Early decisions in lymphoid development
Min Ye1,2and Thomas Graf3
Recent research suggests that lymphoid progenitors in the
bone marrow comprise a heterogeneous cell population. This
population first loses megakaryocyte/erythroid, and then
granulocyte/macrophage, potential before committing to
lymphoid lineages. B and T cells can originate by way of
different pathways that appear to be used with varying
frequencies in the animal. In the bone marrow, B cell
of transcription factors and IL-7 signaling. In the thymus,
multipotent progenitors become committed to the T-cell
lineage through the action of Notch1. The activated
intracellular form of Notch1 suppresses transcription
factors that can instruct myeloid cell fates, thereby directly
coupling extracellular signaling with changes in
transcriptional networks. In conclusion, although a lot is
known about B and T cell commitment, more work
needs to be done to clarify the earliest steps in
1Department of Developmental and Molecular Biology, Albert Einstein
College of Medicine, New York-Bronx, NY 10461, USA
2Harvard Institutes of Medicine, Room 954, 77 Avenue Louis Pasteur,
Boston, MA 02115, USA
3ICREA and Center for Genomic Regulation (CRG), Dr. Aiguader 88,
08003 Barcelona, Spain
Corresponding author: Graf, Thomas (firstname.lastname@example.org)
Current Opinion in Immunology 2007, 19:123–128
This review comes from a themed issue on
Edited by James Hagman and Dietmar Kappes
Available online 15th February 2007
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# 2006 Elsevier Ltd. All rights reserved.
The developmental origin of lymphoid cells from a
hematopoietic ancestor has been controversial. According
to a scheme proposed by Akashi, Kondo, Weissman and
co-workers [1,2], lymphoid cells originate from multi-
potent progenitors (MPPs) within the Lin?Sca-1+c-Kithi
(LSK) fraction, which in turn are derived from hemato-
poietic stem cells (HSCs). MPPs branch into common
towards B and T cells , and common myeloid progeni-
tors (CMPs) , which give rise to megakaryocyte/ery-
throid progenitors (MEPs) and granulocyte/macrophage
progenitors (GMPs) .
In this article, we will discuss recent work that has
challenged this simple scheme and replaced it by a more
complex model. We will also review the roles of various
hematopoietic transcription factors, as well as IL-7 recep-
tor and Notch signaling, at the initiation of the B and
T cell pathways.
New insights into lymphoid–myeloid
Approximately one and a half years ago, a study by
Adolfsson et al. [3??] reported a heterogeneity within
the MPP compartment: FMS-like tyrosine kinase 3low
(Flt3lo) MPP cells exhibited megakaryocyte/erythrocyte
(MegE), granulocyte/macrophage (GM) and lymphoid
potentials, whereas Flt3hiMPP cells lacked significant
MegE potential but still exhibited B and T lymphoid as
well as GM potential. Based on these findings, it was
proposed that in a first restriction step MPPs lose their
MegE potential, becoming lymphoid-primed MPPs
(LMPPs). This scheme also raised the possibility that
GM cells can be generated by way of two separate routes:
one through MPPs and another through LMPPs. Exper-
iments using reporter mice for Ikaros — a zinc finger
DNA-binding protein expressed in hematopoietic stem
cells and progenitors that plays an essential role in early
hematopoiesis — support this new scheme [3??]. In these
mice the IkaroshiLSK fraction, which is also Flt3hi, has
GM and T cell but no MegE potential [4?]. However, the
MPP–LMPP scheme was challenged yet again in an
article by Forsberg et al. [5?], which described that
Flt3+LSK cells retain MegE potential after transplan-
tation into irradiated mice. Although the reason for the
apparent discrepancy between the Adolfsson and the
Forsberg studies is not clear, it might be explained by
differences in the analyses of the in vivo assays used.
A recent study by Lai and Kondo [6??] showed that MPP
cells can be sub-divided into Flt3lovascular cell adhesion
molecule-1+(VCAM-1+), Flt3hiVCAM-1+and Flt3hiV-
CAM-1?cells. These subsets, called MPP, LMPP1
part of a continuum. It is probable that the LMPP1 and
LMPP2 cells correspond to the LMPP cells described by
Adolfsson et al. [3??]. When tested by transplantation into
irradiated hosts, the three subsets have similar lymphoid
potentials but lose their ability to give rise to MegE cells
before losing their GM-forming capacity [6??] (indicated
by the wedges in Figure 1). Because LMPP2 cells already
express the lymphoid genes Rag1 and IL-7R at low levels,
they overlap with early lymphoid progenitors, described
on the basis of Rag1 promoter-driven green fluorescent
protein (GFP) expression .
Current Opinion in Immunology 2007, 19:123–128
Predominant physiological contributions of
multilineage progenitors in T-cell
The physiological significance of CLPs in the generation
of T cells has been questioned by the finding that the
predominant progenitors in the circulation that have T
lineage potential have an LSK phenotype [8,9]. The high
T cell forming efficiency of circulating LSK cells com-
pared with that of CLPs strengthens this argument . In
addition, using clonal assays with thymocytes from
CCR9–GFP mice, a subset of early T lineage progenitors
(ETPs) was identified to have T, B, natural killer (NK)
and GM potential [10?]. Other work showed that a subset
of DN1 cells (CD44hiCD25?) that resemble ETPs can
form macrophages and myeloid dendritic cells at low
frequencies [11,12]. Therefore, early T-cell progenitors
in the postnatal thymus appear to be multipotent, indi-
cating that lymphoid restriction is not necessary for thy-
mus entry. Because MegE potential has never been
reported for thymic precursors it is possible that TSCs
predominantly correspond to the more restricted LMPPs.
Ikaros and PU.1 as initiators of the lymphoid
Hallmarks of lymphoid commitment in the bone marrow
arethegradualup-regulation ofFlt3andIL-7Raaswell as
of Rag1 and Rag2 [3??,6??]. The fact that none of these
genes are expressed in hematopoietic progenitors of
Ikaros-null mice [4?,13] and that their 50regulatory
regions contain functional Ikaros binding sites [14,15]
suggests that Ikaros is an important initiator of the lym-
phoid program. Consistent with this, Ikaros-null mice, or
mice that express a dominant-negative form of Ikaros,
lack all B cells, NK cells and T cells . However,
LMPPs can still be detected in Ikaros?/?mice. These
cells lack B-cell potential and exhibit strongly reduced
T-cell potential, although retaining GM potential [4?].
Therefore, Ikaros is crucial for the initiation of the lym-
phoid program but not for the generation of LMPPs,
although it still needs to be shown that reintroduction
of Ikaros can rescue their B-cell potential.
precursors, but at different levels: high in GM cells, inter-
mediate in B cells, low in LSK cells and undetectable in
pre-T cells [4?,17–19]. PU.1-null mice die around birth,
contain no B, T, NK or myelomonocytic cells in the fetal
liver, and have dramatically reduced numbers of LMPPs
(here defined as Kit+AA4.1+Flt3+IL-7R+) . However,
because PU.1?/?animals show a defect in HSC self-
renewal , it is still possible that the absence of LMPPs
is secondary to a reduction in HSC numbers.
Proposed scheme of early lymphocyte development in the adult mouse. Starting with hematopoietic stem cells (HSCs), multipotent progenitors
(MPPs) within the Lin–Sca-1+c-Kithi(LSK) cell fraction (red boxes) gradually first lose megakaryocyte/erythroid (MegE) and then granulocyte/
macrophage (GM) potential before becoming lymphoid-committed. Lymphoid-primed MPP 2 (LMPP2) cells have minimal MegE and GM potential
and overlap with early lymphoid progenitors (ELPs). Common lymphoid progenitors (CLPs) and early B cell progenitors have NK and T cell
differentiation potentials. LSK cells are continuously released from the bone marrow and seed the thymus through the circulation. Thymic seeding
cells (TSCs) with T as well as B, NK, myeloid and dendritic cell (DC) differentiation potentials become early T lineage progenitors (ETPs) within
the thymus and have an LSK phenotype (indicated by the red box). TSCs and ETPs, in turn, are the predominant subsets that have T-cell
potential within the DN1 fraction (Lin–CD44hiCD25–CD4lo). Black arrows denote differentiation; wedges indicate decreasing probability of
differentiation potential. The asterisks indicate points of B and T cell commitment. The dashed red lines indicate low contributions of progenitors
Current Opinion in Immunology 2007, 19:123–128 www.sciencedirect.com
Reflecting the different expression levels of PU.1 in
lymphoid and GM cells, the role of this factor in lineage
specification is dosage-dependent. Thus, expression of
high levels of PU.1 in PU.1+/?fetal liver progenitors
generates macrophages, whereas lower levels induce
B-cell formation . PU.1 dosage might also be import-
ant for the restriction of MPPs because PU.1 expression
increases slightly during the transition between HSCs/
MPPs and LMPPs [4?]. It is possible that this increase
inhibits MegE formation through the known antagonism
of PU.1 towards GATA-1 — a transcription factor essen-
tial for MegE development [23,24]. In support of this,
Flt3+CMPs, which have no MegE potential, show ele-
vated levels of PU.1, whereas Flt3–CMPs with high
MegE potential express lower or similar levels of PU.1
as in HSCs .
PU.1 is not strictly required for B-cell development
because B lineage cells can be grown out of PU.1?/?fetal
liver cells on S17 stromal cells in the presence of stem cell
factor and IL-7. These cells, which are B220?CD19+,
express IL-7R and transcription factors E2A and early B
cell factor (EBF) at levels comparable to wild-type B-cell
precursors. However, PU.1?/?B-lineage cells develop at
low efficiencies and with delayed kinetics compared with
wild-type cells [26?]. PU.1 therefore facilitates B-cell
development, possibly by accelerating the up-regulation
the rapid onset of EBF expression in PU.1?/?fetal liver
precursors, and both EBF and IL-7Ra genes contain PU.1
binding sites in their upstream regulatory regions . A
recent study on transcriptional activation of myeloid
specific gene colony-stimulating-factor 1 (CSF-1) receptor
gene (c-fms) indicates that PU.1 regulates c-fms expression
in a two-step mechanism. In HSCs and early progenitor
cells it binds to c-fms promoter region, keeping c-fms in a
and other factors to the c-fms enhancer region in differ-
entiated myeloid cells, causing full activation of c-fms
[27??]. The similarities in lymphoid defects between
observations that Ikaros is expressed in PU.1-deficient
and LMPPs (Katia Georgopoulos, personal communi-
cation) do not support such a relationship. It is therefore
more likely that they have non-redundant functions in the
same or parallel pathways.
Roles of E2A, EBF and IL-7R in B-cell
The specification of B-cell development involves the
expression of the pre-B cell receptor components VpreB,
l5 and mb-1 and the initiation of D–J rearrangements at
the IgH locus.The transcription factors E2A and EBF are
essential for these events. In the absence of either E2A or
EBF, B-cell development is arrested before the onset of
D–J heavy chain rearrangements and the up-regulation of
VpreB, l5, and mb-1. However, surprisingly, conditional
deletion of E2A in pre-B cells does not cause a dramatic
decrease in the expression of its target genes VpreB, l5,
mb-1, B29 and IgH [28?], suggesting that E2A, like PU.1,
is required for the initiation but not for the maintenance
of B-cell gene expression.
A key player in B-cell specification is EBF. Thus,
enforced expression of EBF can activate the B lineage
program in the absence of E2A  or PU.1 , raising
the possibility that the primary function of E2A (and of
PU.1) in B-cell development is to facilitate EBF up-
regulation. In support of this theory, an E2A binding site
has been identified in the upstream regulatory region of
EBF , and E2A expression in a macrophage-like cell
line induces EBF up-regulation . The fact that no
B-cell development can be detected in Ikaros-null mice
might be caused by a lack of EBF expression, predicting
that EBF can rescue B-cell development in these mice. A
negative outcome would suggest that the two genes
function in different pathways or that Ikaros requires
additional factors to induce B-cell development.
A detailed study of B cell specific gene regulation  has
demethylation and chromatin remodeling of the mb1
promoter. DNA demethylation in turn is necessary for
the transcriptional activation of mb-1 by Pax5. The con-
tingency of Pax5 on EBF activity explains the observation
the B-cell differentiationdefectofhematopoietic progeni-
specification is reinforced by IL-7 signaling: B-cell devel-
(B220+CD19?CD43+BP1?HSA?) in IL-7- and IL-7Ra-
deficient mice, and both types of mutant progenitors lack
EBF expression [33?,34?]. Stimulating these IL-7?/?pro-
pre-pro-B cells using activated signal transducer and
activator of transcription 5 (Stat5, a downstream effector
of the IL-7 receptor) induces rapid up-regulation of EBF.
Moreover, enforced expression of EBF in IL-7R- or IL-7-
deficient HSCs or CLPs rescues their capacity to differ-
entiate along the B-cell lineage [33?,34?]. Together, these
data indicate that Ikaros andPU.1 initiate theformationof
lymphoid precursors, whereas E2A, IL-7R, EBF and Pax5
are involved in B-lineage specification .
Hierarchical roles of E2A and Pax5 in B-cell
B-cell commitment is characterized by the extinction of
alternative lineage potentials in B-cell progenitors. Thus,
whereas pre-pro-B cells still have T and NK potentials,
CD19+pro-B cells are fully restricted [34?,35]. E2A and
Pax5 mediate this step, as pro-B cells deficient in either
gene promiscuously express non-B lineage genes. In
Early decisions in lymphoid development Ye and Graf 125
Current Opinion in Immunology 2007, 19:123–128
addition, when injected into lethally irradiated mice, they
can give rise to T-lineage cells, macrophages, granulo-
cytes and erythrocytes [36,37]. However the two factors
differ in their MegE suppressing potentials. Irradiated
mice transplanted with E2A?/?pro-B cells develop sig-
nificant numbers of erythroid progenitors already after
one month . By contrast, in Pax5?/?pro-B cell trans-
planted mice, erythrocytes can only be detected four
suggest that E2A suppresses MegE as well as GM and T
potentials, whereas Pax5 predominantly inhibits GM and
T potentials. The hierarchical roles of E2A and Pax5
broadly support the progressive specification model
shown in Figure 1. They also raise the possibility that
E2A, like PU.1, functions between the MPP and the
LMPP stages, with Pax5 acting mainly at or after the
pro-B cell stage. The lineage-repressive functions of Pax5
and E2A in B-cell commitment are probably caused by
the inhibitionoftranscription factors that have alternative
lineage instructive capacities. Consistent with this,
enforced expression of C/EBPa can reprogram CLPs
and committed B-cell precursors into macrophages
[39,40], and GATA-1 can convert CLPs into MegE cells
Notch signaling and T-cell commitment
T-cell commitment occurs following interaction of T-cell
precursors with Notch ligands presented by the thymus
epithelium. The observation that ETPs are absent from
the thymus when Notch signaling is inhibited, whereas
circulating LSK progenitors are unaffected, suggests that
crucial Notch signaling occurs soon after MPPs colonize
the thymus [42??]. An earlier study showed that inacti-
vation of Notch1 leads to the expansion of B lineage cells
in the thymus at the expense of T-cell development .
This could be either because multipotent T-cell progeni-
tors differentiate along an alternative route or because
circulating B-cell precursors seed newly available niches.
Surprisingly, however, B-cell production in the thymus
was not augmented after Notch signal inhibition induced
by the expression of a dominant-negative inhibitor of
CSL in HSCs or transgenic expression of lunatic fringe in
T-lineage cells [42??,44??]. These observations are con-
sistent with the proposal that low-level Notch activity is
sufficient to block the B-cell potential of TSCs whereas
higher levels are required to promote ETP and DN cell
proliferation . They also suggest that targets of
Notch1 that mediate weak signaling effects in TSCs
remain to be discovered.
In a culture system that mimics the thymus microenvir-
onment — the OP9-delta like 1 (OP9-DL1) stromal cell
line — multipotent fetal liver hematopoietic progenitors
differentiate along the T-cell lineage . In this system,
expression of the Notch target hairy and enhancer of split
1(HES1), as well as the T cell transcription factors GATA-
and three days in culture. In spite of this, at least some
progenitors cultured for a whole week on OP9-DL1
stroma still retained B potential when placed in B lineage
permissive conditions, indicating that T-cell commit-
ment is a prolonged process that requires continuous
Notch signaling [47?].
Although Notch1 and GATA-3 are expressed in ETP and
DN2 cells, T-cell commitment does not occur until the
DN3 stage, coincident with the down-regulation of PU.1
[48,49] and of C/EBPa [50??]. This raises the possibility
that T-cell commitment requires the down-regulation of
both PU.1 and C/EBPa to prevent differentiation along
myeloid pathways. This is supported by the observation
that overexpression of PU.1 in fetal liver progenitors
blocks T-cell development and activates Mac-1 .
Likewise, in DN3 and DN4 cells, PU.1 induces the
formation of myeloid dendritic cells whereas C/EBPa
induces the generation of macrophages [50??]. The pic-
ture emerging is that Notch signaling initiates T-cell
commitment in early T-cell progenitors through down-
regulation of PU.1 and C/EBPa, thereby suppressing
alternative differentiation potentials.
Scheme of developmental flows within the hematopoietic lineage tree. This tree is highly speculative and includes some assumptions not
discussed in the review. Several cell types are represented incompletely (e.g. NK, DCs) or not at all (e.g. eosinophils, mast cells). Coloured lines
indicate pathways leading to the formation of different lineages, with thickness of lines indicating flow rates (i.e. the frequency of pathway usage
under physiological conditions). Abbreviations: CMP, common myeloid progenitor; DN, double-negative cell; DP, double-positive cell.
Current Opinion in Immunology 2007, 19:123–128 www.sciencedirect.com
The data reviewed suggest that the controversy about
early lymphoid lineage decisions is beginning to subside.
MPPs are heterogeneous, and early restriction points
might represent a spectrum of commitment probabilities
rather than simple binary decisions (Figure 1). Commit-
ment is mediated by transcription factor interactions that
can be modulated by IL-7R signaling in the bone marrow
and Notch1 in the thymus. The lineage potential of
progenitors in culture or after transplantation does not
necessarily reflectthefrequency withwhichthis potential
is realized under physiological conditions. In an attempt
to visualize the flow and the dynamics of the adult
hematopoietic system, we have drawn in Figure 2 a
diagram that resembles the delta of a river, where differ-
ent branches carry different qualities and amounts of
water. Future work will be needed to further address
the physiological importance of alternative developmen-
We thank Katia Georgopoulos for providing unpublished data and
Avinash Bhandoola, Catherine V Laiosa and Matthias Stadtfeld for
comments on the manuscript. This work was supported by
National Institutes of Health (NIH) grants R01 CA89590-01 and R01
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This article describes a Flt3+subset of ETPs in the thymus that exhibits
weak B-lineage potential. The generation and subsequent differentiation
of ETPs was found to require Notch signaling. In contrast, circulating T-
cell precursors were still detectable, even in the absence of Notch
signaling, suggesting that Notch signals crucial for T-lineage develop-
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43. Wilson A, MacDonald HR, Radtke F: Notch 1-deficient common
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J Exp Med 2001, 194:1003-1012.
In contrast to the previously described increased B-cell formation in
Notch-knockout mice , this study reports that the B-cell potential
of ETPs is not augmented under conditions that limit Notch1 activation. It
suggests that weak Notch signals are sufficient to suppress B-cell
production by thymus seeding cells before reaching the ETP stage
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Using clonal switch-culture analyses, this study showed that, for as long
as one week after culturing fetal liver progenitors on OP9-DL1 stroma,
progeny of some pluripotent progenitors still maintained B-cell potential.
This suggests that continuous Notch signaling is required for the main-
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This study shows that expression of the macrophage-associated tran-
scription factors C/EBPa and PU.1 in fully committed pre-T cells converts
the cells into functional macrophages or myeloid dendritic cells, respec-
tively. The induced differentiation is inhibited by Notch signaling, indicat-
ing a reciprocal antagonism between C/EBPa and PU.1 on one hand and
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