E. Cimanis A. Rapp U.R. Serfling E. Mol. Cell Biol. 1997; 17: 4381-4389Crossref PubMed Scopus (46) Google Scholar), IL-16, and Fas promoters (12Li X.R. Chong A.S. Wu J. Roebuck K.A. Kumar A. Parrillo J.E. Rapp U.R. Kimberly R.P. Williams J.W. Xu X. J. Biol. Chem. 1999; 274: 35203-35210Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 13Bannert N. Avots A. Baier M. Serfling E. Kurth R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1541-1546Crossref PubMed Scopus (58) Google Scholar), and transcription of IL-7Rα (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar, 15Dekoter R.P. Schweitzer B.L. Kamath M.B. Jones D. Tagoh H. Bonifer C. Hildeman D.A. Huang K.J. J. Biol. Chem. 2007; 282: 14194-14204Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recently, we showed that GABP is a key component of a gene regulatory network programming B lineage commitment and differentiation by directly regulating Pax5 gene expression (16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In contrast to extensive studies on GABPα, the roles of the GABPβ subunit in vivo have not been investigated. The most studied GABPβ isoforms are those encoded by the Gabpb1 allele, which generates two protein products, GABPβ1L (originally named GABPβ1 or GABPβ1-1) and GABPβ1S (originally named GABPβ2 or GABPβ1-2), via alternative splicing (17Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (346) Google Scholar, 18de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar). The N-terminal 332 amino acids of both GABPβ1L and GABPβ1S isoforms are identical, but their C termini differ in length and sequence. GABPβ1L has a longer C-terminal tail (50 amino acids), which adopts a leucine zipper-like structure, forming homodimers and even an α2β2 GABP tetramer when two Ets motifs are adjacent or brought into proximity (17Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (346) Google Scholar, 19Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 20Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (239) Google Scholar). In contrast, the C terminus of the shorter isoform, GABPβ1S, is 15 amino acids long, lacks the leucine zipper-like structure, and thus cannot form homodimers or tetramers. Nevertheless, both GABPβ1L and GABPβ1S heterodimerize with GABPα with similar affinity (21Suzuki F. Goto M. Sawa C. Ito S. Watanabe H. Sawada J. Handa H. J. Biol. Chem. 1998; 273: 29302-29308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It has been disputed over whether the leucine zipper-like structure in GABPβ1L has a unique role in transactivation of GABP target genes. In a recent report, we specifically targeted GABPβ1L by deleting exon 9 of the Gabpb1 gene that encodes the entire leucine zipper-like structure without eliminating the GABPβ1S isoform (22Xue H.H. Jing X. Bollenbacher-Reilley J. Zhao D.M. Haring J.S. Yang B. Liu C. Bishop G.A. Harty J.T. Leonard W.J. Mol. Cell Biol. 2008; 28: 4300-4309Crossref PubMed Scopus (13) Google Scholar). In contrast to a pre-implantation lethality in a GABPα-null mutant, GABPβ1L–/– mice were viable. On the other hand, targeting both GABPβ1L and GABPβ1S caused embryonic lethality prior to embryonic day 12.5 (22Xue H.H. Jing X. Bollenbacher-Reilley J. Zhao D.M. Haring J.S. Yang B. Liu C. Bishop G.A. Harty J.T. Leonard W.J. Mol. Cell Biol. 2008; 28: 4300-4309Crossref PubMed Scopus (13) Google Scholar). These findings indicate that the Gabpb1 gene products are required for normal embryogenesis, whereas loss of GABPβ1L expression can be compensated for by other GABPβ isoforms including GABPβ1S. A third GABPβ isoform, GABPβ2 (originally named GABPβ2-1), is encoded by Gabpb2 (18de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar) and is 414-amino acids long with its N-terminal ankyrin repeats (amino acids 1–130) sharing 87% identity with GABPβ1 isoforms. GABPβ2 also has a long C terminus, with its 317–366 amino acid residues sharing 70% identity with that of GABPβ1L and also adopting a leucine zipper-like structure. The 367– 414 amino acid residues in GABPβ2 are unique. The C terminus of GABPβ2 cannot only mediate the formation of GABPβ2 homodimers but also mediates the heterodimerization of GABPβ2 and GABPβ1L (18de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar). Since its initial cloning, GABPβ2 has not been studied, and its function is completely unknown. The structural similarity raised the possibility that GABPβ isoforms may have distinct and overlapping roles in regulating lymphocyte development and functional responsiveness. As one of the first steps aiming to dissect the exact roles of each GABPβ isoform, we have inactivated the Gabpb2 gene using a gene trap strategy. 5′-RACE and RT-PCR—Splenic B cells were negatively selected using EasySep B cell enrichment kits (StemCell Technology), and total RNA was extracted from purified B cells or total thymocytes using the RNeasy Mini kit (Qiagen), and 5′-RACE was done with the GeneRacer kit (Invitrogen). Racer and nest racer primers were supplied in the kit, and antisense primers complementary to mouse GABPβ2 cDNA (5′-GCAGCTTCTAGCAGCCTCTTC and 5′-TTCCCCAAGTCCACCAGAGAC) were used in RT-PCRs (Fig. 1A). Nested PCR was performed to increase the specificity of amplification. PCR products were subcloned into the pCR4-TOPO vector with a TOPO TA cloning kit (Invitrogen) and then sequenced. Generation of GABPβ2-targeted Mice—An ES clone (RRJ488) was obtained from the International Gene Trap Consortium through the Mutant Mouse Regional Resource Center of the University of California at Davis. Mapping of the insertion site of the reporter gene is done using the Expand Long Template PCR System (Roche Applied Science). The ES cells were microinjected into C57BL/6 blastocysts, and male chimeras were identified to achieve germline transmission. GABPβ2+/tp mice were interbred to obtain GABPβ2tp/tp and littermate controls. All experiments with mice followed protocols approved by the Institutional Animal Care and Use Committee, University of Iowa. Generation of GABPβ2 Antisera and Western Blotting—To generate antisera that are specific to GABPβ2 with no cross-reaction with GABPβ1, we used a recombinant GST fusion protein expressing GABPβ2 C-terminal amino acids 275– 414 as an antigen, because the N termini of all GABPβ isoforms contain highly conserved ankyrin repeat domains. To this end, we PCR-amplified cDNA corresponding to the GABPβ2 C-terminal fragment in a pGEX-4T-1 vector and expressed the fusion protein in BL21 Star competent cells (Stratagene). The fusion protein was expressed at high abundance with expected molecular weight and purified with magnetic resin-based Mag-neGST particles (Promega). The expression of GABPβ2 was confirmed by MALDI-TOF mass spectrometry, and the purified protein was used to immunize rabbits for antibody production (Bio-synthesis). To test the specificity of the GABPβ2 anti-serum, we cloned GABPβ1L and GABPβ2 cDNA in pCruz-HA vector (Santa Cruz Biotechnology) so that an HA tag is fused to the N terminus of each expressed protein. We then expressed these proteins in 293 HEK cell lines, and subjected the cell lysates to immunoblot with anti-HA antibody, which detected both GABPβ1L and GABPβ2 of expected molecular weight (Fig. 2B). In contrast, the GABPβ2 antiserum detected GABPβ2 at a 1:2000 dilution but did not cross-react with GABPβ1 (Fig. 2C). For detection of GABPβ2 expression, whole cell extracts were prepared from thymocytes, splenocytes, and splenic B cells as described previously (23Xue H.H. Fink Jr., D.W. Zhang X. Qin J. Turck C.W. Leonard W.J. Int. Immunol. 2002; 14: 1263-1271Crossref PubMed Scopus (24) Google Scholar). Lysate protein (30 μg) was separated on SDS/PAGE gels, transferred to nitrocellulose, and immunoblotted with the GABPβ2 antiserum (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar). An anti-GABPα antibody (H180, Santa Cruz Biotechnology) was used to detect GABPα expression, and an antiserum raised against the N terminus of GABPβ1 (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar) was used to detect both GABPβ1L and GABPβ1S. Flow Cytometry and CFSE Staining—Single cell suspensions were prepared from thymuses, spleens, and bone marrow, and stained with fluorochrome-conjugated antibodies, as described (16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). All fluorochrome-conjugated antibodies were from BD PharMingen or eBiosciences. Negatively selected splenic B cells were labeled with CFSE as described (16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), stimulated with 10 μg/ml of anti-IgM, and dilution of CFSE was determined on different days by flow cytometry. [3H]Thymidine Uptake—Splenic T and B cells were purified by negative selection using EasySep T and B cell enrichment kits (StemCell Technology), respectively, and the purity of isolated cells was ∼95%. T cells (1 × 105/well) were stimulated with plate-bound anti-CD3 (0.25 or 0.5 μg/ml, clone 145-2C11, BD Biosciences) in the absence or presence of anti-CD28 (1 μg/ml, clone 37.51, BD Biosciences). B cells (0.6 × 105/well) were stimulated with 2.5 μg/ml anti-IgM μ chain (Jackson ImmunoResearch Laboratories), 5 μg/ml anti-CD40 (BD PharMingen), 5 μg/ml LPS (Sigma), or 1 μg/ml CpG oligonucleotides (ODN1862, Invivogen). The cells were cultured in 96-well plates in triplicate for each condition for 48–60 h and pulsed with 1 μCi of [3H]thymidine (PerkinElmer) for the last 12 h of culture. Radioactivity incorporated into cells was collected on a UniFilter-96 (GF/B, PerkinElmer) with a cell harvester and was counted using a TopCount.NXT microplate scintillation and a luminescence counter (Packard). Immunization and Enzyme-linked Immunosorbent Assay (ELISA)—To determine the immune response to a T-independent antigen, mice were immunized intraperitoneally with 100 μg of TNP(52)-AECM-Ficoll (Biosearch Technologies), and the sera were collected on day 8 after immunization. For immune responses to a T-dependent antigen, mice were intraperitoneally injected with 100 μg of Imject Ovalbumin (Pierce) mixed with Imject Alum (Pierce, volume 1:1), boosted 1 week later with the same regimen, and sera collected after another week. The sera were diluted in 1:2 series to a total of 12 points, and TNP- or ovalbumin-specific immunoglobulins (Igs) were measured by ELISA. In brief, high binding plates (Immulux, Dynex) were coated with either 20 μg/ml of TNP(38)-BSA (Bioresearch Technologies) or 5 μg/ml of Imject Ovalbumin to absorb antigen-specific Igs, which were then detected with biotin-conjugated rat anti-mouse antibodies specific for murine IgM, IgG1, and IgG3. Visualization of the antigen-antibody complexes was revealed with the avidin-horseradish peroxidase and TMB substrate set (BD PharMingen), and absorbance at 450 nm was read on an ELX800 microplate reader (Bio-TEK). Linear absorbance readings were observed within one particular range of serum dilutions, and these absorbance readings were used to compare antigen-specific Ig titers in WT and GABPβ2-deficient mice. Sheep Red Blood Cell (SRBC) Immunization and Immunohistochemistry—Mice were i.p. injected with 0.2 ml of 10% v/v SRBC suspension (Colorado Serum Company) in phosphate-buffered saline, and spleens were harvested on days 8, 12, and 18 postimmunization. Splenocyte suspensions from half of the spleen were stained with anti-B220 mAb along with fluorescein isothiocyanate-conjugated peanut agglutinin (PNA, Vector Laboratories), and analyzed for GC B cell responses by flow cytometry. The other half of the spleen was used to determine GC structures by immunohistochemistry as described (24Shinall S.M. Gonzalez-Fernandez M. Noelle R.J. Waldschmidt T.J. J. Immunol. 2000; 164: 5729-5738Crossref PubMed Scopus (123) Google Scholar). Structure of 5′-Untranslated Regions in the Gabpb2 Gene—In our previous studies, we used ES clones that have a Gabpa allele inactivated by a gene trap strategy to generate GABPα-deficient mice (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar, 16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In this method, ES cells were retrovirally transduced with a reporter gene, such as βgeo, which is a fusion of β-galactosidase and a neomycin resistance gene. When the reporter gene is integrated into the ES genome and inserted in an intron of a gene, the gene is thus trapped. Because a strong splice acceptor is placed immediately 5′ of the reporter gene after the trapped gene is transcribed, the splicing will occur between the upstream introns and the reporter gene, resulting in truncation and/or inactivation of the trapped gene. By searching the data base of the International Gene Trap Consortium, we identified an ES clone (named RRJ488) which has an insertion of the βgeo reporter gene in the Gabpb2 locus. The insertion presumably occurred in an intron that is downstream of a non-coding exon at the 5′-end of the locus. The information on the Gabpb2 gene structure suggests existence of three 5′ non-coding exons and alternative usage of the exons. If this approach is to be used to study the effect of Gabpb2 inactivation in lymphocytes obtained from RRJ488 ES clone-derived mice, it is important to determine whether these exons are utilized in B and T cells. We therefore used rapid amplification of cDNA 5′-end assay (5′-RACE) to determine the transcription initiation sites and the usage of 5′ non-coding exons in the Gabpb2 gene. We performed nested RT-PCRs on splenic B cells (Fig. 1A) and observed three major transcripts (Fig. 1B). Analysis of the sequences of each transcript revealed that transcription of the Gabpb2 gene can be initiated from three different sites. If we arbitrarily define a location 12,480 bp upstream of exon 2 as “+1,” transcription can be initiated from +959, +1042, and +1118 bp, and that the three 5′ non-coding exons are utilized differently in each transcript (Fig. 1C). We also characterized Gabpb2 transcription initiation sites in thymocytes and observed two major transcripts that correspond to Transcripts 1 and 3 in B cells (data not shown). These results indicate that although transcription of the Gabpb2 gene can be initiated from different locations and there is different usage of 5′ non-coding exons, the protein product from these transcripts is not altered. GABPβ2 Expression Is Completely Abolished in GABPβ2tp/tp Mice—We then mapped the insertion site of the βgeo reporter gene in the RRJ488 ES cells using long template PCR. Based on the sequence tags provided for the ES clone, sense primers were designed based on the genomic sequence starting from exon 1a, and an antisense primer was based on the βgeo coding sequence. Sequence analysis of the PCR product revealed that the insertion occurred between exons 1b and 1c (Fig. 2A). As shown in Fig. 1C, because at least one part of the first non-coding exon (exon 1a) is used in all transcripts, we hypothesized that the insertion of the βgeo reporter between exons 1b and 1c will interfere with normal splicing and inactivate the Gabpb2 gene. ES cells were injected into blastocysts to generate GABPβ2+/tp mice. Germline-transmitted GABPβ2+/tp mice gave birth to GABPβ2tp/tp mice at a normal Mendelian ratio and were grossly normal. To determine GABPβ2 protein expression, we raised an antiserum specific for GABPβ2 using its C-terminal portion as an antigen, which can specifically recognize GABPβ2 but does not cross-react with GABPβ1L (Fig. 2, B and C). By Western blotting, we found that GABPβ2 expression is easily detected in both thymocytes and splenocytes in WT mice and is not detectable in GABPβ2tp/tp mice (Fig. 2D). This is in contrast to the situation of GABPαtp/tp embryos, which had hypomorphic expression of GABPα protein (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar, 16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The difference is likely ascribed to the location of reporter gene insertion and/or the gene context. We further confirmed that ablation of GABPβ2 did not alter the expression of GABPβ1L, GABPβ1S, and GABPα (Fig. 2E). Loss of GABPβ2 Expression Does Not Perturb T and B Cell Development—Previously we have demonstrated that GABPα is critically required for IL-7Rα expression in T cells as well as for normal B cell development (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar, 16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). T cells develop in the thymus, with the most immature thymocytes being double negative (DN) for CD4 and CD8 expression. DN thymocytes then develop into CD4 and CD8 double positive (DP) cells, which further mature to CD4+ or CD8+ single positive cells (25Rothenberg E.V. Taghon T. Annu. Rev. Immunol. 2005; 23: 601-649Crossref PubMed Scopus (223) Google Scholar). The thymocyte, splenocytes, splenic T and B cells numbers between GABPβ2tp/tp and WT control were comparable (Fig. 3A). Staining of thymoyctes with anti-CD4 and anti-CD8 antibodies revealed that the percentages of DN, DP, CD4+, and CD8+ subset were not altered in GABPβ2tp/tp mice (Fig. 3B). DN thymocytes can be further divided into four developmental stages based on CD25 and CD44 expression, i.e. DN1 (CD44+CD25–), DN2 (CD44+CD25+), DN3 (CD44–CD25+), and DN4 (CD44–CD25–). Fractionation of DN thymocytes with CD25 and CD44 staining showed that all four DN subsets appeared at similar frequencies between WT and GABPβ2tp/tp mice (Fig. 3C). These results suggest that the loss of GABPβ2 expression does not affect T cell development, including early stages. In the periphery, splenic CD4+ and CD8+ T cells in GABPβ2tp/tp mice appeared at a normal ratio (Fig. 3D), and IL-7Rα expression on GABPβ2tp/tp CD4+ and CD8+ T cells was not diminished (Fig. 3E and data not shown for CD8 cells), indicating that GABPβ2 is dispensable for IL-7Rα expression on peripheral T cells. We also examined B cell development in GABPβ2tp/tp mice. Fractionation of bone marrow cells reveals three populations of sequentially developing B cells, i.e. pro-B and pre-B (B220+IgM–), immature (B220medIgM+), and recirculating B cells (B220highIgM+) (26Hardy R.R. Hayakawa K. Annu. Rev. Immunol. 2001; 19: 595-621Crossref PubMed Scopus (939) Google Scholar). All three populations in the bone marrow of GABPβ2tp/tp mice were detected at similar percentages to those observed in WT controls (Fig. 3F). In the periphery, the frequency of total splenic B cells and marginal zone B cells (CD21highCD23dim) was comparable between WT and GABPβ2tp/tp mice (Fig. 3H). The CD23brightB220+ subset is heterogeneous and can be further fractionated to sequentially maturation stages, transitional 1 (CD24highCD21low), transitional 2 (CD24highCD21high), and mature follicular (CD24dimCD21dim)B cells (27Loder F. Mutschler B. Ray R.J. Paige C.J. Sideras P. Torres R. Lamers M.C. Carsetti R. J. Exp. Med. 1999; 190: 75-89Crossref PubMed Scopus (691) Google Scholar), and these maturing B cells showed similar frequency in both WT and GABPβ2tp/tp mice (Fig. 3I). These data collectively demonstrate that inactivation of GABPβ2 expression did not detectably perturb B cell development in the bone marrow or further maturation in the spleen. T and B Cell Proliferation in the Absence of GABPβ2—To determine if mature T and B cells that developed in the absence of GABPβ2 are functional, we isolated splenic T and B cells by negative selection. We stimulated T cells with different doses of plate-bound anti-CD3 in the presence and absence of anti-CD28 mAb and found that both WT and GABPβ2-deficient T cells proliferated similarly (Fig. 4A). In contrast, when the purified B cells were stimulated with anti-IgM, which cross-links the B cell receptors (BCRs), GABPβ2-deficient B cells showed increased proliferation in the presence or absence of IL-4 (Fig. 4B). However, anti-CD40-elicited proliferation was comparable in both WT and GABPβ2tp/tp B cells (Fig. 4C). In addition to the clonally rearranged BCRs, B cells express nonclonal pattern recognition receptors, notably Toll-like receptors (TLRs) including TLR4 and TLR9 (28Bernasconi N.L. Onai N. Lanzavecchia A. Blood. 2003; 101: 4500-4504Crossref PubMed Scopus (583) Google Scholar, 29Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3428) Google Scholar, 30Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4824) Google Scholar). We used lipopolysaccharide (LPS) to stimulated TLR4, and ODN1826 (a synthetic oligonucleotide containing unmethylated CpG) to stimulated TLR9. WT and GABPβ2-deficient B cells proliferated similarly in response to both stimulants (Fig. 4D). These data collectively suggest that B cells lacking GABPβ2 have an enhanced proliferative response specifically to BCR stimulation. We next tested if the increased B cell proliferation is a result of suppressed apoptosis or increased cell division. We activated B cells with anti-IgM and monitored apoptosis using Annexin V and 7-AAD staining on various days poststimulation. We did not find any consistent differences in percentages of Annexin V+7-AAD+ apoptotic cells (data not shown). In contrast, when labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE) before anti-IgM stimulation, GABPβ2tp/tp B cells showed moderately accelerated CFSE dilution on all days examined (Fig. 4E). These results indicated that BCR-activated B cells can proliferate faster in the absence of GABPβ2. GABPβ2-deficient B Cells Showed Increased in Vivo Responses to Immunization—To investigate if the increased B cell division is associated with enhanced humoral immune responses, we immunized the mice with both T-independent (TNP-Ficoll) and T-dependent (ovalbumin) antigens. Although TNP-specific IgM and IgG3 were produced at similar levels in WT and GABPβ2-deficient mice (Fig. 5A), production of ovalbumin-specific IgM and IgG1 in GABPβ2tp/tp mice was ∼1.5-fold of those in littermate controls (Fig. 5B). In the immune response to protein antigens, B cells migrate into the follicle in lymphoid organs and form germinal centers (GCs), where antigen-specific B cells further expand and undergo class-switch recombination. To further delineate if loss of GABPβ2 expression can enhance GC responses to protein antigens, we immunized the mice with SRBC and examined GC formation in the spleen. Confocal microscopic analysis showed that both WT and GABPβ2tp/tp mice can form GCs 8 days after immunization, and no apparent alteration in GC size was observed (data not shown). To quantitatively determine the SRBC-elicited GC responses, we used fluorochrome-conjugated PNA, which selectively binds to GC B cells. In most cases, B220+PNAhigh cells were detected at an increased frequency in GABPβ2tp/tp mice (Fig. 5C). On average, we observed a 33 and 40% increase in GC B cell frequency on day 8 and day 12 postimmunization, respectively (Fig. 5D). Similarly, the absolute numbers of splenic GC B cells were also increased by 51% on day 8 and by 31% on day 12 (Fig. 5E), albeit the difference did not reach statistical significance. These data collectively suggest that GABPβ2-deficient B cells exhibit moderately increased responses to protein antigens. A functional GABPα/β complex requires two subunits, GABPα and GABPβ. The DNA binding Ets domain of GABPα directs the GABP complex to its target genes and determines binding specificity. Activation or repression of GABP target genes is mediated through the C terminus of GABPβ. As revealed in crystal structure studies of GABP, the interaction between GABPα and GABPβ augments and stabilizes the DNA binding (31Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar). In contrast to embryonic lethality caused by ablation of GABPα (4Ristevski S. O'Leary D.A. Thornell A.P. Owen M.J. Kola I. Hertzog P.J. Mol. Cell Biol. 2004; 24: 5844-5849Crossref PubMed Scopus (113) Google Scholar, 14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar), we show here that mice deficient for GABPβ2 were viable. Previously we have demonstrated that GABPα is required for IL-7Rα expression in peripheral T cells (14Xue H.H. Bollenbacher J. Rovella V. Tripuraneni R. Du Y.B. Liu C.Y. Williams A. McCoy J.P. Leonard W.J. Nat. Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (123) Google Scholar) and normal B cell development (16Xue H.H. Bollenbacher-Reilley J. Wu Z. Spolski R. Jing X. Zhang Y.C. McCoy J.P. Leonard W.J. Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In GABPβ2-deficient mice, both T and B cells developed normally and the expression of IL-7Rα was not affected. These results indicate that GABPβ2 is dispensable for normal embryogenesis and lymphocyte development. These observations are quite similar to those in GABPβ1L–/– mice as we recently reported (22Xue H.H. Jing X. Bollenbacher-Reilley J. Zhao D.M. Haring J.S. Yang B. Liu C. Bishop G.A. Harty J.T. Leonard W.J. Mol. Cell Biol. 2008; 28: 4300-4309Crossref PubMed Scopus (13) Google Scholar). Given the structural similarity between GABPβ1L and GABPβ2, loss of one GABPβ isoform is likely compensated for by the other. Indeed, we have shown that both GABPα/GABPβ1L and GABPα/GABPβ2 heterodimers can bind to the Ets motif in the IL-7Rα promoter region (22Xue H.H. Jing X. Bollenbacher-Reilley J. Zhao D.M. Haring J.S. Yang B. Liu C. Bishop G.A. Harty J.T. Leonard W.J. Mol. Cell Biol. 2008; 28: 4300-4309Crossref PubMed Scopus (13) Google Scholar). In contrast to the possible redundancy between GABPβ1L and GABPβ2 in lymphocyte development and T cell activation, GABPβ2 appears to have a distinct role in moderately restraining B cell proliferation and humoral responses to protein antigens. GABPβ2-deficient B cells manifested enhanced proliferation specifically by BCR stimulation but not by CD40 ligation or TLR stimulation. This observation suggests that GABPβ2 may have a specific role in negatively regulating BCR signaling, rather than signals that are derived from CD40-CD40 ligand interaction, TLR4 (activated by LPS), or TLR9 (activated by CpG nucleotides). Ligation of BCRs by antigens leads to rapid phosphorylation and activation of Syk, which further activate downstream signal components, including the phosphoinositide 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (Erk), and nuclear factor-κB (NF-κB) pathways, all three of which are known to be crucial for the survival and proliferation of B cells (32Niiro H. Clark E.A. Nat. Rev. Immunol. 2002; 2: 945-956Crossref PubMed Scopus (524) Google Scholar). We probed BCR signaling pathways by stimulating purified B cells with anti-IgM and measuring phosphorylation of tyrosines 519/520 in Syk, serine 473 in Akt, threonine 202 and tyrosine 204 in p44/42 Erk, and serine 32 in IκBα. However, no apparent changes were observed with the potency and duration of these signal pathways, and the protein levels of these signaling molecules were similar between WT and GABPβ2-deficient B cells (data not shown). The precise molecular mechanism by which GABPβ2 negatively regulates BCR signaling awaits further investigation. Consistent with increased BCR-stimulated proliferation of GABPβ2-deficient B cells in vitro, GABPβ2-deficient mice displayed increased IgM and IgG1 antibody levels after ovalbumin immunization and a heightened GC response after challenge with SRBC. The increase is somewhat moderate, and a possible explanation is that only BCR-elicited responses were enhanced in the absence of GABPβ2, which may be blunted by similar responses derived from CD40-CD40L interaction. It is noteworthy that mice lacking GABPβ2 did not show apparent difference in antibody levels after a challenge with TNP-Ficoll. This can be explained by the somewhat modest proliferation of B cells observed in vivo after immunization with a T-independent polysaccharide antigen, and the restriction of expansion to the first few days after exposure (33Garcia de Vinuesa C. O'Leary P. Sze D.M. Toellner K.M. MacLennan I.C. Eur. J. Immunol. 1999; 29: 1314-1323Crossref PubMed Google Scholar). In contrast, antigen-selected B cells undergo marked proliferation for extended periods in GCs after T-dependent challenge (34Camacho S.A. Kosco-Vilbois M.H. Berek C. Immunol. Today. 1998; 19: 511-514Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), allowing for a longer period of time during which the absence of GABPβ2 can generate a higher number of antigen-specific clones. In summary, our data revealed that GABPβ2 has both redundant and distinct roles in the immune system. Lack of GABPβ2 did not affect B cell development but showed increased B cell proliferation and humoral responses to protein antigens. Thus, manipulation of GABPβ2 expression may be a useful approach to modulate B cell responses without interfering with normal B cell development. We thank Dr. Gail Bishop for useful advice and critical reading of this manuscript, Dr. Jian-Qiang Shao of the Central Microscopy Research Facility for technical support on microsection and fluorescence microscopy, and Lorraine Tygrett for technical help with SRBC immunization and flow cytometric analysis. We thank Dr. Yalan Li at the Proteomics Facility, University of Iowa, for performing MALDI-TOF mass spectrometric analysis of the recombinant GST-GABPβ2 fusion protein and help with data analysis.