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Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger Center, University of Erlangen-Nürnberg, Erlangen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5, and the
transmembrane signal molecules Ig
and Ig
. One way to explain how
maturation signals are initiated in late progenitor B cells is that the
pre-BCR is transported to the cell surface and interacts from there
with a ligand on stroma cells. To address this hypothesis, we first
produced soluble Fab-like pre-BCR and BCR fragments, as well as SL
chain, in baculovirus-infected insect cells. Flow cytometry revealed
that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds
to the surface of stroma and several other adherent cell lines, but not
to B and T lymphoid suspension cells. The specific binding of the
soluble pre-BCR to stroma cells is saturable, sensitive to trypsin
digestion, and not dependent on bivalent cations. The binding of
pre-BCR seems to be independent of the H chain of IgM (µH chain),
because SL chain alone was able to interact with stroma cells. Finally,
soluble pre-BCR specifically precipitated a 135-kDa protein from ST2
cells. These findings not only demonstrate for the first time the
capacity of a pre-BCR to specifically bind to a structure on the
surface of adherent cells, but also suggest that the pre-BCR interacts
via its SL chain with a putative ligand on stroma
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of mature B lymphocytes from hemopoietic stem cells is characterized by stepwise and stage-specific DNA rearrangements of Ig V, (D), and J gene segments, resulting in the generation of Ig genes encoding IgH and IgL chains (reviewed in Ref. 9). V(D)J rearrangement begins at the H chain locus in early progenitor B (pro-B)3 cells. Pro-B cells harboring a functional V(D)J exon synthesize the transmembrane form of a functional IgH chain of the µ-isotype (µH chain) and develop first into large early precursor B (pre-B) cells and finally into small pre-B cells, in which rearrangement at the IgL locus begins (10). Once a small pre-B cell produces an L chain able to pair with µH chain, it will develop into a surface IgM-positive immature B cell. Besides generating functional Ig genes, V(D)J recombination can also give rise to either nonproductive Ig genes (9) or genes that, for example, encode µH chains unable to pair with conventional IgL chains (11, 12). Therefore, checkpoints must exist at which B lymphoid cells are screened for the presence of functional Ig chains, i.e., those able to form a functional Ag receptor.
An early checkpoint has been identified at the transition from the late
pro-B to the early pre-B cell stage, at which B-lineage cells are
screened for the presence of a functional membrane-bound µH chain,
that is, one capable of forming the so-called pre-B cell receptor
(pre-BCR) with an IgL-like surrogate L (SL) chain (13, 14, 15, 16)
and the signal transducer Ig/ complex (pre-BCR) (Fig. 1
) (reviewed in Ref. 17).
The SL chain is composed of the two noncovalently associated invariant
Ig-like polypeptides VpreB and 5 (reviewed in Ref. 17).
Both VpreB and 5 are encoded by genes that do not undergo gene
rearrangement and are expressed in early pro-B cells before V(D)J
recombination begins (18). Computer modeling, using as
reference the structure of a conventional L chain, revealed that VpreB
contains one V-like and 5 one C-like Ig fold domain (18, 19). In addition to Ig-like sequences, VpreB and 5 contain at
their C-terminal and N-terminal end, respectively, a unique tail
without sequence similarities to any known protein. When compared with
the number of -strands in Ig domains of a conventional IgL chain,
VpreB lacks the seventh -strand (7) of a typical
VL region and 5 contains, in addition to all
-strands found in a conventional C region, one extra -strand. The
extra -strand in 5 seems to replace the missing -strand in
VpreB, since this extra -strand in 5 is required for the
noncovalent association of 5 with VpreB (20).
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(21)-, VpreB1/2 (22)-, 5
(23)-, and µm-deficient
mice (24). Furthermore, full-length µH chains that fail
to assemble with either SL chain (11) or the B
lymphoid-specific signal-transducing Ig complex (25)
do not rescue B cell development in transgenic mice unable to rearrange
their endogenous IgH gene segments. Pre-BCR-mediated signals have also
been implicated in the clonal expansion of early pre-B cells
synthesizing a functional µH chain (26, 27), the
termination of further rearrangements at the IgH locus
(28, 29, 30), and the redirection of the V(D)J recombinase
from the IgH to the IgL locus in late pre-B cells (31, 32). Although it is generally accepted that signals initiated by a functional pre-BCR are critical for B-lineage cells to efficiently pass the checkpoint between the late pro-B and early pre-B stage (reviewed in Ref. 33), little is known about the mode of signaling of this receptor. Several models to explain how pre-BCR-mediated signals are initiated are discussed in the literature (reviewed in Refs. 5 and 34 and discussed in Ref. 26). For example, the assembled pre-BCR might already constitutively signal either from the endoplasmic reticulum (ER) or from the cell surface. Alternatively, signals might only be initiated from the cell surface after interaction of the pre-BCR with a ligand either presented on or deposited by stroma cells in the extracellular matrix.
To start identifying pre-BCR-interacting structures on stroma cells, we have produced soluble Fab-like pre-BCR fragments consisting of the SL chain and a truncated µH chain containing a V and the first C domain of a µH chain (Fdµ chain). We show in this manuscript that Fab-like, soluble pre-BCR fragments bind in a specific and saturable manner to stroma and other adherent cell lines, but not to several B and T lymphoid suspension cell lines. Furthermore, this binding does not seem to rely on the presence of a µH chain, because the SL chain alone is also capable of interacting with a stroma cell line. We conclude from these findings that the pre-BCR interacts, presumably via its SL chain, with a structure found on the surface of stroma cells, and speculate that this structure is part of a pre-BCR ligand.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The surface IgM-positive mouse B cell lines NYC (35) and WEHI231 (36), the Abelson leukemia virus-transformed mouse pre-B cell lines TK and TK.µ (37), the mouse plasmacytoma J558L (38), the mouse T cell lines EL-4 (ATCC TIB-39; American Type Culture Collection, Manassas, VA) and DO11.10, the mouse stroma lines ST2 (39) and PA6 (40), the mouse fibroblast cell line NIH3T3 (41), the human embryonic kidney line 293T (42), the human cervix carcinoma line HeLa (ATCC CCL-2), the human epidermal larynx carcinoma line HEp-2 (CCL-23; American Type Culture Collection), the human T cell leukemia Jurkat (ATCC TIB-152), and the Chinese hamster ovary line CHO (ATCC CCL-61) were all maintained at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, 5% FCS, 1 mM sodium pyruvate, and 2 mM L-glutamine. Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (43) were maintained as adherent cells in TC100 medium (Life Technologies, Karlsruhe, Germany) supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Antisera and mAbs
Rat IgG mAbs directed against mouse VpreB (clone VP245)
(44), 5 (clone LM34) (44), and the pre-BCR
(clone SL156) (44), the monoclonal hamster anti-mouse
5 Ab, FS1 (37, 45), and unpurified total anti-VpreB
rabbit serum generated against a GST-mouse VpreB-fusion protein were
previously described (37). FITC-conjugated as well as
unconjugated affinity-purified goat Abs against mouse µ- and
-chain were purchased from Southern Biotechnology Associates
(Birmingham, AL); the mouse IgG anti-penta-His mAb from Qiagen
(Hilden, Germany); the FITC-conjugated goat
F(ab')2 anti-mouse IgG (H and L
chains), goat anti-rat IgG (Fc
specific), rabbit
anti-goat IgG (H and L chains), and the HRP-conjugated rabbit
anti-hamster IgG Abs from Dianova (Hamburg, Germany); and
the HRP-conjugated goat anti-rabbit IgG (H and L chains) and goat
anti-mouse IgG (H and L chains) Abs from Bio-Rad (München,
Germany).
Construction of baculovirus transfer vectors
DNA fragments encoding the mature form of mouse VpreB, 5,
L chain as well as the V (VH) and first C
domain (Cµ1) of the µH chain were amplified by PCR from plasmids
containing the corresponding cDNA sequences with the following primers:
for VpreB,
5'-TAGGCCTACCATGAAGTTCTTAGTAAACGTAGCATTAGTATTCATGGTAGTATACATAAGCTACATATACGCACAGCCCATGGTGCATC-3'
and 5'-TTCTAGAAGAGCCTAAGATCCCAAATCTG-3'; for 5,
5'-TCCCGGGACCATGAAGTTCTTAGTAAACGTAGCATTAGTATTCATGGTAGTATACATAAGCTACATATACGCAGAAAGGAGCAGAGCTGTG-3'
and 5'-TACTAGTCCCTAAGAACACTCAGCAGGTG-3'; for Fdµ,
5'-TGGATCCACCATGAAGTTCTTAGTAAACGTAGCATTAGTATTCATGG
TAGTATACATAAGCTACATATACGCAAAGGTCCAGCTGCAGCAG-3' and
5'-TTCTAGACTAGTGATGGTGATGGTGATGTGGAATGGGCACATGCAG-3';
and L chain,
5'-TCCCGGGACCATGAAGTTCTTAGTAAACGTAGCATTAGTATTCATGGTAGTATACATAAGCTACATATACGCAAGATGTGACATCCAGATG-3'
and 5'-TCTCGAGCTA ACACTCATTCCTGTTGAAG-3'. All PCR
primers contained specific restriction sites (underlined) to allow
cloning into appropriate vectors. The sequence encoding the honeybee
melittin leader (46) was engineered into each of
the 5' primers downstream of the restriction site, and the
sequence for six histidines was engineered into the C terminus of the
µH chain 3' primer. PCR products were purified, ligated into the
pCR2.1 cloning vector (Invitrogen, Groningen, The Netherlands), and
verified by DNA sequencing using the Big-Dye sequencing kit from
PerkinElmer Biosystems (Warrington, U.K.). A
SmaI/XhoI L chain fragment was isolated from
the respective pCR2.1 vector and ligated into the transfer vector
pFASTDual (Life Technologies). An Fdµ fragment was isolated as a
BamHI/XbaI fragment and ligated into both the
transfer vector pFASTBac1 (Life Technologies) and pFASTDual-L
vector. A StuI/XbaI VpreB fragment was isolated
from the pCR2.1 vector and ligated into the pFASTDual vector. The
transfer vector pFASTDual-VpreB/5 was constructed by inserting a
SmaI/SpeI 5 fragment from the respective
pCR2.1 vector into the pFASTDual-VpreB transfer vector. To assemble a
vector encoding VpreB, 5, and Fdµ (pFAST-VpreB/5/Fdµ), a
SnaBI/AvrII fragment containing the SV40 poly(A)
site and the melittin-Fdµ sequence under the control of the
polyhedrin promoter was isolated from pFASTBac1-Fdµ, blunt ended with
Klenow enzyme, and ligated into the SnaBI site of the
pFASTDual-VpreB/5 transfer vector.
Production of recombinant proteins in insect cells
Recombinant virus was produced by first transforming competent DH10BAC Escherichia coli cells with recombinant pFAST transfer vectors, as described in the instruction manual from Life Technologies for the BAC-TO-BAC Baculovirus Expression System. Recombinant virus was produced by transfecting Sf21 cells with recombinant baculovirus DNA isolated from recombinant DH10BAC E. coli clones. Virus titer in the growth medium was determined by the end point dilution. Monolayer cultures of Sf21 cells from S. frugiperda were maintained at 27°C in TC100 medium supplemented with 10% heat-inactivated FCS (Life Technologies) and infected with recombinant virus with a multiplicity of infection of 5–10. Cells and cell culture supernatants were harvested 3 days after infection. Cell pellets were solubilized in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 250 mM NaCl, 1 mM PMSF, 0.5% Nonidet P-40) for 30 min on ice. Lysed cells were centrifuged at 13,000 x g for 10 min, and supernatants (lysates) were collected. Cell culture supernatants were supplemented with 1 mM PMSF protease inhibitor.
Metabolic labeling, immunoprecipitation, gel electrophoresis, and Western blotting
Metabolic labeling was performed as described (47).
Briefly, 107 ST2 cells in 15 ml of
methionine-free labeling medium were incubated overnight with 50
µCi/ml 35S TransLabel (ICN Biomedicals,
Eschwege, Germany). Cells were lysed for 30 min on ice in 1 ml
of lysis buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM
EDTA, 0.5% Nonidet P-40, 14 µg/ml aprotinin, 1 mM PMSF, 1 µg/ml
pepstatin, and 2 µg/ml leupeptin, and spun for 10 min at 13,000 rpm
in a microfuge. Cellular lysates were incubated with 1 ml of cell
medium from uninfected, Fab virus-infected, or pre-BCR virus-infected
insect cells for 3 h on ice. Immunocomplexes were precipitated
with either the FS1 mAb (anti-5) or goat anti- Abs
for 2 h on ice, followed by protein G-Sepharose (Pierce, Rockford,
IL), separated on a 10% SDS-Laemmli polyacrylamide gel, and detected
by fluorography.
For Western blotting, proteins were precipitated from 1000 µl of cell culture supernatant of uninfected or infected insect cells with the respective primary Ab for 2 h on ice, followed by incubation with immobilized protein G-Sepharose beads. Proteins in cell lysates, cell culture supernatants, and immunoprecipitates were separated on 12% SDS-Laemmli polyacrylamide gels and analyzed by Western blot analysis, as previously described (47). Briefly, separated proteins were transferred onto Protean nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked with 5% nonfat Carnation dry milk (Nestle, Glendale, CA) in TBS, incubated first with appropriate unconjugated primary Abs, followed by secondary HRP-conjugated Abs, and developed with the ECL method (Amersham, Freiburg, Germany).
Flow cytometry
Adherent cells were first detached with accutase (PAA Laboratories, Colbe, Germany) at 37°C for 15 min and washed once in ice-cold PBS. Cells (5 x 105) were membrane stained for 2 h on ice with 100 µl of supernatant of uninfected or infected insect cells. Cells were then washed, then incubated with either unconjugated primary or fluorochrome-conjugated Abs for 30 min on ice. Unconjugated Abs were detected with appropriate fluorochrome-conjugated secondary Abs. Finally, cells were washed and fluorescence was determined by flow cytometric analysis with a FACSCalibur (BD Biosciences, Mountain View, CA). Flow diagrams were obtained by analyzing the primary data with the CellQuest software program (The Scripps Research Institute, San Diego, CA). After each staining, cells were washed three times with ice-cold PBSF (PBS supplemented for flow cytometry with 0.1% NaN3 and 1% BSA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 as well as a Fdµ chain (consists of a
VH and the first C domain (Cµ1) of the µ
gene) into the same baculovirus-based transfer vector. We then
expressed the genes in insect cells, produced soluble pre-BCR
molecules, and determined by flow cytometry whether the soluble pre-BCR
interacts with lymphoid and stroma-derived cell lines. Production of recombinant Fab-like soluble pre-BCR and BCR fragments
Several attempts to produce soluble pre-BCR molecules in insect
cells by infecting them stepwise or simultaneously with recombinant
VpreB, 5, and Fdµ viruses failed. Therefore, we inserted
expression cassettes for the mature forms of VpreB, 5, and Fdµ
into the same baculovirus-based transfer vector between two bacterial
transposon elements (Fig. 2A).
Similarly, we constructed recombinant baculovirus DNA encoding a
Fab-like BCR fragment by inserting expression cassettes for a
conventional L chain and a Fdµ chain into the same baculovirus
transfer vector (Fig. 2B). To increase the secretion of
heterologous proteins in insect cells (46), we replaced
the mouse leader sequences of all four chains with the 21-aa-long
honeybee melittin leader (Fig. 2C). Recombinant viruses were
produced, amplified, and used to infect Sf21 insect cells, as described
in Materials and Methods. Three bands with expected
molecular masses of 30 kDa for Fdµ, 21 kDa for 5, and 16 kDa for
VpreB could be detected under reducing conditions by immunoblotting
with Abs reactive against (His)5 (detects
recombinant Fdµ chains), VpreB, and 5, respectively, in cellular
lysates and culture media of infected cells (Fig. 3Aa), but not of uninfected
cells (data not shown). When we infected insect cells with virus
encoding only one of the pre-BCR constituents, we detected with the
corresponding Abs signals for Fdµ, 5, and VpreB on immunoblots in
cellular lysates, but not in culture media (data not shown), indicating
that none of the pre-BCR components, when synthesized alone, can
correctly fold and pass the stringent ER quality control.
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L (
27
kDa) and Fdµ chains (30 kDa) in cellular lysates and culture
medium from cells infected with recombinant L/Fdµ virus (Fig. 3
Ab), but not from uninfected cells (data not shown).
However, in contrast to VpreB or 5, L chains are secreted in the
absence of Fdµ production (data not shown). However, this result was
not surprising, because many L chains are secreted by plasma cell lines
in the absence of IgH chains (reviewed in Ref. 48).
To determine whether VpreB, 5, and Fdµ form trimeric complexes, we
incubated the culture medium with a mAb that recognizes only the
assembled pre-BCR (clone SL156), but not free SL chain
(44), separated the immunoprecipitated proteins under
reducing conditions, and analyzed the composition of the
immunoprecipitates with Abs against each individual chain by
immunoblotting. VpreB, 5, and Fdµ could be detected with the
corresponding Abs on immunoblots in electrophoretically separated
anti-pre-BCR precipitates from culture medium of cells infected
with VpreB/5/Fdµ virus, but not from medium of uninfected cells
(Fig. 3Ba). Similar results were obtained when we analyzed
BCR fragments immunoprecipitated from culture medium of cells infected
with L/Fdµ virus under nonreducing conditions, that is, Abs
against L and Fdµ chains coprecipitated Fdµ and L chains,
respectively (Fig. 3Bb).
We also found that SL chains are disulfide linked via 5 with Fdµ
chains, because immunoblot analysis of culture medium from cells
infected with VpreB/5/Fdµ virus revealed under nonreducing
conditions a single band of 50 kDa that reacted with Abs against
µH chain and 5 (data not shown). Similarly, Abs against µH and
L chain detected an 55-kDa band in medium from cells infected
with L/Fdµ virus (data not shown).
From these findings, we conclude that insect cells infected with
recombinant VpreB/5/Fdµ and L/Fdµ virus produce and secrete
trimeric Fab-like pre-BCR and dimeric Fab-like BCR molecules,
respectively.
Soluble Fab-like pre-BCR fragments bind to adherent, but not to suspension, cells
If an interaction of a pre-BCR with a stroma-derived ligand is
critical for early B cell maturation, one would expect that a soluble
pre-BCR molecule binds to stroma lines, but not to B or T lymphoid cell
lines. To address this question, we incubated several bone
marrow-derived stroma and lymphoid line cells with culture medium from
insect cells infected with VpreB/5/Fdµ virus (produce soluble
Fab-like pre-BCR fragments). Pre-BCR binding was then analyzed by flow
cytometry after staining the cells with rat mAbs against VpreB (clone
Vp245), 5 (clone LM34), or pre-BCR (clone SL156), followed by
fluorochrome-conjugated secondary anti-rat Abs. The
anti-pre-BCR Ab SL156 reacts only with a pre-BCR consisting of
VpreB, 5, and µH chain, but not with SL chain alone
(44) and should, therefore, detect only binding of
pre-BCR, but not binding of free SL chain. Culture media from
uninfected insect cells and from cells infected with recombinant
L/Fdµ virus (produce soluble Fab-like BCR fragments) served as
controls to assess the specificity of the pre-BCR binding.
Flow cytometric analysis revealed binding of Fab-like pre-BCR, but not
of Fab-like BCR fragments, to the adherent mouse stroma cell lines ST2
and PA6 and the stroma/fibroblast line NIH3T3 (Fig. 4A). The binding of the
pre-BCR was detectable regardless of whether we used Abs against 5
(Fig. 4A) and VpreB (for ST2, see Fig. 6B) or the
anti-pre-BCR Ab SL156 recognizing an epitope of the complete
pre-BCR (Fig. 4A). In contrast, specific binding of Fab-like
BCR fragments could not be detected with any of the lines (Fig. 4A). Based on these findings and the fact that the Ab SL156
detected binding of the pre-BCR, but not of SL chain alone (compare
Fig. 6Bc with 6Bf), we conclude that soluble
pre-BCR molecules bind specifically to ST2 cells.
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B, pre-BCR binding could not be detected on
lymphoid suspension lines, such as the immature B cell lines WEHI231
and NYC31, the pre-B cell line TK, the plasmacytoma J558L (data not
shown), and the T cell lines Jurkat, EL4, and DO11.10. These findings
indicate that soluble pre-BCR fragments bind to adherent lines from
various species, but not to lymphoid suspension lines.
One could speculate that suspension cells did not bind soluble pre-BCR
fragments, because these cells were, in contrast to adherent lines, not
treated with accutase before staining. However, accutase treatment of
suspension lines TK, DO11.10, and NYC before staining did not result in
pre-BCR binding (Fig. 4B). In addition, pre-BCR binding
could still be detected with ST2 cells that were detached from the
flask by treatment with EDTA (Fig. 4Cc). Therefore, the
accutase treatment is not a prerequisite for pre-BCR/ligand
engagement.
In contrast to Abs against VpreB, 5, and pre-BCR, Abs against µH
chain or the (His)5 tail at the C-terminal end of
the Fdµ chain did not detect pre-BCR binding to ST2 and other
adherent cell lines (data not shown). We were surprised at this
finding, because both anti-µH chain and
anti-(His)5 Abs were able to precipitate
pre-BCR molecules from cell culture medium (data not shown). One
explanation for the failure of anti-µ and
anti-(His)5 Abs to detect pre-BCR binding
could be that the corresponding anti-µ epitopes in the C region
of Fdµ are masked in cell-associated pre-BCR.
Several stroma cell-derived polypeptides whose binding to pre-B cells
depends on the presence of divalent cations have been identified
(49). However, these polypeptides can be excluded as
potential ligands for the pre-BCR, because pre-BCR binding could still
be detected in the presence of EDTA, a chelator for divalent cations
(compare Fig. 4Ca with Fig. 4Cd). The binding of
pre-BCR to ST2 cells depends on the presence of a trypsin-sensitive
structure, because, in contrast to accutase (50), trypsin
treatment abolished the binding of pre-BCR to ST2 cells (Fig. 4Cb). Based on these findings, we conclude that the
interaction of pre-BCR and ST2 cells does not require divalent cations
and is mediated by a trypsin-sensitive structure on ST2 cells.
One important characteristic of a specific receptor/ligand interaction
is that the binding of the ligand with its cognate receptor can be
saturated. To determine whether the binding of pre-BCR to ST2 cells is
saturable, ST2 cells were incubated with serial dilutions of culture
medium from insect cells infected with VpreB/5/Fdµ virus. Pre-BCR
binding was detected by flow cytometry with the anti-5 mAb LM34
(Fig. 5, A and B),
and mean fluorescence values were plotted vs the amount of pre-BCR
added to ST2 cells (Fig. 5C). The titration analysis yielded
a saturation curve typical for a specific receptor/ligand interaction;
that is, mean fluorescence values increased at first and then tapered
off at a pre-BCR concentration of 50 ng/ml (Fig. 5C). In
contrast, BCR binding could hardly be detected and did not increase
significantly by increasing the concentration of soluble BCR fragments
(Fig. 5C). We conclude from these findings that the
interaction of the Fab-like soluble pre-BCR fragment with ST2 stroma
cells is saturable and specific.
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Pre-BCR complexes harbor usually different VH region sequences. Therefore, pre-BCR/ligand interactions should be independent of the utilized VH region and mediated by an invariant structure of the pre-BCR. If this structure is associated with the invariant SL chain, one would expect that SL chain binds to ST2 cells in the absence of µH chain.
To address this question, we first produced soluble SL chain by
infecting Sf21 cells with a recombinant virus encoding the mature forms
of VpreB and 5. Both SL chain components could be detected on
immunoblots with the corresponding Abs in lysate and growth medium from
insect cells infected with recombinant virus (Fig. 6A), but not in lysate and
medium from uninfected cells (data not shown). When we performed a
combined immunoprecipitation and immunoblot analysis similar to that
described in Fig. 3, we found that VpreB and 5 form a noncovalently
associated dimer in the culture medium of cells infected with
recombinant VpreB/5 virus (data not shown). Therefore, as a
conventional L chain, SL chain correctly folds in insect cells and
passes the ER quality control in the absence of a µH chain.
To determine whether SL chain binds to adherent cells, ST2 stroma cells
were incubated with culture medium from insect cells infected with
VpreB/5 virus. Binding of SL chain was detected with rat mAbs
against pre-BCR components, followed by fluorochrome-conjugated
secondary anti-rat Abs. Flow cytometric analysis revealed the
binding of SL chain to ST2 cells using rat mAbs against 5 (Fig. 6Ba) and VpreB (Fig. 6Bb), but not with the rat
mAb against the pre-BCR (Fig. 6Bc). In contrast, all three
Abs detected binding of pre-BCR fragments to ST2 cells (Fig. 6B, d–f). Based on these findings, we conclude
that both the soluble pre-BCR as well as free SL chain interact with
ST2 cells, and that this interaction is at least in part mediated by an
invariant epitope on the SL chain and a trypsin-sensitive structure on
ST2 cells.
Soluble pre-BCR specifically precipitates a 135-kDa polypeptide from ST2 lysate
To start identifying a pre-BCR-binding molecule, we performed an
immunoprecipitation analysis of metabolically labeled proteins from ST2
cells with cell culture medium from cells infected with the
VpreB/5/Fdµ virus (pre-BCR). Medium from uninfected cells (-) and
from cells infected with Fdµ/L virus (BCR) served as negative
controls. Immunocomplexes were then precipitated either with
anti-5 (clone FS-1) or anti- Abs. Electrophoretic
analysis of pre-BCR/anti-5-precipitated
35S-labeled proteins from ST2 cells revealed a
band with an apparent molecular mass of 135 kDa (Fig. 7, lane
4). However, this band was not
detectable in anti-5 (Fig. 7, lane 1),
BCR/anti-5 (Fig. 7, lane 2), and BCR/anti-
precipitates (Fig. 7, lane 3). Based on these findings, we
conclude that the pre-BCR specifically interacts with a ST2-derived
polypeptide with a molecular mass of 135 kDa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To search for a stroma-derived pre-BCR ligand, we first produced
soluble Fab-like pre-BCR fragments consisting of a truncated µH chain
(Fdµ) and a covalently associated bona fide SL chain in insect cells
using the baculovirus-based expression system. This approach has
successfully been used to produce complete Abs (51) and
Fab-like IgG fragments (52), as well as soluble pre-BCR
molecules consisting of a Fdµ and, in contrast to our studies, a
single chain VpreB-5 fusion protein (53). The first
major finding of our study is that insect cells infected with a
recombinant VpreB/5/Fdµ baculovirus secrete a trimeric pre-BCR
complex consisting of a Fdµ- and a disulfide-linked bona fide SL
chain. This is the first time that a heterologous protein complex
consisting of three distinct polypeptide chains has successfully been
synthesized in the baculovirus-based insect expression system. The
soluble pre-BCR fragments behave biochemically like a pre-BCR produced
by pre-B cells. For example, 5 is disulfide linked to µH chain in
pre-BCR of pre-B cells (13, 14, 15) and in Fab-like pre-BCR
fragments secreted by infected insect cells (data not shown). In
addition, the mAb SL156, which recognizes an epitope formed by all
three pre-BCR chains (44), immunoprecipitated pre-BCR
fragments from insect cells infected with a virus encoding Fdµ,
VpreB, and 5 (Fig. 3B). Therefore, we believe that the
soluble pre-BCR fragment secreted by insect cells has a structure
similar to that of the corresponding part of a pre-BCR produced by
pre-B cells.
The second and more important finding is that the soluble pre-BCR
interacts in a specific and saturable fashion with adherent cell lines,
including the widely used stroma lines PA6 and ST2, but not with
several B and T lymphoid suspension cells. This finding shows for the
first time that the pre-BCR binds specifically to a structure found on
the surface of adherent cell lines, and supports the idea that a
pre-BCR ligand might exist on stroma cells. SL chain could participate
in this binding, either directly by interacting with a cross-linking
ligand, or indirectly by inducing a conformational ligand-interacting
epitope in the µH chain. We favor the first possibility, since SL
chain alone interacts with ST2 cells in the absence of a µH chain
(Fig. 6). However, we cannot completely exclude that certain epitopes,
for example in the V region of a µH chain, can influence the
interaction of a pre-BCR with stroma cells. Indeed, pre-B cells using
VH12 µH chains with particular CDR3 regions
seem to be trapped at the pro-B cell stage, despite the fact that these
µH chains assemble with SL chain and are transported to the cell
surface of a transformed pre-B line (54). One explanation
for the inability of some VH12 µH chains to
foster maturation could be that some VH12 regions
change the conformation of the SL chain and thus prevent binding of the
pre-BCR to its cognate ligand.
If ligand-induced cross-linking of the pre-BCR is a prerequisite to initiate a maturation signal in pre-B cells, pre-BCR complexes should be found on the surface of normal pre-B cells and cross-linking of surface pre-BCR should modulate intracellular signal pathways. In fact, low amounts of surface pre-BCR complexes can be detected on freshly isolated pre-B cells (16, 44, 55), and cross-linking of surface pre-BCR complexes on normal and transformed pre-B cell lines induces signal pathways similar to that observed in mature B cells after cross-linking of the BCR (reviewed in Ref. 34). For example, cross-linking of the pre-BCR on transformed pre-B cell lines with anti-µH chain Abs resulted in intracellular calcium mobilization (56, 57, 58), protein tyrosine phosphorylation (59, 60), and translocation of µH chains together with proximal tyrosine kinases and adapter proteins, such as Syk and BLNK, into lipid microdomains (60). The fact that stroma cells are required for pre-BCR-induced proliferation of pre-B cells in an in vitro culture system, in which the expression of a transgenic µH chain gene can be regulated in freshly isolated pro-B cells by a tetracycline-controllable transcription factor (26), further supports the idea that the interaction between a putative stroma cell ligand and a pre-BCR might be a prerequisite to initiate pre-BCR-mediated signals in pre-B cells.
However, three sets of experiments seem to challenge the existence of a
pre-BCR ligand. First, transgenic mutated µH chains lacking either
the VH domain (61) or the
VH, Cµ1, and Cµ2 domains (62)
still promoted the transition of B lymphoid cells from the pro-B to the
pre-B stage in 5-deficient mice, indicating that SL chain is not
required for initiating maturation signals. However, the authors
acknowledged that SL chain-independent maturation signals could also be
triggered by self aggregation of the truncated µH chains, which would
present an artificial situation not found during normal B cell
maturation (34). In support of this idea, the
membrane-anchored viral oncogene v-erb, a homolog of the
epidermal growth factor receptor, is capable of transforming cells,
despite the fact that it lacks the extracellular ligand binding site
(reviewed in Ref. 63). Second, pre-BCR-positive pre-B
cells that originated in an in vitro culture system from normal pro-B
cells divide several times in the absence of stroma cells or IL-7,
suggesting that a pre-BCR ligand on stroma cells is not required for
inducing growth of pre-BCR-positive pre-B cells. However, the authors
cannot exclude that a pre-BCR cross-linking molecule, present in either
the culture medium or the coat of the culture dish, is responsible for
this effect. Third, mAbs against the complete pre-BCR (clone SL156) and
the pre-BCR components VpreB (clone VP245), 5 (clone LM34), and µH
chain did not block the development of pro-B into pre-B cells in a
fetal liver organ culture system (64), suggesting that the
interaction of a pre-BCR with an extracellular ligand is not critical
for this maturation step. However, it is possible that the
anti-pre-BCR mAbs used in that study, which are identical to the
one used in this work, do not interfere with the binding between the
pre-BCR and its cognate ligand. This seems to be the case, since the
mAbs VP245 (anti-VpreB), LM34 (anti-5), and SL156
(anti-pre-BCR) still reacted with soluble cell-associated pre-BCR
(Figs. 4 and 6). One explanation for why Abs against µH chain did not
interfere with pre-B cell maturation in an in vitro stroma cell culture
system could be that either some epitopes in the C region of µH chain
are not accessible for anti-µ Abs in cell-associated pre-BCR, or
anti-µH chain Abs do not prevent the interaction between pre-BCR
and its corresponding ligand. Our findings that polyclonal goat Abs
against the C region of µH chain neither reacted with cell-associated
pre-BCR nor blocked the binding of soluble pre-BCR to ST2 cells (data
not shown) support both ideas.
A major question not resolved in this study is the identity of the
stroma cell-associated structure that interacts with the pre-BCR. The
pre-BCR-interacting structure is present on all adherent lines used in
these studies, but not on lymphoid suspension cells. One feature of
most adherent cell lines is that they produce many proteoglycans as
well as molecules involved in the biosynthesis and turnover of the
extracellular matrix and cell-cell interactions (65).
Proteoglycans are good examples of multifunctional surface molecules
that participate in cell-cell and cell-matrix interactions, organize
cell-matrix adhesion and signaling, serve as coreceptors for
extracellular ligands, enhance protein-protein interactions, regulate
the activity of growth factors, or concentrate growth factors on the
cell surface (reviewed in Refs. 66 and 67).
The ligand could either be a member of the large proteoglycan family or
be part of the extracellular matrix; therefore, it might not be
surprising that pre-BCR binds to many adherent cell lines. The binding
of the pre-BCR to ST2 stroma cells does not require bivalent cations,
since the addition of the chelator EDTA did not change the binding
behavior of soluble pre-BCR fragments. This finding excludes several
stroma-derived surface proteins, such as biglycan, osteonectin,
collagen type I, clusterin, and matrix glycoprotein sc1 as potential
binding partners, since the binding of these factors to pre-B cells
requires bivalent cations (49). However, the structure
must be part of a molecule containing a protease-sensitive polypeptide,
since treatment of trypsin abolished the binding of the pre-BCR (Fig. 4C), and culture medium from insect cells infected with
pre-BCR virus, but not with BCR virus, precipitated a 135-kDa
polypeptide from ST2 lysates. To speculate about the nature of the
135-kDa polypeptide is beyond the scope of this work. However, the
135-kDa polypeptide is not identical with a previously identified SL
chain-associated nonclassical cadherin of 130 kDa, since the message
for this protein could not be detected in ST2, PA6, and NIH3T3 cells
(68). Future studies will identify the 135-kDa polypeptide
and determine whether the binding site for the pre-BCR harbors
carbohydrate or lipid sequences and whether the structure is part of an
integral membrane protein or an extracellular matrix component.
In summary, we have identified a novel saturable interaction of a
soluble pre-BCR fragment with a trypsin-sensitive structure on the
surface of adherent cell lines and described a pre-BCR-interacting
135-kDa polypeptide, which provided the first experimental evidence
that a pre-BCR ligand might exist. Furthermore, we have shown that this
interaction occurs at least in part via a not yet identified structure
associated with SL chain. Candidates for the pre-BCR binding motif
could be the unique C- or N-terminal tails of VpreB and 5,
respectively, since these sequences present the only clear structural
difference between a SL chain and a conventional L chain
(18, 19). Therefore, SL chain not only serves as a folding
template (11, 37) to facilitate the assembly and transport
of a pre-BCR to a signal-competent compartment, but could also
participate directly in the signaling process by interacting with a
cross-linking ligand either anchored on the surface of stroma cells or
deposited by these cells in the extracellular matrix.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hans-Martin Jäck, Division of Molecular Immunology, Nikolaus-Fiebiger Center, University of Erlangen-Nürnberg, Glückstrasse 6, D-91054 Erlangen, Germany. E-mail address: HJAECK{at}molmed uni-erlangen.de
3 Abbreviations used in this paper: pro-B, progenitor B; pre-B, precursor B; pre-BCR, pre-B cell receptor; ER, endoplasmic reticulum; µH chain, H chain of IgM; SL, surrogate L.
Received for publication July 16, 2001. Accepted for publication October 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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