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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 9-23
REVIEW ARTICLE
Fates of human B-cell precursors
Tucker W. LeBien
From the Department of Laboratory Medicine and Pathology, University
of Minnesota Cancer Center, and Center for Immunology, University of
Minnesota, Minneapolis.
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Abstract |
Development of mammalian B-lineage cells is characterized by
progression through a series of checkpoints defined primarily by
rearrangement and expression of immunoglobulin genes. Progression through these checkpoints is also influenced by stromal cells in the
microenvironment of the primary tissues wherein B-cell development
occurs, ie, fetal liver and bone marrow and adult bone marrow. This
review focuses on the developmental biology of human bone marrow
B-lineage cells, including perturbations that contribute to
the origin and evolution of B-lineage acute lymphoblastic leukemia and
primary immunodeficiency diseases characterized by agammaglobulinemia.
Recently described in vitro and in vivo models that support development
and expansion of human B-lineage cells through multiple checkpoints
provide new tools for identifying the bone marrow stromal cell-derived
molecules necessary for survival and proliferation. Mutations in genes
encoding subunits of the pre-B cell receptor and molecules involved in
pre-B cell receptor signaling culminate in X-linked and non-X-linked
agammaglobulinemia. A cardinal feature of these immunodeficiencies is
an apparent apoptotic sensitivity of B-lineage cells at the pro-B to
pre-B transition. On the other end of the spectrum is the apoptotic resistance that accompanies the development of B-lineage acute lymphoblastic leukemia, potentially a reflection of genetic
abnormalities that subvert normal apoptotic programs. The triad of
laboratory models that mimic the bone marrow microenvironment,
immunodeficiency diseases with specific defects in B-cell development,
and B-lineage acute lymphoblastic leukemia can now be integrated to
deepen our understanding of human B-cell development.
(Blood. 2000;96:9-23)
© 2000 by The American Society of Hematology.
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Introduction |
Development of mature blood cells from
lymphohematopoietic progenitors is a complex process governed by
sequential changes in gene expression and external cues emanating from
lymphohematopoietic microenvironments, such as fetal liver and bone
marrow (BM). The last decade has witnessed dramatic progress in
elucidating the molecular mechanisms that govern blood cell
development. Mice with alterations in gene content (transgenics,
knockouts, knockins) have been extraordinarily useful in elucidating
the role of transcription factors, cytokines, and cytokine receptors in
blood cell development. This review focuses on the developmental
biology of human BM B-lineage cells and on perturbations in development
that can contribute to the progression of B-lineage acute lymphoblastic
leukemia (ALL) and immunodeficiency diseases characterized by
agammaglobulinemia. My objective is to provide an update on key issues
in human B-cell development and, where appropriate, compare and
contrast B-cell development in mouse and human. The discussion of
B-lineage ALL and immunodeficiency diseases will consider the
developmental biology of these diseases as they constitute a deviation
from normal programs.
The terminology used in this review is largely consistent with the
terminology used by other laboratories studying human B-cell development. Pro-B cells are those B-lineage cells that express cell-surface CD19 but do not express cytoplasmic or cell-surface µ heavy chains (HCs). Pre-B cells express cell-surface CD19 and cytoplasmic µHCs, and variably express cell-surface µHCs associated with surrogate light chains ( LCs) ie, the pre-B cell receptor (pre-BCR). Immature B cells express cell-surface CD19 and
cell-surface µHCs associated with  or  LCsie, the B-cell
receptor (BCR). B-cell precursors include all B-lineage cells prior to
immature B cells expressing the BCR.
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Sites of B-cell development |
Human B-lineage cells are present in multiple tissue sites in early
fetal development. However, from midgestation through the eighth decade
of life, the BM is the exclusive site of B lymphopoiesis. Pre-B cells
are present in 7- to 8-week gestational age fetal liver1
and 10-week gestational age fetal omentum.2 A thorough analysis of 18- to 20-week fetal tissues revealed that B-cell development is multifocal; CD19+/surface
µHC B-cell precursors and
CD19+/surface µHC+ immature B cells are
present in BM, liver, lung, kidneys, and spleen.3 The
frequency of B-cell precursors as a percentage of the total lymphoid
cell pool is much higher in fetal BM compared with adult
BM.3,4 Adult BM also differs from fetal BM by the presence
of recirculating CD19+/surface µ,  HC+
mature B cells in the former.3 Similar levels of
recombinase-activating gene (RAG)-1, RAG-2, and terminal
deoxynucleotidyl transferase (TdT) are detectable by reverse
transcriptase-polymerase chain reaction (RT-PCR) in pro-B cells from
18-week fetal BM and 62-year adult BM, underscoring the functional
integrity of BM B-cell development throughout life.3 It is
noteworthy that recent studies of T-cell development indicate that
T-cell receptor (TCR) gene rearrangements actively occur in thymocytes
from individuals in their sixth decade of life.5 Thus,
ongoing development of B and T lymphocytes throughout life complements
the existence of memory B and T lymphocytes in maintaining a functional
immune system.
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Developmental stages of B-lineage cells |
Analysis of gene expression in developing lymphoid cells can be
accomplished by multiparameter flow cytometry,
immunohistochemistry/fluorescence microscopy, and RT-PCR. Immunologic
phenotyping of B-lineage ALL using monoclonal antibodies (mAbs) and
flow cytometry has been conducted in many laboratories, and it is not
my intent to summarize the many published reports. The reader is
referred elsewhere for an in-depth review.6
Although lymphoid progenitors are the descendants of hematopoietic stem
cells (HSCs) with the capacity to develop into all lymphohematopoietic
lineages, the earliest populations of lineage-restricted lymphoid
progenitors are poorly characterized. Figure
1 shows potential relationships between
so-called common lymphoid progenitors (CLPs) and progenitors with
increased fitness to form specific lymphoid lineages. The CLP is
defined as a progenitor with the capacity to develop into T, B, or
natural killer (NK) cells, but little or no capacity to develop into
nonlymphoid lineages, such as myeloid/erythroid cells. A cell with the
developmental potential of a CLP has been included in the blood cell
developmental schemes of hematology textbooks for many years. However,
data supporting its existence have only recently been
published.7-10 Galy and colleagues used
fluorescence-activated cell sorting (FACS) to purify
CD34+/CD10+/CD45RA+ BM progenitors
that do not express T, B, or NK lineage surface markers (ie, CD2, CD4,
CD8, CD16, CD19, CD20, and CD56). A battery of in vitro assays and the
severe combined immunodeficiency (SCID)-hu (human) mouse were employed
to demonstrate that
CD34+/CD10+/CD45RA+ progenitors are
capable of developing into B, T, NK, and lymphoid dendritic cell (DC)
lineages, but not myeloid/erythroid lineages.7 However,
this study did not clarify whether T- and B-lineage cells are derived
from a single progenitor. A subsequent study by Ryan and colleagues
showed that CD34+/CD19 lymphoid
progenitors expressing the interleukin 7 receptor (IL-7R) (CD127) gave
rise to CD19+ cells in a colony-forming assay.8
The CD34+/IL-7R+ lymphoid progenitors were
uniformly TdT+, and the majority expressed CD10. RT-PCR
analysis indicated the expression of RAG-1, immunoglobulin (Ig)
(CD79b), and the paired domain transcription factor
PAX-5.8 Ryan and colleagues did not assay for T, NK, or DC potential, but the
CD34+/IL-7R+/CD19 population
contained granulo-monocytic colony-forming units at a
frequency 10-fold to 100-fold lower than
IL-7R/CD19 cells.8 A
recent report indicated that CD34+ BM cells expressing the
CXCR4 chemokine receptor for stromal cell-derived factor-1 (discussed
in more detail below) could develop into B- and T-lineage cells, but
not myeloid/erythroid cells.9 It seems likely that the 3 reports7-9 described a human lymphoid progenitor with
similar developmental potential. A population of IL-7R+
adult murine BM cells are also developmentally restricted to become T,
B, and NK cells10 and may be the murine counterpart of the
lymphoid progenitors isolated from human BM.7-9
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| Fig 1.
Developmental relationship between hematopoietic stem
cells (HSCs), common lymphoid progenitors (CLPs), and putative early-B
or T/NK/dendritic cell (DC) progenitors.
HSCs include all primitive CD34+/lineage
hematopoietic developmental stages prior to the CLP, shown
schematically as 3 cells. Arrows with solid lines indicate
developmental flow culminating in increased lineage restriction. Dashed
arrows indicate possible cellular targets of IL-7 signaling or an
unknown (?) ligand. Numbers on the cell surface indicate CD antigens
useful in distinguishing the developmental compartments. Although not
shown in this figure, the 3 reports that described the cell surface
phenotype of CD19 lymphoid progenitors revealed
considerable heterogeneity.7,8,13 For example, CD7 and CD33
were detected on a minority of the lymphoid progenitors in each
study.7,8,13 There is no known surface marker that
distinguishes the CLP from the early-B cell. It is also likely that
IL-7R expression and signaling vary both within and between the
lymphoid progenitor compartments.
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The model in Figure 1 proposes that CLP can differentiate into 1 of 2 lymphoid progenitor intermediates: early-B cells or T/NK/DC tri-lineage
cells. Early-B cells are characterized by the initiation of
DJH rearrangements and the expression of B-lineage specific
proteins such as VpreB and Ig (CD79a). Support for the existence of
an early-B cell comes from reports showing that DJH rearrangements,11,12 cytoplasmic Ig
protein,13 and VpreB protein14 are present in
CD19 lymphoid progenitors. The CLP could also
differentiate into a T/NK/DC tri-lineage progenitor possibly defined by
the acquisition of CD7. Either the CLP or the T/NK/DC progenitor could
migrate to the thymus and undergo subsequent development.15
It is unknown whether specific signals transduced by BM stromal
cell-derived molecules can promote CLP differentiation into an early-B
cell or a T/NK/DC progenitor.
Murine CLPs and possibly human CLPs are particularly sensitive to IL-7
stimulation. Signaling through the IL-7R is essential for the
development of murine CLPs, although not by virtue of inducing
self-renewal.16 A role for IL-7 signaling in the
development of human CD19+ B-lineage cells from
CD10+/TdT+/CD19 lymphoid
progenitors has been demonstrated in vitro,8 but whether
IL-7 exerts an effect on CLPs and/or early-B cells is unknown. The
relationship between signaling pathways activated following IL-7
stimulation of CLPs and the developmental fate of CLPs are unknown.
IL-7 activation of phosphatidylinositol (PI)-3 kinase/protein kinase B
activation is essential for survival/proliferation of human T-cell
precursors, whereas IL-7 activation of STAT5 favors T-cell differentiation.17 It will be interesting to
determine whether PI-3 kinase, STAT5, or other IL-7
signaling pathways activated in human CLPs can lead to different
developmental fates. The CLP early-B cell step cannot
require IL-7 (see below), and other cytokines or BM stromal
cell-derived compensatory signals must be important.
Figure 2 shows the stages of human B-cell
development and counterpart stages in murine B-cell development.
Several classification schemes have been developed for the
mouse,18 but Figure 2 shows the A through E fractions
originally described by Hardy and colleagues.19 Figure 2
proposes the existence of an early-B cell (as discussed above) that
does not express the cell surface protein CD19. Early-B cells have
initiated DJH rearrangements and express cytoplasmic Ig
(and to some degree Ig) as well as VpreB proteins. I would emphasize
that this is a tentative definition of an early-B cell, since no cell
surface markers that could distinguish early-B cells from CLP have been
reported. This early-B cell may be similar to the
CD19 B-lineage fractions A1 and/or
A2 recently described by Hardy and
colleagues,20 or the
lin/c-kitlo
and lin/c-kit progenitors
described by Payne and colleagues.21 Human
pro-B cells are a well-characterized population expressing CD10, CD34, and CD19.22 The vast majority of pro-B cells express
TdT,22-24 and V-to-DJH rearrangements are
easily detected.11,12 However, single-cell PCR analysis
indicated that a minority of CD19+/CD34+ pro-B
cells have both HC alleles in germ-line configuration.24 Thus, assigning the early-B cell population a DJH
rearrangement status and the pro-B cell population a VDJH
rearrangement status is probably an oversimplification. A difference of
opinion exists as to whether CD19+/CD34+ pro-B
cells express cytoplasmic µHC. Two groups have concluded that pro-B
cells express neither cytoplasmic nor low cell-surface µHC.24,25 In contrast, using FACS-purified
CD19+/CD34+ pro-B cells, we can reproducibly
detect cytoplasmic µHC in 5% to 10% of pro-B cells.26
This result is consistent with the existence of readily detectable
VDJH rearrangements in pro-B cells.11,12 The
human pro-B cell may correspond to Hardy fraction B ± fraction C,
based on analysis of VDJH rearrangements.24,27
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| Fig 2.
Stages in human B-cell development.
Six stages beginning with the CLPs and culminating with immature B
cells are shown as one model of B-cell development in human BM. The
letters in parentheses represent an approximation of the counterpart
stages in murine B-cell development, using the nomenclature of Hardy
and colleagues.19,20 Numbers on the cell surface indicate
CD antigens frequently used to define the individual stages in human
B-cell development. Patterns of gene expression inside the cells have
been determined by RT-PCR and/or flow cytometry. Dashed arrows indicate
possible cellular targets for positive (+) and negative ( )
growth regulators/chemotactic factors produced by BM stromal cells.
Cells in a particular developmental stage are not necessarily uniform
in the expression of a specific receptor. For example, only 10% to
20% of the pre-BI plus the pre-BII cells express the µ-LC
pre-BCR.
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A functional VDJH rearrangement is essential for normal
pro-B cell differentiation into the pre-BI compartment (Figure 2). Pro-B cells that fail to make a functional VDJH
rearrangement undergo apoptosis and are probably phagocytized by BM
macrophages. Pro-B cell differentiation into pre-BI cells is
characterized by loss of CD34 and TdT, and acquisition of cytoplasmic
µHC in more than 95% of the cells.22-24 Similarly to the
mouse,18 human pre-B cells can be generally subdivided into
large proliferating cells (designated pre-BI in Figure 2) and small
postmitotic cells (designated pre-BII in Figure 2) on the basis of
cell-cycle analysis.24 The human pre-BI cells would
partially overlap with Hardy fraction C.19,20 Pre-BII cells
are actively undergoing LC rearrangements.24 In general,
rearrangement precedes rearrangement, and pre-BII cells that
fail to make a functional rearrangement can proceed to rearrange
the LC locus. Interestingly, a very small percentage (approximately
1%) of immature B cells in human BM and peripheral blood express and LC on individual cells.28-30 This dual LC expression
may reflect immature B cells undergoing receptor editing.
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The pre-BCR and related structures |
Mammalian B-lineage cells must traverse several critical checkpoints
on the road to becoming functional antigen-specific B cells. The cell
surface molecular complex appearing at a critical initial checkpoint is
the pre-BCR. The pre-BCR is a complex of proteins consisting of
µHC, LC, and the Ig/Ig signal transducing heterodimer.31 The mammalian LC consists of 2 proteins
generally referred to as VpreB and 5. The genes encoding these 2 proteins were originally discovered in the mouse (Melchers et
al32 and references therein) and their organization differs
between mouse and human (Minegishi et al33 and references
therein) VpreB and 5 proteins are noncovalently associated on the
surface of B-cell precursors and together form a LC-like structure.
In turn, 5 is covalently coupled to the CH1 domain of
µHC via a carboxy-terminal cysteine. Readers are referred to an
earlier review for details on the original identification and
characterization of the VpreB and 5 genes and their encoded
proteins.32
Analysis of the structure, expression, and function of the human
pre-BCR has been facilitated by the development of mAbs against recombinant LC proteins.34-39 The initial panel of mAbs
made against the human LC was used to characterize cytosolic and
cell-surface µHC/LC complexes and surface expression of
LC.34,40 A major conclusion in the original
study34 was that surface µHC/LC expression was
restricted to the pre-B cell compartment (ie,
CD19+/CD34 cells in Figure 2); this
suggests a critical role for the pre-BCR at a relatively late stage of
B-cell development. Subsequent studies using other mAbs yielded
conflicting results.24,35,36 A major difference was the
identification of normal and leukemic pro-B cells (ie,
CD19+/CD34+/µHC) that
stained with anti-VpreB mAb.24,35,36 The low levels of
cell-surface LC, differences in the subtype of the mAb (ie, IgM
versus IgG1), and differences in epitope recognition by various anti-VpreB mAbs were among the explanations offered for the conflicting results.
A series of papers describing new antihuman VpreB mAbs37-39
has provided some resolution to past discrepancies. The recent vintage
of anti-VpreB mAbs includes mostly IgG1 subclass mAbs, thereby
eliminating potential problems that can confound the use of IgM
reagents. The anti-VpreB mAb produced by Wang and colleagues binds to
the surface of pre-B cell lines, but only binds to pro-B cell lines
following permeabilization.37 As expected, normal CD19+ human B-lineage cells coexpressed low levels of cell
surface µHC and VpreB, and approximately 20% of the
CD19+/VpreB+ cells were weakly
CD34+.37 Interestingly, cytoplasmic
VpreB+ is expressed in
CD34+/CD19 lymphoid progenitors at a
stage prior to V-to-DJH rearrangement,37 possibly the early-B cells proposed in Figure 2. Tsuganezawa and colleagues generated mAbs that recognized human VpreB, human 5, or
an epitope expressed only on the assembled pre-BCR.38 Their anti-VpreB mAb binds to the surface of pre-B ALL cell lines but not
pro-B ALL cell lines.38 However, cytoplasmic VpreB or 5 were detected in the majority of pro-B ALL cell lines and freshly isolated pro-B ALL.38 In contrast, the anti-VpreB mAb
produced by Gauthier and colleagues39 binds to the surface
of some µ pro-B ALL cell lines.25 One
of these pro-B ALL cell lines (designated JEA2) was shown to
express cell surface VpreB in association with poorly characterized
molecules of approximately 105 to 130 kd. Interestingly, cross-linking
VpreB on the µ JEA2 pro-B ALL cell line led to an
increase in Ca++ flux, suggesting that VpreB was one
component of a putative signaling receptor on the surface of at least
some pro-B ALL cells.25 These authors also detected
cell-surface VpreB on CD19+/CD34+ normal pro-B
cells and used this data to argue for the existence of 2 distinct
populations:
CD19+/CD34+/µHC/VpreB+
and
CD19+/CD34/µHC+/VpreB+.
However, there is no biochemical evidence that VpreB is associated with
a protein (or proteins) other than µHC on the surface of normal human
B-cell precursors. We have used one of the anti-VpreB mAbs (VpreB8)
made by Wang and colleagues37 to analyze VpreB expression
in fetal BM B-lineage cells. Our results indicate that VpreB is
expressed on the surface of 5% to 10% of the B-cell precursors (ie,
pro-B plus pre-BI plus pre-BII in Figure 2). Furthermore, within the VpreB+ population, approximately 90% of the
cells are CD19+/CD34, and approximately
10% are CD19+/CD34+. Results from our
laboratory show that CD34+/VpreB+ cells also
express cell surface µHC (J. A. R. Pribyl, N. Shah, F. E. Bertrand,
T. W. LeBien, 1999, unpublished data). Thus, we believe that most, if not all,
CD19+/CD34+/VpreB+ normal B-lineage
cells express the conventional pre-BCR. Since the vast majority of
surface VpreB+ cells are CD34, I show
the expression of the pre-BCR on pre-BI and pre-BII cells only (Figure
2). The VpreB+ cells that weakly express CD34 could be
developmentally more similar to pre-BI cells than pro-B cells, but this
has not been tested. The vast majority of B-lineage ALLs express
cytoplasmic or surface VpreB,25,38 probably reflecting the
general phenotypic similarity between normal and leukemic B-cell precursors.
The importance of the LC to normal B-cell development was first
elucidated in a classic study demonstrating that mice with a targeted
disruption in the 5 locus exhibit a block at the pro-B to pre-B
transition.41 The block in B-cell development probably occurs because cells at this transition fail to receive a positive selection signal through the pre-BCR. However, the block is not absolute since the number of B cells in secondary lymphoid tissues gradually increases over time, probably owing to the emergence of B
cells that rearranged LC genes prior to µHC
genes.42,43 The importance of the LC in human B-cell
development has been underscored by the discovery of an
agammaglobulinemia patient with mutations in both 5
alleles33 (see below). Immunologic analysis of this single
patient indicated a disruption in B-cell development more severe than
what occurs in 5-deficient mice. Since only a single patient with a
mutation in the 5 locus has been described to date, it is unclear
whether the gradual recovery of B cells observed in 5-deficient mice
would occur in humans.
How does the pre-BCR perform its critical role at the
pro-Bpre-B transition? Despite heuristic appeal, the notion
that the pre-BCR functions as a receptor for a specific ligand in the
BM or fetal liver microenvironment has not been supported by
experimental evidence. What has become clear is that only about one
half the µHC proteins encoded by functional VDJH
rearrangements are capable of pairing with LC in the
mouse.44-46 Circumstantial evidence suggests that a similar
type of preferential pairing of µHC and LC also occurs in human
pre-B cells.47,48 This pairing is essential for pre-BCR
assembly and expression on the cell surface. Formation of the pre-BCR
heralds a sequence of events, including (1) suppression of RAG-1/RAG-2
expression to ensure allelic exclusion at the µHC locus, (2) a rapid
burst of proliferation in cells expressing the pre-BCR, and (3)
reexpression of RAG-1/RAG-2 and initiation of LC gene rearrangement.
Are there separable roles for LC and µHC in pre-BCR function? This
is controversial. Shaffer and Schlissel reported that transgenic mice
expressing a truncated murine µHC (incapable of pairing with LC)
could still transduce signals leading to changes in surface markers,
transcription, and retargeting of the recombinase ensemble in B-lineage
cells.49 The authors concluded that the LC
functions as a chaperone to facilitate pre-BCR assembly and
expression, but plays no direct role in signal transduction. They also
argued that the capacity of truncated µHC to mediate B-cell
differentiation in the absence of LC ruled against the
pre-BCR/ligand model. However, truncated µHC may undergo enhanced
aggregation compared with full-length µHC, thereby leading to
increased constitutive signaling.50
Pre-BCR cross-linking in vitro initiates signaling events, including
Ca++ flux and protein tyrosine kinase
activation.51,52 How could this occur in vivo in the
absence of an external cross-linking ligand? Elegant recent studies
have provided new insight into the complexity of pre-BCR subunit
protein-protein interactions involving µHC, VpreB, and
5.39,53 These studies suggest mechanisms to explain
pre-BCR assembly, VH repertoire selection, and cell signaling. We might assume that signaling through the pre-BCR minimally
requires the dimerization/aggregation of at least 2 distinct pre-BCR
molecular complexes on the cell surface. With the realization that the
LC is a non-transmembrane-spanning polypeptide complex
disulfide-linked to the µHC and exhibiting VpreB-VH
interactions, it seems possible (as proposed by Melchers54)
that the LC itself could assume a ligand function for the µHC.
Other protein complexes with potentially similar functions to the
pre-BCR have been described. Murine pro-B cell lines express the LC
associated with several proteins ranging from 65 to 200 kd, including a
predominant protein of 130 kd.55 These pro-B cell lines do
not express µHC.55 The protein or proteins associated with LC in these cells have been occasionally referred to as the
surrogate HC. A recent study using a human µ pro-B
leukemic cell line (JEA2) showed that VpreB is noncovalently associated
with a p105 and possibly several other proteins on the cell
surface.25 However, there is no evidence that VpreB associates with p105 (or any other protein other than µHC) on normal
human B-cell precursors. The identity of the murine and human
LC-associated surface proteins is unknown. A novel complex that has
been referred to as the "calnexin pre-BCR"56 was
recently described by Nagata and colleagues.57 The calnexin
pre-BCR consists of Ig/Ig noncovalently associated with the
molecular chaperone calnexin, and was detected on the surface of
pro-B cell lines and early B-lineage cells from RAG-2-deficient
mice.57 Cross-linking Ig on these cells induced the
tryosine phosphorylation of syk, ERK, and PI-3 kinase in vitro, and
pro-B to pre-B cell differentiation in vivo. A potential
µ-independent role for Ig was discovered when mice with a targeted
disruption of the Ig gene were shown to exhibit a block in B-cell
development prior to V-to-DJH rearrangement.58 These
results57,58 suggest a role for
Ig+/µHC molecular complexes in the
earliest stages of murine B-cell development.
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The IL-7 story |
An unresolved issue in studies of human B-cell development is the
identity of the molecule(s) essential for the growth of normal B-cell
precursors. Much has been written of IL-7, and some historical
perspective is warranted. Following the initial cloning and
characterization of IL-7 from murine BM stromal cells more than 10 years ago,59 IL-7 was shown to be crucial for the
proliferation and development of murine B-cell precursors. IL-7 has
been cast as a survival, proliferation, or differentiation factor
depending upon the experimental system being employed.60,61
Mechanistic insight has been gleaned from studying the effect of single
amino acid substitutions on IL-7R chain function. Corcoran and
colleagues showed that a Y-to-F mutation at amino acid residue 449 abrogated the capacity of B-cell precursors to undergo IL-7-induced
proliferation through a PI-3 kinase-dependent pathway.62
Interestingly, functional studies of the IL-7R chain harboring this
mutation uncovered a novel signaling pathway (PI-3 kinase
independent?) that triggered IgH rearrangements and
subsequent B-cell differentiation.62 Recent studies from
the same group indicated that IL-7R signaling can alter recombinase
accessibility of 5' VH genes.63 The
criticality of IL-7 for normal murine B-cell development has been
elucidated in gene-targeted mice. Targeted disruptions in the genes
encoding IL-7,64 the IL-7R chain,65 the
 c subunit of the receptors for IL-2, 4, 7, 9, and
15,66,67 and the Jak3 tyrosine kinase68,69 all
lead to severe impairment in B-cell development. Thymocyte and T-cell
development are also impaired, reflecting the multiple actions these 5 cytokines exert on lymphopoiesis. IL-7 is, however, not the only
cytokine implicated in the regulation of murine B-cell development.
Kincade and colleagues have suggested that at least 16 distinct stromal
cell products can exert positive effects on murine B-cell
development.61 One recent addition to the list is thymic
stromal lymphopoietin (TSLP). TSLP was originally isolated from a
murine thymic stromal cell line.70 TSLP reportedly has the
capability to replace IL-7 in supporting murine B-cell development in
vitro71 and promote the development of surface
IgM+ immature B cells from surface IgM
precursors.72 Interestingly, the TSLP receptor complex
consists of the IL-7R chain and a second subunit distinct from the
c. Furthermore, TSLP induced the activation of
STAT5 but not any of the known Jak kinases.72
Whether IL-7 and TSLP work in a hierarchical or cooperative manner in
regulating murine B-cell development is unknown. Human TSLP has been
cloned (S. Lyman, PhD, Immunex, written communication,
May 1999), but there are no published reports on its bioactivity on
human B-cell precursors.
The role of IL-7 in human B-cell development is strikingly different
from its role in murine B-cell development. In fact, there is a
widespread misconception that IL-7 stimulates the proliferation of
human B-cell precursors. Part of the difficulty in determining IL-7
effects can be attributed to differences in the assay systems employed
and the biological endpoints measured. Initial studies by
us73 and others74,75 demonstrated that
recombinant human IL-7 could exert weak proliferative effects on normal
human B-cell precursors in vitro. However, it was difficult to exclude
the possibility that IL-7 was simply enhancing survival. A stronger effect of IL-7 was observed when normal B-cell precursors were cultured
on human BM stromal cells.26,73,75,76 Under these conditions, CD19+ B-lineage cell numbers increased by
10-fold to 20-fold over 2 to 3 weeks in vitro, but underwent rapid cell
death thereafter.
Biological insight into IL-7 function in human B-cell development has
been gleaned from congenital immunodeficiency patients. Patients with
X-linked severe combined immunodeficiency (XSCID) have
mutations in the c subunit of the receptors for IL-2,
IL-4, IL-7, IL-9, and IL-15. XSCID patients have severe defects in
development of T and NK cells, but have normal or even elevated numbers
of peripheral blood B cells.77 Furthermore,
immunodeficiency patients with autosomal recessive mutations in the
Jak3 tyrosine kinase exhibit a developmental phenotype
indistinguishable from XSCID, including normal numbers of peripheral
blood B cells.78,79 Likewise, 2 patients with mutations in
the IL-7R chain that led to defective expression also had normal
numbers of peripheral blood B cells.80 These collective
experiments of nature clearly indicate that IL-7 signaling is not
essential for at least the numerically normal development of human B
lymphocytes. My laboratory used a human BM stromal cell culture system
to show that HSCs could develop into B-lineage cells independently of
IL-7 stimulation.81 Our results are in accord with B-cell
development occurring in patients with XSCID. However, extensive
proliferation of the pro-B cell compartment does not
occur,81 and it is presumptuous to assume that this in
vitro model completely mimics B-cell development occurring in patients
with mutations in the IL-7 pathway.
Although not essential for human B-cell development, IL-7 does
transduce signals that lead to specific changes in gene expression. Proliferation of CD19+/CD34+ pro-B cells on
human BM stromal cells is enhanced by inclusion of exogenous
IL-7.26 IL-7 stimulation induces a specific increase in
cell-surface CD19 on human pro-B cells82,83 and a decrease in RAG-1, RAG-2, and TdT messenger RNA (mRNA) levels.83
There may be physiologic relevance to these results. For example, IL-7 expression in situ has been detected by RT-PCR analysis of human BM
biopsies.84 The identity of the human BM cell producing
IL-7 in vivo is unknown, although small amounts (less than 2 pg/mL) of
IL-7 can be detected in supernatants from vascular cell adhesion molecule-1 (VCAM-1) (CD106)+ BM stromal cells
in vitro.76,81,85 Similarly, purified VCAM-1+
murine BM reticular cells express cytoplasmic IL-7
protein.86
The complete identity of the human BM stromal cell-derived molecule
(or molecules) that transduce signals essential for human B-cell
development (including the counterpart of the murine IL-7 "signal") is unknown. Part of the difficulty in identifying an IL-7 alternative is that IL-7 could act on at least 3 distinct stages
of lymphoid cell development: CLPs, early-B cells, and pro-B cells
(Figure 2). The cell-cycle disposition and self-renewal capacity of
these compartments have not been determined. However, we do know that
human CD19 progenitors8 and pro-B
cells26 are IL-7 responsive. Figure 3 shows several cytokines that could
regulate human B-cell development. The cytokine-responsive target cells
would include CLPs, early-B cells, and pro-B cells. The BM stromal cell
molecules that regulate development could be secreted or cleaved from
the stromal cell surface. The secreted or cleaved products could in
turn be bound to stromal cell proteoglycans such as heparan sulfate
proteoglycans (HSPGs). Several candidates come to mind. Namikawa and
colleagues reported that a combination of IL-7, IL-3, and Flt3 ligand
is superior to IL-7 alone in supporting human pro-B cell growth on murine BM stromal cells.87 My laboratory has confirmed
their observation using human BM stromal cells (J.A.R. Pribyl and T.W. LeBien, unpublished observations, February 1999). Flt3
ligand is produced by BM stromal cells88,89 (also in our
unpublished observations, February 1999) and could
potentially stimulate similar cellular compartments and transduce
similar signals to IL-7. Oritani and Kincade developed a cloning
strategy to identify murine BM stromal cell gene products that bind to
murine pre-B cells.90 One of the molecules identified was a
secreted extracellular matrix glycoprotein designated
SC1/ECM2.90 Soluble and immobilized SC1/ECM2 enhance the
growth of IL-7-dependent murine pre-B cells,90,91 but the
mechanism of enhancement is unknown. Interestingly, the carboxy
terminus of SC1/ECM2 has high amino acid sequence homology to
osteonectin/SPARC,92 which reportedly can bind cytokines, such as platelet-derived growth factor.93 A human homologue of SC1/ECM2 (designated hevin) has been cloned from high endothelial venules,94 but it is not known whether hevin has any effect on the growth of human B-cell precursors. HSPGs play a critical role in
"presenting" cytokines to survival/growth factor receptors on
lymphohematopoietic cells. HSPGs expressed on murine B-lineage and BM
stromal cells can bind IL-7 and enhance the growth of IL-7-dependent murine pre-B cell lines.95 Furthermore, heparan sulfate is
important for cytokine-mediated expansion of human
long-term-culture-initiating cells.96 Therefore, a
currently unknown molecule produced by BM stromal cells could bind to
HSPGs and mediate survival/growth of human B-cell precursors (Figure
3).
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| Fig 3.
BM stromal cell-derived molecules that could transduce
survival/growth, differentiation, or chemotactic signals to CLP,
early-B, or pro-B cells.
HSPG indicates heparan sulfate proteoglycan. The dashed arrow indicates
that the membrane-bound form of Flt3 ligand is cleaved at the stromal
cell surface. The question mark indicates the unknown growth factor
that could bind to HSPG. IL-7 and the unknown growth factor are shown
binding directly to their cognate receptors, or binding HSPG followed
by binding to their cognate receptors.
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A BM stromal cell product shown in Figure 3 that potentially provides a
unique function in B-cell development is the chemokine stromal
cell-derived factor-1 (SDF-1). Mice with targeted disruptions in the
genes encoding SDF-1 and its receptor CXCR4 exhibit perinatal mortality
owing to perturbations in organ vascularization and lymphohematopoiesis.97-99 B lymphopoiesis and myelopoiesis
are severely impaired,97-99 and a recent study concluded
that a functional CXCR4 receptor is essential for retention of B-cell
precursors in the BM microenvironment.100 CXCR4 has a
complex pattern of expression on CD34+ lymphohematopoietic
cells and CD19+ B-lineage cells.9,100-102 CXCR4
is expressed at all stages of B-cell development, but
CD19+/CD34/LC pre-B
cells and mature B cells express higher levels than
CD19+/CD34+ pro-B cells.102,103
Interestingly, SDF-1-mediated signaling pathways leading to calcium
mobilization and chemotaxis are more rigorously activated in less
mature B-lineage cells expressing lower levels of cell surface
CXCR4.102,103 Thus, BM SDF-1 may trigger signaling pathways
that regulate chemotaxis of B-cell precursors to "preferred
sites" of proliferation within the stromal cell milieu. It is
noteworthy that SDF-1 is expressed in human fetal liver biliary ductal
plate epithelial cells, in apposition to lymphoid cells expressing
VpreB.104
| |
Models of human B-cell development |
Establishment and characterization of in vitro culture systems that
at least partially mimic the in vivo BM microenvironment have been
extremely important for advancing our understanding of human B-cell
development. Progress in this area has been facilitated by the use of
BM stromal cells as a supportive microenvironment. Adherence of B-cell
precursors to BM stromal cells is essential for normal murine and human
B-cell development (reviewed in Kincade et al,61 Jarvis et
al,105 and Ryan et al106). Binding of very late
antigen-4 (VLA-4) (CD49d/CD29) expressed on human B-cell precursors to
VCAM-1 on human BM stromal cells is the primary molecular interaction
that facilitates adhesion of these 2 cell types.107-109
Cytokines can regulate levels of BM stromal cell surface VCAM-1,
thereby influencing the capacity of these cells to support B-cell
precursor adhesion.108 There is substantial evidence that cross-linking VLA-4 with VCAM-1 or the CS-1 domain of fibronectin can
trigger a protein tyrosine kinase cascade in B-lineage cells. However,
there is no evidence that VLA-4 triggers a reciprocal activation of
VCAM-1 culminating in a signal transduced in BM stromal cells. Lymphoid
cell contact with human BM stromal cells can transduce signals leading
to protein tyrosine kinase activation110 and tyrosine
phosphorylation of focal adhesion kinase, paxillin, and
ERK2.111 This signaling pathway is independent of
VCAM-1.110 To what degree these bi-directional (B-cell
precursor  BM stromal cell) signaling events might
influence B-cell developmental fates in vivo is unknown.
Prompted by our success in establishing in vitro human BM stromal cell
culture conditions that support the adhesion108 and short-term growth26,73 of B-cell precursors, we asked
whether a more expanded model of B-cell development could be
established. In an effort to foster physiologic relevance, we
FACS-purified fetal BM CD34++/CD19 HSC
and plated them onto third-passage, nontransformed human fetal BM
stromal cells.81 CD34++/CD19
HSCs underwent commitment and differentiation into the B-lineage over a 3-week period.81 A hierarchy of developmental
changes consonant with B-cell development in vivo occurs in this in
vitro model, including (1) loss of CD34 expression, (2) a continuum of
increase in cell surface CD19, (3) emergence of cytoplasmic µHC+ pre-B cells, including some expressing the
cell-surface pre-BCR, and (4) emergence of immature B cells expressing
µ/ or µ/ BCR. As discussed above, several lines of evidence
rule out a mandatory role for IL-7 in this culture
system.81 The fact that
CD34++/CD19 HSC can differentiate all
the way to immature B cells demonstrates that human fetal BM stromal
cells can provide the developmental cues necessary to traverse the
major checkpoints defined by rearrangement of HC and LC genes. This
human BM stromal cell culture does not support a dramatic numerical
expansion of any specific compartment of B-lineage cells, probably
attributable to the exclusion of fetal bovine serum and exogenous
cytokines. It is also conceivable that the BM stromal cells in this
culture (which are exclusively adventitial
reticular/fibroblastlike cells by third passage) do not represent the
totality of BM stromal cell components essential for optimal
proliferation in vivo.
At the time we were developing our human BM stromal cell culture,
Rawlings and his colleagues were developing an in vitro culture using
the murine S17 stromal cell line.112 They originally showed
that enriched CD34+ cord blood cells would develop into
CD19+ B-lineage cells after 3 to 4 weeks.112
The CD19+ B-lineage cells were at a very early stage of
B-cell development since bulk culture analysis by Southern blotting
indicated no rearrangements at the IgH locus.112 These
CD19+ B-lineage cells could be expanded following transfer
to fresh S17 stromal cells, but did not proliferate following
stimulation with IL-7 or stem cell factor (SCF). A
follow-up report showed that inclusion of Flt3 ligand enhanced the
development of CD19+ B-lineage cells by twofold to
threefold.113 We have recently shown that inclusion of Flt3
ligand at the initiation of our human BM stromal cell culture also
enhances the development of CD19+ B-lineage cells
(J.A.R. Pribyl and T.W. LeBien, unpublished
observations, February 1999), providing additional
support for a role of Flt3 ligand in human B-cell development (Figure
3).
Given the differences in the tempo and degree of B-cell development in
the 2 models, we conducted a side-by-side comparison of murine S17
stromal cells and human fetal BM stromal cells.114 When
human fetal BM CD34+ HSCs were cultured on human fetal BM
stromal cells or human skin fibroblasts, robust differentiation to the
immature B-cell stage occurred within 3 weeks.114 In
contrast, CD19+ B-lineage cells emerging on S17 stromal
cells within the same time frame had twofold to fourfold higher levels
of cell-surface CD19, but no cells expressing the BCR.114
Human and murine S17 stromal cells therefore differ in their capacity
to support human B-cell differentiation under the conditions in which
we compared them. The identity of the soluble or membrane-bound stromal
cell molecules important in both cultures is unknown. When
CD34+ cord blood HSCs are cultured on S17 stromal cells for
6 to 8 weeks, small numbers of cytoplasmic and surface
µHC+ cells can be detected.115 Furthermore,
transferring these 6- to 8-week cultures onto CD40 ligand
(CD154+) fibroblasts supplemented with IL-4
and IL-10 results in terminal human B-cell differentiation to
Ig-secreting cells.115 Murine stromal cell lines other than
S17 also support the development of CD19+ human B-lineage
cells from CD34+ cord blood HSCs.116-119 Two of
these studies showed that a combination of SCF and granulocyte
colony-stimulating factor would enhance the outgr |
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Abstract
Introduction