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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4336-4346
The Human ( L+µ ) proB Complex: Cell
Surface Expression and Biochemical Structure of a Putative Transducing
Receptor
By
Bénédicte Lemmers,
Laurent Gauthier,
Valérie Guelpa-Fonlupt,
Michel Fougereau, and
Claudine Schiff
From the Centre d'Immunologie de Marseille-Luminy, Marseille,
France.
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ABSTRACT |
The surrogate light chain ( L) associates with µ and Ig -Ig
chains to form the preB-cell receptor that plays a critical role in
early B-cell differentiation. Discrepancies exist in human concerning
the existence of L+µ proB cells and
the biochemical structure of such a proB-cell complex remains elusive.
Among new antihuman VpreB monoclonal antibodies (MoAbs), 5 of the
  isotype bound to recombinant and native VpreB protein with high
affinity. They recognized 4 discrete epitopes, upon which 2 were in the
extra-loop fragment. Such MoAbs detected the L at the cell surface
of either preB or on both proB and preB cells. The previously reported
SLC1/SLC2 MoAbs recognize a conformational epitope specific for the
µ/L association in accordance with their preB-cell reactivity.
Using the proB/preB 4G7 MoAb, L cell surface expression was detected
on normal bone marrow, not only on
CD34CD19+ preB but also on
CD34+CD19+ proB cells. Futhermore, this
MoAb identified L+µ fresh proB
leukemic cells of the TEL/AML1 type. Biochemical studies showed that,
at the proB stage, the L is associated noncovalently with two
proteins of 105 and 130 kD. Triggering of this complex induces
intracellular Ca2+ flux, suggesting that the L may be
involved in a new receptor at this early step of the B-cell differentiation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE FIRST STEPS OF B-cell differentiation
take place in the bone marrow, where precursor hematopoietic stem cells
become proB, preB, and then immature B lymphocytes. This period of
differentiation is antigen-independent and is essentially devoted to
the generation of the basic Ig repertoire. This results from a complex
sequence of events that involves multiple gene rearrangements. These
processes are strictly coordinated, with at least two quality control
checkpoints, one dependent on the preB receptor at the transition from
large to small preB cells and the second due to the B-cell receptor at
the immature B-cell stage.
All of these steps are defined by Ig gene rearrangements
(H  ) and the expression of a well-defined set
of surface molecules, among which, in humans, are CD34, CD10, CD19, and
the surrogate light chain (L) markers. The L is composed of two
polypeptides encoded by -like1,2 (or 5 in
mice3) and VpreB4,5 genes, both related to C
and V Ig light chain domains, respectively. Theoretical models of
the µ/L structure have been proposed,6,7 based on the
fact that 5/-like and VpreB interact with each other and with the
heavy chain in a way somewhat similar to a regular Fab fragment. In
these models, the 5 polypeptide contributes the equivalent of the CL
domain, whereas the VpreB, together with a short segment of -like,
may be considered a VL equivalent domain. In addition, it was also
proposed6,7 that the COOH-terminal region of VpreB (25 aa)
and the NH2-terminal portion of -like (50 aa) loop out from the 2 main Ig domains.
In humans, CD34CD10+CD19+
preB cells express at the cell surface a small amount of the preB
receptor composed of the µ/L complex associated with the
Ig -Ig transducing module.8-11 Although this receptor
is expressed at a very low level at this stage, several reports showed
its implication in signal transduction.8,12,13 It is now
well established that this receptor is required to drive (1) the
transition from large to small preB, (2) the repertoire selection of
preB cells,14-18 (3) the amplification of preB
cells, and (4) the control of allelic exclusion at the H chain
locus.19
In addition to being expressed at the cell surface of preB cells, the
L has been detected on µ murine proB-cell lines
in association with proteins of 200, 130, 105, and 65-35 kD.20 In humans, expression of L on
CD34+CD10+CD19+µ
proB cells remains controversial. Using anti-L monoclonal antibodies (MoAbs; SLC1, SLC2), Lassoued et al10,21 concluded that
L cell surface expression was restricted to the preB stage of B-cell differentiation. In contrast, using an anti-VpreB MoAb (688), Sanz and
De La Hera22 detected L on the surface of both
µ proB and µ+ preB-cell lines.
Moreover, 75% of normal cells labeled with this MoAb were
CD34+, most of them being
CD10brightCD19dull, pointing to their proB
status.22,23 In a previous publication, we also reported
L cell surface expression on a human proB-cell line and on normal
CD34+CD38+ proB cells using anti-VpreB
MoAbs.24
Identification of cells expressing L not associated to µ chains
obviously raises the question of a possible function for the proB
complex. In 5/ knockout mice, the preB
compartment is affected, but a normal number of proB cells are
present,25 suggesting a L-independent development of
proB cells. However, the 5/ phenotype may
not be identical to that obtained upon inactivation of the entire L
(ie, 5 and VpreB), and the possible function of a proB-cell complex
remains unanswered.
In addition to the above-mentioned discrepancies regarding L cell
surface expression at the proB-cell stage that are presumably due to
subtle differencies in MoAb specificity, it was difficult to clearly
identify the components associated with L in this proB complex,
partially due to the fact that most MoAbs were of the µ isotype.
By using a new series of antihuman VpreB MoAbs of the isotype
with a high affinity for the VpreB protein, the cell surface expression, biochemical structure, and transduction capacity of the
proB-cell complex was investigated. The fine specificities of these
MoAbs were compared with those of SLC1/SLC2 MoAbs, definitively establishing the existence of surface L chain-expressing
compartments in both human proB and preB cells.
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MATERIALS AND METHODS |
Cells
RS4.11,26 JEA2,24 and REH (kindly provided by
Dr H.G. Drexler, German Collection of Microorganisms and Cell Cultures, Department of Cell Cultures, Braunschweig, Germany) are
µ proB-cell lines. NALM627 and LAZ
22128 are µ+ preB-cell lines and Namalwa
(NWA) is a µ+/+ mature B-cell line. Cells
were maintained at 37°C in 7% CO2 in RPMI medium
supplemented with penicillin, streptomycin, 10% fetal calf serum
(FCS), 2 mmol/L L-glutamine, and 1 mmol/L sodium pyruvate. JEA2
proB-cell line was cultivated for 4 days in RPMI medium containing 20 ng/mL of recombinant human interleukin-7 (IL-7; Immugenex Corp, Los
Angeles, CA).
After we received informed parental consent, normal human bone marrow
was obtained by iliac crest aspiration from 3 young donors (6 months to
3 years of age) and from femurs of a 24-week-old fetus. Bone marrow
from 12 patients suffering from acute lymphocytic leukemia (ALL) with a
TEL-AML1 translocation were obtained from the Timone Hospital
(Marseille, France). These leukemias are characterized by a B-cell
precursor phenotype
CD34+CD10+CD19+cTdT+cIgM.
For normal and ALL bone marrows, the mononuclear cells were isolated by
centrifugation over Ficoll-Hypaque gradients.
Preparation of Antihuman VpreB MoAbs
MoAbs were prepared against the human VpreB protein. Balb/c mice were
first immunized with 25 µg of soluble recombinant VpreB protein in
CFA. The mice were boosted intraperitonealy with the same dose in IFA
(at weeks 2 and 4). At 6 weeks, the last injection was performed in
phosphate-buffered saline (PBS). Four days later, spleen cells were
fused to the mouse myeloma X63-Ag8.653 in the presence of PEG 1500. Supernatants of hybridomas were tested on flat-bottom microtiter plates
coated with rabbit anti-VpreB antibodies24 onto which was
adsorbed the human recombinant VpreB. As negative controls, plates
coated with rabbit anti--like antibodies and with recombinant human
-like protein were used. All antibodies selected by enzyme-linked
immunosorbent assay (ELISA) were further characterized by surface
plasmon resonance using the BIAcore apparatus (Pharmacia Biosensor,
Saint Quentin-Yvelines, France) and by immunoprecipitation, cell
surface staining, and Western blotting.
Antibodies
Surface staining of cell lines, bone marrow, and ALL cells was
performed using the following antibodies: fluorescein isothiocyanate (FITC)-labeled anti-CD10 or anti-CD19 and phycoerythrin (PE)-labeled anti-VpreB (4G7) or irrelevant PE-labeled IgG1 (Immunotech, Marseille, France); PE-labeled polyclonal goat antimouse IgG+IgM (H+L; Jackson Immunoresearch, West Grove, PA); peridinin chlorophyll
(PerCP)-labeled anti-CD34 and Streptavidin-PerCP (Becton
Dickinson & Co, Moutain View, CA); FITC-labeled anti-CD24 (TEBU, CLB,
Amsterdam, The Netherlands); FITC-labeled anti-CD40 (SBA Southern
Biotechnology Associates, Birmingham, AL); and polyclonal
FITC-conjugated rabbit antihuman IgM F(ab')2,
polyclonal FITC-conjugated rabbit F(ab')2 negative control, and FITC-conjugated rabbit antihuman F(ab')2 and anti- F(ab')2
(Dakopatts, Glostrup, Denmark). The anti-VpreB and irrelevant anti-HEL
MoAbs (kindly provided by P. Machy, CIML, Marseille, France) were purified from ascitic fluid on a protein A column (Pharmacia, Uppsala, Sweden). Antihuman surrogate light chain MoAbs
(SLC1 and SLC2) were kindly provided by K. Lassoued and M. Cooper.10,21
Immunoprecipitations were performed using mouse anti-IgM MoAb
(Immunotech), anti-VpreB (4G7), and anti-HEL 1 irrelevant control. In Western blot experiments, the VpreB was detected by the anti-VpreB 4G7 MoAb followed by horseradish peroxidase
(HRPO)-conjugated goat antimouse IgG (Sigma, Saint Quentin
Fallavier, France), and the µ chain was detected using the
HRPO-conjugated anti-µ MoAb (SBA Southern Biotechnology Associates).
Calcium flux stimulations were performed with mouse anti-CD19 and
anti-IgM MoAbs (Immunotech) followed by cross-linking with a PE-labeled
goat F(ab')2 fragment antimouse IgG (H+L; Jackson Immunoresearch).
Flow Cytometric Analysis
For direct staining of cell lines, 106 cells were incubated
at 4°C for 20 minutes in 70 µL PBS, 0.2% bovine serum albumin
(BSA), 0.1% sodium azide, and 0.1% normal mouse serum containing 0.25 µg of PE-labeled anti-VpreB 4G7. For indirect staining, the same incubation conditions were used for the anti-VpreB 10G5, 14G3, and
15D3. Staining was shown with the PE-labeled goat
F(ab')2 fragment antimouse IgG (H+L). PE-labeled IgG1
and anti-HEL IgG1 served as isotype-matched control MoAbs. Staining
using SLC1 or SLC2 was performed using 50 µg/mL of MoAbs and the
revelation was performed as described above. Direct staining of cell
surface µ chain was achieved using FITC-labeled polyclonal rabbit
antihuman IgM F(ab')2.
Normal bone marrow and ALL cells triple-staining was performed using
PerCP-labeled anti-CD34, FITC-labeled anti-CD19, and PE-labeled
anti-VpreB or PerCP-labeled anti-CD34, PE-labeled anti-CD19, and either
FITC-labeled anti-CD10, anti-CD40, or anti-CD24 MoAbs.
For the detection of cytoplasmic µ and / chains in ALL, cells
were first stained with PE-labeled anti-CD19 and PerCP-labeled anti-CD34 and then fixed and permeabilized using the reagent and protocol supplied with IntraPrep Permeabilization Reagent kit from
Immunotech. The µ and / chain detection was performed using FITC-labeled polyclonal rabbit antihuman IgM F(ab')2
or anti- and antibodies. A polyclonal FITC-conjugated rabbit
F(ab')2 served as negative control. Stained cells
were analyzed using a Becton Dickinson FACScan device.
Recombinant Proteins
Human VpreB, scL (-like and VpreB covalently joined by a linker),
two Fdµ (VH-CH1) from NAML6 and 1E8 preB-cell lines, and the two
corresponding Fab-like (Fdµ-scL) recombinant proteins were
produced in baculovirus as described previously.18
BIAcore Analysis
Surface plasmon resonance measurements were performed on a BIAcore
apparatus (Pharmacia Biosensor, Saint Quentin-Yvelines, France).
Recombinant VpreB, scL, Fab-like NALM6, and Fab-like 1E8 were
immobilized covalently to carboxyl groups on the dextran layer of a
Sensor Chip CM5.18 MoAbs (20 µL at 20 µg/mL) were injected on sensor chips, and the amount of bound MoAb to
immobilized antigen was monitored. All measurements were
performed with a continuous flow of 5 µL/min of HEPES-buffered saline
(HBS) buffer. At the end of each cycle, the surface of the
sensor chip was regenerated by 10-µL injections of 10 mmol/L NaOH for
1 minute.
Cell-Surface Biotinylation and Immunoprecipitation
Viable cells (50 × 106) in 2 mL ice-cold PBS were
incubated with 1 mg of Sulfo-NHS-LC-Biotin (Pierce Chemical Co,
Rockford, IL) for 30 minutes at 4°C. After 5 washes with ice-cold
PBS, labeled cells were lysed with 1 mL of NP-40 lysis buffer (1%
NP-40, 150 mmol/L NaCl, 20 mmol/L Tris, pH 8, 1 mmol/L phenylmethyl
sulfonyl fluoride [PMSF], pepstatin, leupeptin, antipain, and
iodoacetamide). After 3 successive incubations with 100 µL of protein
A-Sepharose saturated with 4% nonfat milk, cell lysates were incubated
for 6 hours at 4°C with the indicated antibodies (10 µg)
preadsorbed overnight on protein G sepharose (50 µL).
Immunoprecipitates were washed, suspended in 70 µL of reducing
Laemmli sample buffer, boiled for 10 minutes, and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 5% to
15%) in reducing conditions. The proteins were transferred to
Immobilon-P membranes (Millipore Corp, Bedford, MA). After blocking
with 5% BSA in PBST (PBS, 0.1% Tween 20) for 1 hour, membranes were
shown by incubation with a 1:5,000 dilution of streptavidin-HRPO
(Amersham, Les Velis, France). On the same membranes, the presence of
VpreB and µ proteins were detected, with the 4G7 anti-VpreB MoAb (1 µg/mL) shown by incubation with a 1:3,000 dilution of HRPO-conjugated
goat antimouse IgG MoAb (Sigma) and with a 1:2,500 dilution of
HRPO-conjugated antihuman µ MoAb (SBA Southern Biotechnology
Associates), respectively. For two-dimensional analysis, precipitates
were run in the first dimension under nonreducing conditions using a
5% to 15% gradient SDS-PAGE. The relevant strips were then cut out,
incubated in Laemmli sample buffer with 20 mmol/L dithiothreitol for 30 minutes, and run in the second dimension on a 5% to 15% gradient
SDS-PAGE. Proteins were transferred to immobilon P membranes
(Millipore) and treated as described above.
Pronase treatment.
After cell surface biotinylation, 50 × 106 cells were
washed 2 times in ice-cold PBS, resuspended in 1 mL of PBS containing 50 µg of pronase (Boehringer Mannheim GmbH, Mannheim, Germany), and
incubated at 37°C for 5 minutes. Enzymatic activity was stopped by
the addition of 1 mL ice-cold PBS containing 5% BSA and 100 µg/mL of
DNase. After 3 washes in PBS 5% BSA, any remaining pronase activity
was inactived by 0.5 mmol/L PMSF for 10 minutes on ice. Cells were then
lysed and subjected to immunoprecipitation procedures, as described above.
PNGase F treatment.
Biotinylated immunoprecipitated proteins (from 50 × 106 cells) were resuspended in 30 µL of nonreducing
sample buffer and boiled for 10 minutes. Released proteins were diluted
4 times in PBS, 2% Triton X100 buffer, followed by the addition of 0.4 U PNGase F (N-glycosidase F; Boehringer Mannheim) and overnight
incubation at 37°C. Proteins were precipitated by 1 mL of acetone
at  20°C, suspended in 70 µL of Laemmli sample buffer, and
analyzed by SDS-PAGE under reducing conditions.
Calcium Flux
JEA2, LAZ 221, and Namalwa cells (5 × 106) in 1 mL of
IMDM medium, pH 7.0, 10 mmol/L HEPES were incubated at 37°C for 30 minutes with 2.5 µmol/L of acetoxymethyl ester indo-1 (Molecular
Probes, Eugene, OR). An equal volume of IMDM medium, pH 7.4, supplemented with 5% FCS was then added and cells were incubated for
30 minutes at 37°C. After centrifugation, cells were transferred in
IMDM medium containing 5% normal goat serum (1 mL per 106
cells). The fluorescence ratio of 405:485 nm emitted from Indo-1/AM was
measured under basal conditions or after stimulation either with
anti-VpreB, anti-CD19, anti-IgM, or irrelevant IgG1 MoAb, followed by
cross-linking with PE-labeled polyclonal goat antimouse IgG on a
FACStar apparatus.
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RESULTS |
Fine Characterization of Antihuman VpreB MoAbs
MoAbs against the human VpreB recombinant protein produced in
Escherichia coli have been described previously.18
In brief, hybridoma supernatants were first tested for their reactivity in ELISA using rabbit anti-VpreB antibodies onto which was adsorbed the
human recombinant VpreB protein and were subsequently tested for
detection of recombinant VpreB protein by Western blotting. Ten MoAbs
were selected, from which 5 (4G7, 4E7, 10G5, 14G3, and 15D3) were
characterized for their fine specificities. All 5 MoAbs had binding
constant values between 107 and
1010 mol/L and detected the intracellular VpreB
protein as a single band at 16 kD (or as a doublet, depending on the
cell lines) from proB-and preB-cell line lysates by Western blotting.
All MoAbs were of the 1 isotype, except 15D3, which was
3.18
Localization of the discrete VpreB epitopes was first aproached by
competitive binding of the various pairs of MoAbs to recombinant VpreB
using the Biacore system. Four discrete epitopes were identified by
4G7/4E7 (2 MoAbs that completely inhibit each other), 10G5, 14G3, and
15D3 MoAbs (data not shown). To localize more precisely the epitopes,
we tested the ability of the COOH-terminal 25-residue-long VpreB
peptide to inhibit the binding of the various MoAbs to the immobilized
VpreB protein. This peptide corresponds to the extra loop of the VpreB
molecule.7 As shown in Fig 1,
fixation of 4G7 and 4E7 was similarly inhibited in a dose-dependent
manner by the peptide, indicating that they were specific of an
extra-loop epitope. Binding of 10G5 was inhibited on a less efficient
basis, suggesting that it defines a closely overlapping epitope.
Absence of inhibition with 14G3 and 15D3 indicate that these MoAbs
recognize epitopes on the Ig-like domain of the VpreB protein.
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| Fig 1.
Binding inhibition of anti-VpreB MoAbs to immobilized
VpreB by the COOH-terminal V-preB peptide, using the BIAcore apparatus.
Anti-VpreB MoAbs (20 µg/mL) were preincubated for 30 minutes in HBS
buffer with serial concentrations of the COOH terminal VpreB peptide (0 to 100 µg/mL) and were injected at a flow rate of 5 µL/min in HBS
buffer on a sensor chip surface containing 1,200 RU of recombinant
human VpreB.
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We further tested the capacity of the 4 different MoAbs to recognize
the VpreB protein at the cell surface of proB-cell (RS4.11, JEA2 and
REH), preB-cell (NALM6, LAZ 221), and B-cell (Namalwa) lines
by flow cytometry (Fig 2). The
Namalwa B-cell line that did not express the VpreB protein
served as a negative control for the different MoAbs. The 14G3 and 15D3
MoAbs that recognize the VpreB Ig-like domain failed to label any cell
line. The lack of 15D3 recognition of preB cells is in agreement with
our previous BIAcore analysis that indicates that this epitope is lost
when L is associated with µ.18 The lack of positive
signal on proB cells suggests that this epitope is also lost when L
is bound to components of the proB-cell complex. Regarding 14G3,
because it was able to interact with the recombinant Fab-like molecule when tested with the BIAcore device, its failure to label precursor cells may result from its lower affinity, as compared with that of
other MoAbs.18 By contrast, MoAbs that were specific for the extra-loop epitopes (ie, 10G5 and 4G7/4E7) gave positive signals in
FACS analysis, but had discrete behaviors. The 10G5 MoAb detected VpreB
only when associated with µ chains on the surface of the two
preB-cell lines, whereas 4G7/4E7 recognized both the proB-cell (JEA2
and REH) and the preB-cell (NALM6 and LAZ 221) lines (Fig 2). The large
increase of VpreB surface expression on JEA2 cell line induced by
IL-724 is exclusively detected by the 4G7/4E7 MoAbs. The
labeling specificity is confirmed by the fact that we obtained a
complete FACS inhibition using as inhibitor the COOH-terminal
25-residue-long VpreB peptide (boxed inset in Fig 2). As previously
observed and despite intracellular expression of the VpreB protein, the
RS4.11 proB-cell line was negative for cell surface labeling with all
anti-VpreB MoAbs.
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| Fig 2.
Surface FACS analysis of VpreB and µ chains on human
proB-cell (RS4.11, JEA2, REH), preB-cell (NALM6, LAZ 221), and B-cell
(NAMALWA) lines, using the anti-VpreB (4G7 and 10G5) and anti-µ
MoAbs. The 4G7 MoAb is PE-labeled, whereas the 10G5 is indirectly shown
by a PE-conjugated goat antimouse IgG+IgM (H+L). Isotype-matched
MoAbs served as negative controls. When indicated (line 3), JEA2 cells
were cultured with 20 ng/mL of IL-7 for 4 days. In that case, the 4G7
anti-VpreB labeling is completely inhibitable by 2 µg/mL of the COOH
terminal VpreB peptide (boxed inset).
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Because 4G7 MoAb recognized the VpreB protein both in the presence or
absence of µ chains, its reactivity was compared with the previously
reported SLC1 (1) and SLC2 (µ) antisurrogate light chain
MoAbs10,21 using FACS and BIAcore analysis
(Fig 3). Immunofluorescence experiments
confirm that both SLC1 and SLC2 MoAbs are preB-specific reagents,
because they recognized only the NALM6 preB-cell line and do not stain
the JEA2 proB-cell line cultivated in absence or presence of IL-7 (Fig
3, top). Using the BIAcore technology, we determined the fine
specificity of these MoAbs on different VpreB-containing recombinant
proteins (Fig 3, bottom). SLC1 and SLC2 MoAbs interact with the two
Fab-like proteins, whereas the free scL or VpreB proteins are not or
poorly recognized by these MoAbs. In the same conditions, the 4G7 bound all the recombinant proteins18 and had a higher affinity
for the two Fab-like MoAbs than the SLC1/SLC2 MoAbs. It
thus appears that SLC1/SLC2 MoAbs recognized conformational epitopes
depending on the association of the scL with the Fdµ fragments in
Fab-like complexes, whereas 4G7 detected the VpreB protein regardless
of whether it was associated or not with the Fdµ
chains. These data are in agreement with the
immunofluorescence analysis and confirm the preB-cell specificity of
the SLC1/SLC2 reagents. By contrast, the 4G7 MoAb detects both cell
surface expression of L+µ+ preB-cell and
L+µ proB-cell complexes.
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| Fig 3.
Comparison of the fine specificities of anti-VpreB MoAbs
by FACS and BIAcore analysis. (Top) Surface FACS analysis of VpreB and µ chains on human proB-cell (JEA2), preB-cell (NALM6), and B-cell
(NAMALWA) lines, using the 4G7 anti-VpreB, SLC1, SLC2,10,21
and anti-µ MoAbs. The 4G7 MoAb is PE-labeled and unconjugated SLC1
and SLC2 are shown by a PE-conjugated goat antimouse IgG+IgM (H+L).
Wherever indicated (line 2), JEA2 cells were cultured with 20 ng/mL of
IL-7 for 4 days. (Bottom) Binding of anti-VpreB MoAbs to immobilized
VpreB-containing recombinant proteins using the BIAcore apparatus. In
two separate experiments, 20 µL (20 µg/mL) of 4G7, SLC1, and SLC2
was injected at a flow rate of 5 µL/min in HBS buffer on four
surfaces containing 700, 410, 1,400, and 900 RU of VpreB, scL,
Fab-like NALM6, and Fab-like 1E8, respectively. The resulting
sensorgrams are superimposed.
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Characterization of proB- and preB-Cell Compartments in Normal
Bone Marrows
Because the 4G7 MoAb labels proB- and preB-cell lines, it was used to
define their normal counterpart in fetal or young donor bone marrows.
We performed three-color immunofluorescence analysis using anti-CD34,
anti-CD19, and anti-VpreB MoAbs on 4 bone marrow samples. As shown in
Fig 4, the 4G7 anti-VpreB, in comparison with the irrelevant 1 MoAb, labeled 5.4% of lymphoid fetal bone marrow cells. All cells, whether CD34+VpreB+
(R1) or CD34VpreB+ (R2), expressed the
CD19 marker (Fig 4, right). This points to the identification of two
distinct populations, ie, proB
(CD34+CD19+VpreB+) and preB
(CD34CD19+VpreB+) cells.
Depending on samples, proB cells ranged from 0.3% to 1.2% (0.84% ± 0.45%) and preB cells ranged from 0.8% to 4.2% (2.64% ± 1.62%) of total bone marrow cells.
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| Fig 4.
Three-color immunofluorescence analysis of normal bone
marrow cells shows cell surface expression of L on both the proB and
preB compartments. Fetal bone marrow lymphoid cells were incubated with
PerCP-labeled anti-CD34, FITC-labeled anti-CD19, and PE-labeled
anti-VpreB 4G7 or PE-labeled irrelevant 1 control MoAbs. Both the
CD34+VpreB+ (R1) and the
CD34VpreB+ (R2) cells were
CD19+, identifying proB- and preB-cell compartments,
respectively.
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Characterization of Surface L+µ
proB-Cell Leukemias
Using the 4G7 MoAb, we characterized two proB
L+µ leukemic cell lines from which
one, REH, expressed the TEL/AML1 fusion transcripts resulting from the
t(12;21)(p13;q22) translocation. This observation prompted us to
analyze fresh TEL/AML1-positive leukemias. This was performed on 12 leukemic cell samples that had been shown previously to express the
fusion transcript by a PCR approach.29 These cells were
considered to be proB, because they were positive for the CD34, CD19,
and CD10 markers. We performed three-color immunofluorescence analysis
using anti-CD34, anti-CD19, and either anti-CD10, anti-CD40, anti-CD24,
and anti-VpreB on intact cells or anti-µ and anti-/ antibodies
on permeabilized cells. One representative analysis is presented for
one patient's bone marrow cells in Fig 5
and indicates that all leukemic blasts (92.2% on gate R1) that
coexpress CD34 and CD19 are surface VpreB+. Moreover, we
confirm that these cells are negative for intracytoplasmic µ or
/ chains (Fig 5, bottom) and are positive for the expression of
the TdT protein (data not shown), thus confirming the proB status of
these leukemic cells. From 12 analyzed cases, 8 had the same phenotype
and 4 expressed the VpreB protein only intracellularly (data not
shown).
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| Fig 5.
Three-color immunofluorescence analysis of TEL/AML1
leukemia bone marrow cells shows cell surface expression of L in the
absence of µ chain. Leukemic blasts coexpressing CD34 and CD19 were
gated (R1) and analyzed for the other markers. Cells were stained with
PerCP-labeled anti-CD34, FITC-labeled anti-CD19, and PE-labeled
anti-VpreB or PerCP-labeled anti-CD34, PE-labeled anti-CD19, and either
FITC-labeled anti-CD10, anti-CD40, or anti-CD24 MoAbs. For µ and
/ intracellular staining, cells were first labeled with
PerCP-labeled anti-CD34 and PE-labeled anti-CD19 and then permeabilized
and treated with FITC-conjugated rabbit antihuman IgM
F(ab')2 or FITC-conjugated rabbit antihuman F(ab')2 plus antihuman F(ab')2.
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Biochemical Analysis of the proB- and preB-Cell Surface Complexes
Besides the identification of the early steps of B-cell differentiation
in normal bone marrow, the 4G7 MoAb was used to identify molecular
components associated to L at the cell surface of proB-cell lines.
It also allowed us to compare the biochemical structure of the proB-
and preB-cell complexes. Biotinylated cell surface proteins from a
series of representative cell lines corresponding to proB, preB, and
mature B stages were immunoprecipitated using anti-VpreB (4G7),
anti-µ, and irrelevant 1 MoAbs; were subjected to SDS-PAGE; were
transfered to membrane filter; and were then shown by streptavidin-HRPO
(Fig 6). In agreement with FACS analysis, we did not observe specific VpreB- or µ-associated complexes using the RS4.11 cell line. By contrast, several proteins were
immunoprecipitated from the JEA2 cell line, using the anti-VpreB (4G7)
MoAb: (1) the VpreB protein, faintly labeled but clearly visible after
immunoblotting; (2) the -like component at 20 kD; (3) a p105 that
was strongly biotinylated; and (4) a p130 that was always faintly
labeled. Other weaker bands could be visualized in the range of 42 to
70 kD in some experiments. As expected with this cell line, no µ protein was detected either at the cell surface or intracellularly. Similar results were observed whenever iodination was used instead of
biotinylation, except that the labeling of the VpreB protein was more
intense (data not shown). Using the anti-µ MoAb on the NALM6 (Fig 6)
or on the LAZ 221 (data not shown) preB-cell lines, preB-cell receptor
components were immunoprecipitated, ie, µ heavy chains and weakly
labeled L. Surprisingly, using the 4G7 MoAb, besides the L
components and the µ chain (that is clearly identified in the µ Western blot), the p105 and the p130 proteins were also immunoprecipitated. Thus, the two proB- and preB-cell complexes were
present at the cell surface of the two preB-cell lines. In some
experiments, the 14G3 anti-VpreB MoAb that does not stain proB- and
preB-cell lines is used as a control and gave the same pattern of
background bands as the 1 irrelevant control (data not shown).
Pronase treatment of biotinylated proteins from all three precursor
cell lines led to the disappearance of labeled proteins upon anti-VpreB
immunoprecipitation without affecting the size of the intracellular
VpreB pool, as demonstrated by the anti-VpreB Western blot (Fig 6).
This confirms that biotinylation had been targeted mainly to cell
surface molecules. Finally, as expected, the Namalwa mature B-cell line
expressed only the conventional µ and chains.
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| Fig 6.
Biochemical characterization of the proB-cell surface
complex on proB- and preB-cell lines. The proB-cell (JEA2, RS4.11),
preB-cell (NALM6), and B-cell (Namalwa) lines were surface labeled
with biotin and lysed with 1% NP-40 lysis buffer. Lysates (5 × 107 cells) were incubated with either IgG1 control,
anti-VpreB (4G7), or anti-µ MoAbs. Immunoprecipitates were submitted
to a gradient SDS-PAGE (5% to 15%) under reducing conditions and
transferred onto immobilon P membrane. Cell surface biotinylated
proteins were detected by streptavidin-peroxidase. The membrane was
then successively incubated with the anti-VpreB 4G7 MoAb, which was
shown by a peroxidase-conjugated goat antimouse IgG and finally with a
peroxidase-conjugated mouse antihuman µ MoAb.
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Further characterization of the surface proB-cell complex was achieved
by testing the N-linked oligosaccharides status of the different
components. The treatment of the JEA2 biotinylated anti-VpreB
immunoprecipitated with PNGase F changed the apparent molecular weight
of the p130 protein to p115 and led to the appearance of a p40 derived
from an unknown protein (Fig 7). Digestion
seemed to be complete, as demonstrated by the complete shift of the µ chains from 76 to 62 kD in the similarly treated NALM6 biotinylated anti-µ immunoprecipitate.
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| Fig 7.
Effect of deglycosylation on components of the proB-cell
complex. Cell surface labeling and immunoprecipitation conditions are
as described in Fig 6. The anti-VpreB 4G7 immunoprecipitates from the
JEA2 proB and the anti-µ immunoprecipitates from the NALM6 preB-cell
lines were incubated with or without PNGase F, submitted to a gradient
SDS-PAGE (5% to 15%) under reducing conditions, and transferred onto
immobilon P membrane. Cell surface biotinylated proteins were detected
by streptavidin-peroxidase and the membrane was incubated with the
anti-VpreB 4G7 MoAb and shown by a peroxidase-conjugated goat antimouse
IgG.
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The presence of disulfide bridges between the different components of
the proB cell complex was investigated by two-dimensional SDS-PAGE
analysis. JEA2 cell surface biotinylated proteins were immunoprecipitated with either 4G7 or irrelevant 1 MoAbs and subjected to nonreducing/reducing two-dimensional SDS-PAGE
(Fig 8). All of the proteins that
coimmunoprecipitated with the VpreB molecule were detectable on the
diagonal, indicating that they were noncovalently associated to L
complexes.
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| Fig 8.
Two-dimensional SDS-PAGE analysis of proteins associated
with L at the cell surface of the JEA2 proB-cell line. JEA2 cell
surface labeling and anti-VpreB (4G7) or IgG1 control
immunoprecipitation conditions are as described in Fig 6.
Immunoprecipitates were run in the first and second dimensions under
nonreducing and reducing conditions, respectively, using for both
dimensions a 5% to 15% gradient SDS-PAGE. After transfer onto
immobilon P membrane, biotinylated proteins were shown by
streptavidin-peroxidase.
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Functional Analysis of the proB-Cell Surface Complex
ProB complex signal transduction ability was investigated by measuring
the release of Ca2+ after anti-VpreB
stimulation of the JEA2 proB-cell line. ProB-cell triggering was
compared with anti-VpreB- and anti-µ-induced Ca2+ flux
in the LAZ 221 preB-cell line that was chosen rather than NALM6, which
did not express CD45, or in the Namalwa B-cell line (Fig 9). For the different cell lines, the
Ca2+ response induced by the anti-CD19 stimulation served
as positive control, whereas background level was determined by an
irrelevant isotype-matched 1 MoAb for JEA2 and LAZ 221 or by the
anti-VpreB (4G7) MoAb for Namalwa. An additional negative control using
the 14G3 anti-VpreB MoAb was also introduced for JEA2 cell line (data not shown). The effect of anti-VpreB stimulation was significantly different in preB or proB cells. In preB cells, Ca2+
release started quickly after anti-VpreB stimulation, peaked, and
decreased within a few minutes. Anti-µ stimulation generated the same
profile, although the signal intensity was stronger than with an
anti-VpreB triggering. In proB cells, a lag was observed before any
Ca2+ release that was of lower intensity and reached a
plateau. Thus, anti-VpreB triggering results in different and specific
responses in B-cell precursors depending on whether L is involved in
a proB- or preB-cell complex. These differences may be due to the nature of the transducing components, because the Ig/Ig complex that is part of the µ+/L+ preB receptor is
present in proB-cell lines but is not associated with the
µ/L+ proB-cell
complex.24
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| Fig 9.
Transduction ability of the proB-cell surface complex as
compared with that of the preB- and B-cell receptor. The proB (JEA2),
preB (LAZ 221), and B (Namalwa) cells were loaded with Indo-1 as
described in Materials and Methods. Anti-VpreB 4G7 (30 µg/mL),
anti-CD19 (30 µg/mL), anti-µ (30 µg/mL), or irrelevant IgG1 (30 µg/mL) MoAbs were added and cross-linked by a goat antimouse
IgG+IgM (H+L) at the times indicated by arrows. Fluorescence
variation of indo-1 was measured by FACstar and the percentage of
activated cells was determined by the MultiTime software
(Phoenix Flow Systems, San Diego, CA).
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By analogy to the preB-cell receptor, these data suggest that Ls in
proB-cell complexes may function as proB-cell receptors.
| |
DISCUSSION |
Among newly generated antihuman VpreB MoAbs of the isotype, five
have been selected for their ability to recognize recombinant and
native intracellular VpreB proteins.18 They identify 4 VpreB epitopes, two on the extra-loop and two on the Ig-like domain of
the VpreB molecule, of which only the former gave signals by FACS
analysis. Furthermore, these MoAbs defined two cell surface reactivities (Fig 2). The 10G5 labeled only the preB-cell lines, suggesting that the corresponding epitope remains accessible within the
preB-cell receptor but is lost in the proB-cell complex. By contrast,
4G7 and 4E7 that define a common epitope on the COOH-terminal VpreB
peptide and have a high affinity (kd
1010 mol/L) for VpreB detect the L at the surface
of both proB and preB cells.
The distinctive behavior of the different anti-VpreB shown by FACS
analysis prompted us to compare these MoAbs with the previously reported SLC1/SLC2 using the same techniques (Fig 3). Biacore analysis
showed that SLC1 and SLC2 fixation to free VpreB or L chain was very
weak, whereas they bound to Fab-like molecules strongly, suggesting
that they recognized predominantly a conformational epitope generated
upon association of L to the µ chain. FACS analysis showed that
this epitope was only detected at the surface of preB cells, confirming
the preB-cell specificity of these reagents.10,21 However,
the SLC1 (1) was reported to immunoprecipitate the L in the
absence of the µ chain after internal labeling of the RS4.11
precursor cell line.10 One way to account for this result is to assume that p98, p60, and p40 coimmunoprecipitated proteins confer to L a conformation similar to that induced upon its
interaction with µ chain, resulting in the expression of the SLC1 epitope.
Taking advantage of the 4G7 antibody, a L+ proB
compartment was detected in normal CD34+CD19+
bone marrow cells (Fig 4). These cells did not contain intracytoplasmic µ chains (data not shown), in agreement with previous data in humans.22-24 Because L cell surface expression was
identified on the REH proB-cell line that is a typical prototype of
TEL/AML1 leukemias, we analyzed 12 fresh TEL/AML1 cell populations.
These leukemias that represent the most common genetic translocation in
the B-lineage childhood ALL exhibited a proB phenotype, because they
expressed the CD34, CD19, CD10, and the TdT markers and were negative
for the expression of intracytoplasmic µ chain. It was indeed found
that all of the 12 tested cases were positive for the 4G7 epitope, with
8 having the proB complex expressed at the cell surface. This
observation, in addition to stressing the common expression of the proB
complex at the cell surface of B precursors, also extends the previous
observation from the cell lines to fresh cells that may be considered
closer to the physiological state in vivo. Altogether, these data
suggest that the L may be a good marker for both normal and
pathological proB cells.
In humans, characterization of proteins that allow L to be expressed
at the cell surface (ie, surrogate H chain) has been limited. Sanz and
De La Hera22 identified a p125 protein that coimmunoprecipitated with other minority components at 200, 100, and
70-40 kD using the 688 anti-VpreB MoAb of the µ isotype and the REH
proB-cell line. Using the same approach with the 4G7 MoAb, we found
that the major protein associated with the L at the cell surface of
JEA2 was a p105 (Fig 6). In addition, we consistently detected a weakly
labeled p130 and, depending on the experiments, components at p60-40
Mw. A p125 protein was also identified by Sanz and De La
Hera22 at the surface of the 697 preB-cell line, suggesting
that the proB-cell complex was present on preB cells. We confirm these
data, because we observed that the 4G7 MoAb was capable of
immunoprecipitating the L/p105p130 proB-cell complex on the NALM6
and LAZ 221 preB-cell lines. However, this MoAb, in contrast to 688, immunoprecipitates the L in both proB and preB complexes at the cell
surface of preB cells.
The pattern of surrogate H chain proteins that we obtained is close but
not identical to that detected in a µ precursor
line in mice.20 In this latter case, a complex containing glycoproteins at 200, 130, 105, and occasional bands at 65-35 kD and
5 dimers and VpreB was identified. The potential homologies between
human and mouse proteins include p130 and p105 proteins that are
noncovalently linked to the L. The mouse p130 is a N-linked glycoprotein with a protein core at approximately 100 kD, but it is the
strongest labeled protein after cell surface iodination of the proB
complex, whereas p105 was the major band in humans after cell surface
biotinylation. These labeling intensity differences are not simply due
to the use of different reagents for cell surface labeling of mouse and
human cell lines, because we obtained similar relative band intensity
for the human p105 and the p130 proteins when iodine was used instead
of biotin. Human and mouse p105 differ also in that the latter exists
as homodimers and is sensitive to PNGase F. The identity of this
protein remains unresolved, but a search for a possible
chaperone30 in the vicinity of this molecular weight failed
to identify the grp 94 chaperone among the associated proteins (data
not shown).
The transducing ability of the proB-cell complex has been demonstrated
for the JEA2 proB-cell line by the capacity of the 4G7 anti-VpreB MoAb
to induce a Ca2+ flux. In mice, a Ca2+ influx
induced by anti-5 triggering were also reported with a
µL+ progenitor B-cell
line.12 By comparison with the preB receptor, proB complex
triggering is characterized by a slow and sustained Ca2+
response, suggesting that the inactivation mechanisms that operate after the initial stimulus are less efficient in proB than in preB-cell
lines. Furthermore, in contrast to the preB-cell
receptor8,12 and in accordance with previous results in the
mouse,30 we did not detect any tyrosine phosphorylation
after stimulation of the proB-cell complex (data not shown). This may
be explained by the difference in sensitivity between calcium and
tyrosine phosphorylation tests. The nature of the transducing module
associated with the proB complex remains to be determined, because
Ig and Ig, although present in the JEA2 proB-cell line, were
shown not to be associated with the L chain.24,31
Besides the biochemical and functional characterization of a proB-cell
complex, its possible functions have to be raised. In
5/ mice, a proB to preB differentiation
block exists and normal amounts of proB cells are
present.25 However, we do not know if the
5/ phenotype may be extrapolated to the
entire L (5, VpreB1, and VpreB2) inactivation. We favor the
hypothesis that this complex might be implicated in the delivery of
positive signals (differentiation and/or proliferation) to the proB
cells by interacting with bone marrow stromal cells. The recent
demonstration that mutation within the -like gene in
humans32 leads to a severe immunodeficiency affecting the
proB/preB-cell transition appears compatible with this proposal.
| |
ACKNOWLEDGMENT |
The technical expertise of N. Brun-Roubereau and M. Barad
(cytofluorometry) and of C. Forneli (PE-MoAb conjugation) is gratefully acknowledged. We thank Drs H. Chambost and J. Gabert for providing us
with the TEL/AML1 leukemic cells and thank C. Fossat, D. Sainty, and C. Arnoulet for their help in cell surface analysis studies. Antihuman
surrogate light chain MoAbs (SLC1 and SLC2) were kindly provided by K. Lassoued and M. Cooper. We also thank A. Trautmann for helping us with
measuring the release of Ca2+ and thank K. Lassoued, E. Meffre, and J. Ewbank for their comments on the manuscript.
| |
FOOTNOTES |
Submitted November 10, 1998; accepted February 13, 1999.
L.G. and V.G.-F. contributed equally to this work.
Supported by Centre National de la Recherche Scientifique (CNRS),
Institut Na |
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ABSTRACT
INTRODUCTION