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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 610-617
IMMUNOBIOLOGY
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
Keiko Nomura,
Hirokazu Kanegane,
Hajime Karasuyama,
Satoshi Tsukada,
Kazunaga Agematsu,
Gyokei Murakami,
Satoru Sakazume,
Masahiro Sako,
Rieko Tanaka,
Yoshinori Kuniya,
Takuya Komeno,
Shigehiko Ishihara,
Keizo Hayashi,
Tadamitsu Kishimoto, and
Toshio Miyawaki
From the Department of Pediatrics at the Faculty of
Medicine, Toyama Medical and Pharmaceutical University, the Toyama Red
Cross Hospital, and the Kamiichi Welfare Hospital, Toyama, Japan; the
Department of Immunology, The Tokyo Metropolitan Institute of Medical
Science, Tokyo, Japan; the Department of Pediatrics, Dokkyo Medical
Koshigaya School, Saitama, Japan; the Department of Pediatrics at Osaka
City General Hospital and Kashiwara Municipal Hospital and the
Department of Molecular Medicine, Osaka University Medical
School, Osaka, Japan; the Department of Pediatrics, Shinshu
University School of Medicine, Matsumoto, Japan; the Japanese
Red Cross Society Wakayama Medical Center, Wakayama, Japan;
the Department of Pediatrics, NTT Sapporo Hospital, Sapporo,
Japan; and the Division of Hematology, Institute of Clinical Medicine,
University of Tsukuba, Tsukuba, Japan.
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Abstract |
Surrogate light chains ( 5/VpreB) are selectively
expressed in early precursors of B cells. B-cell defects in
X-linked agammaglobulinemia (XLA) are caused by mutations in the
gene for Bruton's tyrosine kinase. To elucidate the nature of early
B-lineage cells in bone marrow (BM), samples from 13 XLA patients
and 24 healthy controls of different ages were comparatively
analyzed using an antihuman VpreB monoclonal antibody. Expression of
surrogate light (SL) and µ-heavy chains were examined after cell
membrane permeabilization because they are mainly expressed in the
cytoplasm of early B-lineage cells. A flow cytometric analysis of
normal BM identified 5 discrete cell types of B cells:
µ SL++ (pro-B [B-cell progenitor]),
µlowSL++ (pre-B1a),
µlowSL+ (pre-B1b),
µlowSL (pre-B2), and
µhighSL (B). The large cells,
presumably in cycling states, were enriched in pre-B1a cells. The
frequencies of B-lineage cells in BM were higher in young children, and
declined with advancing age. In contrast, XLA showed a profound
reduction in BM B-lineage cells. In XLA BM, an expansion of pro-B cells
with some small pre-B1a cells was marked, but other cells were
negligible. These observations illustrate a B-cell maturation defect in
XLA as well as a normal human B-cell differentiation pathway. The
results suggest that the genetic defect in XLA may impede the evolution
of pro-B cells beyond the earlier pre-B stage into the later stage of
pre-B cells in B-cell development.
(Blood. 2000;96:610-617)
© 2000 by The American Society of Hematology.
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Introduction |
In mammals, the generation of B lymphocytes from
multipotent hematopoietic stem cells is found first in the fetal
liver.1 After birth, the bone marrow (BM) becomes the major
source of B-cell precursors and produces mature B cells throughout
life.2-4 Many studies have demonstrated that
the intracytoplasmic and surface expression of various developmentally
regulated gene products is associated with progression along the B-cell
differentiation pathway.3-10 Early B-cell progenitors
(pro-B cells) express the enzyme terminal deoxynucleotidyl transferase
(TdT), which functions in the completion of immunoglobulin (Ig)
gene rearrangements.5 Pre-B cells were
originally discriminated from mature B cells or other hematopoietic
cells on the basis of an intracytoplasmic µ-heavy (µH) chain and
a lack of cell surface IgM.3 One surface marker identified on early B-lineage cells is the Ig gene
superfamily molecule, CD19, which is expressed until the latest stage
of B cells.6,7 Other cell surface molecules defining early
developmental stages of B cells include CD34, CD10, CD20, and
CD227 and the surrogate light (SL) chains.8-10
Researchers have focused increasing interest on the SL
chains and their biological significance in early B-cell development. The chains are composed of 2 polypeptides encoded by the
511,12 and VpreB13 genes,
which can associate with the µH chain to form the pre-B-cell
receptor.14,15 A crucial role of the pre-B-cell receptor
in early B-cell development has been verified by the findings that
mutations in 1 of the pre-B-cell receptor genes cause development
arrest at the pro-B-cell stage, thereby resulting in a severe
impairment of B-cell generation.16-19 Production of the SL
chains begins at the stage of pro-B cells, that is, before the
formation of pre-B-cell receptors; maintains through the pre-B-cell stage; and halts at the immature B-cell stage.8-10 Thus,
expression properties of the SL chains restricted to both pro-B-cell
and pre-B-cell stages can serve as the potential marker for
elucidation of distinct steps in early B-cell
development.10,20-22
X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency
caused by mutations in the gene coding for Bruton's tyrosine kinase
(Btk) protein.23,24 XLA is characterized by the early onset
of bacterial infection, very low serum Ig levels of all isotypes, and
severely decreased numbers of peripheral B
lymphocytes.25,26 The Btk protein consists of 5 functional
domains: the pleckstrin homology (PH), the Tec homology (TH), the Src
homology 3 (SH3), SH2, and the kinase domain (SH1).25,27
Although the role of this molecule in B-cell development and activation
remains poorly understood, an international registry for XLA shows that
mutations in all 5 domains of the Btk gene cause the
disease.28 The majority of Btk mutations have been
shown to result in deficient expression of the Btk protein because of a
reduction in Btk mRNA or an instability of the produced
protein.23,29-31
We describe a rapid and simple method for detection of patients with
XLA; the deficient status of the Btk protein expression in their
monocytes is easily demonstrable by a flow cytometric assay using the
anti-Btk monoclonal antibody (mAb).31 This method has
identified atypical XLA patients who exhibited mild or even no clinical
symptoms until adult life, despite the paucity of circulating B cells
with hypogammaglobulinemia.32 Regarding the B-cell defect
in XLA, confusing observations have been obtained from earlier studies
using XLA BM samples.33-37 Some studies33,34 have found that XLA patients show normal numbers of pre-B cells in BM,
whereas other studies34,35 have found that pre-B cells are
seen in only low or undetectable numbers. Furthermore, investigations using early B-cell markers, such as TdT, CD19, CD10, and CD34, have
disclosed that normal or increased frequencies of pro-B cells are
present in XLA BM.19,35-39
In the present study, we employed an mAb specific for the human VpreB,
as a component of the SL chains, to examine the distribution of early
B-lineage cells in BM samples from XLA patients. The patients were
diagnosed by demonstration of both Btk protein deficiencies and
Btk gene mutations compared with healthy controls of different ages. Flow cytometric analysis was used to understand the normal B-cell
differentiation pathway in humans and the nature of the Btk
mutation that caused the B-cell maturation defect in XLA.
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Patients, materials, and methods |
Subjects
After receiving informed consent, we obtained BM from 13 patients
with XLA and from 24 healthy individuals of various ages as controls.
Each XLA patient had received care in one of our clinics. Clinical
features and Btk mutations of the XLA patients are summarized
in Table 1. The present age of patients
with XLA ranged from 4 months to 30 years. Patients P6, P7, and P8 were analyzed for Btk mutations in a previous study,29
and patients P4 and P12 were diagnosed by a flow cytometric
demonstration of the deficient Btk expression in monocytes in
another study.31 Btk mutations in the latter 2 patients were detected according to the method described
previously.29 The remaining 8 patients were diagnosed as
XLA+ by a flow cytometric demonstration of a monocyte
Btk deficiency and a genetic analysis of Btk mutations
(Kanegane et al, unpublished data, April 1999).
The healthy controls comprised 10 infants and toddlers (5 months to 3 years), referred to in this report as infants; 9 children (5-10 years); and 5 adults (21-30 years). All of the children underwent
diagnostic BM aspiration for medical indications, and all had
morphologically normal BM. None of them had received cytotoxic drugs.
During this study, we had the chance to see a 4-month-old girl with
severe combined immune deficiency (SCID) who lacked both T and B cells
and showed no mutations in the recombination activation genes,
RAG-1 and RAG-2. We included this patient as 1 subject
in our study.
Cell preparation
Approximately 0.5 mL BM was aspirated from the iliac crest into a
heparinized syringe and mixed with 5 mL Roswell Park Memorial Institute
medium (RPMI 1640) with 10% fetal calf serum (FCS) and antibiotics.
Mononuclear cells were separated from diluted BM by centrifugation on a
Ficoll-Hypaque gradient (Histopaque 1077; Sigma Chemical Co, St Louis,
MO), followed by lysis of contaminated red blood cells (RBCs) using
0.83% ammonium chloride buffer, and washed 3 times in
phosphate-buffered saline (PBS). BM cells were then resuspended in a
staining buffer (PBS with 1% FCS and 0.1% sodium azide). Cell
viability, assessed by trypan blue dye exclusion, was more than 95% in
all the experiments.
Antibodies
The hybridoma producing the mouse mAb specific for human VpreB
(HSL96) was generated as described previously.22 Purified anti-VpreB mAb was labeled with phycoerythrin (PE) (PharMingen, San
Diego, CA). Other mAbs and antibodies (Abs) employed for
3-color immunofluorescence analysis of BM were: PE-cyanin 5.1-labeled (PC5-labeled) anti-CD19 (Immunotech, Marseilles, France) and
fluorescence isothiocyanate-labeled (FITC-labeled) TdT (Dako Japan,
Kyoto, Japan) mouse mAbs; FITC- and PE-labeled goat F(ab')2
antihuman µH chain Abs (Southern Biotechnology Associates, Inc,
Birmingham, AL); and the corresponding fluorochrome-labeled irrelevant
mAbs or Abs (Dako Japan and Immunotech) as controls.
Flow cytometric analysis
We used 3-color immunofluorescence analysis to identify various
B-lineage cells in the BM. In most experiments, the BM cells were fixed
and then permeabilized to analyze the cell surface antigens and
intracellular molecules simultaneously. In brief, the BM cells were
fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd,
Osaka, Japan) in PBS for 15 minutes at room temperature and then
treated with 0.5% saponin (Sigma) in a staining buffer for 15 minutes
on ice. These fixed permeabilized cells were incubated with a
combination of PC5-labeled anti-CD19 mAbs and other FITC-labeled or
PE-labeled mAbs or Abs for 20 minutes on ice and then washed twice in a
staining buffer. In some experiments, viable BM cells were just stained
for cell surface antigens. In other instances, the BM cells were
stained for certain cell surface antigens prior to cell membrane
permeabilization and then subjected to intracellular staining. The
stained cells were analyzed by flow cytometry (EPICS XL-MCL; Beckman
Coulter KK, Tokyo, Japan).
Statistic analysis
The unpaired Student t test was used to analyze data.
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Results |
Flow cytometric analysis of B-lineage cells in BM defined by
VpreB expression
SL chains are mainly expressed in the cytoplasm of early B-lineage
cells, but at very low levels in their cell
surface.8,9,21,22 In this study, the VpreB expression in BM
was evaluated after fixation and permeabilization of BM cells in the
3-color immunofluorescence assay. In addition to surface CD19 staining,
this treatment conveniently resulted in the simultaneous staining of
TdT, as well as of the µH chain, intracellularly expressed in early
B-precursor cells. Figure 1 shows
representative staining profiles of BM samples from 3 controls of
different ages, 2 XLA patients, and 1 B-cell SCID
patient. Although CD19 is conventionally used as an earliest marker for
B-lineage cells, evidence has suggested that the commitment to
B-lineage cells appears to precede CD19 expression.40,41 Apparently confirming this evidence, analysis of CD19 and VpreB expression by BM lymphoid cells indicated that some cells expressing VpreB intensely tended to decrease or lack CD19 expression, which was
marked in XLA or SCID BM samples (Figure 1A).
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| Fig 1.
Three-color immunofluorescence analysis of early
B-lineage cells in BM based on intracellular VpreB expression.
BM cells from 3 controls of different ages, 2 XLA patients (P3 and
P12), and 1 B-cell SCID patient were fixed,
permeabilized, and then stained with PE-labeled anti-VpreB and
PC5-labeled anti-CD19 mAbs in combination with FITC-labeled anti-TdT
mAb or antihuman µH chain Ab. The corresponding fluorochrome-labeled
irrelevant mAbs or Abs were used as controls. The cells were gated for
lymphoid cells by light scatter characteristics upon a flow cytometer
and analyzed in the 3-color manner. (A) As demarcated in the figure,
the combined use of anti-VpreB and anti-CD19 mAbs appeared to detect
the presumably whole populations of B-lineage cells in BM. (B) The
relation between VpreB and the µH chain or TdT expressed by
BM-lineage cells, which were gated by a combined use of CD19 and VpreB
expression. The number indicates the percentage of cells in each
quadrant.
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As illustrated in Figure 1A, a combination of CD19 and VpreB expression
ensured that the analysis was capable of covering the
vast majority of B-lineage cells in BM cells. Figure 1B shows the
relationship of VpreB to the µH chain and TdT expression by the
B-lineage cells gated on the basis of CD19 and VpreB expression. Smaller populations of µSLhigh cells,
seemingly coexpressing TdT, were identified in normal BM, whereas a
large proportion of B-lineage cells in XLA BM and the vast majority of
the cells in SCID BM were identified as
µVpreB+ and
TdT+VpreB+. The
µSLhigh cells in BM lymphoid cells
appeared to represent the pro-B-cell compartment. A question was
raised on whether discrimination between pre-B cells and B cells in
permeabilized BM samples was possible. We then performed a comparative
analysis between intracellular expression of the µH chain and
intracellular VpreB or surface µH chain expression in B-lineage cells
in BM of controls and XLA patients (Figure
2). The figure indicates that mature B
cells expressed the µH chain at higher levels than the presumed
pre-B-cell population without surface µH chain expression.
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| Fig 2.
Comparative expression analysis of the
intracellular µH chain, intracellular VpreB, or cell surface µH
chain by BM B-lineage cells.
The analysis compared a 5-year-old healthy child and an XLA patient
(P3). The intracellular staining of BM cells for VpreB and the µH
chain in combination with CD19 was performed as described in the legend
of Figure 1. For the intracellular versus cell surface expression of
the µH chain by BM B-lineage cells, viable BM cells were first
treated with a PE-labeled antihuman µH chain Ab, fixed,
permeabilized, and subsequently stained with a PC5-labeled anti-CD19
mAb and FITC-antihuman µH chain Ab. The open square denotes mature B
cells that showed more intense expression of the µH chain than the
presumed pre-B-cell population.
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Analysis of various stages of B-lineage cells in BM
As described above, a cellular gating based on both CD19 and VpreB
expression in the permeabilized cell samples helped identify the whole
population of B-lineage cells in BM. Using this system, we next
examined whether various stages of B-lineage cells in BM could be
discriminated by the degree of VpreB and µH chain expression. Figure
3A shows typical profiles of VpreB and µH
chain expression by BM B-lineage cells in a control and an XLA patient. The µ cells expressing VpreB intensely
(µSL++) were considered to be pro-B
cells (R1). The VpreB cells expressing higher levels
of the µH chain (µhighSL) were
assessed to contain mature B cells (R5), although some mature B cells
overlapped with a small fraction of pre-B cells. The
pre-B-cell population with the lower µH chain expression
was distributed from the VpreB+ (R2) to
VpreB (R4) populations. We designated the
VpreB+ pre-B-cell population
(µlowSL+~++) as pre-B1
cells (R2 and R3), and the VpreB pre-B-cell
population (µlowSL) was designated as
pre-B2 cells (R4). The VpreB+ pre-B cells were further
divided into the 2 populations, namely, the cells with intense VpreB
expression (µlowSL++, R2) and the ones with
decreasing VpreB expression (µlowSL+, R3). We
termed the former pre-B1a cells and the latter pre-B1b cells. This
nomenclature was justified by the cell size analysis of each population
in normal BM (Figure 3B). In normal BM, large cells were more enriched
in pre-B1a cells as compared with pre-B1b cells (n = 7;
29.2% ± 5.3% versus 12.9% ± 4.0%
[P < .001]). The other B-lineage cells were largely
composed of small-sized cells. In XLA BM, the pre-B1a cells were
identifiable, whereas the pre-B1b cells was markedly decreased
(Figure 3A). It should be stressed that in contrast to normal BM, the
pre-B1a cells in XLA BM were largely made up of small cells (n = 5;
large cells, 7.0% ± 3.6%) (Figure 3B).
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| Fig 3.
Three-color immunofluorescence analysis of different
stages of B-lineage cells in BM.
BM cells from an 11-month-old healthy infant and an XLA patient (P6)
were fixed, permeabilized, and then stained with PE-labeled anti-VpreB
and PC5-labeled anti-CD19 mAbs in combination with an FITC-labeled
antihuman µH chain Ab. B-lineage cells in BM lymphoid cells were
gated by a combination of VpreB and CD19 expression, as described in
Figure 1. (A) According to the degree of VpreB and µH chain
expression, BM B-lineage cells were arbitrarily divided into 5 fractions: µSL++ (R1),
µlowSL++ (R2),
µlowSL+ (R3),
µlowSL (R4), and
µhighSL (R5) cells. R1, R2, R3, R4,
and R5 fractions comprise 7.6%, 9.9%, 19.0%, 38.8%, and 24.6%,
respectively, of the normal BM or 76.8%, 14.8%, 3.9%, 3.1%, and
1.0% of the XLA BM. (B) The cell size of each fraction of BM-lineage
cells was assessed by forward scatter. In normal BM, large cells were
more enriched in the R2 fraction. By contrast, all fractions were
mainly composed of small cells in XLA BM.
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Analysis of pro-B, pre-B, and B cells in BM of XLA patients and
controls
These observations indicated that there was maturation block in the
B-cell differentiation in XLA patients. We accumulated data on BM
samples, including B-lineage cells, from XLA patients and controls
(Tables 2 and 3). The analysis of normal BM
indicated that the relative frequencies of total B-lineage cells in BM
lymphoid cells were higher in all ages of children and declined with
advancing age. In XLA patients, the frequencies of total B-lineage
cells in BM lymphoid cells were significantly lower compared with the frequencies in children. Each B-lineage cell in BM was similarly identified in infants, children, and adults, reflecting the function of
BM as the provider of B cells after birth. It was noted that the
percentages of pro-B cells within B-lineage cells in XLA BM were
significantly higher than those in controls of all ages. Variable
proportions of pre-B1a cells were identified in XLA BM, but their
percentages were not so increased. The frequencies of pre-B1b, pre-B2,
and B cells were extremely reduced in XLA BM.
The relative frequencies of B-lineage cells among BM lymphoid
cells must be more informative in elucidating the nature of B-cell
defects in XLA patients, and the frequencies were further calculated
from the results of Table 3 (Table
4). Interestingly, age-dependent changes in the distribution of respective B-lineage cells
in BM were seen in the controls. All stages of B-lineage cells, from
pro-B cells to B cells, were present in abundant numbers in the BM
of children of all ages, indicating the active B lymphopoiesis during
these periods in human life. On the other hand, adult BM exhibited the
predominance of B cells and pre-B2 cells, which is concomitant with a
decrease in other earlier B-lineage cells. Characteristically, pro-B
cells in XLA BM were markedly expanded compared with the controls of
any ages, and the number of pre-B1a cells was reduced in
XLA patients compared with the number in younger children. Importantly,
this expansion of pro-B cells was also found in XLA patients in the
adult period (P11-P13). However, adult controls exhibited lower
frequencies of all BM lineage cells than children controls. Later
stages of B-lineage cells (pre-B1b, pre-B2, and B cells) were
negligible, although identifiable, in XLA patients. Figure
4 depicts the mean percentages of
respective B-lineage cells among BM lymphoid cells in XLA patients and
controls. This figure clearly demonstrates an expansion of pro-B cells
in XLA BM as well as age-dependent changes in BM B-lineage cells. The
results suggest that the transition of pro-B cells beyond the earlier
pre-B-cell stage to the later stage of pre-B cells might be severely
impaired in XLA patients.
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Table 4.
Frequencies of B-lineage cells among BM lymphoid cells
in XLA patients and healthy controls of various ages
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| Fig 4.
Relative frequencies of different stages of
B-lineage cells among BM lymphoid cells in XLA patients and in infant,
children, and adult controls.
The mean values shown in Table 4 are figured.
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Discussion |
Many studies in mice and humans have examined how B cells are
committed from hematopoietic stem cells and generated through discrete
developmental stages that are defined by sequential Ig gene
rearrangements and intracellular or cell surface expression of various
B-cell-related molecules. However, the phenotypic criteria and
nomenclatures for distinguishing the different stages of B-cell development, especially in humans, remain controversial.42
Besides B cell lines, B-cell neoplasmas, and normal BM, the genetic
deficit of B-cell development would provide some complementary
information in the understanding of human B-cell development.
In this study, we attempted to subcharacterize the B-cell lineage cells
in human BM using VpreB, a component of the SL chains, which seemed a
suitable marker to specifically delineate early B-cell affiliation. The
cell surface expression of the SL chains is usually expressed at very
low levels and seen, even if detectable, only in a small proportion of
BM B-lineage cells. The chains are intracellularly synthesized in
plentiful amounts in early B-lineage cells before the cell surface
expression of the µH chain appears.8,9,21,22 Thus, we
investigated the distribution of different stages of BM B-lineage cells
defined by VpreB expression in the permeabilized BM cell samples. We
disclosed that a combination of VpreB and CD19 expression could
identify the presumably whole population of B-lineage cells in BM.
Consistent with previous observations,43 mature B cells
were found to express the µH chain at higher levels than the
pre-B-cell population did. We also observed that these presumed mature
B cells with enhanced levels of µH chain, but not other BM B-lineage
cells, expressed L chains (data not shown). Although a
small fraction of mature B cells was, to some extent, overlapped with
pre-B cells, the density of the µH chain expression appeared to
discriminate between both populations, even in permeabilized BM cells.
The flow cytometric analysis of BM samples from controls of different
ages and from XLA patients revealed that the B-lineage cells in BM were
divided into several populations at discrete stages. The earliest
B-lineage cells, termed pro-B cells, which were seemingly
TdT+, expressed relatively high levels of VpreB and lacked
the µH chain expression.
Some other cell surface antigens, such as CD34 and CD10, have been used
to identify the pro-B cells in BM.7,19,35-39 Our preliminary observations indicated that the putative pro-B cells in XLA
BM appeared to express CD34 as well CD10 relatively intensely, whereas
normal BM pro-B cells showed variable expression of both antigens. The
combined use of intracellular VpreB with the cytoplasmic µH chain
might be useful in identifying the pro-B-cell population in BM.
According to the classical criteria,2 the pre-B cells were
defined here as the cells containing the cytoplasmic µH chain. The
flow cytometric analysis showed that the expression of VpreB in the pre-B-cell population tended to decline with maturation. We
demonstrated that pre-B cells without VpreB production were present in
BM. These pre-B cells lacking VpreB expression have been identified in
murine BM.8 We distinguished the pre-B-cell population
with VpreB expression, named pre-B1 cells, from the pre-B2 cells
without VpreB expression. It seemed that pre-B1 cells could further be
divided into 2 subpopulations, termed pre-B1a or pre-B1b cells with
VpreB expression comparable to or lower than that seen in pro-B cells.
The cell size analysis showed that pre-B1a cells were more enriched
with large cells as compared to pre-B1b cells, indicating the active
cycling state in the former cells.
Although the synthesis of the L chains associated with L chain gene
rearrangement is generally believed to begin beyond the stage of pre-B
cells,42 Guelpa-Fonlupt et al10 have proposed that the L chains can be coexpressed on a minor population of µ+VpreB+ B-lineage cells in BM, possibly at
the later stage of pre-B cells. However, our flow cytometric evaluation
revealed that the expression of the L chains and VpreB seemed to occur
independently in BM B-lineage cells (data not shown). This discrepancy
may be partially due to a difference of antibodies used. A tentative
model of the human B-cell differentiation pathway is depicted in Figure
5.
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| Fig 5.
Tentative model of the B-cell differentiation pathway in
healthy human BM and the B-cell defect in XLA BM.
The arrow indicates the major blockage in XLA patients.
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Despite recent availability of early B-cell specific markers, the
ontogenic features of B lymphopoiesis in human BM remains to be
precisely evaluated. Nuñez et al43 have used CD19 as the pan-B-cell marker, in addition to TdT and the µH chain, to examine the frequencies of pro-B, pre-B, and B cells in fetal tissues
and BM samples from controls. They have elegantly
demonstrated that BM in humans functions as the major site of B-cell
generation after birth.43 They have also described that the
relative frequencies of the B-lineage cells in BM decline with
advancing age; mature B cells predominate in adult BM, resulting from
an age-dependent reduction in the ratio of B precursors to mature B
cells.43 Such an age-dependent decrease in total BM
B-lineage cells, with the increasing frequencies of mature B cells, has
also been observed by other investigators.20 Similarly, we
demonstrated that B lymphopoiesis was more active in young children
than in adults. We found that B precursors (or pro-B cells) identified
as µSL++ cells were obviously reduced
in frequency in adult BM. Such information about the age-related
changes in the compartment of BM B-lineage cells would be very helpful
in evaluating the specific block of B-cell differentiation in primary
B-cell defect conditions.
There is now increasing evidence that Btk
participates in signal transduction after B-cell receptor-mediated
activation of mature B cells.44 But the issue of how the
genetic defect in XLA affects early B-cell development is somewhat
confusing. Earlier studies have used fluorescence microscopy to examine
BM pre-B cells containing the cytoplasmic µH chain in a
gammaglobulinemic males with presumed XLA. Pearl et al33
described XLA patients as having a normal frequency of pre-B cells in
BM and speculated that the pre-B-cell to B-cell transition might be
impaired in XLA. In contrast, later studies demonstrated that there is
more heterogeneity in the number of pre-B cells in XLA BM than earlier recognized.34,35 While pre-B cells are more or less
detectable in XLA BM, it has been shown that the majority of XLA
patients have a substantial number of pro-B cells in BM, resulting in
an increased ratio of pro-B to pre-B cells.19,35-39 Conley
et al45 have proposed that the defect in XLA might not be
limited to the pre-B-cell to B-cell transition, and instead, the
defect might interfere with multiple stages of B-cell differentiation.
Mutations in genes other than Btk result in a clinical
phenotype resembling XLA.18,19,37-39,46 Although
approximately 90% of males with presumed XLA have mutations in the
Btk gene,47 it is likely that a heterogeneity in
XLA BM might be partly due to the inclusion of some non-XLA cases in
previous studies.
In this study, we enrolled the XLA patients, all of whom had
identified Btk mutations, to analyze the B-lineage cells in XLA BM. The flow cytometric analysis disclosed that while the frequencies of total B-lineage cells were appreciably decreased, pro-B cells were
markedly expanded in XLA BM, regardless of the present age. We detected
variable numbers of pre-B cells in XLA BM. It should be noted that the
pre-B cells seen in XLA BM were largely composed of the earliest pre-B
population, namely, pre-B1a cells. In contrast to the controls, pre-B1a
cells in XLA BM were constituted with small cells. Similarly, previous
studies have observed that pre-B cells identified in XLA patients were
mainly nonproliferating small cells.35 The important point
was that the relative frequencies of pre-B1a cells in XLA BM were
significantly lower than those seen in normal BM. The later stages of
B-lineage cells, including pre-B1b as well as pre-B2 cells, were
markedly reduced in XLA BM. As depicted in Figure 5, these results
suggest that the genetic defect in XLA might interrupt the
proliferation and survival of pre-B1a cells, resulting in an expansion
of pro-B cells with a few pre-B1a cells in BM.
A leaky generation of mature B cells is often seen in most XLA
patients. Supporting this fact, the Epstein-Barr virus (EBV) is capable
of rescuing these leaked B cells.48-51 While most of the
EBV-transformable B cells from XLA patients produce IgM, Anker et
al50 revealed that an EBV infection could establish B cell lines expressing all Ig isotypes in XLA. Nonoyama et al52
demonstrated that leaky B cells in XLA have the capability to undergo
the final differentiation into the cells producing all Ig subclasses in vivo as well as in vitro. In addition, some XLA cases have been shown
to exhibit a mild phenotype, which is manifested with delayed disease
recognition, nonlethal bacterial infection, and higher than expected
concentrations of serum Ig.32,53,54 In contrast to typical
XLA, the murine Btk mutations, as seen in Btk-null as
well as Xid mice, caused the milder phenotype, which exhibited a generation of near normal numbers of B cells with substantial Ig
levels in the serum.55-57 Although unable to make
antibodies to T-independent antigens, mice with Btk mutations
respond to the specified T-dependent antigens.67-69 Kerner
et al57 demonstrated the existence of an impaired expansion of pro-B cells in Btk-deficient mice. They proposed that the
role of Btk in early B-cell development is the same in humans
and mice, and the consequences of Btk deficiency differ only
qualitatively between both groups. It is tempting to suppose that
B-cell differentiation may occur in Btk-dependent and
Btk-independent manners, and the largest proportion of B-cell
generation in humans, unlike mice, may depend on the molecular function
of Btk.
In conclusion, the results presented in this study have delineated both
the nature of the B-cell defect caused by a Btk mutation as
well as the human B-cell differentiation pathway. It appears that
Btk mutations in XLA impede the maturational evolution of pro-B
cells beyond the earlier pre-B-cell stage to the later stage of pre-B
cells, resulting in a marked reduction in mature B cells. The major
B-cell defect in XLA may exit at the transition of pro-B cells to
earlier pre-B cells. This is verified by observations that there is a
random inactivation of the X chromosome among pro-B cells in female XLA
carriers, whereas the carriers usually exhibit skewing of X
inactivation in peripheral B cells.58 Given that the
pre-B-cell receptor is essentially involved in proliferation and
survival of early B precursors, there is a possibility that Btk may
contribute to intracellular signaling downstream of the pre-B-cell
receptor. This intriguing point will be clarified by further studies
using pro-B cell lines derived from XLA BM.
| |
Acknowledgments |
We thank all the patients and families for their generous cooperation
in this study, Drs Yuichi Adachi and Shoichi Koizumi for helpful
discussion, and Chikako Sakai and Hitoshi Moriuchi for technical assistance.
| |
Footnotes |
Submitted December 28, 1999; accepted March 7, 2000.
Supported by Grant-in-Aid for Scientific Research 10470176 from the Ministry of Education, Science and Culture of Japan, Japan; a
grant from the Ministry of Health and Welfare of Japan, Japan; and a
grant from the Uehara Foundation, Tokyo, Japan.
Reprints: Toshio Miyawaki, Department of Pediatrics,
Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan; e-mail:
toshio65{at}ms.toyama-mpu.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Kincade PW.
Formation of B lymphocytes in fetal and adult life.
Adv Immunol.
1981;31:177-245[Medline]
[Order article via Infotrieve].
2.
Osmond DG.
B cell development in the bone marrow.
Semin Immunol.
1990;2:173-180[Medline]
[Order article via Infotrieve].
3.
Gathings WE, Lawton AR, Cooper MD.
Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans.
Eur J Immunol.
1977;7:804-810[Medline]
[Order article via Infotrieve].
4.
Kamps WA, Cooper MD.
Development of lymphocyte subpopulations identified by monoclonal antibodies in human fetuses.
J Clin Immunol.
1984;4:36-39[Medline]
[Order article via Infotrieve].
5.
Desiderio SV, Yancopoulos GD, Paskind M, et al.
Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.
Nature.
1984;311:752-755[Medline]
[Order article via Infotrieve].
6.
Nadler LM, Anderson KC, Marti G, et al.
B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes.
J Immunol.
1983;131:244-250[Abstract].
7.
Loken MR, Shah VO, Dattilio KL, Civin CI.
Flow cytometric analysis of human bone marrow. II, Normal B lymphocyte development.
Blood.
1987;70:1316-1324[Abstract/Free Full Text].
8.
Karasuyama H, Rolink A, Shinkai Y, Young F, Alt FW, Melchers F.
The expression of VpreB/l5 surrogate chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.
Cell.
1994;77:133-143[Medline]
[Order article via Infotrieve].
9.
Lassoued K, Nuñez CA, Phillips L, et al.
Expression of surrogate light chain receptors is restricted to a late stage in pre-B cell differentiation.
Cell.
1993;73:73-76[Medline]
[Order article via Infotrieve].
10.
Guelpa-Fonlupt V, Tonelle 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.
1994;24:257-264[Medline]
[Order article via Infotrieve].
11.
Sakaguchi N, Melchers F.
Lamda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes.
Nature.
1986;324:579-582[Medline]
[Order article via Infotrieve].
12.
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 U S A.
1989;86:5552-5556[Abstract/Free Full Text].
13.
Kudo A, Melchers F.
A second gene, VpreB in the 5 locus, which appears to be selectively expressed in pre-B lymphocytes.
EMBO J.
1987;6:2267-2272[Medline]
[Order article via Infotrieve].
14.
Karasuyama H, Kudo A, Melchers F.
The proteins encoded by the VpreB and 5 pre-B specific genes can associate with each other and with µ heavy chain.
J Exp Med
1990;172:969-972[Abstract/Free Full Text].
15.
Tsubata T, Reth M.
The products of pre-B cell specific genes (5 and VpreB) and immunoglobulin chain form a complex that is transported onto the cell surface.
J Exp Med.
1990;172:973-976[Abstract/Free Full Text].
16.
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.
1991;350:423-426[Medline]
[Order article via Infotrieve].
17.
Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K.
A critical role of 5 protein in B cell development.
Cell.
1992;69:823-831[Medline]
[Order article via Infotrieve].
18.
Yel L, Minegishi Y, Coustan-Smith E, et al.
Mutations in the mu heavy chain gene in patients with agammaglobulinemia.
N Engl J Med.
1996;335:1486-1493[Abstract/Free Full Text].
19.
Minegishi Y, Coustan-Smith E, Wang Y-H, Cooper MD, Campana D, Conley ME.
Mutations in the human 5/14.1 gene result in B cell deficiency and agammaglobulinemia.
J Exp Med.
1998;187:71-77[Abstract/Free Full Text].
20.
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.
1996;184:2217-2229[Abstract/Free Full Text].
21.
Wang Y-H, Nomura J, Faye-Petersen OM, Cooper MD.
Surrogate light chain production during B cell differentiation: differential intracellular versus cell surface expression.
J Immunol.
1998;161:1132-1139[Abstract/Free Full Text].
22.
Tsuganezawa K, Kiyokawa N, Matsuo Y, et al.
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.
Blood.
1998;92:4317-4324[Abstract/Free Full Text].
23.
Tsukada S, Saffran DC, Rawlings DJ, et al.
Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.
Cell.
1993;72:279-290[Medline]
[Order article via Infotrieve].
24.
Vetrie D, Vorechovsky I, Sideras P, et al.
The gene involved in X-linked agammaglobulinemia is a member of the src family of protein-tyrosine kinases.
Nature.
1993;361:226-233[Medline]
[Order article via Infotrieve].
25.
Sideras P, Smith CIE.
Molecular and cellular aspects of X-linked agammaglobulinemia.
Adv Immunol.
1995;59:135-223[Medline]
[Order article via Infotrieve].
26.
Conley ME, Cooper MD.
Genetic basis of abnormal B cell development.
Curr Opin Immunol.
1998;10:399-406[Medline]
[Order article via Infotrieve].
27.
Tsukada S, Rawlings DJ, Witte ON.
Role of Bruton's tyrosine kinase in immunodeficiency.
Curr Opin Immunol.
1994;6:623-630[Medline]
[Order article via Infotrieve].
28.
Vihinen M, Kwan SP, Lester T, et al.
Mutations of the human BTK gene coding for Bruton tyrosine kinase in X-linked agammaglobulinemia.
Hum Mutat.
1999;13:280-285[Medline]
[Order article via Infotrieve].
29.
Hashimoto S, Tsukada S, Matsushita M, et al.
Identification of Bruton's tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan.
Blood.
1996;88:561-573[Abstract/Free Full Text].
30.
Gaspar HB, Lester T, Levinsky RJ, Kinnon C.
Bruton's tyrosine kinase expression and activity in X-linked agammaglobulinemia (XLA): the use of protein analysis as a diagnostic indicator of XLA.
Clin Exp Immunol.
1998;111:334-338[Medline]
[Order article via Infotrieve].
31.
Futatani T, Miyawaki T, Tsukada S, et al.
Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.
Blood.
1998;91:595-602[Abstract/Free Full Text].
32.
Hashimoto S, Miyawaki T, Futatani T, et al.
Atypical X-linked agammaglobulinemia (XLA) diagnosed in adult.
Internal Med.
1999;38:722-725.
33.
Pearl ER, Vogler LB, Okos AJ, Crist WM, Lawton AR, Cooper MD.
B lymphocyte precursors in human bone marrow: an analysis of normal individuals and patients with antibody-deficiency states.
J Immunol.
1978;120:1169-1175[Abstract/Free Full Text].
34.
Landreth KS, Engelhard D, Anasetti C, Kapoor N, Kincade PW, Good RA.
Pre-B cells in agammaglobulinemia: evidence for disease heterogeneity among affected boys.
J Clin Immunol.
1985;5:84-89[Medline]
[Order article via Infotrieve].
35.
Campana D, Farrant J, Inamdar N, Webster ADB, Janossy G.
Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia.
J Immunol.
1990;145:1675-1680[Abstract].
36.
Milili M, Le Deist F, de Saint-Basile G, Fischer A, Fougereau M, Schiff C.
Bone marrow cells in X-linked agammaglobulinemia express pre-B-specific genes (-like and V pre-B) and present immunoglobulin V-D-J gene usage strongly biased to a fetal-like repertoire.
J Clin Invest.
1993;91:1616-1629.
37.
Meffre E, LeDeist F, de Saint-Basile G, et al.
A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.
Immunol Lett.
1997;57:93-99[Medline]
[Order article via Infotrieve].
38.
Minegishi Y, Coustan-Smith E, Rapalus L, Ersoy F, Campana D, Conley ME.
Mutations Ig (CD79a) result in a complete block in B-cell development.
J Clin Invest.
1999;104:1115-1121[Medline]
[Order article via Infotrieve].
39.
Minegishi Y, Rohrer J, Coustan-Smith E, et al.
An essential role for BLNK in human B cell development.
Science.
1999;286:1954-1957[Abstract/Free Full Text].
40.
Bertrand FE III, Billips LG, Burrows PD, Gartland GL, Kubagawa H, Schroeder HW Jr.
Ig DH gene segment transcription and rearrangement before surface expression of the pan-B-cell marker CD19 in normal human bone marrow.
Blood.
1997;90:736-744[Abstract/Free Full Text].
41.
Dworzak MN, Fritsch G, Froschl G, Printz D, Gadner H.
Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny.
Blood.
1998;92:3203-3209[Abstract/Free Full Text].
42.
Burrows PD, Cooper MD.
B cell development and differentiation.
Curr Opin Immunol.
1997;9:239-244[Medline]
[Order article via Infotrieve].
43.
Nuñez C, Nishimoto N, Gartland GL, et al.
B cells are generated throughout life in humans.
J Immunol.
1996;156:866-872[Abstract].
44.
Kurosaki T.
Genetic analysis of B cell antigen receptor signaling.
Ann Rev Immunol.
1999;17:555-592[Medline]
[Order article via Infotrieve].
45.
Conley ME.
B cells in X-linked agammaglobulinemia.
J Immunol.
1985;134:3070-3074[Abstract].
46.
Conley ME, Sweinberg SK.
Females with a disorder phenotypically identical to X-linked agammaglobulinemia.
J Clin Immunol.
1992;12:139-143[Medline]
[Order article via Infotrieve].
47.
Conley ME, Mathias D, Treadaway J, Minegishi Y, Rohrer J.
Mutations in Btk in patients with presumed X-linked agammaglobulinemia.
Am J Hum Genet.
1998;62:1034-1043[Medline]
[Order article via Infotrieve].
48.
Mensink EJ, Schuurman RK, Schot JD, Thompson A, Alt FW.
Immunoglobulin heavy chain gene rearrangements in X-linked agammaglobulinemia.
Eur J Immunol.
1986;16:963-967[Medline]
[Order article via Infotrieve].
49.
Levitt D, Ochs H, Wedgwood RJ.
Epstein-Barr virus-induced lymphoblastoid cell lines derived from the peripheral blood of patients with X-linked agammaglobulinemia can secrete IgM.
J Clin Immunol.
1984;4:143-150[Medline]
[Order article via Infotrieve].
50.
Anker R, Conley ME, Pollok BA.
Clonal diversity in the B cell repertoire of patients with X-linked agammaglobulinemia.
J Exp Med.
1989;169:2109-2119[Abstract/Free Full Text].
51.
Timmers E, Kenter M, Thompson A, et al.
Diversity of immunoglobulin heavy chain gene segment rearrangement in B lymphoblastoid cell lines from X-linked agammaglobulinemia patients.
Eur J Immunol.
1991;21:2355-2363[Medline]
[Order article via Infotrieve].
52.
Nonoyama S, Tsukada S, Yamadori T, et al.
Functional analysis of peripheral blood B cells in patients with X-linked agammaglobulinemia.
J Immunol.
1998;161:3925-3929[Abstract/Free Full Text].
53.
Bykowsky MJ, Haire RN, Ohta Y, et al.
Discordant phenotype in siblings with X-linked agammaglobulinemia.
Am J Hum Genet
1996;58:477-483[Medline]
[Order article via Infotrieve].
54.
Kornfeld SJ, Haire RN, Strong SJ, et al.
A novel mutation (Cys145- ->Stop) in Bruton's tyrosine kinase is associated with newly diagnosed X-linked agammaglobulinemia in a 51-year-old male.
Mol Med.
1996;2:619-623[Medline]
[Order article via Infotrieve].
55.
Rawlings DJ, Saffran DC, Tsukada S, et al.
Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice.
Science.
1993;261:358-361[Abstract/Free Full Text].
56.
Khan WN, Alt FW, Gerstein RM, et al.
Defective B cell development and function in Btk-deficient mice.
Immunity.
1995;3:283-299[Medline]
[Order article via Infotrieve].
57.
Kerner JD, Appleby MW, Mohr RN, et al.
Impaired expansion of mouse B cell progenitors lacking Btk.
Immunity.
1995;3:301-312[Medline]
[Order article via Infotrieve].
58.
Conley ME, Parolini O, Rohler J, Campana D.
X-linked agammaglobulinemia.
Immunol Rev.
1994;138:5-21[Medline]
[Order article via Infotrieve].
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Abstract
Introduction