Download Light Chain λ and Ig κ Immature B Cell Stage in Mice Without Ig

Block in Development at the Pre-B-II to
Immature B Cell Stage in Mice Without Ig κ
and Ig λ Light Chain
This information is current as
of June 18, 2017.
Xiangang Zou, Tony A. Piper, Jennifer A. Smith, Nicholas
D. Allen, Jian Xian and Marianne Brüggemann
J Immunol 2003; 170:1354-1361; ;
doi: 10.4049/jimmunol.170.3.1354
http://www.jimmunol.org/content/170/3/1354
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References
The Journal of Immunology
Block in Development at the Pre-B-II to Immature B Cell
Stage in Mice Without Ig␬ and Ig␭ Light Chain1
Xiangang Zou, Tony A. Piper, Jennifer A. Smith, Nicholas D. Allen, Jian Xian, and
Marianne Brüggemann2
Silencing individual C (constant region) ␭ genes in a ␬ⴚ/ⴚ background reduces mature B cell levels, and L chain-deficient
(␭ⴚ/ⴚ␬ⴚ/ⴚ) 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. The Journal of Immunology, 2003, 170: 1354 –1361.
B
Laboratory of Developmental Immunology, The Babraham Institute, Babraham,
Cambridge, United Kingdom
Received for publication October 10, 2002. Accepted for publication November
18, 2002.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a European Union program grant and by the
Babraham Institute.
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@bbsrc.ac.uk
Copyright © 2003 by The American Association of Immunologists, Inc.
inefficient. However, despite the lack of ␬ 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 ␮ H chain can pair with a surrogate L chain,
consisting of VpreB and ␭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 ␮ 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 –17). Cell surface expression of the preBCR induces proliferation, and after several divisions large preB-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 ␣-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 ␮ 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).
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.
0022-1767/03/$02.00
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cells express surface Ig with either ␬ or ␭ L chain, a
choice termed isotype exclusion. The proportion of Abs
containing a ␬ or ␭ L chain varies considerably in the
different species, but in the mouse only a few percent of Abs carry
␭ (1). L chain genes are encoded by two different loci, and in the
mouse there are an extensive number of V ␬ gene segments upstream of five J ␬ and one C␬ gene (2). The mouse ␭ 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-C3J1-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 ␬ locus is ⬎10 times larger than the ␭ locus, with
⬎100 V genes, this extensive complexity is not regarded as the
reason why most mouse Abs carry a ␬ L chain. It may be that the
mouse ␬ locus is simply more efficient in DNA rearrangement,
which is supported by the finding that in the majority of cells with
rearranged V␬ the ␭ locus is still in germline configuration, while
in most cells expressing ␭ L chain the ␬ locus is either nonproductively rearranged or deleted (6).
Several mouse strains with silenced ␬ L chain locus have been
described. They were generated by homologous integration of a
selectable marker gene in C␬ or targeted removal of C␬ or J␬
(7–11). Silencing expression of ␬ L chain shed light on isotype
exclusion and L chain activation, and it was concluded that ␬ and
␭ expression are separate and independent events. Although homozygous ␬⫺/⫺ mice compensate for the ␬ deficiency with increased
␭ production, their splenic B cells and ␮⫹ cells in the bone marrow
are reduced compared with those in normal mice (7, 9), which may
suggest that ␭ L chain rearrangement and expression are relatively
The Journal of Immunology
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
Targeting constructs
A phage ␭ 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␭ and C␭ probe
(clone 505; provided by M. Neuberger, Medical Research Council), which
identified several clones containing V␭ and, separately, C␭ 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␭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␭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.
FIGURE 1. Targeted integration and deletion of the mouse ␭ L chain locus. A, The ␭ 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– 6) that identify wild-type configuration, targeted integration, and C␭ 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⌬⫹/⫺ and ES1.3⌬⫺/⫺) with digests and probes (A–D) indicated. PCR
analysis of tail DNA identified the configuration of the Ig␭ 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.
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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␣/Ig␤ 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␣, or
Ig␤ 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 ␥
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 ␮ 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,
1355
1356
Analysis of homologous integration
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. ␭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 ␭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 ⫻ 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 ␭ES3.1⌬⫺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: ␭C1⫺/⫺ (mouse 130 ⫽ ES1.3); ␭C1⫺/⫺ and
␭C3⫺/⫺ (mouse 1.3 ⫽ ES1.3⌬); ␭C1⫺/⫺, ␭C2⫺/⫺, and ␭C3⫺/⫺ (mouse
50 ⫽ ES2.4); and deletion (⌬) of ␭C1⫺/⫺, ␭C2⫺/⫺, ␭C3⫺/⫺, and ␭C4⫺/⫺
(mouse 1.3–2.4⌬). Crossing into the ␬⫺/⫺ background established four
mouse strains with different functional L chain content: ␭1⫺/⫺␬⫺/⫺,
␭1.3⫺/⫺␬⫺/⫺, ␭1.3.2⫺/⫺␬⫺/⫺, and ␭1–2⌬⫺/⫺␬⫺/⫺.
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), FITCconjugated monoclonal rat anti-mouse IgM (␮ 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 (␮-chain specific;
02082D; BD PharMingen), FITC-conjugated anti-mouse IgD (02214D;
BD PharMingen), biotin- or FITC-conjugated anti-mouse Ig␭ (02172D,
02174D; BD PharMingen), and/or PE-conjugated anti-mouse Ig␬ (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).
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), ␬⫺/⫺, ␭1⫺/⫺␬⫺/⫺, ␭1.3⫺/⫺␬⫺/⫺, ␭1.3.2⫺/⫺␬⫺/⫺, and ␭1–2⌬⫺/⫺␬⫺/⫺ 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 allophycocyaninconjugated anti-B220. Spleen cells were stained with biotin-conjugated anti-IgM, FITC-conjugated anti-IgD, biotin- or FITC-conjugated anti-␭, and/or
PE-conjugated anti-␬ and allophycocyanin-conjugated anti-B220 for setting the B lymphocyte gate.
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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 ␭ES3.1⌬-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 ␭ 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⬘-GGA
GATCAGGAATGAGGGACAAAC-3⬘ (4⬎); 3⬘tkNeorev, 5⬘-CTCGACGG
ATCCGTCGAGGAATTCC-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.
L CHAIN KNOCKOUT MICE
The Journal of Immunology
1357
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 antimouse IgM (␮-chain specific, 04-6811; Zymed), PE-conjugated antimouse CD45R (B220; 01125A, 01129A; BD PharMingen), and biotinconjugated 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 ␮m) of spleens fixed with cold acetone, blocked with
10% rat serum, and stained with biotinylated anti-␮ (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.
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
(␮-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 ␮
(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
Silencing of the mouse ␭ L chain locus
To investigate B cell development without L chain we produced
mice with a deleted Ig␭ locus. The ␭⫺/⫺ mice were crossed with
animals carrying a nonfunctional Ig␬ locus, ␬⫺/⫺ mice, also obtained by gene targeting (11). The mouse ␭ L chain locus contains
three V region genes, four J segments, and four C region genes that
can independently rearrange and express three different ␭ L chains
Block in development at the immature B cell stage
The different ␭ knockout mice, crossed to homozygosity with ␬⫺/⫺
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 ␮ 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 proand 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 ␬⫺/⫺ 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 ␭1⫺/⫺␬⫺/⫺ and ␭1.3⫺/⫺␬⫺/⫺
mice, and this drop in expression was in line with the observation that
C1 is the most frequently expressed ␭ L chain (4). This highlights the
Table I. Cell numbers in spleen and bone marrow of normal, ␬⫺/⫺, and C␭ deletion micea
Organ
NM
␬⫺/⫺
␭1⫺/⫺ ␬⫺/⫺
␭1.3⫺/⫺ ␬⫺/⫺
␭1.3.2⫺/⫺ ␬⫺/⫺
␭1–2⌬⫺/⫺ ␬⫺/⫺
Bone marrow
Total cell no. ⫻ 106b
c-Kit⫹, B220⫹ pro/pre-B-I cells
B220⫹, CD43⫹ pro/pre-B-I cells
B220⫹, CD25⫹ pre-B-II B cells
B220⫹, IgM⫹ immature/mature B cells
9.5 ⫾ 1.9
0.5 ⫾ 0.3
0.4 ⫾ 0.2
1.2 ⫾ 0.4
0.9 ⫾ 0.3
8.9 ⫾ 1.4
0.4 ⫾ 0.2
0.4 ⫾ 0.2
1.1 ⫾ 0.3
0.4 ⫾ 0.2
8.5 ⫾ 2.1
0.4 ⫾ 0.2
0.3 ⫾ 0.2
1.1 ⫾ 0.3
0.2 ⫾ 0.1
9.2 ⫾ 1.4
0.3 ⫾ 0.2
0.4 ⫾ 0.2
0.9 ⫾ 0.4
0.2 ⫾ 0.1
8.9 ⫾ 1.5
0.4 ⫾ 0.2
0.4 ⫾ 0.2
1.0 ⫾ 0.3
Not
detectablec
8.8 ⫾ 1.8
0.4 ⫾ 0.3
0.4 ⫾ 0.2
1.0 ⫾ 0.5
Not
detectablec
Spleen
Total cell no. ⫻ 107
B220⫹
IgM⫹
5.6 ⫾ 1.2
2.9 ⫾ 0.6
2.5 ⫾ 0.5
5.0 ⫾ 1.4
2.2 ⫾ 0.8
2.0 ⫾ 0.7
2.9 ⫾ 1.1
1.2 ⫾ 0.7
0.9 ⫾ 0.4
2.6 ⫾ 1.4
1.2 ⫾ 0.4
1.0 ⫾ 0.6
IgD⫹
2.2 ⫾ 0.5
1.8 ⫾ 0.7
0.8 ⫾ 0.4
0.9 ⫾ 0.6
⫹
2.9 ⫾ 0.7
2.1 ⫾ 0.9
1.2 ⫾ 0.5
1.2 ⫾ 0.4
1.3 ⫾ 0.7
(4 –7 ⫻ 104)
Not
detectablec
Not
detectablec
Not
detectablec
1.2 ⫾ 0.7
(3– 6 ⫻ 104)
Not
detectablec
Not
detectablec
Not
detectablec
IgL
a
Four to six mice (⬎3 mo old) were used, and cells were stained with relevant Abs for the listed features (see Materials and Methods) and analyzed by FACS. Total cell
numbers were determined by trypan blue staining.
b
Cells were from one femur.
c
Levels were ⬍103.
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Protein analysis
(Ref. 6 and refs. therein). C4 has not been found to be expressed.
Silencing of the ␭ locus was conducted in four successive steps by
targeted introduction of three loxP sequences into the C1 and, separately, C2 region of the ␭ 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: ␭C1⫺/⫺; ␭C1⫺/⫺, and
␭C3⫺/⫺; ␭C1⫺/⫺, ␭C2⫺/⫺, and ␭C3⫺/⫺; and deletion of ␭C1⫺/⫺,
␭C2⫺/⫺, ␭C3⫺/⫺, and ␭C4⫺/⫺. These mouse strains were crossed
into the ␬⫺/⫺ background, and the resulting homozygous animals
were termed according to their silenced or deleted (⌬) C genes:
␭1⫺/⫺␬⫺/⫺, ␭1.3⫺/⫺␬⫺/⫺, ␭1.3.2⫺/⫺␬⫺/⫺, and ␭1–2⌬⫺/⫺␬⫺/⫺.
Deletion of the ␭ 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.
1358
rather inefficient expression of ␭ L chain genes (39, 40) and shows
that the lack of L chain in ␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺ 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).
␮ H chain polypeptide is retained in the cytoplasm of immature
B cells
FIGURE 3. Cytoplasmic staining of CD25⫹ bone marrow B cells from
␭1–2⌬⫺/⫺␬⫺/⫺ and normal (NM) mice. A, Separation of B cells according
to their size. B, Cytoplasmic staining with FITC-coupled anti-␮.
and expression should have been initiated, we wondered how H chain
expression and maintenance would be affected. Cytoplasmic staining
⫹
of ␮ H chain (Fig. 3B) revealed that CD25 B220⫹ gated lympho⫺/⫺ ⫺/⫺
cytes in ␭1–2⌬ ␬
mice stained with intensity similar to that of
control cells from normal mice. To further evaluate the size and quantity of cytoplasmic ␮ H chain products, bone marrow cells were lysed,
and H chain was captured with anti-␮ coupled to Sepharose and gelfractionated. Fig. 4 shows that the m.w. of single cytoplasmic ␮ H
chain was indistinguishable in normal and L chain-silenced mice,
while no ␮ chains were secreted or released into the serum of ␭1–
2⌬⫺/⫺␬⫺/⫺ mice. These results emphasize that H chain rearrangement and ␮ 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.
B cell reduction upon C␭ 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 ⫻ 105 IgM⫹ B cells were
found in the bone marrow of one femur, but this was reduced
to ⬃4 ⫻ 105 in ␬⫺/⫺ mice, to ⬃2 ⫻ 105 in ␭1⫺/⫺␬⫺/⫺ and
␭1.3⫺/⫺␬⫺/⫺ mice, and to undetectable levels in both ␭1.3.2⫺/
⫺/⫺
⫺␬
and ␭1–2⌬⫺/⫺␬⫺/⫺ 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
␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺ 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 ␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺ animals. Remaining
expression of C3 and/or C2 in ␭1⫺/⫺␬⫺/⫺ and ␭1.3⫺/⫺␬⫺/⫺ mice
reduced the levels of Ig⫹ B cells to about half the number in ␬⫺/⫺
mice (Table I). Unexpectedly, ␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺
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 ␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺ mice did
not elicit Ab production or H chain release (data not shown). The
lack of serum Ig in ␭1.3.2⫺/⫺␬⫺/⫺ mice confirmed that C␭4 is a
pseudogene and that the remaining V␭ genes cannot be expressed
using an as yet unknown C gene in the ␭ locus. Furthermore,
silencing of the L chain loci established that B cell development is
FIGURE 4. The ␮ H chain is retained in the cytoplasm, but not secreted.
Abs from ␭1–2⌬⫺/⫺␬⫺/⫺ and normal (NM) mice in serum and from lysed
bone marrow cells were captured with anti-␮ coupled to Sepharose, gelfractionated, and visualized by incubation with biotinylated anti-␮ and
chemiluminescent detection.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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 ␬ or ␭ L
chain, which can associate with a ␮ 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 ␭1–2⌬⫺/⫺␬⫺/⫺,
␬⫺/⫺ and normal mice. As this implies that developmental progression was fully sustained up to the stage when L chain rearrangement
L CHAIN KNOCKOUT MICE
The Journal of Immunology
1359
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 ␭1–2⌬⫺/⫺␬⫺/⫺ mouse were
stained for B and T cell populations with biotinylated
anti-␮, visualized with TRITC-labeled streptavidin
HRP- and FITC-labeled anti-CD4.
L chain dependent and that there are no other genes that compensate for L chain deficiency.
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-␮ 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-␮ 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 ␮⫹ B cells from the bone marrow is prohibited.
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 ␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺ mice. Cells were
stained with PE-conjugated anti-CD5 and allophycocyanin-conjugated
anti-B220.
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– 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 ␭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 ␮ transmembrane exons were rendered nonfunctional, which prevented
surface IgM expression (14). This caused a block in development
in ␮MT 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, ␮MT 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 (␭1.3.2⫺/⫺␬⫺/⫺ and ␭1–2⌬⫺/⫺␬⫺/⫺), which
show H chain rearrangement and cytoplasmic Ig␮ 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␬, had a moderate
effect on B cell development and is well tolerated, while silencing
of BCR components such as Ig␤ 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 ␭⫺/⫺␬⫺/⫺ 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 –20, 44).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Complete lack of mature B cells
Discussion
1360
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␥ 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 ␭ 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.
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