The Protean Nature of Cells
in the B Lymphocyte Lineage
Richard R. Hardy,1,* Paul W. Kincade,2and Kenneth Dorshkind3
1The Division of Basic Sciences, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
2The Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
3The Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
The subdivision of bone marrow (BM) with surface markers and reporter systems and the use of mul-
tiple culture and transplantation assays to assess differentiation potential have led to extraordinary
progress in defining stages of B lymphopoiesis between the hematopoietic stem cell and B cell
receptor (BCR)-expressing lymphocytes. Despite the lack of standard nomenclature and a series
of technical issues that still need to be resolved, there seems to be a general consensus regarding
tean and able to alter their differentiation potential during embryogenesis and after birth in response
to infections suggests that a full understanding of B cell development and how it is regulated has not
yet been attained.
The Early Events
Hematopoietic stem cells (HSCs), defined by their exten-
sive self-renewal capacity and potential to generate all
blood-cell lineages, become restricted to the B lympho-
cyte lineage through progressive stages of differentiation
that have been characterized by several groups. HSCs
are identified by expression of high amounts of the recep-
tor for stem cell factor, c-kit (CD117), and the absence
of cell-surface proteins expressed on differentiated mye-
loid, erythroid, and T lineage cells. This lineage-negative
(Lin–), CD117hifraction of BM cells is highly enriched for
long-term repopulating HSCs (Spangrude et al., 1988).
Recently, expression of the CD150 member of the SLAM
family of molecules has been shown to greatly enrich for
HSCs within this CD117hiLin–fraction (Kiel et al., 2005).
Other correlates to transition from HSCs to short-term
repopulating stem cells and primitive progenitors include
loss of the adhesion molecule VCAM-1 and CD90 (Thy-1)
along with acquisition of the growth-factor receptor Flk-2
(Flt-3) and CD27.
Correlating changes in cell lineage potential, assayed
in vitro or in vivo, with cell-surface phenotype has been
a powerful tool used to resolve the earliest stages of B
cell development from HSCs (Figure 1). Primitive cells,
termed ‘‘early lymphoid progenitors’’ (ELPs), within the
CD117hiLin–fraction that exhibit lymphoid or B lineage
bias have been described based on their expression of
genes considered lymphocyte restricted. Thus, transcrip-
tion of terminal deoxynucleotidyl transferase (TdT), a gene
that mediates nontemplated nucleotide additions during
antigen receptor recombination, initiates very early. Acti-
vation of the lymphocyte-restricted recombinase activat-
ing genes, particularly Rag-1, initiates in a fraction of the
TdT+ELPs, and such cells appear particularly lymphoid
biased (Igarashi et al., 2002). High sensitivity to steroid
hormones also helps to distinguish early, lymphoid-spec-
ified progenitors from cells dedicated to other lineages
(Medina et al., 2001). Much more needs to be learned
on timely expression of the Ikaros, PU.1, E2A, and EBF1
transcription factors (Busslinger, 2004).
ELPsdonot expressthe receptorfor IL-7,CD127,butin
the BM they progress to generate CD127+common lym-
phoid progenitors (CLPs). CLPs were originally described
asbeing lymphoid-committed cellsthathadlostthe ability
to generate most other hematopoietic lineages (Kondo
et al., 1997). Textbook models of hematopoietic develop-
ment view them as a branch-point between the B and T
lineages, but more recent work has suggested that the
thymus is likely seeded by more primitive progenitors (All-
man et al., 2003). Furthermore, the presence of extensive
Ig heavy-chain DJ rearrangements in CLPs (Rumfelt et al.,
2006) and their rapid progression to early stages of B lin-
eage development suggest that most are early B cell pro-
genitors that retain residual potential for generation of
alternate lineages. In addition to B cells, CLPs as originally
defined by Kondo and colleagues (Lin–Sca1+CD117+
CD127+) also represent major intermediates en route to
becoming NK cells (Hirose et al., 2002; Kondo et al.,
1997; Welner et al., 2007).
Lineage Stability Is Acquired Gradually
Lineage potential of precursors can be tested in vivo by
cell transfer or in vitro by a diverse array of assays. The
most widely used in vivo assay for assessing hematopoi-
etic lineage potential is competitive repopulation of le-
thally irradiated mice with donor cells from Ly5 disparate
congenic animals (Uchida et al., 1994). An advantage of
this approach is that differentiation potentially occurs in
Immunity 26, June 2007 ª2007 Elsevier Inc.
a physiologic environment, with the caveats that irradia-
tion may alter cell homing patterns and substantially per-
turb delicate microenvironmental niches. This approach
has established the broad outlines of progressive restric-
tion to distinct hematopoietic lineages, including the CLP
and common myeloid progenitor (CMP) stages (Terskikh
et al., 2003).
The oldest procedure for determining the capacity of
progenitors to generate diverse hematopoietic cell fates
This procedure, together with fetal thymic organ culture
and long-term BM culture (‘‘stromal cell culture’’), are the
classic approaches for differentiation of all blood-cell line-
ages. An interesting recent development has been the
application of a Delta1-transduced stromal cell line that
permits generation of T lineage cells without the complex
procedure of fetal organ culture (Schmitt and Zuniga-
Pflucker, 2002) and that can be readily applied in single-
cell analysis (Rumfelt et al., 2006). Various single-cell
procedures have been developed that enable the lineage
potential of smaller numbers of highly purified progenitors
to be analyzed.
Traditional modelsofhematopoiesis depictitasarather
However, the use of increasingly sophisticated in vitro
assays has revealed that lineage biases, read out in vivo
as absolute, may not actually be so. For example, analysis
of CLP-stage cells that appear lymphoid restricted when
assayed in vivo reveals some degree of myeloid potential
in single-cell stromal-cell culture (Balciunaite et al., 2005;
Rumfelt et al., 2006). This view has been corroborated
by use of fluorescent reporters that assess activation of
and development. In this regard, genes such as Rag1 and
Rag2 are first expressed in cells of the ELP (Igarashi et al.,
2002) and multilineage progenitor (MLP) (Rumfelt et al.,
2006) stages that retain substantial myeloid potential. In
parison of data on lineage restriction of variously delin-
eated precursor populations by in vivo and in vitro assays
reveals a more stochastic picture in which lymphoid spec-
ification occurs as a gradual rather than abrupt process.
Thus, cells at early stages exhibit a lymphoid ‘‘bias’’ rather
than ‘‘commitment’’ in that they are efficient in generating
Figure 1. A Framework for Delineating Progressive Stages in Development of B Lineage Cells in Mouse Bone Marrow, Based
on Ordered Changes in Cell-Surface Molecules
Cell-surface expression shown in the beige box. Gene expression assessed by analysis of mRNA, reporter constructs, or cytoplasmic staining is
shown in the violet box. Note that the ELP cell fraction, identified by cytoplasmic expression of Tdt or a Rag1 GFP reporter, is contained within
the MLP subset,identified by cell-surface analysis. Abbreviations: HSC, hematopoietic stem cells; MLP,multilineage progenitor; ELP, early lymphoid
progenitor; NF B, newly formed B cell; Fo B, follicular B cell.
Immunity 26, June 2007 ª2007 Elsevier Inc.
B and T cells, but retain some potential for production of
alternatehematopoietic celllineages,particularly myeloid.
It seems clear that the process of progressive B lineage
specification is being established at the earliest stages of
hematopoiesis, including in cells, defined below, termed
MLP (Figure 1). As differentiation progresses from the ELP
through the CLP stage, much, but not all, of the potential
for production of myeloid lineage cells is lost. Precisely
how B lymphoid specification and commitment are estab-
lished remains unclear. An emerging area ripe for further
study is the role played by microRNAs. These small (19–
at the post-transcriptional level by directing cleavage or
repression of messenger RNAs (Kluiver et al., 2006). It is
intriguing that B lineage-restricted miRNAs have been de-
scribed (Monticelli et al., 2005). Much better understood is
the activation and repression of groups of genes in devel-
oping B lineage cells by a network of transcription factors
that include PU.1, E2A, Ebf1, Bc11a, and PBX1 (Medina
et al., 2004). It is now well established that doses, combi-
nations, and cross competition between these transcrip-
tion factors influence how lineage-specified progenitors
become firmly committed to particular fates. In addition,
there is evidence that immature lymphoid precursors ex-
press low amounts of mRNA for genes characteristic of
diverse cell lineages in a process termed ‘‘priming’’ that
presumably reflects opening of chromatin and expression
of a set of transcription factors at low levels (Ye et al.,
2003). This process eventually culminates in expression
of the transcription factor Pax5 that is crucial in suppress-
ing alternate cell fates in developing B lineage cells (Nutt
et al., 1999). Thus, CD19 expression, activated by Pax5,
is a marker for B lineage-committed cells. The Pax5 and
Ebf1 transcription factors provide an interesting example
of feed-forward regulation: Pax5 expression is Ebf1 de-
pendent, but Pax5 induces more Ebf1 expression (Roess-
ler et al., 2007). This may account in part for why Pax5
sustains commitment to the B lineage. Additional details
regarding the transcriptional regulation of B lymphopoie-
sis can be found in the article by Nutt and Kee in this issue
(Nutt and Kee, 2007).
The sustained expression of transcription factors such
as Pax5 is necessary to maintain B lineage fate decisions,
and there are many examples where inhibition resulting
from transformation or experimental manipulation results
in the conversion of lymphoid progenitors into myeloid
cells (Iwasaki et al., 2003; Iwasaki-Arai et al., 2003; Kondo
et al., 2000; Xie et al., 2004). A particularly dramatic illus-
tration of what is sometimes referred to as ‘‘latent’’ poten-
tial in B cells comes from studies in which cessation of
Pax5 expression was induced (Busslinger, 2004; Mikkola
et al., 2004). Well-differentiated B lineage cells became
undifferentiated on conditional targeting of the gene for
this essential transcription factor, and the cells reacquired
the potential for generating multiple non-B cell types.
process. In fact, it has been suggested that myelopoiesis
represents a default pathway utilized when some tran-
scription factors needed for lymphopoiesis are absent
(Laiosa et al., 2006). The reverse conversion of myeloid
progenitors to lymphocytes was observed only when con-
retroviral vector (Baba et al., 2005). This represents an ar-
tificial stimulation of the canonical Wnt-signaling pathway.
There is less evidence that lineage conversion occurs
under physiological conditions, and a distinction needs
to be made between mature cells and progenitors. Chaos
would seem to result from instability in the former, and in-
deed celiac disease was recently reported to result from
cytotoxic T cells that somehow acquired NK cell proper-
ties (Meresse et al., 2006). Another report showed that
BM plasmacytoid dendritic cells (pDCs) generated con-
ventional DCs as a consequence of lympho-choriomenin-
gitis virus infection (Zuniga et al., 2004), but this transition
might potentially be beneficial. Recently described inter-
feron-producing killer dendritic cells (IKDCs) display hy-
brid properties between dendritic and NK cells, but may
not actually be capable of conversion between these two
cell types (Chan et al., 2006; Taieb et al., 2006). Rather,
they could be a highly specialized cell type with functions
previously ascribed to the other lineages. In fact, there is
now evidence that IKDCs arise from a unique differentia-
tion pathway (Welner et al., 2007). As another possible ex-
ample of conversion between mature cell types, B-1 cells
have been reported to have potential for macrophage dif-
rello and Phipps, 1995). Much more work is required to
learn the normal extent and relative merit of such conver-
sions and whether or not they underlie hematopoietic
Resolution and Characterization of Developing
B Lineage Cells
B lineage cells prior to complete immunoglobulin (Ig)
heavy-chain rearrangement were first identified by coex-
pression of ‘‘B220,’’ the relatively B lineage-restricted
high-molecular-weight isoform of Ptprc (CD45), together
with leukosialin (sialophorin), CD43 (Hardy et al., 1991).
These early B lineage cells can be further subdivided
based on expression of two developmentally regulated
surface proteins, CD24, the heat-stable antigen (HSA),
and BP-1, a zinc-dependent cell-surface metallopepti-
dase also known as aminopeptidase A. Later stages of
developing B cells downregulate CD43 and can be sub-
divided into surface Ig–pre-B cells and two fractions of
ing IgD+cells. IgM–cells in BM were also subdivided by
another group based on expression of c-kit (CD117),
IL-7Ra (CD127), and IL2Ra (CD25) (Rolink et al., 1994).
The earliest stages are CD117+CD127+, with CD117 grad-
ually downregulated and CD25 expressed later. The for-
mer separation scheme delineates 6 (or 7) fractions of
cells that are labeled alphabetically from A to F, whereas
the latter approach identifies stages termed pre-BI and
pre-BII (Figure 1). Corresponding fractions have also
been described in the fetal liver (Li et al., 1993).
The 220,000 kDa product of the Ptprc(Cd45) gene is
commonly referred to as ‘‘B220,’’ but it is important to
Immunity 26, June 2007 ª2007 Elsevier Inc.
note that different antibodies that identify B220 actually
recognize distinct epitopes, with some differences in
expression. CD45 undergoes extensive cell- and differen-
tiation-stage-specific RNA splicing, with the isoform con-
taining exon A present in the 220 kDa protein considered
to be relatively B lineage specific. The 14.8 and RA3-2C2
antibodies recognize CD45-transfected cells containing
exon A (Johnson et al., 1989), staining B lineage cells,
and a subset of T cells (Luqman et al., 1991). Another
widely used B220-specific antibody, RA3-6B2, binds to
the 220 kDa product of CD45 but does not stain exon
A-transfected cells, presumably because of an additional
requirement for post-translational modification that does
not occur in the transfected cells (Johnson et al., 1997).
As a consequence, the ‘‘6B2’’ epitope is not found on the
resting CD8+T cells stained by 14.8 or 2C2. Yet even the
6B2 epitope is expressed on cell types other than B line-
age cells, including subsets of NK, T, and dendritic cells.
This is particularly a problem when dealing with relatively
rare cell fractions, such as very early B cell precursors in
BM, that constitute less than 1% of the total cell popula-
tion. For example, although most B220+cells coexpress
CD19, the small fraction that does not is enriched for
very early B lineage precursors. However, in addition to
B cell precursors, this B220+CD19–subset also includes
NK cells, pDCS, and their precursors. Most, but not all,
of the ‘‘contaminating’’ cells in this subset could be elimi-
nated by excluding cells that failed to express AA4 (also
known as CD93) (Li et al., 1996), a fetal stem cell marker
that is also found on pre-B cells (Petrenko et al., 1999).
Later it became clear that even further selection is re-
quired, such as absence of Ly6C-, NK1.1-, and DX5-ex-
pressing cells, to purify a relatively homogenous fraction
One of our laboratories has extensively characterized
BM B lineage stages among the ‘‘Lineage–’’ cells that bear
CD93 (Rumfelt et al., 2006). Simultaneous analysis for ex-
pression of CD19 divides such cells into later (CD19+) and
earlier (CD19–) fractions. Further subdivision based on ex-
pression of CD117, CD127, and B220 delineates at least
three stages of early CD19–hematopoietic cells that in-
clude the CD117hiCD127–B220–, CD117+CD127+B220–,
and CD117+CD127+B220+populations. The CD19+IgM–
of CD24 and CD43 into CD24+CD43+, CD24hiCD43+,
and CD24hiCD43–fractions, similar to previous work
2001; Rolink et al., 1998, 1999, 2004). These cell fractions,
based on changes in cell-surface phenotype, can be ar-
ranged in a linear differentiation scheme, similar to the
one originally suggested for B220+cell stages, but now in-
cluding two earlier precursor fractions, corresponding to
the CD117hiCD127–and CD117+CD127+stages. Based
on their capacity to competitively repopulate hematopoi-
etic lineages in vivo (Rumfelt et al., 2006), these fractions
can be referred to as multilineage progenitor (MLP) and
common lymphoid progenitor (CLP). CLPs defined in this
way identify all of the Lin–CD117+CD127+cells in BM and
so are presumably functionally equivalent to the Lin–
CD117+CD127+Sca-1+CLP cells originally described by
Kondo and Weissman (Kondo et al., 1997). However,
the same name has also been used to refer to Lin–
the lack of conformity in delineating precursor populations
that may lead to confusion. A standardized nomenclature,
established by a broadly representative international
workshop, should help to address this issue. In any case,
the B220+fraction of CD19–stage cells, long referred to
as Fraction (Fr) A, is extensively depleted of alternate line-
age contamination by this staining procedure. It corre-
sponds to a stage that has been called pre-pro-B and is
CD19+IgM–cells can be further subdivided into Fr B and C
(pro-B), Fr C-prime (early or large pre-B), and Fr D (late or
These stages can be used as a framework for further
characterization of developing B lineage cells in BM. For
example, single-cell DNA PCR analysis revealed that
very substantial amounts of Ig heavy-chain DJ rearrange-
chain alleles are rearranged by the CD19+pro-B stage
ysis of lineage potential suggests some revision to previ-
ous models. For example, detectable myeloid potential
remains in CLP-stage cells and decreases sharply in Fr
A, whereas T lineage potential is still readily detected in
Fr A cells (Rumfelt et al., 2006). In the ‘‘lineage potential’’
sense, both CLP and Fr A are ‘‘lymphoid progenitors,’’
and cells resembling Fr A have been referred to as CLP-2
(Martin et al., 2003). Both CLP and Fr A express the IL-7
receptor, CD127, but it seems likely that their immediate
CD127–precursors, identifiable by transcription of the
Rag1 locus, may be activating a broader lymphoid-speci-
fying program. Such ELPs have been isolated with a Rag1
GFP-reporter mouse strain (Igarashi et al.,2002), currently
tained within the MLP fraction.
As alluded to previously, the potential of cells that we
consider ‘‘B lineage specified’’ to generate T cells under-
lies current uncertainty as to the identity of the BM cell(s)
that seeds the thymus and sustains cell production in
that organ (Bhandoola and Sambandam, 2006). The cen-
tral issue is the degree to which cells at the CLP or down-
stream stages of development enter the circulation and
lodge in the thymus (Krueger and von Boehmer, 2007).
In addition, even if they do seed the thymus, the efficiency
with which they produce thymocytes is also an issue
(Petrie, 2007). Clonal analyses that compare in parallel
lations need to be performed in order to measure the num-
Our view is that one should distinguish potential from pre-
from CLP onward are progressing along the B (and NK) lin-
eages (Perry et al., 2006). Particularly in light of mutations
that reduce CLP (and later B lineage stages) while sparing
Immunity 26, June 2007 ª2007 Elsevier Inc.
T lineage development (Allman et al., 2003), it seems prob-
ablethatanearlier hematopoietic progenitor normallycolo-
becomingB lineage specifiedthatis notyetB lineage com-
mitted. Such cells likelyoccupymicroenvironmental niches
in the BM that tightly regulate their progression, although
this issue needs to be rigorously examined. Please see fur-
ther discussion of this complex and important topic else-
where in this issue (Bhandoola et al., 2007; Rothenberg,
Alternative Pathways of B Cell
Development: B-1 and B-2
It is reasonable to conclude that the majority of B lympho-
cytes are produced via the pathway just described,
termed ‘‘B-2’’ (Hardy and Hayakawa, 2001). However, this
lymphopoiesis that parallel this apparently predominant
CLP-to-B-cell route of development exist. For example,
the common myeloid progenitor (CMP), despite its name,
has been reported to retain residual B lymphocyte devel-
opmental potential (Akashi et al., 2000; D’Amico and Wu,
2003). When CMPs were subdivided based on Flt3 ex-
pression, both Flt3+and Flt3?fractions produced myeloid
cells, but only the Flt3+cells generated B lymphocytes.
This latter potential does not appear to be due to contam-
ination of CMPs with more immature hematopoietic pre-
cursors, because T cell developmental potential was not
observed from any CMP fraction (D’Amico and Wu,
2003). It remains to be determined whether CMP-derived
B cells are functionally equivalent to their CLP-derived
B lymphocytes and macrophages are generated has been
described (Montecino-Rodriguez and Dorshkind, 2002).
Precisely where this intermediate should be placed in
schemes of hematopoiesis is not clear. One possibility is
that these B macrophage progenitors are derivatives of
ELPs, CLPs, or even pre-pro-B cells that retain residual
myeloid potential. A second possibility, for which we
must stress no evidence exists, is that they are generated
independently from HSCs or early hematopoietic interme-
diates. A third possibility is that they represent the vestige
of a fetal developmental program that persists into post-
natal life. One reason for proposing this is that B macro-
phage progenitors have been described as a feature of
fetal hematopoiesis (Cumano et al., 1992; Lacaud et al.,
1998). In addition, the Lin?CD45Rlo/?CD19+cells with
which the B macrophage activity is associated can gener-
ate B-1 but not B-2 B cells after transfer in vivo (Monte-
cino-Rodriguez et al., 2006; Tung et al., 2006), and B-1
progenitors are produced most efficiently during embryo-
genesis (Hayakawa et al., 1985).
hematopoiesis are not identical processes (Hayakawa
et al., 1994; Ikuta et al., 1990; Kincade et al., 2002; Li
et al., 1993; Traver et al., 2001). In this regard, the initial
red blood cells produced in the embryo express fetal he-
moglobin, and fetal, but not adult, stem cells can generate
Vg5+gd T cells (Godin and Cumano, 2005). It has also
been proposed that B-1 B cell progenitors are preferen-
tially produced in the embryo and could thus be consid-
ered as part of this fetal developmental program (Hardy
and Hayakawa, 2001; Hayakawa and Hardy, 2000). B-1
B cells account for ?5% of B cells in the mouse, where
they participate in T-independent responses and consti-
tute a high proportion of B cells in the peritoneal and pleu-
ral cavities. In those sites, they exhibit a distinctive sIgMhi
sIgDloCD11b+CD5+B-1a or sIgMhisIgDloCD11b+CD5?
B-1b phenotype (Dorshkind and Montecino-Rodriguez,
2007; Hardy, 2006). Early reports demonstrated the supe-
rior capacity of fetal tissues, in contrast to adult BM, to
generate B-1 B cells and supported their fetal or neonatal
origin, relatively independent of adult B cell development
(Hardy and Hayakawa, 1991; Hayakawa et al., 1985;
Solvason et al., 1991). However, an alternative hypothesis
proposed that B-1 cells develop by distinctive antigen
activation of follicular (‘‘B-2’’) B cells (Cong et al., 1991;
reviewed in Dorshkind and Montecino-Rodriguez, 2007;
genitor sources preferentially generate B-1a (i.e., CD5+) B
cell progeny (Hardy and Hayakawa, 1991; Kantor et al.,
1992), and there is recent evidence that Lin?CD45Rlow?
CD19+cells with B-1, but not B-2, B cell developmental
potential appear before B-2 B cell progenitors in BM
(Montecino-Rodriguez et al., 2006; Tung et al., 2006). It
is also clear that antigen-dependent selection is also re-
quired for the production and likely maintenance of the
mature B-1 B cell pool (Hayakawa et al., 1999). An issue
that remains to be settled is whether comparable B cell
antigen receptor crosslinking results in identical conse-
quences for all newly formed B cells or instead whether
distinctive ‘‘B-1 type’’ cells become mature B-1 B cells
and ‘‘B-2 type’’ cells experience negative consequences.
Experiments with a BCR transgenic model have provided
evidence for this latter alternative (Hayakawa et al., 2003).
Adult BM retains some potential to repopulate B-1 B
cells, although B-1a B cells are reconstituted at a much
lower efficiency than B-1b cells (Hayakawa et al., 1985).
At least some of this activity is attributable to the low
BM.However,theoriginof thesecellsremains unclear.Al-
though they could be the progeny of adult precursors, it is
tempting to speculate, based on recent reports that fetal
HSC survive into postnatal life, that they represent the
remnants of a fetal program. One study reported that
fetal-derived stem cells survive until at least 2 weeks after
birth (Kikuchi and Kondo, 2006), whereas another report
has raised the possibility that, based on differences in
cyclingpotential, BMstem cellsretain fetal characteristics
until at least 3 to 4 weeks after birth (Bowie et al., 2006).
Taken together, these observations suggest that for at
least some time after birth, the BM consists of a develop-
mentally heterogeneous population of HSCs. This obser-
vation in turn raises the possibility that B-1 progenitors
in postnatal BM are derived from such fetal HSC. Confir-
mation, or refutation, of this hypothesis will depend on
testing the potential of putative fetal HSC isolated from
Immunity 26, June 2007 ª2007 Elsevier Inc.
postnatal marrow to recapitulate fetal developmental pro-
grams. It seems evident that fetal and adult HSC are de-
velopmentally distinct, but the conditions in the fetus
and adult that are responsible for their generation have
not been defined. This issue is currently a topic of intense
research, and it is logical to propose that comparing the
fetal and adult environments may provide clues.
The Hematopoietic Microenvironment
and Cell Fate
Blood-cell development is localized to tissues in which
populations of stromal cells provide cellular and soluble
cues that regulate the growth and differentiation of hema-
topoietic stem and progenitor populations. For example,
B cell development in adult BM is associated with a popu-
lation of sessile stromal cells present within the medullary
cells are associated are CXCL12?IL-7+stroma suggests
that distinct cellular nichesthat regulatethe growth,differ-
entiation, and/or survival of developing B lineage cells
exist (Nagasawa, 2006).
The dependence of hematopoiesis on specialized mi-
croenvironmental milieus raises the possibility that the
niche in which an HSC or more differentiated B lineage
progenitor develops or lodges may in turn trigger epige-
netic changes and distinct patterns of gene expression
that dictate whether a fetal or adult developmental pro-
gram will ensue. In this case, the hematopoietic microen-
vironment would function in a deterministic manner. The
precise way in which the adult hematopoietic microenvi-
ronment regulates cell fate decisions is not understood,
and even less is known about how the fetal environment
functions. The dissection of this aspect of fetal hemato-
poiesis will be complicated by the fact that blood-cell de-
velopment, and the potential to generate B cells, is asso-
ciated with distinct intra- and extraembryonic tissues
at different times during embryogenesis (Godin and
Nevertheless, information about the microenvironmen-
tal signals involved in fetal and postnatal B lymphopoiesis
is emerging. For example, IL-7 has been considered to be
an obligate B lymphopoietic factor based on the finding
that B lymphopoiesis does not occur in IL-7-deficient
mice (von Freeden-Jeffry et al., 1995). However, a subse-
quent report demonstrated that B-1 B cells are produced
(Carvalho et al., 2001), and indications are that this is due
in part to the effects of thymic stromal lymphopoietin
(TSLP) on fetal B cell progenitors (Vosshenrich et al.,
2003, 2004). Additional cytokines such as Flt ligand may
also play a role in fetal B lymphopoiesis (Sitnicka et al.,
2003). A major goal will be to determine how the environ-
for the initiation of different B lineage developmental pro-
grams. It will be particularly interesting in this regard to
determine whether differences in PU.1 expression, which
is required for B-2 but not B-1 development, are regulated
by environmental cues (Rosenbauer et al., 2006; Ye et al.,
There is a growing appreciation that exogenous stimuli
may influence HSCs and B lineage progenitor fate in post-
natal BM as well. As mentioned above, artificial delivery of
Wnt signals or targeting of the Pax5 gene in B cell progen-
itors causes them to lose differentiated properties and
become multipotential (Baba et al., 2005; Mikkola et al.,
2004). In another study, newly formed B cells downregu-
lated a number of genes when the antigen receptor was
conditionally targeted (Tze et al., 2005). It is not yet known
whether any normal cues can reverse differentiation in
BM, but it certainly seems possible. Moreover, progeni-
tors normally express receptors that can signal dramatic
changes in their fates.
In addition to signals from the hematopoietic microenvi-
duction to a degree that has only recently been revealed.
Although the use of antigen-specific receptors expressed
that will become effective contributors to the adaptive im-
mune system has been extensively studied, it has only re-
cently been appreciated that hematopoietic progenitors
express functional Toll-like receptors (TLR) and associ-
ated signaling molecules (Kim et al., 2005; Nagai et al.,
2006; Sioud et al., 2006). This observation challenged the
long-held assumption that stem cells were incapable of
self-nonself recognition, and we now know that they can
genitors, and long-term repopulating HSCs in the mouse
express relatively high amounts of TLR2, TLR4, MD-2,
TLR9, and CD14 (Nagai et al., 2006; P.W.K., unpublished
Exposure of stem cells to ligands for either of two TLRs
(lipopeptide for TLR2 and LPS for TLR4) drives them into
cycle and promotes acquisition of lineage markers. Mye-
loid progenitors are normally dependent on growth and
differentiation factors (M-CSF, G-CSF, GM-CSF, etc.),
but they become activated macrophages after stimulation
with just TLR ligands. This change happens within 72 hr in
tralizing antibodies to cytokines were added (Nagai et al.,
2006). In contrast, the influence of TLR ligands on CLPs
is quite different. In that case, B lineage differentiation in
culture is totally blocked, and all progenitors are directed
to a myeloid dendritic cell phenotype (Nagai et al., 2006).
More recent studies have shown that a TLR9 ligand redi-
rects lymphoid differentiation toward production of den-
dritic cells and this phenomenon occurs in virus-infected
mice (P.W.K., unpublished data). Human CD34+hemato-
poietic cells express TLR2, TLR4, TLR7, TLR9, and
TLR10 (Kim et al., 2005; Sioud et al., 2006). Although it is
not yet clear whether TLR ligands alter human lymphopoi-
esis, the receptors are functional, transmitting signals for
chemokine and cytokine production as well as increased
The fate of highly purified lymphocyte progenitors is
altered when they are directly exposed to TLR ligands in
serum-free, stromal cell-free cultures, but only if they
express the corresponding TLR and necessary signaling
molecules (Nagai et al., 2006; P.W.K., unpublished data).
Immunity 26, June 2007 ª2007 Elsevier Inc.
Furthermore, TLR-defective cells are not influenced as
‘‘bystanders’’ when present in the same cultures. This
suggests that induction and autocrine responsiveness to
growth factors is not essential for redirection of differenti-
types in response to microbial and viral products. As one
example, cells corresponding to late stages of B lympho-
poiesis are mobilized from the BM when any of a series of
substances are placed in the peritoneal cavity (Jyonouchi
et al., 1981; Nagaoka et al., 2000; Ueda et al., 2004, 2005).
This phenomenon is highly dependent on production of
tumor necrosis factor and downregulation of the chemo-
influence what cells are made in the BM but also how long
they are retained. Lymphoid progenitors can be directly
affected by TLR signals as well as by cytokines released
in their immediate environment.
In addition to pathogen products, many endogenous
substances are thought to be TLR ligands (Tsan and Gao,
2004). It is easy to imagine that their release from dam-
aged marrow cells during chemotherapy or irradiation
would alter patterns of blood-cell formation. There is as
yet no indication from gene-targeting studies that TLR or
related molecules are essential for building the immune
system. Rather, their expression by hematopoietic cells
could primarily provide threat-response capability. That
is, patterns of blood-cell formation in, and mobilization
from, BM could quickly change in order to boost re-
sponses to serious pathogens. It is too early to know
whether either the suppression of lymphopoiesis or aug-
mentation of dendritic cell production is protective during
mechanism has deleterious long-term effects on stem
cells or other components of the immune system. For ex-
ample, chronic stimulation might exhaust the self-renewal
potential of HSC, causing them to prematurely age. Den-
dritic cells have been implicated in autoimmune disease,
erbate symptoms. These new findings add to other infor-
mation suggesting that all B lymphocytes may not be pro-
duced through precisely the same pathways and at the
same rates. Rather, the marrow may be more responsive
to circumstances than previously thought.
Progression to Mature Functional B Cell
After production in the BM, newly formed B cells are func-
tionally immature and migrate to the spleen where they
either die or undergo further maturation. Maturing splenic
B cells can be subdivided by differential expression of
several cell-surface proteins, including CD21, CD23,
CD24 (HSA), and CD93 (AA4.1), into ‘‘transitional B cells,’’
cells that progress from T1 (CD93+CD21–CD23–) to T2
(CD93+CD21–CD23+), culminating in downregulation of
IgM, loss of CD93, and upregulation of CD21 to yield the
mature follicular (Fo) phenotype (Allman et al., 2001). An-
other transitional stage, considered intermediate between
T2 and Fo (termed T3), has recently been suggested to
harbor cells undergoing negative selection because of
self-reactivity (Merrell et al., 2006), but the relative propor-
tions of bona fide transitional cells compared to such
‘‘anergic’’ B cells remain to be established. Transitional
stage cells are short-lived, as shown by bromodeoxyuri-
dine incorporation (Hsu et al., 2002), and are not function-
ally competent, as shown by the fact that they fail to
proliferate after BCR crosslinking (Allman et al., 2001).
Furthermore, such BCR crosslinking of immature B cells
results in B cell tolerance rather than an immune response
(King and Monroe, 2000). Studies with transgenic models
of self-reactivity have shown that these B cells can be de-
leted, undergo receptor editing, or rendered functionally
unresponsive (anergic) by BCR signaling (Chen et al.,
1997b; Erikson et al., 1991; Goodnow et al., 1988; Nema-
zee and Burki, 1989; Tiegs et al., 1993).
Most immature B cells lacking such self-reactivity enter
These cells are termed follicular (Fo) B cells because of
their localization to the B cell follicle region (Rolink et al.,
2004). Entry into this anatomical site constitutes the final
stage in maturation for developing BM B cells, because
competition for this site is compromised in several trans-
genic models of B cell tolerance (Cyster et al., 1994; Man-
dik-Nayak et al., 1999). These cells do not proliferate but
persist in the resting state for several months. A condi-
tional knockout study, eliminating expression of the BCR
by V region gene deletion, revealed that expression of
the BCR is required for cell survival (Lam and Rajewsky,
1998). It remains to be determined whether this is due to
‘‘tonic signaling’’ resulting from simple assembly of the
BCR signaling complex or instead reflects a kind of ‘‘con-
stitutive positive selection’’ signaling by low-affinity bind-
ing to crossreactive self-determinants.
Marginal zone (MZ) B cells are a second population of
mature splenic B lymphocytes. Along with specialized
macrophages, they are localized in a distinct anatomical
region of the spleen that represents the major antigen fil-
tering and scavenging area (Martin and Kearney, 2000;
Martin et al., 2001). It appears that MZ B cells, like B-1 B
cells, are preselected to express a BCR repertoire that is
biased toward bacterial cell-wall constituents and senes-
cent self-components such as oxidized low-density lipo-
proteins. Also similar to B-1 B cells, MZ B cells respond
very rapidly to antigenic challenge, likely independently of
T cells, and also participate in the early phase of T-depen-
B-1 B cells are abundant in the peritoneal cavity, but are
also found in spleen (Hayakawa and Hardy, 1988). Al-
though they constitute a low proportion of the total B cell
pool in spleen, their total number is comparable with that
in the peritoneal cavity. Unlike Fo B cells, they persist
cells, as shown in cell-transfer studies (Hayakawa et al.,
1986). The most distinctive feature of CD5+B-1a B cells
is their elaboration of autoantibodies for certain self-spec-
ificities that include branched carbohydrates, glycolipids,
tidylcholine (PtC), the Thy-1 glycoprotein, viral coat
Immunity 26, June 2007 ª2007 Elsevier Inc.
proteins, and bacterial cell-wall constituents (Baumgarth
et al., 2000; Hardy et al., 1989; Hayakawa et al., 1984,
tive antibodies are not pathogenic, but rather are termed
tigation, but, as with MZ B cells, at least some are thought
to function in clearance of senescent cells or proteins and
to provide an initial immunity to common bacterial or viral
pathogens, serving as a kind of ‘‘hard-wired’’ memory B
cell population (Baumgarth et al., 2005).
Even though B-1, MZ, and Fo B cells may develop from
distinct precursors (Hardy and Hayakawa, 1991; Haya-
kawa et al., 1985; Kantor et al., 1992; Montecino-Rodri-
guez et al., 2006), signaling through the BCR is emerging
as a common mechanism that guides their transition to
maturity after the appearance of surface IgM (Casola
et al., 2004; Hayakawa et al., 1999, 2003; Lam and Rajew-
sky, 1999; Tze et al., 2005; Wen et al., 2005). Various
transgenic models of self-reactivity have shown that al-
though most BCR signaling at the immature stage results
in negative consequences (Goodnow et al., 1988; Hartley
et al., 1991; Nemazee and Burki, 1989; Tiegs et al., 1993),
some specificities can guide at least some B cells into the
B-1 pool (Arnold et al., 1994; Chumley et al., 2000; Haya-
kawa et al., 1999, 2003; Wasserman et al., 1998), particu-
larly natural autoantibodies such as anti-PtC and ATA
(anti-thymocyte autoantibody). Study of one ATA, specific
for CD90 (Thy-1), is particularly informative because the
self-antigen is a protein that can be readily manipulated
(Hayakawa et al., 1990). Thus, B cells with this ATA BCR
can be studied in the normal CD90+context, where it is
a natural autoantibody, and also in a CD90-deficient con-
text, where it is not. Transgenic mice on a CD90 wild-type
background that expresses a BCR specific for ATA de-
velop a B-1 B cell population and have high amounts of
ATA in serum, whereas such animals on a CD90-deficient
B cells expressing the ATA BCR in these mice develop
predominantly to the Fo B cell fate, consistent with the
model that B-1 cells are produced through a process of
positive selection whereas Fo B cells require little (or no)
selection by antigen.
While selection by antigen is now well established for
B-1 B cells, the role of such selection in MZ B cells is more
Studies with a VH81X transgenic mouse revealed striking
BCR usage, pairing a single light chain with the heavy-
chain transgene in all B cells of the MZ B phenotype, likely
indicating antigen-driven selection (Chen et al., 1997a).
However, a different group of investigators studying mu-
tant mice where BCR signaling was weakened proposed
a diametrically different model of MZ B cell development,
suggesting that these cells are uniquely unreactive to self
or environmental antigens (Cariappa et al., 2001). More
recently, work with the anti-CD90 ATA model, where
transgenic B cells developed in the presence of different
amounts of self-antigen, provided support for antigenic
selection in the origin of (at least some) MZ B cells (Wen
et al., 2005). That is, although ATA B cells developing in
a CD90-deficient background became Fo B cells, such
cells predominantly adopted a MZ B cell phenotype when
developing in the presence of very low amounts of CD90.
Furthermore, these MZ B phenotype cells localized to the
marginal zone of spleen and showed rapid response to
BCR signaling, typical of normal MZ B cells.
Studies with anti-CD90 ATA BCR transgenic mice sup-
port a model where different amounts of signaling at the
immature B cell stage are critical for development of the
three functionally distinct mature B cell populations (Fig-
ure 2). This signaling can arise from crosslinking of the
BCR, but it can also derive from other microenvironmental
sources, specifically via B cell-activating factor (BAFF) or
Notch2 signaling (Saito et al., 2003; Schiemann et al.,
2001; Schneider et al., 2001). Thus, Fo B cell development
is critically dependent on BAFF signaling, rather than
BCR crosslinking, while B-1 development is absolutely
dependent on BCR crosslinking and independent of
BAFF (Schiemann et al., 2001). MZ B cell development
uniquely requires Notch2 signaling (Saito et al., 2003;
Tanigaki et al., 2002). Entry into these mature B cell pop-
ulations will therefore depend on the cell’s capacity to ex-
perience BAFF or Notch2 signals, i.e., receptor expres-
sion, in addition to the level of its BCR signal (Lam and
Rajewsky, 1999). Furthermore, it seems likely that both
BCR diversity and cellular context will differ between fetal
(B-1) and adult (B-2) B cell development (Benedict et al.,
2000; Benedict and Kearney, 1999; Hao and Rajewsky,
2001; Hayakawa et al., 1994; Marshall et al., 1998). This
model posits a gradation of signaling, with BCR predom-
inant for B-1 B cells, weaker BCR (plus Notch2) for MZ
B cells, and BAFF alone (or with very weak BCR signaling)
for Fo B cells. This graded development is consistent with
B-1 cell generation in an ‘‘immature’’ microenvironment,
the neonatal spleen, with Fo production dependent on
a fully mature adult microenvironment.
Concluding Remarks and Remaining Questions
Throughout this review, we have summarized the tremen-
dous advances that have been made in our understanding
of B cell development and highlighted many of the issues
that still remain. The mouse has been used to develop
much of this current understanding of B lymphopoiesis,
Figure 2. Reciprocal Dependence on BCR and BAFF
Signaling of Three Mature B Cell Populations and
Predominant Developmental Timing
Immunity 26, June 2007 ª2007 Elsevier Inc.
andithasservedwellasamodel system.Indeed, theprin-
ciples learned from that species have provided a founda-
tion for studies of human HSC and B cell biology. Never-
theless, numerous gaps in our understanding of human
B cell development remain (see commentary by Payne
and Crooks, 2007, in this issue). For example, although
B-1 B cellshave been shown to be associated with certain
autoantibodies (Casali et al., 1987; Hardy et al., 1987) and
autoimmune conditions (Dauphinee et al., 1988) in hu-
mans, little is known about the developmental origins of
these cells. Our understanding of how human B lympho-
poiesis is regulated also remains incomplete. Thus, al-
though B-2 B cell development in the mouse is absolutely
dependent on IL-7, this cytokine does not function as an
obligate B lymphopoietic factor in humans. Whether there
exists a single factor on which human B cell development
is dependent or whether instead multiple cytokines regu-
late this process remains to be determined. Such informa-
tion is relevant to the development of strategies to boost
therapy or, possibly, to the generation of new lympho-
cytes from human embryonic stem cells. Finally, although
age-associated declines in B cell production in mice are
well documented, the effects of senescence on human
B cell development remain largely unexplored (Rossi
et al., 2003). Thus, although studies with mice will provide
studies of human Blymphopoiesis shouldbe encouraged.
Work discussed here was supported by NIHgrants toR.R.H. (AI 26782
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