VpreB1/VpreB2/λ5 Triple-Deficient Mice Show Impaired Development but Functional Allelic Exclusion of the IgH Locus

This information is current as Takeyuki Shimizu, Cornelia Mundt, Steve Licence, Fritz of September 27, 2021. Melchers and Inga-Lill Mårtensson J Immunol 2002; 168:6286-6293; ; doi: 10.4049/jimmunol.168.12.6286 http://www.jimmunol.org/content/168/12/6286 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

VpreB1/VpreB2/␭5 Triple-Deficient Mice Show Impaired B Cell Development but Functional Allelic Exclusion of the IgH Locus1

Takeyuki Shimizu,2* Cornelia Mundt,† Steve Licence,† Fritz Melchers,3* and Inga-Lill Mårtensson†

At the precursor B cell stage during bone marrow B cell development, Ig ␮H chain associates with surrogate L (SL) chain, which is encoded by the three VpreB1, VpreB2, and ␭5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or ␭5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either ␭5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either ␭5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically Downloaded from excluded and thus the SL chain appears not to be involved in allelic exclusion. The Journal of Immunology, 2002, 168: 6286Ð6293.

evelopment of B lymphocytes from early progenitors is Surface deposition of the pre-BCR allows the cell to be stimu- ordered by the stepwise rearrangements of the V(D) and lated to between two and five rounds of cell division (17). Such J segments of the IgH and L chain loci (1–4). In proliferating large pre-BII cells thereby expand the number of cells

D http://www.jimmunol.org/ mice and humans, the IgH locus is rearranged before the ␬ and ␭ which produce SL chain-pairing ␮H chains. All other cells, either

L chain loci (5, 6). It has been proposed that the product of a nonproductively VDJH rearranged on both IgH alleles or produc- successful rearrangement at one IgH allele signals the developing ing only nonpairing ␮H chains, do not expand, and may even be B cell to turn off rearrangements at the other allele of the same induced to apoptose due to their lack of surface expression of the locus, thereby ensuring that one B cell produces only one type of pre-BCR (18). A quantification by single-cell PCR analysis of the ␮ IgH (7–9). Precursor (pre-) B lymphocytes first rearrange DH to JH two H chains in VDJH-rearranged cells in the pre-BII and mature segments on both IgH alleles before they enter VH to DJH rear- B cell compartments and the repertoire suppression of the SL ␮ ␮ rangements (3, 10). If the H chain resulting from a productive VH chain-nonpairing VH domains of H chains produced from the 4 to DJH rearrangement can pair with the surrogate L (SL) chain, a productively rearranged alleles (and not seen in the nonproduc- pre-B cell receptor (pre-BCR) is formed, in combination with SL tively rearranged alleles) support such a scenario (16, 19). by guest on September 27, 2021 chain, on the cell surface. The SL chain is formed by the nonco- Experimentally induced, as well as naturally occurring, muta- valent association of the VpreB and ␭5 , and is assumed to tions in the genes encoding the SL chain support the view that the form a ␭ L chain-like structure capable of disulfide-bound associ- pre-BCR signals proliferative expansion of pre-BII cells. Targeted ation with a ␮H chain (11–15). Approximately half of all produc- disruption of the ␭5 gene in mice as well as a naturally occurring tively rearranged IgH loci have been found to produce ␮H chains deletional mutation in humans both abolish proliferative expansion capable of combining with the SL chain (16). Nonproductive VDJ of pre-BII cells and reduce the rate of B cell generation compared

rearrangements and productive rearrangements which result in with that expected from a normal number of DJH/DJH-rearranged nonpairing ␮H chains appear to change neither the phenotype nor pre-BI cells (20–22). In bone marrow without selective expansion the functional stage of the cell and they allow rearrangement at the of SL chain-pairing ␮H chain-producing precursors, subsequent second IgH allele. In contrast, pairing ␮H chains cause allelic ex- sites of L chain rearrangements and production finally lead to se- clusion by preventing the rearrangement of the second allele. lectable surface IgMϩ immature B cells. These results also clearly indicate that an incomplete VpreB/␮H chain pre-BCR-like com- plex (23) is not sufficient to signal proliferative expansion. Fur- *Basel Institute for Immunology, Basel, Switzerland; and †Developmental Immunol- ogy, Babraham Institute, Cambridge, United Kingdom thermore, targeted disruption of one of the two VpreB genes (in Received for publication November 13, 2001. Accepted for publication April 3, 2002. mice two genes encode the VpreB1 and VpreB2 proteins which are 98% structurally identical (12, 24)), i.e., VpreB1, is not sufficient The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance to induce a marked defect in the generation of B cells, demon- with 18 U.S.C. Section 1734 solely to indicate this fact. strating that VpreB2 alone, together with ␭5, is sufficient to gen- 1 The Basel Institute for Immunology was founded and supported by F. Hoffmann-La erate a functional SL chain and pre-BCR (25). However, the tar- Roche Ltd. (Basel, Switzerland). C.M., S.L., and I.-L.M. are supported by the Bio- geted disruption of VpreB1 and VpreB2 on the same technology and Biological Sciences Research Council. gave rise to a B cell generation-defective phenotype which is ap- 2 Address correspondence and reprint requests to Dr. Takeyuki Shimizu at the current address: Division of Biosignaling, Research Institute for Biological Science, Tokyo parently indistinguishable from that obtained following disruption University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan. E-mail ad- of the ␭5 gene (26). This result suggests that there are no other dress: [email protected] proteins which can substitute for VpreB1 and VpreB2. 3 Current address: Department of Cell Biology, Biozentrum of the University of Notably, both the ␭5-deficient as well as the VpreB1/VpreB2 Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. double-deficient mice showed allelic exclusion of the IgH locus in 4 Abbreviations used in this paper: SL, surrogate L; neo, neomycin; NP, 4-hydroxy- 3-nitrophenylacetyl; BCR, B cell receptor; TD, T cell dependent; TI, T cell indepen- the immature/mature B cell compartments (19, 26, 27), question- dent; ES, embryonic stem. ing the idea that the pre-BCR-like complex, composed of ␮H chain

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 6287

and one of the SL chain components, signals allelic exclusion in SL chain expression pre-B cells. Total RNA was prepared from two million bone marrow cells from 3-wk- To delete all genes encoding the SL chain, we have generated a old mice using RNAzol (Tel-Test, Friendswood, TX). cDNA was synthe- triple-deficient mouse strain in which both VpreB genes as well as sized using SuperScript II reverse transcriptase-polymerase and random the ␭5 gene have been disrupted by targeted integration of deleting primers (Life Technologies). PCR primers and reaction conditions for the ␭ elements on the same chromosome. Interestingly the triple-mutant VpreB genes were described previously (24). Primer sequences for 5 exon 2 were: 5Ј-GTTTTGGTATGTCTTTGGTGGTGGG-3Ј and ␭5 exon 3: 5Ј- mice showed the same B cell generation defect as reported for the GGTCTGTTTGGAGGGTTGGGTTG-3Ј. Conditions were as follows: 30 ␭5- and the VpreB1/VpreB2-deficient mice. Surprisingly, how- cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 30 s. ever, immature and mature B cells in these mice still showed IgH One million bone marrow cells from 3-wk-old mice were cultured on allelic exclusion. irradiated stromal cells (S17) in RPMI 1640 medium (Life Technologies) supplemented with 50 ␮M 2-ME, antibiotics, 10% FCS, and 10% IL-7. One week later, the cultured cells were collected and expression of the SL chain on the cell surface and in the cytoplasm was analyzed by FACS. Materials and Methods FACS analysis Targeting vectors Cell suspensions were prepared by conventional methods and stained in ␭ A targeting vector for the VpreB1- 5 locus was made as follows. A 5.5-kb HBSS (Life Technologies) supplemented with 10 mM HEPES and 3% XbaI-HindIII fragment which contains the second and third exons and the FCS. After staining, the cells were suspended in the same medium con- Ј ␭ 3 region of the 5 gene was isolated from phage clone 7pB12-2 (28). This taining propidium iodide (0.25 ␮g/ml) to exclude dead cells. To analyze fragment was cloned between the XbaI-HindIII sites of pBluescript II the allotype of IgM, bone marrow cells were stained with PE-labeled anti- (Stratagene, CA). The thymidine kinase gene cassette was inserted down- IgMa, biotin-labeled anti-IgD, and allophycocyanin-labeled anti-B220 Abs Downloaded from ␭ stream of the 5 gene. In a separate vector, the neomycin (neo) resistance in the presence of 5% rat serum. After washing, the cells were stained with gene cassette was inserted between the SalI-XhoI sites of pBluescript II. A FITC-labeled anti-IgMb Abs and tricolor-labeled streptavidin. Cytoplasmic Ј 1.6-kb SacII-BglII fragment, which contains the region 5 of the VpreB1 staining (for detection of SL chain) was performed according to the man- gene, was isolated from pUCE5.6X (24) and inserted between the SacII- ufacturer’s instructions (Caltag Laboratories, Burlingame, CA). The Abs Ј BamHI sites. The resulting 5 VpreB1-neo gene hybrid was cut out using used were as described previously (26). All data were acquired on a FACS- SacII-XhoI and inserted into the blunt-ended XbaI site of the vector which Calibur and analyzed using the CellQuest program (BD Biosciences, contained the 3Ј end of the ␭5 gene and the thymidine kinase gene. As a Mountain View, CA). http://www.jimmunol.org/ result, the entire region of the VpreB1 gene and the first exon of the ␭5 gene were deleted in the targeting construct. The neo gene was cloned in the Immunization and ELISA opposite transcriptional orientation to VpreB1 and ␭5. The targeting vector for VpreB2 has been described previously and uses hygromycin as a se- Control and homozygous mutant littermates were immunized by i.p. in- lection marker (26). In addition, the neo cassette has a loxP site on either jection of either 100 ␮g of NP-chicken ␥-globulin in alum or 5 ␮gof side whereas the hygromycin gene has a loxP site on only one side (see 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll. Two weeks after immuniza- Fig. 1). tion, the mice were bled and sera were prepared and analyzed by ELISA as previously described (26). The sera were analyzed as 5-fold serial dilu- tions. Immunizations were performed under PPL 80/1263. Gene targeting in ES cells and establishing SL chain-deficient mice Results by guest on September 27, 2021 Establishment of SL chain-deficient mice E14 embryonic stem (ES) cells (strain 129) were cultured on irradiated embryonic fibroblasts (Incyte Genomics, St. Louis, MO) in DMEM (Life The three genes encoding the SL chain (VpreB1, VpreB2, and ␭5) Technologies, Grand Island, NY) with 50 ␮M 2-ME (Sigma-Aldrich, St. are all located on mouse chromosome 16 (29, 30); therefore, tar- Louis, MO), 1000 U/ml ESGRO (Life Technologies), gentamicin, and 15% geting of these three genes in ES cells was performed in two steps. ␭ FCS. The VpreB1- 5 targeting vector was linearized with NotI. The ES In the first step, the VpreB1 gene and the first exon of the ␭5 gene cells were transfected and cultured with 300 ␮g/ml G418 (Life Technol- ogies) and 2 ␮M gancyclovir (Roche, Basel, Switzerland). Single colonies were replaced with the gene encoding neomycin resistance (Fig. were screened for VpreB1-␭5 homologous recombination by Southern 1). Two correctly targeted ES cell clones (clones 522 and 515, data blotting. Two homologously targeted ES cell clones (522 and 515) were not shown) were used for the second step in which the VpreB2 transfected with the linearized VpreB2 targeting vector and selected in 300 gene was substituted by the hygromycin resistance gene (Fig. 1). ␮g/ml hygromycin (Roche Molecular Biochemicals, Mannheim, Germa- ny). Single colonies were screened for VpreB2 homologous recombination The double-targeted ES cell clones were injected into blastocysts by PCR (26). Positive clones were confirmed by Southern blotting. and four clones gave rise to germline transmission. In two of these Four ES clones targeted at both the VpreB1-␭5 and VpreB2 loci were (522-H2A3, 515-3H11), the two targeting events were found to used to establish chimeric mice. Two of these (515-3H11 and 522-H2A3) segregate together, demonstrating that they were on the same chro- had the two mutations integrated on the same chromosome, whereas the mosome. The heterozygous mutant mice were bred for homozy- other two ES clones (522-N3C4 and 515-4F4) had integrated the two mu- tations on separate . The agouti mice (129 ϫ C57BL6 back- gosity which was confirmed by Southern blotting as shown in Fig. ground) with targeted mutations from 515-3H11 and 522-H2A3 ES clones 1. Both strains showed the same phenotype and the homozygous were intercrossed to establish homozygous SL chain-deficient mice. The VpreB1Ϫ/ϪVpreB2Ϫ/Ϫ␭5Ϫ/Ϫ mice are hereafter called SL genotypes of the mice were determined by PCR using previously described chain-deficient mice. primers (26) and/or Southern blotting with probes A and B shown in Fig. 1. Homozygous SL chain-deficient mice were also crossed with mice ex- Lack of VpreB and ␭5 expression in SL chain-deficient mice pressing cre recombinase ubiquitously. These were then intercrossed to obtain homozygous SL chain-deficient mice with a deletion of the neo Total RNA from bone marrow cells of SL chain-deficient as well cassette on both alleles (SL⌬neo-deficient) but with the hygromycin cas- as control mice was prepared and reverse transcribed. The exis- sette intact. These mice were screened by several PCRs to confirm that the tence of the SL chain gene mRNA was first analyzed using primers genotype was correct using a combination of primers detecting VpreB1, VpreB2, ␭5, neo, and hygromycin. In addition, they were also screened for specific for VpreB1 and VpreB2 (Fig. 2A). In wild-type and het- the presence of an ϳ700-bp PCR product indicative of the neo deletion erozygous mutant mice, expression of both genes was detected, using one primer located just upstream of the recombined loxP site (5Ј- while in the homozygous mutant mice, no message was found. TTGTCTCATTATGTAGCCAAGGCC-3Ј) and another primer down- Since mRNA encoding a ubiquitously expressed gene (hypoxan- stream of the XbaI site in ␭5 intron 1 (5Ј-CAAGGTCTTCTTGACT GGGT-3Ј). Conditions were as follows: 30 cycles of 94°C for 30 s, 55°C thine guanine phosphoribosyl transferase) was detected, we con- for 20 s, and 72°C for 20 s. Mice were established and analyzed under PPL cluded that the VpreB genes are not expressed in the SL 80/1143 and PPL 80/1501. chain-deficient mice. 6288 COMPLETE SL CHAIN-DEFICIENT MICE

FIGURE 1. Establishment of SL chain-deficient mice. Genomic organization of the VpreB1-␭5 (top) and VpreB2 (bottom) loci are shown on the left. The partial restriction enzyme maps of the loci before (wt) and after (ko) targeting are shown. For the VpreB1-␭5 locus, the map after neo cassette deletion (⌬neo) is also shown. Restriction enzyme sites: B, BglII; Ba, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SacII; X, XbaI. The BglII and XbaI sites were destroyed during cloning. The ar- rows under the neo and hygro indicate the orientation of these genes, which are opposite to those of the SL chain genes. Southern blottings of tail DNA are shown on the right. DNA was digested with EcoRI and the membrane hybridized with probe A (top). The same DNA was di- gested with BamHI and the membrane was hybridized with probe B (bottom). Wild-type (wt) and homologous recombinant (ko) bands are indicated with arrows. The genotypes of the mice are shown at the top. Downloaded from

Thereafter, we analyzed for ␭5 message using primers specific homozygous mutant cells, whereas an actin probe gave a signal of for exon 2 and exon 3, since these are still present after the tar- the same strength in both RNA preparations (data not shown). geting event. As shown in Fig. 2B, there is a properly spliced Thus, the truncated ␭5 mRNA levels were too low to be detected message, as judged by the size of the band, present in the homozy- by Northern blotting. gous mutant mice, with levels around 10-fold decreased as com- The ␭5 exon 2/3 mRNA in the homozygous mutant mice is http://www.jimmunol.org/ pared with that of the control. Northern blot analysis on total RNA probably the result of cryptic transcription initiation sites just up- prepared from in vitro-cultured pre-BI cells (the same cultures as stream of exon 2 in intron 1: we did not detect any transcripts were used for analysis, see below) was then performed. A starting further upstream in the intron (data not shown). The level ␭5 cDNA probe gave rise to a signal in RNA from control but not of exon 2/3 expression might be due to sequences in the neo cas- sette. Especially since this was cloned in the opposite direction, positioning the enhancer in the cassette just upstream of the po- tential cryptic initiation sites in the ␭5 intron. To test whether the enhancer in the neo cassette had any effect on the ␭5 exon 2/3 transcripts, we deleted the neo cassette by crossing SL chain-de- by guest on September 27, 2021 ficient mice with transgenic mice expressing cre recombinase ubiquitously. The offspring were intercrossed resulting in SL chain-deficient mice with the neo cassette deleted on both alleles. These mice are hereafter termed SL⌬neo-deficient mice. We an- alyzed bone marrow RNA from these mice by RT-PCR, again using primers for ␭5 exon 2/3. As shown in Fig. 2B (⌬Neo), by deleting the enhancer in the neo cassette the transcripts are no longer detectable in the homozygous mutant mice. Thus, the en- hancer in the neo cassette was the cause of the truncated ␭5 transcripts. We also searched for possible translation starts sites and open reading frames to determine whether a truncated ␭5 protein could be translated from the exon 2/3 transcripts. Even if the transcript starts in intron 1, it would have to be closer than 159 bp from the splice site as determined by RT-PCR assays (data not shown). There are no translation start sites with an open reading frame that could encode a protein in this region. The few ATG present all result in a stop codon after a few amino acids. Thus, even though the SL chain-deficient mice transcribe exon 2/3, this could not give rise to a protein. We then tested for the presence/absence of SL chain protein in the SL chain-deficient mice. Bone marrow cells from wild-type, FIGURE 2. Analysis of SL chain expression. A and B, Total RNA was heterozygous, and homozygous SL chain-deficient mice were cul- ⌬ prepared from bone marrow cells of SL chain- and/or SL Neo-deficient tured in vitro for 1 wk on irradiated stromal cells in the presence (⌬Neo) mice of the indicated genotype and analyzed by RT-PCR using of IL-7. Under these conditions, it is possible to expand and main- primers specific for VpreB1 and VpreB2 (A; NC, negative control without cDNA), or ␭5 exon 2/3 (B). The expression of SL chain protein on the cell tain pre-BI cells which express the SL chain on the cell surface surface (C) and in the cytoplasm (D) of in vitro-cultured pre-BI cells from along with BILL-cadherin and other proteins (31–33). Surface ex- heterozygous and homozygous SL chain-deficient mice was analyzed by pression of the SL chain was analyzed using mAbs specific for FACS using Abs recognizing VpreB and ␭5. Histograms show isotype VpreB and ␭5 (Fig. 2C). Pre-BI cells from wild-type and heterozy- control (thin line) overlaid with the specific stain (thick line). gous mutant mice expressed both ␭5 and VpreB on the surface, The Journal of Immunology 6289

Reduced numbers of pre-BII cells in the bone marrow of SL chain-deficient mice To investigate the effect of SL chain deficiency on B cell devel- opment, we analyzed the different bone marrow B cell compart- ments by FACS (Fig. 3A). As there was no obvious difference between wild-type and heterozygous mutant mice (data not shown), data from these genotypes were pooled and are shown as controls in the figures and tables. Table I summarizes the number of B lineage cells. Compared with control mice, the total number of nucleated cells in bone marrow was decreased in SL chain- deficient mice (ϳ80%) as were B220ϩ cells (ϳ50%). The B220ϩ B lineage cells were thereafter analyzed in detail. The pro-/pre-BI cell population (B220ϩc-kitϩ) was increased ϳ2-fold compared with control mice, whereas the pre-BII cell population (B220ϩCD25ϩ) was severely decreased in SL chain-deficient mice. The SL chain-mutant mice showed a 25-fold decrease in young (3 wk) and a 12-fold reduction in 8- to 9-wk-old mice as compared with control mice. In wild-type mice, pre-BII cells have Downloaded from productively rearranged the IgH loci and they express cytoplasmic ␮ FIGURE 3. FACS analysis of SL chain-deficient and control mice. H chain, some of which can pair with the SL chain to form a Bone marrow (A) and spleen (B) cells were prepared and analyzed by pre-BCR and signal cell proliferation. This population can be di- FACS for expression of the indicated cell surface markers. Viable lym- vided into large (cycling) and small (resting) cells. By collecting a phocytes were gated. In the IgM/Ig␬ dot plots in B, the cells were first large number of bone marrow cells from 8- to 9-wk-old mice, we ϩ gated on B220 cells. Cells were stained with either anti-B220 or anti-IgM determined that the proportion of large cells among the Ab in combination with Abs specific for the molecules indicated above B220ϩCD25ϩ cells was 26% in control and 41% in SL chain- http://www.jimmunol.org/ each figure. The percentages of relevant populations in the lymphocyte gate deficient mice (data not shown). From this, we calculated the num- are shown. ber of large and small pre-BII cells. Compared with control mice, SL chain-deficient mice showed an ϳ7-fold decrease in the large pre-BII cell population and an ϳ15-fold decrease in the small pre- while cells from homozygous mutant mice failed to express these BII population. These data suggested that in SL chain-deficient proteins. Cells from all cultures expressed B220, CD19, and c- mice the generation of pre-BII cells and/or the proliferative ex- (data not shown), confirming that the cells were indeed pre-BI pansion of large pre-BII cells is impaired.

cells. These results showed that in SL chain-deficient mice, none by guest on September 27, 2021 of the SL chain components are present on the cell surface. Reduced numbers of immature and mature B cells in bone We also analyzed the same cultures for expression of SL chain marrow and spleen of SL chain-deficient mice proteins in the cytoplasm. As shown in Fig. 2D, whereas both The number of both immature (IgMϩIgDϪ) and mature VpreB and ␭5 were readily detectable in the cytoplasm of cultured (IgMϩIgDϩ) B cells in bone marrow was markedly reduced in SL wild-type cells, none of these proteins was found in the cytoplasm chain-deficient mice. Depending on age, the difference in IgMϩ of cells from homozygous mutant mice. In addition, we analyzed cells was 20- and 9-fold in young (3 wk) and old (8–9 wk) mice, ex vivo cells from homozygous SL⌬Neo-deficient and control respectively. However, the ratio of small pre-BII to immature B mice by staining the cell surface with B220 and c-kit followed by cells, as analyzed in 8- to 9-wk-old animals, was 2:1 in control and staining of the cytoplasm with either the VP245 or the LM34 Ab 1:1 in SL chain-deficient mice (Table I). This suggested that the in combination with Abs recognizing the ␮H chain. By gating on absence of the SL chain, while impairing the transition from pre-BI the B220ϩc-kitϩ (pro-/pre-BI) cells, it was clear that the control to pre-BII cells, does not influence the transition from pre-BII to cells stained positive for VpreB and ␭5, whereas the homozygous immature B cells. SL⌬Neo-deficient cells were negative (data not shown). Thus, Immature B cells generated in the bone marrow migrate to the pre-B cells from SL chain-deficient as well as SL⌬Neo-deficient spleen where they mature. Therefore, spleen cells of SL chain- mice do not express any detectable SL chain proteins. deficient mice were analyzed to study the effect of the mutations on

Table I. Absolute numbers of B lineage cells in bone marrow and spleena

Age Organ (wk) Genotype No. Total B220ϩ B220ϩc-kitϩ B220ϩCD25ϩ B220ϩIgMϩ B220ϩIgDϩ

Bone marrow 3 Control 5 22.41 Ϯ 2.19 8.33 Ϯ 1.14 1.00 Ϯ 0.12 5.75 Ϯ 1.04 2.14 Ϯ 0.29 0.91 Ϯ 0.10 Ϫ/Ϫ 5 18.77 Ϯ 0.76 3.27 Ϯ 0.39 2.27 Ϯ 0.24 0.23 Ϯ 0.02 0.11 Ϯ 0.02 0.05 Ϯ 0.01 8–9 Control 6 35.15 Ϯ 4.91 9.03 Ϯ 1.30 1.18 Ϯ 0.19 3.72 Ϯ 0.42 3.90 Ϯ 0.77 2.75 Ϯ 0.79 Ϫ/Ϫ 5 29.80 Ϯ 2.55 4.65 Ϯ 0.55 1.76 Ϯ 0.23 0.32 Ϯ 0.09 0.44 Ϯ 0.11 0.25 Ϯ 0.05 Spleen 3 Control 5 214.42 Ϯ 17.4 102.71 Ϯ 6.83 83.34 Ϯ 6.77 84.16 Ϯ 7.90 Ϫ/Ϫ 5 57.06 Ϯ 3.42 6.04 Ϯ 0.94 3.82 Ϯ 0.63 3.52 Ϯ 0.57 8–9 Control 6 170.40 Ϯ 14.27 108.03 Ϯ 10.10 96.21 Ϯ 10.31 98.49 Ϯ 9.03 Ϫ/Ϫ 5 70.10 Ϯ 8.11 21.90 Ϯ 8.11 20.32 Ϯ 2.38 19.58 Ϯ 2.03

a The mean and SEM of the absolute numbers (ϫ106) are shown for indicated populations in bone marrow (two femurs) and spleen of control and homozygous SL chain-deficient (Ϫ/Ϫ) mice. 6290 COMPLETE SL CHAIN-DEFICIENT MICE the peripheral B cell populations (Fig. 3B and Table I). The total of different genotypes were bled and the sera analyzed by ELISA number of nucleated cells was 4- and 2-fold decreased in young to determine the amount of IgM. Wild-type and heterozygous and old SL chain-deficient mice, respectively. The B220ϩ cell mutant mice both demonstrated levels of 1.4 mg/ml and ho- population was also affected. In young mice, an almost 15- to mozygous mutant mice reached levels of 1.6 mg/ml (three an- 20-fold reduction was observed whereas in older mice the differ- imals per group). Thus, there was no significant difference in ence was less pronounced (5-fold). The majority of IgMϩ cells the total IgM concentration when comparing control and ho- were also IgDϩ (mature B cells) independent of genotype. The mozygous mutant mice. number of mature B cells was also decreased 20- to 25-fold in The responses against T cell-dependent (TD) and -independent 3-wk-old and 5-fold in 8- to 9-wk-old SL chain-deficient mice. (TI) Ags were also analyzed. Eight-week-old mice were immu- This suggested that the SL chain does not affect the differentiation nized with either NP-chicken ␥-globulin (TD Ag) or NP-Ficoll (TI from immature to mature B cells. The ratio of Ig␬ vs Ig␭ did not Ag). Two weeks later, serum samples were prepared and Ag-spe- vary between the genotypes. cific Abs were measured by ELISA. Immune responses to TD Ags, both against the hapten and the carrier, were similar in SL chain- SL⌬Neo-deficient mice deficient and control mice (Fig. 4 and data not shown). The Ab The data presented in Fig. 2B demonstrate the presence of a trun- response against the TI Ag was reduced 5- to 10-fold in mice cated ␭5 transcript (exon 2/3) in the bone marrow of homozygous lacking the SL chain as compared with control mice (Fig. 4). Thus, SL chain-deficient mice, but this was not detectable in bone mar- despite reduced numbers of B lymphocytes in the periphery, the row from SL⌬Neo-deficient mice. The sequence data indicated SL chain-deficient mice were able to mount an immune response that it would not be possible to make a truncated protein from this against both T cell-dependent and -independent Ags, although not Downloaded from RNA. In addition, the FACS analyses in Fig. 2 showed that no ␭5 at the same level as the control, implying that the peripheral B cells protein was detected in the homozygous SL chain-deficient pre-BI are functionally normal. cells. Even so, it may be argued that a truncated ␭5 protein is produced that is not recognized by the LM34 Ab. We therefore Allelic exclusion in SL chain-deficient mice analyzed the SL⌬Neo-deficient mice, where we performed the To investigate whether allelic exclusion of the IgH locus was func- same analyses as above both on bone marrow and spleen cells tioning in the absence of the SL chain, the frequency of single http://www.jimmunol.org/ from homozygous mutant and wild-type mice. We did not detect immature and mature B cells expressing two IgH allotypes on the any obvious difference in any of these analyses when compared cell surface was analyzed. SL chain-deficient and control mice with those described above for the SL chain-deficient mice (data heterozygous for the IgMa and IgMb alleles were selected. Bone not shown). We therefore conclude that the SL chain-deficient marrow cells from these mice were stained with Abs recognizing mice are “true” knockouts and show the phenotype of mice lacking B220, IgD, IgMa, and IgMb and analyzed by FACS (Fig. 5). Both the entire SL chain, i.e., VpreB1, VpreB2, and ␭5. Therefore, with IgDϪ and IgDϩ cells were analyzed and no difference was ob- the exception of IgH allelic exclusion, the following experiments served (data not shown). In both control and SL chain-deficient were performed only on the SL chain-deficient mice. mice, ϳ55–60% of the IgMϩ cells expressed IgMa and ϳ40–45% b ϩ by guest on September 27, 2021 B-1a B cells in the peritoneum of SL chain-deficient mice expressed IgM . In control mice, 0.7% of the IgM cells expressed both IgMa and IgMb and in SL chain-deficient mice this percentage In the peritoneum, the B-1a B cells can be distinguished from was 0.5%. In a separate experiment, bone marrow cells from these conventional B cells by the expression of CD5. To investigate the mice were stained for cytoplasmic IgMa and IgMb. The results potential effect on the B-1a B cells, peritoneal cells were isolated showed IgH allelic exclusion independent of the genotype (data from SL chain-deficient and control mice and analyzed by FACS. not shown). This demonstrated that in the absence of SL chain The absolute number of cells is summarized in Table II. In very ϩ ϩ allelic exclusion of IgH remains intact in bone marrow immature young mice (10 days), the number of B-1a B cells (B220 CD5 ) and mature B cells. We also analyzed the SL⌬neo-deficient mice ϳ was reduced 12-fold, while at 8 to 9 wk of age, SL chain-defi- in the same way and similar results were obtained. Thus, the lack cient mice had comparable numbers of B-1a B cells to control of the entire SL chain does not affect the mechanism of IgH allelic mice. The number of conventional B cells was still reduced in 8- exclusion. to 9-wk-old SL chain-deficient mice. In contrast, non B lineage Ϫ ϩ B220 CD5 cells were not affected either in young or in older SL Discussion chain-deficient mice. Thus, the number of B-1a B cells is also At the transition from DJH/DJH-rearranged pre-BI to VDJH-rear- affected by the lack of SL chain. ranged pre-BII cells, pre-BCRs are deposited on the surface mem- IgM serum levels and immune responses brane of those cells in which a productive rearrangement has given rise to a ␮H chain capable of pairing with the SL chain. This To investigate whether the decreased number of B lymphocytes in the periphery had any effect on the level of IgM in the serum, mice

Table II. Reduction in B-1a B cells in SL chain-deficient micea

Age Population Control Ϫ/Ϫ

10 days B1-a 0.72 Ϯ 0.15 0.06 Ϯ 0.02 B220ϩCD5Ϫ 0.96 Ϯ 0.26 0.04 Ϯ 0.01 B220ϪCD5ϩ 0.18 Ϯ 0.05 0.10 Ϯ 0.02 9 wk B1-a 24.43 Ϯ 3.79 31.26 Ϯ 14.34 B220ϩCD5Ϫ 17.30 Ϯ 2.52 5.00 Ϯ 0.21 B220ϪCD5ϩ 13.50 Ϯ 1.40 11.33 Ϯ 1.93 FIGURE 4. Immune responses. Mice of the indicated genotype were immunized with TD or TI Ags, and the amount of Ag-specific Abs in the a The mean and SEM of absolute numbers (ϫ104) are shown for B-1a (B220ϩCD5ϩ), B220ϩCD5Ϫ and B220ϪCD5ϩ lymphocyte populations in the peri- sera were analyzed by ELISA. The titers of hapten (NP)-specific IgM and toneum of mice (three to five per group) of indicated genotype and age. IgG are shown. CGG, Chicken ␥-globulin. The Journal of Immunology 6291

to the conventional L chain which is generated later (37, 38). Therefore, in SL chain-mutant mice, many more pre-BI cells

would have to enter VH to DJH rearrangements to produce an equal number of cells (as compared with wild-type mice) producing well-fitting ␮H chains in a given period of time. According to this view, B cell differentiation from the SL chain-mutant cells is not leaky, but simply inefficient. ␮ The original repertoire of VH domains expressed in H chains as a consequence of VH to DJH rearrangements at the transition of pre-BI to pre-BII cells in the bone marrow is influenced in a major

way by the SL chain (16). The VH domains that cannot pair with FIGURE 5. Allelic exclusion of the IgH locus. Bone marrow cells were SL chains, notably VH81X within the VH7183 family, the VHQ52 a prepared from control and SL chain-deficient mice heterozygous for IgM domains, and nearly half of all VHJ558 domains are suppressed and IgMb. Surface expression of the IgM allotypes was analyzed by FACS. from the ␮H chain repertoire of pre-BII and all subsequent B lin- B220ϩIgDϪ cells were gated and the expression of IgMa and IgMb was eage cells; the cells that express them cannot form a pre-BCR and plotted. The percentages of single- and double-expressing cells, as a per- therefore are not expanded in the same way as those ␮H chain ϩ centage of total IgM cells, are shown. ␭ Ϫ/Ϫ producers that contain pairing VH domains. In 5 mice, the pre-BII compartment still contains all of these nonpairing ␮H chain producers. We predict that future single-cell PCR/pairing Downloaded from surface expression of pre-BCR promotes between two to five analyses of the ␮H chain repertoires expressed in single pre-BII rounds of division, thus expanding the number of cells producing cells of the SL chain-deficient mice will also show the same non- ␮ ␭ Ϫ/Ϫ H chain between 4- and 32-fold. When the pre-BCR cannot be suppressed VH repertoire as that observed in 5 pre-BII cells. deposited on the surface, there is no proliferative expansion of the As L chain becomes expressed at the transition from pre-BII ␮ pre-BII compartment (17, 34). As we have predicted from our cells to mature B cells, the VH repertoire of H chain-producing Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ previous observations in ␭5 and VpreB1 VpreB2 mice cells becomes suppressed in ␭5 cells, as it is in wild-type cells http://www.jimmunol.org/ (17, 21, 26), the SL chain-deficient, VpreB1Ϫ/ϪVpreB2Ϫ/Ϫ␭5Ϫ/Ϫ when the SL chain is testing the ␮H chain at the transition from mice also lack pre-BII cell proliferative expansion and show a B pre-BI to pre-BII cells. A future single-cell PCR/pairing analysis is

cell immunodeficiency (Fig. 3 and Table I). Since the SL chain- likely to show the same VH repertoire change for SL chain-defi- and the SL⌬neo-deficient mice show the same phenotype, they are cient cells at the transition from pre-BII to mature B cells. throughout the discussion termed SL chain-deficient mice. In terms A second functional role has been proposed for the pre-BCR, of kinetics of development, defective cellularity of the immature namely, to signal a pre-BII cell, which has made one productive ␮ and mature B cell compartments (Table I) and response to T cell- VDJH rearrangement and produced a H chain that can pair with dependent Ags (Fig. 4), all seem indistinguishable in the single the SL chain and form a pre-BCR on the surface, to turn off the ␭5Ϫ/Ϫ, the double VpreB1Ϫ/ϪVpreB2Ϫ/Ϫ (20, 26), and the SL rearrangement machinery (recombination-activating gene 1, re- by guest on September 27, 2021 chain-deficient mice. The pre-BI population, as in the other SL combination-activating gene 2, and TdT), and to close the second ϩ chain-mutant mice, is 2- to 3-fold larger than in wild-type litter- DJH-rearranged allele (39). Thereby, the resulting VDJ /DJH pre- mates, probably because the cells cannot exit this compartment by BII cell is allelically excluded and prevented from possible VDJ

further B cell differentiation (Table I). The ratio of the sizes of the rearrangement at the second allele. Since VH to DJH rearrange- small pre-BII compared with the immature B cell compartment, as ments occur randomly in- and out-of-frame, two-thirds of all orig- well as that of the small pre-BII compared with the mature B cell inal rearrangements are out-of-frame and unable to produce a ␮H compartment is similar in wild-type, ␭5Ϫ/Ϫ, VpreB1Ϫ/Ϫ chain. Consequently, no pre-BCR is made and the cell rearranges VpreB2Ϫ/Ϫ, and SL chain-deficient mice (Table I) (21, 26). All the second allele. Of these, one-third will be productive, thus gen- these observations suggest that the major measurable defect intro- erating a VDJϪ/VDJϩ-rearranged cell. This cell is allelically ex- duced by mutations of the SL chain subunits is the lack of prolif- cluded without the need of suppression of any further VDJ rear-

erative expansion at the transition from pre-BI to pre-BII cells. rangement, but might have to be suppressed for secondary VH Although we have not yet measured the kinetics of in vitro dif- replacements (40). Therefore, it cannot be excluded that the ma- ϩ ferentiation of surface IgM B cells from SL chain-deficient chinery involved in VH replacements should be turned off in these pre-BI cells, we would predict from our observations of the B cell cells, and the VDJ-rearranged loci should become inaccessible for ␭ Ϫ/Ϫ developmental defects that these kinetics, as in 5 pre-BI cells VH replacements. (21), would also be indistinguishable between SL chain-deficient A defect in signaling for allelic exclusion should be readily vis- and wild-type pre-BI cells. Hence, expression of the SL chain be- ible as an increase in the number of “double producers” which are fore expression of the ␮H chain in pre-BI cells (35, 36), possibly immature and mature B cells which deposit IgM with ␮H chain with BILL-cadherin (32, 33) and other associated proteins, appears produced from both alleles on a single cell. If the defect is com- ϩ not to influence the capacity of these cells to enter VH to DJH plete, the VDJ /DJ-rearranged cells should continue to rearrange rearrangements and later VL to JL rearrangement and differentiate the second allele. Hence, they should disappear from the repertoire to pre-BII, immature, and mature B cells. From an unaltered dif- of pre-BII and all subsequent cells. It was already surprising that ferentiation capacity of the ␭5Ϫ/Ϫ pre-BI cells in vitro (21), we ␭5Ϫ/Ϫ pre-B and B cells had a ratio of VDJ/DJ to VDJϩ/VDJϪ conclude that they have the same unaltered capacity to differentiate cells which was indistinguishable from that in wild-type B cells a b in vivo. Hence, the developmental defect of SL chain-mutant pre- (19). Furthermore, newly generated B cells of F1 IgM /IgM mice BII cells is most likely their inability to proliferate, leading to a of wild-type as well as ␭5Ϫ/Ϫ mice had Ͻ0.5% double producers reduction in the number of ␮H chain-producing pre-BII cells in (27). It should be noted that both wild-type and ␭5Ϫ/Ϫ pre-BII and which subsequent L chain gene rearrangement can take place. Fur- mature B cells contained comparable numbers (between 4 and 8% thermore, those cells producing ␮H chains which best fit the SL of all VDJ/VDJ cells) of VDJϩ/VDJϩ double ␮H chain-producing chain are expanded most, and these ␮H chains should also fit best cells. However, in these cells it is always the case that only one of 6292 COMPLETE SL CHAIN-DEFICIENT MICE

the two ␮H chains produced could be deposited on the surface as 3. Osmond, D. G., A. Rolink, and F. Melchers. 1998. Murine B lymphopoiesis: a pre-BCR (19). This might be expected from the previous finding towards a unified model. Immunol. Today 19:65. 4. Meffre, E. R., R. Casellas, and M. C. Nussenzweig. 2000. regulation of that although approximately half of all originally produced ␮H B cell development. Nat. Immunol. 1:379. chains can pair with the SL chain, the other half cannot (16). This 5. Alt, F., N. Rosenberg, S. Lewis, E. Thomas, and D. Baltimore. 1981. Organiza- ␮ tion and reorganization of immunoglobulin genes in A-MULV-transformed cells: suggests that nonpairing H chains which cannot form a pre-BCR rearrangement of heavy but not chain genes. Cell 27:381. are also unable to turn off the rearrangement machinery and close 6. Alt, F. W., G. D. Yancopoulos, T. K. Blackwell, C. Wood, E. Thomas, M. Boss, the second allele. R. Coffman, N. Rosenberg, S. Tonegawa, and D. Baltimore. 1984. Ordered re- arrangement of immunoglobulin heavy chain variable region segments. EMBO J. Although it was still conceivable that a partially defective pre- 3:1209. BCR, composed of VpreB and ␮H chain, might signal allelic 7. Nussenzweig, M. C., A. C. Shaw, E. Sinn, D. B. Danner, K. L. Holmes, exclusion (23), the results obtained from VpreB1Ϫ/ϪVpreB2Ϫ/Ϫ H. C. Morse III, and P. Leder. 1987. Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin ␮. Science 236:816. double-mutant mice now rule out this possibility. These double- 8. Manz, J., K. Denis, O. Witte, R. Brinster, and U. Storb. 1988. Feed back inhi- mutant B cells still allelically exclude the IgH locus as well as bition of immunoglobulin gene rearrangement by membrane ␮, but not by se- ␮ wild-type B cells (26). creted heavy chains. J. Exp. Med. 168:1363. 9. Kitamura, D., and K. Rajewsky. 1992. Targeted disruption of ␮ chain membrane Although it has been observed previously that ␭5 protein cannot exon causes loss of heavy-chain allelic exclusion. Nature 356:154. bind ␮H chain in the absence of VpreB protein, as it appears in- 10. Rolink, A., J. Andersson, P. Ghia, U. Grawunder, D. Haasner, H. Karasuyama, E. ten Boekel, T. H. Winkler, and F. Melchers. 1995. B-cell development in capable of forming a disulfide-bonded complex (23), the possibil- mouse and man. Immunologist 3:125. ity existed that a noncovalent, weak interaction would suffice to 11. Sakaguchi, N., and F. Melchers. 1986. ␭5, a new light-chain-related locus selec- form a pre-BCR-like complex with the capacity to signal allelic tively expressed in pre-B lymphocytes. Nature 324:579.

12. Kudo, A., N. Sakaguchi, and F. Melchers. 1987. Organization of the murine Downloaded from exclusion. The results reported here for SL chain-deficient mice Ig-related ␭5 gene transcribed selectively in pre-B lymphocytes. EMBO J. 6:103. now rule this out: immature and mature B cells are still allelically 13. Karasuyama, H., A. Kudo, and F. Melchers. 1990. The proteins encoded by the a b ␭ ␮ excluded in F IgM /IgM SL chain-deficient mice. Although anal- VpreB and 5 pre-B cell-specific genes can associate with each other and with 1 heavy chain. J. Exp. Med. 172:969. ysis of the V repertoire in the developing B cells and the contri- ␭ H 14. Tsubata, T., and M. Reth. 1990. The products of pre-B cell specific genes ( 5 and ␮ bution of pairing and nonpairing ␮H chains in these repertoires has VpreB) and the immunoglobulin chain form a complex that is transported onto yet to be conducted, we conclude from the results presented in this the cell surface. J. Exp. Med. 172:973. 15. Melchers, F., H. Karasuyama, D. Haasner, S. Bauer, A. Kudo, N. Sakaguchi, paper that the SL chain, and hence the pre-BCR, is not required for B. Jameson, and A. Rolink. 1993. The surrogate light chain in B-cell develop- http://www.jimmunol.org/ allelic exclusion of the IgH locus. ment. Immunol. Today 14:60. 16. ten Boekel, E., F. Melchers, and A. Rolink. 1997. Changes in the VH gene rep- Allelic inclusion is observed in B cells, in which one IgH allele ertoire of developing precursor B lymphocytes in mouse bone marrow mediated has been mutated in the transmembrane portion of the ␮H chain to by the pre-B cell receptor. Immunity 7:357. prevent its membrane deposition (9). Hence, membrane-bound ␮H 17. Rolink, A. G., T. Winkler, F. Melchers, and J. Andersson. 2000. Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the chain signals allelic exclusion, but not together with the SL chain. bone marrow or fetal liver environment. J. Exp. Med. 191:23. Three other possible partners have been suggested: the heat shock 18. Melchers, F., E. ten Boekel, T. Seidl, X. C. Kong, T. Yamagami, K. Onishi, protein 70 chaperon H chain binding protein (41), the 8HS20- T. Shimizu, A. G. Rolink, and J. Andersson. 2000. Repertoire selection by pre- B-cell receptors and B-cell receptors, and genetic control of B-cell development encoded VpreB3 (42), and prematurely expressed L chains (43– from immature to mature B cells. Immunol. Rev. 175:33. 45). VpreB3 has 36% amino acid homology with VpreB1 and is 19. ten Boekel, E., F. Melchers, and A. G. Rolink. 1998. Precursor B cells showing by guest on September 27, 2021 associated with ␮H chain in pre-B cell lines. The function of H chain allelic inclusion display allelic exclusion at the level of pre-B cell re- ceptor surface expression. Immunity 8:199. VpreB3 is still unknown. However, as the VpreB3 gene is located 20. Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, and K. Rajewsky. on mouse chromosome 10 (46), it would be both possible and 1992. A critical role of ␭5 protein in B cell development. Cell 69:823. 21. Rolink, A., H. Karasuyama, U. Grawunder, D. Haasner, A. Kudo, and interesting to cross VpreB3-deficient mice with the SL chain-de- F. Melchers. 1993. B cell development in mice with a defective ␭5 gene. Eur. ficient mice. We have not observed N region insertions in V-J J. Immunol. 23:1284. joints of L chains from mature B cells of ␭5Ϫ/Ϫ mice (47). These 22. Minegishi, Y., E. Coustan-Smith, Y. H. Wang, M. D. Cooper, D. Campana, and M. E. Conley. 1998. Mutations in the human ␭5/14.1 gene result in B cell defi- should have been detected if the L chain genes had been rear- ciency and agammaglobulinemia. J. Exp. Med. 187:71. ranged before the IgH genes, since IgH from wild-type as well as 23. Seidl, T., A. Rolink, and F. Melchers. 2001. The VpreB protein of the surrogate ␭5Ϫ/Ϫ mice have N regions inserted at VDJ joints (47). In addi- light-chain can pair with some ␮ heavy-chains in the absence of the ␭5 protein. ␬ Eur. J. Immunol. 31:1999. tion, we have not observed -chain expression in pre-BI cells of 24. Dul, J. L., V. Argon, T. Winkler, E. ten Boekel, F. Melchers, and I.-L. Mårtens- Ϫ Ϫ ␭5 / mice expressing ␮H chain in the cytoplasm, nor have we son. 1996. The murine VpreB1 and VpreB2 genes both encode a protein of the detected VJ-rearranged L chain loci in such cells by single-cell surrogate light chain and are co-expressed during B cell development. Eur. J. Im- munol. 26:906. PCR. Therefore, we do not favor the BCR as signal transducers for 25. Mårtensson, A., Y. Argon, F. Melchers, J. L. Dul, and I.-L. Mårtensson. 1999. allelic exclusion in SL chain-mutant mice. Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in VpreB1-deficient mice. Int. Immunol. 11:453. In the search for genes encoding the signal transduction pathway 26. Mundt, C., S. Licence, T. Shimizu, F. Melchers, and I.-L. Mårtensson. 2001. Loss for allelic exclusion at the IgH locus of pre-B cells, the SL chain- of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double- deficient ES cells and SL chain-deficient pre-BI cells should be deficient mice. J. Exp. Med. 193:435. 27. Lo¨ffert, D., A. Ehlich, W. Mu¨ller, and K. Rajewsky. 1996. Surrogate light chain valuable tools for additional mutations which might affect allelic expression is required to establish immunoglobulin heavy chain allelic exclusion exclusion. during early B cell development. Immunity 4:133. ␭ 28. Kudo, A., and F. Melchers. 1987. A second gene, VpreB in the 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO Acknowledgments J. 6:2267. We are grateful to Drs. Bru¨ggemann and Zou for the cre-recombinase 29. Kudo, A., D. Pravtcheva, N. Sakaguchi, F. H. Ruddle, and F. Melchers. 1987. transgenic mice. We thank T. Saunders and M. George for blastocyst in- Localization of the murine ␭5 gene on chromosome 16. Genomics 1:277. jections and our colleagues for reading and discussing this manuscript. 30. Bauer, S. R., L. A. D’Hoostelaere, and F. Melchers. 1988. Conservation of the organization of the pre-B cell specific VpreB1/VpreB2/␭5 loci in the genus Mus. Curr. Top. Microbiol. Immunol. 137:130. References 31. Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, and F. Melchers. 1991. Long- 1. Rolink, A., U. Grawunder, T. H. Winkler, H. Karasuyama, and F. Melchers. term proliferating early pre B cell lines and clones with the potential to develop 1994. IL-2 receptor ␣ chain (CD25, TAC) expression defines a crucial stage in to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. pre-B cell development. Int. Immunol. 6:1257. 10:327. 2. ten Boekel, E., F. Melchers, and A. G. Rolink. 1995. The status of Ig loci rear- 32. Karasuyama, H., A. Rolink, and F. Melchers. 1993. 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