Involvement of Twisted Gastrulation in T Cell-Independent Plasma Cell Production Sotiris Tsalavos, Katerina Segklia, Ourania Passa, Anna Petryk, Michael B. O'Connor and Daniel Graf This information is current as of September 26, 2021. J Immunol 2011; 186:6860-6870; Prepublished online 13 May 2011; doi: 10.4049/jimmunol.1001833 http://www.jimmunol.org/content/186/12/6860 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Involvement of Twisted Gastrulation in T Cell-Independent Plasma Cell Production

Sotiris Tsalavos,* Katerina Segklia,* Ourania Passa,* Anna Petryk,†,‡ Michael B. O’Connor,‡ and Daniel Grafx

Bone morphogenetic protein (BMP) signaling is increasingly implicated in immune cell differentiation and function; however, direct in vivo evidence for such a role is still missing. In this article, we report that Twisted gastrulation (TWSG1), an extracellular reg- ulator of BMP signaling, is expressed in activated B cells and regulates T-independent B cell responses in the mouse. Twsg1-deficient B cells mount stronger T-independent type 2 responses reflected as increased IgM levels and numbers of Ag-specific IgM-secreting cells. BCR stimulation of Twsg1-deficient B cells results in hyperproliferation, hyperresponsiveness, and decreased apoptosis, whereas TLR stimulation results in hyperproliferation and increased IgG3 production. These changes are reflected on the molecular level by increased transcription of Bcl-6, Pax5, and the BMP-responsive gene Id-2. The TWSG1 effects on B cells Downloaded from appear to be cell intrinsic, suggesting that Twsg1 expression in B cells serves to interpret BMP signals on a per-cell basis. In summary, our observations on the role of TWSG1 in B cell function is opening new paths toward the exploration of the role of BMP signaling in immunological processes. The Journal of Immunology, 2011, 186: 6860–6870.

roduction of Abs by B cells is one of the key elements of amounts of IgM within 3–4 d after antigenic stimulation (7).

an immune response toward the elimination of invading In vitro, a variety of stimuli induce B cells to differentiate to Ig- http://www.jimmunol.org/ P extracellular pathogens. Depending on the nature of the producing plasma cells such as LPS that via engagement of TLR4 Ag, Ab production can be divided into T cell dependent (TD) and (8) induce plasma cells that switch to IgG2b and IgG3 (9). T cell independent (TI). In TD responses, B cell activation through Bone morphogenetic proteins (BMPs) are members of the TGF- the BCR requires a costimulatory signal through CD40, which is b superfamily of secreted signaling molecules. BMPs were orig- provided by CD40L on activated T cells (T cell help) (1). B cell inally named for their ability to induce ectopic bone formation stimulation through repetitive Ags like polysaccharides on bac- (10) but are actually widely involved in development, homeosta- terial cell walls do not require T cell help and are therefore termed sis, and repair of many tissues (11–13), including the hemato- TI responses (2). TI type I (TI-1) and TI type II immune responses poietic system (14–17). Several BMPs are expressed in adult he- (TI-2) can be distinguished on the basis of the Ag. In vivo, LPS (3) matopoietic and lymphoid cell lines, implying BMP signaling in by guest on September 26, 2021 induces TI-1 responses, whereas Ficoll (4) drives TI-2 responses. the regulation of immune function (16). With respect to B cells, Ab production against TI Ags is rapid. TI Ags have been shown to BMP2 can induce growth arrest in the mouse B cell hybridoma localize to the splenic marginal zone (5, 6), thus triggering mainly line HS-72 (18, 19), and BMP6 has been implied in fine-tuning marginal zone B cells (MZB) and B1 cells that produce large the balance between proliferation, apoptosis, and differentiation of developing human B cells (20, 21). After binding of BMPs to their cognate receptors (BMP type I and II receptor heterodimer), *Institute of Immunology, Biomedical Sciences Research Center Alexander Fleming, Smad-dependent and -independent signaling cascades are being 16672 Vari, Greece; †Department of Pediatrics, University of Minnesota, Minneap- olis, MN 55455; ‡Department of Genetics, Cell Biology and Development, Univer- activated (22). In Smad-dependent signaling, Smad1/5/8 are sity of Minnesota, Minneapolis, MN 55455; and xInstitute of Oral Biology, Faculty of phosphorylated and translocated to the nucleus to exert their Medicine, University of Zurich, 8032 Zurich, Switzerland cellular effects. Known target genes of Smad-dependent signaling Received for publication June 2, 2010. Accepted for publication April 7, 2011. are the inhibitor of differentiation (Id) gene family (23, 24). Id This work was supported by the Association for International Cancer Research and proteins are important regulators of lymphocyte development (25, the European Union 6th Framework Program, Network of Excellence MUGEN 26). With respect to adult B cells, Id3 knockout mice show im- (LSHG-CT-2005-005203). paired B cell proliferation and immune responses (18), whereas S.T. planned, performed research, analyzed data, and wrote the manuscript; K.S. and O.P. performed research and analyzed data; A.P. and M.B.O. contributed vital new Id2 regulates MZB differentiation (27), suppresses IgE class reagents; and D.G. designed the project, performed experiments, analyzed data, and switch recombination, and associates with Pax5, thus regulating wrote the manuscript. AID activity (28). These requirements for Id activity indicate the Address correspondence and reprint requests to Dr. Daniel Graf, Institute of Oral potential involvement of BMP signaling in the regulation of im- Biology, Faculty of Medicine, University of Zurich, Plattenstrasse 11, 8032 Zurich, Switzerland. E-mail address: [email protected] mune functions. However, direct in vivo evidence for BMP func- The online version of this article contains supplemental material. tion on lymphocytes is still missing, likely a reflection of the early and complicated phenotypes observed in most BMP knockout Abbreviations used in this article: ASC, Ab-secreting cells; A.U., arbitrary unit; BM, bone marrow; BMP, bone morphogenetic protein; cKO, conditional knockout; FDG, mice (29). fluorescein di-b-D-galactopyranoside; GC, germinal center; Id, inhibitor of differen- BMP signaling is highly regulated in the extracellular space tiation; MHC-II, MHC class II; MLN, mesenteric lymph node; MZB, marginal zone B cell; rBmp2, recombinant BMP2; rChordin, recombinant Chordin; rTwsg1, where BMPs interact with other secreted proteins, mostly antag- recombinant Twsg1; T1, transitional stage 1; TD, T cell dependent; TI, T cell in- onists such as Gremlin, Chordin, Noggin, and Twisted gastrulation dependent; TI-1, TI type I immune response; TI-2, TI type II immune response; (TWSG1). TWSG1 synergistically interacts with chordin or TWSG1, Twisted gastrulation; wt, wild-type. chordin-like molecules to regulate BMP activity (30). Depending Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 on the context, TWSG1 can modulate BMP activity in a positive www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001833 The Journal of Immunology 6861 or negative manner (31–34). Several in vitro studies point toward Immunizations an important role of TWSG1 in the regulation of the immune Eight- to 12-wk-old mice were injected i.p. with 25 mg of the hapten DNP system. TWSG1 expressed by developing thymocytes in a TCR- coupled to LPS (DNP-LPS) in PBS for the TI-1 and with 25 mg DNP3- dependent manner synergizes with Chordin to block BMP2/4 that Ficoll in PBS for the TI-2 (both from Biosearch Technologies). Sera were negatively regulate thymocyte proliferation and differentiation collected at 7 and 14 d. (17). TWSG1 expressed in mature T cells in a Tob-dependent ELISA and ELISPOT manner has been shown to inhibit proliferation and cytokine production of alloreactive CD4+ T cells (35). The in vivo require- For Ig baseline, relative levels from serum and peritoneal cavity Nunc plates were coated with 2 mg/ml goat anti-mouse Ig(H+L). Igs were detected ments for Twsg1 are background dependent. Whereas it has been using HRP-labeled goat anti-mouse IgM, IgG, IgG1, IgG2a, IgG2b, IgG3, reported that Twsg1-deficient mice are viable but show impaired IgA (all from Southern Biotechnology). The same procedure was also lymphocyte development in some mice (36), Twsg1 null mutants carried out for the detection of IgM, IgG2b, and IgG3 in the in vitro on the C57Bl6 background die in utero and display craniofacial cultures. Specific anti-DNP Igs were detected from collected sera by coating the plates with 1 mg/ml DNP-BSA (Calbiochem). Sera obtained malformations of variable severity (37) (D. Graf and O. Passa, from mice and culture supernatants were serially diluted and added in each unpublished observations). well. OD490 values were converted to arbitrary units (A.U.) by comparison We have found that Twsg1 is also expressed in activated B cells. with a pool of sera from the control group. Purified B cells from immu- Using a conditional gene ablation strategy, we investigated its nized mice with DNP-Ficoll were incubated overnight at 37˚C in plates function specifically in B cells in vivo. We found that B cell- coated with 1 mg/ml DNP-BSA. Ab-secreting cells (ASCs) were detected with biotinylated anti-mouse IgM (Southern Biotechnology) followed by derived TWSG1 is not required for B cell development per se, streptavidin-HRP and detected using 3-amino-9-ethylcarbazole substrate but rather that it is involved in the regulation of TI responses (Sigma-Aldrich). Spots were counted on A.EL.VIS Eli.Expert 4-Plate Downloaded from in vivo. Twsg12/2 B cells show enhanced proliferation, activation, ELISPOT Reader (Automated ELISA-Spot Assay Video Analyis Sys- and IgG3 production after stimulation in vitro. This indicates tems). a role for TWSG1 in regulating plasma cell production and im- B cell purification and in vitro stimulation plies for the first time, to our knowledge, the presence of a BMP CD19+ fraction was purified from erythrocyte-depleted splenocytes by signaling network to regulate B cell function. MACS using anti-mouse CD19 beads (Miltenyi Biotech), according to the

manufacturer’s instructions. Purity was .95% as assessed by FACS. Pu- http://www.jimmunol.org/ rified B cells were stimulated as follows: anti-CD40 (FGK-45, kindly Materials and Methods provided by A. Rolink, University of Basel, Basel, Switzerland), anti-IgM Mice F(ab9)2 (Southern Biotechnology), LPS (Sigma-Aldrich), 100 ng/ml recombinant BMP2 (rBmp2; PeproTech), recombinant Twsg1 (rTwsg1), Heterozygote Twsg1-lacZ reporter mice (Twsg1+/lacZ) have been described f/+ and 1 mg/ml recombinant Chordin (rChordin; R&D Systems). For mea- Twsg1 5 elsewhere (38). Mice carrying a conditional allele ( ) (37) were suring proliferation, 2 3 10 purified B cells in RPMI were plated in 96- crossed with FlpCre mice to remove the neo cassette and were sub- well, flat-bottom plates, and proliferation was measured after 72-h culture sequently backcrossed into C57BL/6 mice for at least six and up to eight by pulsing the cells with 0.5 mCi [3H]thymide 16 h before harvesting. For generations. The F1 progeny homozygous for the floxed allele (Twsg1f/f) B cell activation, cells were stimulated with 5 mg/ml anti-IgM F(ab9) or 1 was crossed with CD19cre mice (39) to generate mice with a conditional 2 f/f +/cre mg/ml LPS. For plasma cell induction, B cells were incubated with 1 mg/ Twsg1 Twsg1 CD19 by guest on September 26, 2021 loss of in the B cell compartment [ ; designated as ml LPS. Secreted Ig in the supernatant were measured by ELISA as de- Twsg1 A -conditional knockout (cKO) in this article] (Supplemental Fig. 2 ). scribed after 7 d of culture. Mice homozygous for the floxed allele (Twsg1f/f) were used as control animals, because they showed no statistical differences in activation assays Semiquantitative RT-PCR to Twsg1f/+Cd19cre/+ mice. Similar (Twsg1f/f) mice were crossed with Vav- Cre mice (40) to generate mice with a conditional loss of Twsg1 in the RNAwas extracted from purified B cells from control and Twsg1-cKO using complete hematopoietic compartment. As a control, we also deleted Twsg1 TRIzol, and concentrations were determined by spectrophotometry. For in the germline, which was perinatally lethal. Mice were maintained at the each fraction, 2 mg RNA was reverse transcribed into cDNA with Super- Biomedical Sciences Research Center Alexander Fleming animal facility Script II (Invitrogen). Semiquantitative RT-PCR was done with serial under specific pathogen-free conditions. Experiments on live animals were dilutions of 1:5 in a solution containing 1.2 ml MgCl2, 4.0 ml mixture approved by the Hellenic Ministry of Rural Development (Directorate of containing 29-deoxynucleoside 59-triphosphates, 1.0 ml primer mix, and Veterinary Services) and by Biomedical Sciences Research Center Alex- 0.5 ml Taq polymerase (Invitrogen). Cycling conditions were as follows: ander Fleming’s Animal Research and Ethics Committee for compliance 94˚C for 5 min, then 35 cycles of 94˚C for 20 s, 57˚C for 30 s, and 72˚C for to the Federation of European Laboratory Animal Science Associations’ 90 s. For primer sequences, see Table I. regulations. Immunohistochemistry and lacZ staining Abs and flow cytometry OCT (BDH)-embedded tissues were “snap frozen” in liquid nitrogen steam. Five- to 6-mm cryostat sections were placed in gelatin-coated slides, Erythrocyte-depleted single-cell suspensions from bone marrow (BM), air-dried, fixed in ice-cold acetone for 10 min, and rehydrated in PBS. spleen, lymph nodes, and peritoneal cavity were stained with the following Sections were incubated overnight with Abs against rabbit anti-Bmp2 9 Abs: FITC and biotin anti-IgM F(ab )2 (Southern Biotechnology); PE-CD5 (PeproTech) and detected with AP-labeled anti-rabbit IgG (Southern (53-7.3), PE-B220 (RA3-6B2), FITC-CD3 (145-2C11), allophycocyanin- Biotechnology) followed by staining with naphthol-AS-MX-phosphate/ CD138 (281-2), biotin-CD43 (S7), streptavidin-FITC, PerCP and allo- fast blue BB. Subsequent staining was performed with biotin-labeled F4/80 phycocyanin (all from BD Biosciences); allophycocyanin-CD25 (PC61), (BM8; BioLegend), detected with streptavidin-HRP (Vector Laboratories), biotin-B220 (RA3-6B2) (all from eBioscience); allophycocyanin-CD21/35 or rat anti-monocytes/macrophages (MOMA-2; Serotec), detected with (7E9), PE-IgD (11-26c.2a), PE-Cy7-CD23 (B3B4), FITC-MHC class II b biotinylated anti-rat IgG (BioLegend) and streptavidin-HRP followed by (MHC-II) I-A (AF6-120.1), PE-Cy7-CD86 (PO3), and PE-Cy7-CD69 staining with 3,39-diamino benzidine tetrahydrochloride. For lacZ staining, (H1.2F3) (all from BioLegend). sections were fixed in cold glutaraldehyde/formaldehyde and incubated For fluorescein di-b-D-galactopyranoside (FDG) detection, cells were with 2 mg/ml 5-bromo-4-chloro-3-indoyl-2-D-galactopyranoside (HT Bio- m incubated for 30 min at 37˚C in culture medium containing 33 M technology, Cambridge, U.K.) at 37˚C overnight. Subsequently, the sections C12FDG (Invitrogen) and subsequently stained for flow cytometry. Apo- were incubated with biotin-B220 (RA3-6B2) followed by streptavidin-HRP. ptotic cells were visualized by incubating the cells with biotin-Annexin V Sections were visualized on a Nikon Eclipse microscope. (BD Biosciences) followed by streptavidin-FITC (BD Biosciences) and propidium iodide (Fluka). For CFSE labeling, purified B cells were labeled Western blotting with 10 mM CFSE in PBS/0.1% BSA for 10 min at 37˚C. All flow cytometry data were collected on a Becton Dickinson FACS Canto II A total of 5 3 106 purified B cells suspended in RPMI/1% FBS were serum cytometer and were analyzed with FlowJo (TreeStar). Statistical analysis starved for 1 h before stimulation with 1 mg/ml LPS + 100 ng/ml rBmp2. was performed using Student t test. Cells were lysed at the various time points with radioimmunoprecipitation 6862 TWISTED GASTRULATION IN B CELL FUNCTION assay cell extraction buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% In thymocytes, pre-TCR–induced TWSG1 antagonizes BMP SDS, 1mM Na3VO4, 1mM PMSF, 13 protease inhibitor mixture; Sigma- activity in an autocrine fashion (17). The activation-induced ex- Aldrich). Ten micrograms cell lysate was loaded on acrylamide gel for pression of Twsg1 suggested a similar function in B cells. To SDS-PAGE electrophoresis. Western blotting carried out on nitrocellulose Cre membrane (Whatman) blocked with PBS/0.1% Tween 20/5% BSA and address this, we crossed a conditional Twsg1 allele to CD19 probed with goat anti-tsg (R&D Systems) overnight in PBS/0.1% Tween to delete Twsg1 specifically in B cells (Supplemental Fig. 2A). 20/1% BSA. Membranes were washed 3 3 10 min with PBS/T and in- Successful deletion was confirmed in LPS-activated B cells by RT- cubated with anti-goat IgG-HRP (Southern Biotechnology). Membranes PCR and Western blot (Fig. 2A,2B, Table I). Twsg1-cKO mice did were visualized using ECL plus detection kit (Amersham) on Biomax film (Kodak). Membranes were striped and reprobed with b-actin (Sigma- not exhibit any gross abnormalities in the lymphoid organs. Total Aldrich), to assess equal protein loading. cell counts of BM, spleen, and lymph nodes were comparable with littermate controls (Fig. 2C). The relative frequencies of B and Results T cells in spleen and peripheral blood were also not altered (Fig. Twsg1 is preferentially expressed in LPS-activated B cells 2D). This was in contrast with the phenotype observed in mice with global deficiency of TWSG1 (36). Vav-Cre–mediated de- Twsg1 is a bona fide BMP modifier, and its expression correlates letion of Twsg1 in all hematopoietic cells also resulted in normal with sites of BMP activity (31, 41). Twsg1 is expressed in several cellularity of the lymphoid organs (Supplemental Fig. 2B), sug- adult tissues including primary and secondary lymphoid organs gesting that it is not the loss of hematopoietic cell-derived Twsg1, (17, 36, 42, data not shown). Because BMP signals have been but rather of BM stroma-derived Twsg1 that is responsible for the implied in the regulation B cell function (20, 21, 43), we hy- block in lymphoid development observed in global TWSG1 de- Twsg1 pothesized that was also differentially expressed in B cells. Downloaded from ficiency (36, 40). Using a Twsg1-lacZ reporter mouse (Twsg1+/lacZ) that allows vi- sualization of Twsg1 gene expression in situ, we observed lacZ expression in the lymphoid follicles of the spleen and mesenteric B cell-specific deletion of Twsg1 does not affect B cell lymph nodes (MLNs) (Supplemental Fig. 1). Staining was ob- development served in some B220+ cells, suggesting a restricted or dynamic We next investigated in more detail the effects of TWSG1 de- expression in B cells. Testing for Twsg1 expression in purified ficiency on the development of B cell subcompartments in BM and http://www.jimmunol.org/ B cells ex vivo, we found that Twsg1 was not expressed in resting spleen. In the BM, similar percentages of pro-B (CD19+B220+ B cells but was induced by LPS (Fig. 1A,1B). No expression of IgM2IgD2c-Kit+CD252) and pre-B (CD19+B220+IgM2IgD2c- other BMP antagonists was observed (Fig. 1C, Table I). BMP kit2CD25+) cells in both control and Twsg1-cKO mice were ob- receptor expression was also induced by LPS similar to Twsg1. served. Similarly, the immature (B220+CD19+IgM+IgDint/2) and Hardly any receptor expression was seen in resting B cells, but mature/recirculating (B220+CD19+IgM+IgDint/2) B cell subsets several BMP type I receptors (Alk1, Alk2, Alk3, Alk6) and BMP showed no significant changes, suggesting normal B cell de- type II receptors (BMPR-II, ActR-II, ActR-IIb) were induced in velopment in Twsg1-cKO mice (Fig. 3A). In Twsg1-cKO spleens, LPS-activated B cells (Fig. 1D, Table I). This suggests that acti- follicular B cell (CD23+CD212) numbers were similar to the 2 + vated B cells are the predominant BMP targets, though the weak wild-type (wt) control, but the numbers of MZB (CD23 CD21 ), by guest on September 26, 2021 expression of Alk-6 and BMPR-II in unstimulated B cells suggests as well as the newly formed/transitional B cells (CD232CD21low), that at least some resting B cells might also be receptive to BMP were reduced (Fig. 3B, top panel). This was also reflected in signals. a small decline in the fraction III population (IgDlowIgMhigh) that

FIGURE 1. Twsg1 is expressed in activated B cells. A and B, Twsg1 promoter activity measured by FACS in FDG-stained purified splenic B cells of wt (Twsg1+/+) and Twsg1+/lacZ mice stimulated with 1 mg/ml LPS at several time points. B, Mean fluorescence intensity (MFI) of the FDG intensity of the panels in A;(A, B) mean and SE of three mice per group. Representative of two independent experiments. C and D, RT-PCR of (C) BMP antagonists and (D) BMP receptors expressed in unstimulated and LPS-stimulated purified splenic B cells. The Journal of Immunology 6863

Table I. RT-PCR primer sequences

Gene Forward Primer Reverse Primer Twsg1 59-AGGGGGCGTGGACATTG-39 59-CTGTGTCTCCCCGTGTCC-39 Noggin 59-CTCTACGCCCTGGTGGTG-39 59-AAGCCCGGGTCGTAGTG-39 Chordin 59-CAAGCCTCAGCGGAAGAA-39 59-CAAGCCCAGCCAATAGAACT-39 Gremlin 59-CTGTTTCCGGCTGGTGTT-39 59-AACAGCCGCACTATCATCAA-39 Alk1 59GGCCGATATGGTGAGGTG-39 59-TGTTGCCGATATCCAGGTAA-39 Alk2 59-GCCCAGCTGCCCACTAA-39 59-CAGCTGCCCCTCCATACTT-39 Alk3 59-AAGGCCGCTATGGAGAAG T-39 59-AGCGGTTAGACAGCATTGG-39 Alk6 59-ACCCGGCCATAAGTGAAG-39 59-CAGATGGGGGTGGAGGTC-39 Bmpr-II 59-AGAAGCCTGGAAAGAAAATAGC-39 59-TGTGGCGTGCAAATGTGT-39 Actr-II 59-GTTCGCCGTCTTTCTTATCTC-39 59-TGGTGCCTCTTTTCTCTGC-39 Blimp1 59-TTGTCGGGACTTTGCGGAG-39 59-TGGAAGACGGAAGGGGACTG-39 Irf4 59-TTGCTGAGCCACCTGGAGAG-39 59-TGGATGGAAGAATGACGGAGG-39 Xbp1 59-GCAGCAAGTGGTGGATTTGG-39 59-TTCTGGGGAGGTGACAACTGG-39 Mitf 59-GCAACGGAACAGCAACGAG-39 59-CAACAGGTGAGAGGGCATCG-39 Bcl6 59-AGTCGGGACATCTTGACGGAC-39 59-TCAGGAACTCTTCACGGGGAG-39 Pax5 59-AGTGGCATCCTGGGCATCAC-39 59-GGGCTCGTCAAGTTGGCTTTC-39 Gapdh 59-TCTTCTTGTGCAGTGCC-39 59-ACTCCACGACATACTCAGC-39 Downloaded from contains MZB and transitional stage 1 (T1)/newly formed cells Increased proliferation and activation in Twsg1-deficient (Fig. 3B, middle panel). The percentages of the B1 cell com- B cells + 2 + + + partment (B220 CD23 IgM CD43 CD5 ) were unchanged (Fig. The Twsg1 expression in LPS-activated B cells indicated a poten- 3B, bottom panel). tial involvement of TWSG1 in B cell activation-dependent pro- To gauge whether B cell-specific ablation of Twsg1 affects Ig cesses. We therefore measured B cell proliferative responses to http://www.jimmunol.org/ production, we assessed the baseline Ig titers. No differences were mitogenic stimuli. Twsg1-cKO purified B cells were hyperpro- observed in blood serum levels for IgM, IgG and its subtypes, and liferative compared with control cells over a range of anti-IgM in IgA in the peritoneal cavity (Fig. 3C). These results are in line and LPS doses (Fig. 4A). In contrast, proliferation induced by anti- with the observation of normal development of spontaneous ger- CD40 was not altered, and CD40 stimulation did not reverse anti- minal centers (GCs) in Peyer’s patches, MLNs, and spleen (not IgM– or LPS-induced hyperproliferation (Fig. 4B). Next, we shown). Overall, no significant alterations toward B cell de- wanted to test whether the hyperproliferation was due to increased velopment and homeostatic Ig production were observed in activation or increased survival. Three days after stimulation, Twsg1-cKO mice, suggesting that TWSG1 is not critically in- control and Twsg1-cKO B cells increased similarly in size and volved in these processes. granularity, as is typical of “blasting” cells (Fig. 4C). Expression by guest on September 26, 2021

FIGURE 2. Ablation of Twsg1 from B cells does not affect lymphocyte development. A, Semiquantitative RT-PCR for Twsg1 from 72-h LPS-stimulated B cells from control and Twsg1-cKO mice. B, Western blot for Twsg1 in control and Twsg1-cKO LPS-activated B cells. b-actin was used to assess equal protein loading. C and D, Assessment of effect of twsg1 B cell deletion in control and Twsg1-cKO mice (C) total cell counts from spleen, MLN, and BM (n = 5 mice/group), and (D) frequencies of B (B220+) and T (CD3+) cells in spleen and peripheral blood analyzed by FACS (means and SE of n = 3 mice/group). Representatives from three independent experiments are shown. 6864 TWISTED GASTRULATION IN B CELL FUNCTION

FIGURE 3. B cell-specific ablation of twsg1 does affect B cell development and Ig production. A and B, FACS analysis of control and Twsg1-cKO cells stained for (A) pro-B (CD252c-Kit+), pre-B (CD25+c- kit2), immature (IgM+IgD+/2) and mature/ recirculating (IgM+IgD+) cells in the BM, pro-B, pre-B were gated as (B220+CD19+ IgM2IgD2) and immature, mature as (B220+CD19+)(B) splenic B cell subsets analyzed with CD23, CD21, IgM, and IgD. Subsets were defined as follicular B cells (FOB; CD23hiCD21int/hi), MZB (CD23lo/2). B1 cells in the spleen were defined as B220+CD232IgM+CD432CD5+. Numbers denote the percentages of cells and SE (n = Downloaded from 3 mice/group). Representative data from two independent experiments. C, Baseline serum levels measured by ELISA for IgG, IgM, and peritoneal cavity IgA (left panel), and IgG subtypes (right panel) from con- trol (open square) and Twsg1-cKO mice http://www.jimmunol.org/ (filled triangle). n = 5 mice/group. F-I, fraction I; F-II, fraction II; F-III, fraction III.

of several activation markers was assessed. CD25 and CD69 levels IgG3 titers were lower at day 7 but recovered by day14. Immu- by guest on September 26, 2021 were increased on Twsg1-cKO cells relative to littermate controls nization with the TI-2 Ag DNP-Ficoll resulted in significantly after 24 h in response to anti-IgM and lesser to LPS (Fig. 4D, left higher IgM and IgG3 levels by day 14 (Fig. 5B). To assess whether panels,4F). To exclude that the increased activation was not due the increased Ig levels corresponded to more Ag-specific B cells, to haploinsufficiency for CD19 because of the use of CD19Cre,we we performed an ELISPOT assay on in vitro restimulated B cells compared the activation profiles between Twsg1f/f and Twsg1f/+ 4 d after DNP-Ficoll immunization. As shown in Fig. 5C, Twsg1- Cd19Cre/+. No significant difference between the controls was cKO mice had more DNP-specific ASCs. These data establish that observed (Supplemental Fig. 3). No alterations in expression ki- TWSG1 is a negative regulator of TI type 2 responses in vivo. netics were observed for MHC-II and CD86 (Supplemental Fig. 4). Increased Pax5, Bcl-6, and Id2 expression and IgG3 Increased CD25 and CD69 levels after IgM stimulation indicated production in Twsg1-cKO B cells that Twsg1-cKO cells might have an increased sensibility to BCR activation and lesser TLR4 activation. This “hyperactivation” was The increased number of ASCs prompted us to investigate whether accompanied by fewer Annexin V+ cells, demonstrating reduced TWSG1 directly affects plasma cell production. For this, purified apoptosis in Twsg1-cKO B cells (Fig. 4E). Having seen an effect B cells were stimulated with LPS for 4 d in vitro. No significant low + both on proliferation and apoptosis, we sought to resolve which differences in the percentage of plasma cells (B220 CD138 ) effect is predominant in Twsg1-cKO B cells. For this, we labeled were detected between Twsg1-cKO and control B cells (Fig. 6A). B cells with CFSE to trace cell divisions. More Twsg1-cKO B cells The expression of several transcription factors controlling plasma underwent cell divisions and more cell divisions were completed cell generation and fate (Blimp1, Irf4, Xbp-1, and Mitf) also when compared with control cells, suggesting that the increased remained unaltered (Fig. 6B, Table I). However, we detected en- activation and proliferation are predominant (Supplemental Fig. 5). hanced expression of Pax-5 and Bcl-6 (Fig. 6B). LPS stimulation Together, these data suggest that TWSG1 is involved in activation, induces IgM, IgG2b, and IgG3 secretion. We thus investigated proliferation, and apoptosis of B cells. whether the levels of these Ig subtypes were altered. IgM and IgG2b titers remained unaltered, but we observed increased IgG3 Enhanced TI Ig production of Twsg1-cKO mice levels in the culture supernatant from Twsg1-deficient B cells The hyperresponsiveness of Twsg1-deficient B cells prompted us 7 d after LPS stimulation (Fig. 6C). to investigate the in vivo responses of Twsg1-cKO mice. Because To gauge whether these differences were the result of altered the hyperresponsiveness was restricted to IgM and LPS stimula- BMP signaling in Twsg1-cKO B cells, we probed for changes in tion, and did not appear to involve CD40 signals, we focused on the expression of BMP target genes. We focused on the Id gene TI-specific Ab responses. Fourteen days after immunization with family (Id1-4) because these molecules are downstream targets of the TI-1 Ag DNP-LPS (DNP-LPS), Twsg1-cKO mice showed BMP signaling and have, at least in part, been implicated in B cell significantly higher Ag-specific IgM responses (Fig. 5A). The function and IgG production (25, 27, 44, 45). Semiquantitative The Journal of Immunology 6865 Downloaded from http://www.jimmunol.org/

FIGURE 4. Enhanced B cell proliferation, activation, and reduced apoptosis in Twsg1-cKO mice. A and B,[3H]Thymidine incorporation in B cells stimulated for 72 h with (A) increasing concentrations of anti-IgM and LPS, and (B) anti-CD40 (10 mg/ml) or combinations of anti-CD40 with anti-IgM (5 mg/ml) or LPS (1 mg/ml). C, FACS plots showing live cells in the forward scatter (FSC)/side scatter (SSC) gate after 72-h stimulation. D, CD25 and CD69 expression after 24 h on LPS (top panels) and anti-IgM (bottom panels) stimulation. E, Apoptotic cells (Annexin V+PI2) in unstimulated, LPS-, and anti-IgM–stimulated B cells after 24-h stimulation. p , 0.05 in anti-IgM–stimulated panel. F, Graphical representation of the percentage of gated cells in D. Percentages in FACS plots are means and SE of triplicate readings from three mice per group. All experiments were performed on purified B cells. n/s, NS. by guest on September 26, 2021

RT-PCR analysis showed increased expression of Id2 in LPS- Presence of BMP signaling network components in B cells activated Twsg1-cKO B cells compared with control cells. Lev- As TWSG1 appeared to modulate BMP signals, it raised the els of Id1 and Id3 were unaltered, and Id4 remained, as expected, question whether BMPs are expressed in B cells or within their undetected. These findings indicate that TWSG1 regulates B cell local environment in secondary lymphoid organs. Several BMPs responsiveness to polyclonal activators, possibly by antagonizing are expressed in primary and secondary lymphoid organs, but no BMP signals as reflected by altered expression levels of Id2. expression was observed either in resting or in LPS-activated

FIGURE 5. Twsg1-cKO mice mount in- creased TI responses. A, DNP-IgM,-IgG2b, -IgG3 in serum from control (n = 8) and Twsg1-cKO (n = 6) mice 7 and 14 d after immunization with DNP-LPS. B, DNP-IgM,-IgG3 in serum from control (n = 6) and Twsg1-cKO (n = 8) mice 14 d after immunization with DNP-Ficoll. C, ELISPOT of DNP-IgM ASCs isolated from control and Twsg1-cKO mice before and 4 d after immunization with DNP-Ficoll (n = 3 mice/group). Representative of two independent experiments. *p , 0.05, Student t test. 6866 TWISTED GASTRULATION IN B CELL FUNCTION Downloaded from http://www.jimmunol.org/

FIGURE 6. Absence of twsg1 enhances IgG3 production and id2 transcription in LPS-activated B cells. A, Plasma cells (B220+CD138low) generated from purified control and Twsg1-cKO B cells after 4-d LPS stimulation. Numbers in the gates denote averages from triplicate readings representative of by guest on September 26, 2021 three independent experiments. B and D, Semiquantitative RT-PCR of RNA from control and Twsg1-cKO B cells stimulated for 72 h with LPS. Repre- sentative of two independent experiments with three mice per group. C, ELISA for IgM, IgG2b, and IgG3 levels in the supernatant of B cell cultures after 7-d stimulation with LPS. Readings are averages of triplicates. Representative of three independent experiments. All experiments were performed with three mice per group. *p , 0.001.

B cells in vitro (data not shown). We thus investigated whether levels 24 h after stimulation because we had established that CD25 BMPs were present in B cell areas of the lymphoid follicles. We levels were increased on activated Twsg1-cKo B cells. rTwsg1 by focused first on BMP2, which is clearly expressed in the spleen itself had no effect on B cell activation (data not shown). Anti- (data not shown). Using double immunohistochemistry, we iden- IgM–induced upregulation of CD25 was not affected by any dose tified BMP2 in the marginal zone of lymphoid follicles in the of rTwsg11 tested, neither in Twsg1-cKO nor control B cells (Fig. spleen (Fig. 7A), which colocalized, at least in part, with F4/80 7D). Because exogenous Twsg1 did not acutely interfere with and Moma-II+ marginal zone macrophages. We thus tested whether BCR signaling, we tested whether Twsg1-deficient B cells were BMP2 could affect plasma cell differentiation directly. For this, already altered in situ and the increased BCR activation observed we stimulated control and Twsg1-cKO B cells with LPS in the in vitro was a reflection of this. To address this, we mixed control presence of BMP2. We did not observe any effects on plasma cell or Twsg1-cKO B cells (CD45.2) with competitor wt (CD45.1) differentiation (Fig. 7B). Similarly, Ab secretion remained un- B cells at a 1:1 ratio and stimulated them with anti-IgM. If the altered and, not surprisingly, was also not affected by the addition Twsg1 effect was mediated by secreted TWSG1, then the com- of Chordin (Fig. 7C). Furthermore, BMP2 had no direct effect on petitor CD45.1 B cells should “rescue” the Twsg1–cKo–B cell B cell activation and proliferation (data not shown). Thus, whereas effect, resulting in decreased CD25 levels in cKO B cells. If the the molecular data indicate that B cells are capable of receiving TWSG1 effect was cell intrinsic, then introduction of competitor BMP signals, we failed to detect any effect in vitro. B cells should have no effect on the activation phenotype (in- creased CD25 levels) observed in Twsg1-cKo B cells. As shown in B cell intrinsic effect of TWSG1 Fig. 7E and 7F, 24 h after stimulation, both control and Twsg1-cKO TWSG1 is predominantly produced in activated B cells rather than B cells displayed their respective CD25 signature independent of resting B cells. Because we failed to detect any direct effect of the presence of competitor B cells. CD25 levels neither decreased BMPs on B cell activation, proliferation, and Ig production, we in Twsg1-cKO B cells nor increased in CD45.1 wt B cells. The wondered whether the TWSG1 was responsible for the effects on observations that rTwsg1 did not affect B cell activation and that wt B cell activation, or whether any other mechanism was in operation. or Twsg1-cKO B cells did not alter their activation profile even if To address this, we tested the effect of rTwsg1 in a range of doses on cocultured suggests that the TWSG1 effect on B cell activation is anti-IgM–stimulated B cells. As readout we used CD25 expression cell intrinsic and predetermined in vivo. The Journal of Immunology 6867 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Presence of BMP2 is not associated with Twsg1-mediated Ig production in vitro. A, Double immunohistochemistry probing for BMP2 (alkaline phosphatase, blue) and macrophage markers F4/80 and MOMA-II in wt spleens (HRP, brown). Original magnification 340 (top panels) and 3200 (bottom panels). B, Plasma cells (B220+CD138low) generated after stimulation of purified control and Twsg1-cKO B cells with LPS and LPS+rBmp2 for 4 d. Numbers in the gates denote averages from triplicate readings representative of three independent experiments. C, ELISA for IgM, IgG2b, and IgG3 levels in the supernatant of B cell cultures after 7-d stimulation with LPS, LPS+rBmp2, and LPS+rBmp2+rChordin. Readings are averages of triplicates. Representative of three independent experiments. D, CD25 expression on purified control and Twsg1-cKO B cells 24 h after stimulation with anti-IgM in the presence of varying concentrations of rTwsg1. Numbers in the gates denote the average from triplicate readings from three mice per group and three independent experiments. E, CD25 expression on control and Twsg1-cKO CD45.2 B cells mixed at a 1:1 ratio with CD45.1 B cells stimulated with IgM for 24 h. F, Mean fluorescence intensity (MFI) for CD25 of the plots shown in E. Shown are averages of three mice from a representative of three independent experiments. *p , 0.001. F, lymphoid follicle.

In summary, in this report, we show that B cells express the it does alter B cell responses. Twsg1-deficient B cells show in- BMP modifier TWSG1 in an activation-dependent manner. B cell- creased TI-2 responses, which is reflected in vitro by increased specific TWSG1 deficiency does not affect B cell development, but activation, proliferation, and Ig secretion (IgM and IgG3). These 6868 TWISTED GASTRULATION IN B CELL FUNCTION effects could not be modulated in vitro, either through addition of In vitro stimulation of purified B cells with anti-IgM or LPS recombinant BMP, BMP antagonists, or rTwsg1 itself. Competitor revealed increased proliferation and reduced apoptosis in Twsg1- experiments established that the Twsg1 effect on B cell activation deficient cells. In contrast, proliferation induced by anti-CD40 was is B cell intrinsic, indicating a role for TWSG1 in a preceding not altered, and CD40 stimulation did not reverse anti-IgM– or B cell maturation/selection step. LPS-induced hyperproliferation. The hyperproliferation was ac- companied by increased expression levels of CD25 and CD69, whereas other activation/differentiation markers such as CD86 and Discussion MHC-II were not altered. It is not clear whether the increased In this report, we have demonstrated that TWSG1, a bona fide BMP CD25 and CD69 levels are a direct effect of TWSG1 deficiency or modulator, regulates B cell activation, proliferation, TI plasma cell whether they are an epiphenomenon associated with the increased production, and Ab secretion. We provide evidence that TWSG1 is proliferation. We used the altered CD25 expression levels to ad- not acting autonomously on B cells, but it might be part of an dress whether the observed hyperproliferation was the result of extracellular BMP signaling network that regulates B cell func- activation-induced TWSG1 acting on the B cells in an autocrine/ tion in the lymphoid follicles of spleen and lymph node. To our paracrine fashion, or whether Twsg1-deficient cells were already knowledge, this is the first report to show an in vivo function of altered in situ and the increased proliferation was only a reflection a member of the BMP signaling machinery in secondary lymphoid of this. Because CD45.1 (competitor)-derived TWSG1 could not organs. affect the hyperactivation of Twsg1-cKO B cells in coculture To date, there is no direct proof that BMP signals regulate experiments, we conclude that the propensity for hyperproli- lymphocyte development and function in vivo. In vitro studies have feration is B cell intrinsic. Therefore, it was not surprising that Downloaded from implied BMP2 and BMP6 as negative regulators of B cell pro- addition of exogenous BMP pathway molecules (rTwsg1, rBmp2, liferation (19, 21). Furthermore, Bmp6 is part of the gene signature rChordin) had no effect on in vitro activated B cells. that defines diffuse large B cell lymphoma with predicted poor The in vivo and in vitro differences pointed toward a pre- outcome (46). TWSG1 is a modifier of BMP signaling, and has dominant role of TWSG1 in TI rather than TD responses. In line been shown to both positively and negatively affect BMP signaling with this, immunization with both TI-1 and TI-2 resulted in in-

(31–34). In developing and mature T lymphocytes, TWSG1 has creased secretion of IgM and, in some subclasses, of IgG, which http://www.jimmunol.org/ been identified as a BMP antagonist. In the thymus, pre-TCR sig- as shown for DNP-Ficoll was accompanied by more Ag-specific naling inducted TWSG1 interacts with stroma-derived Chordin to ASCs. In contrast, no significant differences to IgM or IgG pro- attenuate BMP2/4 binding, which inhibits thymocyte proliferation duction were observed in TD responses, and the size of GCs or the and differentiation (17). In mature T cells, TWSG1 regulates cy- percentage of GC B cells were comparable (data not shown). TI tokine production in CD4+-alloreactive T cells in vitro (35). In- responses are thought to be initiated by MZB cells, because they terestingly, both reports suggest that TWSG1 acts in an autocrine have the ability to respond very rapidly to TI Ags because of their manner. low activation threshold allowing them to cope with low Ag In mice, TWSG1 deficiency affects lymphocyte development concentrations, thus enhancing cellular responses (50). Although (36). This phenotype is not fully penetrant and is not observed spleens of Twsg1-cKO mice contain lower numbers of MZB, our by guest on September 26, 2021 on all genetic backgrounds. Whereas it is seen on a mixed data suggest that their ability to respond to TI-1 and TI-2 Ags is C57Bl6:129 background (O. Passa and D. Graf, unpublished enhanced. observations), TWSG1 deficiency on the C57Bl6 background is LPS stimulation of Twsg1-cKO B cells in vitro resulted in in- perinatally lethal and accompanied by a series of severe cranio- creased production of IgG3, indicating a possible role of TWSG1 facial malformations (37, 47) (O. Passa and D. Graf, unpublished in the class switch to IgG3. These higher IgG3 levels did not observations). Because of this background dependency, we have correspond to a global increase in plasma cells or in the altered chosen to perform our studies on mice backcrossed at least six expression of the main transcription factors that drive plasma cell generations to C57Bl6 background. differentiation. We did observe, however, an increase in the ex- After B cell-specific deletion of Twsg1, primary or secondary pression of the Bcl-6, Pax5, and Id2. Bcl-6 and Pax5 are known for lymphoid organs had normal B cell counts, in contrast with their key roles in B cell proliferation, maturation, and plasma cell a global TWSG1 deficiency in mice. Similarly, conditional de- differentiation (51–54). Gonda et al. (28) have proposed that the letion in all hematopoietic cells using the Vav-Cre (40) resulted modulation of the relative abundance of Pax5, E2A, and Id pro- in normal cellularity of the lymphoid organs. This indicated that teins provides a mechanism for subtle regulation of the effects of BM stroma-derived TWSG1 rather than hematopoietic cell-derived Pax5 and E2A in B cell activation, and these differential activities TWSG1 might be responsible for the lymphoid defects observed determine the activation of the master switch regulator, AID. in Twsg1 null mice. Based on our data, we propose that the enhanced expression levels More detailed analysis of Twsg1-cKO spleens revealed a 30% of Pax5 and Id2, regulated by TWSG1 activity, are shifting the bal- reduction of MZB. This reduction was also reflected in the IgDlow ance toward the germline transcription of the Cg3 locus, thus en- IgMhigh fraction, which contains newly formed/T1 and MZB cells. hancing IgG3 production in activated B cells under specific conditions. Interestingly, these B cell populations reside in the outer ring of TWSG1 is a bona fide modulator of BMP activity, and no the marginal zone, the very region where functional defects are function for TWSG1 without BMP interaction has been reported. In being observed (increased TI responses) (48, 49). The transition of the search of a BMP signaling network that could be regulated by newly formed immature B cells to follicular cells and MZB is TWSG1 in situ, we identified BMP2 in the vicinity of the splenic regulated by multiple signaling pathways. Of interest are Id2-de- marginal zone ring. The question is whether TWSG1 modulates ficient mice, in which newly formed/T1 and MZB populations BMP2 activity in vivo. The differences observed in Twsg1-cKo are also impaired with a shift toward follicular cells (27). Id2 is B cells on IgM and Ig subtype kinetics suggest that such a BMP a BMP target gene; thus, it is conceivable that a BMP signal signal could affect B cell activation and subsequent Ig responses regulates this aspect of B cell differentiation. In line with this, based on the fact that a common integrator of BCR and BMP reduced percentages in newly formed/T1 and MZB populations signaling are the Id proteins (25). Interestingly, Twsg1-deficient are also observed in Twsg1-cKO mice. B cells have increased expression levels of Id2. The Journal of Immunology 6869

In vitro experiments failed to reveal any direct effect of exog- 13. Wagner, D. O., C. Sieber, R. Bhushan, J. H. Bo¨rgermann, D. Graf, and P. Knaus. 2010. BMPs: from bone to body morphogenetic proteins. Sci. Signal. 3: mr1. enous BMP2 on B cell activation (data not shown), proliferation, 14. Bhatia, M., D. Bonnet, D. Wu, B. Murdoch, J. Wrana, L. Gallacher, and and Ig production in either control or Twsg1-cKO cells. Ig pro- J. E. Dick. 1999. Bone morphogenetic proteins regulate the developmental duction was also not affected by Chordin, a known interaction program of human hematopoietic stem cells. J. Exp. Med. 189: 1139–1148. 15. Durand, C., C. Robin, K. Bollerot, M. H. Baron, K. Ottersbach, and E. Dzierzak. partner of TWSG1 (33). Because BMP receptors are hardly 2007. Embryonic stromal clones reveal developmental regulators of definitive expressed on resting B cells but are strongly upregulated after hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 104: 20838–20843. activation in vitro, this could provide a likely explanation for the 16. Detmer, K., T. A. Steele, M. A. Shoop, and H. Dannawi. 1999. Lineage-restricted expression of bone morphogenetic protein genes in human hematopoietic cell earlier observations. Although we failed to identify the mecha- lines. Blood Cells Mol. Dis. 25: 310–323. nism and exact circumstances by which BMP signals affect 17. Graf, D., S. Nethisinghe, D. B. Palmer, A. G. Fisher, and M. Merkenschlager. B cells, we cannot exclude that the in vitro hyperresponsiveness 2002. The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development. J. observed in the Twsg1-deficient B cells is the result of an en- Exp. Med. 196: 163–171. dogenous BMP signal “preprogramming” the B cell for an altered 18. Ishisaki, A., K. Yamato, S. Hashimoto, A. Nakao, K. Tamaki, K. Nonaka, P. ten response in vitro. Dijke, H. Sugino, and T. Nishihara. 1999. Differential inhibition of Smad6 and Smad7 on bone morphogenetic protein- and activin-mediated growth arrest and By dissecting the role of TWSG1 in B cell activation and TI apoptosis in B cells. J. Biol. Chem. 274: 13637–13642. responses, the results show for the first time, to our knowledge, a 19. Yamato, K., S. Hashimoto, T. Imamura, H. Uchida, N. Okahashi, T. Koseki, potential in vivo role of BMP signaling in B cell differentiation. A. Ishisaki, M. Kizaki, K. Miyazono, Y. Ikeda, and T. Nishihara. 2001. Acti- vation of the p21(CIP1/WAF1) promoter by bone morphogenetic protein-2 in B cells deficient in the BMP modulator TWSG1 are hyperre- mouse B lineage cells. Oncogene 20: 4383–4392. sponsive to BCR and TLR4 stimulation, resulting in increased 20. Kersten, C., G. Dosen, J. H. Myklebust, E. A. Sivertsen, M. E. Hystad,

IgG3 production and enhanced TI-2 responses. The observed E. B. Smeland, and E. Rian. 2006. BMP-6 inhibits human bone marrow B Downloaded from lymphopoiesis—upregulation of Id1 and Id3. Exp. Hematol. 34: 72–81. phenotypes in Twsg1-cKO mice do not show an absolute re- 21. Kersten, C., E. A. Sivertsen, M. E. Hystad, L. Forfang, E. B. Smeland, and quirement for TWSG1 in these processes, rather pointing to a J. H. Myklebust. 2005. BMP-6 inhibits growth of mature human B cells; in- second level of control toward fine-tuning the B cell’s response to duction of Smad phosphorylation and upregulation of Id1. BMC Immunol. 6: 9. 22. Sieber, C., J. Kopf, C. Hiepen, and P. Knaus. 2009. Recent advances in BMP Ag encounter. Unraveling the mechanisms that regulate BMP receptor signaling. Cytokine Growth Factor Rev. 20: 343–355. signaling and how the cells respond to such signals will be another 23. Korchynskyi, O., and P. ten Dijke. 2002. Identification and functional charac- terization of distinct critically important bone morphogenetic protein-specific step toward understanding the regulatory complexity behind B cell http://www.jimmunol.org/ response elements in the Id1 promoter. J. Biol. Chem. 277: 4883–4891. differentiation and Ab production. 24. Lo´pez-Rovira, T., E. Chalaux, J. Massague´, J. L. Rosa, and F. Ventura. 2002. Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene. J. Biol. Acknowledgments Chem. 277: 3176–3185. D.G. is grateful to the Biomedical Sciences Research Center Alexander 25. Engel, I., and C. Murre. 2001. The function of E- and Id proteins in lymphocyte Fleming, at which the project was initiated and most of the experiments development. Nat. Rev. Immunol. 1: 193–199. 26. Sugai, M., H. Gonda, Y. Nambu, Y. Yokota, and A. Shimizu. 2004. Role of Id were performed. We thank Eftychia Lekka, Georgia Ikonomou, Rainhard proteins in B lymphocyte activation: new insights from knockout mouse studies. Maier, Vinko Tosevski, and Asja Guzman for technical assistance, Aris J. Mol. Med. 82: 592–599. N. Economides for the Twsg1-lacZ reporter, Alexandre J. Potocnik for 27. Becker-Herman, S., F. Lantner, and I. Shachar. 2002. Id2 negatively regulates Cre CD45.1 mice, Klaus Rajewsky for the CD19 mice, Mark Coles, and B cell differentiation in the spleen. J. Immunol. 168: 5507–5513. by guest on September 26, 2021 Petra Knaus for critical comments on the manuscript. 28. Gonda, H., M. Sugai, Y. Nambu, T. Katakai, Y. Agata, K. J. Mori, Y. Yokota, and A. Shimizu. 2003. The balance between Pax5 and Id2 activities is the key to AID gene expression. J. Exp. Med. 198: 1427–1437. Disclosures 29. Graf, D., and A. N. Economides. 2008. Dissection of BMP signaling using ge- nome engineering tools. In Bone Morphogenetic Proteins: From Local to Sys- The authors have no financial conflicts of interest. temic Therapeutics. S. Vukicevic, and K. T. Sampath, eds., eds. Birkhauser, Basel, Switzerland, p. 115–139. 30. Larraı´n, J., M. Oelgeschla¨ger, N. I. Ketpura, B. Reversade, L. Zakin, and References E. M. De Robertis. 2001. Proteolytic cleavage of Chordin as a switch for the dual 1. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, and R. J. Noelle. 1996. Immune activities of Twisted gastrulation in BMP signaling. Development 128: 4439– 4447. regulation by CD40 and its ligand GP39. Annu. Rev. Immunol. 14: 591–617. 31. Oelgeschlager, M., J. Larraı´n, D. Geissert, and E. M. De Robertis. 2000. The 2. Fagarasan, S., and T. Honjo. 2000. T-independent immune response: new aspects ¨ evolutionarily conserved BMP-binding protein Twisted gastrulation promotes of B cell biology. Science 290: 89–92. BMP signalling. Nature 405: 757–763. 3. Baker, P. J., J. R. Hiernaux, P. W. Stashak, and J. A. Rudbach. 1985. Cyclic 32. Chang, C., D. A. Holtzman, S. Chau, T. Chickering, E. A. Woolf, development of immunological memory to bacterial lipopolysaccharide. Infect. L. M. Holmgren, J. Bodorova, D. P. Gearing, W. E. Holmes, and Immun. 48: 1–6. A. H. Brivanlou. 2001. Twisted gastrulation can function as a BMP antagonist. 4. Vos, Q., A. Lees, Z. Q. Wu, C. M. Snapper, and J. J. Mond. 2000. B-cell acti- Nature 410: 483–487. vation by T-cell-independent type 2 antigens as an integral part of the humoral 33. Ross, J. J., O. Shimmi, P. Vilmos, A. Petryk, H. Kim, K. Gaudenz, immune response to pathogenic microorganisms. Immunol. Rev. 176: 154–170. S. Hermanson, S. C. Ekker, M. B. O’Connor, and J. L. Marsh. 2001. Twisted 5. Amlot, P. L., D. Grennan, and J. H. Humphrey. 1985. Splenic dependence of the gastrulation is a conserved extracellular BMP antagonist. Nature 410: 479–483. antibody response to thymus-independent (TI-2) antigens. Eur. J. Immunol. 15: 34. Scott, I. C., I. L. Blitz, W. N. Pappano, S. A. Maas, K. W. Cho, and 508–512. D. S. Greenspan. 2001. Homologues of Twisted gastrulation are extracellular 6. Harms, G., M. J. Hardonk, and W. Timens. 1996. In vitro complement-dependent cofactors in antagonism of BMP signalling. Nature 410: 475–478. binding and in vivo kinetics of pneumococcal polysaccharide TI-2 antigens in 35. Tzachanis, D., L. Li, E. M. Lafuente, A. Berezovskaya, G. J. Freeman, and the rat spleen marginal zone and follicle. Infect. Immun. 64: 4220–4225. V. A. Boussiotis. 2007. Twisted gastrulation (Tsg) is regulated by Tob and 7. Martin, F., A. M. Oliver, and J. F. Kearney. 2001. Marginal zone and B1 B cells enhances TGF-beta signaling in activated T lymphocytes. Blood 109: 2944– unite in the early response against T-independent blood-borne particulate anti- 2952. gens. Immunity 14: 617–629. 36. Nosaka, T., S. Morita, H. Kitamura, H. Nakajima, F. Shibata, Y. Morikawa, 8. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. Y. Kataoka, Y. Ebihara, T. Kawashima, T. Itoh, et al. 2003. Mammalian twisted 4: 499–511. gastrulation is essential for skeleto-lymphogenesis. Mol. Cell. Biol. 23: 2969– 9. Kearney, J. F., M. D. Cooper, and A. R. Lawton. 1976. B lymphocyte differ- 2980. entiation induced by lipopolysaccharide. III. Suppression of B cell maturation by 37. Petryk, A., R. M. Anderson, M. P. Jarcho, I. Leaf, C. S. Carlson, J. Klingensmith, anti-mouse immunoglobulin antibodies. J. Immunol. 116: 1664–1668. W. Shawlot, and M. B. O’Connor. 2004. The mammalian twisted gastrulation 10. Wozney, J. M., V. Rosen, A. J. Celeste, L. M. Mitsock, M. J. Whitters, gene functions in foregut and craniofacial development. Dev. Biol. 267: 374– R. W. Kriz, R. M. Hewick, and E. A. Wang. 1988. Novel regulators of bone 386. formation: molecular clones and activities. Science 242: 1528–1534. 38. Zakin, L., and E. M. De Robertis. 2004. Inactivation of mouse Twisted gastru- 11. Chen, D., M. Zhao, and G. R. Mundy. 2004. Bone morphogenetic proteins. lation reveals its role in promoting Bmp4 activity during forebrain development. Growth Factors 22: 233–241. Development 131: 413–424. 12. Kishigami, S., and Y. Mishina. 2005. BMP signaling and early embryonic pat- 39. Rickert, R. C., J. Roes, and K. Rajewsky. 1997. B lymphocyte-specific, Cre- terning. Cytokine Growth Factor Rev. 16: 265–278. mediated mutagenesis in mice. Nucleic Acids Res. 25: 1317–1318. 6870 TWISTED GASTRULATION IN B CELL FUNCTION

40. de Boer, J., A. Williams, G. Skavdis, N. Harker, M. Coles, M. Tolaini, T. Norton, limits apoptosis in the distal region of the mandibular arch in mice. Dev. Biol. K. Williams, K. Roderick, A. J. Potocnik, and D. Kioussis. 2003. Transgenic 328: 13–23. mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. 48. Saito, T., S. Chiba, M. Ichikawa, A. Kunisato, T. Asai, K. Shimizu, Immunol. 33: 314–325. T. Yamaguchi, G. Yamamoto, S. Seo, K. Kumano, et al. 2003. Notch2 is pref- 41. Dale, L. 2000. Pattern formation: a new twist to BMP signalling. Curr. Biol. 10: erentially expressed in mature B cells and indispensable for marginal zone B R671–R673. lineage development. Immunity 18: 675–685. 42. Graf, D., P. M. Timmons, M. Hitchins, V. Episkopou, G. Moore, T. Ito, 49. Pillai, S., A. Cariappa, and S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. A. Fujiyama, A. G. Fisher, and M. Merkenschlager. 2001. Evolutionary con- servation, developmental expression, and genomic mapping of mammalian Immunol. 23: 161–196. Twisted gastrulation. Mamm. Genome 12: 554–560. 50. Levy, S., S. C. Todd, and H. T. Maecker. 1998. CD81 (TAPA-1): a molecule 43. Seckinger, A., T. Meissner, J. Moreaux, H. Goldschmidt, G. M. Fuhler, involved in signal transduction and cell adhesion in the immune system. Annu. A. Benner, M. Hundemer, T. Re`me, J. D. Shaughnessy, Jr., B. Barlogie, et al. Rev. Immunol. 16: 89–109. 2009. Bone morphogenic protein 6: a member of a novel class of prognostic 51. Shaffer, A. L., X. Yu, Y. He, J. Boldrick, E. P. Chan, and L. M. Staudt. 2000. factors expressed by normal and malignant plasma cells inhibiting proliferation BCL-6 represses genes that function in lymphocyte differentiation, in- and angiogenesis. Oncogene 28: 3866–3879. flammation, and cell cycle control. Immunity 13: 199–212. 44. Kee, B. L., R. R. Rivera, and C. Murre. 2001. Id3 inhibits B lymphocyte pro- 52. Max, E. E., Y. Wakatsuki, M. F. Neurath, and W. Strober. 1995. The role of genitor growth and survival in response to TGF-beta. Nat. Immunol. 2: 242–247. BSAP in immunoglobulin isotype switching and B-cell proliferation. Curr. Top. 45. Sugai, M., H. Gonda, T. Kusunoki, T. Katakai, Y. Yokota, and A. Shimizu. 2003. Microbiol. Immunol. 194: 449–458. Essential role of Id2 in negative regulation of IgE class switching. Nat. Immunol. 53. Usui, T., Y. Wakatsuki, Y. Matsunaga, S. Kaneko, H. Koseki, and T. Kita. 1997. 4: 25–30. Overexpression of B cell-specific activator protein (BSAP/Pax-5) in a late B cell 46. Rosenwald, A., G. Wright, W. C. Chan, J. M. Connors, E. Campo, R. I. Fisher, is sufficient to suppress differentiation to an Ig high producer cell with plasma R. D. Gascoyne, H. K. Muller-Hermelink, E. B. Smeland, J. M. Giltnane, et al; Lymphoma/Leukemia Molecular Profiling Project. 2002. The use of molecular cell phenotype. [Published erratum appears in 1999 J. Immunol. 163: 1091.] J. profiling to predict survival after chemotherapy for diffuse large-B-cell lym- Immunol. 158: 3197–3204. phoma. N. Engl. J. Med. 346: 1937–1947. 54. Wakatsuki, Y., M. F. Neurath, E. E. Max, and W. Strober. 1994. The B cell- 47. MacKenzie, B., R. Wolff, N. Lowe, C. J. Billington, Jr., A. Peterson, B. Schmidt, specific transcription factor BSAP regulates B cell proliferation. J. Exp. Med. Downloaded from D. Graf, M. Mina, R. Gopalakrishnan, and A. Petryk. 2009. Twisted gastrulation 179: 1099–1108. http://www.jimmunol.org/ by guest on September 26, 2021