Survival of B Cell Development, Differentiation, and Stages Element

Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival This information is current as of September 25, 2021. Hui-Chen Chen, John C. Byrd and Natarajan Muthusamy J Immunol 2006; 176:2208-2218; ; doi: 10.4049/jimmunol.176.4.2208 http://www.jimmunol.org/content/176/4/2208 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival1

Hui-Chen Chen,† John C. Byrd,*§¶ and Natarajan Muthusamy2*†‡¶ CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser119-ala phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed ϳ70% increase in pre-B1 (CD43؉B220؉CD24؉(int)) and ϳ60% decreased pre-BII (CD43؉B220؉CD24؉؉(high)) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed ϳ4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice

revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into Downloaded from -RAG-2؊/؊ mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43؉B220؉CD24high population that is nor mally absent in RAG-2؊/؊ mice. In the periphery, the Tg mice exhibited decreased CD21dimCD23highIgM؉ follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the ﬁrst evidence for a differential role for CRE-binding proteins in multiple stages of B cell development,

functional maturation, and B1 and B2 B cells. The Journal of Immunology, 2006, 176: 2208–2218. http://www.jimmunol.org/

yclic AMP response element binding protein-1 belongs Activation of resting murine B and T cells through antigen re- to the basic leucine zipper family of transcription factors. ceptors leads to the phosphorylation of CREB-1 at critical Ser133, C CREB-1 binds to CRE, which is composed of which is essential for the transcriptional activation function of the TGANNTCA sequences as homo- and heterodimers in association protein (22–24). Stimulation through the BCR resulted in a dose- with members of the CREB/activating transcription factor (ATF)3 dependent induction of CREB-1-binding activity that is subjected family of proteins, including ATF-1 and cAMP response element to negative regulation by IFN-␥ in a STAT-1-dependent manner modulator, as well as AP-1 family members such as c-Jun (1–5). (25). Consistent with a role for CREB-1 in the regulation of cell 133 The activation-induced phosphorylation of CREB-1 at Ser (or growth, the expression of immediate early growth response genes, by guest on September 25, 2021 Ser119) in an alternatively spliced form increased the DNA-binding such as c-fos and junB, and cell cycle regulatory genes, such as the activity of the protein in some but not other cell types (4–7). Phos- proliferating cell nuclear Ag (PCNA), is regulated by Ser133-phosphorylation at Ser133 activates CREB-1, at least in part, by facil- phorylated CREB-1 through direct binding and activation of the itating its binding to CREB-binding protein (CBP). This, in turn, promoter region of these genes (26–30). interacts with and activates the components of basal transcriptional A requirement for CREB-1 in mature B cell survival is impli- machinery (8–13). Multiple signaling pathways, including cAMP- cated by the CREB-1-dependent induction of the bcl-2 gene in responsive protein kinase A, protein kinase C, calcium/calmodu- human B cell lines and transgenic (Tg) mice (31–33). Ser133-phos- lin-dependent CaM kinases II and IV, and a Ras-dependent serine/ phorylated CREB-1 binds to the CRE element in promoter regions threonine kinase RSK2, mediate phosphorylation and activation of of antiapoptotic genes such as Bcl-2 and Mcl-1 and regulate their CREB-1 in different cell types (14–22). expression. CREB-1Ϫ/Ϫ mice die shortly after birth, thus preclud- ing the analysis of the immune function of mature B cells (34). The lack of a strong phenotype in some CREB family member knock- *Division of Hematology and Oncology, Department of Internal Medicine, †Molec- ular Virology, Immunology and Medical Genetics, ‡Veterinary BioSciences, §Divi- out studies suggested the functional redundancy among the mem- sion of Medicinal Chemistry, College of Pharmacy, and ¶OSU Comprehensive Can- bers of this family (35). To determine the in vivo role of CRE- cer Center, The Ohio State University, Columbus, OH 43210 binding proteins in early B cell development and functional Received for publication April 28, 2005. Accepted for publication October 18, 2005. maturation, we generated four independent Tg mice lines overex- The costs of publication of this article were defrayed in part by the payment of page pressing a dominant-negative phosphomutant CREB-1 in devel- charges. This article must therefore be hereby marked advertisement in accordance oping B cells in the bone marrow. Characterization of these Tg with 18 U.S.C. Section 1734 solely to indicate this fact. mice revealed a critical role for CRE binding proteins in different 1 This work was supported by grants from the American Cancer Society (to N.M.), Leukemia and Lymphoma Society of America (to J.C.B.), and The D. Warren Brown stages of B cell development and functional maturation. Foundation (to J.C.B. and N.M.). H.-C.C. is a recipient of a Raymon E. Mason Foundation Fellowship for graduate research. 2 Address correspondence and reprint requests to Dr. Natarajan Muthusamy, Chronic Materials and Methods Lymphocytic Leukemia, Experimental Therapeutics Laboratory, Division of Hema- Animals tology and Oncology, 1132C, James Cancer Hospital, 300 West 10th Avenue, Co- lumbus, OH 43210. E-mail address: [email protected] Mutant CREB-1-Tg animals described were generated at the OSU CCC/CCRI 3 Abbreviations used in this paper: ATF, activating transcription factor; CBP, CREB- transgenic facility. The Tg expression vector pBH was constructed by clon- binding protein; PCNA, proliferating cell nuclear Ag; Tg, transgenic; KLH, keyhole ing the EcoRI fragment of pEPB splice-neo (a gift from Drs. W. Muller and limpet hemocyanin; TNP, trinitrophenyl; NTg, nontransgenic; mCRE, mutant CRE K. Rajewsky, Institute for Genetics, University of Cologne, Cologne, Ger- oligonucleotide; HPRT, hypoxanthine phosphoribosyltransferase. many) containing the Ig H chain promoter and intronic enhancer and the

SV40 splice and poly(A) into the EcoRI site of pBluescript KS II(ϩ). The moved by purification in G50 Sephadex spin columns. The binding reactions dominant-negative CREB-1 cDNA was produced by PCR-mediated mu- were conducted, at room temperature, with 3 ␮g of nuclear extract, 30,000 tagenesis of the human ⌬CREB-1 cDNA at Ser119 (Ser119 to Ala) (30). The dpm (0.1–0.5 ng) of radiolabeled oligonucleotide probe, in 5ϫ Ficoll-binding hCREB-1Ser119-Ala was cloned into the BglII site of the pBH Tg vector. buffer (10 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and 4% Ficoll), 250 Four independent founder lines were identified by PCR using primers 5Ј- ng of poly(deoxyinosinic-deoxycytidylic acid) in 75 mM KCl and double- Ј Ј ␮ AAAACCACTTCTTCAAACCACAGC-3 and 5 -CTGCTGGAGAA distilled H2O to make the volume to 15 l. The DNA-protein complexes were GAAGGGACATC-3Ј corresponding to the Ig H chain intronic enhancer fractionated by electrophoresis in 4% nondenaturing polyacrylamide gel, run and the Ig H chain promoter, which amplified ϳ700 bp fragment in the Tg in 0.25ϫ Tris-borate-EDTA, at 4°C. The gel was then dried on 3M Whatman tail DNA. The lines were further confirmed by Southern blot analysis. paper and subjected to autoradiography. In vitro transcribed and translated Recombination activating gene-2-deficient (Rag-2Ϫ/Ϫ) mice were obtained CREB-1 protein generated using rabbit reticulolysate system was used as a originally from Dr. F. Alt (Harvard Medical School, Boston, MA). Female positive control (30). FVB/N mice were obtained from Harlan Sprague Dawley. All animal studies described were approved by the Institutional Animal Care and Use Cell preparation and culture Committee. Mice (4–8 wk old) were sacrificed by cervical dislocation. Bone marrow Reagents cells were obtained from the femur. Peritoneal cells were collected by flushing the peritoneal cavity with PBS. Splenic B cells were prepared by Goat anti-mouse IgM (Jackson Immuno Research Laboratories) and PMA, incubating the RBC-depleted splenocytes for 20 min on ice in an anti-Lyt2 ionomycin, and DNP conjugated to keyhole limpet hemocyanin (KLH) (3.155) and anti-L3T4 (R7-172.4) Ab mixture, followed by 30-min incu- were purchased from Calbiochem. [32P]dCTP and dGTP were obtained bation with rabbit complement at 37°C. The cells were then washed and from Amersham Life Sciences. Rabbit complement was purchased from resuspended at 107 cells/ml in RPMI 1640 medium. The live B cells were Pel-Freeze Biologicals. Ficoll-Paque, LPS, trinitrophenyl (TNP)-LPS, and purified using Ficoll-Paque density gradient method. Single-cell suspen- Hoechst no. 33342 were purchased from Sigma-Aldrich. Anti-human sions were maintained in RPMI 1640 medium supplemented with 5% heat- Downloaded from CREB-1 Ab (24H4B) was obtained from Santa Cruz Biotechnology. The inactivated FCS, 50 ␮M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, LumiGLO chemiluminescent kit was purchased from Kirkegaard & Perry 100 ␮g/ml streptomycin, and 0.1 mM nonessential amino acid. The cells

Laboratories. Fluorochrome-labeled anti-B220 (RA3-6B2), anti-IgM (R6- were cultured in a 37°C incubator with 5% CO2. 60.2), anti-MAC-1 (M1/70), anti-IgD (AMS9.1), anti-CD5 (53-7.3), anti- CD43 (S7), anti-CD24 (30-F1), anti-CD4 (RM4–5)m and anti-CD8 (53- Western blot analysis 6.7) were purchased from BD Pharmingen. The Vybrant CFDA SE cell ϫ 6 ␮ Tracer kit (V12883) was purchased from Molecular Probe. The total cellular protein was extracted by boiling 4 10 cells in 50 l of 1ϫ SDS sample buffer. The samples were separated on 10% SDS- http://www.jimmunol.org/ Southern blot analysis polyacrylamide gel by electrophoresis and blotted onto a nitrocellulose membrane (Schleicher & Schuell Microscience). After blocking with PBS The tail biopsies from Tg or nontransgenic (NTg) mice were digested over- containing 5% nonfat dry milk powder, the blots were probed with 1 ␮g/ml night with lysis buffer (50 mM Tris (pH 8), 100 mM EDTA, 0.5% SDS, mouse-anti-human CREB-1 Ab. After washing the membrane three times and 350 ␮g of proteinase K), and the genomic DNA samples were prepared with PBS ϩ 0.1% Tween 20, the blots were further incubated with the by phenol/chloroform extraction followed by ethanol precipitation. appropriate goat anti-mouse Ig-HRP-conjugated secondary Ab. The blots Genomic DNA (5–10 ␮g) digested with EcoRI was separated on 0.7% ϩ were developed using LumiGLO Chemiluminescent Substrate System as agarose gel, transferred onto Immunobilon-Ny transfer membrane (Mil- per the manufacturer’s instructions. lipore), cross-linked by UV, and then baked in a vacuum oven at 80°C for 30 min. The membrane was subsequently prehybridized for 30 min at 42°C Flow cytometry

in hybridization solution (2.5 mg of salmon sperm DNA, 50% formamide, by guest on September 25, 2021 4.8ϫ SSC, 8 mM Tris (pH 7.5), 5ϫ Denhardt’s solution, 0.2% SDS, and Single-cell suspensions from the bone marrow, spleen, lymph node, thy- 5% dextran sulfate), followed by fresh hybridization solution containing mus, or peritoneum were washed twice with PBS containing 0.1% BSA. denatured radiolabeled probe (a transgene-specific 0.35-kb PstI/BamHI Cells were incubated with indicated fluorochrome-labeled Abs on ice for fragment spanning the SV40 splice and poly(A) region of the Tg construct) 30 min, washed twice, and then resuspended in PBS containing 0.02% overnight. The blots were then washed and exposed to x-ray film. sodium azide. Dual and multiparameter flow cytometric analysis was performed on ELICS ELITE ESP Coulter Counter. The data were acquired in Northern blot analysis list mode and analyzed using the Windows Multiple Document Interphase (WinMDI) program developed by J. Trattor at The Scripps Institute (La RNA was isolated using TRIzol reagent. Total RNA (10 ␮g) was separated ϩ Jolla, CA). The pre-BI and pre-BII B cells were sorted using FACS fol- on 1% RNA agarose gel, transferred onto nylon Hybond-N membrane lowing staining with indicated fluorochrome-labeled Abs. (Amersham Biosciences), and then cross-linked by UV. The membrane was then baked in a vacuum oven at 80°C for 30 min and prehybridized for RT-PCR analysis 2 h at 42°C in hybridization solution (5% standard saline citrate phosphate/ EDTA, 5% Denhardt’s solution, 0.5% SDS, 50% formamide, 10% dextran RNA samples from sorted pre-BI or pre-BII cells were isolated using sulfate, and 3 ␮g of salmon sperm DNA), followed by fresh hybridization TRIzol reagent with 20 ␮g of glycogen as the carrier. RNA (100–200 ng) solution containing a denatured radiolabeled transgene-specific 0.3-kb was used for first-strand cDNA synthesis. The reverse transcription was XhoI/BamHI fragment derived from the CD2 poly(A) region. performed using random primer oligonucleotides as primers and mouse mammary tumor virus reverse transcriptase as described in the manufac- Preparation of nuclear extract turer’s protocol (Invitrogen Life Technologies). One microliter of reverse transcriptase product (cDNA) was amplified using primer pairs specific for Splenic B cells from Tg and NTg mice were washed in cold PBS, resus- ␮ ␮ ␮ tested genes. PCR were performed in a 25- l volume containing 1 lof pended in 100 l of buffer A (10 mM HEPES, 1.5 mM MgCl2,10mM cDNA, 1ϫ PCR buffer, 200 ␮g of four deoxynucleotide triphosphates, 6 KCl, 0.5 mM DTT, and 0.2 mM PMSF/aprotinin), and incubated on ice for ␮ ␮ M of each of the sense and antisense primers, 2 mM MgCl2, and 2.5 U 10 min and centrifuged. The cell pellet was resuspended in 25 l of buffer of Taq polymerase (Invitrogen Life Technologies), using PTC-100 Pro- C (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM grammable Thermal Controller (MJ Research). The PCR cycle was re- EDTA, 0.5 mM DTT, and 0.2 mM PMSF/aprotinin) and incubated on ice peated 26–32 cycles. The PCR products were separated on 1.5% agarose for 20 min and centrifuged at 12,000 rpm for 2 min at 4°C. The concen- gel containing the ethidium bromide and photographed, or a semiquanti- tration of the nuclear protein in the supernatant was determined by the tative multiplex RT-PCR in the same tube was designed to compare the Bradford assay (Bio-Rad). The nuclear extract was aliquoted into (5 ␮l) Ϫ RT-PCR products of genes of interest along with hypoxanthine phospho- volumes and frozen at 70°C until further use. ribosyltransferase (HPRT) gene products to determine the relative levels of Double-stranded oligo probe preparation and EMSA expression of these genes in each of these samples. The density of each band was quantified using ImageQuant 5.0 (Molecular Dynamics). All the The following double-stranded oligonucleotides were used: CRE oligonucle- oligonucleotide primers were designed according the published sequences otide (CRE element underlined), 5Ј-gatcGCCTCCTTGGCTGACGT of the respective genes. The primers were designed to be specific to the CAGAGAG-3Ј (forward); and mutant CRE oligonucleotide (mCRE), 5Ј- respective genes and do not possess a significant match to any other mouse gatcGCCTCCTTGGCTCAGCACAGAGAG-3Ј (forward). Annealed double- sequence in the GenBank database. The oligonucleotide primers were cus- stranded oligo nucleotides (100 ng) were labeled with 32p-nucleotides using tom-synthesized by Invitrogen Life Technologies. The primer sequences nick translation buffer as described previously (30). The free probe was re- used for PCR, with the expected product size in brackets are listed as 2210 DIFFERENTIAL ROLE FOR CREB-1 IN B1 AND B2 B CELL DEVELOPMENT

follows: HPRT (176 bp) sense, 5Ј-CCAGCAAGCTTGCAACCTTA a potential role for CREB-1 in B cell development and/or func- ACCA-3Ј, and antisense, 5Ј-GTAATGATCAGTCAACGGGGGAC-3Ј; tional maturation (Ref. 25; H.-C. Chen and N. Muthusamy, un- Ј Ј Mb-1 (310 bp) sense, 5 -GCCAGGGGGTCTAGAAGC-3 , and antisense, published observation). To test this directly, we generated Tg mice 5Ј-TCACTTGGCACCCAGTACAA-3Ј; c-Jun (692 bp) sense, 5Ј-GGG GAAGCACTGCCGTATGGAG-3Ј, and antisense, 5Ј-CCCGGGTTGA overexpressing a dominant-negative CREB-1 transcription factor AGTTGCTGAGGTT-3Ј; c-Myc (506 bp) sense, 5Ј-CTCGCCGCCGCT in developing B cells in the bone marrow. Schematic representa- GGGAAACTT-3Ј, and antisense, 5Ј-CACCGCCGCCGTCATCGTCTT- tion of a Tg vector-encoding the mutant CREB-1 in which the 3Ј; JunB (660 bp) sense, 5Ј-CGCCCGGATGTGCACGAAAATG-3Ј, and critical Ser119 had been changed to alanine is shown in Fig. 1a. antisense, 5Ј-CGGAAGCGCCACGACTCAAACC-3Ј; c-Fos (584 bp) sense, 5Ј-GGGTTTCAACGCCGACTACGA-3Ј, and antisense, 5Ј- Previous studies have shown that such a mutant CREB-1 protein GGGCTGCCAAAATAAACTCCA-3Ј; PCNA (800 bp) sense, 5Ј-TCCT bound to DNA but abrogated transcription activation of target TGGTACAGCTTACT-3Ј, and antisense, 5Ј-TGCTAAGGTGTCAGC genes, thus serving as a dominant-negative inhibitor of CREB-1 in ATT-3Ј; and VpreB (376 bp) sense, 5Ј-CTGCTGTCCTGCTCATGCT-3Ј, vitro and in vivo (8, 28, 30, 32). Human ⌬CREB-1 cDNA with Ј Ј and antisense, 5 -ACGGCACAGTAATACACAGCC-3 . Ser119 to alanine mutation was generated by PCR and cloned into Cell cycle analysis a B cell-specific pBH expression vector containing the Ig ␮ H chain promoter and intronic enhancer (Fig. 1a). Southern blot anal- Bone marrow cells (4 ϫ 106) were stained with anti-B220-R-PE-cyanine 5 (PE-Cy5, BD CyChrome), anti-CD43-R-PE, and anti-CD24-FITC for 20 ysis of tail DNA from four independent founders, using a probe min on ice and subsequently washed twice with PBS-0.01% BSA buffer. from the unique region of the Tg vector shown in Fig. 1a, identi- The cells were fixed in 1% paraformaldehyde for 10 min, washed twice fied ϳ5, 13, 38, and 15 copies of the transgene (Fig. 1b). A rep- with PBS, and then permeabilized with freeze-cold 70% ethanol for 10 min resentative Northern blot analysis of total RNA isolated from the on ice. The cells were spun down and resuspended in 2 ml of 2 ␮g/ml bone marrow of each of the Tg lines and a NTg littermate from one Downloaded from Hoechst 33342 staining solution for 30 min at room temperature. The cell cycle analysis was performed on ELICS ELITE ESP flow cytometer. The of the lines using the human CREB-1-specific probe is shown in data were acquired in list mode and analyzed using WinMDI version 2.8 Fig. 1c. The expression of the human CREB-1 transgene was con- and Modfit LT cell cycle analysis software. firmed by Western blot analysis of the protein isolated from each B Cell proliferation assay of the Tg lines using anti-human CREB-1 Ab (Fig. 1d) and by EMSA (Fig. 1e). Purified splenic B cells (2.5 ϫ 105) were cultured in 96-well tissue culture http://www.jimmunol.org/ plates (Costar), with 10 ␮g/ml goat anti-mouse IgM and 0.1 ␮g/ml PMA Ϯ Defective development of mature B cells associated with pre-BI ␮ 0.5 g/ml ionomycin as indicated. Cells were cultured for 40 h in 5% CO2 to pre-BII transitional block in bone marrow of mCREB-1 Tg at 37°C and then pulsed with 1 ␮Ci of [3H]thymidine (sp. act. 2Ci/mM) (Amersham Biosciences) for 8 h. [3H]Thymidine incorporation was ana- mice lyzed in a Packard liquid scintillation counter (Packard). Representative Expression of CREB-1 throughout the B cell developmental stages results of multiple experiments are presented as geometric mean of re- suggested a role for CREB-1 in B cell development. To directly sponses from triplicate cultures with SE of mean. test whether overexpression of dominant-negative CREB-1 in the Immunization and ELISA bone marrow interfered with normal B cell development, bone Groups of four to six wild-type and mCREB-1 Tg mice (6–8 wk old) were marrow cells from Tg and NTg littermates were analyzed by flow immunized i.p. with 50 ␮g of DNP-KLH, 50 ␮g of TNP-LPS, or 10 ␮gof cytometry using fluorochrome-labeled anti-IgM and anti-B220 by guest on September 25, 2021 TNP-Ficoll in sterile PBS. The mice were bled before (preimmune sera) Abs. The B220ϩ/IgMϪ population, which represents the pro-B and and after immunization at weekly intervals. Four weeks after the primary ϳ ␮ pre-B population, was decreased 65% in the Tg bone marrow. immunization with DNP-KLH, the mice were challenged with 50 gof Ϯ Ϯ DNP-KLH and were bled 5 days later for secondary immune sera. Anti- (7.56 0.89% in NTg mice vs 3.5 1.1% in Tg mice; Fig. 2a and TNP Abs were determined using isotype-specific ELISA as described pre- Table I). Furthermore, in comparison to the NTg littermates, the viously (36). Each of the wells in the ELISA plates were coated with 20 ␮g mCREB-1 Tg mice revealed ϳ65% decrease in B220ϩ/IgMϩ B of TNP and blocked with 10% FBS in PBS. Sera were serially diluted, and cells that represent the immature and mature B cells in the bone the Ag specific Ab titer was detected with peroxidase-conjugated rabbit marrow (4.4 Ϯ 0.98% in NTg vs 1.4 Ϯ 0.19% in Tg mice (Fig. 2a, anti-mouse isotype-specific Abs using SBA clonotyping system (Southern Biotechnology Associates). The results are presented as mean serum con- left panel, and Table I)). This defective development is unique to centrations of indicated isotypes Ϯ SD from 5 to 10 mice/group. B cells as no detectable defects in the development of T cells in the thymus, spleen, or lymph node in Tg mice were observed (Fig. 2a, Adoptive transfer analysis right panel, and data not shown). Development of B cells from Groups of four recipient RAG-2Ϫ/Ϫ mice were sublethally irradiated with lymphoid precursors in the bone marrow can be distinguished into 137 6 300 rad from a Cs source. Donor bone marrow cells (10 ϫ 10 ) sus- pro-B (B220ϩCD43ϩCD24Ϫ), pre-BI (B220ϩCD43ϩCD24ϩ(int)), pended in PBS were injected i.v. into the recipient animals 1 day after ϩ ϩ ϩϩ(high) ϩ irradiation. Four weeks after the transfer, the bone marrow and splenic cells early pre-BII (B220 CD43 CD24 ), late pre-BII (B220 Ϫ ϩϩ(high) ϩ Ϫ ϩϩ ϩ were analyzed by flow cytometry. The donor-derived cells in the recipients CD43 CD24 ), immature (B220 CD43 CD24 IgM ), ϩ Ϫ Ϫ ϩ were identified by CD43ϩB220ϩCD24high populations that are not nor- and mature (B220 CD43 CD24 IgM ) B cell stages based on Ϫ Ϫ mally found in RAG-2 / mice (37). the differential expression of B220, CD43, and CD24 surface molecules. Decreased B220ϩIgMϩ and B220ϩIgMϪ B cell in the Results bone marrow suggested a possible developmental block at or be- Production of Tg mice overexpressing dominant-negative mutant Ser1193Ala fore the sIgM expressing immature B cell stage. To define the CREB-1 (CREB-1 ) in bone marrow specific stage at which overexpression of mCREB-1 results in Activation of B cells through the AgR (BCR) induced phosphor- early B cell developmental defects, bone marrow cells from Tg and ylation of CREB-1 on the Ser119 residue that is known to be re- NTg littermates were examined by multiparameter flow cytometry quired for the transcriptional activation of CREB-1 (15). We have using anti-B220 (CyChrome), anti-CD43 (PE), and anti-CD24 shown recently that activation of B cells through BCR induced (FITC) Abs. As shown in Fig. 2b, ϳ70% decrease in dose-dependent CRE-binding activity, with minimal alteration in B220ϩCD43Ϫ cells and a moderate, yet consistent decrease the levels of the CREB-1 protein. Furthermore, BCR induced (ϳ15%) in B220ϩCD43ϩ cells was observed. Thus, B cell devel- CREB-1-binding activity is subjected to IFN-␥-mediated regula- opment appears to be blocked as early as the B220ϩCD43ϩ stage tion in B cells. These observations along with the constitutive ex- in the Tg mice. The B220ϩCD43ϩ cells can be further distin- pression pattern of CREB-1 in the bone marrow B cells suggested guished into pro-B (B220ϩCD43ϩCD24Ϫ), pre-BI (B220ϩ The Journal of Immunology 2211

CD43ϩCD24ϩ(int)), and early pre-BII (B220ϩCD43ϩCD24ϩϩ(high)) B cells based on the expression of CD24 (38, 39). Although three- color ﬂow cytometric analysis revealed no difference in B220ϩCD43ϩCD24Ϫ cells, ϳ70% increase pre-BI cells (39% in NTg vs 67% in Tg mice), and ϳ60% decrease in pre-BII cells (46% in NTg vs 20% in Tg mice) was observed in the bone marrow from Tg mice (Fig. 2, b and c, and Table I), indicating a developmental block in pre-BI to pre-BII transition in mCREB-1 Tg mice.

The developmental defect in mCREB-1 bone marrow is cell intrinsic In addition to the B cell developmental defects, signiﬁcant reduction in the bone marrow cells due to development of osteopetrosis was observed in Tg mice (29.6 ϫ 106Ϯ 4.6 ϫ 106 in NTg vs 5.9 ϫ 106Ϯ 1.6 ϫ 106 in Tg mice) (Fig. 2d) (M. Bayoumy, H.-C. Chen, and N. Muthusamy, manuscript in preparation). The bone marrow environment is critical for B cell development. To determine whether the developmental block observed in the mCREB-1 Tg bone marrow B cells is due to cell intrinsic defects, bone marrow Downloaded from cells from either wild-type or Tg littermates were adoptively transferred i.v. into sublethally irradiated RAG-2Ϫ/Ϫ mice. RAG-2Ϫ/Ϫ mice lack B220ϩCD43ϩCD24high population due to developmental block at pro-B/pre-BI stage of the B cell development (37). Analysis of B220ϩCD43ϩCD24high pre-BII population in the Ϫ/Ϫ RAG-2 recipients 4 wk after transfer exhibited ϳ2-fold in- http://www.jimmunol.org/ crease in B220ϩCD43ϩCD43high pre-BII cells in mice transferred with the wild-type bone marrow cells. However, no increases in B220ϩCD43ϩCD24high cells were observed in the Tg bone marrow recipients (Fig. 2e). Furthermore, consistent with the defective B cell progression, RAG-2Ϫ/Ϫ recipients that received the Tg bone marrow cells exhibited ϳ50% reduction in more mature B220ϩCD43Ϫ B cells compared with those that received the same number of wild-type bone marrow cells (data not shown). No osteopetrosis was observed in the recipient RAG-2Ϫ/Ϫ mice in the 4 by guest on September 25, 2021 wk time period of the study.

Deregulated expression of c-Jun and JunB associated with defective S-phase entry of pre-BII B cells in the mCREB-1 Tg mice PhosphoCREB-1 has been shown to be involved in regulation of bcl-2 gene expression in human B cell line and murine B cells (32, 33). RT- PCR analysis of RNA from Tg and NTg pre-BI and FIGURE 1. Production of Tg mice with dominant-negative mutant Pre-BII cells revealed comparable levels of bcl-2 transcription. It A119 CREB-1 transgene. a, Schematic representation of the CREB-1 trans- is likely that the B cell developmental abnormalities in the gene. The Ig H chain promoter, enhancer, and the SV40 splice and poly(A) mCREB-1 Tg mice could be due to potential abnormal down- site are shown. The mutant hCREB-1 was produced by PCR-mediated modulation of bcl-2 in other stages of developing B cells. To test mutagenesis of the human ⌬CREB cDNA at Ser119 (Ser119 to Ala119), thereby creating a nonphosphorylatable dominant-negative form of this directly, mCREB-1 mice were crossed with Tg mice that over- CREB-1 (CREB-1A119). The hCREB-1A119 was cloned into the BglII site express bcl-2 in B cells. Overexpressed Bcl-2 failed to rescue the of the pBH transgene vector as described in Material and Methods. b, bone marrow B cell developmental abnormalities in the double Tg Southern blot analysis of tail DNA from wild-type (WT) and four inde- mice (data not shown), suggesting bcl-2-independent CREB-1-dependent Tg founder lines. Tail DNA was probed with probe unique to the pendent regulatory pathways in the observed B cell developmental Tg vector (shown in a). The numbers shown in the bottom of each lane defect. Expression of immediate early cell growth regulatory genes represent the different founder lines and WT. c, Northern blot analysis of such as c-fos, junB, and PCNA have been shown to be regulated by RNA from bone marrow cells from WT and four different founder lines of Ser119/133-phosphorylated CREB-1 (27, 28). Furthermore, protein Tg mice. Expression levels of the transgene transcript were identified using RNA prepared from bone marrow cells probed with probe specific to the transgene as described in Materials and Methods. Top panel, The expression of transgene transcripts. A comparable 28s ribosomal RNA level in of protein loading in each of the lanes is shown (control). The numbers each of the lanes is shown as loading control (bottom panel). The numbers shown in the bottom of each lane represent the different founder lines and shown in the bottom of each lane represent the different founder lines and WT control. e, EMSA of nuclear extract from NTg and Tg mice. Nuclear WT control. d, Western blot analysis of transgene expression in bone mar- extract prepared from splenic B cells from two Tg (Tg#1 and Tg#2) and a row cells from WT and four different founder lines of Tg mice. The ex- NTg mice were analyzed by EMSA using radiolabeled WT CRE (lanes pression level of Tg human CREB-1 protein was analyzed in protein ex- 2–4) or mutant CRE (lanes 5–7) oligonucleotides as described in Materials tract prepared from WT and four different founder lines of Tg mice, using and Methods. In vitro transcribed and translated recombinant CREB-1 pro- an Ab specific to human CREB-1. The presence of equivalent levels tein (IVT-CREB) was used as positive control (lane 1). 2212 DIFFERENTIAL ROLE FOR CREB-1 IN B1 AND B2 B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/

FIGURE 2. Defective pre-BI to Pre-BII developmental progression in mCREB-1 Tg mice. a, left panel, Bone marrow cells from NTg and Tg littermates were stained with PE-conjugated anti-B220 and FITC-conjugated anti-IgM. Right panel, Thymocytes from NTg and Tg littermates were stained with by guest on September 25, 2021 PE-conjugated anti-CD4 and FITC-conjugated anti-CD8. The results shown are representative of more than eight experiments. The cells representing various populations are presented as percentage in each of the quadrants. b, Bone marrow cells from NTg (top panel) and Tg (lower panel) littermates were stained with CyChrome-conjugated anti-B220, PE-conjugated anti-CD43, and FITC-conjugated anti-CD24 Abs. The expression levels of CD24 in the B220ϩCD43ϩ cells are shown in the right of the panel. Pro-B cells (B220ϩCD43ϩCD24Ϫ), pre-BI cells (B220ϩCD43ϩCD24ϩ(int)), and early pre-BI cells (B220ϩCD43ϩCD24ϩϩ) are distinguished based on the expression levels of CD24 in the B220ϩCD43ϩ cells. The numbers represent the percentage of cells in bone marrow (left panel) and the percentage of cells in the B220ϩCD43ϩ cells (right panel). The results shown are representative of four independent experiments. c, The percentage of pre-BI and early pre-BII in the bone marrow from NTg and Tg littermates were analyzed as mentioned in b. The numbers p Ͻ 0.02; unpaired Student’s t test). d, Decreased bone marrow cells ,ءء ;p Ͻ 0.01 ,ء) .represent the mean of percentage of cells Ϯ SD from four mice in the Tg mice. The total bone marrow cells from NTg and Tg mice femur were enumerated using a hemocytometer from four mice. Numbers represent mean Ϯ SD. Right panel, X-ray analysis (top) and histological analysis (H&E staining) of Tg and NTg littermates exhibiting dense skeletal structures in the Tg mice compared with NTg littermates. e, Bone marrow cells (10 ϫ 106/mice) from NTg and Tg mice were injected i.v. into sublethally irradiated (300 rad) RAG-2Ϫ/Ϫ mice. Four weeks after the transfer, B cells in the bone marrow from the recipients were analyzed by FACS as mentioned in b. The fold change in the percentage of B220ϩCD43ϩCD24high (early pre-BII cells) in the recipients compared with the control mice are shown. Ⅺ, The control RAG-2Ϫ/Ϫ mice without transfer with the cells (PBS control). f, The recipient mice with NTg bone marrow cells. u, The recipient mice with Tg bone marrow cells. kinase-mediated signaling events have also been shown to regulate NTg littermates were analyzed by reverse transcriptase-mediated c-Jun, JunB, and JunD (40–42). To test whether deregulation of PCR analysis. Interestingly, pre-BII B cells from Tg mice revealed any of these potential phospho-CREB-1 target genes could con- a consistent increase in c-Jun and JunB transcripts compared with tribute for the defective expansion of pre-BII cells in the Tg mice, wild-type littermate controls (Fig. 3a). No detectable differences RNA from sorted pre-BII B cells from the bone marrow of Tg and were observed in expression of PCNA, Mb-1, and vpreB or HPRT

Table I. B cell populations in bone marrow of mCREB Tg micea

Total B220ϩIgMϩ B220ϩIgMϪ Pre-BII Pre-BI

# ϫ 106 # ϫ 106 %#ϫ 106 %#ϫ 106 %#ϫ 106 %

NTg 29.6 Ϯ 4.63 1.31 Ϯ 0.38 4.41 Ϯ 0.98 2.24 Ϯ 0.49 7.56 Ϯ 0.89 0.38 Ϯ 0.05 1.29 Ϯ 0.11 0.32 Ϯ 0.019 1.1 Ϯ 0.14 ءءϮ 0.42 1.87 ءϮ 0.005 0.096 ءϮ 0.2 0.55 ءϮ 0.01 0.03 ءϮ 1.1 3.5 ءϮ 0.017 0.19 ءϮ 0.19 1.48 ءϮ 0.29 0.08 ءTg 5.92 Ϯ 1.65

a Four NTg and four Tg 6- to 10-wk-old mice were analyzed by ﬂow cytometry. Numbers represent mean Ϯ SD for absolute cell numbers or percentage of bone marrow .(p Ͻ 0.02 (unpaired Student’s t test ,ءء ;p Ͻ 0.01 ,ء .(%) cells The Journal of Immunology 2213

transcripts (Fig. 3a). The increased junB and c-jun expression was conﬁrmed independently by multiplex PCR analysis using same RNA preparations analyzed with respective test gene primers along with HPRT internal control primers. A representative result of this analysis and summary of four independent experiments are shown in Fig. 3b. JunB and c-Jun have been shown to play antagonistic roles in cell cycle regulation (40, 41). Both c-Jun and JunB have been

implicated in G1-S phase transition (42, 43). Given that the pre-BII B cells in the normal bone marrow are actively proliferating cells, we tested whether the decrease in the pre-BII population is due to

a block in G1 to S phase progression. The cell cycle proﬁle of pre-BII B cells from Tg and their littermate NTg controls were tested in vitro. Consistent with previous observations, pre-BII B cells showed increased cycling status at 48 h in culture (Fig. 3c, top panel). However, cell cycle analysis of pre-BII cells from Tg mice exhibited a consistent decrease in S-phase entry compared with wild-type littermates (Fig. 3c (41 vs 11% in NTg and Tg

mice, respectively). This is further reﬂected by accumulation of Downloaded from

cells in G0-G1 and G2-M phase of the cell cycle.

Abnormal splenic B cell profiles in the mutant CREB-1 Tg mice CREB-1 has been implicated to play a role in mature T and B cell signaling and development (22, 23, 25, 30, 33). To determine whether the abnormal B cell development is further reflected in the http://www.jimmunol.org/ peripheral lymphoid organs, splenocytes, and lymph node cells from Tg and NTg mice were analyzed. When compared with NTg mice, the mCREB-1 Tg mice revealed ϳ40% decrease in the IgMϩB220ϩ cells (29.3 Ϯ 0.96% in NTg vs 17.7 Ϯ 2.9% in Tg mice), with minimal change in CD4 or CD8 T cell population (Fig. 4a). This phenotypic distribution is further reflected in significant decrease in the absolute number of mature B cells in the spleen Ϯ ϫ 6 Ϯ ϫ 6 Ͻ (36 3.6 10 in NTg vs 22 1.9 10 in Tg mice, p 0.01). by guest on September 25, 2021 Interestingly, Tg mice revealed alterations in specific B cell populations in the spleen. Thus, when compared with NTg mice, mCREB-1 Tg mice revealed ϳ45% decrease in CD21dimCD23high follicular B cells with minimal change in the CD21highCD23dim marginal zone B cell (16.5 Ϯ 1.4 ϫ 106 in NTg vs 9.5 Ϯ 0.92 ϫ 106 cells in Tg mice p Ͻ 0.01 (Fig. 4c and Table II)).

Defective humoral immune responses in mCREB-1 Tg mice CREB/AP-1 family of proteins has been shown to regulate the expression of Ig gene (44). To test whether overexpression of dominant-negative CREB-1 in the mice alters the serum Ig, ELISA analysis of sera from 8-wk-old naive NTg and Tg mice was performed. Although minimal differences were observed in the levels of serum IgA, IgG, IgG2a, IgG2b, and IgG3, a consistent yet moderate increase (ϳ30%) in total IgM levels in Tg mice compared with NTg Ϯ Ϯ ␮ Ͻ FIGURE 3. Increased junB and c-jun expression associated with defec- littermates (38 5vs29 3 g/ml, p 0.001) (Fig. 5a). tive S phase entry of Tg pre-BII B cells. a, Total RNA was prepared form To test the potential differences in immune responses in vivo, Tg, sorted pre-BII cells using the TRIzol method. The expression levels of and NTg mice were immunized with DNP-KLH (T-dependent Ag), indicated gene transcripts were determined by RT-PCR analysis as men- TNP-LPS (T-independent type 1 Ag), or TNP-Ficoll (T-independent tioned in the Materials and Methods. HPRT was used as an internal control to normalize the expression levels of the genes tested. The data shown here are representative of four independent experiments. b, Relative levels of junB and c-jun expression in NTg and Tg pre-BII B cells. Multiplex RT- PCR analysis of JunB and c-Jun transcripts using the respective primers mice were stimulated with media for 48 h. The cells were stained with and HPRT primer sets in the same reaction to eliminate variabilities is CyChrome-conjugated anti-B220, PE-conjugated anti-CD43, and FITC- shown on the top two panels. The mean fold changes relative to the NTg conjugated anti-CD24 Abs. After ﬁxation and permeabilization with 1% controls (set at 1) in four independent experiments is shown in the bottom paraformaldehyde and 70% ethanol, cells were stained with 2 ␮g/ml p Ͻ 0.01, when compared with NTg con- Hoechst 33343, followed by FACS analysis. The data were acquired in ,ءء p Ͻ 0.001, and ,ء).panels trols). HPRT was used as an internal control to normalize the expression list-mode, and cell cycle analysis was performed using WinMDI Ver2.8 levels of the genes tested. c, Defective S-phase entry of pre-BII cells from and Modﬁt LT Verity cell cycle analysis software. The data shown are a mCREB-1 Tg mice. Bone marrow cells (1 ϫ 106/ml) from NTg and Tg representative of three independent experiments. 2214 DIFFERENTIAL ROLE FOR CREB-1 IN B1 AND B2 B CELL DEVELOPMENT

type 2 Ag). Ag-specific (DNP/TNP-specific Ab response was evalu- ated in the preimmune, 7-day postprimary immune and in 5-day post- secondary immune sera. Tg and NTg mice exhibited comparable levels of anti-DNP-specific IgM, IgG2, IgG2b, and IgG3 levels during the primary immune response to DNP-KLH immunization. However, dramatic defects in secondary immune responses to DNP-KLH were observed in the Tg mice. Thus, in spite of the presence of comparable Ag-specific serum IgM levels, Tg mice exhibited significant reduction in Ag-specific IgG1 (5.7 Ϯ 2.9 vs 2.2 Ϯ 1.3 ␮g/ml ( p Ͻ 0.005)), IgG2b (1.5 Ϯ 0.66 vs 0.3 Ϯ 0.16 ␮g/ml ( p Ͻ 0.005)), and IgG3 (0.23 Ϯ 0.06 vs 0.099 Ϯ 0.06 ␮g/ml ( p Ͻ 0.005)) Ab levels compared with age and sex matched controls (Fig. 5c). Thus, the expression of the IgG1, IgG2a, IgG2b, and IgG3 were reduced ϳ62, 91, 80, and 61%, respectively. Interestingly, a consistent increase in IgA levels during primary (ϳ40%) (0.018 Ϯ 0.005 vs 0.025 Ϯ 0.008 ␮g/ml ( p Ͻ 0.05)) and secondary (Ͼ100%) (0.011 Ϯ 0.003 vs 0.024 Ϯ 0.0069 ␮g/ml ( p Ͻ 0.001)) responses in Tg mice compared with NTg mice (Fig. 5c). Furthermore, a significant decrease in IgG2a levels

was noticed in the Tg mice in primary ((0.32 Ϯ 0.14 vs 0.09 Ϯ 0.05 Downloaded from ␮g/ml ( p Ͻ 0.001)) and secondary IgG2a (1.6 Ϯ 0.5 vs 0.4 Ϯ 0.09 ␮g/ml ( p Ͻ 0.001)) immune responses to T-dependent Ag (Fig. 5, b and c). In contrast to T-dependent Ag, Ag specific immune responses to T-independent TNP-LPS or TNP-Ficoll were comparable in the both Tg and NTg controls (Fig. 5d). http://www.jimmunol.org/ Abnormal accumulation of B-1 B cells that are resistant to apoptosis in mCREB-1 Tg mice In contrast to the bone marrow and spleen, a consistent increase in total peritoneal cells was observed in Tg mice compared with NTg littermates (23.8 Ϯ 5.2 ϫ 105 and 37.9 Ϯ 2.6 ϫ 105 in NTg and Tg mice, ( p Ͻ 0.01) (Table III)). Furthermore, flow cytometric analysis of peritoneal cells revealed significant increase in B220ϩIgMϩ population in Tg mice compared with NTg (22 Ϯ 3 and 33 Ϯ 2.6% in NTg and Tg mice, p Ͻ 0.01; Table III). The by guest on September 25, 2021 increased cell number and the abnormal distribution reflected ϳ3- fold increase in IgMϩB220ϩ peritoneal B cells in the Tg mice (13 Ϯ 0.01 ϫ 105) compared with the wild-type littermate (5 Ϯ 0.01 ϫ 105) controls. The IgMϩB220ϩ cells were further confirmed to be B1 B cells based on the (IgMhighIgDlowMac-1ϩ) staining (Fig. 6a). B1 B cells can be further divided into CD5ϩ B-1a and CD5Ϫ B1b cells based on the expression of CD5 surface marker. Flow cytometric analysis of peritoneal B cell with anti- B220-(PE-Cy5), anti-Mac-1-(PE), and anti-CD5-(FITC) showed ϳ3- to 4-fold increase in both B-1a (CD5ϩ) and B-1b (CD5Ϫ) cells in Tg mice compared with NTg littermates (Fig. 6b and Table III). FIGURE 4. Decreased IgMϩB220ϩ B cells associated with reduced fol- To determine whether the increased peritoneal B1 B cells is due licular but not marginal zone B cells in mCREB-1 Tg mice. a, Splenocytes (top panel) and lymph node cells (bottom panel) from NTg and Tg litter- to active proliferation and expansion, peritoneal cells were stained mates were stained with PE-conjugated anti-B220 and FITC-conjugated with CFSE and cultured in the presence or absence of LPS, anti- anti-IgM or PE-conjugated anti-CD4 and FITC-conjugated anti-CD8. The IgM, or PMA. Flow cytometric analysis of CFSE dye reduction in numbers represent the percentage of cells within the quadrants (left panel) activated peritoneal B cells failed to reveal any differences in cell or the boxes (right panel). The results shown are representative of four division over a period of 24–72 h tested (data not shown). To independent experiments with similar observations. b, Splenocytes from determine whether the increased B1 B cells in the mCREB Tg NTg and Tg littermate were stained with CyChrome-conjugated anti-B220 mice is due resistance to apoptosis, peritoneal cells from Tg and and biotinylated anti-CD23 and FITC-conjugated anti-CD21 followed by NTg mice were cultured in vitro for 24 h, and the apoptosis of B staining with PE-conjugated streptavidin. The expression levels of CD21 and CD23 of B220ϩ gated cells are shown. The numbers represent the percentage of cells in the B220ϩ population. The results shown are a representative of four independent experiments with similar outcomes. c, The ␮g/ml), or PMA (0.1 ␮g/ml) ϩ ionomycin (0.5 ␮g/ml) in 200-␮l volume absolute cell number of marginal zone (MZ) (CD21highCD23dim) and fol- for 40 h. The cells were then pulsed for another 8 h with 1 ␮Ci of [3H]thy- licular (FO) (CD21dimCD23high) B cells from NTg and Tg mice are shown. midine. The cells were harvested and the [3H]thymidine incorporation was The cells were identified as described in b and extrapolated with total measured as described in Materials and Methods. The data are represented splenocytes. Numbers represent mean Ϯ SD from four independent exper- as fold induction in response to activation compared with unstimulated p Ͻ 0.01; unpaired Student’s t test). d, Splenic B cells (2.5 ϫ conditions. Numbers represent the mean of fold induction Ϯ SD from six ,ء) iments .(p Ͻ 0.001; unpaired Student’s t test ,ء) from NTg or Tg mice were stimulated with medium, anti-IgM (10 independent experiments (105 The Journal of Immunology 2215

Table II. B cell populations in spleen of mCREB Tg micea

Total B220ϩIgMϩ Marginal Zone B Cells Follicular B Cells B220ϩCD5ϩ CD4ϩ

# ϫ 106 # ϫ 106 %#ϫ 106 %#ϫ 106 %#ϫ 106 %#ϫ 106 %

NTg 123 Ϯ 13.5 36 Ϯ 3.6 29.3 Ϯ 0.9 10.4 Ϯ 1.78 8.4 Ϯ 0.65 16.5 Ϯ 1.43 12.4 Ϯ 1.43 7.1 Ϯ 0.96 5.78 Ϯ 0.68 34.4 Ϯ 1.8 28 Ϯ 2.5 Ϯ 4.3 30 Ϯ 2.4 38 ءϮ 0.06 3.83 ءءϮ 0.74 5 ءϮ 1.06 8.45 ءϮ 0.76 9.51 Ϯ 0.95 7.7 ءϮ 1.81 9.75 ءϮ 2.9 17.7 ءTg 126 Ϯ 18.8 22 Ϯ 1.9

a Four NTg and four Tg 6- to 10-wk-old mice were analyzed by flow cytometry. Mean Ϯ SD for absolute cell numbers (#) or percentage (%) of splenocyte populations are .(p Ͻ 0.03 (unpaired Student’s t test ,ءء ;p Ͻ 0.01 ,ء shown cells was determined by using B220/annexin V and propidium tive pre-BCR assembly, although a role for CREB-1 in pre-BCR- iodide staining. Peritoneal B220ϩ B cells from Tg mice showed 3- mediated signaling events remains to be tested. Nevertheless, the to 4-fold decrease in spontaneous apoptosis compared with the defective S-phase entry in mCREB-1 pre-BII B cells is consistent NTg littermates. (Fig. 6c). with a role for abnormally expressed JunB and c-Jun in the inhibition of cell cycle progression. Consistent with this hypothesis, Discussion overexpression of JunB has been shown to mediate an inhibitory The studies described in this report have identified differential role effect on cell cycle progression through blocking cyclin D1, which for CREB-1 in multiple stages of B cell development and func- in turn is required for the G1-S phase transition (40). The observed Downloaded from tional maturation in vivo. Thus, overexpression of dominant -neg- results emphasize the complex nature of gene regulation by AP-1 ative CREB-1 in B cells resulted in pre-BI to pre-BII B cell pro- family members by CREB-1. For example, Jun family members gression in the bone marrow, decreased follicular B cells in the exhibit different functional properties as transcription factors. Spe- spleen, increased B1 B cells in the peritoneum, and defective T- cifically, JunB is known to be a weak AP-1 transactivator as com- dependent Ab responses. The observed defects were noted in four pared with c-Jun. Additional junB overexpression is known to an-

independent lines, as well as in both FVB/N and (FVB/N ϫ tagonize some of the transcriptional activities of c-Jun (53, 54). http://www.jimmunol.org/ C57BL/6) mixed backgrounds (data not shown). Also, the effect of junB and c-jun depend on the promoter context, Signiﬁcant reduction in mature, immature, and pre-BII B cells their phosphorylation status and cell type (55). associated with an increase in pre-BI B cells suggests an early- The observed developmental defects in the mCREB-1 Tg mice stage-speciﬁc role for CREB-1 in pre-BI to pre-BII transition. The are selective for B but not T cells. The decrease in mature B cells reduction in pre-BII cells in the mCREB-1 mice suggested a pos- in the spleen could be attributed to the developmental block in the sible cell survival or proliferative defect in these cells. Prosurvival bone marrow (56, 57). Alternatively, it is likely that the cells that genes such as Bcl-2 or Bcl-xL are known to be involved in B cell had escaped the development block in the bone marrow could be development in the bone marrow (45, 46). Pre-BI to pre-BII tran- still susceptible to CREB-1-dependent deregulation of cell prolif- sitional block in the mCREB-1 Tg mice is not due to deregulation eration. Consistent with this the mCREB-1 splenic B cells exhib- by guest on September 25, 2021 of these antiapoptotic genes as analysis of sorted pre-BI and pre- ited a moderate yet consistent decrease in proliferation in response ϩ BII B cells revealed comparable levels of Bcl-2 and Bcl-xL ex- to anti-IgM or PMA ionomycin (Fig. 4d). This conclusion is pression in Tg and NTg mice. Consistent with this, expression of further supported by a possible role for CREB-1 in the prolifera-

Bcl-2 or Bcl-xL transgenes in the mCREB-1 Tg mice failed to tion of mature B cells (24, 25, 33). The defective development of rescue neither the accumulated pre-BI nor the reduced pre-BII phe- mature peripheral B cell population in the mCREB-1 Tg mice notype in the double Tg mice (data not shown). The failure of could be attributed to the change in BCR signaling strength, the

Bcl-2 and Bcl-xL transgenes to rescue the defective pre-BII B cell specificity, and the repertoire usage in different immune compart- expansion suggests possible involvement of other cell survival ments (58). genes such as Mcl-1, XIAP, Bax, and Bad. Consistent with this The defective secondary immune responses in the mCREB-1 Tg hypothesis Mcl-1 gene promoter has been shown to be regulated mice are intriguing. The selective decrease in Ag specific IgG1, by Ser133-phosphorylated CREB-1 in myeloid cell lines (47). IgG2a, IgG2b, and IgG3 Ab levels during secondary immune re- Several lines of evidences indicate a potential role for CREB-1 sponses could be due to the defective responsiveness of Ag-spe- in the cell cycle regulation of pre-B cells. First, RT-PCR analysis cific B cells to T cell-derived cytokine. Consistent with this hy- of RNA from pre-BI and pre-BII B cells revealed the reciprocal pothesis, recently we demonstrated a critical role for CREB-1 in B regulation of JunB in pre-BI and pre-BII B cells. Thus, the in- cell responsiveness to IFN-␥ during AgR-mediated B cell activa- creased JunB in pre-BII B cells can account for the decreased tion (25). Alternatively, CREB-1 may regulate expression of dis- S-phase entry as reported previously (40–43, 48). Consistent with tinct Ab isotypes. This is consistent with the CREB binding sites this hypothesis, the mCREB-1 Tg pre-BII B cells exhibited defec- in several Ig promoters, including IgA and IgG1 (44, 59, 60). tive cell cycle progression associated with decreased S-phase entry The increased B1 B cells in the peritoneum can be attributed to (Fig. 3c). It is likely that the defective B cell development found in several possibilities. The abnormal B1 B cell population may be the mCREB-1 Tg bone marrow could be attributed to the defective attributed to the abnormal development of B1 B cells from the fetal IL-7 signaling due to CREB-1-dependent deregulation of the AP-1 liver precursor (61). Alternatively cells blocked in pre-BI stage in family members such as c-Fos and c-Jun (49, 50). Consistent with the bone marrow, could contribute to abnormal development, mi- this, mice lacking IL-7, IL-7R␣, or the common ␥c chain show a gration and/or subsequent development into B1 B cells in the peri- block of B cell development, which results in a reduction of pre-B toneum. Acquisition of B1 B cell surface phenotype by B2 B cells, cell populations (51, 52). Alternatively, a role for CREB-1 in IL- through modulation of surface molecules upon Ag receptor en- 7-independent activation pathways involving stromal cell compart- gagement by multivalent cross-linking of surface IgM, has been ment cannot be ruled out. Comparable levels of expression of observed in vitro (62). Two lines of evidences indicate that the vpreB, ␭5, and mb-1 transcripts from sorted pre-BII cells indicate increased B1 B cells in the peritoneal cavity are not due to abnor- that the defective pre-BII development is less likely due to defec- mal proliferation. First, CFSE-labeled B1 B cells from Tg and NTg 2216 DIFFERENTIAL ROLE FOR CREB-1 IN B1 AND B2 B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Defective humoral immune response in the mCREB-1 Tg mice. a, Comparable serum Ab levels in Tg and NTg mice. Levels of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs in preimmune sera from unimmunized NTg (E) and mCREB-1 Tg mice (F) is shown. The results represent mean serum concentration of indicated isotype Ϯ SD from 10 mice per group. b, Primary immune response to T-dependent Ag, DNP-KLH. NTg mice (E) and mCREB-1 Tg mice (F) were injected with DNP-KLH (50 ␮g/mice) i.p. (IP). Seven days after immunization, the levels of TNP-KLH-specific IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs to the primary immune response were analyzed by ELISA as described in Materials and Methods. The results represent mean serum concentration of indicated isotype Ϯ SD from 10 to 13 mice/group. c, Secondary immune response to T-dependent DNP-KLH. NTg (E) and mCREB-1 Tg mice (F) were injected i.p. with DNP-KLH (50 ␮g/mice) 4 wk after the primary immunization. Five days after secondary immunization, the level of TNP-KLH-specific IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs were analyzed by ELISA as described in Materials and Methods. The results represent mean serum concentration of indicated isotype Ϯ SD from six to eight mice per group. d, Immune response to T-independent type 1(TNP-LPS) and type II (TNP-Ficoll) Ags. NTg (E) and mCREB-1 Tg mice (F) were injected i.p. with TNP-LPS (50 ␮g/mice). Seven days after immunization, the levels of Ag-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 Abs were analyzed by isotype-specific ELISA as described in Materials and Methods. IgM response to T-independent type 2 Ag TNP-Ficoll (10 ␮g/ml i.p) is shown in the lower right most panel. The results represent mean serum concentration of indicated isotype Ϯ SD from four to six mice per group.

Table III. B cell populations in peritoneum of mCREB Tg micea

Total B220ϩIgMϩ B1 B Cells B1a B Cells B1b B Cells

# ϫ 105 # ϫ 105 %#ϫ 105 %#ϫ 105 %#ϫ 105 %

NTg 23.8 Ϯ 52 5.2 Ϯ 1.4 21.58 Ϯ 3.04 3.9 Ϯ 0.8 13.1 Ϯ 3.18 0.9 Ϯ 0.2 4.25 Ϯ 0.79 1.9 Ϯ 0.5 8.29 Ϯ 1.98 ءϮ 3.0 16.3 ءϮ 1.6 5.99 ءϮ 1.18 7.98 ءϮ 0.5 3.0 ءϮ 2.75 24.6 ءϮ 1.0 9.3 ءϮ 2.6 32.8 ءϮ 1.5 12.5 ءTg 37.9 Ϯ 2.6

a Four NTg and four Tg 6- to 10-wk-old mice were analyzed by ﬂow cytometry. Numbers represent mean Ϯ SD for absolute cell numbers (#) or percentage (%) of peritoneal .(p Ͻ 0.01 (unpaired Student’s t test ,ء .cells The Journal of Immunology 2217

optosis mediated through up-regulation of expression of antiapoptotic genes such as Bcl-2 and Mcl-1. Thus, the increased resistance to apoptosis by B1 B-cells from Tg mice could be attributed to altered expression of any of the known or hitherto unknown antiapoptotic genes or their regulators. Consistent with this, CREB-1 has been shown to bind and regulate expression of the promoter regions of antiapoptotic genes such as Mcl-1 and Bcl-2 in vitro. In this context, it is less expected that dominant-negative CREB-1 in Tg mice rendered resistance to apoptosis in peritoneal B cells. The mechanistic basis for differential role of CREB-1 in B1 and B2 B cell is not clear. It is possible that phosphorylation events that are constitutively active in B1 but not B2 B cells, as evidenced by constitutively active STAT3, ERK, and NF-AT may attribute to dominant-negative CREB-1-dependent resistance to death (63, 64). It is likely that the ultimate CREB-1-dependent positive or negative physiological outcome on growth and viability of a cell type is dependent on the activation status of multiple signaling pathways, including protein kinase C, protein kinase A, CaMKi-

nase, and Ras that regulate CREB-1 and its dimerization partners Downloaded from such as ATF-1. It is also likely the differential effect of CREB-1 in B1 and B2 B cells could be attributed to differential competition of phosphomutant CREB-1 to other CRE binding proteins such as ATF-1, ATF-2, or the cAMP response element modulator. Re- cently CREB-1 has been implicated to play a role in myeloid cell

transformation (65, 66). Ongoing experiments aimed to identify http://www.jimmunol.org/ natural CREB-1 targets in vivo using the mutant CREB-1 B cells will deﬁne the role for CREB-1 in B1 and B 2 B cell growth and survival and Ab responses. Acknowledgments We thank Cindy McAllister for the help with the ﬂow cytometry. Excellent secretarial support provided by Kathy Porter is greatly appreciated. We thank Drs. Carol Whitacre, Michael Caligiuri, and Azad Kaushik for help-

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