|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4317-4324
Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage
of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific
to Human Pre-B-Cell Receptor
By
Keiko Tsuganezawa,
Nobutaka Kiyokawa,
Yoshinobu Matsuo,
Fujiko Kitamura,
Noriko Toyama-Sorimachi,
Keisuke Kuida,
Junichiro Fujimoto, and
Hajime Karasuyama
From the Department of Immunology, The Tokyo Metropolitan Institute
of Medical Science, Tokyo; Biomedical R&D Department, Sumitomo Electric
Industries, Ltd, Yokohama; the Department of Pathology, National
Children's Medical Research Center, Tokyo; and Fujisaki Cell Center,
Hayashibara Biochemical Labs Inc, Okayama, Japan.
 |
ABSTRACT |
Three novel monoclonal antibodies (MoAbs) have been
established that recognize distinct epitopes of a human pre-B-cell
receptor (pre-BCR) composed of a µ heavy (µH) chain and a
 5/VpreB surrogate light (SL) chain. HSL11 reacts with 5 whereas
HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each
component of the pre-BCR but does bind to the completely assembled
pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of
a panel of human cell lines showed that HSL11 and HSL96 specifically
stained cell lines derived from the pro-B and pre-B-cell stages of
B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre-B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the
determination of the cell lineage and developmental stage of acute
lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs
(B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed
were stained in the cytoplasm with these three MoAbs, the vast majority
of non-B- and non-T-ALLs (53 out of 56 cases) were found positive for
either 5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for
cytoplasmic µH chain, indicative of pre-B-cell origin.
Interestingly, 6 out of these 19 pre-B-ALL cases were found negative
for cytoplasmic staining with HSL2. From these results, we propose a
novel classification of B-ALL in which five subtypes are defined on the
basis of the differential expression of SL chain, µH chain, pre-BCR,
and light chain along the B-cell development.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE LYMPHOBLASTIC leukemia (ALL) is the
most common malignancy in children and accounts for approximately 80%
of childhood leukemia.1,2 ALL seems to be a heterogeneous
group of disorders with subgroups that have distinct clinical and
prognostic features. Several attempts have been made to establish the
classification of ALL cells that can be correlated with clinical
characteristics and the behavior of the disease such as response to
therapy.1,2
The French-American-British (FAB) classification is based on morphology
of leukemic cells and identifies three subgroups of ALL, namely L1, L2,
and L3.3 However, this classification does not tell the
cell lineage and maturational stage from which leukemia arises. Indeed,
the vast majority of childhood ALLs are classified as L1. On the other
hand, the immunophenotypic classification is based on expression of
certain antigens on the surface of leukemic cells and seems more
clinically relevant in case of ALL.4,5 Because normal
lymphocytes express specific antigens in an orderly fashion through
their different stages of development, the expression of certain
antigens on the surface of ALL cells can be considered to indicate the
specific stage of lymphocyte development where the malignant
transformation occurred. Based on reactivity with a panel of
antibodies, ALL is broadly classified as having a T- or B-cell origin.
B-lineage ALL (B-ALL) is further subdivided into three distinct
subtypes: early pre-B cell (or B precursor), pre-B cell, and B
cell2,4,5; or into four subtypes: pro-B (B-I), common
(B-II), pre-B (B-III), and mature-B (B-IV).6 Approximately
85% to 90% of childhood ALL and 75% to 80% of adult ALL is assigned
to be of B-cell lineage, of which nearly 70% falls into the early
pre-B-cell subtype and less than 5% into the mature B-cell subtype.
Markers such as CD10, CD19, CD20, CD21, CD22, CD23, CD24, and HLA-DR
are commonly used to characterize B-ALL. However, one must be cautious
in use of these markers, because no marker is absolutely lineage
specific and stage specific. For example, CD19 can be found in at least
50% of acute myeloid leukemia (AML) with t(8;21),7 and
CD10 in T-lineage ALL (T-ALL) as well.8 Moreover,
approximately 5% to 10% of childhood ALL and 30% of adult ALL cases
express myeloid markers.9,10 The rearrangement patterns of
immunoglobulin (Ig) and T-cell receptor (TCR) genes have also been used
as markers for clonality and origin of leukemic cells. However, the
molecular technique to detect these patterns is not as easy and fast as
immunophenotyping, and also has a limitation in the determination of
the cellular origin. TCR rearrangements are often observed in B-ALL and
also in AML, but at a lower frequency.11,12 IgH gene
rearrangements occur in 10% to 15% of T-ALL and in about 20% of AML
cases.12,13 Thus, there is a clear need to discover more
reliable markers to define the cellular origin of leukemic cells.
Recent studies have shown that pre-B-cell receptor (pre-BCR) plays
critical roles in early B-cell development,14-17 and the lack of its expression resulted in severe impairment of B-cell production.18-21 The pre-BCR is composed of a µ heavy
(µH) chain and surrogate light (SL) chain,22,23 and its
expression is restricted to the pre-B-cell stage of B-cell
development.24,25 The SL chain is composed of 5 and
VpreB,26-30 both of which are already produced before the
µH chain is expressed, that is, before the formation of pre-BCR.
Thus, the expression of the SL chain is restricted to both pro-B and
pre-B-cell stages of B-cell development.24,25 These
lineage- and stage-specific expression patterns of the SL chain and
pre-BCR make them very attractive as potential markers to determine the
cell lineage and developmental stage of ALL cells.
In the present study, we have established three monoclonal antibodies
(MoAbs) with distinct specificity to the human SL chain and pre-BCR.
Analysis of a panel of human leukemia samples has shown that the flow
cytometry with those MoAbs is indeed an easy, fast, and reliable way to
diagnose the cell lineage and developmental stage of ALL. Based on this
analysis, we will propose a novel classification of B-ALL.
Several different nomenclatures are used by researchers to distinguish
different stages of B-cell development and subtypes of
B-ALL.2,4-6,25,31 To avoid possible confusion, in this study we use the following nomenclature for the stages of B-cell development: pro-B cells, pre-B cells, and B cells as H
chain-L chain--, H chain+L
chain--, and H chain+L
chain+-B-lineage cells, respectively.
| |
MATERIALS AND METHODS |
Cell lines.
Human pro-B-cell lines include RS4;11,32
REH,33 NALM16,34 NALM19,35
NALM20,36 and NALM27.37 Human pre-B-cell lines include NALM6,38 NALM17,37 697,39
HPB-NULL,37 and SMS-SB.40 LBW-4,41
Daudi,42 and GM60743 are human B-cell lines
whereas Jurkat44 and MOLT-445 are human T-cell
lines. All the cell lines were maintained in RPMI 1640 supplemented
with 10% fetal calf serum (Equitech-Bio, Inc, Ingram, TX), 100 U/mL of
penicillin-streptomycin (GIBCO-BRL, Grand Island, NY), 2 mmol/L
L-glutamine (GIBCO-BRL), and 5 × 10-5 mol/L
2-mercaptoethanol.
Leukemia samples.
Sixty-three childhood leukemia samples which had been diagnosed by
conventional phenotypic analyses and stocked in liquid nitrogen were
analyzed in this study. They included 56 samples of non-B- and
non-T-ALL, 3 of mature B-ALL (L3 of FAB classification), 2 of T-ALL,
and 2 of AML. All the non-B- and non-T-ALL samples had been
categorized into L1 of FAB classification and assigned to be of
B-lineage origin based on the expression of CD19 on their surface, but
further subtyping had not been performed. Sources of samples were
mainly bone marrow or peripheral blood, but in 1 case of T-ALL the
sample was obtained from pleural effusion, and in all 3 cases of mature
B-ALL the samples were taken from lymph nodes. Immediately before
immunofluorescence staining, frozen samples were thawed, washed, and
resuspended in staining buffer. The research followed the tenets of the
Declaration of Helsinki.
Antibodies.
Mouse MoAbs specific to human CD10, CD19, CD22, CD79b,  L chain and
L chain were purchased from Pharmingen (San Diego, CA). A mouse MoAb
specific to human µH chain was purified from culture supernatants of
a hybridoma clone M-2E6 (American Type Culture Collection,
HB138).46 Phycoerythrin (PE)-conjugated goat anti-mouse antibody was purchased from Southern Biotechnology Associates Inc
(Birmingham, AL).
Establishment of transfectants expressing a human SL chain and a
human/mouse-chimeric pre-BCR.
cDNAs encoding human 5 and VpreB were cloned from a human
pre-B-cell line NALM6 by reverse transcription polymerase chain reaction (RT-PCR) based on the published nucleotide
sequences.28-30 Ig-nonproducing X63.Ag8-653 myeloma
cells47 were transfected with the expression vector
BCMGSHyg22 carrying either the 5 or VpreB cDNA as
described previously.48 Ltk-µC37 fibroblast
cells expressing mouse µH chain22 were transfected with
BCMGSHyg carrying both human 5 and VpreB cDNAs in the same way as
described previously22 to establish
Ltk-pre-BCR transfectants that secrete a soluble form of
chimeric pre-BCR (mouse µH chain/human SL chain complex).
Establishment of MoAbs specific to human 5, VpreB,
and pre-BCR.
The chimeric pre-BCR complexes were purified by absorption to
M4149 (anti-mouse µH MoAb)-conjugated Sepharose beads
(Pharmacia Biotech, Uppsala, Sweden) from culture supernatants of the
Ltk-pre-BCR transfectants. BALB/c mice (6-week-old female;
Japan SLC, Hamamatsu, Japan) were immunized intraperitoneally seven
times at 1-week intervals, each time with approximately 1 µg of the complex absorbed on beads, and then administered a final boost with
intravenous injection of 30 µg of the complex eluted from the
M41-conjugated beads. Three days after final injection, spleen cells
were fused with PAI myeloma cells50 and cultured in medium containing hypoxanthine, aminopterin, thymidine
(GIBCO-BRL) and recombinant interleukin-6.51
Culture supernatants of resulting hybridomas were initially screened by
enzyme-linked immunosorbent assay for reactivity to the chimeric
pre-BCR but not to human IgM (µ and µ). Supernatants of
selected cultures were further characterized by cell staining as well
as immunoprecipitation and cloned twice by limiting dilution. All the
MoAbs established and described here were of the IgG1 subclass and had
L chains as determined by the isotyping kit (Pharmingen).
Immunoprecipitation.
Cells were biosynthetically labeled with [35S]-methionine
(Dupont, Wilmington, DE) for 4 hours and lysed with 1% NP-40 lysis buffer as described previously.48 Cell lysates were
precleared with protein G-Sepharose beads (Pharmacia Biotech AB,
Uppsala, Sweden) and then reacted for 2 hours with protein G-Sepharose beads preincubated with hybridoma culture supernatants or purified antibodies. Immunoprecipitates were electrophoresed on 13% sodium dodecyl sulfate (SDS) polyacrylamide gel, the gel was dried, and the
radioactive bands were visualized by the phosphoimager Fuji BAS2000
(Fuji Photo Film Co Ltd, Tokyo, Japan).
Cell-surface and cytoplasmic staining.
For cell-surface staining, cells suspended in staining buffer
(phosphate-buffered saline with 0.1% bovine serum albumin,
0.05%NaN3) were incubated with 1 µg/mL of mouse MoAbs
for 30 minutes on ice and then with 2 µg/mL of PE-conjugated goat
anti-mouse antibodies. For cytoplasmic staining, cells were
suspended in staining buffer containing 0.05% saponin (Sigma Chemical
Co, St Louis, MO) and 10 mmol/L HEPES, pH 7.3, and stained as for
cell-surface staining. Throughout staining and washing procedures,
0.05% of saponin was kept in buffer. Stained cells were applied to the
flow cytometer FACSCalibur (Becton Dickinson, Mountain View, CA). Cells
present in the lymphocyte gate defined by light scatter were analyzed.
| |
RESULTS |
Establishment of MoAbs specific to 5, VpreB, or a
conformational epitope of the human pre-BCR complex.
Three MoAbs, designated HSL2, HSL11, and HSL96, were established as
described in Materials and Methods. All the MoAbs precipitated from
NALM6 pre-B cells three proteins of 70 kD, 22 kD, and 18 kD
corresponding to µH chain, 5, and VpreB, respectively, as did an
anti-µH chain MoAb (Fig 1A).
Intriguingly, HSL11 and HSL96 precipitated the 5/VpreB SL chain
complex from µH chain-negative RS4;11 pro-B cells whereas HSL2 did
not (Fig 1B). To identify the specificity of each MoAb further,
X63.Ag8-653 cells transfected with either the 5 or VpreB gene were
used for immunoprecipitation. HSL96 precipitated an 18-kD protein from
the VpreB transfectant but not from the 5 transfectant, indicating
its specificity to VpreB (Fig 1C). On the other hand, HSL11
precipitated 22-kD and 16-kD proteins from the 5 transfectant but
not from the VpreB transfectant, indicating its specificity to 5
(Fig 1C). Though the exact nature of the 16-kD protein coprecipitated
with 5 remains to be elucidated, it has been reported that a similar
16-kD protein was detected in the immunoprecipitates of human pre-BCR
complexes.25,52 It should be related to 5 because it
could be detected only in the 5-expressing cells (NALM6, RS4;11, the
5-transfectant) but not in the VpreB transfectant. In contrast to
HSL11 and HSL96, HSL2 precipitated neither 5 nor VpreB from the 5
and VpreB transfectants (Fig 1C). It also did not precipitate IgM
(µ and µ) from human B-cell lines Daudi and LBW-4 (data not
shown). Thus, HSL2 reacted with the complete pre-BCR complex but not
with each component of the pre-BCR complex or the 5/VpreB SL chain.
This indicates that HSL2 is a unique MoAb that recognizes a
conformational epitope formed only when the µH chain and 5/VpreB
SL chain associate with each other to make the pre-BCR complex. On the
other hand, HSL11 and HSL96 reacted with 5 and VpreB, respectively,
in free form, in the form of the 5/VpreB SL chain, or in the form of the complete pre-BCR complex.
View larger version (44K):
[in this window]
[in a new window]
| Fig 1.
Specificity of the newly established MoAbs HSL11, HSL96,
and HSL2 for human 5, VpreB, and pre-BCR, respectively. A
pre-B-cell line NALM6 (A), a pro-B-cell line RS4;11 (B), and
X63.Ag8-653 cells transfected with either the 5 or VpreB gene (C)
were biosynthetically labeled with [35S]-methionine for 4 hours and lysed with 1% NP-40 lysis buffer. Detergent-soluble lysates
were incubated with indicated MoAbs, and immunoprecipitates were
analyzed by 13% SDS polyacrylamide gel electrophoresis under reducing
conditions. The positions of µH, 5, and VpreB are indicated by
arrows, and a 16-kD band precipitated by HSL11 together with 5 is
indicated by an arrow head.
|
|
Differential reactivity of the MoAbs to human pro-B and pre-B-cell
lines.
The reactivity of the three MoAbs to a panel of human cell lines was
examined by immunofluorescence flow cytometry on the cell surface as
well as in the cytoplasm (Fig 2 and
Table 1). For cytoplasmic staining, the
cell membrane was permealized with 0.05% of saponin added in the
staining buffer. HSL11, HSL96, and HSL2 were all found to stain the
cytoplasm and cell surface of all five µH+ pre-B-cell
lines analyzed (HPB-NULL in Fig 2 and Table 1; 697, SMS-SB, NALM6, and
NALM17 in Table 1). The intensity of the surface staining was 7 to 10 times less than that of the cytoplasmic staining, indicating that the
majority of pre-BCR was retained in the cytoplasm. None of the MoAbs
stained mature B-and T-cell lines (LBW-4 in Fig 2 and Table 1; Daudi,
GM607, Jurkat, and MOLT-4 in Table 1), confirming no cross-reactivity
to Ig or TCR.
View larger version (51K):
[in this window]
[in a new window]
| Fig 2.
Flow cytometric analysis of human B-lineage cell lines
with the MoAbs. A pro-B-cell line RS4;11, a pre-B-cell line HPB-NULL,
and a B-cell line LBW-4 were stained by the indicated MoAbs in the
cytoplasm (A) and on the cell surface (B). For cytoplasmic staining,
0.05% of saponin was added to the staining buffer. Cells were first
incubated with the MoAbs and then with PE-anti-mouse L chain
antibody. The fluorescence intensity of the stained cells was analyzed
by a flow cytometer. The thin dotted-line histograms indicate control
staining with an isotype-matched control MoAb whereas the thick
solid-line histograms indicate staining with MoAbs specific to 5,
VpreB, pre-BCR, µH chain, L chain, or L chain. All data
including other cell lines are summarized in Table 1.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
The Lineage- and Differentiation Stage-Specific
Expression of SL Chain and Pre-BCR Detected by the MoAbs in a Panel
of Human Cell Lines
|
|
When µH-negative pro-B-cell lines were analyzed, the three MoAbs
showed differential reactivity. HSL11 and HSL96 stained cytoplasm of
RS4;11 pro-B-cell line whereas HSL2 did not (Fig 2). This was also the
case for other pro-B-cell lines NALM16, NALM19, NALM20, and NALM27
(Table 1). One pro-B-cell line, REH, was found to be stained by HSL11
but not by HSL96 or HSL2 (Table 1), in agreement with the result of
RT-PCR analysis that showed the presence of 5 transcripts but not
VpreB transcripts in REH (data not shown). The lack of reactivity of
HSL2 with µH- pro-B-cell lines is in line with the
conclusion drawn from the immunoprecipitation experiments that HSL2
reacts with the complete pre-BCR complex but not with each component of
the complex or 5/VpreB SL chain. Notably, none of the MoAbs stained
the cell surface of the pro-B-cell lines even though 5 and VpreB
proteins were detected intracellularly by HSL11 and HSL96,
respectively. This suggests that only completely assembled pre-BCR
complexes can be expressed on the cell surface in humans, in agreement
with a previous report,25 but in contrast to the
observation in mouse pro-B cells.24,48
Collectively, HSL11 and HSL96 specifically reacted with cell lines
derived from both pro-B- and pre-B-cell stages when analyzed by the
cytoplasmic staining. In contrast, HSL2 exclusively recognized cell
lines derived from the pre-B-cell stage but not from the earlier or
later stages of B-cell development. Thus, flow cytometric analysis with
the combination of these MoAbs seems to be a convenient and reliable
way to distinguish the developmental stage of B-lineage cells.
Flow cytometric analysis of human acute leukemia cells.
The analysis of human cell lines described above strongly suggested a
possible clinical application of the MoAbs to determine the cellular
origin of human leukemia cells. Therefore, we next performed flow
cytometric analysis of 63 childhood leukemia samples that had been
diagnosed by conventional phenotypic analyses and stocked in liquid
nitrogen. These included 56 samples of non-B- and non-T-ALL, 3 of
mature B-ALL, 2 of T-ALL, and 2 of AML (Fig 3 and Table 2). All the non-B-, non-T-ALL
samples had been assigned to be of B-lineage origin based on the
expression of CD19 on their surface, but further subtyping had not been
performed. Because the analysis of human cell lines showed that the
surface expression of pre-BCR was extremely low as compared with its
cytoplasmic expression, we focused on the cytoplasmic staining in the
following experiments. The MoAbs used were HSL11 (anti-5), HSL96
(anti-VpreB), HSL2 (anti-pre-BCR), anti-µH chain, anti-L chain,
anti-L chain, anti-CD19, anti-CD22, and anti-CD79b. Staining
profiles of representative samples are shown in Fig 3, and all data are
summarized in Tables 2 and 3.
View larger version (52K):
[in this window]
[in a new window]
| Fig 3.
Flow cytometric analysis of human acute leukemia samples
by using the MoAbs HSL11, HSL96, and HSL2. Frozen samples of childhood
acute leukemia were thawed and stained in the cytoplasm (in a few cases
on the cell surface as well) by the MoAbs specific to 5, VpreB,
pre-BCR, µH chain, L chain, L chain, CD19, CD22, or CD79b in
the same way as in Fig 2. Staining profiles with these MoAbs (thick
solid line) and staining profiles with an isotype-matched control MoAb
(thin dotted line) are overlaid in the histograms. Though data are not
shown, the T-ALL sample was positive for CD3, CD4, and CD8 whereas the
AML sample was positive for CD13 and CD33. All data including other
leukemia samples are summarized in Tables 2 and 3. Sources of leukemia
samples: BM, bone marrow; PB, peripheral blood; LN, lymph node; PE,
pleural effusion.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3.
A Novel Classification of Non-B-, Non-T-ALL Based on
the Expression of SL Chain, µH Chain, and Pre-BCR, and the
Incidence of Each Group Observed in This Study
|
|
None of the mature B-ALL, T-ALL, and AML samples were positive for
5, VpreB, or pre-BCR as detected by HSL11, HSL96, and HSL2,
respectively (three rows from the bottom in Fig 3 and Table 2). Among
56 samples of non-B-, non-T-CD19+ ALL analyzed, 19 were
found positive for cytoplasmic µH chain, indicative of pre-B-cell
origin, whereas the rest (37 samples) were negative, indicative of
pro-B-cell origin (Table 2). Whereas none of these non-B-, non-T-ALL
samples expressed L chains ( or ), 82%, 88%, and 23% of them
expressed 5, VpreB, and pre-BCR, respectively (Table 2). The vast
majority (95%) of the non-B-, non-T-ALL samples were found to
express at least one of the 5 and VpreB components of the SL chain
(Table 3). In the remaining 3 cases (5%), no components of 5,
VpreB, µH chain, and L chain were detected (for example, top row in
Fig 3). Nevertheless, these three "null" samples were found to
express not only CD19 but also CD22 and CD79b in the cytoplasm (for
example, top row in Fig 3), indicative of B-lineage origin.
Thirty-four cases (61%) of the non-B-, non-T-ALL samples expressed
SL chain (5 and/or VpreB) but not µH chain (Table 3): 24 cases were positive for both 5 and VpreB (for example, second row in
Fig 3), 3 were positive for 5 only, and 7 were positive for VpreB
only. All the 19 µH+ pre-B-ALL samples were found to
express SL chain (5 and/or VpreB; Table 3): 18 were positive
for both 5 and VpreB (for example, third and fourth rows of Fig 3),
and 1 was positive for 5 only. When the expression of pre-BCR was
analyzed by HSL2, 13 out of the 19 µH+ pre-B-ALL samples
were positive for pre-BCR (for example, fourth row in Fig 3) whereas
the remaining 6 samples were negative for pre-BCR (for example, third
row in Fig 3) as summarized in Table 3. In a few cases in which enough
cell numbers were available, cell-surface staining was also performed.
The surface expression of pre-BCR in pre-B-ALL samples was found to be
extremely low when compared with the surface expression of IgM in
mature B-ALL samples (for example, compare the expression of surface
µH chain in fourth and fifth rows of Fig 3).
| |
DISCUSSION |
The differentiation of B cells from hematopoietic stem cells progresses
through a series of successive stages that are defined by sequential
rearrangements of Ig loci, first in IgH locus and then in IgL locus,
and expression of various stage-associated markers including Ig H and L
chain proteins.31,53 Recent studies have clarified our
understanding of mechanisms by which the Ig gene rearrangements are
controlled and how Ig gene products participate in the regulation of
the B-cell differentiation program.15 Pre-BCR composed of
µH chain and SL chain has been shown to transduce signals for cell
proliferation and differentiation as well as the allelic exclusion at
IgH chain loci during the pre-B-cell stage of B-cell
differentiation.14-17 The importance of pre-BCR in human
B-cell development was highlighted by the discovery of B-cell-deficient patients in which the gene coding for µH chain or
SL chain had a deletion or mutation.20,21
Besides the physiological importance of pre-BCR, its expression pattern
restricted to an early stage of B-cell development makes it a hallmark
of early B-cell precursors. Indeed, it has been shown that the
expression of VpreB and 5 genes coding for SL chain could be used as
a specific marker for B-cell precursor leukemias.54,55
However, the detection of the gene expression by Northern blotting as
shown in previous studies would not be a routine laboratory
work.54,55 Though the RT-PCR technique might be more
sensitive to detect the expression and more convenient as a routine
work,55 a possible drawback of this sensitive technique is
to have false positive signals derived from normal B-cell precursor cells contaminated in leukemia samples.
In the present study, we showed that flow cytometric analysis with the
combination of the SL chain- and pre-BCR-specific MoAbs was an easy,
fast, and reliable way to diagnose leukemias derived from early B-cell
precursors. Because the expression of SL chain and pre-BCR on the cell
surface was found to be extremely low and often undetectable,
cytoplasmic staining was needed for definite determination of their
expression in leukemic cells. Fortunately, we figured out that
cytoplasmic staining of leukemia samples could be performed as easily
as cell-surface staining by just adding 0.05% of saponin in staining
buffer without any other treatment of cells such as fixation with
paraformaldehyde or ethanol. Though several laboratories have
established MoAbs specific to either human 5 or VpreB
protein,25,56-58 no study has been reported that uses those
MoAbs for analysis of leukemia specimens as far as we know. Because all
those MoAbs are of IgM subclass in contrast to our MoAbs (IgG1
subclass), they might not be adequate for cytoplasmic staining. In
accordance with the previous studies analyzing the gene expression
using Northern blot and RT-PCR,54,55 the vast majority of
non-B-, non-T-ALL (53 out of 56 cases) analyzed in the present study
were found to express 5 protein, VpreB protein, or both. On the
contrary, none of mature B-ALL, T-ALL, and AML analyzed so far
expressed these proteins, indicating the lineage and stage fidelity of
SL chain expression even in leukemic cells.
The flow cytometric analysis using the MoAbs established in this study
seems to be extremely useful for the precise classification of B-ALL
according to the stages of B-cell development from which leukemic cells
have arisen. In the course of B-cell development, the production of
µH chains starts at the pre-B-cell stage and continues until the
mature B-cell stage, whereas the production of SL chains already starts
at the pro-B-cell stage and ceases before B-cell
stage.24,25 Therefore, the B-cell differentiation process
can be divided at least into three stages: the pro-B-cell stage
expressing SL chains but not µH chains; the pre-B-cell stage expressing both SL and µH chains, hence pre-BCR; and the B-cell stage
expressing µH chains and conventional L chains in place of SL chains.
In the present study, we found that the expression patterns of 5 and
VpreB proteins were parallel in most of the cell lines and leukemic
samples though some cases expressed only either of these proteins. To
avoid complexity in the ALL classification and make it as simple as
possible, we decided to score cells as positive for SL chain when 5
or VpreB or both were detected in them by HSL11 and HSL96 MoAbs (Table
3). By these criteria, among 56 non-B-, non-T-CD19+ ALL
samples analyzed in the present study, 34 cases (61%) are assigned to
pro-B ALL whereas 19 cases (34%) are assigned to pre-B ALL. The
remaining 3 cases (5%) were found negative for both SL and µH
chains. Because these 3 expressed not only CD19 but also CD22 and CD79b
(B29, Ig ) in their cytoplasm, it is most likely that they were
derived from very early progenitor cells that had been committed to
B-cell lineage. Therefore, we would like to propose to designate this
group as pro-B-I-ALL and the SL chain+µH
chain- group as pro-B-II-ALL (Table 3).
Intriguingly, not all of the 19 µH chain+ pre-B-ALL
cases were stained by the MoAb HSL2 which recognizes pre-BCR: 13 cases were positive for HSL2 staining whereas 6 cases were negative (Table
3). In 1 case of the HSL2-negative pre-B-ALL group, the reason for
nonreactivity with HSL2 was easily understandable because that case
lacked VpreB expression. In contrast, the remainder (5 cases) of the
HSL2-negative pre-B-ALL cases expressed all the components of pre-BCR
detected by the specific MoAbs. Because HSL2 recognizes a completely
assembled pre-BCR complex but not each component of the pre-BCR, one
may assume that in these 5 cases both µH chain and 5/VpreB SL
chain were produced but not associated with each other as efficiently
as to form pre-BCR. Though limited cell numbers in stocked leukemic
specimens could not allow us to test this assumption by biochemical
analyses, a recent study on the repertoire of variable
region of IgH chains (VH) of mouse bone marrow precursor B
cells suggests this assumption is plausible. The study showed that only
half of µH chains expressed in early pre-B cells (c-kit+
cytoplasmic µH+ cells) had the capacity to associate with
SL chains to form a pre-BCR.59 Though such an analysis has
not yet been performed for human counterparts, our observation in human
pre-B-ALL samples strongly suggests that the same thing may happen in
an early pre-B-cell stage of normal human B-cell development. The
study on mouse pre-B cells predicted distinct fates of
pre-BCR+ and pre-BCR- pre-B cells in B-cell
development: the former can differentiate further toward B cells
whereas the latter cannot unless another rearrangement or
VH gene replacement takes place to replace µH chains.59 Based on these, we would like to propose to
categorize pre-B-ALL into two groups, pre-B- (pre-BCR+)
ALL and pre-B- (pre-BCR-) ALL, which can be easily
distinguished by the reactivity with HSL2 (Table 3). We believe that
transitional pre-B-ALL, which was recently reported to express µH
chains on the surface but not with conventional L chains,60
will likely fall into the pre-B- (pre-BCR+) ALL category
of our classification when tested. As far as we analyzed, none of the
leukemia samples expressed both SL chains and conventional L chains
simultaneously (Tables 2 and 3), in accordance with the observation in
normal human precursor B cells.25,61
In conclusion, the flow cytometric analysis with the combination of the
MoAbs established in this study made it possible to categorize B-ALL
into five subgroups: pro-B-I-ALL, pro-B-II-ALL, pre-B-
(pre-BCR-) ALL, pre-B- (pre-BCR+) ALL, and
B-ALL. This classification is based on the differential expression of
SL chain, µH chain, pre-BCR, and L chain along normal B-cell
development. Therefore, it would improve the precision of diagnosis of
ALL by providing specific information regarding the lineage and stage
of differentiation of the malignant cells. A large-scale survey of
leukemia cases will be necessary to correlate each subtype of B-ALL
defined by the novel classification with prognosis or response to
treatment. Flow cytometric analysis with the MoAbs might also
facilitate the detection of minimal residual leukemia. Furthermore, the
MoAbs established in this study could be useful tools to study normal
early B-cell development in human bone marrow as well.
| |
ACKNOWLEDGMENT |
We thank Mariko Yamagishi for excellent secretarial assistance.
| |
FOOTNOTES |
Submitted June 22, 1998;
accepted August 1, 1998.
Supported in part by grants-in-aid from the Ministry of Education,
Science, Sports and Culture, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hajime Karasuyama, MD, PhD, Department of
Immunology, The Tokyo Metropolitan Institute of Medical Science,
3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan; e-mail:
karasuyama{at}rinshoken.or.jp.
| |
REFERENCES |
1.
Pui CH:
Childhood leukemias.
N Engl J Med
332:1618, 1995[Free Full Text]
2.
Cortes JE, Kantarjian HM:
Acute lymphoblastic leukemia. A comprehensive review with emphasis on biology and therapy.
Cancer
76:2393, 1995[Medline]
[Order article via Infotrieve]
3.
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C:
The morphological classification of acute lymphoblastic leukaemia: Concordance among observers and clinical correlations.
Br J Haematol
47:553, 1981[Medline]
[Order article via Infotrieve]
4.
Pui CH, Behm FG, Crist WM:
Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia.
Blood
82:343, 1993[Abstract/Free Full Text]
5.
Jennings CD, Foon KA:
Recent advances in flow cytometry: Application to the diagnosis of hematologic malignancy.
Blood
90:2863, 1997[Free Full Text]
6.
Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, van't Veer MB:
Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL).
Leukemia
9:1783, 1995[Medline]
[Order article via Infotrieve]
7.
Kita K, Nakase K, Miwa H, Masuya M, Nishii K, Morita N, Takakura N, Otsuji A, Shirakawa S, Ueda T, Nasu K, Kyo T, Dohy H, Kamada N:
Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: Frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34.
Blood
80:470, 1992[Abstract/Free Full Text]
8.
Pui CH, Rivera GK, Hancock ML, Raimondi SC, Sandlund JT, Mahmoud HH, Ribeiro RC, Furman WL, Hurwitz CA, Crist WM, Behm FG:
Clinical significance of CD10 expression in childhood acute lymphoblastic leukemia.
Leukemia
7:35, 1993[Medline]
[Order article via Infotrieve]
9.
Wiersma SR, Ortega J, Sobel E, Weinberg KI:
Clinical importance of myeloid-antigen expression in acute lymphoblastic leukemia of childhood.
N Engl J Med
324:800, 1991[Abstract]
10.
Guyotat D, Campos L, Shi ZH, Charrin C, Treille D, Magaud JP, Fiere D:
Myeloid surface antigen expression in adult acute lymphoblastic leukemia.
Leukemia
4:664, 1990[Medline]
[Order article via Infotrieve]
11.
Hara J, Benedict SH, Champagne E, Mak TW, Minden M, Gelfand EW:
Relationship between rearrangement and transcription of the T-cell receptor  , , and  genes in B-precursor acute lymphoblastic leukemia.
Blood
73:500, 1989[Abstract/Free Full Text]
12.
Oster W, Konig K, Ludwig WD, Ganser A, Lindemann A, Mertelsmann R, Herrmann F:
Incidence of lineage promiscuity in acute myeloblastic leukemia: Diagnostic implications of immunoglobulin and T-cell receptor gene rearrangement analysis and immunological phenotyping.
Leuk Res
12:887, 1988[Medline]
[Order article via Infotrieve]
13.
Felix CA, Reaman GH, Korsmeyer SJ, Hollis GF, Dinndorf PA, Wright JJ, Kirsch IR:
Immunoglobulin and T cell receptor gene configuration in acute lymphoblastic leukemia of infancy.
Blood
70:536, 1987[Abstract/Free Full Text]
14.
Melchers F, Rolink A, Grawunder U, Winkler TH, Karasuyama H, Ghia P, Andersson J:
Positive and negative selection events during B lymphopoiesis.
Curr Opin Immunol
7:214, 1995[Medline]
[Order article via Infotrieve]
15.
Rajewsky K:
Clonal selection and learning in the antibody system.
Nature
381:751, 1996[Medline]
[Order article via Infotrieve]
16.
Burrows PD, Cooper MD:
B-cell development in man.
Curr Opin Immunol
5:201, 1993[Medline]
[Order article via Infotrieve]
17.
Karasuyama H, Rolink A, Melchers F:
Surrogate light chain in B cell development.
Adv Immunol
63:1, 1996[Medline]
[Order article via Infotrieve]
18.
Kitamura D, Roes J, Kuhn R, Rajewsky K:
A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene.
Nature
350:423, 1991[Medline]
[Order article via Infotrieve]
19.
Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K:
A critical role of 5 protein in B cell development.
Cell
69:823, 1992[Medline]
[Order article via Infotrieve]
20.
Yel L, Minegishi Y, Coustan-Smith E, Buckley RH, Trubel H, Pachman LM, Kitchingman GR, Campana D, Rohrer J, Conley ME:
Mutations in the µ heavy-chain gene in patients with agammaglobulinemia.
N Engl J Med
335:1486, 1996[Abstract/Free Full Text]
21.
Minegishi Y, Coustan-Smith E, Wang YH, Cooper MD, Campana D, Conley ME:
Mutations in the human 5/14.1 gene result in B cell deficiency and agammaglobulinemia.
J Exp Med
187:71, 1998[Abstract/Free Full Text]
22.
Karasuyama H, Kudo A, Melchers F:
The proteins encoded by the VpreB and 5 pre-B cell-specific genes can associate with each other and with m heavy chain.
J Exp Med
172:969, 1990[Abstract/Free Full Text]
23.
Tsubata T, Reth M:
The products of pre-B cell-specific genes (5 and VpreB) and the immunoglobulin µ chain form a complex that is transported onto the cell surface.
J Exp Med
172:973, 1990[Abstract/Free Full Text]
24.
Karasuyama H, Rolink A, Shinkai Y, Young F, Alt FW, Melchers F:
The expression of VpreB/5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.
Cell
77:133, 1994[Medline]
[Order article via Infotrieve]
25.
Lassoued K, Nunez CA, Billips L, Kubagawa H, Monteiro RC, LeBlen TW, Cooper MD:
Expression of surrogate light chain receptors is restricted to a late stage in pre-B cell differentiation.
Cell
73:73, 1993[Medline]
[Order article via Infotrieve]
26.
Sakaguchi N, Melchers F:
5, a new light-chain-related locus selectively expressed in pre-B lymphocytes.
Nature
324:579, 1986[Medline]
[Order article via Infotrieve]
27.
Kudo A, Melchers F:
A second gene, VpreB in the 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.
EMBO J
6:2267, 1987[Medline]
[Order article via Infotrieve]
28.
Bauer SR, Kudo A, Melchers F:
Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.
EMBO J
7:111, 1988[Medline]
[Order article via Infotrieve]
29.
Hollis GF, Evans RJ, Stafford-Hollis JM, Korsmeyer SJ, McKearn JP:
Immunoglobulin light-chain-related genes 14.1 and 16.1 are expressed in pre-B cells and may encode the human immunoglobulin  light-chain protein.
Proc Natl Acad Sci USA
86:5552, 1989[Abstract/Free Full Text]
30.
Schiff C, Bensmana M, Guglielmi P, Milili M, Lefranc MP, Fougereau M:
The immunoglobulin -like gene cluster (14.1, 16.1 and Fl1) contains gene(s) selectively expressed in pre-B cells and is the human counterpart of the mouse 5 gene.
Int Immunol
2:201, 1990[Abstract/Free Full Text]
31.
Uckun FM:
Regulation of human B-cell ontogeny.
Blood
76:1908, 1990[Free Full Text]
32.
Stong RC, Korsmeyer SJ, Parkin JL, Arthur DC, Kersey JH:
Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics.
Blood
65:21, 1985[Abstract/Free Full Text]
33.
Rosenfeld C, Goutner A, Choquet C, Venuat AM, Kayibanda B, Pico JL, Greaves MF:
Phenotypic characterisation of a unique non-T, non-B acute lymphoblastic leukaemia cell line.
Nature
267:841, 1977[Medline]
[Order article via Infotrieve]
34.
Kohno S, Minowada J, Sandberg AA:
Chromosome evolution of near-haploid clones in an established human acute lymphoblastic leukemia cell line (NALM-16).
J Natl Cancer Inst
64:485, 1980
35.
Matsuo Y, Ariyasu T, Adachi T, Tsubota T, Imanishi J, Minowada J:
Establishment and characterization of a bi-phenotypic leukemic cell line, NALM-19, from a patient with acute leukemia.
Hum Cell
4:257, 1991[Medline]
[Order article via Infotrieve]
36.
Matsuo Y, Ariyasu T, Ohmoto E, Kimura I, Minowada J:
Bi-phenotypic t(9;22)-positive leukemia cell lines from a patient with acute leukemia: NALM-20, established at the onset; and NALM-21, NALM-22 and NALM-23, established after relapse.
Hum Cell
4:335, 1991[Medline]
[Order article via Infotrieve]
37.
Matsuo Y, Drexler HG:
Establishment and characterization of human B cell precursor-leukemia cell lines.
Leuk Res
22:567, 1998[Medline]
[Order article via Infotrieve]
38.
Hurwitz R, Hozier J, LeBien T, Minowada J, Gajl-Peczalska K, Kubonishi I, Kersey J:
Characterization of a leukemic cell line of the pre-B phenotype.
Int J Cancer
23:174, 1979[Medline]
[Order article via Infotrieve]
39.
Findley HW Jr, Cooper MD, Kim TH, Alvarado C, Ragab AH:
Two new acute lymphoblastic leukemia cell lines with early B-cell phenotypes.
Blood
60:1305, 1982[Abstract/Free Full Text]
40.
Smith RG, Dev VG, Shannon WA Jr:
Characterization of a novel human pre-B leukemia cell line.
J Immunol
126:596, 1981[Abstract]
41.
Hendershot L, Levitt D:
Differential regulation of membrane and secretory µ chain synthesis in human B cell lines. Regulation of membrane µ or secreted µ.
J Exp Med
156:1622, 1982[Abstract/Free Full Text]
42.
Klein E, Klein G, Nadkarni JS, Nadkarni JJ, Wigzell H, Clifford P:
Surface IgM- specificity on a Burkitt lymphoma cell in vivo and in derived culture lines.
Cancer Res
28:1300, 1968[Abstract/Free Full Text]
43.
Dolby TW, Devuono J, Croce CM:
Cloning and partial nucleotide sequence of human immunoglobulin µ chain cDNA from B cells and mouse-human hybridomas.
Proc Natl Acad Sci USA
77:6027, 1980[Abstract/Free Full Text]
44.
Schneider U, Schwenk HU, Bornkamm G:
Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma.
Int J Cancer
19:521, 1977
45.
Minowada J, Onuma T, Moore GE:
Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes.
J Natl Cancer Inst
49:891, 1972
46.
Forghani B, Myoraku CK, Schmidt NJ:
Production of monoclonal antibodies to human IgM for assay of viral IgM antibodies.
J Virol Methods
5:317, 1982[Medline]
[Order article via Infotrieve]
47.
Kearney JF, Radbruch A, Liesegang B, Rajewsky K:
A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.
J Immunol
123:1548, 1979[Abstract/Free Full Text]
48.
Karasuyama H, Rolink A, Melchers F:
A complex of glycoproteins is associated with VpreB/5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines.
J Exp Med
178:469, 1993[Abstract/Free Full Text]
49.
Leptin M:
Monoclonal antibodies specific for murine IgM. II. Activation of B lymphocytes by monoclonal antibodies specific for the four constant domains of IgM.
Eur J Immunol
15:131, 1985[Medline]
[Order article via Infotrieve]
50.
Stocker JW, Forster HK, Miggiano V, Stahli C, Staiger G, Takacs B, Staehelin T:
Generation of 2 new mouse myeloma cell lines `PAI' and `PAI-O' for hybridoma production.
Res Discl
217:155, 1982
51.
Tohyama N, Karasuyama H, Tada T:
Growth autonomy and tumorigenicity of interleukin 6-dependent B cells transfected with interleukin 6 cDNA.
J Exp Med
171:389, 1990[Abstract/Free Full Text]
52.
Kerr WG, Cooper MD, Feng L, Burrows PD, Hendershot LM:
µ heavy chains can associate with a pseudo-light chain complex ( L) in human pre-B cell lines.
Int Immunol
1:355, 1989[Abstract/Free Full Text]
53.
Tonegawa S:
Somatic generation of antibody diversity.
Nature
302:575, 1983[Medline]
[Order article via Infotrieve]
54.
Bauer SR, Kubagawa H, Maclennan I, Melchers F:
VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.
Blood
78:1581, 1991[Abstract/Free Full Text]
55.
Schiff C, Milili M, Bossy D, Tabilio A, Falzetti F, Gabert J, Mannoni P, Fougereau M:
-like and V preB genes expression: An early B-lineage marker of human leukemias.
Blood
78:1516, 1991[Abstract/Free Full Text]
56.
Guelpa-Fonlupt V, Tonnelle C, Blaise D, Fougereau M, Fumoux F:
Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression.
Eur J Immunol
24:257, 1994[Medline]
[Order article via Infotrieve]
57.
Sanz E, de la Hera A:
A novel anti-VpreB antibody identifies immunoglobulin-surrogate receptors on the surface of human pro-B cells.
J Exp Med
183:2693, 1996[Abstract/Free Full Text]
58.
Meffre E, Fougereau M, Argenson JN, Aubaniac JM, Schiff C:
Cell surface expression of surrogate light chain ( L) in the absence of µ on human pro-B cell lines and normal pro-B cells.
Eur J Immunol
26:2172, 1996[Medline]
[Order article via Infotrieve]
59.
ten Boekel E, Melchers F, Rolink AG:
Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor.
Immunity
7:357, 1997[Medline]
[Order article via Infotrieve]
60.
Koehler M, Behm FG, Shuster J, Crist W, Borowitz M, Look AT, Head D, Carroll AJ, Land V, Steuber P, Pullen DJ:
Transitional pre-B-cell acute lymphoblastic leukemia of childhood is associated with favorable prognostic clinical features and an excellent outcome: A Pediatric Oncology Group study.
Leukemia
7:2064, 1993[Medline]
[Order article via Infotrieve]
61.
Ghia P, ten Boekel E, Sanz E, de la Hera A, Rolink A, Melchers F:
Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.
J Exp Med
184:2217, 1996[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y. Watanabe, T. Takahashi, A. Okajima, M. Shiokawa, N. Ishii, I. Katano, R. Ito, M. Ito, M. Minegishi, N. Minegishi, et al.
The analysis of the functions of human B and T cells in humanized NOD/shi-scid/{gamma}cnull (NOG) mice (hu-HSC NOG mice)
Int. Immunol.,
July 1, 2009;
21(7):
843 - 858.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Takada, H. Kanegane, A. Nomura, K. Yamamoto, K. Ihara, Y. Takahashi, S. Tsukada, T. Miyawaki, and T. Hara
Female agammaglobulinemia due to the Bruton tyrosine kinase deficiency caused by extremely skewed X-chromosome inactivation
Blood,
January 1, 2004;
103(1):
185 - 187.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. G. Noordzij, N. S. Verkaik, M. van der Burg, L. R. van Veelen, S. de Bruin-Versteeg, W. Wiegant, J. M. J. J. Vossen, C. M. R. Weemaes, R. de Groot, M. Z. Zdzienicka, et al.
Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint in bone marrow
Blood,
February 15, 2003;
101(4):
1446 - 1452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Taguchi, N. Kiyokawa, K. Mimori, T. Suzuki, T. Sekino, H. Nakajima, M. Saito, Y. U. Katagiri, N. Matsuo, Y. Matsuo, et al.
Pre-B Cell Antigen Receptor-Mediated Signal Inhibits CD24-Induced Apoptosis in Human Pre-B Cells
J. Immunol.,
January 1, 2003;
170(1):
252 - 260.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-C. Rissoan, T. Duhen, J.-M. Bridon, N. Bendriss-Vermare, C. Peronne, B. d. S. Vis, F. Briere, and E. E. M. Bates
Subtractive hybridization reveals the expression of immunoglobulinlike transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells
Blood,
October 16, 2002;
100(9):
3295 - 3303.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. G. Noordzij, S. de Bruin-Versteeg, N. S. Verkaik, J. M. J. J. Vossen, R. de Groot, E. Bernatowska, A. W. Langerak, D. C. van Gent, and J. J. M. van Dongen
The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins
Blood,
August 28, 2002;
100(6):
2145 - 2152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
R. P. Stephan, E. Elgavish, H. Karasuyama, H. Kubagawa, and M. D. Cooper
Analysis of VpreB Expression During B Lineage Differentiation in {lambda}5-Deficient Mice
J. Immunol.,
October 1, 2001;
167(7):
3734 - 3739.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nomura, H. Kanegane, H. Karasuyama, S. Tsukada, K. Agematsu, G. Murakami, S. Sakazume, M. Sako, R. Tanaka, Y. Kuniya, et al.
Genetic defect in human X-linked agammaglobulinemia impedes a maturational evolution of pro-B cells into a later stage of pre-B cells in the B-cell differentiation pathway
Blood,
July 15, 2000;
96(2):
610 - 617.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. E. Donohoe, G. B. Beck-Engeser, N. Lonberg, H. Karasuyama, R. L. Riley, H.-M. Jack, and B. B. Blomberg
Transgenic Human {lambda}5 Rescues the Murine {lambda}5 Nullizygous Phenotype
J. Immunol.,
May 15, 2000;
164(10):
5269 - 5276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction