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The Journal of Immunology, 2003, 170: 1354-1361.
Copyright © 2003 by The American Association of Immunologists

Block in Development at the Pre-B-II to Immature B Cell Stage in Mice Without Ig{kappa} and Ig{lambda} Light Chain 1

Xiangang Zou, Tony A. Piper, Jennifer A. Smith, Nicholas D. Allen, Jian Xian and Marianne Brüggemann2

Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Silencing individual C (constant region) {lambda} genes in a {kappa}-/- background reduces mature B cell levels, and L chain-deficient ({lambda}-/-{kappa}-/-) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B cells express surface Ig with either {kappa} or {lambda} L chain, a choice termed isotype exclusion. The proportion of Abs containing a {kappa} or {lambda} L chain varies considerably in the different species, but in the mouse only a few percent of Abs carry {lambda} (1). L chain genes are encoded by two different loci, and in the mouse there are an extensive number of V {kappa} gene segments upstream of five J {kappa} and one C{kappa} gene (2). The mouse {lambda} L chain locus contains within an ~200-kb region 2 sets of V, J, and C genes that can independently rearrange: V2-Vx-J2-C2-J4-C4 and V1-J3-C3-J1-C1 (3). C1 appears to be predominantly expressed, followed by C2 and then C3, which are less frequently expressed (4), while C4 is not found to be expressed due to a lack of functional splice sites (5). Although the {kappa} locus is >10 times larger than the {lambda} locus, with >100 V genes, this extensive complexity is not regarded as the reason why most mouse Abs carry a {kappa} L chain. It may be that the mouse {kappa} locus is simply more efficient in DNA rearrangement, which is supported by the finding that in the majority of cells with rearranged V{kappa} the {lambda} locus is still in germline configuration, while in most cells expressing {lambda} L chain the {kappa} locus is either nonproductively rearranged or deleted (6).

Several mouse strains with silenced {kappa} L chain locus have been described. They were generated by homologous integration of a selectable marker gene in C{kappa} or targeted removal of C{kappa} or J{kappa} (7, 8, 9, 10, 11). Silencing expression of {kappa} L chain shed light on isotype exclusion and L chain activation, and it was concluded that {kappa} and {lambda} expression are separate and independent events. Although homozygous {kappa}-/- mice compensate for the {kappa} deficiency with increased {lambda} production, their splenic B cells and µ+ cells in the bone marrow are reduced compared with those in normal mice (7, 9), which may suggest that {lambda} L chain rearrangement and expression are relatively inefficient. However, despite the lack of {kappa} L chain, these mice are healthy and can mount an efficient immune response (11).

During B cell development gene segments encoding Ig H chains rearrange first by D (diversity) to JH recombination at the pro-B cell stage. This is followed by VH to D-JH recombination at the pre-B-I stage; if a µ H chain can pair with a surrogate L chain, consisting of VpreB and {lambda}5 protein, this forms a surface expressed pre-B cell receptor (pre-BCR)3 at the pre-B-II differentiation stage. Ordered rearrangement of Ig genes appears to direct B cell development and provides important checkpoints to establish the successful completion of BCR expression (reviewed in Refs.12 and 13). Thus, the µ H chain provides vital functions, some in association with the surrogate L chain, in controlling allelic exclusion and establishing a survival signal to allow progression through the B-lineage pathway (14, 15, 16, 17). Cell surface expression of the pre-BCR induces proliferation, and after several divisions large pre-B-II cells differentiate into small resting pre-B-II cells (18). The pre-B-II stage with a defined ratio of large and small pre-B cells has been identified by surface expression of the IL-2R {alpha}-chain, CD25 (19). At the pre-B-II stage, L chain V-J rearrangement occurs (20, 21), and the cells can leave the bone marrow for further differentiation into mature B cells and, upon Ag encounter, into plasma cells or memory cells in secondary lymphoid organs such as spleen or lymph nodes. The importance of H and L chain polypeptides in B cell development has also been analyzed in recombination-activating gene (RAG-1 or RAG-2)-deficient mice (15, 16). RAG deficiency is regarded as nonleaky, and the mice cannot rearrange their H or L chain gene. The experiments confirmed that µ H chain is required to promote pro-B cell maturation, but, interestingly, it appeared that despite the lack of L chain, a substantial proportion of activated B cells were able to migrate to the spleen (22). RAG-deficient mice carrying an introduced H chain should have very similar functional activity to mice lacking L chain expression, but it cannot be established whether other rearranging genes or L chain family members identified on other chromosomes can substitute L chain function (23 and http://www.ensembl.org/mus_musculus/familyview?family = ENSF00000000042 and ENSF00000000595; using the algorithm identification described in Ref.24).

B cell development without L chain is debatable. The BCR consists of two Ig H chains, each associated with one Ig L chain in conjunction with the Ig{alpha}/Ig{beta} coreceptor (25). These six chains must assemble correctly in the endoplasmic reticulum (ER) to facilitate the transport and cell surface expression of IgM necessary for B cell development to progress. Immature B cells without L chain have not been observed, and a lack of surface IgH, Ig{alpha}, or Ig{beta} expression leads to reduced signal transducer activity, which can arrest B cell maturation (26). H chain, synthesized before L chain, is chaperoned and retained in the cytoplasm, but if L chain association fails, single H chains, unlike L chains, undergo rapid intracellular degradation as a result of inefficient transport from the ER to the Golgi (27). However, single mutated H chains accumulate in heavy chain disease (28), and it has been discovered that {gamma} H chains, lacking the CH1 domain, are assembled routinely as fully functional H2 IgG Abs without L chains in camelids (29). Although it is not known how these H chain-only Abs develop, it appears that they use particular VH genes, and they may skip µ H chain expression in development (30).

Here we show that mice with silenced L chain loci are immunodeficient. They do not produce B-1 or B-2 cells in the periphery, and B cell development is compromised at the immature B cell stage, with a complete block at the stage of differentiation when L chain rearrangement should have been completed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Targeting constructs

A phage {lambda} library derived from embryonic stem (ES) cell DNA, a gift from A. Smith and T. Rabbitts (Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.), was hybridized with a V{lambda} and C{lambda} probe (clone 505; provided by M. Neuberger, Medical Research Council), which identified several clones containing V{lambda} and, separately, C{lambda} genes. Part of the C2-C4 and C3-C1 regions were subcloned in pUC19 to assemble the constructs and obtain gene probes. This allowed blunt end insertion of loxP from pGEM-30 (31) in the HindIII site 3' of J3, loxP insertion in tkNeo (Stratagene, La Jolla, CA), blunt end insertion of tkNeo-loxP into C{lambda}1, and loxP-tkNeo, derived from pGH-1 (pGEM-30 and pGH-1 were gifts from H. Gu, Institute for Genetics, University of Cologne, Cologne, Germany), into C{lambda}2 (see Fig. 1). The ~14-kb C3-C1 targeting construct was obtained by XhoI and HindIII digest, and the ~13-kb C2-C4 targeting construct was obtained by XhoI excision in the internal and polylinker site. Restriction sites for integration of tkNeo (SacI and BamHI) or loxP (HindIII) in the targeting constructs were not maintained.



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  		FIGURE 1. Targeted integration and deletion of the mouse {lambda} L chain locus. A, The {lambda} locus is ~200 kb with two sets of J-C genes (J2-C2—J4-C4 and J3-C3—J1-C1) separated by ~110 kb (58 ). Two V genes, V2 and Vx, are located ~75 and ~56 kb upstream of C2, respectively, and V1 is located ~20 kb upstream of C3. Bars below the line indicate probes A, B, C, and D. B, Targeted integration of C3-C1 inserts tkNeo-loxP into C1 and loxP 3' of J3, which allows deletion of C3, J1, and the 5' region of C1. The C2-C4 targeting construct inserts loxP-tkNeo into C2. Both targeting constructs disable all functional C genes. Oligonucleotides (1 2 3 4 5 6 ) that identify wild-type configuration, targeted integration, and C{lambda} deletion are indicated below the line. C, Upon Cre-mediated deletion an ~120-kb region between C2 and C1 is removed. D, Analysis of targeted integration (C3-C1 and C2-C4) by Southern blot and Cre-mediated deletion (C2-C1 deletion) by PCR. Southern hybridization of normal mouse DNA (NM), ES cell DNA from clones with homologous integration in C3-C1 (ES1.3) and C2-C4 (ES2.4), and deletion of C3-C1 (ES1.3{Delta}+/- and ES1.3{Delta}-/-) with digests and probes (A–D) indicated. PCR analysis of tail DNA identified the configuration of the Ig{lambda} locus before and after Cre deletion. For all reactions a mixture of oligonucleotides 1–6 was used. The resulting bands were a product of the oligonucleotide combinations (oligos) indicated.

 
Analysis of homologous integration

Electroporation of targeting constructs and ES cell selection has been described (11). The C3-C1 construct was integrated in HM-1 (32), and C2-C4 was integrated in {lambda}ES3.1{Delta}-5 ES cells. Targeting of C3-C1 was identified with a 0.4-kb HindIII fragment (probe A, all probes are marked in Fig. 1A) and SacI digest of ES cell DNA and was verified with a 2-kb XbaI-HindIII fragment (probe B) and SacI, HindIII, and BamHI digests, which also allowed identification of C3-C1 Cre-loxP deletion. Homologous integration in C2-C4 was identified with a 0.7-kb HindIII-XbaI fragment (probe C, the XbaI site is immediately 5' of SacI) and a 1.2-kb HindIII-BamHI fragment (probe D), and HindIII and BamHI digests of ES cell DNA. To obtain deletion of the {lambda} locus, the Cre plasmid pBS185 (10347-011; Life Technologies, Paisley, U.K.) was transiently integrated by electroporation (11). Clones were tested by PCR using the following oligonucleotides (arrows 1–6 in Fig. 1B): C1rev, 5'-GCCTTTCCCATGCTCTTGCTGTCAGGG-3' (< 1); C1for, 5'-CCAAGTCTTCGCCATCAGTCACCC-3' (2>); 3'J3for, 5'-CCCAGGTGCTTGCCCCACAGGTTTAGG-3' (3>); 5'C2for, 5'-GGAGATCAGGAATGAGGGACAAAC-3' (4>); 3'tkNeorev, 5'-CTCGACGGATCCGTCGAGGAATTCC-3' (<5 neo); and tkNeofor, 5'-ATGGCCGATC CCATATTGGCTGCAGGG-3' (neo 6>). Oligos 1–2 identified the wild-type configuration; oligos 1–6 and, separately, 4–5 identified construct integration; while the combination of oligos 1–3 and 1–4 identified partial or complete C gene deletion. PCR reactions were performed under the following conditions: two initial cycles of 45 s at 97°C, 30 s at 60°C, and 60 s at 72°C, followed by 30 cycles with 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C, then 10 min at 72°C to complete the reaction.

Derivation of mice

Chimeric mice and germline transmission were obtained as previously described (33, 34). The mice have been derived, bred, and investigated according to Home Office project license PPL 80/1469. {lambda}1.3 mice, in a 129/Ola x BALB/c background, were mated with 129/Ola mice for five generations and crossed with mice expressing Cre recombinase and each other to obtain homozygous {lambda}1.3-/- animals. For the derivation of transgenic mice expressing Cre protein ubiquitously, the Cre plasmid pBS185 was linearized with ScaI and purified using a DNA purification kit (28304; Qiagen, Crawley, U.K.). DNA was microinjected into the male pronucleus of F1 embryos (CBA x C57BL/6) according to standard methods (34), and several founders were produced, two of which showed a high gene/locus deletion rate when crossed with loxP mice. For the derivation of ES cells, blastocysts were collected and cultured on mitomycin C-treated feeder cells (33). Several ES cell lines were obtained, and {lambda}ES3.1{Delta}-5, a female line, was used for integration of the C2-C4 targeting construct. Homologous integration was obtained, and chimeric mice and subsequently germline transmission mice were produced. Homologous integration and locus deletion were identified, and animals with the following genes silenced were used for further breeding: {lambda}C1-/- (mouse 130 = ES1.3); {lambda}C1-/- and {lambda}C3-/- (mouse 1.3 = ES1.3{Delta}); {lambda}C1-/-, {lambda}C2-/-, and {lambda}C3-/- (mouse 50 = ES2.4); and deletion ({Delta}) of {lambda}C1-/-, {lambda}C2-/-, {lambda}C3-/-, and {lambda}C4-/- (mouse 1.3–2.4{Delta}). Crossing into the {kappa}-/- background established four mouse strains with different functional L chain content: {lambda}1-/-{kappa}-/-, {lambda}1.3-/-{kappa}-/-, {lambda}1.3.2-/-{kappa}-/-, and {lambda}1–2{Delta}-/-{kappa}-/-.

Flow cytometric analysis

For analysis of B cell populations by flow cytometry, cells from the different tissues were prepared and stained with various combinations of differently labeled Abs against cell surface markers (see Fig. 2). The labeled Abs for bone marrow cells were PE-conjugated anti-mouse c-kit (CD117; 09995B; BD PharMingen, San Diego, CA), PE- or allophycocyanin-conjugated anti-mouse CD45R (B220; 01125A, 01129A; BD PharMingen), biotin-conjugated anti-mouse CD25 (01092A; BD PharMingen), FITC-conjugated monoclonal rat anti-mouse IgM (µ chain specific; 04-6811; Zymed, San Francisco, CA) and/or biotin-conjugated anti-mouse CD43 (01602D; BD PharMingen). The labeled Abs for spleen cells were PE- or allophycocyanin-conjugated anti-mouse CD45R (B220; 01125A, 01129A; BD PharMingen), Biotin-conjugated anti-mouse IgM (µ-chain specific; 02082D; BD PharMingen), FITC-conjugated anti-mouse IgD (02214D; BD PharMingen), biotin- or FITC-conjugated anti-mouse Ig{lambda} (02172D, 02174D; BD PharMingen), and/or PE-conjugated anti-mouse Ig{kappa} (559940; BD PharMingen); for peritoneal cells they were PE-conjugated anti-mouse CD5 (Ly-1; 01035A; BD PharMingen) and allophycocyanin-conjugated anti-mouse CD45R (B220; 01125A, 01129A; BD PharMingen).



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  		FIGURE 2. Block in B cell development at the pre-B-II to immature transition stage. Flow cytometric analysis of bone marrow (A) and splenic (B) B cell populations from normal (NM), {kappa}-/-, {lambda}1-/-{kappa}-/-, {lambda}1.3-/-{kappa}-/-, {lambda}1.3.2-/-{kappa}-/-, and {lambda}1–2{Delta}-/-{kappa}-/- mice. The profiles are representative for results obtained for at least five mice per group and show staining of gated bone marrow lymphocytes for pro- and pre-B cell markers: PE-conjugated c-kit, biotin-conjugated anti-mouse CD43, biotin-conjugated anti-mouse CD25, or biotin-conjugated anti-IgM in combination with PE- or allophycocyanin-conjugated anti-B220. Spleen cells were stained with biotin-conjugated anti-IgM, FITC-conjugated anti-IgD, biotin- or FITC-conjugated anti-{lambda}, and/or PE-conjugated anti-{kappa} and allophycocyanin-conjugated anti-B220 for setting the B lymphocyte gate.

 
For cytoplasmic staining, bone marrow B cells were pretreated using a fix and perm cell permeabilization kit (GSA-004; Caltag, Burlingame, U.K.) and then were stained with FITC-conjugated monoclonal rat anti-mouse IgM (µ-chain specific, 04-6811; Zymed), PE-conjugated anti-mouse CD45R (B220; 01125A, 01129A; BD PharMingen), and biotin-conjugated anti-mouse CD25 (01092A; BD PharMingen) according to the manufacturer’s protocol. Binding of biotinylated Ab was developed with streptavidin-Quantum Red (S2899; Sigma-Aldrich, St. Louis, MO) or strepavidin-Tri-color (SA1006; Caltag).

Histology

The method of Doody et al. (35) was used to analyze fresh-frozen cryostatic sections (10 µm) of spleens fixed with cold acetone, blocked with 10% rat serum, and stained with biotinylated anti-µ (04-6811; Zymed), followed by tetramethylrhodamine isothiocyanate (TRITC)-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) and FITC-labeled anti-CD4 (090004D; BD PharMingen). Mounted sections were viewed with a BX40 epifluorescence microscope (Olympus, New Hyde Park, NY) with appropriate filters, and the images were recorded digitally with a high resolution CCD camera (F-View) using the analySIS 3.1 image analysis software (SIS, Munster, Germany). Gray-scale images were captured using separate filter sets for FITC and TRITC, pseudo-colored, and merged using Adobe Photoshop 6.0 software.

Protein analysis

Serum Abs were identified by ELISA as previously described (11). For separation on acrylamide gels, digitonin lysates of bone marrow cells (36) and, separately, serum, were incubated for 1 h at 4°C with anti-mouse IgM (µ-chain specific; The Binding Site, Birmingham, U.K.) coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia LKB, Uppsala, Sweden) as described (37). Samples were fractionated on 4–15% precast gels (161-1104; Bio-Rad, Hemel Hempstead, U.K.) and, after transfer to nitrocellulose membranes, incubated with biotinylated anti-mouse µ (B-9265; Sigma-Aldrich) for 1 h at room temperature and then placed in streptavidin-biotinylated HRP solution (RPN 1051; Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min on a rocker. Bands were visualized with SuperSignal West Pico chemiluminescent substrate (34080; Pierce, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Silencing of the mouse {lambda} L chain locus

To investigate B cell development without L chain we produced mice with a deleted Ig{lambda} locus. The {lambda}-/- mice were crossed with animals carrying a nonfunctional Ig{kappa} locus, {kappa}-/- mice, also obtained by gene targeting (11). The mouse {lambda} L chain locus contains three V region genes, four J segments, and four C region genes that can independently rearrange and express three different {lambda} L chains (Ref.6 and refs. therein). C4 has not been found to be expressed. Silencing of the {lambda} locus was conducted in four successive steps by targeted introduction of three loxP sequences into the C1 and, separately, C2 region of the {lambda} locus (Fig. 1). Introduction of the C3-C1 targeting construct (Fig. 1B, right) silenced C1, and germline transmission mice were produced that, upon mating with ubiquitous Cre expressers, had C3-C1 deleted on both alleles. Such mice, bred into the 129/Ola background, were used for the derivation of ES cells, which allowed homologous integration and silencing of C2 (Fig. 1B, left). Germline transmission mice were obtained and bred with the Cre expressers and each other, which resulted in homozygous animals with a C2 to C1 deletion of ~120 kb (Fig. 1C). Analysis of ES cells and mice by Southern blot and PCR, with representative examples shown in Fig. 1D, identified homologous integration and locus deletion and resulted in separate animals with the following genes silenced: {lambda}C1-/-; {lambda}C1-/-, and {lambda}C3-/-; {lambda}C1-/-, {lambda}C2-/-, and {lambda}C3-/-; and deletion of {lambda}C1-/-, {lambda}C2-/-, {lambda}C3-/-, and {lambda}C4-/-. These mouse strains were crossed into the {kappa}-/- background, and the resulting homozygous animals were termed according to their silenced or deleted ({Delta}) C genes: {lambda}1-/-{kappa}-/-, {lambda}1.3-/-{kappa}-/-, {lambda}1.3.2-/-{kappa}-/-, and {lambda}1–2{Delta}-/-{kappa}-/-. Deletion of the {lambda} locus was verified by sequencing of the 686-bp PCR fragment shown in Fig. 1D, which contained the 3'J2 and 3'C1 region separated by loxP following Cre recombination.

Block in development at the immature B cell stage

The different {lambda} knockout mice, crossed to homozygosity with {kappa}-/- mice, were analyzed to establish the effect of reduced numbers of L chain genes on B cell development (Fig. 2 and Table I). For this, bone marrow cells were stained for B220 in combination with c-Kit, CD43, CD25, and µ H chain and were analyzed by flow cytometry (Fig. 2A). This identified very similar ratios of early B cell levels compared with normal mice, which established that pro- and pre-B cell development was little affected by partial or complete loss of L chain expression. However, a dramatic change was visible at the stage when L chain rearrangement should have been completed (38), and in {kappa}-/- mice the level of IgM+ B220+ cells was reduced by >50% compared with that in normal mice. A further decrease of >50% was seen in both {lambda}1-/-{kappa}-/- and {lambda}1.3-/-{kappa}-/- mice, and this drop in expression was in line with the observation that C1 is the most frequently expressed {lambda} L chain (4). This highlights the rather inefficient expression of {lambda} L chain genes (39, 40) and shows that the lack of L chain in {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice blocks development at the pre-B-II to immature B cell transition stage when immature B cells fail to express surface IgM. As a consequence of this complete block in B cell maturation, no surface Ig+ cells were found in the spleen of L chain-deficient mice (Fig. 2B).


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  		Table I. Cell numbers in spleen and bone marrow of normal, {kappa}-/-, and C{lambda} deletion micea

 
µ H chain polypeptide is retained in the cytoplasm of immature B cells

At the pre-B-II to immature B cell transition stage, CD25 expression is revoked, surrogate L chain is no longer expressed, and BCR formation is initiated by productive rearrangement of a {kappa} or {lambda} L chain, which can associate with a µ H chain. During this stage large CD25+ pre-B-II cells differentiate after several divisions into small CD25+ resting pre-B-II cells, which are in the process of rearranging their L chain genes (13). The arrest in development at the pre-B-II transitional stage in mice with silenced L chain genes raises the question of whether the levels of large and small resting CD25+ B cells are maintained. To analyze the distribution of CD25+ B220+ lymphocytes, cell numbers were plotted against cell size. Fig. 3A shows that the levels of large and small pre-B-II cells were adequately maintained and very similar in {lambda}1–2{Delta}-/-{kappa}-/-, {kappa}-/- and normal mice. As this implies that developmental progression was fully sustained up to the stage when L chain rearrangement and expression should have been initiated, we wondered how H chain expression and maintenance would be affected. Cytoplasmic staining of µ H chain (Fig. 3B) revealed that CD25+ B220+ gated lymphocytes in {lambda}1–2{Delta}-/-{kappa}-/- mice stained with intensity similar to that of control cells from normal mice. To further evaluate the size and quantity of cytoplasmic µ H chain products, bone marrow cells were lysed, and H chain was captured with anti-µ coupled to Sepharose and gel-fractionated. Fig. 4 shows that the m.w. of single cytoplasmic µ H chain was indistinguishable in normal and L chain-silenced mice, while no µ chains were secreted or released into the serum of {lambda}1–2{Delta}-/-{kappa}-/- mice. These results emphasize that H chain rearrangement and µ polypeptide production is L chain independent and that normal B cell development is fully maintained up to the differentiation stage when H and L chain associate as BCR.



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  		FIGURE 3. Cytoplasmic staining of CD25+ bone marrow B cells from {lambda}1–2{Delta}-/-{kappa}-/- and normal (NM) mice. A, Separation of B cells according to their size. B, Cytoplasmic staining with FITC-coupled anti-µ.

 


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  		FIGURE 4. The µ H chain is retained in the cytoplasm, but not secreted. Abs from {lambda}1–2{Delta}-/-{kappa}-/- and normal (NM) mice in serum and from lysed bone marrow cells were captured with anti-µ coupled to Sepharose, gel-fractionated, and visualized by incubation with biotinylated anti-µ and chemiluminescent detection.

 
B cell reduction upon C{lambda} gene removal

Silencing individual L chain genes initiated a sharp decline in IgM+ B cells. In normal mice (kept in the same pathogen-free conditions as the L chain mutants), ~9 x 105 IgM+ B cells were found in the bone marrow of one femur, but this was reduced to ~4 x 105 in {kappa}-/- mice, to ~2 x 105 in {lambda}1-/-{kappa}-/- and {lambda}1.3-/-{kappa}-/- mice, and to undetectable levels in both {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice (Table I and Fig. 2A). Although IgM+ cells, present at reduced levels, continued to mature, they did not fully reconstitute B cell numbers in secondary lymphoid organs; the levels of Ig+ B cells in the spleen of mice with depleted L chain genes were significantly reduced and were undetectable in {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice (Fig. 2B). The total cell numbers (and weights, data not shown) of the spleen were also markedly reduced in the L chain-depleted mice, by more than half for the {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- animals. Remaining expression of C3 and/or C2 in {lambda}1-/-{kappa}-/- and {lambda}1.3-/-{kappa}-/- mice reduced the levels of Ig+ B cells to about half the number in {kappa}-/- mice (Table I). Unexpectedly, {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice derived from heterozygous females or foster mothers had significant Ab titers in serum, still detectable by ELISA 6 wk after weaning. However, serum analyses from such mice older than 3 mo showed that no Abs remained, and furthermore, that rigorous immunizations of {lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice did not elicit Ab production or H chain release (data not shown). The lack of serum Ig in {lambda}1.3.2-/-{kappa}-/- mice confirmed that C{lambda}4 is a pseudogene and that the remaining V{lambda} genes cannot be expressed using an as yet unknown C gene in the {lambda} locus. Furthermore, silencing of the L chain loci established that B cell development is L chain dependent and that there are no other genes that compensate for L chain deficiency.

Complete lack of mature B cells

The diminution (reduction in cell numbers and size) of the spleens of L chain-depleted mice can be presumed to result from their lack of mature B cells, as demonstrated in Fig. 5. Spleen sections of normal and L chain-deficient mice were stained with biotinylated anti-µ and were visualized by streptavidin coupled to TRITC and FITC-labeled anti-CD4. In L chain-deficient mice no IgM+ cells could be identified, and precipitate of the anti-µ Ab formed yellow/brown spots, whereas staining of IgM+ cells was accomplished in normal mouse sections performed in parallel. Thus, there was a distinct lack of follicular and marginal zone B cells, while staining for T cells appears to be normal. This is in agreement with the more quantitative analysis of B cell numbers made by flow cytometry (Fig. 2) and provides evidence for the proposition that B cell migration to the spleen is BCR mediated (41). Further analysis of secondary lymphoid organs, such as the peritoneum, established the lack of both B-1 and B-2 cells, while other cell populations, such as T cells, remained at normal levels (Fig. 6). This general lack of mature B cells confirmed that the L chain is an essential requisite to B cell development and that migration of µ+ B cells from the bone marrow is prohibited.



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  		FIGURE 5. There was a lack of IgM+ cells in the spleen of L chain-deficient mice. Splenic cryosections from a normal mouse and a {lambda}1–2{Delta}-/-{kappa}-/- mouse were stained for B and T cell populations with biotinylated anti-µ, visualized with TRITC-labeled streptavidin HRP- and FITC-labeled anti-CD4.

 


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  		FIGURE 6. The peritoneum of L chain-negative mice is depleted of B cells. Flow cytometric analysis (50,000 cells/profile) of B and T cell populations in the peritoneum of {kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/- mice. Cells were stained with PE-conjugated anti-CD5 and allophycocyanin-conjugated anti-B220.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In L chain-deficient mice, B cell development is aborted at the pre-B-II to immature B cell stage when surface IgM receptor expression should have been accomplished. The BCR is required to initiate emigration from the bone marrow to the periphery (42, 43, 44). This complete block in development at an important checkpoint prevents B cell maturation, and mice without Ig L chain are immunodeficient regarding Ab-expressing B cells. The surrogate L chain encoded by VpreB and {lambda}5 does not sustain B cell development, and with the failure to express L chain polypeptides, B cell differentiation ceases exactly at the stage when L chain rearrangement should have been completed (20, 45). This re-emphasizes the importance of L chain for immune development and that, at least in the mouse, there is no gene or rescue event that can compensate for L chain deficiency.

B cell development in the mouse has been extensively studied by gene targeting, and in one of the early experiments the µ transmembrane exons were rendered nonfunctional, which prevented surface IgM expression (14). This caused a block in development in µMT mice, leading to the accumulation of pre-B-I cells and the disappearance of pre-B-II cells. With the lack of pre-BCR assembly and surface IgM expression, no differentiation into immature or mature B cells was obtained, although DNA rearrangement was maintained. Indeed, µMT mice do rearrange H and L chain genes, while mice with deleted JH segments only maintain L chain rearrangement (20). This is in agreement with the results of our L chain-deficient mice ({lambda}1.3.2-/-{kappa}-/- and {lambda}1–2{Delta}-/-{kappa}-/-), which show H chain rearrangement and cytoplasmic Igµ expression, and thus reiterate that H and L chain rearrangement and expression are independent events. The critical importance of the BCR in signaling and normal progression of development through the different B cell maturation stages was further analyzed by gene targeting of individual BCR components (reviewed in Ref.26). The results showed that silencing of some genes, such as C{kappa}, had a moderate effect on B cell development and is well tolerated, while silencing of BCR components such as Ig{beta} blocks any progress in development (11, 20, 42, 46, 47). The block in B cell development was frequently accompanied by the accumulation of cells before the stage of differentiation when the silenced gene should be active (20, 42, 48, 49). This is not seen at any pro- or pre-B cell stage in the {lambda}-/-{kappa}-/- mice, and the levels of CD25+ large and small B cells immediately before the block in development are very similar to those in normal mice. A reason for this may be that the cells entering the pre-B-II stage and those undergoing apoptosis at this developmental checkpoint, perhaps half the CD25+ cells generated in the bone marrow, die without maturing into IgM+ B cells, allowing fairly constant cell levels to be maintained (18, 19, 20, 44). In L chain-deficient mice, pro- and pre-B cell levels are indistinguishable from the control levels, which suggests that L chain expression, or rather the lack of it, at these early stages does not interfere with B cell development up to the immature stage. Rajewsky and co-workers (20) argue that early L chain expression may at least in principle allow CD43+ B cells to express surface IgM and develop into immature B cells by omitting passage through the small pre-B-II stage. A major decrease in pre-B-II cells was also found in surrogate L chain-deficient mice, but impaired transition from the pre-B-I to the pre-B-II cell stage did not appear to influence the transition to the immature stage (46). At the immature B cell stage, L chain expression appears to compensate for the deficiency induced by the lack of surrogate L chain; however, it may also be possible that early L chain rearrangement in pro- or pre-B-I cells allows their rapid expansion independently of the pre-BCR (Ref.13 and references therein). This hypothesis can now be tested by the introduction of a rearranged L chain gene into the L chain-deficient mouse strain.

The importance of L chain expression was studied in RAG-1- and RAG-2-deficient mice where B cell development is arrested at the B220+CD43+ pro-B cell stage (15, 16). Complementation with a rearranged membrane µ H chain gene allowed progression in development to the small pre-B cell stage, but Igµ was only expressed in the cytoplasm and not on the cell surface. This is in agreement with the observation that L chain is crucial in facilitating dissociation of H chain binding protein and transport to the cell surface (50, 51). However, it appears that H chain-only B cell precursors can be matured under enforced Bcl-2 expression, which allows surface µ expression and accumulation of mature B cells in the periphery (22). In the bone marrow of RAG-1-/-{lambda}5-/- mice carrying a rearranged H chain, transition from pro-B- to pre-B cell and surface IgM expression was only seen when either {lambda}5 or a rearranged L chain was introduced (52). Nussenzweig and colleagues (52) argued that when neither {lambda}5 nor conventional L chain are expressed, B cell development cannot proceed past the pro-B cell stage. This is not seen in our L chain-deficient mice, where development progresses to the pre-B-II stage, nor in surrogate L chain-deficient animals, which show near normal B cell maturation (46).

Interestingly, an important role has been attributed to the surrogate L chain in allelic exclusion at the pre-B cell stage, and it has been suggested that while normal mice allelically exclude, {lambda}5-deficient mice allelically include and produce increased levels of productive VHDJH rearrangements on the second allele (53). However, others found that both H chain alleles are productively rearranged in a minority of B cells, but in {lambda}5-deficient as well as normal mice only one allele is expressed on the surface (54). This raises the question of whether mice deficient for surrogate and normal L chain, which cannot express a pre-BCR or BCR, do accumulate larger numbers of cells with two productive H chain polypeptides. Cross-breeding of the L chain-deficient ({lambda}-/-{kappa}-/-) and surrogate L chain-deficient strains (46) will answer this and may support the suggestion that allelic exclusion after successful rearrangement of both H chain alleles, which with an incidence of a few percent is a relatively rare event, is mediated by deposition of a pre-B or B cell receptor on the surface (54).

Ab development without L chain has been reported in the camel, where about half of all Ig is in the H2 form (29). Indeed, immunizations of camelids generated highly specific H2 IgGs, which can also be expressed in the mouse (30) (M. Brüggemann and X. Zou, unpublished observation). In the camel it appears that µH chain rearrangement and expression, and possibly switching, are circumvented when H2 IgG is expressed (30). Although little is known about B cell development in the camelidae, the structure of camel H2 IgG reveals remarkable information about how H chain expression without L chain could be achieved. Particular V genes have hydrophobic to hydrophilic amino acid substitutions that prohibit L chain association; however, these VHH genes rearrange to the same D-J segments that allow conventional H2L2 Ig to be expressed. In addition, several camel C{gamma} genes have a point mutation in the 3'C721 splice consensus sequence that precludes the use of this exon (55). Expression without CH1 prevents H chain retention in the cytoplasm (56, 57), and this may allow B cell development without L chain. In other words, V gene mutation and removal of CH1 may allow H chain-only B cell development, which is perhaps an ancient process to produce Ab repertoires without L chain.


   Acknowledgments
 
We thank Geoff Morgan for help with the FACS analysis, Michael Neuberger and Ursula Storb for {lambda} clones and information about locus layout, John Coadwell for computation, Sarah Bell and Fatima Santos for help with staining and analysis of tissue sections, and Lill Martensson and Martin Turner for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by a European Union program grant and by the Babraham Institute. Back

2 Address correspondence and reprint requests to Dr. Marianne Brüggemann, Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge, U.K. CB2 4AT. E-mail address: marianne.bruggemann{at}bbsrc.ac.uk Back

3 Abbreviations used in this paper: BCR, B cell receptor; C, constant region gene; D, diversity segment; ER, endoplasmic reticulum; ES, embryonic stem; H, Ig heavy chain; RAG, recombination-activating gene; TRITC; tetramethylrhodamine isothiocyanate. Back

Received for publication October 10, 2002. Accepted for publication November 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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