A VH12 Transgenic Mouse Exhibits Defects
in Pre-B Cell Development and Is Unable to
Make IgM+ B Cells
This information is current as
of June 18, 2017.
Hongsheng Wang, Jian Ye, Larry W. Arnold, Suzanne K.
McCray and Stephen H. Clarke
J Immunol 2001; 167:1254-1262; ;
doi: 10.4049/jimmunol.167.3.1254
http://www.jimmunol.org/content/167/3/1254
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References
A VH12 Transgenic Mouse Exhibits Defects in Pre-B Cell
Development and Is Unable to Make IgMⴙ B Cells1
Hongsheng Wang,2 Jian Ye,2,3 Larry W. Arnold, Suzanne K. McCray, and Stephen H. Clarke4
T
he first Ig receptor expressed by B-lineage cells is the
pre-B cell Ag receptor (pre-BCR).5 It is composed of at
least three parts: the Ig ␮H-chain, the surrogate L chain
components ␭5 and VpreB (1–5), and the transmembrane signal
transduction molecules Ig␣ and Ig␤ (6, 7). Not all pre-B cells
express a pre-BCR complex upon productive rearrangement of the
H chain VH, D, and JH gene segments. The inability of a pre-B cell
to display a pre-BCR results in cell death, be it due to the inability
to make an H chain that can pair with surrogate L chain, the inability to make the surrogate L chain, or the inability to signal
through the pre-BCR (8 –13). The pre-BCR is also essential for
mediating allelic exclusion (11, 12, 14), and for initiating changes
associated with differentiation to a pre-BII cell, including L chain
gene rearrangement (15–17). Whether all of these events occur as
a result of just one signal by the pre-BCR or multiple signals is
unknown.
B cell development follows a set pathway involving changes in
expression of a variety of cell surface and cytoplasmic proteins,
and rearrangement of Ig H and L chain genes (14, 18 –20). Pre-BI
cells are the first B-lineage cells to have undergone an Ig gene
rearrangement. These cells have a D to JH rearrangement on one or
both H chain alleles, but lack VH and VL gene rearrangements (18,
21). They undergo VH to DJH rearrangement, and those that ac-
Department of Microbiology and Immunology, University of North Carolina, Chapel
Hill, NC 27599
Received for publication May 17, 2001. Accepted for publication May 23, 2001.
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 by National Institutes of Health Grants AI29576 and
AI43587 and a grant from the Arthritis Foundation to S.H.C.
2
H.W. and J.Y. contributed equally to this work.
3
Current address: Curagen Corporation, 322 East Main Street, Branford, CT 06405.
4
Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department
of Microbiology and Immunology, CB#7290, 804 Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599. E-mail address: [email protected]
5
Abbreviations used in this paper: BCR, B cell Ag receptor; BrdU, 5-bromo-2⬘deoxyuridine; CDR3, third complementarity-determining region; PtC, phosphatidylcholine; Tg, transgenic; RAG, recombination-activating gene.
Copyright © 2001 by The American Association of Immunologists
quire a productive (in-frame) rearrangement express cytoplasmic
␮ (22, 23). Not all H chains are able to associate with surrogate L
chain (11, 24), but those cells that have a H chain that can associate
with surrogate L chain express pre-BCRs on their surface. These
cells are pre-BII cells and they are the most abundant pre-B cell
type in the mouse bone marrow (20). Cells that enter this compartment are initially pre-BCR⫹, large, and cycling, but transition
into smaller noncycling pre-BCR⫺ cells as they mature (18, 25).
Small pre-BII cells undergo L chain gene rearrangement (18, 25),
and those that express an L chain that can pair with the H chain
express surface IgM and are defined as immature B cells. These
cells exit the bone marrow and migrate to the spleen, where they
differentiate to mature recirculating B cells.
We have followed the differentiation of B cells expressing a
single VH gene segment, VH12, because VH12 B cells provide an
unusual window on B cell development. Most VH12 B cells in
adult mice bind the common phospholipid phosphatidylcholine
(PtC) and are B-1 (26, 27). All VH12 H chains from these cells
have a 10/G4 third complementarity-determining region (CDR3)
and pair with V␬4/5H L chains (26 –28). There is a strong bias for
the differentiation of VH12 B cells with the ability to bind PtC.
First, the majority (⬃95%) of VH12-expressing cells are selectively lost during the transition from pre-BI to pre-BII (29). VH12
pre-B cell survival appears to be dependent on the structure of the
H chain CDR3; those with a 10/G4 CDR3 are favored for survival,
while those of other CDR3 sequences (designated non-10/G4) are
generally disfavored. Non-10/G4 VH12 H chains can associate
with surrogate L chain and be expressed on the cell surface as a
pre-BCR in cells of a pre-B cell line (29), indicating that the inability to support pre-B cell differentiation is not necessarily due to
an inability to form a pre-BCR. Second, 10/G4 VH12 H chains are
unable to associate with most L chains (30), and V␬4/5H is one of
the few L chains with which it will pair. This bias in association in
part creates a high frequency of B cells that can bind PtC. These
cells are then selected into the B-1 subset (26, 31, 32). The combination of selection for VHCDR3 and V␬4/5H strongly implies
that PtC-specific B-1 cells have important survival value. Indeed,
anti-PtC Abs have been demonstrated to provide protection against
certain bacterial infections (33).
0022-1767/01/$02.00
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VH12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine.
Selection begins with the VH third complementarity-determining region (CDR3) at the pre-B cell stage, in which most VH12 pre-B
cells are selectively eliminated, enriching for those with VHCDR3s of 10 aa and a fourth position Gly (designated 10/G4). To
understand this selection, we compared B cell differentiation in mice of two VH12 transgenic lines, one with the favored 10/G4
VHCDR3 and one with a non-10/G4 VHCDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small
pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B
cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor
expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that VH12 H chains have evolved such
that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells. The Journal of
Immunology, 2001, 167: 1254 –1262.
The Journal of Immunology
To understand the events that affect the loss of non-10/G4 preBII cells, we compared B cell differentiation between 10/G4 and
non-10/G4 VH12 transgenic (Tg) mice. The 10/G4 VH12 Tg mice
(6-1 mice) were previously generated and described (26), and produce B cells of both the conventional and B-1 subsets (26). We
report in this work that non-10/G4 VH12 Tg mice carrying an 8/G0
VH12 rearrangement are similar to 10/G4 VH12 Tg mice in that
both drive differentiation to the pre-BII cell stage, exclude endogenous gene rearrangement, and initiate L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are
deficient in cell division at the large pre-BII cell stage and are
unable to generate Tg-expressing B cells. Thus, 8/G0 B cell differentiation is deficient at both the pre-B and B cell stages.
Materials and Methods
1255
Analysis of transcripts
Bone marrow pre-B cells were purified in a two-step process using magnetic beads. Bone marrow cells were stained with biotin anti-IgM and
incubated with streptavidin-coated Dynabeads M-280 magnetic beads (Dynal, Lake Success, NY). The IgM⫹ cells were removed by magnet. Bone
marrow cells depleted of IgM⫹ cells contained ⬍1% IgM⫹ cells. These
cells were incubated with anti-B220-coated beads (Miltenyi Biotec, Auburn, CA) and B220⫹IgM⫺ cells separated by magnet. These cells were
⬎95% pure based on flow cytometry analysis. Total RNA was extracted
from purified cells, and RT-PCR of C␮ transcripts was done by 5⬘-RACE
(Life Technologies, Gaithersburg, MD), according to the manufacturer’s
instructions. cDNA was prepared using an oligonucleotide complementary
to the first exon of C␮ (5⬘-ATCCTTGAAGGTTCAG-3⬘). The PCR was
performed using a poly(G) oligonucleotide supplied by the manufacturer
and a C␮ oligonucleotide (5⬘-TTCACCTGGAACTACCAGAAC-3⬘),
which is internal to the C␮ oligonucleotide used to generate the cDNA. The
RT-PCR products were cloned into the pAMP vector (Life Technologies)
and subject to DNA sequencing, as described previously (29).
Mice
Cell transfection
To assess pre-BCR formation, a ␮-chain-deficient pre-B cell line Bine 4.8,
kindly provided by H.-M. Jack (Loyola University of Chicago, Maywood,
IL), was transfected with H chain constructs, as described previously (29,
30). Briefly, cells were washed with PBS and resuspended in a Gene Pulser
cuvette (0.4-cm electrode) in 0.45 ml PBS with 10 ␮g DNA linearized with
SfiI (Life Technologies). Electroporation was done using a Bio-Rad Gene
Pulser apparatus (Hercules, CA). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin,
and streptomycin for 24 h and then plated in 24-well plates in the presence
of 0.6 mg/ml G418 (Life Technologies). After 7–9 days, cells from individual wells were used for analysis of H chain expression and pre-BCR
formation.
To assess the ability of VH12 H chains to associate with conventional L
chains, L chain-only hybridoma cells were transfected with the 8/G0 construct, as described (30). In the case of V␬4/5H and V␬21C, the 8/G0
construct was cotransfected with L chain constructs into P3-X63-Ag8.653,
as described (30). The bulk cultures of G418-selected cells were used for
testing secretion of IgM molecules.
ELISA
Antibodies
mAbs against B220 (RA3-6B2), IgMa (DS-1), IgMb (AF7-78), CD43 (S7),
CD25 (7D4), CD2 (RM2-5), and c-kit (2B8) were obtained from BD
PharMingen (San Diego, CA), and were either directly conjugated to FITC,
R-PE, or Cy-Chrome, or were biotinylated. Unlabeled and FITC-conjugated goat anti-mouse ␮- or ␬-chain Abs were purchased from Southern
Biotechnology Associates (Birmingham, AL).
Flow cytometry
To detect membrane molecules, single cell suspensions were prepared in
HBSS (without Ca2⫹, Mg2⫹, and phenol red) containing 3% FCS and 0.1%
sodium azide (CHBSS). FcR were blocked by incubation with mAb 2.4G2
(purified from 2.4G2 hybridoma culture supernatant). Cells were then
stained with appropriate concentrations of the above Abs in a volume of 50
␮l and incubated at 4°C in the dark for 20 min. Biotinylated mAbs were
revealed with streptavidin-conjugated Cy-Chrome (BD PharMingen). For
the detection of intracellular ␮H- or ␬L-chains, bone marrow cells were
first stained with B cell phenotype-specific Abs, followed by fixation with
1% paraformaldehyde. Cells were then permeabilized with 0.04% saponin
(Sigma, St. Louis, MO) in 0.5% BSA/PBS buffer and stained with FITCconjugated goat anti-mouse ␮- or ␬-chain Abs for 30 min at 4°C. After
washing twice with saponin buffer and once with CHBSS, cells were analyzed using a FACScan (BD Biosciences, Mountain View, CA) with acquisition computer and software from Cytomation (Fort Collins, CO). All
data represent cells falling within the lymphocyte gate determined by forward and 90°C light scatter. All contour plots are 5% probability.
For cell-sorting experiments, 5–10 ⫻ 107 adult (8 –20 wk) bone marrow
cells were stained with Abs recognizing B220, IgMa/b, and CD43. B220⫹
IgMa/b⫺ CD43⫺ fraction D cells were sorted on a MoFlo high speed sorter
(Cytomation). Sorted populations were always ⬎95% pure. The cells were
then fixed with 70% ethanol and stained with a buffer containing 100 ␮g/ml
propidium iodide and 250 ␮g/ml RNase A (Boehringer Mannheim, Indianapolis, IN) overnight at 4°C. The DNA content was analyzed by FACScan, as described.
To quantify the expression levels of cytoplasmic ␮H-chains in transfected
pre-B cell lines, a cell lysate ELISA was used, as described previously (29).
Briefly, 96-well microtiter plates coated with polyclonal goat anti-mouse
␮H-chain (Southern Biotechnology Associates) were incubated with 5000
cells in lysis buffer containing 1% Nonidet P-40, 10 mM Tris, pH 7.4, 10
mM NaCl, 0.3 mM MgCl2, 200 ␮g/ml PMSF, and 2 ␮g/ml aprotinin. After
extensive washes, the plates were incubated with alkaline phosphataselabeled polyclonal goat anti-mouse ␮H-chain Ab (Southern Biotechnology
Associates), followed by p-nitrophenyl phosphate (Sigma) to develop the
reaction. OD readings were determined by an automated plate reader (Molecular Devices, Sunnyvale, CA).
To test whether a complete Ig molecule was formed by L chain-only cell
lines, supernatant was subjected to ELISA using microplates coated with
polyclonal goat anti-mouse ␮H-chain (Southern Biotechnology Associates) and alkaline phosphatase-labeled polyclonal goat anti-mouse ␬Lchain (Southern Biotechnology Associates) to develop the reaction (30). In
those cases in which Ig secretion was not detected, the production of H and
L chains was confirmed by ELISA using cell lysates and the polyclonal
goat anti-mouse ␮H- or ␬L-coated plates, as above. The former were developed with phosphatase-labeled polyclonal goat anti-mouse ␮H-chain to
detect H chain, and the latter were developed with phosphatase-labeled
polyclonal goat anti-mouse ␬L-chain to detect L chain (30). OD readings
were determined with an automated plate reader (Molecular Devices).
5-bromo-2⬘-deoxyuridine (BrdU) labeling
Adult mice were BrdU labeled in vivo using the method of Allman et al.
(35). Briefly, BrdU (Sigma) was administered in drinking water at 0.5
mg/ml with 1 mg/ml dextrose continuously for 2–3 days, or injected i.p. at
0.6 mg per mouse every 12 h for 24 h. At each time point, mice were
sacrificed and bone marrow cells were isolated for staining with anti-IgM
PE and anti-B220 CyChrome, as described above. Subsequent permeabilization followed by treatment with DNase (Sigma) and staining with antiBrdU-FITC (BD Biosciences) allowed use of FACS analysis to assess the
fraction of BrdU-labeled B cells.
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VH12 (10/G4 (6-1)) (26) and 2-12H (34) Tg mice have been previously
described. V1 H chain Tg mice were kindly provided by J. Kenny (National Cancer Institute, Frederick, MD), and recombination-activating gene
(RAG)-1⫺/⫺ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The 8/G0 Tg mice were generated using a non-10/G4 VH12-DJH1 construct designated 8/G0. The construct used is identical with that
used to make 10/G4 (6-1) Tg mice, except that the CDR3 is 8 aa in length
and lacks a Gly (29). The 8/G0 Tg mice were produced by the University
of North Carolina Transgenic Mouse Facility by microinjection of the construct into fertilized eggs of (C57BL/6 ⫻ SJL)F2 mice. Mouse lines carrying the 8/G0-C␮ were identified by PCR analysis of tail DNA using an
oligonucleotide complementary to a sequence of the VH12 (5⬘-CTTCCT
TACCTGCTCTATTACTGGTTTCC-3⬘) and an oligonucleotide complementary to a sequence 3⬘ of JH1 exon (5⬘-TGAGGAGACGGTGACCGT
GGTC-3⬘). DNA was prepared by incubating tail snips in 50 mM Tris-HCl
(pH 8), 100 mM EDTA, 100 mM NaCl, and 1% SDS with 1 ␮g/␮l proteinase K at 55°C overnight. PCR was performed as described (34). The
10/G4 (6-1) and 8/G0 Tg mice have been maintained by backcrossing male
Tg⫹ mice with female C.B17 mice. Mice were bred and maintained in our
own pathogen-free mouse colony at University of North Carolina.
1256
VH12 Tg MICE
FIGURE 2. The 8/G0 splenic B cells do not express transgene H chains.
Spleen cells from newborn and adult mice were stained with CyChromeconjugated anti-B220, PE-conjugated anti-IgMa, and FITC-conjugated antiIgMb. Contour plots are gated on the lymphocyte gate (adult) or B220⫹
cells in the lymphocyte gate (neonatal). Twenty thousand cells were analyzed from each mouse. The percentage of gated cells in each box is given
for adult mice, and the percentage of cells in each of the indicated quadrants is provided for neonatal mice.
The 8/G0 H chains are unable to support B cell development
Adult peripheral B cells in 8/G0 Tg mice were examined for expression of endogenous (IgHb allotype) and transgene (IgHa allotype) H chains. There are no detectable IgHa B cells in the bone
marrow of these mice (Fig. 1A), nor are there B cells expressing
either allotype in the neonatal spleen (Fig. 2). To exclude the possibility that the absence of B cell development is due to a lack of
8/G0 H chain expression, cells were stained for cytoplasmic ␮Hchain. As shown in Fig. 3, nearly all small 8/G0 pre-B cells stain
brightly for cytoplasmic ␮H-chain. This level is not different from
that in small pre-BII cells of non-Tg littermates. Anti-allotypic
FIGURE 1. The 8/G0 pre-B cells differentiate to the pre-BII cell stage.
Bone marrow cells from 8/G0 Tg, 10/G4 (6-1) Tg, and non-Tg littermate
mice were stained with FITC-conjugated anti-B220, PE-conjugated antiIgMa or IgMb, and biotinylated anti-c-kit, CD2, CD25, or CD43. Biotinylated reagents were visualized with CyChrome streptavidin. A, Cells
stained with B220 and IgMa or IgMb (top row) were gated on lymphocytes
according to forward and side light scatter. All other histograms are also
gated on IgM⫺ cells. Gates R1 through R6 indicate the criteria used to
measure cell numbers in each subset presented in Table II. The percentage
of cells in gates R2 and R3 of total lymphocytes is given. B, Size of cells
in the pre-BII compartment. B220⫹, IgMa/b⫺, and CD25⫹ cells were gated
(R4 in A). The horizontal line is in the same position in all three histograms. C, CD43 expression level by pre-BII cells. The pre-BII cells were
gated on B220⫹, IgMa/b⫺ cells (R1 in A).
Statistical analysis
The paired Student’s t test and the independent Student’s t test were used
to assess the significance of the observed differences in the number of B
cells in each subpopulation and in pre-BCR expression levels. p ⬍ 0.05
was considered significant.
Results
To understand the loss of non-10/G4 VH12 pre-B cells, we have
generated Tg mice using an 8/G0 VH12-D-JH1 construct. The 8/G0
H chains can associate with both the ␭5 and VpreB components of
the surrogate L chain, and are expressed on the cell surface of a
pre-B cell line (29). Comparison of B cell development between
these mice and our previously generated 10/G4 (6-1) Tg mice permits the identification of developmental differences responsible for
FIGURE 3. The 8/G0 pre-B cells express cytoplasmic transgene H
chains. Bone marrow cells from 8/G0 Tg, 8/G0/RAG-1⫺/⫺ Tg, and non-Tg
littermate mice were stained with CyChrome-conjugated anti-B220 and
PE-conjugated anti-IgMa and IgMb, followed by permeabilization and
staining with FITC-conjugated anti-␮H-chain Abs. The histograms are
gated on small B220⫹, IgMa/b⫺ cells, with the exception of those from
RAG-1⫺/⫺ mice, which are gated on both large and small B220⫹, IgMa/b⫺
cells. The percentage of cytoplasmic ␮H-chain-positive cells (indicated by
the horizontal bar) is given.
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the loss of most non-10/G4 VH12 pre-B cells. Two 8/G0 founder
mice were generated and backcrossed to C.B17 (IgHb) mice. The
characteristics of one are described in detail in this work.
The Journal of Immunology
1257
Table I. Origin of cDNA transcripts from 8/G0 pre-B mRNA
8/G0 Tg
Endogenous ␮
Othera
a
25
0
9
These cDNA lack a VH and are of transcripts that initiate 5⬘ of DH, JH, or C␮.
The 8/G0 H chains mediate allelic exclusion
Although 8/G0 H chains do not support B cell development, they
appear to mediate allelic exclusion. The 8/G0 Tg mice have only
small numbers of IgHb-expressing B cells in adult bone marrow
and neonatal spleen relative to non-Tg littermates (Figs. 1A and 2).
The small size of these populations, coupled with the fact that
these cells do not coexpress the IgHa allotype, suggests that the
FIGURE 4. The V1 transgene rescues B cell development in 8/G0 Tg
mice. Spleen cells from V1-only Tg, 8/G0 Tg, and V1/8/G0 Tg mice were
stained and analyzed, as described in Fig. 2. The percentage of IgMa,
B220⫹ cells is provided for each histogram.
The 8/G0 pre-B cells differentiate to small pre-BII cells, but
undergo limited cell division at the large pre-BII cell stage
To examine 8/G0 pre-B cell development, comparison was made between B-lineage cells of 8/G0, 10/G4 (6-1), and non-Tg mouse bone
marrow. The cell surface markers used to distinguish pro/pre-BI and
pre-BII cells include CD43, CD25, CD2, and c-kit. Pro- and pre-BI
cells are CD43high, CD25⫺, CD2⫺, and c-kit⫹, while pre-BII cells are
CD43low/⫺, CD25⫹, CD2⫹, and c-kit⫺ (14, 18, 25).
Like non-Tg mice, 8/G0 and 10/G4 (6-1) Tg mice have B220⫹,
CD43high pro/pre-BI bone marrow cells (Fig. 1A). The remaining
pre-B cells in mice of both 8/G0 and 10/G4 (6-1) exhibit a pre-BII
phenotype. They are CD2⫹, CD25⫹, and c-kit⫺ (Fig. 1A). Although the significance of this is not yet understood, the levels of
CD43 expression by 8/G0 and 10/G4 pre-B cells are intermediate
(CD43int) to pre-BI and pre-BII of wild-type mice (Fig. 1C). Also,
the 8/G0 and 10/G4 pre-BII cells are equivalent in size to the
pre-BII cells of non-Tg mice (Fig. 1B). Thus, both 8/G0 and 10/G4
pre-B cells differentiate to the small pre-BII cell stage.
The sizes of the pre-B cell populations are shown in Table II
and, with one exception, are not significantly different. The one
significant difference is that while control mice have equal numbers of pro/pre-BI cells and large pre-BII cells, 8/G0 Tg mice have
57% fewer large pre-BII cells than pro/pre-BI cells (Table II; p ⬍
0.01). This indicates that the large pre-BII population of 8/G0 Tg
mice is unusually small. This is also evident by comparison of the
proportion of large and small pre-BII cells in these mice (Table
III). The 8/G0 Tg mice have a smaller percentage of large pre-BII
cells ( p ⬍ 0.01) and a larger percentage of small pre-BII cells
( p ⬍ 0.01) than either non-Tg or 10/G4 (6-1) Tg mice (Table III).
A third H chain Tg mouse, 2-12H, that expresses a J558 H chain
has a frequency of large and small pre-BII cells equivalent to those
of non-Tg and 10/G4 (6-1) Tg mice. Because most large pre-BII
cells from normal mice are in cell cycle (18, 25), the proportions
of cycling pre-B cells in these mice were determined. As shown in
Table III, ⬃30% of pre-BII cells of non-Tg, 10/G4 (6-1), and 2-12
Tg mice are in the S/G2M phase of the cell cycle, consistent with
previous reports using non-Tg mice (18, 25). However, only
12.5% of 8/G0 pre-BII cells are in cycle, consistent with the reduced frequency and number of large pre-BII cells in these Tg
mice (Tables II and III). This difference in distribution of pre-BII
cells between large and small is also evident between 8/G0 Tg and
10/G4 (6-1) Tg mice that lack RAG-1 expression, indicating that
this difference is due to the 8/G0 H chain and not to coexpression
of an endogenous H chain. Together these data suggest that there
is less clonal expansion at the large pre-BII cell stage in 8/G0 Tg
mice than in 10/G4 (6-1) Tg mice.
The turnover rate of small 8/G0 pre-B cells is also slower than
that of 10/G4 (6-1) and non-Tg mice, as measured by BrdU incorporation. One day after BrdU treatment, 40% fewer 8/G0 small
pre-BII cells are BrdU labeled than 10/G4 or non-Tg small pre-BII
cells (Fig. 5). The BrdU levels are equivalent in these populations
by day 3. Thus, 8/G0 pre-B cells undergo less clonal growth and
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reagents do not recognize cytoplasmic H chains of pre-B cells (36),
and therefore cannot be used to confirm that these H chains are of
transgene origin. However, using RT-PCR on purified B220⫹
IgM⫺ bone marrow cells, we could only detect 8/G0 transcripts
(Table I), suggesting that the cytoplasmic ␮H-chain in these mice
is of 8/G0, not endogenous, origin. That 8/G0 H chains are produced
was definitively demonstrated using 8/G0/RAG-1⫺/⫺ mice. As these
mice cannot undergo VDJ rearrangement, any ␮ protein present must
be transgene encoded. As shown in Fig. 3, 8/G0/RAG-1⫺/⫺ small
pre-B cells have the same cytoplasmic ␮H-chain level as non-Tg littermate mice. Therefore, we conclude that the 8/G0 transgene encodes
the majority, if not all, cytoplasmic ␮H-chains in 8/G0 pre-B cells.
To exclude the possibility that a B cell deficiency could be due
to integration of the 8/G0 transgene into an essential B cell-specific
developmental gene, we generated Tg mice that express 8/G0
along with a second H chain transgene. The 8/G0 Tg mice were
bred with mice carrying the rearranged V1 gene of the S107 family
(37). Providing a second H chain rearrangement bypasses the allelic exclusion barrier imposed by the 8/G0 transgene (see paragraph below), thereby allowing B cell development to be driven by
a second H chain. Unlike 8/G0 Tg mice, V1-only Tg mice have
large numbers of Tg-expressing B cells (Fig. 4). If the integration
position of 8/G0 transgene disrupts B cell development, then mice
Tg for both H chains should not develop B cells. Conversely, if
there is no effect on development, the number of B cells will be
equal to that in V1-only Tg mice. As shown in Fig. 4, the spleens
of double Tg and V1-only Tg mice have essentially equal frequencies of B cells, indicating that integration position is not responsible for the absence of 8/G0 B cells. Thus, the 8G0 H chain is
unable to support B cell development. Because 10/G4 (6-1) mice
expressing a 10/G4 VH12 H chain generate large numbers of Tgexpressing B cells (26), we conclude that VHCDR3 determines the
differentiative potential of VH12 B cells.
8/G0 H chain is an excellent excluder of endogenous H chain gene
rearrangement. We cannot exclude the possibility that IgHb rearrangements occur and that cells coexpressing both H chains are
eliminated, but we think this is unlikely because we find no evidence of IgHb transcripts in 8/G0 pre-B cells (Table I). Thus, the
most likely basis for the absence of IgHb-expressing B cells in
8/G0 Tg mice is allelic exclusion induced by 8/G0 H chains. The
number of IgHb B cells in adult spleen (Fig. 2) increases with age
(data not shown), suggesting that a few IgHb B cells are generated
by the bone marrow and that they accumulate in the spleen
over time.
1258
VH12 Tg MICE
Table II. B cell development in bone marrow of adult VH12 Tg micea
No. Cells in the Lymphocyte Gate (⫻103)
Pre-BIIb
Mice
B220⫹ cells
Pro/pre-B1c
Large
Small
Small:larged
Immaturee
Recirculatingf
C.B17
8/G0
10/G4(6-1)
21.1 ⫾ 2.1
12.1 ⫾ 1.0
13.5 ⫾ 6.0
2.8 ⫾ 0.4
2.8 ⫾ 0.4
1.5 ⫾ 0.2
3.1 ⫾ 0.8
1.2 ⫾ 0.2g
1.5 ⫾ 0.3
6.7 ⫾ 0.8
7.7 ⫾ 0.6
5.2 ⫾ 1.8
2.2
6.4
3.5
5.0 ⫾ 0.4
0.4 ⫾ 0.1
4.3 ⫾ 1.3
3.4 ⫾ 0.4
0.05 ⫾ 0.01
0.7 ⫾ 0.3
a
Bone marrow cells were analyzed by flow cytometry. Cell numbers were relative numbers of 50,000 bone marrow granulocytes. Data are mean ⫾
SE of five mice per group.
b
B220⫹ IgMa/b⫺ CD43low (R6 in Fig. 1A and then by size).
c
B220⫹ IgMa/b⫺ CD43⫹ (R5 in Fig. 1A).
d
The ratio of small to large pre-BII cells.
e
B220⫹ IgMa/b⫹ (R2 in Fig. 1A).
f
B220high IgMa/b⫹ (R3 in Fig. 1A).
g
p ⬍ 0.01 compared with 8/G0 pro/pre-BI cells.
persist at the small pre-BII cell stage longer than either 10/G4 or
non-Tg pre-B cells.
H chain association with surrogate L chain is critical to pre-BII cell
differentiation (11–13). We have previously shown that the 8/G0 H
chain can associate with surrogate L chain in a pre-B cell line (29),
and the fact that 8/G0 Tg mice have a pre-BII cell population
indicates that it can associate with surrogate L chain in vivo. To
more carefully assess the ability of the 8/G0 and 10/G4 H chains
to form pre-BCRs, the 8/G0 and 10/G4 H chain constructs were
transfected into the pre-B cell line Bine 4.8. As an H chain control,
the J558 2-12H construct was also transfected. The Bine 4.8 pre-B
cell line produces surrogate L chains, but lacks H chains, and
therefore cannot produce a pre-BCR. Multiple cell lines from three
independent transfections were compared for each H chain. As
shown in Fig. 6 and Table IV, cells transfected with 10/G4 and
2-12H constructs have roughly twice as much cell surface preBCR as those transfected with the 8/G0 construct ( p ⬍ 0.001).
Comparison was made only between cell lines producing equal
amounts of cytoplasmic H chain (Fig. 6 and Table IV). Thus, cell
lines producing 8/G0 H chains are significantly less efficient at
formation of the pre-BCR than those producing 10/G4 H chains.
L chain gene rearrangement in 8/G0 Tg mice
L chain rearrangement is initiated at the small pre-BII cell stage,
and ␬L-chains are present in the cytoplasm of a significant perTable III. Pre-B II cell analysis in adult VH12 Tg mice
Mice
C57BL/6c
2-12H
C.B17c
10/G4(6-1)
8/G0
10/G4(6-1)/RAG-1⫺/⫺
8/G0/RAG-1⫺/⫺
% Large Pre-BIIa
% Small Pre-BIIa
% in S⫹G2-Mb
29.6 ⫾ 1.6
29.7 ⫾ 1.5
30.6 ⫾ 1.4
23.4 ⫾ 1.3
12.1 ⫾ 0.6d
39.4 ⫾ 1.9
22.0 ⫾ 3.4f
70.4 ⫾ 1.6
70.3 ⫾ 1.5
69.4 ⫾ 1.4
76.6 ⫾ 1.3
87.9 ⫾ 0.6d
59.2 ⫾ 2.2
76.0 ⫾ 5.0f
32.4 ⫾ 1.9
28.6 ⫾ 2.6
33.3 ⫾ 2.4
28.6 ⫾ 1.7
12.5 ⫾ 1.6e
NDg
ND
a
The large and small pre-BII cells were defined as B220⫹ IgM⫺ CD43low within the lymphocyte gate (gate R6 in Fig. 1A).
The data (mean ⫾ SE) represent five independent experiments with the exception of that from 10/G4(6-1)/RAG-1⫺/⫺ and
8/G0/RAG-1⫺/⫺ mice, which represent four independent experiments.
b
The cell cycle analysis was performed on sorted B220⫹ IgM⫺ CD43low cells and represents three independent experiments
(mean ⫾ SE).
c
2-12H Tg mice were backcrossed with C57BL/6 mice. 6-1 and 8/G0 Tg mice were backcrossed with C.B17 mice. Therefore, both C57BL/6 and C.B17 mice are included as littermate controls.
d
p ⬍ 0.01 compared with 10/G4(6-1) Tg mice.
e
p ⬍ 0.05 compared with 10/G4(6-1) Tg and control C.B17 mice.
f
p ⬍ 0.01 compared with 10/G4(6-1)/RAG-1⫺/⫺ Tg mice.
g
ND, Not done.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Pre-BCR formation
centage of pre-BII cells of normal mice (38). To determine
whether 8/G0 pre-BII cells initiate V␬ gene rearrangement, 8/G0
pre-B cells were examined for the presence of cytoplasmic ␬Lchains. Approximately 14% of IgM⫺ B220⫹ cells of non-Tg littermate mice are cytoplasmic ␬L-chain positive (Fig. 7) in agreement with Pelanda et al. (39), and an equivalent number of 8/G0
pre-B cells are cytoplasmic ␬L-chain positive (Fig. 7). J␬ rearrangement was verified by ligation-mediated PCR (data not
shown). Thus, the pre-BII cells of 8/G0 Tg mice have initiated L
chain gene rearrangement, despite the fact that essentially none
reach the immature B cell stage. We rule out the possibility that the
␬L-chain gene rearrangement in 8/G0 pre-BII cells is driven by
expression of an endogenous ␮H-chain, because no endogenous
␮H-chain transcripts are detected among 8/G0 pre-B cells (Table
I), and all 8/G0 small pre-BII cells express the transgene H chain
(Fig. 3).
That some 8/G0 pre-B cells undergo ␬L-chain gene rearrangement and can express ␬L-chain proteins raises the possibility that
the absence of 8/G0 B cells is due to an inability of the 8/G0 H
chain to associate with ␬L- or ␭L-chains. The ability of 8/G0 H
chains to associate with conventional L chains was tested by transfection of the 8/G0 H chain gene construct into cells of L chainonly cell lines, or along with an L chain gene construct into cells
of an H and L chain-loss cell line. IgM␬ or IgM␭ Ab could be
detected in the supernatants of only one of nine 8/G0 transfectants,
suggesting that 8/G0 H chains are unable to associate with most L
The Journal of Immunology
FIGURE 5. The 8/G0 small pre-BII cells have a slower turnover rate
than 10/G4 small pre-BII cells. BrdU incorporation into small B220⫹,
IgM⫺ cells of 8/G0 Tg, 10/G4 (6 –1) Tg, and non-Tg littermate mice was
measured at 1, 2, and 3 days following initiation of treatment, as described
in Materials and Methods. Each time point represents three or more mice.
The 8/G0 are significantly different from 10/G4 (6-1) and non-Tg pre-B
cells at day 1 (ⴱ, p ⬍ 0.001) and at day 2 (#, p ⬍ 0.01). The 10/G4 (6-1)
pre-B cells incorporate BrdU significantly faster than non-Tg B cells (ⴱⴱ,
p ⬍ 0.05).
Discussion
We have examined mice carrying an 8/G0 VH12 H chain transgene
to understand the basis for the absence of non-10/G4 VH12 Blineage cells from the pre-B and B cell repertoires. Comparison of
10/G4 and 8/G0 Tg mice indicates deficiencies at both the pre-B
and B cell stages. The 8/G0 H chains, like 10/G4 H chains, are
excellent allelic excluders and are able to drive differentiation to
the small pre-BII cell stage, where they initiate L chain gene rearrangement. However, 8/G0 Tg mice have significantly fewer
pre-BII cells in cell cycle than do 10/G4 Tg mice, and the small
8/G0 pre-BII cells have a slower turnover rate than small 10/G4 Tg
pre-BII cells. Most significantly, 8/G0 pre-B cells are unable to
differentiate to the B cell stage. We suggest that these defects are
due to a deficiency in 8/G0 pre-BCR function and to the inability
to make an 8/G0 BCR. Interestingly, the 8/G0 phenotype is similar
to that of ␥2b Tg mice (40, 41). As with the 8/G0 H chain, the ␥2b
H chain mediates allelic exclusion, but not B cell development.
This is attributed to a deficiency with the ␥2b C region. In contrast,
the differences in B cell development between 10/G4 and 8/G0 Tg
mice that we observe must be due to differences in VHCDR3.
The low percentage of large cycling pre-BII cells in 8/G0 Tg
mice relative to 10/G4 Tg mice is not due to an inability to express
an 8/G0 pre-BCR in vivo. First, 8/G0 pre-BCRs can be expressed
by cells of a pre-B cell line, albeit at lower than normal levels
(Table IV). Second, 8/G0 pre-B cell differentiation advances beyond the pre-BI cell stage, and 8/G0 H chains are excellent excluders of endogenous H chain gene rearrangement. Neither can
occur in the absence of a functional pre-BCR (9, 10, 42). Third,
8/G0 Tg, RAG-1⫺/⫺ mice have the same pre-BII-like population
(Table III and data not shown), formally excluding the possibility
that an endogenous H chain is responsible for pre-BII cell development. Nor is there a signaling pathway defect downstream of the
FIGURE 6. The 8/G0 pre-BCR is expressed poorly by cells of a pre-B
cell line. The ␮H-chain-loss pre-B cell line Bine 4.8 was transfected with
8/G0, 10/G4, or 2-12H constructs. The G418-resistant cells from separate
wells were analyzed by flow cytometry. Staining with FITC-conjugated
polyclonal goat anti-mouse IgM Abs revealed the pre-BCR expression levels on transfected cells. The intracellular Tg expression levels were measured by staining paraformaldehyde-fixed and membrane-permeabilized
cells with the same Ab. The data shown are of representative lines. The
light line is the unstained control, and the dark line is the staining by the
anti-IgM Ab.
pre-BCR that blocks all B cell development regardless of the H
chain, because like V1-only Tg mice, 8/G0-V1 double Tg mice
produce large numbers of B cells. Thus, we conclude that an 8/G0
pre-BCR is formed and expressed in vivo, but that it is deficient in
some functions.
Our data suggest that the deficiency in 8/G0 pre-BCR function
is due to a low expression level. The 8/G0 pre-BCRs are expressed
at only half the level of 10/G4 pre-BCRs in a pre-B cell line (Table
IV and Fig. 6). We presume that this is due to a poor ability of the
8/G0 H chain to associate with the surrogate L chain, and therefore
that VHCDR3 structure determines the ability of VH12 H chains to
form a pre-BCR. The most significant defect in 8/G0 pre-B cells is
the reduced percentage of large pre-BII cells in cycle. The preBCR is required for large pre-B cells to undergo clonal expansion
(9, 43– 45), but this is the first demonstration of suboptimal clonal
expansion in vivo. Assuming that the 8/G0 pre-BCR is expressed
at subnormal levels in vivo, these data argue that the strength of the
clonal growth signal is a function of the pre-BCR expression level.
Less clonal growth of large pre-BII cells would explain why there
are fewer large pre-BII cells than pro/pre-BI cells in 8/G0 Tg mice
(Table II). The prolonged t1/2 of small 8/G0 pre-BII cells is probably related to the reduced input of new cells from the large preBII compartment. It is suggested that small pre-BII cells do not
differentiate to immature B cells until the small pre-BII compartment has been filled (19). Thus, a slower input of cells into this
compartment would necessitate a longer t1/2.
The pre-BCR is able to deliver signals through multiple pathways, providing a possible mechanism for partial pre-BCR
function in vivo (46). Iritani et al. (47) have demonstrated that
there is more than one signaling pathway leading from the preBCR. They find that constitutively activated Raf-1 drives B cell
differentiation, but not allelic exclusion. The activation of these
pathways could be dependent on the strength of the signals
through the pre-BCR. Additional sites on a signaling molecule
or additional components of a pathway may be phosphorylated
with increasing strength of the pre-BCR signal, as seen in the
␨-chain of the TCR complex and in an FcR (48, 49). We suggest
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
chains (Table V). As previously reported, 10/G4 H chains associate with three of these nine L chains (30) and therefore are similarly deficient. Secreted IgM could be detected in all nine control
2-12 H chain gene transfectants. Although H and L chains could be
detected in the cell lysates of 8/G0 and 10/G4 transfectants at
levels equivalent to those in 2-12H transfectants, no cytoplasmic
IgM␬ or IgM␭ Abs were detected (data not shown). Thus, VH12 H
chains, particularly 8/G0 H chains, are exceptionally limited in
their ability to associate with conventional L chains.
1259
1260
VH12 Tg MICE
Table IV. Expression of 8/G0, 10/G4, and 2-12H ␮ H chains in the Bine 4.8 pre-B line
Expt.a
H Chains
No. of Cell Lines
Cytoplasmic ␮b
Cell Surface ␮b
1
2-12H
10/G4
8/G0
10G4
8/G0
2-12H
10/G4
8/G0
6
5
4
5
5
6
6
8
387.6 ⫾ 6.6 (365.2–410.9)
101.0 ⫾ 10.6 (73.7–122.9)
260.1 ⫾ 16.6 (231.4–303.7)
0.57 ⫾ 0.06 (0.39–0.71)
0.62 ⫾ 0.11 (0.41–0.96)
0.28 ⫾ 0.04 (0.17–0.41)
0.23 ⫾ 0.04 (0.17–0.32)
0.27 ⫾ 0.04 (0.18–0.39)
25.2 ⫾ 0.8
8.4 ⫾ 0.5
4.9 ⫾ 0.5c
5.1 ⫾ 0.5
1.6 ⫾ 0.1d
13.3 ⫾ 1.3
15.4 ⫾ 2.5
7.0 ⫾ 0.7d
2
3
a
Three independent transfection experiments are shown. Cells were from independently derived cell lines.
The cytoplasmic ␮H-chain expression was assayed by flow cytometry and presented as mean fluorescent intensity (Expt.
1) or by ELISA and presented as the mean OD (Expt. 2 and 3). The range of values is given in parentheses. The cell surface
␮H-chain expression was analyzed by flow cytometry and presented as mean fluorescent intensity. The median fluorescence
intensities are absolute values by subtraction of the background. All results are presented plus and minus their SE.
c
p ⬍ 0.005 compared with 10/G4 lines.
d
p ⬍ 0.001 compared with 10/G4 and 2-12H lines.
b
FIGURE 7. Some 8/G0 pre-BII cells express cytoplasmic ␬L-chains. Bone
marrow cells from 8/G0 Tg, 8/G0/RAG-1⫺/⫺ Tg, and non-Tg littermate mice
were stained for B220IgMaIgMb, and cytoplasmic ␬L-chains. Histograms are
gated on B220⫹ IgMa⫺ IgMb⫺ cells. The percentage of cells inside the indicated gates representing the cells that express ␬L-chains is given.
The poor ability of 8/G0 H chains to associate with conventional
L chains indicates that the VHCDR3 limits the differentiation of
VH12 pre-BII cells to immature B cells. This is consistent with the
function of the pre-BCR to perform a quality control function for
L chain association (50). Thus, the absence of 8/G0 B cells in
either the bone marrow or spleen may be due to the inability of
8/G0 H chains to associate with most conventional L chains. However, we cannot exclude other explanations for the inability of
8/G0 to drive differentiation to the B cell stage, because 8/G0 H
chains can associate with at least one V␬10 L chain. Even a low
frequency of B cell development, as in ␭5T mice (9), results in
accumulation of significant numbers of splenic B cells. In addition,
10/G4 H chains are deficient in ability to associate with L chains,
yet 10/G4 Tg mice (6-1) generate large numbers of B cells of both
the conventional and B-1 subsets (26, 30). Thus, the absence of
8/G0 B cells could also be due to a defect at the pre-BII cell stage
that blocks B cell differentiation despite an ability for 8/G0 H
chains to associate with at least some L chains. For example, poor
expression of the 8/G0 pre-BCR may be unable to mediate positive
selection and turn off an ongoing programmed cell death pathway,
as suggested previously (23). Such a possibility is supported by the
observation that a ␥2b transgene shows a similar deficit in B cell
production (40), despite a demonstrated ability to associate with
conventional L chains (40, 41). Efforts are currently underway to
resolve these two possibilities.
The VHCDR3 selection at the pre-B cell stage favors the
development of the VH12 B-1 cell repertoire. Although 10/G4
pre-BII cell development appears to be normal, we have previously demonstrated a limitation at the pre-BII to immature B
Table V. VH12 H chain association with L chaina
␭1
V␬RF
V␬4/5H
V␬31
V␬8T 1-12b
V␬8T 2E10b
V␬1A
V␬10
V␬21C
8/G0
10/G4
2-12H
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
a
The allotype-specific ELISAs were performed on tissue culture supernatant.
Negative values were ⬍4 times the background OD reading, and in all cases cytoplasmic ␮ and L chain were detected. Positive values were 10 –30 times background
OD. The 10/G4 and 2-12H data were taken from Tatu et al. (Ref. 30).
b
V␬8T 1-12 and V␬8T 2E10 are two different members of the V␬8 family (Ref. 52).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
that the separate pathways are differentially sensitive to receptor level, making it possible for the 8/G0 pre-BCR to signal
allelic exclusion and differentiation to pre-BII, but not normal
clonal growth. A greater sensitivity to induction of allelic exclusion than to cell division would ensure that only allelically
excluded pre-B cells continue differentiation, thereby preventing the production of B cells with multiple H chains. Equal
sensitivity to allelic exclusion and differentiation would likely
result in an unacceptable frequency of pre-B cells in which
complete differentiation is promoted in the absence of allelic
exclusion, and resulting in the production of B cells that express
two H chains.
The normal cycling observed for 10/G4 pre-BII cells, but not
8/G0 pre-BII cells, is consistent with the selective advantage of
10/G4 VH12 pre-B cells over non-10/G4 VH12 pre-B cells in
non-Tg mice. However, the ability of 8/G0 H chains to drive differentiation to the pre-BII cell stage does not explain the tremendous loss of non-10/G4 VH12 pre-B cells in non-Tg mice (29). One
possible explanation is that other non-10/G4 VH12 H chains are
less efficient at driving large pre-BII proliferation than are 8/G0 H
chains, or are unable to support differentiation beyond the pre-BI
cell stage. This possibility is supported by the observation that
Bine 4.8 pre-B cells transfected with two other non-10/G4 H chain
expression constructs (14/G7 and 12/G3, 7) express less surface
pre-BCR than cells transfected with the 8/G0 expression construct
(29). These H chains may be poorer at association with surrogate
L chain than 8/G0 H chains, and thus unable to support pre-BII cell
differentiation, similar to H chains that cannot form a pre-BCR
(11–13, 24). Thus, non-10/G4 H chains may exhibit a range of
abilities to drive pre-BII cell differentiation based on their ability
to associate with surrogate L chain, and thereby account for the
absence of most non-10/G4 pre-BII cells from the repertoire (29).
The Journal of Immunology
Acknowledgments
We gratefully acknowledge the Flow Cytometry Facility and the Transgenic Mouse Facility at the University of North Carolina for their assistance with this work. We are also indebted to Dr. James Kenny for his V1
Tg mice, and Anne Wolthusen for her assistance with mouse breeding and
genetic analysis.
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cell checkpoint for these cells (30). The 10/G4 VH12 B cells
that express a V␬4/5H L chain rearrangement are favored at this
transition due to a limited repertoire of L chains with which
10/G4 H chains can associate (30), and to selective maturation
to the B-2 cell stage of V␬4/5H-expressing 10/G4 B cells in the
spleen (51). The inability of 8/G0 H chains to associate with
V␬4/5H L chains (Table IV) indicates that the ability to associate with this L chain is determined by VHCDR3. Thus, there
is synergy between the selection for VHCDR3 at the pre-BII cell
stage and for the L chain at the immature B cell stage. This
focuses the VH12 B cell repertoire to a combination of H and L
chain that can bind PtC. PtC-specific B cells are selected into
the B-1 subset from B-2 cell precursors (26, 31, 32) and are
responsible for a high level of anti-PtC Abs in circulation (21).
This Ab, like other Abs produced by B-1 cells, probably provides an important early defense during bacterial infections, as
anti-PtC Abs are protective against bacterial infections in acute
peritonitis (33). We suggest that this extraordinary selection to
produce anti-PtC VH12 B cells with a 10/G4 VHCDR3 and a
V␬4/5H L chain is the underlying evolutionary basis for the
selective loss of non-10/G4 VH12 pre-B cells. Thus, VH12 H
chains have evolved such that association with surrogate L
chain and conventional V␬4/5H L chains is most efficient with
a 10/G4 CDR3.
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VH12 Tg MICE
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