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Development of the diverse repertoire of B lymphocytes, which useimmunoglobulin molecules expressed on their surfaces as antigenreceptors, occurs in a stepwise way at discrete stages of B cell devel-opment1. First immunoglobulin heavy (H) chains, then light (L)chains, are made. Precursor B cells, called ‘preB-I’ cells, with two H-chain alleles with rearranged diversity and joining heavy chainregions (DHJH) serve as precursors in which variable heavy chain(VH)-to-DHJH rearrangements are induced. The product of an in-frame VHDHJH-rearranged H-chain allele, the µ chain, is assessed forits fitness to associate with a surrogate light chain (SLC) and to forma preBCR on the cell surface2. Once formed and deposited on thesurface, the ‘fit’ preBCRs signal the preB-II cells to proliferate and atthe same time to turn off the expression of the SLC. Therefore, therepertoire of well fit µ chains is expanded, whereas those that fit lessor not at all are not favored or are eliminated. The proliferating cellscome to rest when they have used up the previously synthesized SLCsfor preBCR formation. When preB-II cells exit the cell cycle andbecome resting cells, VL-to-JL rearrangements at the L-chain geneloci are induced, and L chains are made that combine with µ chainsto form antigen-specific BCRs on immature B cells. Hence, preBCRsare essential in the development of the antigen-recognizing reper-toire of BCRs.

The SLC is composed of VpreB and λ5 proteins that pair with the µchain2. The assembly of the preBCR takes place in several steps and atdefined stages of the development of B lymphoid cells. It begins withthe coordinated synthesis and assembly of the SLC, from either of twoVpreb genes and one λ5 gene in early lymphoid progenitors1,3,4.Although the three-dimensional structures of VpreB and λ5 have notyet been determined, their amino acid sequence homologies haveallowed predictions of their structures. VpreB seems to have a V

region–like structure; however, it lacks the last β-strand (β7) of a typ-ical V domain5,6. Instead it has a C-terminal domain with nosequence homologies to any known protein. In this nonIg portion ofVpreB, twelve of the charged amino acids (arginine, lysine, asparticacid and glutamic acid residues) are evolutionarily conserved7. Theλ5 protein has high structural homologies with the λ-light-chainconstant region at its C-terminus (Cλ5), where it can be bound to thefirst constant region domain of µ chains by a disulfide bond. The J-region sequence of the λ light chain also has a region with highhomology to the λ5 protein (Jλ5), which seems to have the structureof a β-strand of a V-region domain. This β-strand of the Jλ5-encodedpart of λ5 seems to complement the missing β7-strand on the VpreBmolecule, allowing noncovalent, strong interactions between λ5 andVpreB8. The nonIg portion of λ5 also contains evolutionarily con-served charged amino acids, in this case seven arginines, as well as aconserved cysteine residue at position 49. Thus it is possible that thecharged amino acid residues in the nonIg portions of VpreB and λ5could interact with each other.

Deposition of the preBCR on the surface initiates signaling thatresults in the down-regulation of SLC expression. This down-regulationis abolished in preB cells deficient in the adapter protein BLNK, alsoknown as SLP65 or BASH, indicating that this adapter protein is anessential part of the preBCR signaling complex1,9,10. Proliferation ofthe preB-II cells1,11,12 is abolished in mice deficient in the µ trans-membrane sequence13, λ5 (ref. 14) or double-deficient in VpreB1 andVpreB2 (ref. 15) and in Ig-α∆C Ig-β∆C mice16. Hence, SLC expres-sion is mandatory for the proliferative expansion of preB-II cells with‘fit’ µ chains.

Proliferation can occur from a single wild-type cell suspended intissue culture in the apparent absence of external ligands for the

1National Institute of Infectious Diseases, Department of Immunology, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. 2Biozentrum, University of Basel,Department of Cell Biology, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Correspondence should be addressed to K.O. (ohnishik@nih.go.jp).

Published online 3 August 2003; doi:10.1038/ni959

The nonimmunoglobulin portion of λ5 mediates cell-autonomous pre-B cell receptor signalingKazuo Ohnishi1 & Fritz Melchers2

The pre-B cell receptor (preBCR), composed of µ immunoglobulin (Ig) and surrogate light chains, signals large ‘preB-II’ cells to proliferate in the apparent absence of ligands or cooperating cells. We deleted the N-terminal, nonimmunoglobulin (nonlg) portion of λ5, or mutated seven arginine residues in it to serine residues. PreBCRs with such mutant λ5 proteins showedincreased cell surface representation and a diminished rate of aggregation and internalization. Tyrosine phosphorylation ofpreBCR complexes containing mutant λ5 proteins was abolished. These results indicate that the nonIg portion of λ5, and theseven arginine residues in it, are needed for signal transduction, and that signaling could be cell autonomous. We propose twomodels to explain the apparently constitutive, ligand-independent signal-transducing capacity of the preBCR.

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preBCR17. The lack of influence of µ-, λ5- or VpreB-specific mAbs orantibodies to self-antigens in fetal liver organ culture of preB-IIcells18, and the dysfunction of VH-deleted µ chains as partners of theSLC in preBCRs19, have all countered the possibility of ligands forpreBCRs with signaling capabilities. However, some experimentshave indicated that ligands for the preBCR may exist and may beexpressed on bone marrow stromal cells in the environment wherepreB cells develop20.

Several observations have indicated that cross-linking for signaltransduction is also mandatory for preBCRs on preB-II cells.PreBCRs on large preB-II cells are apparently down-regulated in aspontaneous and constitutive manner21. Tyrosine phosphorylation ofpreBCR complex–associated proteins in these preB-II cells alsooccurs constitutively22. Spontaneous aggregation of preBCRs, per-haps even inside the cells, has been noted23. However, it has remainedunclear what cross-links preBCRs.

In naturally occurring and experimentally constructed19,24 µ chaindiseases, the VH domain has been deleted on the expressed IgH allele.Such truncated µ chains still form preBCRs and BCRs. As B cell devel-opment remains normal with such mutant receptors, it indicates thatsignaling for proliferation and differentiation of precursor B as well asimmature and mature B cells can be mediated by mutant forms ofpreBCRs. This phenomenon has been explained by the apparent sticki-ness of such VH-depleted µ chains, which induces multimer formationand, hence, self cross-linking on cell membranes. Here, we test thefunction of the nonIg portion of λ5 in the surface representation,aggregation and internalization of the preBCR, and its function in sig-nal transduction of the preBCR.

RESULTSGeneration of mutant λ5-expressing preB cell linesTo determine the influence of the nonIg portion on the surfacedeposition, rate of internalization and tyrosine phosphorylation ofthe preBCR complex in these preB cells, we first establishedAbelson virus–transformed, µ chain-expressing preB cell lines ofλ5-deficient (λ5–/–) mice. We selected one of these cell lines basedon the capacity of its µ chain to pair with the wild-type form of λ5and with VpreB to form a preBCR deposited on the cell surface. Wethen tested the effect of preBCRs reconstituted with wild-type λ5or with five structural variants of λ5. We generated these variantsby deletion of and site-specific mutations within the nonIgsequences of λ5 (Fig. 1). In the first variant, we deleted the totalsequence of the nonIg portion, leaving a gene encoding only the Jλ5and Cλ5 parts (JC mutant). In the second variant, we mutated theconsensus cysteine at position 49 (Cys49, shared with rat andhuman λ5; Fig. 1b) to serine (CS mutant). In the third variant, wemutated one consensus arginine at position 48 and one arginine atposition 53, as well as Cys49, to serine residues (CS_2R2S mutant).In the fourth variant, we mutated six consensus arginine residues atpositions 12, 14, 26, 35, 43 and 48, and one at position 53, to serineresidues (7R7S mutant). In the fifth variant, we mutated the sevenarginine residues as well as Cys49 to serine residues (CS_7R7Smutant). We cloned all the variant forms of λ5, as well as the wild-type form, into a retroviral vector that carries the gene encodinggreen fluorescent protein (GFP) as a marker for expression intransfected cells. As a control, we cloned Igk from SP6 hybridomainto the same vector.

850 VOLUME 4 NUMBER 9 SEPTEMBER 2003 NATURE IMMUNOLOGY

Figure 1 Structures of wild-type and mutantforms of λ5. An array of arginine residues isevolutionarily conserved in the nonIg domain ofλ5. (a) Comparison of the domain structures ofconventional light chain and λ5. L, leadersequence; V, variable domain; C, constantdomain; J, joining region; CDR, complementarity-determining region. The cysteine (c) residuesinvolved in intra- and intermolecular disulfidebonds are shown. The λ5 protein has a nonIgdomain (green box) at its N terminus. (b) Alignment of the nonIg domains of λ5 frommouse, rat and human. Arginine (R) and cysteine(C) residues are marked with red and yellow,respectively. The numbers on the arginine andcysteine residues are for mouse λ5. Upper-caseletters indicate consensus amino acids.

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Figure 2 Surface expression of preBCR in Abelsonvirus–transformed λ5–/–pre-B cells. A λ5–/– pre-Bcell clone was infected with GFP-positiveretrovirus containing either wild-type (WT) ormutated λ5 or IgK genes. On day 2 aftertransfection, cells were stained with mAbsspecific for λ5, µ chain or κ chain and analyzedby flow cytometry. Horizontal axes, GFPexpression; vertical axes, λ5 (mAb LM34), λ5complexed with µ chain (mAb SL156), µ chain(mAb M41) and κ chain (mAb 187.1). µ-SL, µ-surrogate light chain. Experiments wererepeated at least five times and representativestaining patterns are shown.

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We then stably transfected Abelson virus–transformed preB cellsof λ5–/– mice, which express VpreB and µ chain, with retrovirusescontaining either the gene encoding GFP alone or together with thegene encoding either the wild-type, JC, CS, CS_2R2S, 7R7S orCS_7R7S forms of λ5, or κ light chain. The stable transfectants allexpressed GFP, as shown by flow cytometry (Fig. 2). We assessed sur-face deposition of preBCR molecules in λ5-deficient preB cell linesexpressing either wild-type or mutated λ5 by staining cells on thesurface with mAb LM34, which recognizes λ5 protein in free and µchain-bound forms25, mAb SL156, which recognizes λ5 protein onlywhen bound to µ chains23, µ chain-specific mAb M41, which recog-nizes the third constant domain (CH3)26 or κ chain-specific mAb187.1. Mock-transfected cells were not stained by any mAb, whereascells transfected with the wild-type form showed low surface stain-ing, especially when the µ chain-specific mAb was used (Fig. 2). TheCS mutant showed a surface representation of λ5 that was indistin-guishable from that of wild-type. In contrast, the JC, CS_2R2S,7R7S, and CS_7R7S mutant forms were expressed in increased abun-dance on the cell surface, ranging from an approximately 5-foldincrease for CS_2R2S to a 10-fold increase for JC, 7R7S andCS_7R7S. These increases were seen with all three mAbs, indicatingthat the λ5 mutant proteins had all associated with µ chains in apreBCR-like conformation as detected by the SL156 mAb. The highfluorescent intensity of κ chain-expressing cells, which expressedsurface BCRs, was similar to that of cells expressing JC, 7R7S andCS_7R7S mutant preBCRs.

We were also able to visualize this difference in surface depositionof the preBCRs by fluorescence confocal microscopy (Fig. 3). Wild-type and CS forms of the preBCR were low in abundance anddeposited in a spotty fashion, which is also true of wild-type preB-IIcells from fetal liver or bone marrow of normal mice27. In contrast,the JC and CS_7R7S mutant forms of preBCR (as well as the 7R7Sform; data not shown), and κ-chain BCR, showed a bright, rim-likefluorescence distributed evenly over the whole cell surface (Fig. 3).

We concluded from these observations that the nonIg portion ofλ5 does not interfere with the assembly of the preBCR, but insteadcontrols its surface representation. Deletion of the nonIg portion, aswell as mutation of the seven consensus arginine residues to serineresidues, allowed increased accumulation of preBCR on the pre-Bcell surface. These data indicated that these charged arginine residuesprevent a higher number of preBCRs to be deposited on the surface.Mutation only of the consensus Cys49 of the nonIg portion did notchange the surface expression of preBCR.

The difference in preBCR deposition on the cell surface couldresult from a difference in the rates with which the mutant and wild-type λ5 forms are synthesized. However, pulse labeling of the trans-fected preB cells with 35S-methionine followed by specificimmunoprecipitation of the preBCR components did not demon-strate any difference in rate of wild-type and mutant λ5 synthesis(data not shown). In addition, all cell lines expressing mutant λ5 aswell as the wild-type λ5-expressing cells produced similar amountsof intracellular endo-N-acetylglucoseaminidase H (Endo H)–sensi-tive µ chains. However, both the wild-type form and CS mutant hadreduced amounts of Endo H–resistant µ chains as compared with theJC and CS_7R7S mutants (Fig. 4a). Endo H–sensitive forms of µchains are considered to be the high-mannose, nonbranched carbo-hydrate-containing precursors of the galactose-, fucose- and neu-raminic acid-containing, Endo H–resistant forms of µ chain that aredeposited on the cell surface28. We concluded from these data thatthe kinetics of assembly and the stability of assembling receptorcomplexes with different forms of the SLC are very similar, whereasonly small (up to 20%) differences exist in the amount of EndoH–sensitive µ chains.

PreBCR internalizationA reduced amount of Endo H–resistant µ chains could be the result ofeither a decreased rate of surface deposition or an increased rate ofinternalization and degradation of these µ chains. Thus, we testedwhether newly made λ5 assembled in preBCRs could be processed dif-ferently in the λ5–/– pre-B cells, depending on the structures of thenonIg portion. We measured the rates of internalization of surface-expressed wild-type and mutant preBCRs. The wild-type and allmutant λ5 proteins formed preBCRs that were internalized at indistin-guishable rates when they were cross-linked by antibody to µ chain(Fig. 4b, right). BCRs formed on κ chain-transfected cells showedsimilar kinetics. Hence, the capacity for preBCR internalization wasnot affected by the mutations in the nonIg portion of λ5. In contrast,the ‘externally uninduced’, steady-state internalization showed signifi-cant differences (Fig. 4b, left). PreBCRs with wild-type or CS mutantλ5 were internalized at a threefold-higher rate than were preBCRscontaining the JC and CS_7R7S mutant forms or κ-chain-containingBCRs. We concluded that a slower rate of internalization of preBCRswith the JC and CS_7R7S mutant forms may explain the increasedsurface accumulation of these preBCRs. Thus, the nonIg portion ofλ5, and specifically its conserved seven arginine residues, caused arapid, constitutive internalization of the preBCR.

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Figure 3 Confocal fluorescence microscopy ofpreBCR deposition on the surface of preB cells.Cells (as in Fig. 2) were stained with Alexa 594Fab SL156 (or Alexa 594 Fab M41 in κ chain-expressing cells). Experiments were repeatedthree times and representative staining patternsare shown. WT, wild-type.

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Tyrosine phosphorylation of the preBCR complexReceptor signaling is usually mediated by ligand binding and receptorcross-linking. Such ligand might not be required for preBCR signal-ing, as it has been suggested that the preBCR signals constitutively inpre-B cells, as evidenced by the active phosphorylation pattern of pro-teins associated with the preBCR signaling complex22. To test the sta-tus of preBCR signaling in λ5–/– preB cell lines expressing eitherwild-type or mutated λ5, we isolated the surface-expressed form of thepreBCR complex and measured the extent of tyrosine phosphoryla-tion by immunoblot analysis. We found distinct differences in phos-phorylation of proteins in regions where the Igα and Igβ components

were present (Fig. 4c, left). Immunoblots with Igα-specific mAbdetected similarly high quantities of protein from wild-type cells andall the mutant transfectants, indicating that preBCRs in all cells wereassociated with similar quantities of Igα (Fig. 4c, bottom). The Igαdetected by the specific mAb (Fig. 4c, bottom) had the same mobilityas one of the phosphorylated proteins (Fig. 4c, left).

Introduction of the wild-type form as well as the CS mutant formof λ5 into the λ5-deficient preB cells increased the constitutive phos-phorylation of Igα and Igβ. Phosphorylated proteins were also visiblein the region around 50–70 kDa. In contrast, neither the JC nor theCS_7R7S mutant nor the κ chain restored this constitutive signaling

852 VOLUME 4 NUMBER 9 SEPTEMBER 2003 NATURE IMMUNOLOGY

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Figure 5 Wild-type preBCR forms large aggregates. (a) Transfectants with wild-type λ5 (WT) or deletion mutant (JC) and µκ-expressing WEHI231 cells weresurface biotinylated and separated into nuclear fractions (filled bars), cytosolic fractions (shaded bars) and membrane fractions (open bars). The biotinylatedsurface µ chains and the total µ chains in each fraction were quantified by ELISA. (b) Discontinuous sucrose density gradient centrifugation of wild-type (WT)and mutant preBCRs. The transfectants and µκ-expressing WEHI231 cells were surface biotinylated, lysed with NP-40 buffer, and separated by sucrosedensity gradient centrifugation at a low speed. Biotinylated µ chains in the fractions were quantified by ELISA. Results are representative of three separateexperiments.

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Figure 4 Comparison of Endo H sensitivity, receptor internalizationrate and tyrosine phosphorylation of wild-type and mutant preBCRs.(a) The ratios of Endo H–sensitive and Endo H–resistant µ chains inwild-type (WT) and mutant preBCRs and µκ BCR. The µ chains fromAbelson virus–transformed λ5–/– pre-B cells transfected with genesencoding the wild-type, CS, JC or CS_7R7S forms of λ5 or κ chainwere immunoprecipitated with mAb to µ, then were left untreated(left; –) or treated with Endo H (right; +Endo H) and thenimmunoblotted with horseradish peroxidase–conjugated antibody to µ.Right margin, the positions of Endo H–resistant (filled triangle) andEndo H–sensitive (open triangle) µ chains. Below, quantification(arbitrary units) of Endo H–resistant (upper) and Endo H–sensitive(lower) µ chains. (b) Internalization of surface-expressed preBCRs andµκ BCR. Transfectants were labeled with Alexa 594 Fab antibody to µ(M41), and internalization rates were determined by fluorescence-activated cell sorting–based internalization assay. Left, steady-state internalization rateswithout receptor cross-linking; right, internalization rates in cross-linked conditions (cells were incubated in the presence of 5 µg/mL F(ab′)2 rabbitpolyclonal antibody to mouse µ chain). **, P < 0.01, compared with wild-type (WT; n = 4). (c) Tyrosine phosphorylation status of preBCR-associated proteinsin the transfectants. Top blots, surface-expressed µ complex was immunoprecipitated, and tyrosine-phosphorylated proteins were detected by immunoblotusing horseradish peroxidase–labeled mAb to phosphotyrosine (4G10). Left, steady-state tyrosine phosphorylation; right, tyrosine phosphorylation in cross-linked conditions as in b. Middle blots, sample loading was normalized and comparable loading amounts were ensured by probing the same blots withhorseradish peroxidase–labeled antibody to µ. Bottom blots, immunoblots with antibody to Igα indicate equivalent amounts of Igα were coprecipitated in thelanes, and identify the Igα band (filled arrowheads in top blots). MWM, molecular weight markers (sizes, left margin). Results are representative of threeseparate experiments.

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capacity. When we tested the antibody ligand–induced, cross-linkedλ5–/– preB cells, transformed with genes encoding the wild-typeform, the CS, or JC, CS_7R7S mutants or κ light chain, for their phos-phorylation status, all cells showed a high phosphorylation patternscharacteristic of the constitutively induced forms of the preBCR. Weused a µ chain-specific antibody to cross-link preBCR molecules withwild-type or mutant λ5 on the surfaces of preB cells. We found highphosphorylation patterns characteristic of constitutively inducedforms of the preBCR in cells expressing wild-type as well as in cellsexpressing mutant λ5 and µκ BCRs (Fig. 4c, right). These datashowed that the preBCR remained inducible by external ligands, suchas µ chain-specific antibody, and was therefore signaling competenteven with the mutant forms of λ5. We concluded from these resultsthat replacement of the seven consensus arginine residues by serineresidues in the N-terminal nonIg portion of λ5, or their completedeletion, abolishes the constitutive signaling capacity of the preBCR,as shown by phosphorylation of proteins within the preBCR complex.

Aggregated forms of the preBCRIn many cases, signaling from surface membrane-bound receptors ismediated by cross-linking of these receptors. Therefore, we analyzedthe apparent molecular weights of preBCRs in the cell lines expressingpreBCRs with either wild-type or mutant forms of λ5. We labeled thesurface-expressed preBCRs with biotin, and then separated cell sampleshomogenized in hypotonic conditions into nuclear, cytosolic andmembrane fractions. We quantified the surface-expressed, biotinylatedµ chains and the total µ chains in each fraction using enzyme-linkedimmunosorbent assay (ELISA; Fig. 5a, left). We recovered surface-expressed, wild-type preBCR molecules mainly from the low-speedcentrifugation pellet with the nuclei; however, we found surface-expressed preBCRs with the deletion-mutant form of λ5 (JC) in themembrane fraction. In contrast, we recovered total µ chains mainlyfrom the membrane fraction for both cell lines (Fig. 5a, right). Theseresults indicated that the complex formed by the wild-type preBCR wasextremely large, so that it was enriched in the low-speed centrifugation

pellet fraction, which is normally discarded during immunoprecipita-tion procedures. To confirm this large aggregate formation by the wild-type preBCR, we used centrifugation at low speed with a discontinuoussucrose gradient. We found large aggregates at the 5–35% sucrose inter-face for wild-type and CS mutant samples, whereas deletion mutant(JC) and substitution mutant (CS_7R7S) samples as well as µκ-expressing WEHI231 cells did not contain such aggregated forms of µchains (Fig. 5b). We concluded from these experiments that the expres-sion of wild-type λ5 in λ5-deficient cells favors the formation of largeraggregates of preBCRs and that on preB cells expressing the JC andCS_7R7S mutant forms of λ5, decreased abundance of smaller aggre-gates formed instead.

Based on these experimental results, we propose two possible mod-els for the function of the nonIg portion of λ5 in the signaling ofpreBCRs for proliferation and down-regulation of SLC expression(Fig. 6). Both assume that the preBCR must be cross-linked to signal.Both propose that signaling is cell autonomous; that is, no externallyprovided ligands are needed to occupy the preBCR. In the first model,the charged amino acids in the nonIg portions of λ5 and VpreB inter-act with cross-linking components provided by the same preB-II cells(Fig. 6a). In the second model, oppositely charged amino acids in λ5and VpreB bind the two proteins together, and cross-link preBCRs in aself-autonomous way (Fig. 6b).

DISCUSSIONThe results presented here are consistent with the hypothesis that thepreBCR signals the pre-B cells constitutively and without apparentexogenous ligands. One result of such signaling could be the constitu-tive internalization, or continuous down-regulation, of the receptorfrom the surface. The charged arginine residues in the N-terminalnonIg portion of λ5 within the preBCR, composed of λ5 and VpreBand µ chain, were required for this constitutive internalization and sig-naling of the preBCR. Hence, once the µ chains have properly pairedwith SLC29, the resulting preBCRs do not have to engage their VHdomains with any external ligands or superantigens30.

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Figure 6 Hypothetical models for preB cellautonomous cross-linking of preBCR byelectrostatic interaction of nonlg domains of λ5.(a,b) The nonlg domain is formed by the nonlgportion of λ5, in which seven evolutionarilyconserved arginine residues are located, and bythe nonlg portion of VpreB, in which six positivecharges and six negative charges are located. Ina, preB cell–derived molecule ‘X’, which mayhave ambivalently charged regions in themolecule, interacts with the oppositely chargedregions of the nonlg domains of λ5 and VpreB tocross-link the neighboring preBCRs. In b, thecharged amino acids of the nonlg domains oftwo neighboring preBCRs directly interact witheach other to create autonomous self-cross-linking of preBCRs.

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Abelson virus–transformed λ5–/– cell lines that express anSLC–pairing µ chain have many advantages that justify their use inevaluating the function of the nonIg portion of λ5 and its protein inpreBCR formation, even though a transformed cell line is only in alimited way representative of the normal preB cell at the stage ofpreBCR expression. These transformed cells are already proliferatingand do not down-regulate SLC gene expression in response topreBCR signals. Nevertheless, the features of preBCRs with differentsurrogate light chains noted here should also be representative of pri-mary cells. All cells express a functional µ chain. This is in contrast tonormal, primary preB-II cells, which are DHJH rearranged on bothIgh alleles and proliferate for many divisions on stromal cells in thepresence of interleukin 7 (IL-7)3. Removal of IL-7 induces VH-to-DHJH rearrangements, but only approximately 40% of the differenti-ated cells are expected to express a µ chain, and only half of these (oreven as few as one in four; H. Karasuyama, personal communication)can pair with the SLC29.

Furthermore, whereas Abelson virus transformation ‘freezes’ thestatus of immunoglobulin gene rearrangement of a precursor B cell(in our case, of a productively rearranged VHDHJH allele), normalprimary differentiating cells continue their rearrangements in vitroto VLDL-rearranged, L chain–expressing cells (that is, BCR-expressing B cells)31. Hence, expression of these L chains wouldobscure analysis of different forms of SLCs in their effects on thesurface representation, internalization, aggregation and signaltransduction of the preBCR.

Finally, primary preB-II cells can be grown on stromal cells in thepresence of IL-7 for long periods of time, but they tend to accumulatenonproductively VHDHJH-rearranged Igh alleles17,32. Hence, the largenumber of cells needed for internalization, aggregation and tyrosinephosphorylation experiments are, at best, extremely difficult toobtain. In addition, whereas proliferating Abelson virus–transformedcell lines are viable in these cultures, differentiating VHDHJH—VLDL-rearranged primary preB and B cells are induced to apoptosis33, fur-ther reducing the number of cells that can be assayed.

As we added no ligands to the preB cells, and as no other types ofcells were present in our experiments, the most likely mode of preBcell stimulation is one that is autonomous for preB cells. We proposetwo models to explain such an autonomous mode of stimulation. Inthe first, the preB cell synthesizes a molecule ‘X’, which has two bind-ing sites for the nonIg portion of λ5. When µ chains combine with theSLC to form the preBCR, the nonIg portion of λ5 becomes availablefor binding to ‘X’ either because λ5 changes its conformation orbecause ‘X’ becomes available for binding. Two binding sites on ‘X’,possibly exposing negatively charged amino acids to complement thepositively charged arginine residues of the λ5 nonIg portion, allowthe cross-linking of two adjacent arms of two preBCR molecules,assuming that the preBCR, like IgM, is a dimeric structure with two µchains and two SLCs. Such cross-linking induces stimulation.

In the second model, the nonIg portion of the SLC of the preBCRcan mediate self-cross-linking through polar noncovalent interactionsof a maximum of seven positively charged, conserved arginineresidues in the nonIg portion of λ5, with a maximum of five negativelycharged, conserved glutamic acid residues at positions within thenonIg portion of the VpreB protein, allowing multimer formation ofpreBCRs in the surface membrane of preB-II cells. The nonIg domainof preBCR, which is formed by nonIg portions of 23 amino acids ofVpreB and of 62 amino acids of λ5, is expected to be positioned inspaces where in normal V regions the CDR3 region is formed. FreeSLC, or SLC in a complex with gp130-gp35-65, which is a proteincomplex on the surface of preB-I cells without an association with µ

chains25, should be in a form that does not allow self cross-linking. Asin the first model, we propose that when µ chain is expressed in preB-II cells and pairs with the SLC, the nonIg portions of the SLC changetheir conformations. We further propose that in the absence of mole-cule ‘X’, the nonIg tail of λ5 of the preBCR molecules can form a polar,noncovalent association with the nonIg portion of VpreB from theneighboring preBCR molecule, and vice versa. The second arm of thepreBCR could do the same with a second neighbor, and so on. Thismodel predicts that deletion of the nonIg portion of VpreB would havethe same effect as a similar deletion from λ5. As a VpreB1, VpreB2, λ5triple-deficient strain of mice has been generated11, this prediction cannow be tested. We also expect that preB cell development will remaindefective in λ5–/– mice carrying the JC or CS_7R7S mutated forms ofλ5 as transgenes.

METHODSCells and antibodies. The antibodies used were: LM34, a rat mAb that recog-nizes free and bound form of λ5 (ref. 25); SL156, a rat mAb that recognizes λ5only when it is bound to µ chain23; M41, a rat mAb that recognizes CH3 domainof mouse µ chain26; 187.1, a rat mAb that recognizes mouse κ-light-chain(Southern Biotechnology); and polyclonal rabbit F(ab′)2 antibody to mouseIgM (Southern Biotechnology). For making the Fab fragment of antibodiesusing immobilized papain (Pierce), biotinylation of the antibodies using sulfo-NHS-LC-biotin (21335; Pierce) and labeling of the antibodies with Alexa 594(Molecular Probes) were done according to the manufacturers’ protocols. ThepreB cell clone from a λ5–/– mouse was established by transforming λ5–/– fetalliver cells with temperature-sensitive Abelson murine leukemia virus34.

Genes, vectors and site-directed mutagenesis. Mouse λ5 and SP6 Igk cDNA (agift from T. Seidl, Institute of Cancer Research, Chester Beatty Laboratories,London, UK) was cloned into pBluescript II KS– (Stratagene). The site muta-tions were generated by the overlap extension method35 as follows: To generatethe CS mutant, PCR 1 (with 5′-ATATAGAGCTCGAGGGACAATGAAGCTCAGAGTAGGACAGACTCTG-3′, the 5′ outside primer located at upstreamand initiation region of λ5, and 5′-CCTATGGGGCGAGGACCTGGGACCTGC-3′, the 3′ CS primer) and PCR 2 (with 5′-CCTATCCGCGGGTCGACATCTAAGAACACTCAGCAGGTGACAC-3′, the 3′ outside primer located attermination and downstream region of λ5, and 5′-GCAGGTCCCAGGTCCTCGCCCCATAGG-3′, the 5′ CS primer) were done using wild-type λ5 cDNA asa template. Subsequently, PCR 3 (overlap extension) was done using the 5′ and3′ outside primers described above and the products of PCR 1 and PCR 2 astemplates. Using the SacI site (introduced by 5′ outside primer) and the HindIIIsite (introduced by the 3′ outside primer), the end product was cloned intopBluescript II KS–. Results were verified by DNA sequencing. For the CS_2R2Smutant, 5′-AGATGGAAGGCTATGGGGCGAGGAGCTGGGACCTGCTCC-3′(the 3′ CS_2R2S primer) and 5′-GGAGCAGGTCCCAGCTCCTCGCCC-CATAGCCTTCCATCT-3′ (the 5′ CS_2R2S primer) were used. For the 7R7Smutant, two rounds of overlap extension PCR were done using the followingprimer sets: For round 1, 5′-GGAAGCAACAGTCCTAGCCTATGGGCCCTTCCCGGCAGTCTCCTGTTCCAGATCATCCCATCGGGAGCAGGT-3′ (the 5′3R primer) and 5′-ACCTGCTCCCGATGGGATGATCTGGAACAGGAGACTGCCGGGAAGGGCCCATAGGCTAGGACTGTTGCTTCC-3′ (the 3′ 3Rprimer); and for round 2, 5′-AGCTCAGCATCAAGTAGCAGTGCTGTGGGC-3′ (the 5′ ERSR primer) and 5′-GCCCACAGCACTGCTACTTGATGCTGAGCT-3′ (the 3′ ERSR primer). For the CS_7R7S mutant, the same PCR as forCS was done, using 7R7S DNA as a template. The PCR products were clonedinto pCR2.1-TOPO (Invitrogen). To delete the N-terminal nonIg region of λ5in the JC mutant, the pELVC vector was used to introduce the leader sequenceof SP6 IgH as described before29. The PCR product of a reaction using 5′-GAT-CATGTCGACTTTGGTGGTGGGACCCAGCTC-3′ (the 5′ JC primer) and 5′-TGAGTGCGGCCGCGAGTAAGCTTCAGGGAGGACTTACCTCTAA-3′ (the3′-JC primer) was cloned into the SalI-NotI site of the pELVC vector. Thismanipulation gave rise the three N-terminal amino acids, QVD, in place oforiginal sequence, WYV, after splicing and translation. The EcoRI-NotI frag-ment, encoding the leader and JC region, was cloned into pCR2.1-TOPO.

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Retrovirus-mediated transfection. The retroviral vector pBIG was constructedfrom Moloney retroviral vector pBMN-Z (a gift from G.P. Nolan, Departmentof Microbiology and Immunology, Stanford University, Stanford, California).The EcoRI-NotI (blunted) fragment, including a multicloning site and a geneencoding enhanced green fluorescent protein (EGFP), was excised frompIRES2-EGFP (Clontech) and inserted into the EcoRI-SalI (blunted) site ofpBMN-Z in place of lacZ. The wild-type and mutated λ5 genes were insertedinto the vector using the SacI (blunted)-SalI site (SacI (blunted)-SacII for the JCmutant). Virus production in the phoenix packaging cell line and the infectionswere done according to a protocol developed by G.P. Nolan and colleagues(Nolan, Department of Microbiology and Immunology, Stanford University,Stanford, California; www.stanford.edu/group/nolan/retroviral_systems).

Flow cytometry and confocal microscopy. For flow cytometry, cells werestained with biotinylated first-step antibodies and second-step phycoerythrin-streptavidin, and were analyzed with a FACSCaliburTM flow cytometer (BDBiosciences). For the laser confocal scanning microscopy, cells were stainedwith an Alexa 594–labeled Fab fragment of SL156 mAb and analyzed with alaser-scanning microsope (LSM510; Zeiss).

Internalization assays. The fluorescence-activated cell sorting–based receptorinternalization assay was done according to a published procedure36. Forsteady-state internalization assessment, cells (5 × 106) were stained on ice for 30 min with Alexa 594–labeled Fab SL156 mAb. After being washed, the cellsuspension was incubated at 37 °C. At each time point, an aliquot of 1 × 106

cells was treated with five volumes of ice-cold acid buffer (PBS and HCl, pH 2.0) for 45 s, neutralized by the addition of 0.2 volumes of 1 M HEPES, pH7.6, and fixed by the addition of 0.5 volumes of 4% paraformaldehyde. Themean fluorescent intensities of Alexa 594 remaining in GFP-positive cells weremeasured with a FACSVantageTM SE flow cytometer (BD Biosciences). Theinternalization rate was calculated by the following equation: Internalized (%)= (At – A0)/(T0 – A0) × 100, where T0 is total bound Alexa 594 on the surface attime 0, A0 is acid-resistant Alexa 594 at time 0 and At is acid-resistant Alexa 594at the time of sampling. For measurement of the internalization rate in receptorcross-linked conditions, polyclonal rabbit F(ab′)2 antibody to µ was added attime 0 at a final concentration of 5 µg/ml.

Immunoprecipitation and immunoblot detection of tyrosine phosphoryla-tion. For collection of the surface-expressed preBCR complexes, cells (5 × 107)were incubated for 30 min on ice with Fab-SL156 (2 µg), washed twice with ice-cold PBS and lysed with digitonin (1%) lysis buffer, as described before37. Aftercentrifugation at 1,500 r.p.m. (F241.5P rotor; Beckman Coulter, Inc.) for 5 min,4 µg antibody to mouse κ (mAb MAR18.5) was added with 20 µl of a gel of pro-tein A–Sepharose 4B to precipitate the surface preBCR complex. After beingwashed, the beads were treated with SDS sample buffer and separated by elec-trophoresis by the Laemmli system, blotted onto a nitrocellulose membrane,and probed with horseradish peroxidase–labeled antibody to phosphotyrosine(4G10) or polyclonal rabbit F(ab′)2 antibody to µ using a chemiluminescentsubstrate (Pierce).

Surface biotinylation, subcellular fractionation and sucrose density gradientcentrifugation. Cells (5 × 107) were washed three times with ice-cold PBS con-taining 0.01% NaN3 (PBS-NaN3), and cell surface molecules were biotinylatedfor 10 min on ice with 1 ml of 0.5 mg/ml sulfo-NHS-LC-biotin dissolved inPBS-NaN3. After this incubation, cells were washed and incubated with NHS-blocking solution (50 mM Tris, pH 7.5, 50 mM glycine and 150 nM NaCl) toremove unreacted sulfo-NHS-LC-biotin. Subcellular fractionation was accom-plished using a published procedure38. Surface-biotinylated cells were resus-pended in a hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2, protease inhibitors, 1 mM NaVO4 and 1 mM NaF) and incu-bated for 10 min on ice. After the addition of 0.25 volumes of a solution of 10mM Tris, pH 7.5, 0.5 mM MgCl2 and 0.6 M NaCl, cells were homogenized by 30strokes with a Dounce homogenizer and were centrifuged at 500g for 5 min.The resultant pellet was designated the nuclear fraction. Next, 0.1 volumes of0.5 M EDTA was added to the supernatant, which was then centrifuged at100,000g for 45 min. The supernatant resulting from this second centrifugationwas designated the cytosolic fraction; the pellet was designated the membranefraction. NP-40 and EDTA were added to the nuclear, cytosolic and membrane

fractions, to final concentrations of 1% and 5 mM, respectively, and volumeswere made equal. Then, samples were sonicated at 0 °C for 10 s at a power of 3.5with a W-225R ultrasonic disruptor (Hear System/Ultrasonics) equipped witha number 419 microtip. To quantify the biotinylated and total µ chain in eachfraction, ELISA plates coated with monoclonal antibody to µ (M41) were incu-bated with serial titrations of each subcellular fraction and results were detectedwith alkaline phosphatase–conjugated streptavidin (for surface µ chain) andalkaline phosphatase–conjugated antibody to mouse IgM (for total µ chain).The recovery of µ chains in each fraction was calculated as follows: µ-chainsrecovered (%) = (absorbance of the specific fraction)/Σ(absorbance of eachfraction) × 100.

For discontinuous sucrose density gradient centrifugation, surface-biotiny-lated cells (2 × 107) were lysed with NP-40 lysis buffer, passed through stainlessmesh to remove ‘fluffy materials’, and then layered on discontinuous layers of60% (1 ml), 35% (1 ml) and 5% (2 ml) sucrose in NP-40 lysis buffer in aBeckman SW50.1 swinging rotor tube. After a 3-min spin at 3,000 r.p.m.(SW50.1 rotor; no brake setting; the rotor stopped after about 10 min), aliquotsof 0.4 ml were taken from the tops and biotinylated µ chains were analyzed byELISA as described above.

ACKNOWLEDGMENTSWe thank H. Karasuyama for the SL156 and LM34 antibodies; and T. Takemoriand K. Karjalainen for advice and discussions. This work was supported in part bygrants to F.M. from the Swiss National Funds (No-3100-066682.01/1) and to K.O.from the Japan Society for the Promotion of Science (KAKENHI 14570290).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 15 May; accepted 8 July 2003Published online at http://www.nature.com/natureimmunology

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