Notch1 engagement by Delta-like-1 promotesdifferentiation of B lymphocytes toantibody-secreting cellsMargarida Almeida Santos*, Leonor Morais Sarmento*, Manuel Rebelo*, Ana Agua Doce*, Ivan Maillard†,Alexis Dumortier‡, Helia Neves*§, Freddy Radtke‡, Warren S. Pear¶, Leonor Parreira*§, and Jocelyne Demengeot*�

*Instituto Gulbenkian de Ciencia, 2781-901 Oeiras, Portugal; †Division of Hematology–Oncology and ¶Department of Pathology and Laboratory Medicine,Abramson Family Cancer Research Institute and Institute for Medicine and Engineering, University of Pennsylvania School of Medicine,Philadelphia, PA 19104; ‡Swiss Institute for Experimental Cancer Research and Swiss Federal Institute of Technology Lausanne,1066 Epalinges, Switzerland; and §Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, 1649-028 Lisboa, Portugal

Edited by Max D. Cooper, University of Alabama at Birmingham, Birmingham, AL, and approved August 15, 2007 (received for review March 30, 2007)

Notch signaling regulates B and T lymphocyte development and T celleffector class decision. In this work, we tested whether Notch activityaffects mature B cell activation and differentiation to antibody-secreting cells (ASC). We show increased frequency of ASC in culturesof splenic B cells activated with LPS or anti-CD40 when providedexogenous Notch ligand Delta-like-1 (Dll1). Our results indicate thatNotch–Dll1 interaction releases a default pathway that otherwiseinhibits Ig secretion upon B cell activation. Thus, Dll1 enhancedspontaneous Ig secretion by naturally activated marginal zone B andB1 cells and reversed the inhibition of ASC differentiation mediatedby B cell receptor crosslinking during LPS. Moreover, suppression ofNotch signaling in B cell expression of either a dominant-negativemutant form of Mastermind-like 1 or a null mutation of Notch1 notonly prevented Dll1-mediated enhancement of ASC differentiationbut also reduced dramatically LPS-induced Ig secretion. Finally, weshow that Dll1 and Jagged-1 are differentially expressed in discreteareas of the spleen, and that the effect of Notch engagement on Igsecretion is ligand-specific. These results indicate that Notch ligandsparticipate in the definition of the mature B cell microenvironmentthat influences their terminal differentiation.

activation � B cells � immunoglobulin � commitment � LPS

Notch receptors and their ligands are a family of conservedtransmembrane proteins that regulate cell fate decisions in

diverse tissues and organisms (1), including the lymphocytelineage. The fundamental finding that Notch1–Delta-like-1(Dll1) interactions condition hematopoietic progenitor differ-entiation by promoting T lineage and preventing B cell devel-opment opened a new field for the understanding of lymphocytedifferentiation. These findings were born out of in vitro studiesbased on the usage of stromal cells transduced to express specificNotch ligands (2–4) and in vivo studies assessing loss- andgain-of-function mutant phenotypes (5, 6). Notch signaling alsoaffects later stages of lymphocyte maturation; it participates inalternative helper T cell differentiation (reviewed in ref. 7) andin transitional B cell progression to a marginal zone (MZ) B cellphenotype. In this latter case, Notch2–Dll1 interactions and thetranscription factor CBF1/Supressor of Hairless/Lag1(CSL) in-duced by Notch signaling have been shown to be specifically andstrictly required (8–10). In contrast, the contribution of Notchsignaling to B cell activation has not yet been systematicallyinvestigated.

B cell activation and subsequent differentiation to effectorstages are tightly regulated, whether T cell-dependent or inde-pendent, during ‘‘natural’’ activation or upon immunization andinfection. Which molecular components instruct or select a givenclonal progeny to differentiate either to an antibody-secretingcell (ASC) or a memory cell, or to remain a nonsecreting blast,is unsolved.

Terminal differentiation to ASC is irreversible and appears toresult from an all-or-nothing decision, because activated B cellseither secrete high levels of Ig or retain the splice variantencoding a membrane-bound Ig. The ‘‘commitment choice’’ anactivated B cell appears to undertake prompted us to assesswhether Notch engagement participates in cell fate decisionduring B cell activation. We used a stromal cell line expressingthe Notch ligands Dll1 or Jagged1 (Jg1), as well as purifiedrecombinant ligands, to evidence that B cell differentiation toASC is regulated by Notch signaling in a ligand- and receptor-specific manner.

ResultsDll1 Enhances LPS-Induced ASC Differentiation. To assess the effectof Notch signaling on B cell differentiation to effector stages, restingsplenic B cells were stimulated with LPS in the presence of S17stromal cells transduced with an expression vector encoding theNotch ligand Dll1 (S17-Dll1) or GFP only (S17-vector) (2). Thenumber of B cells recovered at day 4 in control and experimentalcultures was not significantly different. In contrast, although 9–10%of the recovered B cells had differentiated to IgM-secreting cells incontrol conditions, the presence of S17-Dll1 increased this fre-quency to 16–19% (Fig. 1A). B cell differentiation to IgG-secretingcells in the presence of LPS was marginal, yet this was significantlyenhanced by Dll1 (Fig. 1A). The titer of secreted IgM and IgG inthe culture supernatants was directly proportional to the number ofIg-secreting cells, indicating that Dll1 controls the frequency of Bcells that differentiate to Ig-secreting cells but not their secretionrate (Fig. 1B). In cultures supplemented with TGF-� or IL-4 toinduce class switch, Dll1 also increased IgA or IgE secre-tion, respectively (Fig. 1C). Finally, immobilized recombinant Dll1(Delta1ext-IgG; ref. 11) enhanced the differentiation of resting B cellsto ASC upon LPS stimulation to a similar extent as S17-Dll1. Takentogether, these results demonstrate that Dll1 directly promotes Bcell terminal differentiation upon LPS stimulation.

Dll1 Operates on Late Stages of B Cell Activation to Promote AntibodySecretion. We next investigated at which step of the activationprocess Dll1 would modify B cells response. Dll1 did not affect

Author contributions: L.P. and J.D. designed research; M.A.S., L.M.S., M.R., A.A.D., I.M., andA.D. performed research; H.N., F.R., W.S.P., and L.P. contributed new reagents/analytictools; M.A.S., L.M.S., L.P., and J.D. analyzed data; and M.A.S., L.M.S., and J.D. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: ASC, antibody-secreting cell; MZ, marginal zone; FDC, follicular dendriticcell; FO, follicular; FOB, follicular B cell.

�To whom correspondence should be addressed. E-mail: jocelyne@igc.gulbenkian.pt.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702891104/DC1.

© 2007 by The National Academy of Sciences of the USA

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B cell survival and did not induce activation in the absence ofLPS [supporting information (SI) Fig. 8]. Moreover, it increasedneither the expression of activation markers (SI Fig. 9) nor theproliferative responses (Fig. 2A and SI Fig. 10) that followsexposure to LPS. Syndecan-1 (CD138) is the surface markeravailable to identify B cells at late stages of activation, althoughnot exclusively at the Ig secreting stage (12). Analysis of CD138expression on B cells stimulated for 2–4 days with LPS in thepresence or not of Dll1 did not reveal significant differences (notshown). To directly test whether Dll1 influences late stages of Bcell activation, cells were first activated with LPS for 3 days in theabsence of stromal cells, purified according to CD138 expres-sion, and seeded in secondary cultures for 24 h in the presenceof stroma cells (Fig. 2B). Exposure of CD138� cells to S17-Dll1instead of S17-Vect resulted in an �2- to 2.5-fold increase in thenumber of recovered Ig-secreting cells (Fig. 2C). This effect wasnot due to differential B cell survival, as determined by TUNELassays (SI Fig. 11). Dll1 also increased the number of IgM-secreting cells recovered from the CD138� cell cultures but didnot modify the proportion of cells that acquired CD138 expres-sion (Fig. 2D). These data indicate that Dll1 influences latestages of ASC differentiation, later than acquisition of theSyndecan marker.

In unimmunized mice, MZB and peritoneal B1 cells display aphenotype of nonsecreting activated cells. Their conversion toASC in vivo requires migration out of the MZ and peritonealcavity, respectively (13), a phenomenon proposed to result fromrelease of local inhibition (14). Culturing MZB and B1 cells inmedium without additional stimuli partially mimics this phe-nomenon, because a significant fraction of these cells convert toASC. This conversion increased �3-fold in the presence ofS17-Dll1 as compared with controls (Fig. 2E). These resultssuggest that activation of Notch signaling by Dll1 releases aninhibitory signal that otherwise maintains B cells in a nonse-creting blast state. An in vitro assay that reveals late inhibition ofASC differentiation is based on BCR cross-linking concomitantwith LPS-mediated stimulation (15). In this assay, ASC inhibi-tion is an ‘‘all-or-nothing’’ phenomenon at the single-cell level,

dependent on BCR signal strength and BCR ligand concentra-tion and independent of proliferation and Ig secretion rate (16).Dll1 induced �1-log decrease in the sensitivity of B cells toantimicron-mediated inhibition of ASC differentiation (Fig. 2F).This result strengthens our hypothesis that Dll1-Notch activationpromotes ASC differentiation by reversion of inhibitory signalsotherwise preventing Ig secretion.

LPS-Induced Ig Secretion Is Suppressed in the Absence of Notch1 andCSL Activity. Dll1 appeared to trigger Notch signaling on activatedB cells, because it led to the transcriptional up-regulation of theNotch target genes Hes-1 (17) and Deltex-1 (18) as revealed bysemiquantitative RT-PCR analyses of day 4 LPS cultures (Fig.3A). The canonical Notch pathway operates through the forma-tion of a complex involving the cleaved intracellular portion ofNotch (ICN), the CSL transcription factor, and the Mastermind-like proteins (MAMLs) (19). Expression of a dominant-negative

Fig. 1. Dll1 enhances B cell differentiation to ASCs. (A–C) Resting B cells werecultured with S17 cells retrotransduced with either the vector alone (Vect) orDll1 construct (Dll1) in the presence of LPS (10 �g/ml) for 4 days. (A) Frequencyof IgM and IgG ASC determined by Spot-Elisa. (B) Concentration of IgM andIgG in the culture supernatant. (C) IgA and IgE concentration in the superna-tant of cultures supplemented in addition with TGF-� or IL-4. (D) Similar to A,except that S17 cells were replaced by coated Fc-Dll1ext-IgG (Fc-Dll1) or the Fcportion of human IgG (Fc). Shown is one representative experiment of six (A)or two (B–D). *, P � 0.05; **, P � 0.01.

Fig. 2. Dll1 operates on late stages of ASC differentiation. (A) Resting B cellswere cultured as in Fig. 1A. Proliferation was monitored by [3H]thymidineincorporation at day 3. (B–D) Splenic CD19� cells were cultured alone for 3days in medium containing 10 �g/ml LPS, purified according to CD138 expres-sion (B), and seeded in secondary cultures together with S17-Vector (Vect) orS17-Dll1 (Dll1) and LPS (10 �g/ml) for 24 h. The frequency of IgM-secreting cellsobtained from the two sorted populations (C) and CD138 expression onoriginally CD138� cells (D) was determined. (E) B1 and MZ B cells weremaintained in cultures containing S17-Vect or S17-Dll1 in medium alone for48 h before measuring the frequency of IgM-secreting cells. (F) Resting B cellswere seeded on transduced S17 cells and treated with various amounts ofantimicron chain mAb 2 h before the addition of 10 �g/ml LPS. On day 4, thenumber of IgM-secreting cells was scored by ESA. Shown are the percentagesof IgM-secreting cells obtained in the respective control cultures (no antimi-cron mAb) from the means of triplicate cultures. The 50% inhibition is indi-cated by the dotted line. One experiment representative of three (A) and oftwo (B–F) independent repeats is shown. *, P � 0.05.

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form of MAML1 (DNMAML) inactivates this complex (20, 21).Follicular (FO) CD23� B (FOB) cells expressing DNMAMLwere prepared from either DNMAMLf or poly(I:C)-treatedMx-Cre� DNMAMLf mice (20). In the former case, DNMAMLexpression was induced in DNMAMLf B cells by Tat-Cretransduction, and cells expressing the DNMAML-GFP fusionprotein (GFP�) were sort-purified. DNMAML-expressing andcontrol cells proliferated equally upon LPS stimulation, irre-spectively of whether they were plated on S17-Dll1 or S17-vect(not shown). However, DNMAML-expressing B cells poorlydifferentiated to ASC, and exogenous Dll1 failed to enhance thisresponse (Fig. 3 B and C). These results confirm that theenhancement of ASC differentiation by Dll1 operates throughthe canonical Notch pathway. More importantly, these experi-ments revealed that plasma cell induction by LPS requires intactNotch/CSL/MAML pathway irrespective of the presence ofexogenous ligand. In turn, this finding predicts that LPS-activated B cells express both the Notch receptor(s) and Notchligand(s) necessary to allow Ig secretion. Real-time RT-PCRanalysis confirmed that resting B cells express Notch2 but littleNotch1 and Delta-1 (ref. 9 and data not shown). Western blotanalysis revealed that resting and activated B cells express Dll1protein to a level comparable to that of the S17 vector (Fig. 4ARight). As described by the supplier, Dll1-specific Ab alsocross-react with Delta-4. The additional specific band detectedby Western blot analysis may correspond to either Delta-4 orposttranscription modification of Dll1. Strikingly, upon LPS-mediated activation, B cells undergo a marked increase inNotch1 (N1) protein expression (Fig. 4A). To directly testwhether N1 is required for ASC differentiation in the absence ofexogenous ligand, splenic FO cells were isolated from poly(I:C)-treated Mx-cre� Notch1f/f (22) or Mx-cre� DNMAMLf/�

mice and respective controls and stimulated with LPS in theabsence of stromal cells. Proliferation monitored at day 3 andcell counts at day 4 confirmed similar expansion between mutantand control cells (not shown). Both DNMAML expression andN1 disruption reduced by �3-fold the frequency of ASC recov-ered at day 4 of culture, when compared with controls (Fig. 4B).Overall, we interpret these results as indicating that LPS-inducedASC differentiation relies on N1 signaling through the CSLtranscription factor. In vitro, in the absence of the exogenous

Notch ligand, this signal may be triggered by presentation of theNotch ligand Dll1 by neighboring B cells. In vivo, several othercell types expressing higher amounts of Dll1 may be responsiblefor local triggering of Notch1 on activated B cells.

Discrete Expression of Delta-1 in Specific Areas of the Spleen. B cellactivation and differentiation to plasma cells take place indefined areas of the spleen and lymph nodes. We assessed thelocation of Dll1 in spleen by immunostaining on serial sections(Fig. 5). Dll1 expressed by FOB cells or S17-vec was undetectableby immunostaining, whereas S17-Dll1 tested strongly positive(not shown). Dll1 expression, however, was readily detectable inthe MZ and the FO dendritic cell (FDC) area of primary andsecondary follicles (Fig. 5A). In contrast, expression of the Notchligand Jg1 was strictly restricted to the MZ (Fig. 5B and data notshown). In the MZ, few Dll1/Jg1 double-expressing cells weredetected (Fig. 5B). Similar data were obtained when analyzingmice immunized with OVA-Alum (not shown). In conclusion,restricted areas of the spleen present either Dll1 alone or Dll1and Jg1 together, and the two ligands appear mostly differen-tially expressed.

Dll1 Enhances B Cell Responses to T Cell-Dependent-Like ActivationSignals. The specific expression of Dll1 by FDCs in primaryfollicles and germinal centers prompted us to test whether Dll1affects plasma cell differentiation after T cell-dependent acti-vation. Stimulation of FOB cells by anti-CD40 mAb in vitromimics partially this path. Dll1 enhanced B cell proliferativeresponse induced by anti-CD40 Ab treatment (Fig. 6A) andconsequently the number of cells recovered (not shown). Inaddition, the frequencies of cells secreting IgM or IgG (Fig. 6B)were increased. Taken together, these results indicated thatDll1-N1 signaling affects differently CD40L/CD40 and LPS/Toll-like-Receptor-4-mediated B cell proliferation, whereas itmay still operate through the same pathway to regulate ASCdifferentiation.

The Notch Ligand Jg1 Inhibits Ig Secretion by LPS-Activated B Cells.The colocalization in the MZ of cells expressing either Jg1 orDll1 prompted us to investigate whether Notch-mediated en-

Fig. 3. Inhibition of Dll1/Notch/CSL signaling abrogates ASC differentiation.(A) Expression of Hes1 and Deltex1 was assessed by conventional RT-PCR oncultures set as in Fig. 1A. Representative of at least two independent samples.(B) CD23� splenic B cells purified from poly (I:C)-induced DNMAMLf Mx-Cre-(Cont) or DNMAMLf Mx-Cre� (DN) BM chimeras were cultured and monitoredas in Fig. 1A. (C) DNMAMLf/�splenic B cells were transduced with Tat-Cre andsorted 24 h later according to GFP expression. GFP� and GFP� cells were platedseparately in secondary cultures as in B (one experiment representative oftwo). *, P � 0.05; ***, P � 0.005.

Fig. 4. LPS-induced ASC differentiation is suppressed in the absence ofNotch1 and CSL activity. (A) Expression of Dll1 (Left) and N1 (Right) proteins inresting (�) and LPS-activated B cells isolated from WT splenocytes revealed byWestern blot. Resting B cells were purified by Percoll gradient. Protein lysatesfrom S17-vector and S17-Dll1 (Upper) or 3T3-vector and 3T3-N1�E (Lower)served as controls. The white lines indicate that intervening lanes have beenspliced out. The additional frame in Upper shows a lighter exposure of theS17-Dll1 extract for Dll1 detection. (B and C) CD23� splenic B cells werepurified from poly(I:C)-induced Mx-Cre � DNMAMLf/� (DN) and Mx-Cre �Notch1f/f (N1KO) mice and from the respective control mice (Cont). B cells werecultured and monitored as in Fig. 1A (one experiment representative ofthree).

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hancement of plasma cell differentiation is ligand-specific. Sur-prisingly, S17-Jg1 cells inhibited both IgM (Fig. 7A) and IgG (notshown) secretion upon LPS activation. The effect seemed mar-ginal but reproducible (1.5- to 2-fold in seven independent

experiments). Similar inhibition was obtained when using coatedFc-Jg1 fusion protein (Fig. 7A). A previous report indicated thatvariation in ligand density could determine the outcome ofNotch signaling during early hematopoiesis (4). To test whether,in our system, variation in ligand density could reproduce thedifferential effect of Jg1 and Dll1, we sort-purified S17-Jg1 andS17-Dll1 according to their GFP fluorescence and used each cellsubset as feeders in cultures of resting B cells stimulated withLPS (Fig. 7C). The numbers of recovered B cells upon LPScultures were similar in all conditions (not shown). In culturesseeded with S17-Dll1, the higher the GFP content in the stromalcells, the higher the frequency of IgM-secreting B cells. In sharpcontrast, in cultures seeded with S17-Jg1, the higher the GFPcontent in the stromal cells, the lower the frequency of IgM-secreting B cells. These results demonstrate that Notch-mediatedregulation of Ig secretion upon LPS activation is ligand-specific,and that ligand titration associates with a quantitative and not aqualitative alteration of the response.

DiscussionIn this work, we reveal that Notch signaling plays an essential rolein mature B cell terminal differentiation to Ig-secreting plasmablast. Our study demonstrates that the Notch ligand Dll1 pro-motes ASC differentiation by a mechanism requiring Notch1 andCSL. Analysis of loss-of-function mutants for either CSL (8),Notch2 (9), or Delta-1 (10) has previously focused on addressingMZB cell differentiation. The first study in addition tested andfailed to evidence a clear role for CSL in either T-dependent orindependent immune responses. Whether the normal Ab re-sponses reported in these MZB-less animals relate to additionalmodifications of B cell physiology associated with altered splenicarchitecture or compensatory mechanisms remains to be eluci-dated. In addition, in CSL-deficient cells but not in DNMAML-expressing cells, the potential repressor activity of CSL (dis-cussed in ref. 7) would be absent and the expression of someNotch target genes derepressed, independently of Notch

Fig. 5. Differential localization of Dll1 and Jg1 in the spleen. Serial sectionsof spleen prepared from nonimmunized C57BL/6 mice were submitted toimmunostainings. (A) PNA staining alone revealed primary follicles (pFO) andgerminal centers (GC), whereas anti-Dll1 was combined with FDCM2 Ab onimmediate adjacent sections. (B) Representative costainings for Dll1 and Jg1.Solid and open arrows indicate FO and MZ areas, respectively. (Originalmagnification, �200.) The additional frames show higher magnification(�800) of the corresponding boxed areas.

Fig. 6. Dll1 increases the frequency of B cells differentiating to ASC inresponse to T-dependent stimulus. Splenic resting B cells were cultured onS17-Dll1 (Dll1) or S17-Vector (Vect) stromal cells and supplemented with either20 �g/ml or the indicated concentrations of anti-CD40 mAb. Incorporation of[3H]thymidine was measured at day 3 (A), and IgM- and IgG-secreting cellfrequency was determined by Spot-ELISA at day 4 (B). Each assay has beenperformed at least twice in triplicate on independent cell samples, and similarresults were obtained. *, P � 0.05; **, P � 0.01.

Fig. 7. Quantitatively but not qualitatively dose-dependent effect of Dll1and Jg1 on ASC differentiation. (A) Resting B cells were cultured for 4 days inthe presence of LPS in plates coated at 10 �g/ml with Fc-Dll1ext-IgG (Fc-Dll1) orFc-Jg1ext-IgG (Fc-Jg) or the Fc portion of human IgG (Fc). (B) S17 cells transducedwith Dll1 or Jd1 constructs were sort-purified according to GFP intensity. Fullgray histogram, before sorting; open histograms, GFP distribution of thesorted populations. (C) Splenic resting B cells were cocultured on sortedS17-Dll1 (Left) or S17-Jg1 (Right) stromal cells and supplemented with 10�g/ml LPS. (A and C) The numbers of IgM-secreting cells were determined atday 4 as in Fig. 1. Each assay has been performed twice on independent cellsamples and yields similar results. *, P � 0.05; **, P � 0.01.

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receptor–ligand interactions. During the preparation of thepresent manuscript, a report was made available indicating thatNotch activity through CSL synergizes with a B cell antigenreceptor and CD40 signals responsible for B cell proliferationand production of IgG1 (23). This study provides further supportto the idea that previous CSL loss-of-function mutants did notallow direct assessment of Notch function during B cell activa-tion. Finally, we document discrete expression of Dll1 in MZ andFDC areas of the spleen consistent with a role for this ligand inthe local regulation of activated B cells during immune re-sponses. The extension of our ex vivo study to physiologicalconditions and its articulation with the role of Notch in MZBdifferentiation will await the analysis of chimeric animals pre-senting normal MZB cells and specific mutation in the Notchpathway restricted to FOB cells.

We further demonstrate that Notch signaling is more than amodulator of activated B cells, because it is required for ASCdifferentiation. Thus, both Notch1-deficient and DNMAML-expressing B cells seeded in the absence of the exogenous ligandrespond to LPS by vigorous proliferation but poor differentia-tion to Ig-secreting cells. These findings support the idea that Bcell differentiation to Ig-secreting plasmablast upon LPS acti-vation is not a default pathway. Transcriptional repressors andactivators are regulated late during the B cell activation processthat leads to Ig secretion (24). Which of these master genes areturned on or off upon Notch signal remains to be functionallyassessed, because many display several CSL prototypical bindingsites in their regulatory regions (unpublished data). Cell auton-omous constitutive activation of the Notch pathway has neverbeen reported. We therefore propose that B cells present to eachother a Notch ligand allowing lateral Notch signaling inductionand progression to terminal differentiation. This proposition isfurther supported by evidence that B cells express constitutivelydetectable levels of Delta1 and, only upon activation, high levelsof Notch1 (Fig. 4). Whether this process of B cell lateralinduction is restricted to culture conditions or also participatesin B cell homeostasis in vivo remains to be addressed.

Our study reveals that Notch1 is involved in the regulation ofB cell differentiation, namely terminal differentiation to Ig-secreting cells. Previous work assessing the role of Notch sig-naling in immature B cell commitment to a MZ B cell stageclearly evidenced a role for Notch2 (9). Notch2 appears consti-tutively expressed by cells of the B lineage (9), whereas Notch1expression is inducible upon activation (Fig. 4). The differentialexpression of Notch receptor at a particular stage of B celldifferentiation/activation must allow the use of common envi-ronmental clues (Notch ligand) for stage-specific regulation. Arecent study revealed that Dll1 modulates the proliferativeresponses of B cells upon BCR and CD40 triggering, through theregulation of the MAPK signaling pathway (23). We hereconfirmed that Dll1 enhances B cell expansion that follows CD40ligation and reveal that its effect on plasma cell differentiationis independent of its role in B cell proliferation and mostprobably of the MAPK pathway. It is conceivable that cell cycleregulation in B cells is under Notch2 control, whereas laterregulation of genes involved in plasma cell differentiation isunder Notch1 control.

In contrast to the B cell lineage, Notch1 had already beenimplicated in T cell differentiation to the effector stage. Thus,both Notch1 and Notch3 are expressed in peripheral CD4 T cells,and several conflicting studies report that the Notch signalaffects, either positively or negatively, T cell differentiation toTh1 and/or Th2 (reviewed in ref. 25). Nevertheless, the geneticprogram that leads an activated B cell to differentiate into aplasma cell in which most metabolic resources serve the pro-duction of secreted Ig probably has little equivalent in T celldifferentiation to helper cells. Moreover, our findings indicatethat Notch1 signal interferes with the pathway that limits plasma

cell differentiation. The genes involved in the progression ofactivated cells to Ig secretion are unlikely to be essential for Tcell differentiation.

Our comparison of Dll1 with another Notch ligand, Jg1,revealed opposite effects on B cell terminal differentiation. Weinterpret these results as indicating that Jg1 does not induce theN1 signal required for ASC differentiation and competes withDll1 for N1 binding, hence preventing B-B lateral induction ofNotch signaling. Inhibitory binding of Jg1 to N1 has beenreported (26) and described to result from interactions of higheraffinity than those involved in the activation signal ensued bybinding of Dll1 to N1. We show expression of both Jg1 and Dll1in the MZ area, suggesting that competition may take place insitu. Moreover, because Jg1 is essential for discriminating Notchsignals for tissue boundary formation and patterning duringearly embryogenesis (1), it may also participate in structuring thesplenic B cell zone.

In conclusion, our findings that Dll1–Notch1 interactionsrelease the inhibition of Ig secretion from LPS- and naturallyactivated B cells through canonical Notch signaling opens ave-nues for the positioning of Notch receptors, Notch ligands, andNotch signaling modulators in effector B cell regulation. In turn,it is expected that manipulation of these molecular componentswill help design alternative therapeutic strategies for the man-agement of autoimmune diseases, multiple myeloma, and poorhumoral protective immune responses.

Materials and MethodsMice. C57BL/6 (B6) mice were originally obtained from TheJackson Laboratory (Bar Harbor, ME). DNMAMLf (20) andNotch1f (22) mice have been described. All mice were bredunder specific pathogen-free conditions at the Instituto Gulben-kian de Ciencia facility, the University of Pennsylvania, and theSwiss Institute for Experimental Cancer Research, respectively,and used at 8–12 weeks of age. Mx-cre transgenic mice (27) wereoriginally obtained from Taconic Farms (Gaithersburg, MD).Mouse experiments have been approved by the respective insti-tute ethical committee and national authorities.

Induction of Cre-Mediated Floxed DNA Deletion. DNMAMLf andNotch1f mice received polyIC (Sigma, St. Louis, MO) as de-scribed (27). Deletion was confirmed by GFP detection in bloodGr-1�CD11b� cells (routinely �98%) and PCR on purified Bcells, respectively. For DNMAML mice, after induction, 5 � 105

to 106 bone marrow cells were transplanted into lethally irradi-ated (900 rads) B6 mice, used at least 12 weeks after transplan-tation. In other experiments, DNMAML expression was inducedin vitro by transduction of purified TAT-Cre (28) to CD19�

DNMAMLf splenic B cells stimulated for 24 h with LPS.

Antibodies, Flow Cytometry, and Cell Purification. APC-conjugatedanti-CD19 (1D3), phycoerythrin (PE)-conjugated anti-IgM(AFG-78), biotinylated anti-Mac-1 (M1/70), CD138 (Syndecan),CD19 (1D3), and CD23 (B3B4) were all purchased from BDBiosciences (Franklin Lakes, NJ). Biotinylated mAbs were re-vealed with Streptavidin-PE. Flow cytometry analysis was per-formed by using FACSCalibur and CellQuest (Becton Dickin-son, Franklin Lakes, NJ) on gated lymph lymphocytes. Splenicresting and naturally activated B cells were separated by Percollgradients as described (29), a preparation that yielded onaverage 98% CD19�CD23� FO cells, and 80% CD21�.MZ Bcells, respectively. B1 cells were purified from peritoneal lavagesaccording to their CD19�IgM�Mac1high phenotype on a MoFlosorter (Cytomation, Fort Collins, CO). Splenic FOB cells wereprepared by magnetic purification on LS� columns (MiltenyiBiotec, Auburn, CA) by using biotinylated anti-CD23 mAb andstreptavidin microbeads. Purity was routinely �97% in allexperiments.

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Cell Culture Conditions and Reagents. Dll1, Jg1, and control retro-virus, their production, and the transduction of S17 cells havebeen described (2). Dll1 and Jg1 expression was confirmed byimmunostaining and inhibition of myoblast differentiation (2)(data not shown). LPS and anti-CD40 induced proliferation andplasma cell differentiation of B cells cultured on a monolayer ofS17 cells, as well as BCR ligation and anti-CD40 stimulationassays, have been described (29). LPS from Salmonella typhi-murium was purchased from Sigma; anti-�b (MB86) and anti-CD40 (FGK) mAbs were produced in-house. Culture superna-tant from the transfected 3T3-IL4 cell line was used at a dilution1/100. TGF-� was purchased from PreProTech (Rocky Hill, NJ)and used at 10 ng/ml. Delta1ext-IgG (11) was a gift from I.Bernstein (Fred Hutchinson Research Center, Seattle, WA),Jaggedext-IgG was purchased from R&D Systems (Minneapolis,MN), and reagent grade purified human Fc-IgG1 was fromSigma-Aldrich. Proteins (10 �g/ml) were immobilized as de-scribed (11). For proliferation assays, [3H]thymidine (ICN,Aurora, OH; sp. Act. 5 Ci/mol) was added for the last 6 h of 3days of culture.

Elisa and Spot-ELISA. IgM- and IgG-secreting cells were revealedby Spot-ELISA (29), and the number of spots was expressed aspercentage of the recovered B cells in the respective cultures.IgM, IgG, IgA, and IgE concentration in culture supernatantswas defined by ELISA.

PCR. RNA was extracted with TRIzol and reverse-transcribed byusing SuperScript II RT and oligo(dT)12–18 primer (Life Tech-nologies, Gaithersburg, MD). Primer sequences are listed in SITable 1. For standard PCR, the reaction was run for 30 cycles ina PTC-100 cycler (MJ Research, Waltham, MA).

Immunoblot Analyses. Immunoblotting was performed as de-scribed (30). Gel loading was normalized to protein concentra-tion and confirmed by �-actin probing. Antibodies specific forDelta-1 (C-20) and �-actin (I-19) were purchased from SantaCruz Biotechnology (Santa Cruz, CA). Intracytoplasmic Notch1

antibody was kindly provided by J. C. Aster (Harvard MedicalSchool, Boston, MA). Membrane-tethered Notch1 �E trans-duced 3T3 cells were used as control (30).

Immunohistology. Tissues were embedded in Tissue-Tek OCT(Sakura Finetek, Zoeterwoude, The Netherlands), and snap-frozen 8-�m-thick sections [Leica (Wetzlan, Germany) CM1850] were collected onto 0.01% poly-L-lysine (Sigma–Aldrich)-coated slides, fixed in cold acetone, and blocked in PBS 3% BSA(Calbiochem, Darmstadt, Germany). Biotinylated anti-mouseFDCM2 (Immunokontact, Abingdon, U.K.) or PNA (VectorLaboratories, Peterborough, U.K.), goat anti-mouse Dll1, orrabbit anti-mouse Jg1 (both from Santa Cruz Biotechnology)were revealed by Avidin-D-Rhodamin (Vector Laboratories);anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA);anti-goat Alexa-488 and/or anti-goat Alexa-594 (both fromMolecular Probes, Eugene, OR). Sections were mounted inFluoromount-G (Southern Biotechnology, Birmingham, AL).Digital images were captured by using a Photometrics CoolSnapHQ camera and Metamorph software from a fluorescencemicroscope (Leica DMRA2). Postacquisition analysis usedImage-J software.

Statistical Analysis. Statistical analysis was performed by using theunpaired Student’s t test.

We thank Andreia Gomes for help with the real-time PCR, Rosa MariaSantos for antibody production, and Alexis Perez for cell sorting. Wethank Irwin Bernstein for the mouse FC-Dll1 fusion protein and JanAndersson, Antonio Coutinho, Jose Faro, and Thiago Lopes-Carvalhofor critical evaluation of this work. This work was supported by theFundacao para a Ciencia e Tecnologia, Portugal, with the coparticipationof Fundo Europeu de Desenvovimento Regional [Grants POCTI/MGI/44111/02 and BCI/37953/01 and fellowships SFRH/BD/6222/2001,SFRH/BPD/14978/2005, and SFRH/BD/2733/2000 (to M.A.S., L.M.S.,and M.R. respectively)]. M.A.S. received a European Federation ofImmunology Societies short-term fellowship. I.M. is supported by theDamon Runyon Cancer Research Foundation (DRG-102-05), W.S.P. bythe National Institutes of Health and a Leukemia and Lymphoma SocietySCOR Award, and F.R by the Swiss National Science Foundation.

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