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5-Deficient Mice1
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*
Division of Developmental and Clinical Immunology, and Departments of
Medicine,
Pediatrics,
Microbiology, and
¶ Pathology, University of Alabama, Birmingham, AL 35294;
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The Howard Hughes Medical Institute, Birmingham, AL 35294; and
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Department of Immune Regulation, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5 surrogate L chain complex is an essential component
of the pre-B cell receptor, the expression of which serves as an
important checkpoint in B cell development. Surrogate L chains also may
serve as components of murine pro-B cell receptors whose function is
unknown. We have produced two new mAbs, R3 and R5, that recognize a
different VpreB epitope than the one recognized by the previously
described VP245 anti-mouse VpreB Ab. These Abs were used to confirm
the expression of surrogate L chains on wild-type pro-B and pre-B cell
lines. Although undetectable on the cell surface, VpreB was found to be
normally expressed within B lineage cells of 5-deficient mice.
Nevertheless, VpreB expression was extinguished at the B cell stage of
differentiation in these mice. The normal pattern of VpreB expression
in 5-deficient mice excludes an essential role for pro-B and pre-B
cell receptors in VpreB
regulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig
, to form a
signal-transducing pre-B cell receptor (BCR) (reviewed in Ref.
2). The SLC is composed of two proteins, VpreB and 5,
extended regions of which exhibit homology with V and C regions of Ig L
chains (3, 4). The pre-BCR has been implicated in the
clonal expansion of pre-B cells, the prevention of apoptosis, mediation
of µHC allelic exclusion, and the induction of L chain gene
rearrangement (5, 6, 7, 8, 9, 10, 11). Mice and humans incapable of
forming the pre-BCR because of mutations in their µHC,
5, Ig, or Ig genes have
impaired B lineage development manifested by a partial or complete
block in differentiation at the pro-B to pre-B cell stage
(12, 13, 14, 15, 16, 17, 18, 19).
VpreB and 5 gene expression is initiated in
pro-B cells (20, 21), and their protein products rapidly
associate to form the SLC in pro-B and pre-B cells (3, 4, 22). The VpreB protein shares significant sequence homology with
the L chain V region, and includes eight of the nine strands of an
Ig fold. However, the 26 carboxyl-terminal amino acids of VpreB have no
homology with Ig L chain, and their tertiary structure is unknown
(4). Reminiscent of this nonhomologous C-terminal portion
of VpreB, the first 62 amino acids of the 5 protein also share no
homology with known proteins, although the remainder of 5 shares
homology with both the L chain J and C1
regions (3). The 5 carboxyl-terminal end forms a
separate Ig domain, while the 7 strand in the amino-terminal 5
region may complete the VpreB Ig domain to allow formation of the
combined VpreB/5 SLC unit (23). The intact SLC can
displace the chaperone H chain binding protein from nascent µHC to
allow completion of pre-BCR assembly, release from the endoplasmic
reticulum, and cell surface expression (reviewed in Ref.
24). Pro-B cells do not produce µHC because they have
not yet undergone productive VDJH gene
rearrangement, but murine pro-B cells may express the SLC with a
surrogate H chain protein complex to form a pro-BCR on the cell surface
(25, 26, 27, 28), and BILL (B lineage, intestine,
leukocytes, and liver) cadherin has been identified as one component of
the pro-BCR complex (29). Although a pro-BCR functional
role has been proposed in pro-B cell development (2, 28),
convincing evidence for this hypothesis has not been forthcoming. The
apparently normal pro-B cell development in
5-/- mice (13) would argue
against an important physiologic role for the pro-BCR. In addition,
whether or not a pro-BCR exists in humans has been difficult to resolve
(30, 31, 32, 33, 34, 35, 36).
Consistent with an essential pre-BCR role in B lineage differentiation,
5-deficient mice have a block in differentiation at the pro-B to
pre-B cell stage. However, this is a leaky phenotype in that B cell
accumulation has been observed with increasing age of the
5-/- mice (13). Another
interesting feature in the characterization of these mice was the
inability to detect VpreB protein in their pro-B cells using the VP245
anti-VpreB Ab (37, 38). This observation suggested
that in the absence of its 5 companion, the VpreB protein may be
rapidly degraded. Alternatively, the VpreB protein produced by the
5-deficient pro-B cells could have eluded detection by the VP245 Ab,
which is the only anti-VpreB Ab previously reported. In the present
studies, we have produced and characterized two new anti-VpreB
mAbs, compared their epitope specificity with the VP245 Ab, and used
this panel of anti-VpreB mAbs to examine VpreB expression by pro-B
cells from wild-type and 5-deficient mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and 5-/- mice were obtained
from The Jackson Laboratory (Bar Harbor, ME), and the CD (Sprague
Dawley) rat was obtained from Charles River Breeding Laboratories
(Wilmington, MA). The SCID7 pro-B, 70Z/3 pre-B, 18-81 pre-B, BC77
transitional pre-B/B, A20 mature B, J558 plasmacytoma, and S1A mature T
cell lines were grown at 37°C with 8% CO2 in
IMEM supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 2 mM
glutamine, and antibiotics. SCID7 and BC77 cell cultures were
supplemented with IL-7 (PeproTech, Rocky Hill, NJ). X63 cells
transfected with the murine VpreB gene (25) were grown
with hygromycin B as the selection agent. Goat Abs to mouse µHC,
FITC-conjugated goat anti-rat IgG, alkaline phosphatase-conjugated
goat Abs to rat IgG, streptavidin-PE, and
streptavidin-allophycocyanin were obtained from Southern
Biotechnology Associates (Birmingham, AL). The unconjugated LM34
(rat anti-mouse 5) and VP245 (rat anti-mouse VpreB) mAbs
were produced as described (25). PE-conjugated
anti-mouse CD19 and unconjugated anti-CD79a mAbs were purchased
from BD PharMingen (San Diego, CA). Unconjugated hamster anti-mouse
Ig (HM79) was a gift from T. Nakamura (University of Tokyo, Tokyo,
Japan). FITC-conjugated rat anti-mouse kLC (187.1) and lLC (JC5)
mAbs were kindly provided by J. Kearney (University of Alabama,
Birmingham, AL).
Production of rVpreB proteins
A full-length mouse VpreB1 DNA was
obtained by PCR amplification of the corresponding cDNA
(provided by F. Alt, Harvard University, Boston, MA) as a
template, using the following primers: upstream,
5'-AATATCGGATCCCAGCCCATGGT-3' and downstream,
5'-CTTGAAGCTTCTAAGATCCCAAATC-3'. A truncated mouse VpreB DNA was
obtained by PCR amplification of the VpreB DNA clone as a template,
using the upstream primer indicated above and
5'-CTTGAAGCTTGGCACAGTAATACAC-3' as the downstream primer. Primer
sets were designed to incorporate BamHI and
HindIII restriction sites on the product ends, and the
amplified products were subcloned into the pQE-30 expression vector
(Qiagen, Hilden, Germany) using the BamHI and
HindIII restriction sites. The construct was sequenced to
confirm insert fidelity before being transformed into Escherichia
coli (M15 strain). After induction with 0.1 mM isopropyl
-D-thiogalactoside, the His-tagged rVpreB
protein was purified from 8 M urea-denatured cell lysates by passage
over a nickel column and elution with a low-pH 8-M urea solution before
extensive dialysis against PBS (pH 7.4).
Hybridoma production
Five weekly injections of rVpreB protein (150 µg) were given before a booster immunization of the CD rat with 70Z/3 pre-B cells 1 day before fusion of regional lymph node cells with the Ag8.653 plasmacytoma (39). Heterohybridomas were grown in hypoxanthine-aminopterin-thymidine medium for 10 days, and the supernatants were screened 5 days later for anti-VpreB activity by an ELISA in which rVpreB was directly coated to the wells. Secondary screening involved immunofluorescence analysis of supernatant reactivity with the 70Z/3 pre-B cell line. Selected hybridomas were subcloned, and the Ig isotype of anti-VpreB Abs was determined by indirect capture ELISA (Zymed Laboratory, South San Francisco, CA). Biotinylation of the anti-VpreB Abs was performed with EZ-link sulfo-NHS-LC-biotin, following the manufacturer’s instructions (Pierce, Rockford, IL).
Immunochemical analysis
For Western blot analysis, recombinant proteins (0.5 mg) or 1% Nonidet P-40 cell lysates were separated on a 13% reducing SDS polyacrylamide gel before protein transfer onto nitrocellulose membranes by electrophoresis. The membranes were blocked with 5% nonfat dry milk in PBS plus 0.1% Tween 20 before incubation with test Abs. Washed membranes were then incubated with goat anti-rat IgG Abs conjugated with alkaline phosphatase for 1–2 h at room temperature, and reactive bands were visualized by Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI). In immunoprecipitation assays, 70Z/3 pre-B cells were preincubated in Met- and Cys-free RPMI 1640 for 2 h, then labeled with 500 mCi of both [35S]Met and [35S]Cys for 5 h before harvesting and lysis in 1% digitonin lysis buffer (22). Cell lysates were incubated in plastic wells coated with control or test Abs, and bound material eluted with Laemmli sample buffer for analysis by SDS-PAGE and autoradiography.
Immunofluorescence analysis
Viable cells incubated with hybridoma supernatant were washed before staining with FITC-conjugated goat anti-rat IgG Abs for immunofluorescence analysis. In anti-VpreB epitope discrimination experiments, viable 70Z/3 cells were preincubated with or without the unlabeled test anti-VpreB Ab (3 µg) before washing and incubation with a biotinylated anti-VpreB Ab. Cell-bound biotinylated Ab was revealed with streptavidin-PE. For intracellular immunofluorescence, cells were fixed in 0.25% paraformaldehyde for 1 h at 4°C before permeabilization with 0.1% saponin in PBS. Subsequent staining and washing procedures were performed in the presence of 0.1% saponin.
Analysis of bone marrow cells expressing surface VpreB used an enhanced
immunofluorescence detection system in which magnetofluorescent
liposomes were conjugated to Fabs of sheep anti-digoxigenin Abs
(40). To block nonspecific FcR binding, viable cells were
preincubated with 200 µg/ml IgG and 20 µg/ml anti-Fc
IIR mAb
in PBS containing 0.5% BSA for 10 min at 4°C. A digoxigen-conjugated
Ab was then added for an additional 15 min before washing and
resuspension in washing buffer containing the
anti-digoxigenin-conjugated liposomes in a final volume of 200
µl. After a 1-h incubation on ice with agitation, the cells were
washed and analyzed by FACS. The liposome-staining specificity was
assessed by preincubation with a 100- to 1000-fold excess of unlabeled
primary Ab (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant VpreB protein was used as an immunogen in rats to
generate six hybridomas that were shown by ELISA to produce
anti-mouse VpreB Abs. Ab reactivity with VpreB was confirmed by
immunofluorescence assessment of reactivity with 70Z/3 cells that
express the pre-BCR. All six of the anti-VpreB mAbs were found to
be of IgG2a isotype. Two of these, R3 (IgG2a, 
) and R5 (IgG2a, ),
were selected for further characterization on the basis of their robust
VpreB reactivity and usage of different L chains.
The R3 and R5 Abs reacted with rVpreB protein and not with control
recombinant proteins by Western blot analysis (Fig. 1
, lanes 6 and 7,
and data not shown). The R3 and R5 Abs were also reactive with a native
protein of expected VpreB molecular size in pre-B cell lysates, but not
with proteins produced by mature B cells or T cells (Fig. 1, lanes 1–4, and data not shown). The R5 Ab was also found to
be reactive with a 
16-kDa protein in lysates of VpreB-transfected
plasmacytoma cells and not with proteins produced by 5-transfected
cells (data not shown). The R3 and R5 Abs were capable of
immunoprecipitating VpreB proteins together with associated µHC,
Ig, and Ig pre-BCR components in pre-B cell lines (Fig. 2), thereby indicating that these Abs can
recognize VpreB within the context of the pre-BCR complex.
Immunofluorescence analysis further indicated that the R3 and R5 Abs
recognize cell surface molecules expressed by early B lineage cells,
including cell lines of pro-B, pre-B, and pre-B/B phenotypes, and do
not recognize cell surface molecules expressed by mature B, plasma
cell, or T cell lines (Fig. 3). The
pattern of cell surface reactivity is thus identical for the R3, R5,
and VP245 anti-VpreB Abs (Fig. 3) (25). Notably,
however, the VP245 Ab of proven VpreB specificity (25)
failed to react with VpreB proteins in the Western blot assays
described above.
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In a competitive binding assay, the R3 and R5 Abs were found to be
capable of reciprocally inhibiting pre-B cell binding, while the VP245
Ab did not inhibit binding by either the R3 or R5 Ab and vice versa
(Fig. 4). Abs directed against the µHC
and 5 components of the pre-BCR had no effect on the pre-B cell
reactivity of the three anti-VpreB Abs (data not shown). These
findings suggested that the R3 and R5 Abs recognize the same epitope or
neighboring VpreB epitopes in the pre-BCR, while the VP245 Ab
recognizes a different epitope.
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A). Selective reactivity with
the intact VpreB protein was confirmed by Western blot assay (Fig. 5B), thereby inferring that the R3 and R5 Abs may recognize
the nonhomologous region of VpreB. In contrast, VP245 Ab reactivity
could not be demonstrated with either the intact or truncated forms of
rVpreB in these assays (Fig. 5). The composite results of these
experiments thus indicate that the VP245 Ab and the R3/R5 Abs recognize
different VpreB epitopes.
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5+/+ and 5-/- B lineage cells
Having demonstrated the distinctive reactivity patterns of the R3,
R5, and VP245 anti-VpreB Abs, we used this panel of anti-VpreB
Abs to examine the intracellular and cell surface expression of VpreB
by 5-deficient and sufficient B lineage cells. In permeabilized
pre-B and pro-B cell lines derived from wild-type mice, all three
anti-VpreB Abs yielded similar staining patterns and intensities
when optimal concentrations of the Abs were used (Fig. 6A and data not shown).
However, when a plasmacytoma cell line transfected with a VpreB
construct was analyzed, the intracellular staining with the VP245 Ab
was much lower relative to staining with the R3 and R5 Abs (Fig. 6B). This result suggests that the VP245 epitope is greatly
enhanced within the context of the intact surrogate L chain complex, a
finding that may help to explain why VP245 does not react well with the
VpreB protein in ELISA or Western blot assays.
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5 protein, they were used to
readdress the issue of VpreB expression by bone marrow B lineage cells
of 5-deficient mice. As previously reported (37, 38),
we could not detect VpreB on CD19+ B lineage
cells from 5-/- mice (Fig. 7A). However, when the
5-/- cells were permeabilized before
staining with the R3 and R5 Abs, VpreB could easily be detected in B
lineage cells within the 5-/- bone marrow
(Fig. 7B). The absence of intracellular VP245 staining in
these cells was confirmed, although all three anti-VpreB Abs
yielded similar staining patterns for permeabilized B lineage cells
from normal mice (data not shown). Intracellular immunofluorescence
analysis further confirmed the VpreB expression by the
CD19+ pro-B cells (µHC-)
and pre-B cells (µHC+) in
5-/- bone marrow (Fig. 7C). In
keeping with previous evidence indicating that B cell differentiation
is not completely blocked in the 5-/- mice
(13), VpreB+ and
VpreB- subpopulations of the µHC-producing
cells were observed, and VpreB synthesis was shown to be normally
extinguished in the bone marrow surface IgM-positive B cells in these
mice (Fig. 7D). These findings indicate that while VpreB is
normally expressed within the pro-B and pre-B cells in the bone
marrow of 5-deficient mice, the VpreB molecules alone do not
associate with surrogate H chains to form pro-BCR or associate with the
µHC to form pre-BCR. Nevertheless, VpreB expression is
extinguished before L chain expression by newly formed B cells even
in 5-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 SLC complex.
This difference in epitope specificity may reflect the different
strategies used to produce these anti-VpreB Abs. The R3
and R5 Abs were produced through fusion of lymphocytes from
a rat immunized with rVpreB protein, whereas the VP245 Ab was produced
by fusion of lymphocytes from a rat immunized with an intact pre-BCR
complex (25).
The issue of cell surface expression of surrogate L chain by B lineage
cells before they express µHC has been complicated by an
inconsistency in the results of mouse and human pro-B cell analyses
(25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 38). Although anti-human VpreB mAbs
against the non-Ig-like domain of recombinant and native VpreB proteins
have failed to identify VpreB on human pro-B cells (23, 34), the present analysis with the R3, R5, and VP245
anti-VpreB Abs confirms previous studies in which the VP245 Ab was
used to demonstrate cell surface VpreB on murine pro-B cell lines and,
at lower levels, on primary pro-B cells from wild-type mice (25, 26) and from Rag-1- and Rag-2-deficient mice (our unpublished
observations). The failure of VpreB to reach the cell surface in the
5-deficient mice thus indicates this component is required for the
surrogate L chain interaction with BILL-cadherin and possibly
other murine pro-BCR components (29).
Because the R3 and R5 Abs recognize VpreB equally well in the presence
or absence of its 5 SLC partner, these Abs could be used to show
that VpreB is expressed normally within early B lineage cells in the
5-deficient mice. VpreB nevertheless failed to reach the cell
surface of B lineage cells in these mice, a finding that indicates the
VpreB/5 SLC complex is required for the assembly and plasma membrane
expression of both the pro-BCR and the pre-BCR. This analysis of
5-deficient mice also reveals that the normal on and off pattern of
VpreB expression during B lineage differentiation is not compromised in
5-deficient mice. In these mice, VpreB expression was found to be
initiated normally during the pro-B cell stage, to persist into the
pre-B cell stage, and to be extinguished before the
IgM+ B cell stage. This normal profile of VpreB
expression indicates that pre-BCR competency is not essential for the
down-regulation of VpreB and, perhaps, 5 as
well, although the latter possibility was not addressed in these
experiments. The Pax-5 and early B cell factor transcription
factors have been implicated in the initiation of VpreB and
5 transcription (41, 42, 43), and both are
expressed through the mature B cell stage, making it unlikely that
either is solely responsible for the termination of SLC transcription
(reviewed in Ref. 44). Although the coordinate regulation
of these genes has been ascribed to a locus control region
(45), the VpreB and 5 regulatory
mechanism is still incompletely defined. A role for signaling via the
pro-BCR and pre-BCR in VpreB and 5 regulation
would appear to be excluded by the present results.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Max D. Cooper, University of Alabama, WTI 378, 1824 Sixth Avenue South, Birmingham, AL 35294-3300. E-mail address: Max.Cooper{at}ccc.uab.edu
3 Abbreviations used in this paper: µHC, Ig µ H chain; BCR, B cell receptor; SLC, surrogate L chain.
Received for publication May 17, 2001. Accepted for publication July 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 324:579.[Medline]
5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO J. 6:2267.[Medline]
5 protein in B cell development. Cell 69:823.[Medline]
. Science 272:411.[Abstract]
/. J. Immunol. 161:252.5/14.1 gene result in B cell deficiency and agammaglobulinemia. J. Exp. Med. 187:71. (CD79a) result in a complete block in B cell development. J. Clin. Invest. 104:1115.[Medline]
5/14.1 and VpreB to produce an intact surrogate light chain. Proc. Natl. Acad. Sci. USA 96:3041.5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines. J. Exp. Med. 178:469.5 antibody FS1 identifies a 130 kDa protein associated with 5 and VpreB on the surface of early pre-B cell lines. Int. Immunol. 6:393.5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transducing molecule. Int. Immunol. 3:1129.
L) in the absence of µ on human pro-B cell lines and normal pro-B cells. Eur. J. Immunol. 26:2172.[Medline]
L+µ-) proB complex: cell surface expression and biochemical structure of a putative transducing receptor. Blood 93:4336.5 gene. Eur. J. Immunol. 23:1284.[Medline]
5 surrogate light chain in early bone marrow precursor B cells of normal and B-cell deficient mutant mice. Cell 77:133.[Medline]
interferon and interleukin-10 on the surface of cytokine-secreting cells. Nat. Med. 6:107.[Medline]
5 promoters as well as the KI and KII sites upstream of the J genes. Eur. J. Immunol. 27:750.[Medline]
5-VpreB1 locus control region. Mol. Cell. Biol. 19:671.This article has been cited by other articles:
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  C. Mundt, S. Licence, G. Maxwell, F. Melchers, and I.-L. Martensson Only VpreB1, but not VpreB2, is expressed at levels which allow normal development of B cells Int. Immunol., January 1, 2006; 18(1): 163 - 172. [Abstract] [Full Text] [PDF]
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 D. Mielenz, C. Vettermann, M. Hampel, C. Lang, A. Avramidou, M. Karas, and H.-M. Jack Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition J. Immunol., March 15, 2005; 174(6): 3508 - 3517. [Abstract] [Full Text] [PDF]
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W. Schuh, S. Meister, E. Roth, and H.-M. Jack Cutting Edge: Signaling and Cell Surface Expression of a {micro}H Chain in the Absence of {lambda}5: A Paradigm Revisited J. Immunol., October 1, 2003; 171(7): 3343 - 3347. [Abstract] [Full Text] [PDF]
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 B. de Andres, P. Gonzalo, S. Minguet, J. A. Martinez-Marin, P. G. Soro, M. A. R. Marcos, and M. L. Gaspar The first 3 days of B-cell development in the mouse embryo Blood, December 1, 2002; 100(12): 4074 - 4081. [Abstract] [Full Text] [PDF]
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Y.-H. Wang, R. P. Stephan, A. Scheffold, D. Kunkel, H. Karasuyama, A. Radbruch, and M. D. Cooper Differential surrogate light chain expression governs B-cell differentiation Blood, April 1, 2002; 99(7): 2459 - 2467. [Abstract] [Full Text] [PDF]
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VpreB) lacking the non-Ig-like region was
examined by ELISA (A) and Western blotting
(B). B, The solid arrow indicates the
location of full-length VpreB protein, and the open arrow indicates
truncated VpreB. Coomassie blue staining of the gel reveals both
proteins, whereas the R5 Ab recognizes only the full-length VpreB
protein in the Western blot assay.
